Abeloff's Clinical Oncology, 4th Edition

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Memorial

Martin D. Abeloff, MD

Martin D. Abeloff, a founding editor of Clinical Oncology, died on September 14, 2007 in Baltimore, Maryland after a courageous battle with cancer, a disease he had dedicated his life to fighting. He was a wonderful and caring clinician, an extremely effective leader, and a beloved mentor to many trainees and young faculty. Marty was the kind of friend you hope to have and the kind of clinician and clinical scientist that you would want to be. He will be sorely missed as a colleague, a physician, a father, a husband, and a friend. Marty was born on April 4, 1942 in Shenandoah, Pennsylvania. He received his BA from The Johns Hopkins University in 1963, and his MD from The Johns Hopkins University School of Medicine in 1966. He spent the next year as an intern at the University of Chicago Hospitals and Clinics. In 1967, he eclipsed his already numerous accomplishments by marrying Diane Kaufman. The two daughters of this union, Alisa and Jennifer, represent Marty’s most enduring and important legacy.

His second legacy, in medicine, was established on his return to Baltimore in 1971, as a fellow in clinical oncology. He would spend the rest of his career at The Johns Hopkins Hospital. He achieved the rank of Professor of Medicine in 1990 and, at various times, served as the training program director, chief of medical oncology, clinical director of the cancer center, oncologist in chief at The Johns Hopkins Hospital, and, since 1992, the director of The Johns Hopkins Oncology Center, later renamed the Sidney Kimmel Comprehensive Cancer Center. During that time, Marty brought to life the idea of a comprehensive, user-friendly textbook of oncology that would be as valuable to the practicing oncologist as to the primary care physician and physicians-in-training. The first edition of Clinical Oncology was published in 1995 to a gratifying response. It is now established as a cornerstone reference for those caring for patients with cancer. In the fourth edition, we mourn the loss of our friend and colleague, but we continue Marty’s vision for a better, unique, and

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user-friendly text. In his honor, this edition has been renamed Abeloff’s Clinical Oncology so that future generations of oncologists will remember his inspiration and leadership. The editors, all of whom were recruited by Marty to work with him on this book, dedicate this text, which is already a tangible aspect of his legacy in medicine, as a living memorial to him. Abeloff’s Clinical Oncology will serve as a reminder to all its users of this

extraordinary person and exemplary physician who went before them. It was a privilege to have worked with him. James O. Armitage, MD John E. Niederhuber, MD Michael B. Kastan, MD, PhD W. Gillies McKenna, MD, PhD

Preface Few specialties in medicine are experiencing the rapid advancement in both laboratory and clinical science that is occurring in clinical oncology. The genetic mechanisms underlying cancer and their downstream effects are quickly becoming understood. This improved understanding of the molecular pathways in specific cancers is being translated into improved therapy. The development of antibodies and small molecules aimed at specific molecular targets that are key in the neoplastic process provide the promise of less toxic and more effective therapeutic options. Cancer prevention as a clinical science is making advances both in eliminating known carcinogens (e.g., tobacco) and in the development of drugs that inhibit carcinogenesis. The accumulative effect of these advances is, for the first time, sustained reduction in the age-adjusted death rate from cancer in the United States. The fourth edition of Abeloff’s Clinical Oncology incorporates these exciting changes. Each chapter begins with a summary highlighting the key points that would, for example, allow one to pass a board exam. In addition to a critical analysis of the literature, authors present their own opinions in specially identified boxes and algorithms. The use of color throughout the text makes the material more easily understood. Our goal is to provide a textbook that is the most useful, understandable, attractive, and thorough in presenting the principles of clinical oncology. It is meant to be equally useful to students and trainees, experts in the various disciplines of oncology, and as a reference text for physicians from other disciplines who also see patients with cancer. It is our hope that readers will find this scholarly textbook properly balanced between the disciplines of science, clinical medicine, and humanism and that it will serve them well in their

efforts to prevent, diagnose, and effectively treat their patients suffering from cancer. The multidisciplinary nature of cancer care is, and will continue to be, reflected in our editors. Specialists in pediatric oncology, surgical oncology, radiation oncology, medical oncology, and hematologic malignancies direct the development of the book. Reflecting the interdisciplinary care necessary for optimal care of patients, many chapters are the joint product of several of these disciplines. The editors are deeply indebted to our outstanding authors who, in a most diligent and thoughtful way, have brought their knowledge and skills to the fourth edition of Abeloff’s Clinical Oncology.

ACKNOWLEDGMENTS This fourth edition represents a highly collaborative and dynamic effort between the editors and Elsevier. We are greatly indebted to Dolores Meloni for her creative input and guidance and for turning the principles behind this text into a reality. Mary Beth Murphy and Nancy Lombardi are acknowledged for their truly exceptional support of this project. The expert support provided by Michele Pass, Elaine Ryan, Simone John, Margaret Hall, and Kim Bennett is also greatly appreciated. Finally, we want to express our gratitude to our outstanding authors for their superb contributions and for their generosity and friendship. James O. Armitage, MD John E. Niederhuber, MD Michael B. Kastan, MD, PhD W. Gillies McKenna, MD, PhD

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Contributors

James L. Abbruzzese, MD

Seena C. Aisner, MD

Chair, University of Texas M.D. Anderson Cancer Center, Houston, TX Cancer of Unknown Primary

Professor of Pathology and Vice-Chair, Pathology and Laboratory Medicine, New Jersey Medical School, Newark, NJ Tumors of the Pleura and Mediastinum

Martin D. Abeloff, MD* Formerly Marion I. Knott Professor and Director, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD Cancer of the Breast

Ghassan K. Abou-Alfa, MD Assistant Attending Physician, Department of Gastrointestinal Medical Oncology, Memorial Sloan–Kettering Cancer Center, New York, NY Liver and Bile Duct Cancer

Rhoda M. Alani, MD Department of Oncology, Johns Hopkins School of Medicine, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD Melanoma

Steven R. Alberts, MD Professor of Oncology, Mayo Clinic College of Medicine; Consultant in Medical Oncology, Mayo Clinic, Rochester, MN Cancer of the Stomach

Janet L. Abrahm, MD Associate Professor of Medicine and Anesthesia, Departments of Medical Oncology and Psychosocial, Palliative Care, Harvard Medical School; Director, Pain and Palliative Care Program, Dana Farber Cancer Institute, Boston, MA Caring for Patients at the End of Life

Jeffery S. Abrams, MD Acting Associate Director, Cancer Therapy Evaluation Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD Structures Supporting Cancer Clinical Trials

Geza Acs, MD, PhD Associate Professor, Department of Oncologic Sciences and Pathology and Cell Biology, University of South Florida; Associate Member, Moffitt Cancer Center, Tampa, FL Cancer of the Endometrium

Richard F. Ambinder, MD, PhD Professor, Departments of Oncology, Medicine, Pathology, and Pharmacology, Johns Hopkins University, Baltimore, MD HIV-Associated Malignancies

Leslie A. Andritsos, MD Department of Internal Medicine, Division of Hematology/ Oncology, The Ohio State University, Columbus, OH Chronic Lymphoid Leukemias

Frederick R. Appelbaum, MD Professor and Head, Medical Oncology Division, University of Washington School of Medicine; Director, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA Acute Myeloid Leukemia in Adults

Sachin Apte, MD, MS Joseph Aisner, MD Professor of Medicine and Occupational Medicine, Chief Medical Officer, Cancer Institute of New Jersey; Chief of Oncological Services, Robert Wood Johnson University Hospital, New Brunswick, NJ Tumors of the Pleura and Mediastinum *Deceased

Assistant Professor of Gynecologic Oncology, University of South Florida; Moffitt Cancer Center, Tampa, FL Cancer of the Endometrium

James O. Armitage, MD Joe Shapiro Professor of Medicine, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE Non-Hodgkin’s Lymphoma

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Deborah Armstrong, MD

Michael R. Bishop, MD

Associate Professor of Oncology, Gynecology, and Obstetrics, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD Ovaries and Fallopian Tubes

Senior Investigator and Clinical Head, Experimental Transplantation and Immunology Branch, National Cancer Institute/ Center for Cancer Research, National Institutes of Health, Bethesda, MD Hematopoietic Stem Cell Transplantation

Mamad M. Bagheri, MD Department of Dermatology, Marshfield Clinic, Hemet, CA Nonmelanoma Skin Cancers: Basal Cell and Squamous Cell Carcinomas

Charles M. Balch Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD Melanoma

Lodovico Balducci, MD Professor of Oncology and Medicine, University of South Florida College of Medicine, Tampa, FL Cancer in the Elderly: Biology, Prevention, and Treatment

Claudia Beghé, MD Associate Professor of Medicine, University of South Florida College of Medicine, Tampa, FL Cancer in the Elderly: Biology, Prevention, and Treatment

Robert Benjamin, MD Professor and Chair, Department of Sarcoma Medical Oncology, MD Anderson Cancer Center, Houston, TX Sarcomas of Soft Tissue

Charles L. Bennett, MD Professor of Medicine, Division of Hematology/Oncology, Northwestern University Feinberg School of Medicine; Associate Director, Midwest Center for Health Services and Policy Research, Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL Economic Analysis of Cancer Treatment

William J. Blot, PhD Professor, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN Cancer of the Lung: Non-Small Cell Lung Cancer and Small Cell Lung Cancer

Leslie Blumgart, MD Attending Surgeon, Hepatobiliary Service, Memorial Sloan– Kettering Cancer Center, New York, NY Liver and Bile Duct Cancer

Guido T. Bommer, MD Department of Internal Medicine, Division of Molecular Medicine and Genetics, University of Michigan School of Medicine, Ann Arbor, MI Progressing from Gene Mutations to Cancer

Michael J. Borowitz, MD Professor of Pathology and Oncology, Johns Hopkins Medical Institutions; Director of Hematopathology and Flow Cytometry, Johns Hopkins Hospital, Baltimore, MD Flow Cytometry in Oncologic Diagnosis

Julie R. Brahmer, MD Assistant Professor of Oncology, Johns Hopkins University School of Medicine; Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD Effusions

Viven H.C. Bramwell, PhD Professor, Department of Oncology, Division of Medical Oncology, University of Calgary, Calgary, Alberta, Canada Sarcomas of Soft Tissue

Malcom V. Brock, MD Ross Stuart Berkowitz, MD Professor of Gynecology, Harvard Medical School; Director of Gynecologic Oncology, Brigham and Women’s Hospital; Dana Farber Cancer Institute, Brigham and Women’s Hospital, Boston, MA Gestational Trophoblastic Disease

Donna Bernstein, MS Certified Genetic Counselor, Memorial Sloan-Kettering Cancer Center, New York; North Shore University Hospital, Manhasset, NY Genetic Factors: Hereditary Cancer Predisposition Syndromes

Associate Professor, Divisions of Thoracic Surgery and Tumor Biology, Johns Hopkins University School of Medicine, Baltimore, MD Cancer of the Esophagus

Ali Bydon, MD Instructor in Neurological Surgery, Johns Hopkins University, Baltimore, MD Spinal Cord Compression

Mitchell S. Cairo, MD Professor of Pediatrics, Medicine, and Pathology, Columbia University, New York, NY Tumor Lysis Syndrome

Contributors

Dario Campana, MD, PhD

Anthony Cmelak, MD

Member, Departments of Oncology and Pathology, Vice Chair for Laboratory Research, Department of Oncology, St. Jude Children’s Research Hospital; Professor of Pediatrics, University of Tennessee Health Science Center, Memphis, TN Childhood Leukemia

Associate Professor, Department of Radiation Oncology, Vanderbilt–Ingram Cancer Center, Nashville; Medical Director, Vanderbilt Cancer Center at Franklin, Franklin, TN Superior Vena Cava Syndrome

Peter F. Coccia, MD David P. Carbone, MD, PhD Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN Cancer of the Lung: Non-Small Cell Lung Cancer and Small Cell Lung Cancer

Ittner Professor and Vice-Chair of Pediatrics; Chief, Section of Pediatric Hematology/Oncology, University of Nebraska Medical Center, Omaha, NE Tumor Lysis Syndrome

Alfred M. Cohen, MD, FACS, FASCRS H. Ballentine Carter, MD Professor of Urology, Brady Urological Institute, Johns Hopkins University School of Medicine, Baltimore, MD Prostate Cancer

Manpreet K. Chadha, MBBS Medical Oncology Fellow, Rosewell Park Cancer Institute, Buffalo, NY Endocrine Complications

Daniel W. Chan, PhD Professor Pathology, Oncology, Urology, and Radiology; Director of Clinical Chemistry Division, Johns Hopkins University, Baltimore, MD Biomarkers for Cancer Diagnostics

Alfred E. Chang, MD Hugh Cabot Professor of Surgery, Chief, Division of Surgical Oncology, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI Acute Abdomen, Bowel Obstruction, and Fistula

Nai-Kong V. Cheung, MD, PhD Associate Professor, Weill Cornell Medical College; Director, Enid Haupt Chair in Pediatric Oncology; Director, Neuroblastoma Program; Head of Robert Steel Laboratory; Member and Attending, Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY Therapeutic Antibodies and Immunologic Conjugates

Michaele Christian, MD Cancer Therapy Evaluation Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, MD Structures Supporting Cancer Clinical Trials

Sonoita, AZ Cancer of the Rectum

Robert E. Coleman, MBBS, MD, FECP, FRCPE Professor of Medical Oncology, University of Sheffield; Honorary Consultant, Weston Park Hospital, Sheffield, UK Bone Metastases

Carolyn Compton, MD, PhD Bethesda, MD Colon Cancer

Linda D. Cooley, MD, MBA Associate Professor, University of Missouri–Kansas City Medical School; Associate Professor, Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital; Director, Cytogenetic Laboratory, Children’s Mercy Hospital, Kansas City, MO Conventional and Molecular Cytogenetics of Neoplasia

Jorge Cortes, MD Professor of Medicine and Internist, Department of Leukemia, University of Texas M.D. Anderson Cancer Center, Houston, TX Chronic Myeloid Leukemia

Sara A. Courtneidge, PhD Professor, The Burnham Institute for Medical Research, La Jolla, CA Intracellular Signaling

Kenneth H. Cowan, MD, PhD Director, Eppley Institute, University of Nebraska Medical Center, Omaha, NE Gene Therapy in Oncology

Michael F. Clarke, MD Professor of Internal Medicine, The Karel and Avice Beekhuis Professor in Cancer Biology; Associate Director, Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford, CA Stem Cells, Cell Differentiation, and Cancer

Daniel J. Culkin, MD Professor and Chair, Department of Urology, University of Oklahoma College of Medicine; Chief, Adult Urology Service, Oklahoma Medical Center; Oklahoma City, OK Cancer of the Penis

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Contributors

Josep Dalmau, MD, PhD

Jeffrey S. Dome, MD

Professor of Neurology, University of Pennsylvania; Attending Neurologist, Department of Neurology, Hospital of the University of Pennsylvania, Philadelphia, PA Paraneoplastic Neurologic Syndromes

Chief, Division of Oncology, Acting Chief, Division of Hematology, Children’s National Medical Center, Washington, DC Pediatric Solid Tumors

Giulio J. D’Angio, MD

John H. Donohue, MD

Professor (Emeritus), University of Pennsylvania School of Medicine; Hospital of the University of Pennsylvania; Children’s Hospital, Philadelphia, PA Second Malignant Neoplasms

Laura Dawson, MD Assistant Professor, Department of Radiation, Oncology, University of Toronto; Staff Radiation Oncologist, Radiation Medicine Program, Princess Margaret Hospital/University Health Network, Toronto, Ontario, CAN Liver Metastases

Steven R. Deitcher, MD Executive Vice President of Drug Development, Chief Medical Officer, Hana Biosciences, Inc., South San Francisco, CA Diagnosis, Treatment, and Prevention of Cancer-Related Venous Thrombosis

Ronald P. DeMatteo, MD Professor, Department of Surgery, Weill Cornell Medical College; Attending Surgeon, Department of Surgery, Memorial Sloan–Kettering Cancer Center, New York, NY Cancer of the Small Bowel

Philip A. DeSimone, MD Professor of Medicine, University of Kentucky, School of Medicine, Lexington, KY Cancer of the Rectum

Theodore L. DeWeese, MD Professor and Chair of Radiation Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD Prostate Cancer

Subba R. Digumarthy, MD Department of Radiology, Massachusetts General Hospital, Boston, MA Cancers of the Cervix, Vulva, and Vagina

Professor of Surgery, Mayo Clinic College of Medicine, Rochester, MN Cancer of the Stomach

James H. Doroshow, MD Director, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD Principles of Molecularly Targeted Therapy: Present and Future; Structures Supporting Cancer Clinical Trials

Jeffery A. Drebin, MD Professor of Surgery, University of Pennsylvania School of Medicine, Abramson Cancer Center, Philadelphia, PA Carcinoma of the Pancreas

Dan G. Duda, DMD, PhD Assistant Professor of Radiation Oncology, Harvard Medical School; Assistant Biologist, Edwin L. Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital, Boston MA Vascular and Interstitital Biology of Tumors

Austin Duffy, MD Special Fellow, Department of Gastrointestinal Medical Oncology, Memorial Sloan–Kettering Cancer Center, New York, NY Liver and Bile Duct Cancer

Linda R. Duska, MD Assistant Professor of Gynecology, Obstetrics, and Reproductive Biology, Harvard Medical School; Assistant in Obstetrics and Gynecology, Massachusetts General Hospital; Gillette Center for Women’s Cancers, Boston, MA Cancers of the Cervix, Vulva, and Vagina

Mario A. Eisenberger, MD Professor of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD Prostate Cancer

Rebecca L. Elstrom, MD, MA Angela Dispenzieri, MD Associate Professor of Medicine, Mayo Clinic, Rochester, MN Multiple Myeloma and Related Disorders

Assistant Professor, Division of Hematology/Oncology, University of Michigan, Ann Arbor, MI Cell Life and Death

Contributors

Janine T. Erler, PhD

Arlene A. Forastiere, MD

Postdoctoral Fellow, Stanford University, Stanford, CA; Team Leader, Institute of Cancer Research, University of London, London, UK The Cellular Microenvironment and Metastases

Professor of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD Cancer of the Esophagus

Michael S. Ewer, MD Professor of Medicine, University of Texas M.D. Anderson Cancer Center, Houston, TX Cardiac Effects of Cancer Therapy

Eric R. Fearon, MD, PhD Department of Internal Medicine, Division of Molecular Medicine and Genetics, University of Michigan School of Medicine, Ann Arbor, MI Progressing from Gene Mutations to Cancer

Leslie A. Fecher, MD

James M. Ford, MD Associate Professor of Medicine, Pediatrics, and Genetics, Division of Oncology and Medical Genetics, Stanford University School of Medicine, Stanford, CA DNA Damage Response Pathways and Cancer

Alison G. Freifeld, MD Professor of Internal Medicine, Director, Immunocompromised Host Infectious Disease Program, University of Nebraska Medical Center, Omaha, NE Infection in the Patient with Cancer

Department of Oncology, Johns Hopkins School of Medicine; The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD Melanoma

Carl E. Freter, MD, PhD

Alessandro Fichera, MD

Arlan F. Fuller, Jr., MD

Assistant Professor, Department of Surgery, University of Chicago Pritzer School, Chicago, IL Cancer of the Small Bowel

Assistant Professor of Gynecology, Obstetrics, and Reproductive Biology, Harvard School of Medicine; Chief, Division of Gynecologic Oncology, Massachusetts General Hospital; Gillette Center for Women’s Cancers, Boston, MA Cancers of the Cervix, Vulva, and Vagina

Alexandra H. Filipovich, MD Professor, Department of Pediatrics, Cincinnati Children’s Medical Center; Director, Clinical Laboratory, Division of Hematology and Oncology, Cincinnati Children’s Hospital Center, Cincinnati, OH Immunodeficiency and Cancer

Professor of Medicine, University of Missouri–Columbia; Ellis Fischel Cancer Center, Columbia, MO Systemic Therapy

Emma E. Furth, MD Professor of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Abramson Cancer Center, Philadelphia, PA Carcinoma of the Pancreas

Karen A. Fitzner, PhD Department of Medicine, Division of Hematology/Oncology, Robert H. Lurie Comprehensive Cancer Center at the Northwestern University Feinberg School of Medicine, Chicago, IL Economic Analysis of Cancer Treatment

Robert L. Foote, MD Professor of Oncology, Mayo Clinic College of Medicine; Consultant, Department of Radiation Oncology, Mayo Clinic, Rochester, MN Oral Complications

Michael C. Garofalo, MD Assistant Professor, Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, MD Cancer of the Rectum

Mark C. Gebhardt, MD Frederick W. and Jane M. Ilfeld Professor of Orthopaedic Surgery, Harvard Medical School; Chief Orthopaedic Surgeon, Department of Orthopaedic Surgery, Beth Israel Deaconess Medical School, Boston, MA Sarcomas of Bone

James M. Foran, MD Assistant Professor of Medicine, Director, Hematology/ Oncology Fellowship Training Program, University of Alabama at Birmingham Comprehensive Cancer Center, Birmingham, AL Myelodysplastic Syndromes

N. Lynn Gerber, MD Professor, Rehabilitation Science, George Mason University, Fairfax, VA; Special Volunteer, Clinical Center, National Institutes of Health, Bethesda, MD Rehabilitation of Individuals with Cancer

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Manish Gharia, MD

Anne Kathryn Goodman, MD

Department of Dermatology, University of Wisconsin; Department of Dermatolgoy, Middleton VA Medical Center, Madison, WI Nonmelanoma Skin Cancers: Basal Cell and Squamous Cell Carcinomas

Associate Professor of Gynecology, Obstetrics, and Reproductive Biology, Harvard School of Medicine; Associate Director, Division of Gynecologic Oncology, Massachusetts General Hospital; Gillette Center for Women’s Cancers, Boston, MA Cancers of the Cervix, Vulva, and Vagina

Amato J. Giaccia, PhD Professor of Radiation Oncology and Cancer Biology, Director of Radiation Oncology, Stanford University, Stanford, CA The Cellular Microenvironment and Metastases

Mark R. Gilbert, MD Professor and Deputy Chair, Department of Neuro-oncology, M.D. Anderson Cancer Center, Houston, TX Neurologic Complications

John Glaspy, MD, MPH Professor of Medicine, Department of Medicine, Division of Hematology and Oncology, University of California at Los Angeles School of Medicine, Los Angeles, CA Disorders of Blood Cell Production in Clinical Oncology

Katrina Y. Glover, MD Assistant Professor, University of Texas M.D. Anderson Cancer Center, Houston, TX Cancer of Unknown Primary

Ziya L. Gokaslan, MD Professor of Neurosurgery, Oncology, and Orthopedics, Vice Chair and Director of Spine Division, Department of Neurosurgery, Johns Hopkins University, Baltimore, MD Spinal Cord Compression

Nicola Gökbuget, MD Leiterin der Studienzentrale, Klinikum der J. W. Goethe Universität, Frankfurt, GER Acute Lymphocytic Leukemia in Adults

Donald Peter Goldstein, MD Clinical Professor of Obstetrics, Gynecology, and Reproductive Biology, Harvard Medical School; Senior Scientist, Department of Obstetrics and Gynecology, Brigham and Women’s Hospital; Co-Director, New England Trophoblastic Disease Center, Boston, MA Gestational Trophoblastic Disease

Ellen Gordon, MD Department of Dermatology, University of Wisconsin; Department of Dermatology, Middleton VA Medical Center, Madison, WI Nonmelanoma Skin Cancers: Basal Cell and Squamous Cell Carcinomas

Daniel M. Green, MD Professor of Pediatrics, State University of New York at Buffalo School of Medicine and Biomedical Sciences; Attending Physician, Department of Pediatrics, Roswell Park Cancer Institute, Buffalo, NY Second Malignant Neoplasms

Michael R. Grever, MD Professor and Chair of Internal Medicine, Charles A. Doan Professor of Medicine, Professor of Pharmacology, The Ohio State University College of Medicine, Columbus, OH Chronic Lymphoid Leukemias

Andrew Grigg Associate Professor, Department of Medicine, University of Melbourne; Deputy Director, Clinical Harmatology and Bone Marrow Transplant Service, Royal Melbourne Hospital, Victoria, AUS Special Issues in Pregnancy

Louise Grochow, MD Senior Director, Global Medical Science, AstraZeneca, Waltham, MA Colon Cancer

Thomas G. Gross, MD, PhD Associate Professor, Department of Pediatrics, The Ohio State University School of Medicine; Chief, Division of Hematology, Oncology, and Bone Marrow Transplant, Children’s Hospital, Columbus, OH Immunodeficiency and Cancer

Adriana Gonzalez, MD Assistant Professor of Pathology, Vanderbilt University Medical School; Attending Pathologist, Vanderbilt University Medical Center, Nashville, TN Cancer of the Lung: Non-Small Cell Lung Cancer and Small Cell Lung Cancer

Stuart A. Grossman, MD Professor of Oncology, Medicine, and Neurosurgery, Sidney Kimmel Cancer Center at Johns Hopkins, Professor, Johns Hopkins Hospital, Baltimore, MD Cancer Pain

Contributors

Leonard L. Gunderson, MD, MS

Ernie Hawk, MD, MPH

Getz Family Professor of Radiation Oncology, Mayo Clinic College of Medicine; Consultant in Radiation Oncology, Mayo Clinic in Arizona; Deputy Director for Clinical Affairs, Mayo Clinical Center, Scottsdale, AZ Cancer of the Stomach

Rockville, MD Colon Cancer

Juliet Gunkel, MD Department of Dermatology, University of Wisconsin; Department of Dermatology, Middleton VA Medical Center, Madison, WI Nonmelanoma Skin Cancers: Basal Cell and Squamous Cell Carcinomas

Martin Gutierrez, MD Staff Clinician, Medical Oncology Branch, National Cancer Institute, Bethesda, MD Principles of Molecularly Targeted Therapy: Present and Future

Thomas M. Habermann, MD Departments of Medicine and Dermatology, Division of Hematology, Mayo Clinic College of Medicine, Rochester, MN Cutaneous T-Cell Lymphoma and Cutaneous B-Cell Lymphoma

Nancy H. Heideman, PharmD, BCPS Assistant Professor, Pharmacy Practice, College of Pharmacy, University of New Mexico; Clinical Pharmacist, Children’s Hospital of New Mexico, Albuquerque, NM Hyponatremia

Richard L. Heideman, MD Professor and Executive Director, Department of Pediatrics, University of New Mexico, Albuquerque, NM Hyponatremia

Lee J. Helman, MD Scientific Director for Clinical Research, Center for Cancer Research, National Cancer Institute, Bethesda, MD Sarcomas of Soft Tissue

Jessica Hochberg, MD Fellow, Division of Pediatric Blood and Marrow Transplantation, Columbia University, New York, NY Tumor Lysis Syndrome

Barrett G. Haik, MD Hamilton Professor and Chair, Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Sciences Center; Director, Ophthalmology Service, St. Jude Children’s Cancer Research Hospital, Memphis, TN Eye, Orbit, and Adnexal Structures

Dieter Hoelzer, MD

John D. Hainsworth, MD Chief Scientific Officer, Sarah Cannon Research Institute, Nashville, TN Nausea and Vomiting

Senior Lecturer, Academic Unit of Clinical Oncology, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, UK Bone Metastases

Dennis Hallahan, MD

Sandra J. Horning, MD

Professor and Chair, Department of Radiation Oncology, Vanderbilt University Medical Center, Nashville, TN Cancer of the Lung: Non-Small Cell Lung Cancer and Small Cell Lung Cancer

Professor of Medicine, Oncology, and Bone and Marrow Transplantation, Stanford University Medical Center, Stanford, CA Hodgkin’s Lymphoma

Nader N. Hanna, MD, FACS, FICS Chief of Surgical Oncology, Associate Professor of Surgery, University of Maryland School of Medicine, Baltimore, MD Cancer of the Rectum

Professor of Oncology, University of Frankfurt, Frankfurt, GER Acute Lymphocytic Leukemia in Adults

Ingunn Holen, MSc, PhD

Kim Huang, MD Department of Radiation Oncology, University of California at San Francisco, San Francisco, CA Brain Metastases and Neoplastic Meningitis

Eleanor E. R. Harris, MD Associate Professor of Radiation Oncology, University of South Florida; Clinical Director and Residency Program Director, Division of Radiation Oncology, Moffitt Cancer Center, Tampa, FL Cancer of the Endometrium

Peter B. Illei, MD Assistant Professor and Director of Immunopathology, Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD Principles of Oncologic Surgical Pathology

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Elaine S. Jaffe, MD

Rosalyn A. Juergens, MD

Clinical Professor of Pathology, George Washington University School of Medicine and Health Sciences, Washington, DC; Chief, Hematopathology Section, Laboratory of Pathology, Center for Cancer Research, Bethesda, MD World Health Organization Classification of Hematologic Malignancies

Assistant Professor, Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD Effusions

Sanjay B. Jagannath, MD Assistant Professor of Medicine, Johns Hopkins Hospital, Baltimore MD Cancer of the Esophagus

Rakesh K. Jain, PhD Andrew Werk Cook Professor of Tumor Biology, Department of Radiation Oncology, Harvard Medical School, Boston, MA; Affiliated Faculty, Harvard–MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA; Director, Edwin L. Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA Vascular and Interstitital Biology of Tumors

William Jarnagin, MD Associate Attending Surgeon and Vice Chair, Department of Surgery, Memorial Sloan-Kettering Cancer Center; Associate Professor of Surgery, Weill Medical College of Cornell University, New York, NY Liver and Bile Duct Cancer

Jeffrey A. Kant, MD, PhD Professor of Pathology and Human Genetics, Director, Division of Molecular Diagnostics, University of Pittsburgh Medical Center, Pittsburgh, PA Molecular Diagnostics

Hagop Kantarjian, MD Professor and Chair, Leukemia Department, M.D. Anderson Cancer Center, Houston, TX Chronic Myeloid Leukemia

Zeynel A. Karcioglu, MD Professor of Ophthalmology, Ocular Oncology, and Orbital Diseases and Surgery, Hamilton Eye Institute, University of Tennessee; Consultant Ophthalmologist, St. Jude Children’s Cancer Research Hospital, Memphis, TN; Professor Emeritus Tulane University School of Medicine; Former Haik/St. Giles Foundation Professor of Ocular Oncology; Member Louisiana Endowment for Eminent Scholars Trust, New Orleans, LA Eye, Orbit, and Adnexal Structures

Danielle M. Karyadi, PhD Staff Scientist, Cancer Genetics Branch, National Human Genome Research Institute, National Institute of Health, Bethesda, MD Genetic Factors: Finding Cancer Susceptibility Genes

Anuja Jhingran, MD Associate Professor of Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX Cancers of the Cervix, Vulva, and Vagina

Norbert Kased, BS Department of Radiation Oncology, University of California at San Francisco, San Francisco, CA Brain Metastases and Neoplastic Meningitis

David H. Johnson, MD Cornelius A. Craig Professor of Medical and Surgical Oncology, Director, Division of Hematology and Oncology, Vanderbilt University School of Medicine; Deputy Director, VanderbiltIngram Cancer Center, Nashville, TN Cancer of the Lung: Non-Small Cell Lung Cancer and Small Cell Lung Cancer; Superior Vena Cava Syndrome

Michael B. Kastan, MD, PhD Cancer Center Director, St. Jude’s Children’s Research Hospital, Memphis, TN DNA Damage Response Pathways and Cancer

Daniel R. Kaul, MD

Department of Radiation Oncology, Hospital of the University of Pennsylvania, Philadelphia, PA Complementary and Alternative Medicine

Assistant Professor, Division of Infectious Disease, Director, Transplant Infectious Disease Service, Assistant Program Director, Internal Medicine Residency Program, University of Michigan School of Medicine, Ann Arbor, MI Infection in the Patient with Cancer

Kevin D. Judy, MD

John Kawaoka, MD

Associate Professor, Department of Radiation Oncology, University of Pennsylvania School of Medicine; Hospital of the University of Pennsylvania, Philadelphia, PA Cancer of the Central Nervous System

Assistant Instructor in Dermatology, The Warren Alpert Medical School of Brown University; Resident, Rhode Island Hospital, Providence, RI Alopecia and Cutaneous Complications

Heather Jones, MD

Contributors

Margaret Kemeny, MD

Janessa Laskin, MD

Professor, Mt. Sinai School of Medicine, New York; Director, Queens Cancer Center, Queens Hospital, Jamaica, NY Liver Metastases

Assistant Professor of Medicine, University of British Columbia; Medical Oncologist, British Columbia Cancer Agency, Vancouver, British Columbia, CAN Superior Vena Cava Syndrome

Nancy Kemeny, MD Professor of Medicine, Weill Cornell Medical College of Cornell University; Attending Physician, Gastrointestinal Oncology Service, Division of Solid Tumor Oncology, and Department of Medicine, Memorial Sloan–Kettering Cancer Center, New York, NY Liver Metastases

Thomas W. Kensler, PhD

Fred Lee, Jr., MD Professor of Radiology, Chief, Abdominal Imagining, Chief, Oncologic Imagining, Department of Radiology, University of Wisconsin–Madison, Madison, WI Colon Cancer

Susanna I. Lee, MD

Professor, Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD Environmental Factors

Instructor in Radiology, Harvard School of Medicine; Staff Radiology, Department of Radiology, Massachusetts General Hospital, Boston, MA Cancers of the Cervix, Vulva, and Vagina

Lawrence R. Kleinberg, MD

Jacqueline Lees, PhD

Associate Professor, Department of Radiation Oncology, John Hopkins University; Co-Director, Stereotactic Radiosurgery, John Hopkins Hospital, Baltimore, MD Cancer of the Esophagus

Boris Kobrinsky, MD Clinical Instructor, New York University School of Medicine; Attending Physician, New York University Medical Center, New York, NY Cardiac Effects of Cancer Therapy

Jeanne Kowalski, PhD Johns Hopkins School of Medicine, Baltimore, MD Biostatistics and Bioinformatics in Clinical Trials

Shivaani Kummar, MBBS Staff Clinician, Medical Oncology Branch, National Cancer Institute, Bethesda, MD Principles of Molecularly Targeted Therapy: Present and Future

Geeta Lal, MD, MSc, FRCSC, FACS

David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge MA Control of the Cell Cycle

Renato Lenzi, MD Associate Professor, University of Texas M.D. Anderson Cancer Center, Houston, TX Cancer of Unknown Primary

Caryn Lerman, PhD Mary W. Calkins Professor, Department of Psychiatry and Annenberg Public Policy Center, Deputy Director, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA Nicotine Dependence: Current Treatments and Future Directions

Allan Lipton, MD Professor of Medicine and Oncology, Milton S. Hershey Medical Center, Hershey, PA Hypercalcemia

Assistant Professor of Surgery, University of Iowa; Attending Surgeon, University of Iowa Hospitals, Iowa City, IA Cancer of the Endocrine System

Charles L. Loprinzi, MD

Paul F. Lambert, PhD

Gerard Lozanski, MD

Professor of Oncology, University of Wisconsin School of Medicine and Public Health; McArdle Laboratory for Cancer Research, Madison, WI Viruses and Human Cancer

Department of Internal Medicine, Ohio State University, Columbus, OH Chronic Lymphoid Leukemias

Mayo Clinic College of Medicine, Rochester, MN Oral Complications

Robert Lustig, MD Julie R. Lange, MD, ScM Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD Melanoma

Associate Professor, Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA Cancer of the Central Nervous System

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Contributors

Mitchell Machtay, MD Department of Radiation Oncology, Kimmel Cancer Center of Thomas Jefferson University, Philadelphia, PA Pulmonary Complications of Anticancer Treatment

Amit Maity, MD Associate Professor, Department of Radiation Oncology, University of Pennsylvania School of Medicine; Hospital of the University of Pennsylvania, Philadelphia, PA Cancer of the Central Nervous System

Uzma Malik, MD, FRCPC Associate, Radiation Oncology Department, Henry Cancer Center, Wilkes-Barre, PA Cancer of the Anal Canal

W. Gillies McKenna, BSc, MD, PhD, FRCR, FMedSci Professor of Radiation Oncology and Biology, Oxford University; Radiobiology Research Institute, Churchill Hospital; Director, Gray Cancer Institute; Honorary Director, MRC Radiation Oncology and Biology Unit, Oxford, UK Basics of Radiation Therapy

Steven Meranze, MD Professor of Radiology and Surgery, Directory, Interventional Radiology, Vanderbilt University Medical Center, Nashville, TN Superior Vena Cava Syndrome

James M. Metz, MD

Department of Urology, Oklahoma University Health Science Center, Oklahoma City, OK Cancer of the Penis

Department of Radiation Oncology, Hospital of the University of Pennsylvania; Abramson Cancer Center, Philadelphia, PA Carcinoma of the Pancreas; Complementary and Alternative Medicine

John C. Mansour, MD

Frank L. Meyskens, MD

Assistant Professor of Surgery, University of Texas Southwestern Medical School; Simmons Comprehensive Cancer Center, Dallas, TX Establishing and Maintaining Vascular Access

Professor of Medicine and Biological Chemistry, University of California–Irvine; Associate Vice Chancellor of Health Sciences, Chao Family Comprehensive Cancer Center, Orange, CA Cancer Prevention, Screening, and Early Detection

C. Scott Manatt, MD

Pierre P. Massion, MD Associate Professor of Medicine and Cancer Biology, Chief, Pulmonary Section, Vanderbilt University School of Medicine, Nashville, TN Cancer of the Lung: Non-Small Cell Lung Cancer and Small Cell Lung Cancer

R. Samuel Mayer, MD

Fabrizio Michelassi, MD Lewis Atterbury Stimson Professor, Chair, Department of Surgery, Weill Medical College of Cornell University, New York, NY Cancer of the Small Bowel

Radha Mikkilineni, MD

Assistant Professor, Department of Physical Medicine and Rehabilitation, Johns Hopkins University of Medicine, Baltimore, MD Rehabilitation of Individuals with Cancer

Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD Melanoma

Beryl McCormick, MD

Formerly Professor and Chair, Department of Health Systems and Outcomes, Director, NIH P30 Center for Collaborative Intervention Research, The Johns Hopkins University School of Nursing; Joint Appointment in Oncology, The Johns Hopkins University School of Medicine; Director of Nursing Research, Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD Fatigue

Attending and Acting Chair, Memorial Hospital; Member, Memorial Sloan–Kettering Cancer Center, New York, NY Cancer of the Breast

Charles J. McDonald, MS, MD Professor and Chair, Department of Dermatology, Warren Alpert School of Medicine, Brown University; Physician in Chief, Department of Dermatology, Rhode Island Hospital, Providence, RI Alopecia and Cutaneous Complications

Ross McDougall, MD, PhD Professor of Radiology and Medicine, Nuclear Medicine Residency Program Director, Stanford University School of Medicine, Stanford, CA Cancer of the Endocrine System

Victoria Mock, PhD, RN, FAAN*

Mohammed Mohiuddin, MD, FRCR, FACR Co-Director, Geisinger Cancer Service Line, Medical Director, GHS/FCCC Henry Cancer Center, Wilkes-Barre, PA Cancer of the Anal Canal

*Deceased

Contributors

James Montie, MD

Kenneth Offit, MD, MPH

Valassis Professor of Urologic Oncology, Department of Urology, University of Michigan, Ann Arbor, MI Carcinoma of the Bladder

Chief, Clinical Genetics Service, Department of Medicine, Memorial Sloan–Kettering Cancer Center, New York, NY Genetic Factors: Hereditary Cancer Predisposition Syndromes

A. Ross Morton, MD, FRCP, FRCPC Professor of Medicine, Queen’s University, Ontario, CAN Hypercalcemia

Anthony J. Murgo, MD, MS Head, Early Clinical Trials Development, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD Principles of Molecularly Targeted Therapy: Present and Future

James R. Neff, MD* Formerly Professor of Orthopedic Surgery and Pathology, University of Nebraska College of Medicine; Professor of Orthopedic Surgery and Pathology, University of Nebraska Medical Center, Omaha, NE Sarcomas of Bone

William G. Nelson, MD, PhD Professor of Oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD Prostate Cancer

Mihaela Onciu, MD Department of Pathology, St. Jude Children’s Research Hospital; University of Tennessee College of Medicine, Memphis, TN Childhood Lymphoma

Eileen M. O’Reilly, MD Associate Attending Physician, Department of Gastrointestinal Medical Oncology, Memorial Sloan–Kettering Cancer Center, New York, NY Liver and Bile Duct Cancer

Elaine A. Ostrander, PhD Chief, Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD Genetic Factors: Finding Cancer Susceptibility Genes

Brian O’Sullivan, MD Professor, Princess Margaret Hospital, Ontario Cancer Institute, Toronto, Ontario, CAN Sarcomas of Soft Tissue

Suzanne Nesbit, PharmD, BCPS Clinical Coordinator, Cancer Pain Service, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins; Clinical Pharmacy Specialist, Pain Management, Department of Pharmacy, Johns Hopkins Hospital, Baltimore, MD Cancer Pain

Drew M. Pardoll, MD, PhD Professor, School of Nursing, Johns Hopkins University, Baltimore, MD Cancer Immunology

Catherine K. Park, MD, MPH John E. Niederhuber, MD Bethesda, MD Colon Cancer; Establishing and Maintaining Vascular Access; Surgical Interventions in Cancer

Tracey O’Connor, MD Assistant Professor of Medicine, State University of New York at Buffalo School of Medicine and Biomedical Science; Assistant Professor of Oncology, Roswell Park Cancer Institute, Buffalo, NY Reproductive Complications

Thomas O’Dorisio, MD Professor of Medicine, University of Iowa College of Medicine; Clinical Attending and Member, Holden Comprehensive Cancer Center, University of Iowa Health Clinics, Iowa City, IA Cancer of the Endocrine System *Deceased

Associate Member, Moffitt Cancer Center, Tampa, FL Cancer of the Endometrium

Freda Patterson, PhD Manager of Research Studies, Department of Psychiatry, University of Pennsylvania, Philadelphia, PA Nicotine Dependence: Current Treatments and Future Directions

Steven Z. Pavletic Head, Graft-Versus-Host and Autoimmunity Unit, Experimental Transplantation and Immunology Branch, National Cancer Institute, Bethesda, MD Hematopoietic Stem Cell Transplantation

Michael C. Perry, MD, MS, MACP Professor and Director, Division of Hematology and Medical Oncology, University of Missouri; Nellie B. Smith Chair of Oncology, Ellis Fischer Cancer Center, Columbia, MO Systemic Therapy

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Contributors

LoAnn C. Peterson, MD

Amy A. Pruitt, MD

Professor of Medicine, Northwestern University Feinberg School of Medicine; Attending Physician, Director of Hematopathology Division, Northwestern Memorial Hospital, Chicago, IL Hairy Cell Leukemia

Associate Professor of Neurology, University of Pennsylvania School of Medicine; Hospital of the University of Pennsylvania, Philadelphia, PA Cancer of the Central Nervous System

Ching-Hon Pui, MD Peter C. Phillips, MD Schoemaker Professor and Director of Pediatric Neurooncology, Children’s Hospital of Philadelphia; University of Pennsylvania School of Medicine, Philadelphia, PA Cancer of the Central Nervous System

Professor of Pediatrics, University of Tennessee Health Science Center; Chair, Department of Oncology, American Cancer Society Professor, St. Jude Children’s Research Hospital, Memphis TN Childhood Leukemia

Steven Piantadosi, MD, PhD

Joe Bill Putnam, MD

Samuel Oschin Comprehensive Cancer Institute at Cedars– Sinai Medical Center, Los Angeles, CA Biostatistics and Bioinformatics in Clinical Trials

Professor and Chair of Thoracic Surgery, Ingram Professor of Surgery, Professor of Biomedical Informatics, Vanderbilt– Ingram Cancer Center, Nashville, TN Cancer of the Lung: Non-Small Cell Lung Cancer and Small Cell Lung Cancer

Robert Pili, MD Associate Professor of Oncology and Urology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD Cancer of the Kidney

Peter W.T. Pisters, MD Professor of Surgery, University of Texas M.D. Anderson Cancer Center, Houston, TX Sarcomas of Soft Tissue

Mark R. Pittelkow, MD Department of Medicine, Division of Hematology, Mayo Clinic College of Medicine, Rochester, MN Cutaneous T-Cell Lymphoma and Cutaneous B-Cell Lymphoma

John P. Plastaras, MD Department of Radiation Oncology, Hospital of the University of Pennsylvania, Philadelphia, PA Second Malignant Neoplasms

Elizabeth A. Platz, ScD, MPH Associate Professor, Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health; Sidney Kimmel Comprehensive Cancer Center; James Buchanan Brady Urological Institute, John Hopkins Medical Institutions, Bethesda, MD Use of Epidemiology in Oncology

Harry Quon Director, Head and Neck Radiation Oncology, Departments of Radiation Oncology and Otorhinolaryngology–Head and Neck Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA Cancer of the Head and Neck

Martin N. Raber, MD Clinical Professor of Medicine, Division of Medicine, M.D. Anderson Cancer Center, University of Texas, Houston, TX Cancer of Unknown Primary

S. Vincent Rajkumar, MD Professor of Medicine, Chair, Myeloma Amyloidosis Dysproteinemia Group, Mayo Clinic, Rochester, MN Multiple Myeloma and Related Disorders

William F. Regine, MD Professor and Chair, Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, MD Cancer of the Rectum

Mark Ritter, MD, PhD Associate Professor, Vice Chair, Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI Colon Cancer

Julian Pribaz, MD

John Robert Roberts

Professor of Surgery, Harvard Medical School; Brigham and Women’s Hospital; Children’s Hospital, Boston, MA Lymphedema

Cardiac and Thoracic Surgery, Surgical Clinic, Sarah Cannon Cancer Center, Nashville, TN Superior Vena Cava Syndrome

Contributors

Leslie Robinson-Bostom, MD

Anthony H. Russell, MD

Associate Professor in Dermatology, The Warren Alpert Medical School of Brown University; Attending Physician, Rhode Island Hospital, Providence, RI Alopecia and Cutaneous Complications

Associate Professor of Radiation Oncology, Harvard School of Medicine; Director of Gynecologic Radiotherapy, Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA Cancers of the Cervix, Vulva, and Vagina

Ronald Rodriguez, MD, PhD Associate Professor of Urology, Medical Oncology, Cellular and Molecular Medicine, and Viral Oncology, Director, Urology Residency Program, Johns Hopkins University School of Medicine, Baltimore, MD Cancer of the Kidney

Charles J. Ryan, MD Assistant Professor of Medicine, University of California at San Francisco Comprehensive Cancer Center, San Francisco, CA Testicular Cancer

Vergilio Sacchini, MD Carlos Rodriguez-Galindo Associate Member, St. Jude Faculty, Medical Director, Mexico Program–International Outreach, St. Jude Children’s Research Hospital, Memphis, TN Pediatric Solid Tumors

Myrna Rosenfeld, MD, PhD Associate Professor, Department of Neurology, Division Chief, Neuro-oncology, University of Pennsylvania, Philadelphia, PA Paraneoplastic Neurologic Syndromes

Nadia Rosenthal, PhD Head, Mouse Biology Unit, EMBL-Monterotondo Outstation, Montereotondo, ITA; Director of Science, Harefield Heart Science Centre, National Heart and Lung Institute, Imperial College, London, UK; Director, Australian Regenerative Medicine Institute, Monash University, Melbourne, AUS Molecular Tools in Cancer Research

James L. Rubenstein, MD

General Surgery, Memorial Sloan–Kettering Cancer Center, New York, NY Cancer of the Breast

Alan B. Sandler, MD Associate Professor of Medicine, Vanderbilt University School of Medicine; Director, Thoracic Oncology, Medical Director, Vanderbilt–Ingram Cancer Center Affiliate Network Program, Vanderbilt University Medical Center, Nashville, TN Cancer of the Lung: Non-Small Cell Lung Cancer and Small Cell Lung Cancer

Howard Sandler, MD, MS Newman Family Professor and Senior Associate Chair, Department of Radiation Oncology, Professor, Department of Urology, University of Michigan Medical School, Ann Arbor, MI Carcinoma of the Bladder

John T. Sandlund, MD

Department of Medicine, University of California at San Francisco, San Francisco, CA Brain Metastases and Neoplastic Meningitis

Department of Hematology/Oncology, St. Jude Children’s Research Hospital; University of Tennessee College of Medicine, Memphis, TN Childhood Lymphoma

Brian P. Rubin, MD, PhD

Victor M. Santana, MD

Assistant Professor of Pathology, University of Washington, Seattle, WA Sarcomas of Soft Tissue

Professor, University of Tennessee; Director, Division of Solid Malignancies, St. Jude Children’s Research Hospital, Memphis, TN Pediatric Solid Tumors

Reena Rupani, MD Assistant Instructor in Dermatology, The Warren Alpert Medical School of Brown University; Resident, Rhode Island Hospital, Providence, RI Alopecia and Cutaneous Complications

Robert A. Schnoll, PhD Assistant Professor, Department of Psychiatry, University of Pennsylvania, Philadelphia PA Nicotine Dependence: Current Treatments and Future Directions

Valerie W. Rusch, MD Professor of Surgery, Cornell University Medical College; Chief of Thoracic Surgery, Memorial Sloan–Kettering Cancer Center, New York, NY Lung Metastases

Daniel M. Sciubba, MD Fellow, Neruo-oncology, Department of Neurosurgery, Johns Hopkins University, Baltimore, MD Spinal Cord Compression

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Contributors

Michael V. Seiden, MD, PhD

Stephen N. Snow, MD

President and CEO, Fox Chase Cancer Center, Philadelphia, PA Cancers of the Cervix, Vulva, and Vagina

Department of Dermatology, University of Wisconsin; Department of Dermatology, Middleton VA Medical Center, Madison, WI Nonmelanoma Skin Cancers: Basal Cell and Squamous Cell Carcinomas

Mikkael A. Sekeres, MD Assistant Professor of Medicine, Cleveland Clinic, Taussig Cancer Center, Cleveland, OH Myelodysplastic Syndromes

William H. Sharfman, MD Department of Oncology, Johns Hopkins University School of Medicine; The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD Melanoma

Ricky A. Sharma, MA, MB, BChir, MRCP(UK), FRCR, PhD

Lori J. Sokoll, PhD Associate Professor of Pathology, Oncology, and Urology, Johns Hopkins University School of Medicine, Baltimore MD Biomarkers for Cancer Diagnostics

Mika A. Sovak, MD, PhD Assistant Professor of Medicine, Cancer Institute of New Jersey; Robert Wood Johnson University Hospital; University of Medicine and Dentistry of New Jersey, New Brunswick, NJ Tumors of the Pleura and Mediastinum

Senior Fellow, Oxford University; Honorary Consultant, Clinical Oncology, Oxford Radcliffe Hospitals NHS Trust, Oxford, UK Basics of Radiation Therapy

James L. Speyer, MD

Kostandinos Sideras, MD

Alex I. Spira, MD

Hematology and Oncology Fellow, Mayo Clinic College of Medicine, Rochester, MN Oral Complications

Division of Hematology/Oncology, Inova Fairfax Hospital, Fairfax, VA Effusions

Kenneth Silver, MD

Dempsey Springfield, MD

Associate Professor, Department of Physical Medicine and Rehabilitation, Vice Chair of Clinical Affairs, Department of Physical Medicine and Rehabilitation, Johns Hopkins University School of Medicine; Medical Director, Department of Physical Medicine and Rehabilitation, Good Samaritan Hospital, Baltimore, MD Rehabilitation of Individuals with Cancer

Visiting Professor in Orthopedics, Harvard School of Medicine; Associate Orthopedic Surgeon, Massachusetts General Hospital, Boston, MA Sarcomas of Bone

Eric J. Small, MD Professor of Medicine, University of California at San Francisco Comprehensive Cancer Center, San Francisco, CA Testicular Cancer

Professor of Medicine, New York University; Medical Director, NYU Clinical Cancer Center, New York, NY Cardiac Effects of Cancer Therapy

Sheri L. Spunt, MD Associate Member, St. Jude’s Faculty, Department of Oncology, Divisions of Cancer Survivorship and Solid Tumors, St. Jude Children’s Research Hospital, Memphis, TN Pediatric Solid Tumors

Daniel Stewart, MD

Professor of Medicine and Urology, University of Michigan School of Medicine, Ann Arbor, MI Carcinoma of the Bladder

Department of Dermatology, University of Wisconsin; Department of Dermatology, Middleton VA Medical Center, Madison, WI Nonmelanoma Skin Cancers: Basal Cell and Squamous Cell Carcinomas

Penny K. Sneed, MD

Paul T. Strickland, MD

Department of Radiation Oncology, University of California at San Francisco, San Francisco, CA Brain Metastases and Neoplastic Meningitis

Professor, Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD Environmental Factors

David C. Smith, MD

Contributors

Bill Sugden, MD

Joseph E. Tomaszewski, PhD

Professor, Department of Oncology, McArdle Laboratory for Cancer Research, University of Wisconsin at Madison, Madison, WI Viruses and Human Cancer

Deputy Director, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD Principles of Molecularly Targeted Therapy: Present and Future

Siobhan Sutcliffe, PhD, ScM, MHS

Suzanne L. Topalian, MD

Assistant Professor, Department of Surgery, Alvin J. Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO Use of Epidemiology in Oncology

Director, Melanoma Program, Department of Surgery, Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD Melanoma

Frank M. Torti, MD Weijing Sun, MD Associate Professor of Medicine, University of Pennsylvania School of Medicine; Abramson Cancer Center, Philadelphia, PA Carcinoma of the Pancreas

Professor and Chair, Department of Cancer Biology, Wake Forest University School of Medicine; Director, Comprehensive Cancer Center at Wake Forest University, Winston-Salem, NC Testicular Cancer

Donald L. Trump, MD Martin S. Tallman, MD Professor of Medicine, Northwestern University Feinberg School of Medicine; Attending Physician, Northwestern Memorial Hospital, Chicago, IL Hairy Cell Leukemia

James E. Talmadge, PhD Professor, University of Nebraska Medical Center, Omaha, NE Gene Therapy in Oncology

Ayalew Tefferi, MD Professor of Medicine, Mayo Clinic, Rochester, MN Myeloproliferative Disorders

Professor of Medicine, State University of New York at Buffalo; President and Chief Executive Officer, Roswell Park Cancer Institute; Professor of Molecular Pharmacology, University of Buffalo, Buffalo, NY Endocrine Complications; Reproductive Complications

Katherine A. Vallis, MBBS, PhD, MRCP, FRCR, FRCPC CR-UK Senior Research Group Leader, Radiation Oncology and Biology; Honorary Consultant Clinical Oncologist, Oxford Radcliffe Hospitals NHS Trust, Oxford, UK Basics of Radiation Therapy

Gauri R. Varadhachary, MD Peter Thom, MS Genetic Counselor, Memorial Sloan–Kettering Cancer Center, New York, NY Genetic Factors: Hereditary Cancer Predisposition Syndromes

Craig B. Thompson, MD Professor of Medicine, Director, Abramson Cancer Center of the University of Pennsylvania, Philadelphia, PA Cell Life and Death

Associate Professor, University of Texas M.D. Anderson Cancer Center, Houston TX Cancer of Unknown Primary

Sreenivas Vemulapalli, MD Chief, Urologic Oncology, Department of Urology, University of Oklahoma College of Medicine, Oklahoma City, OK Cancer of the Penis

Kala Visvanathan, MB, BS, FRACP, MHS

Aston University, School of Life and Health Sciences, Birmingham, UK Cachexia

Assistant Professor, Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Sidney Kimmel, Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD Use of Epidemiology in Oncology

Kensei Tobinai, MD, PhD

Nina D. Wagner-Johnston, MD

Chief, Hematology and Stem Cell Transplantation Division, National Cancer Center Hospital, Tokyo, JAPAN Adult T-Cell Leukemia-Lymphoma

Post-Doctoral Fellow, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD HIV-Associated Malignancies

Michael J. Tisdale, BSc, PhD, DSc

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Contributors

Richard L. Wahl, MD

Wyndham H. Wilson, MD, PhD

Professor of Radiology and Oncology, Henry N. Wagner, Jr. Professor of Nuclear Medicine, Director of Nuclear Medicine/ PET Facility Vice Chair, Radiology for Technology and Business Development, Johns Hopkins University School of Medicine, Baltimore, MD Imaging

Chief, Lymphoma Theraputics Section, Metabolism Branch, Center for Cancer Research, National Cancer Institute, Bethseda, MD Non-Hodgkin’s Lymphoma

Toshiki Watanabe, MD Institute of Medical Sciences, University of Tokyo, Tokyo, JAPAN Adult T-Cell Leukemia-Lymphoma

Barbara L. Weber, MD Vice President, Discovery and Translational Medicine, Department of Oncology, GlaxoSmithKline, Collegeville, PA Cancer of the Breast

Sharon Weber, MD Associate Professor of Surgery, University of Wisconsin Hospital, Madison, WI Liver and Bile Duct Cancer

Antonio C. Wolff, MD Associate Professor of Oncology, Johns Hopkins Kimmel Cancer Center, Baltimore, MD Cancer of the Breast

Sandra L. Wong, MD Assistant Professor of Surgery, University of Michigan Health Systems, Ann Arbor, MI Acute Abdomen, Bowel Obstruction, and Fistula

Gary S. Wood, MD Johnson Professor and Chair, Department of Dermatology, University of Wisconsin, Madison, WI Nonmelanoma Skin Cancers: Basal Cell and Squamous Cell Carcinomas

Lance S. Wyatt, MD Ronald J. Weigel, MD, PhD Professor and Head, Department of Surgery, University of Iowa Roy J. and Lucille A. Carver College of Medicine; Department of Surgery, University of Iowa Hospitals and Clinics; Iowa City, IA Cancer of the Endocrine System

Irving L. Weissman, MD Director, Stanford Institute for Stem Cell Biology and Regenerative Medicine; Director, Comprehensive Cancer Center; Professor of Pathology and Developmental Biology; Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford, CA Stem Cells, Cell Differentiation, and Cancer

Private practice, Los Angeles, CA Lymphedema

Anaadriana Zakarija, MD, MS Instructor, Northwestern University Feinberg School of Medicine; Director, Northwestern Center for Bleeding Disorders, Northwestern Memorial Hospital, Chicago, IL Hairy Cell Leukemia

Tal Z. Zaks, MD, PhD Adjuvant Assistant Professor, University of Pennsylvania, Philadelphia; Director, Oncology Medicine Development Center, GlaxoSmithKline, Collegeville, PA Cancer of the Breast

William Westra, MD

Jason A. Zell, DO, MPH

Associate Professor of Pathology, Johns Hopkins University School of Medicine; Associate Professor of Pathology, Otolaryngology–Head and Neck Surgery, and Dermatology, Associate Director, Division of Surgical Pathology, Johns Hopkins Hospital, Baltimore, MD Principles of Oncologic Surgical Pathology

Assistant Professor, Department of Medicine, University of California at Irvine School of Medicine, Irvine, CA Cancer Prevention, Screening, and Early Detection

Kathleen S. Wilson, MD Associate Professor, Department of Pathology, University of Texas Southwestern Medical Center; Faculty, McDermott Center for Growth and Development; Faculty, Southwestern Graduate School of Biomedical Sciences; Associate Director, Cytogenetics Laboratory, Dallas, TX Conventional and Molecular Cytogenetics of Neoplasia

A. BIOLOGY AND CANCER

1

Molecular Tools in Cancer Research Nadia Rosenthal

S U M M ARY • Our understanding and treatment of cancer have always relied heavily on parallel developments in biological research. Molecular biology provides the basic tools to study genes that are involved in cancer growth patterns and tumor suppression. An advanced understanding of the molecular processes that govern cell growth and differentiation has revolutionized the diagnosis and prognosis of malignant disorders.

O F

K EY

P OI NT S

• Current cancer research seeks to integrate the complex interaction of the cell’s genome with its environment and emphasizes the need for a systematic approach to the analysis of gene function and dysfunction in the context of the intact organism. Discovery of the mechanisms that are responsible for genetic instability may lead to more reliable tests for hereditary susceptibility to cancer and, ultimately, to more effective therapies.

INTRODUCTION Since the previous edition of this book was published, advances in our understanding of the basic mechanisms of cancer have continued to inform and refine clinical approaches to prevention and therapy. New prognostic and predictive markers derived from molecular biology can now pinpoint specific genetic changes in particular tumors or detect occult malignant cells in normal tissues, leading to improved technologies for tumor screening and early detection. Diagnostic approaches have expanded from morphologic criteria and single-gene analysis to whole-genome technologies imported from other biological disciplines. A new systemic vision of cancer is emerging, in which the importance of individual mutation has been superseded by an appreciation for the new parameters set by geneenvironment interactions, with profound influences on a tumor cell’s transcriptional profile and function. Results from these cross-disciplinary applications underscore the complexity of carcinogenesis and promise to streamline the design of strategies for both cancer prevention and advanced cancer therapy. This overview will serve as a foundation of conceptual and technical information for understanding the exciting new advances in cancer research that will be described in subsequent chapters. Since the discovery of oncogenes, which provided the first concrete evidence of cancer’s genetic basis, applications of advanced molecular techniques and instrumentation have yielded new insights into normal cell biology as well. A basic fluency in molecular biology will soon be a necessary prerequisite for clinical oncologists, since many of the new diagnostic and prognostic tools that are in use today and will be in use tomorrow will rely on these fundamental principles of gene, protein, and cell function.

OUR UNSTABLE HEREDITY Cancer genetics has classically relied on the candidate gene approach, detecting acquired or inherited changes in specific genetic loci accu-

• This introductory chapter relates basic principles of molecular biology to emerging perspectives on the origin and progression of cancer and explains newly developed laboratory techniques, including whole-genome analysis, expression profiling, and refined genetic manipulation in animal models, providing the conceptual and technical background necessary to grasp the central principles and new methods of current cancer research.

mulated in a single cell, which then proliferates to produce a tumor composed of its identical clonal progeny. During the early steps of tumor formation, mutations that lead to an intrinsic genetic instability allow additional deleterious genetic alterations to accumulate. These genetic changes confer selective advantages on tumor cell clones by disrupting control of cell proliferation. The identification of specific mutations that characterize a tumor cell has proved invaluable for analyzing the neoplastic progression and remission of the disease. Methods for mutation detection all rely on the manipulation of DNA, the basic building block of heredity in the cell. DNA consists of two long strands of polynucleotides that twist around each other clockwise in a double helix (Fig. 1-1). Nucleic acid bases attached to the sugar groups of each strand face each other within the helix, perpendicular to its axis. These comprise only four bases: the purines adenine and guanine (A and G) and the pyrimidines cytosine and thymine (C and T). During assembly of the double helix, stable pairings of nucleotides from either strand are made between A and T or between G and C. Each base pair forms one of the billions of rungs in the long, unbroken ladder of DNA that forms a chromosome. The functional unit of inherited information in DNA—the gene—is most often represented by a discrete section of sequence that is necessary to encode a particular protein structure. Gene expression is initiated by forming a copy of the gene, messenger RNA (mRNA), which is constructed base by base from the DNA template by a polymerase enzyme. Once the sequence is transcribed, an RNA transcript is modified at both ends and then undergoes a highly regulated process called splicing. In higher organisms, most protein-coding gene sequences are interrupted by stretches of noncoding sequences, called introns. The genetic machinery must remove these introns to form a continuous chain of coding sequences, or exons, which subsequently undergo translation into protein. The splicing process requires absolute precision because the deletion or addition of a single nucleotide at the splice junction would throw the three-base coding sequence out of frame.

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Part I: Science of Clinical Oncology Cell nucleus containing 23 pairs of chromosomes

Genes

DNA strand Chromosomes Sugar Bases

Cytosine thymine Bases

Adenine and guanine Phosphate P group

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P

P H H

CH2

CH2

CH2

P

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Figure 1-1 • DNA (deoxyribonucleic acid) is the cell’s genetic material, contained in single compacted strands comprising chromosomes within the cell nucleus. In the DNA double helix, the two intertwined components of its backbone are composed of sugar (deoxyribose) and phosphate molecules that are connected by pairs of molecules called bases. The sequence of four bases (guanine, adenine, thymine, and cytosine) in the DNA helix determines the specificity of genetic information. The bases face inward from the sugar-phosphate backbone and form pairs with complementary bases on the opposing strand for specific recognition. The arrangement of chemical groups is unique for each base pair, allowing base pairs to be specifically targeted by transcription factors, polymerases, restriction enzymes, and other DNA-binding proteins.

H

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The biologic importance of RNA splicing is not entirely understood, but many medically relevant genes have alternative splice patterns in which different combinations of exons are chosen for the final mRNA transcript, such that one gene can encode many different proteins (Fig. 1-2). The choice of protein isoform to be expressed from a gene with multiple splicing possibilities is a decision that can be perturbed in disease. Since protein synthesis occurs in the cytoplasm, genetic information is transported out of the nucleus by mRNA. In the cytoplasm, proteins are then synthesized, or translated, in macromolecular complexes called ribosomes that read the mRNA sequence and convert the nucleic acid code, based on threebase segments or codons, into a 20-amino-acid code to form the corresponding protein. The complete set of DNA sequences carried on all the chromosomes is known as the genome. Although the general map of the genome is shared by all members of a species, the recent sequencing of the human genome has given us new tools to reveal the more subtle variations that arise between individuals. These variations are critical, both as a natural engine driving heterogeneity within a species and

as a source of predisposition to cancer types. The most common forms of human genetic variations arise as single-nucleotide polymorphisms (SNPs). Because these allelic dissimilarities are abundant, inherited, and dispersed throughout the genome, SNPs can be used to track racial diversity, personal traits, and susceptibility to common forms of cancer (Fig. 1-3). How do SNPs arise between individuals? One source of variation in DNA sequence derives from deviations in the strict base-pairing rule underlying the structure, storage, retrieval, and transfer of genetic information. The duplicated genetic information in the two strands of DNA not only permits the repair of a damaged coding sequence, but also forms the basis for the replication of DNA. During cell division, polymerase enzymes unwind the DNA strands and copy them, using the base sequences as a template for constructing a new helix so that the dividing cell passes its entire genetic content on to its progeny. Errors in this process are rare, and person-to-person differences make up only about 0.5% of the human genome. SNPs are inherited if they occur in the germline. Most genetic variation is of no obvious functional consequence, occurring in regions that do not

Molecular Tools in Cancer Research • CHAPTER 1

Gene

RNA

Exon

Exon

Exon

Exon

Exon

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2

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Alternative splicing

Figure 1-2 • Alternative splicing produces multiple related proteins, or isoforms, from a single gene. (Adapted from Guttmacher AE, Collins F: Genomic medicine: A primer. N Engl J Med 2002;347:1512–1520.)

RNA

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Translation

Translation

Protein A

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encode protein or alter the regulation of nearby genes. Given the disruptive effects that even subtle genetic changes can have on cell function, it is important to distinguish SNPs that represent mutations from benign polymorphisms. Our ability to monitor hundreds of thousands of SNPs simultaneously is one of the most important advances in modern medical

Tens to hundreds of changes between primary and secondary tumors

Primary tumor

5

genetics. Relatively simple genotyping technologies for SNP detection rely largely on the polymerase chain reaction (PCR). In this procedure, two chemically synthesized single-stranded DNA fragments, or primers, are designed to match chromosomal DNA sequences flanking the segment in which an SNP is positioned. The strands of genomic DNA are separated by heating and, after cooling, the primer

Primary tumor

Figure 1-3 • Using SNPs to determine cancer susceptibility. Millions of single-nucleotide polymorphisms (SNPs) exist between individuals, as depicted by the red arrows and the SNP density map of human chromosome 11 (right). By contrast, point mutations, deletions, insertions, and rearrangements between normal tissues and tumors or between primary and secondary tumors probably number in the tens to hundreds (or potentially thousands), as depicted by the spectral karyotype image at the bottom of the figure. Because the constitutional genetic polymorphisms are present in all of the tissues of the body, it might be possible to distinguish differences in metastatic versus nonmetastatic tumors and in nontumor tissue before they ever happen to develop a solid tumor. (Adapted from Hunter K: Host genetics influence tumour metastasis. Nat Rev Cancer 2006;6:141–146.)

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11p15.5 11p15.4 11p15.3 11p15.2 11p15.1 11p14.3 11p14.2 11p14.1 11p13 11p12 11p11.2 11p11.12 11p11.11 11q11 11q12.1 11q12.2 11q12.3 11q13.1 11q13.2 11q13.3 11q13.4 11q13.5 11q14.1 11q14.2 11q14.3 11q21 11q22.1 11q22.2 11q22.3 11q23.1 11q23.2 11q23.3 11q24.1 11q24.2 11q24.3 11q25

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Part I: Science of Clinical Oncology

Primers

Primers Heat

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Figure 1-4 • Amplification of DNA by PCR. The DNA sequence to be amplified is selected by primers, which are short, synthetic oligonucleotides that correspond to sequences flanking the DNA to be amplified. After an excess of primers is added to the DNA, together with a heat-stable DNA polymerase, the strands of both the genomic DNA and the primers are separated by heating and allowed to cool. The polymerase elongates the primers on either strand, thus generating two new, identical double-stranded DNA molecules and doubling the number of DNA fragments. Each cycle takes just a few minutes and doubles the number of copies of the original DNA fragment.

binds to its matching sequence in the genomic DNA. With the addition of nucleotide building blocks and a heat-stable DNA polymerase, the primer pairs, or amplicons, initiate synthesis of new DNA strands using the chromosomal material as a template. Each successive copying cycle, initiated by “melting” the resulting double-stranded products

with heat, doubles the number of DNA segments in the reaction (Fig. 1-4). The technique is exceptionally sensitive; millions of identical DNA copies can be generated in a matter of hours with PCR using a single DNA molecule as the starting material. Other novel methods for large-scale SNP detection include single nucleotide primer extension, allele-specific hybridization, oligonucleotide ligation assay, and invasive signal amplification, which detect polymorphisms directly from genomic DNA without the requirement of PCR amplification. Regardless of the method that is used to characterize them, the collective SNPs in a selected genomic region characterize a haplotype, or specific combination of alleles at multiple linked genetic loci along a chromosome that are inherited together. Even when the SNPs within a given haplotype are not directly involved in a disease, they provide markers for clonality and for the loss or rearrangement of specific chromosomal segments in growing tumors. In the human nucleus, each of the 23 tightly compacted chromosomes has a characteristic size and structure and a distinctive base sequence that carries unique protein coding information. Other noncoding DNA sequences are used for directing the transcription of neighboring genes through complex regulatory circuits that involve protein binding and modification of the DNA itself or shifting of its chromosomal packaging. Although genomic instability is generally considered a consequence of tumor formation rather than the initial trigger of cancer, the loss, gain, or rearrangement of chromosomal segments through deletion or translocation is a common form of neoplastic mutation, as protein-coding segments from different genes are combined or regulatory sequences are brought into new proximity to genes that they do not normally control. Gross changes in DNA arrangement can be detected by cytogenetic analysis of chromosomal features on metaphase spreads. Fluorescent in situ hybridization provides greater resolution by localizing specific chromosomal DNA sequences corresponding to fluorescently labeled probes (Fig. 1-5) and can be used to track specific alterations in chromosomal structure where known genes are involved. Although cytogenetic techniques are useful in detecting consistent, nonrandom structural abnormalities of clonal tumor cells, they

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Figure 1-5 • Detection of chromosomal translocations in interphase cells by fluorescent in situ hybridization. This technology uses a labeled DNA segment as a probe to search homologous sequences in interphase chromosomes for the t(9;22)(q34;q11) translocation, associated with chronic myeloid leukemia. On the left, patient nuclei were hybridized with probes for chromosome 9 (labeled with SpectrumRed fluorophore) and chromosome 22 (labeled with SpectrumGreen). (Republished with permission of AlphaMed Press, from Oncologist, Varella-Garcia M. Molecular cytogenetics in solid tumors: laboratorial tool for diagnosis, prognosis, and therapy. 2003;45–58; permission conveyed through Copyright Clearance Center, Inc.)

Molecular Tools in Cancer Research • CHAPTER 1

require cell culture, which can limit their usefulness, particularly in analyzing solid tumors. Particularly when chromosomal alterations are too small to be detected by cytogenetic analysis, exploitation of the exquisite sequence specificity of certain bacterial DNA endonucleases, called restriction enzymes, allows the systematic cleavage of very large DNA molecules isolated from tumor samples into predictable, manageable subfragments. These can be identified by hybridization of a short, specific DNA or RNA oligonucleotide probe to its complementary base sequence, or target, in the fractionated genomic material. In most applications, the enzyme-digested sample of DNA is size fractionated by gel electrophoresis and transferred or “blotted” onto a nylon membrane to which labeled probe is then applied. In this procedure, DNA fragments containing sequences that hybridize with radioactively labeled probe can be detected by autoradiography or, alternatively, by nonisotopic colorimetric or chemiluminescent systems (Fig. 1-6). When a SNP occurs in the recognition site of a restriction enzyme, lost or rearranged DNA produces a change in the enzyme cleavage pattern, and these restriction-fragment-length polymorphisms can be detected by blotting analysis. PCR-based analysis using flanking DNA sequences as primers is also used to produce fragments, changes in the size of which, due to addition or loss of DNA bases, can be detected by gel electrophoresis. Subtler single base mutations can be identified by automated DNA-sequencing techniques. The plethora of data that arise from genome-wide association studies using currently available techniques poses particular challenges to cancer researchers. Discerning the causal genetic variants among genotype-phenotype associations requires extensive replication, control for underlying genetic differences in population cohorts, and consistent classification of clinical outcomes. New technologies must be met with equivalently sophisticated and rigorous analytical methodologies for the true genetic cause of cancer to be teased out from our variable and often unstable heredity.

ENGINEERING GENES The engineering of genes by recombinant DNA technology evolved from methods that were initially devised to provide sequences in amounts sufficient for biochemical analysis. The original protocol involves clipping the desired segment from the surrounding DNA and inserting it into a bacterial or viral vector, which is then amplified millions of times in a host bacterium. Using recombinant DNA technology, genetic engineering can routinely produce industrial quantities of pure, clinically useful products in a cost-effective way. For diagnostic purposes, it is easier and faster to amplify a known genomic DNA sequence directly from a patient sample with PCR, but the classic approach is still applied to the construction of recombinant DNA libraries. To be useful, a DNA library must be as complete as possible, with recombinant members, or clones, sufficiently numerous to include all the sequences in an individual genome. For certain kinds of gene linkage analysis that require long, uninterrupted stretches of DNA, special vectors, such as bacterial or yeast artificial chromosomes, can carry foreign DNA fragments of enormous lengths. Chromosomal segments represented in genomic DNA libraries can contain the structure of an entire gene, including the information that regulates its expression and formed the starting material for sequencing the human genome. For some applications, construction of partial libraries, which contain only the DNA sequences transcribed by a particular tissue or type of cell, is sufficient. The starting material in this case is mRNA. For cloning purposes, the enzyme reverse transcriptase can convert mRNA into complementary DNA (cDNA). Advanced techniques for acquiring full-length copies of RNA transcripts include rapid amplification of cDNA ends. The cDNAs are then incorporated into bacterial vectors. The number of clones in a cDNA library is much smaller than that in a genomic library, since a cDNA library repre-

sents only the genes that are expressed by the tissue of interest and contains exclusively the coding portion of genes. Screening DNA libraries for a specific gene classically relied on mass growth of bacterial hosts on agar, transfer of replicates to nylon filter and exposure to a specific DNA probe. The probe’s unique sequence of nucleotides ensures that it hybridizes only to a nucleic acid molecule with the complementary sequence, which marks the position of the target clone. Cloning vectors can also be modified to drive the expression of their payload, producing “expression” libraries in bacteria that can be screened for protein production using specific antibodies, a technique that becomes increasingly important in large-scale screening of protein products.

LOSING CONTROL OF THE GENOME Mutations that lead to oncogenic transformation of a cell invariably affect the expression of its genetic information that specifies functional products, either RNA molecules or proteins used for various cellular functions. The primary level of gene control is the transcription of DNA into RNA. Gene regulation, or the control of RNA synthesis, represents a complex process that is frequently a target of neoplastic mutation. DNA regulatory sequences do not encode a product. Yet without them, a cell could not coordinate the expression of the hundreds of thousands of genes in its nucleus, select only certain genes for expression, and activate or repress them in response to precise internal or external signals. These control centers of the genome contain binding sites for multiple proteins, called transcription factors, which interact to form regulatory networks that control gene transcription. Their function can be altered by signals that induce modifications such as phosphorylation, or by interactions with other regulators such as steroid hormones. Many of the cell’s responses to a wide variety of external stimuli, such as neurotransmitters, antigens, cytokines, and growth factors, are mediated through transcription factors binding to DNA regulatory sequences. Certain regulatory DNA sequences common to many genes are positioned upstream of the transcription start site (Fig. 1-7). Collectively called the promoter of a gene, these proximal sequences comprise binding sites for the RNA polymerase and its numerous cofactors. Whereas the position of the promoter with regard to the transcription start site is relatively inflexible, other DNA regulatory elements, known as enhancers, occur in unpredictable locations, often at a considerable distance from the genes they control. Some transcription factors bind to particular regions of enhancers and drive their associated genes in many types of cells, whereas others, which are active in only a limited variety of cells, maintain a tissue-specific pattern of gene expression. Enhancers are often responsible for the aberrant expression of genes induced by chromosomal translocationassociated specific forms of cancer; a normally quiescent gene promoting cell growth that is dislocated to a position near a strong enhancer may be activated inappropriately, resulting in loss of growth control. Enhancers and promoters have been assigned specific roles by means of cell culture assays or in transgenic animals in which putative regulatory DNA sequences are linked to test or “reporter” genes and are examined for their ability to activate expression of the reporter gene in response to the appropriate signals. By assessing the effects of deleting, adding, or changing DNA sequences within the regulatory element, the precise nucleotides that are critical for recognition by transcription factors can be determined. The interaction between protein and DNA is also used to identify transcription factor-binding sites in a regulatory region. Whereas electrophoretic mobility shift assays, or DNA footprinting, were once standard techniques for determining protein-DNA interactions, emerging genome-wide technologies, such as chip sequencing (see Fig. 1-13 later in the chapter), are revolutionizing the way in which we see the simultaneous interaction of a transcription factor complex

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Molecular Tools in Cancer Research • CHAPTER 1 Figure 1-6 • A, Analysis of DNA by gel electrophoresis and Southern blotting. Genomic DNA is cut with restriction enzymes into fragments before being separated according to size by gel electrophoresis. The four lanes on the gel represent the digestion of the DNA with four different restriction enzymes. In Northern blotting, total cellular RNA, including messenger RNA, can also be separated according to size. After electrophoresis, the nucleic acids in the gel are transferred directly onto a charged nylon filter, to which they are tightly bound. Thus, the filter contains a precise replica of the nucleic acid distribution in the gel. The filter is then hybridized in a rotating sealed chamber with a DNA or RNA probe specific for the target of interest (in this case, sequences in a microbial pathogen). Probes have traditionally been radioactively labeled with nucleotides containing phosphorus 32; however, the use of nonradiolabeled probes is becoming more common. After the probe has hybridized to its target sequence, the nonhybridized probe is washed away, and the filter is exposed to x-ray film. A DNA sequence complementary to the probe is seen as a dark band on the developed film. The position of the hybridized target sequence in each lane is unique to the restriction enzyme that is used to digest the DNA. This procedure is termed Southern blotting when DNA is analyzed and Northern blotting when RNA is analyzed. B, Cytogenetic and molecular analyses of tumor cells. Three methods of detecting the specific genetic alterations shared by all the neoplastic cells in a tumor are shown. (1) If the genetic alteration is large enough, as in the deletion of a region of DNA between loci A and B, cytogenetic analysis can detect grossly visible karyotypic changes. (2) Southern blot analysis can detect small changes in gene structure that routine karyotyping studies cannot find. In this example, a probe to locus A normally detects a large DNA restriction fragment, as shown by the band for the normal DNA sample. Because of the deleted DNA segment in the tumor cells, the probe for the region between loci A and B hybridizes to a smaller, rearranged, tumor-specific restriction fragment. The normal, larger band shown on the blot is from DNA contributed by nonneoplastic stromal and reactive cells. (3) In many applications, the polymerase chain reaction (PCR) can detect alterations in DNA structure with the highest degree of sensitivity. Here, the primers that anneal to loci A and B in normal DNA are too far apart to yield an amplified PCR product. The deletion shown in the tumor DNA brings the two annealing sites close to one another, allowing the generation of a novel amplified PCR product. (A, Modified from Naber SP: Molecular pathology-diagnosis of infectious disease. N Engl J Med 1994;331:1212– 1215. B, From Naber SP: Molecular pathology-detection of neoplasia. N Engl J Med 1994;331:1508–1510.)

with virtually all of its potential genomic targets in a particular cell state. Our appreciation of oncogenic perturbations, either by mutation of regulatory protein-coding genes or in the target sequences these proteins recognize, has recently extended to include epigenetic lesions, although these mechanisms have been more difficult to define. Multiple levels of control are necessary to ensure correct gene expression, which is so central to the normal function of the cell. Epigenetics refers generally to control information that is inherited during cell division along with the DNA sequence itself. A key component of epigenetic regulation, chromatin, wraps DNA into coils with scaffolding proteins such as histones as a necessary component of chromosomal compaction but also plays a critical role in gene accessibility (Fig. 1-8). Active genetic loci are associated with loosely configured euchromatin, whereas silent loci are condensed in heterochromatin. The formation of chromatin configurations both controls and is controlled by patterns of methylation on specific DNA sequences, relating the underlying genetic information to its higher-order structure that determines whether a particular gene regulatory element is available to transcription factors. These epigenetic modifications of the nuclear environment that determine the accessibility of a gene can persist during cell division, as inherited patterns of methylation provide permanent marks for altered chromatin configuration in daughter cells. Recent research has linked rearrangement of chromatin and associated DNA methylation with the inactivation of tumor suppressor genes and neoplastic transformation. Defects that could lead to

cancer involve perturbations in the “epigenotype” of a particular locus through the silencing of normally active genes or activation of normally silent genes, which are associated with changes in DNA methylation, histone modification, and chromatin proteins (Fig. 1-9). Changes in the number or density of heterochromatin proteins associated with cancer-related genes such as EZH2 or of euchromatic proteins such as trithorax in leukemia can also be associated with abnormal patterns of methylation in gene promoter regions as well as with higher-order chromosomal structures that are only beginning to be understood. Finally, it is increasingly evident that interactions between the “epigenome,” the genome, and the environment are a common target for mutation and can have profound effects on the gene expression readout of a cancer cell.

PROFILING TUMORS Monitoring global gene expression patterns of cells represents one of the latest breakthroughs in developing a molecular taxonomy of cancer. Genome-wide profiling of gene expression in tumors delivers an unprecedented view into the biological processes underlying tumor progression by following the changes in a tumor cell’s transcriptional landscape. Relying on two-color fluorescence-based microarray technology (DNA microarray), simultaneous evaluation of thousands of gene transcripts and their relative expression can provide a snapshot of the “transcriptome,” the full complement of RNA transcripts that are produced at a specific time during the progression of malignancy. Transcriptional profiling using microarrays typically involves screens of mRNA expression from two sources (such as tumor and normal cells), using complementary cDNA or oligonucleotide libraries that are arranged in extremely high density on microchips. These are probed with a mixture of fluorescently tagged cDNAs generated from the tumor and normal samples, which results in differential staining of each gene spot. The relative intensity of the two different colors reflects the RNA expression level of each gene in each sample as analyzed with a laser confocal scanner (Fig. 1-10). By using microarrays, single genes that constitute diagnostic, prognostic, or therapeutically relevant markers can be systematically monitored. Alternatively, the entire set of expressed genes can be collectively analyzed by using powerful statistical methods to classify tumors by their transcriptional profile. Microarray analysis has already dramatically improved our ability to explore the genetic changes that are associated with cancer etiology and development and is providing new tools for disease diagnosis and prognostic assessment. For example, DNA microarray analysis of multiple primary breast tumor transcriptomes has revealed a reproducible 70-gene expression signature that was recently cleared by the U.S. Food and Drug Administration for a PCR-based application in which expression analysis of a relatively small gene group can predict the prognosis of early-stage breast cancers. When applied on a larger scale, these assays can predict response to chemotherapy or optimize pharmaceutical intervention by targeting therapeutic approaches to specific patient populations and, ultimately, to individualized therapy. Serial analysis of gene expression (SAGE) provides a simultaneous, comprehensive evaluation of multiple mRNA species. Unlike microarray analysis, SAGE does not require prior knowledge of the genes of interest and provides quantitative and qualitative data of potentially every transcribed sequence in a particular tissue or cell type. Furthermore, SAGE can quantify low-abundance transcripts and can reliably detect relatively small differences in transcript concentrations between cell populations. The SAGE method generates a short sequence tag that functions as a unique identifier of a transcript, derived from a defined location within that transcript. Many transcript tags are concatenated into a single molecule and then sequenced, revealing the identity of multiple tags simultaneously. The relative presentation of each member of a SAGE tag library is proportional to the corresponding mRNA abundance in the original transcript

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Part I: Science of Clinical Oncology Gene structure Exon 1 Enhancer

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Figure 1-7 • Mammalian gene structure and expression. The DNA sequences that are transcribed as RNA are collectively called the gene and include exons (expressed sequences) and introns (intervening sequences). Introns invariably begin with the nucleotide sequence GT and end with AG. An AT-rich sequence in the last exon forms a signal for processing the end of the RNA transcript. Regulatory sequences that make up the promoter and include the TATA box occur close to the site where transcription starts. Enhancer sequences are located at variable distances from the gene. Gene expression begins with the binding of multiple protein factors to enhancer sequences and promoter sequences. These factors help to form the transcription-initiation complex, which includes the enzyme RNA polymerase and multiple polymerase-associated proteins. The primary transcript (pre-mRNA) includes both exon and intron sequences. Post-transcriptional processing begins with changes at both ends of the RNA transcript. At the 5′ end, enzymes add a special nucleotide cap; at the 3′ end, an enzyme clips the pre-mRNA about 30 base pairs after the AAUAAA sequence in the last exon. Another enzyme adds a polyA tail, which consists of up to 200 adenine nucleotides. Next, spliceosomes remove the introns by cutting the RNA at the boundaries between exons and introns. The process of excision forms lariats of the intron sequences. The spliced mRNA is now mature and can leave the nucleus for protein translation in the cytoplasm. (Adapted from Rosenthal N: Regulation of gene expression. N Engl J Med 1994; 331:931–932.)

Translation into protein

population. Comparative expression profiles can then be deduced by comparing the abundance of individual tags within each sample set. This allows changes in global expression profiles of normal or malignant tissues under different therapeutic conditions to be rapidly evaluated. These technologies can be applied to the analysis of noncoding RNA species as well. Beside the 20,000 protein-coding transcripts that are used to classify a wide variety of human tumors, hundreds if not thousands of small, noncoding interference RNA species have recently been discovered with critical functions in multiple biological processes, many of which are directly or indirectly involved in the

control of cell proliferation. Known as microRNAs (miRNAs), these short transcripts arise from primary genome-encoded transcripts of variable sizes that are processed into 70- to 100-nucleotide hairpinshaped precursors, which are processed into mature miRNAs of 21to 23-base-pair RNA molecules (Fig. 1-11). miRNAs function by base-pairing with specific mRNAs to inhibit translation or to promote mRNA degradation. In the context of cancer, miRNAs could act in concert with other effectors such as p53 to inhibit inappropriate cell proliferation. A global decrease in miRNA levels is often observed in human cancers, indicating that small RNAs could have an intrinsic function in tumor suppression. The utility of monitoring the expres-

Molecular Tools in Cancer Research • CHAPTER 1

Figure 1-8 • Chromatin packaging of DNA. The 4 meters of DNA in every human cell must be compressed in the nucleus, reaching compaction ratios of 1:400,000. This is achieved by wrapping the DNA (blue) around histone protein complexes (green), forming nucleosomes that are connected by a thread of free linker DNA. Each nucleosome, together with its linker, packages about 200 base pairs (66 nm) of DNA. The nucleosomes are then coiled into chromatin, a rope of nucleoprotein about 30 nm thick (bottom left electron micrograph). To allow DNA to be accessed by transcription and replication apparatus, chromatin is relaxed (bottom right electron micrograph). (Courtesy of Jakob Waterborg www.umkc.edu/sbs/waterborg/ chromat/chromatn.html © 1998 Jakob Waterborg.)

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Figure 1-9 • Gene accessibility through epigenetics. The nature of epigenetic lesions. The cartoon depicts known and possible defects in the epigenome that could lead to disease. A, X is a transcriptionally active gene with sparse DNA methylation (brown circles), an open chromatin structure, interaction with euchromatin proteins (green protein complex), and histone modifications such as H3K9 acetylation and H3K4 methylation (green circles). Y is a transcriptionally silent gene with dense DNA methylation, a closed chromatin structure, interaction with heterochromatin proteins (red protein complex), and histone modifications such as H3K27 methylation (pink circles). B, The abnormal cell could switch its epigenotype through the silencing of normally active genes or activation of normally silent genes, with the attendant changes in DNA methylation, histone modification, and chromatin proteins. In addition, the epigenetic lesion could include a change in the number or density of heterochromatin proteins in gene X (such as EZH2 in cancer) or euchromatic proteins in gene Y (such as trithorax in leukemia). There may also be an abnormally dense pattern of methylation in gene promoters (shown in gene X) and an overall reduction in DNA methylation (shown in gene Y) in cancer. The insets show that the higher-order loop configuration may be altered, although such structures are currently only beginning to be understood. (Adapted from Feinberg AP: Phenotypic plasticity and the epigenetics of human disease. Nature 2007;447:433–440.)

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Figure 1-10 • Microarray-based expression profiling of breast tumor tissue. A, Reference RNA and tumor RNA are labeled by reverse transcription with different fluorescent dyes (green for the reference cells and red for the tumor cells) and are hybridized to a cDNA microarray containing robotically printed cDNA clones. B, The slides are scanned with a confocal laser scanning microscope, and color images are generated with RNA from the tumor and reference cells for each hybridization. Genes that are upregulated in the tumors appear red; those with decreased expression appear green. Genes with similar levels of expression in the two samples appear yellow. Genes of interest are selected on the basis of the differences in the level of expression by known tumor classes (e.g., BRCA1-mutation-positive and BRCA2-mutation-positive). Statistical analysis determines whether these differences in the gene expression profiles are greater than would be expected by chance. C, The differences in the patterns of gene expression between tumor classes can be portrayed in the form of a color-coded plot, and the relationships between tumors can be portrayed in the form of a multidimensional-scaling plot. Tumors with similar gene expression profiles cluster close to one another in the multidimensional-scaling plot. D, Particular genes of interest can be further studied through the use of a large number of arrayed, paraffin-embedded tumor specimens, referred to as tissue microarrays. E, Immunohistochemical analyses of hundreds or thousands of these arrayed biopsy specimens can be performed to extend the microarray findings. (From Hedenfalk I, Duggan D, Chen Y, et al: Gene expression profiles in hereditary breast cancer. N Engl J Med 2001;344:539–548.)

sion of miRNAs in human cancer is just now being explored, but preliminary findings reveal an extraordinary level of diversity in miRNA expression across cancers and the large amount of diagnostic information that is encoded in a relatively small number of miRNAs. Significant technologic advances facilitating the profiling of the miRNA expression patterns in normal and cancer tissues hint at the unexpected greater reliability of miRNA expression signatures than the respective signatures of protein-coding genes in classifying cancer types. Along with their potential diagnostic value, miRNAs are also being tested for their prognostic use in predicting clinical behaviors of cancer patients. Although Northern blot analysis is a reliable technique to detect gene expression at the mRNA level, it has some limitations, such as unequal hybridization efficiency of individual probes and difficulty in detecting multiple miRNAs simultaneously. For cancer studies, it is important to be able to compare the expression pattern of all

known miRNAs between cancer cells and normal cells. Thus, DNA microarrays are used to detect the full complement of miRNA expression at a single point in time. Since probe specificity in miRNA microarray analysis can be problematic, owing to the small target size, hybridization can first be performed in solution and then quantified by using multicolor flow sorting. Real-time PCR can also be employed to quantify specific miRNA sets or to capture a more detailed picture of their changing expression profiles in tumor progression. Identification of the miRNAs that are involved in tumor pathogenesis and elucidation of their action in a specific cancer will be the next necessary steps for their manipulation in a therapeutic setting.

THE CANCER PROTEOME The term proteome describes the entire complement of proteins that are expressed by the genome of a cell, tissue, or organism. More

Molecular Tools in Cancer Research • CHAPTER 1

Protein-coding gene

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Figure 1-11 • MicroRNA production and gene regulation in animal cells. Mature functional microRNAs of approximately 22 nucleotides are generated from long primary microRNA (pri-microRNA) transcripts. First, the pri-microRNAs, which usually contain a few hundred to a few thousand base pairs, are processed in the nucleus into stem-loop precursors (pre-microRNA) of approximately 70 nucleotides by the RNase III endonuclease Drosha and DiGeorge syndrome critical region gene 8 (DGCR8). The pre-microRNAs are then actively transported into the cytoplasm by exportin 5 and Ran-GTP and further processed into small RNA duplexes of approximately 22 nucleotides by the Dicer RNase III enzyme and its partner Loqacious (Loqs), a homolog of the human immunodeficiency virus transactivating response RNA-binding protein (TRBP). The functional strand of the microRNA duplex is then loaded into the RNA-induced silencing complex (RISC). Finally, the microRNA guides the RISC to the target messenger RNA (mRNA) target for translational repression or degradation of mRNA. (Adapted from Chen C-J: MicroRNAs as oncogenes and tumor suppressors. N Engl J Med 2005;353: 1768–1771.)

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specifically, it is used to describe the set of all the expressed proteins at a given time point in a defined setting, such as a tumor. Like RNA transcription, the synthesis of proteins is a highly regulated process that contributes to the specific proteome of a particular cell and can be perturbed in diseases such as cancer. Advances in protein analytical techniques over the last decade have progressed to the point at which even small numbers of specific proteins expressed in tissues can be used to predict the prognosis of a cancer. The improvement of protein-based assays has made it possible to identify and examine the expression of most proteins and to envision large-scale protein analysis on the level of gene-based screens.

Translational repression

Various systematic methodologies contributed to the current explosion of information on the proteome and are being compared for their ability to provide suitable platforms for generating databases on protein structural features, interaction maps, activity profiles, and regulatory modifications. The yeast two-hybrid system is a popular genetics-based approach for detecting protein-protein interactions inside a cell (Fig. 1-12). One protein that is fused to the DNA binding domain (bait) and a different protein that is fused to the activation domain of a transcriptional activator (prey) are expressed together in yeast cells. If the bait and prey interact, transcription of a reported gene is induced and

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Figure 1-12 • Exploring protein-protein interactions with the yeast two-hybrid system. Two-hybrid technology exploits the fact that transcriptional activators are modular in nature. Two physically distinct functional domains are necessary to get transcription: a DNA-binding domain that binds to the DNA of the promoter and an activation domain that binds to the basal transcription apparatus and activates transcription. A, The known gene encoding protein A is cloned into the “bait” vector, fused to the gene encoding a DNA-binding domain from some transcription factor. When placed into a yeast system with a reporter gene, this fusion protein can bind to the reporter gene promoter, but it cannot activate transcription. B, Separately, a second gene (or a library of cDNA fragments encoding potential interactors), protein B, is cloned into the “prey” vector, fused to an activation domain of a different transcription factor. When placed into a yeast strain containing the reporter gene, it cannot activate transcription because it has no DNA-binding domain. C, When the two vectors are placed into the same yeast, a transcription factor is formed that can activate the reporter gene if protein B, made by the second plasmid, binds to protein A. D, Screening a yeast two-hybrid library. The plate on the left holds 96 different yeast strains in patches (or colonies), each of which expresses a different bait protein (top). The plate on the right holds 96 patches, each of the same yeast strain (prey strain) that expresses a protein fused to an activation domain (prey). The plate of bait strains and the plate of prey strains are pressed to the same replica velvet, and the impression is lifted with a plate containing YPD medium. After 1 day of growth on the YPD plate, during which time the two strains mate to form diploids, the YPD plate is pressed to a new replica velvet, and the impression is lifted with a plate containing diploid selection medium and an indicator such as X-Gal. Blue patches (dark spots) on the X-Gal plate indicate that the lacZ reporter is transcribed, suggesting that the prey interacts with the bait at that location. (C, Text after http://www.invitrogen. com/catalog_project/cat_hybrid.html [July, 2000]; figure retrieved from http:// www.nature.com/nature/journal/v403/n6770/pdf/403601a0.pdf. D, From Bartel PL, Fields S (eds): The Yeast Two-Hybrid System. New York, Oxford University Press, 1997; Finley RL Jr, Brent R: Two-hybrid analysis of genetic regulatory networks. Retrieved from http://www.genetics.wayne.edu/finlab/YTHnetworks.html.)

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detected, typically by a color reaction that reflects the transactivation of the reporter gene and, by proxy, the interaction of the two test proteins. The method can also be used for large-scale protein interactions, RNA-protein interactions, and protein-ligand binding. As a complementary proteomics tool, mass spectrometry is an accurate mass measurement of charged peptides that are isolated by two-dimensional gel electrophoresis, producing a mass-to-charge ratio of charged samples under vacuum that can be used to determine the sequence identity of peptides. Combined with a specific proteolytic cleavage step, mass spectroscopy can be used for peptide mass mapping. Automation of this process has made mass spectroscopy the analytic tool of choice for many proteomics projects. Monoclonal antibodies (mAbs) have been a cornerstone of protein analysis in cancer research and more recently have risen to prominence as cancer therapeutics based on their exquisite specificity for protein targets and their potent interference with protein function. Laboratory mice have been the animal model of choice for generating a ready source of diverse high-affinity and high-specificity mAbs; however, the use of rodent antibodies as therapeutic agents has been restricted by the inherent immunogenicity of mouse proteins in a human setting. The more recent application of transgenic mouse technology to introduce variable regions encoded by human sequences into the corresponding mouse immunoglobulin genes has enabled

the generation of “humanized” therapeutic mAbs with reduced immunogenicity. Numerous of these mAb-based agents are currently in trial or in use as therapeutics for cancer, and the potential for further optimization of mAbs through genetic engineering promises to open new avenues for in vivo therapy. From an epigenetics perspective, new techniques are enabling the genome-wide characterization of protein-DNA interactions that can uncover novel transcription factor targets, histone modifications, and DNA methylation patterns within a cancer cell. Combining chromatin immunoprecipitation (ChIP) with microarray (ChIP-on-chip) allows genome-wide screening for the binding position of protein factors to their gene targets. In ChIP-on-chip assays, a cross-linking reagent is applied in vivo to proteins associated with DNA in the nucleus, which then can be coimmunoprecipitated with specific antibodies to the protein under analysis. The bound DNA and appropriate controls are then fluorescently labeled and applied to microscopic slides for microarray analysis, rendering a simultaneous profile of all the binding positions of specific proteins in the cancer cell’s genome (Fig. 1-13). After a decade of development, proteomics is still primarily a basic research activity, yet in the near future, this technology is likely to have a profound impact on medicine. By defining the collective protein-protein interactions in a cancer cell (its “interactome”), func-

Molecular Tools in Cancer Research • CHAPTER 1 Antibody Binding sites

TF

TF 000000000000000000 000000

0000

Chr1 ENr231 Probe Repeats TAFii250 RNAP

000

00000000

Figure 1-13 • ChIP-on-chip is a technique for location, isolation, and identification of the DNA sequences occupied by specific DNA-binding proteins in cells. These binding sites may indicate functions of various transcriptional regulators and help to identify their target genes during development and disease progression. The types of functional elements that one can identify using ChIP-on-chip include promoters, enhancers, repressor and silencing elements, insulators, boundary elements, and sequences that control DNA replication. (Adapted from Ren lab: www.chiponchip. org/Images/scheme_800x600_crop.jpg.)

000

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Binding site identification

tional relationships between disease-promoting genes could be revealed that would provide novel candidates for intervention. Networks of disorder-gene associations are already being built that offer a platform for describing all known phenotype and disease gene associations, often indicating the common genetic origin of many diseases. A precise diagnosis of cancer using proteomics could be envisioned, based on highly discriminating patterns of proteins in easily accessible patient samples. Proteomics information also promises to provide sophisticated mathematical models of the molecular events underlying a process as complex as neoplastic transformation, which will capture the dynamics of the disease with unprecedented power.

MODELING CANCER IN VIVO Once the mechanistic underpinnings of a particular cancer have been described, creating an animal model to test that mechanism becomes critical to understanding the pathophysiology and to design therapeutic strategies for treatment. Recent advances in manipulating the mouse genome have resulted in more sophisticated models of human cancer. These methodologies can circumvent embryonic death by targeted alteration of gene expression only after a critical period in development and reduce the complexity of gene functional analysis by restricting its pattern of activation. Inducible gene expression or silencing also allows acute, as opposed to chronic, effects to be assessed. Integrating an oncogene that causes malignancy into the genome of a mouse without altering the mouse’s own genes generates a transgenic, cancer-prone mouse that transmits this trait to its offspring with a dominant pattern of inheritance. Although species differences in tumor susceptibility and disease remission exist between mouse and human, the tools for genetic manipulation in mouse are superior to those in other mammals, and useful information about the function of oncogenes can be gained by targeted expression of mutant protein products in mouse tissues. The technology for producing transgenic mice joins recombinant DNA methodology with standard techniques that are used today by in vitro fertilization clinics, relying on our understanding of mammalian reproduction and the development of protocols to harvest, manipulate, and reimplant eggs and early embryos (Fig. 1-14). The transgene is constructed so that the gene product will be expressed under appropriate spatial and temporal control. In addition to all the standard signals that are necessary for efficient transcription and translation of the gene, transgenes contain a promoter, or regulatory region, that drives transcription in either a ubiquitous or tissue-

000 00000000

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Array data analysis

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Genomic arrays

restricted pattern. This requires an extensive knowledge of genetic regulation in the target cells. A recent advance that circumvents this requirement involves embedding the transgene inside another gene locus that is expressed in the desired pattern. Held in a bacterial artificial chromosome for easier manipulation, this long stretch of DNA surrounding the host gene is likely to carry all the necessary regulatory information to guarantee a predictable expression pattern of the introduced transgene. The transgene DNA is then injected into the male pronucleus of a fertilized mouse egg, obtained from a female mouse in which hyperovulation has been hormonally induced. The injected eggs are cultured to the two-cell stage and then implanted in the oviduct of another recipient female mouse. Transgenic pups are identified by the presence of the transgene in their genomic DNA (obtained from the tip of the tail and analyzed by PCR). Typically, several copies of the transgene are incorporated in a head-to-tail orientation into a single random site in the mouse genome. About 30% percent of the resulting pups will have integrated the transgene into their germline DNA and constitute the founders of the transgenic lines. RNA analysis of their progeny determines the level of transgene expression and whether the transgene is being expressed in the desired location or at the appropriate time. Given the variability in transgene number and chromosomal location, transgene expression patterns and levels can diverge considerably among different founder lines carrying the same transgene. In general, transgenesis is optimal for modeling oncogenic mutations that cause a gain of function, producing disease even when they occur in only one of a gene’s two alleles. For example, an activating mutation in a growth factor that causes abnormal cell proliferation can be mimicked by introducing a transgenic version of the mutated growth factor gene under the control of an appropriate regulatory sequence for expression in the tissue of interest. The relative susceptibility of such a transgenic mouse to tumorigenesis can help to distinguish between a primary and secondary role of the mutant factor, and established lines of these animals can be used for testing new therapeutic protocols. The genetic construction of cancer-prone transgenic mice with the capacity to induce oncogene expression in vivo provides a new avenue to modeling the role of oncogenes in tumor generation and maintenance. This technology relies on conditional mutagenesis. Producing conditional mutations in mice requires a DNA recombinase enzyme that does not recognize any mouse sequence but rather targets short, foreign recognition sequences to catalyze recombination between them. By strategic placement of these recognition sequences in appropriate orientations either beside or within a mouse gene, the

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Promoter

Figure 1-14 • Generation of transgenic mice. The transgene containing the DNA sequences necessary for the expression of a functional protein is injected into the male (larger) pronucleus of uncleaved fertilized eggs through a micropipette. The early embryos are then transferred into the reproductive tract of a female mouse that has been rendered “pseudopregnant” by hormonal therapy. The resulting pups (founders) are tested for incorporation of the transgene by assaying genomic DNA from their tails. Founder animals that have incorporated the transgene (+) are mated with nontransgenic mice, and their offspring are mated with each other to confirm germline integration and to establish a line of homozygous transgenic mice. Several transgenic lines that have incorporated different numbers of transgenes at different integration sites (and thus express various amounts of the protein of interest) are usually studied. UT, untranslated. (Adapted from Shuldiner AR: Transgenic animals. N Engl J Med 1996;334:653–655.)

3' Flanking Coding region 2' UT region

5' UT

Transgene

Collection of fertilized eggs from a superovulated donor mouse

Injection of transgene into male pronucleus of uncleaved fertilized egg

Transfer of early embryos into reproductive tract of a pseudopregnant mouse

– – + + – + Assay of genomic DNA from tails of founder animals for incorporation of the transgene

Sequential matings to determine germline integration Study of phenotype

recombination results in deletion, insertion, inversion, or translocation of associated genomic DNA (Fig. 1-15). Two recombinase systems are currently in use: the Cre-loxP system from bacteriophage P1 and the Flp-FRT system from yeast. The 34–base-pair loxP or FRT recognition sequences do not occur in the mouse genome, and both Cre and Flp recombinases function autonomously, without the need for cofactors. Cre- or Flp-mediated recombination is not distance or cell-type dependent and can occur in proliferating or differentiated tissues. The general scheme involves two mouse lines, one carrying the recombinase either as a transgene driven by inducible regulatory elements or knocked into one allele of a gene expressed in the desired tissue. The other mouse line harbors a modified gene target, including recognition sequences. Mating the two lines results in progeny carrying both the target gene and the recombinase, which interacts with the target gene only in the desired tissue. A popular conditional methodology is based on the activation of nuclear hormone receptors to control gene expression. Two current systems involve activation of a mammalian estrogen receptor, an estrogen analog 4-hydroxy-tamoxifen, or an insect hormone receptor with the corresponding ligand ectodysone. Although several variations on these hormone-receptor systems are currently in use, the underlying principle is the same. The Cre recombinase gene or another regulatory protein, such as a transcription factor, is fused with the ligand-binding domain from a nuclear hormone receptor protein. The resulting chimeric transgene is placed under the control of a promoter that directs expression to the tissue of interest, and transgenic animals are generated. In the absence of the hormone or an analog, the fusion protein accumulates in the desired tissue but is rendered inactive through its association with resident heat shock proteins. Administered hormone, either systemically or topically, binds to ligand-binding domain moiety of the fusion protein, dissociates it from the heat shock protein, and allows the transcriptional regulatory component to find its natural DNA targets and promote lox-P mediated recombination or, in the case of an inducible transcription factor, activate expression of the corresponding genes. If the ligand-binding domain is fused to a recombinase, administration of hormone leads to the rearrangement of target sequences. This reaction is not reversible but lends additional temporal control over recombinase-based mutation. If the ligand-binding domain is fused to a transcription factor, removal of hormone leads to inactivation of the fusion protein and gene downregulation. Another inducible method in use is the tetracycline (tet) regulatory system. In the classic design (tTA or tet-off), a fusion protein combining a bacterial tet repressor and a viral transactivation domain drives expression of the target transgene by binding to upstream tet operator sequences flanking the transgene transcription start site. In the presence of the antibiotic inducer, the fusion protein is dissociated from the operator sequences, inactivating the transgene. In a

Molecular Tools in Cancer Research • CHAPTER 1

Cell type specific promoter

Cre

loxP

Target gene

loxP

X

Cre

Cre

A

Special cell type

CMV-β actin promoter

βgeo

All other cells

3PA

loxP

MODELS OF RECESSIVE GENE MUTATIONS IN CANCER EGFP

loxP Cre

CMV-β actin promoter EGFP

B

Conditional expression systems have already been developed to generate hematopoietic, leukemogenic, and lymphomagenic mutations in the mouse, as well as solid tumors. These inducible cancer models can be exploited to identify oncogenic signals that influence host-tumor interactions, to establish the role of a given oncogenic lesion in advanced tumors, and to evaluate therapies targeted toward cancer-causing mutations. Potential clinical application of inducible systems include targeting virally delivered transgene expression to malignant tissues by the use of specific inducible regulatory elements, restricting the expression of transgenes exclusively to affected tissues, and increasing the therapeutic index of the vectors, particularly in the context of solid tumors. In all cases, a basic knowledge of the specific mutations that are involved in the molecular genetics of malignancies is required, since it is often unclear that the causal mutation underlying the genesis of neoplasia continues to play a central role in the progression to the fully transformed state. This is particularly important in modeling cancers that are characterized by genetic plasticity, in which drug resistance can arise subsequent to primary tumor formation.

loxP

Figure 1-15 • Conditional mutagenesis schemes demonstrated with the Cre-loxP system. A, Two mouse lines are required for conditional gene deletion: a conventional transgenic mouse line with Cre targeted to a specific tissue or cell type and a mouse strain that embodies a target gene (endogenous gene or transgene) flanked by two loxP sites in a direct orientation (“floxed gene”). Recombination (excision and consequently inactivation of the target gene) occurs only in cells that express Cre recombinase. Hence, the target gene remains active in all cells and tissues that do not express the Cre recombinase. B, The Z/EG double reporter system. These transgenic mice constitutively express lacZ under the control of the cytomegalovirus enhancer/chicken actin promoter. Expression is widespread, notable exceptions being liver and lung tissue. Expression is observed throughout all embryonic and adult stages. When crossed with a Cre recombinase-expressing strain, lacZ expression is replaced with enhanced green fluorescent protein expression in tissues that express Cre. This double reporter system makes it possible to distinguish a lack of reporter expression from a lack of Cre recombinase expression while providing a means to assess Cre excision activity in live animals and cells. (A, Courtesy of Kay-Uwe Wagner, National Institutes of Health. B, From Novak A, Guo C, Yang W, et al: Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 2000;28:147–155.)

complementary design, called reverse tTA (rtTA or tet-on), structural modification of the tet repressor makes the antibiotic an active requirement for binding of the fusion protein to the operator sequences, such that its administration activates transgene expression at any time during the life span of the mouse, whereas withdrawal results in downregulation of the gene. It is important that the transgene integrate into a genomic locus that permits proper tTA or rtTA regulation so that the system exhibits minimal intrinsic leakiness and good antibiotic responsiveness.

In contrast to dominantly acting oncogenes, recessive genetic disorders, such as loss-of-function mutations in a tumor suppressor gene, require both copies (alleles) of a gene to be inactivated. The methods that are needed to produce animal models of recessive genetic disease differ from those that are used in studying dominant traits. Gene knockout technology has been developed to generate mice in which one allele of an endogenous gene is removed or altered in a heritable pattern (Fig. 1-16). Gene disruption or replacement is first engineered in pluripotent cells, termed embryonic stem (ES) cells, which are genetically altered by introduction of a replacement gene that is inactive or mutant. To reduce random integration of the foreign DNA, the replacement gene is embedded into a long stretch of DNA from its native locus in the mouse, which targets the recombination event to the homologous position in the ES cell genome. Inclusion of selectable markers along with the replacement gene allows selection of the cells in which homologous recombination has taken place. Site-specific recombinase systems combined with gene-targeting techniques in ES cells can also be used to inducing recessive single point mutations or site-specific chromosomal rearrangements in a tissue- and timerestricted pattern. In a variation on this theme called knock-in, a foreign gene, such as one encoding a marker, can be placed in the locus of an endogenous gene. The engineered ES cells are then microinjected into the cavity of an intact mouse blastocyst sufficiently early in gestation that these cells can, in principle, populate all the tissues of the developing chimeric embryo. This is rarely the case, so contribution of ES cells to the resulting animal is most often assessed by using ES cells and blastocysts whose genes for coat color differ. If the ES cells contribute to the germ cells of the founder mouse, their entire haploid genome can be passed on to subsequent generations. By mating subsequent progeny of the founder mouse, both alleles of the mutated gene can be passed to a single animal. Overlapping genetic functions can also be defined by crossbreeding mice with mutations in different genes. In this way, it is possible to study the combinatorial effects of oncogene and tumor suppressor gene mutations. These experimental systems are of great value in dissecting the pathogenesis of many tumor types. In some knockout studies, the phenotype of the mutated gene is anticipated by prior knowledge of the gene’s function. However, unexpected mutant phenotypes may help clarify the mechanism of the underlying neoplasia. Pharmacologic manipulation of transgenic knockout animal models of cancer will prove useful in screening therapeutic agents with

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Embryonic stem cell Tumor suppressor gene 5' Homologous region Intron

3' Homologous region Cellular gene

Embryonic stem cell culture

pgk-neo

pgk-tk

Plasmid DNA Knockout vector

Homologous recombination

Cellular gene replaced

Selection by neomycin and glanciclovir

Injection of embryonic stem cells into host blastocyst

Implantation of chimeric blastocyst in foster mother

Germline offspring

Chimeric offspring

Figure 1-16 • Gene knockout strategy for generating mice that lack a tumor suppressor gene. Embryonic stem cells (upper left panel) contain the tumor suppressor cellular gene (upper right panel), which consists of exon 1 (olive green, a 5′ noncoding region), an intron, and exon 2 (red, a protein-coding region, and yellow, a 3′ noncoding region). A knockout vector consisting of a collinear assembly of a DNA flanking segment 5′ to the cellular gene (blue), the phosphoglycerate kinase-bacterial neomycin gene (pgk-neo, violet), a 3′ segment of the cellular gene (yellow), a DNA flanking segment 3′ to the cellular gene (green), and the phosphoglycerate kinase-viral thymidine kinase gene (pgk-tk, orange) is created and introduced into the embryonic stem cell culture. Double recombination occurs between the cellular gene and the knockout vector in the 5′ homologous regions and the 3′ homologous regions (dashed lines), resulting in the incorporation of the inactive knockout vector, including pgk-neo but not pgk-tk, into the cellular genomic locus of the embryonic stem cell. The presence of pgk-neo and the absence of pgk-tk in these replaced genes will allow survival of these embryonic stem cells after positive-negative selection with neomycin and ganciclovir. The clone of mutant embryonic stem cells is injected into a host blastocyst, which is implanted into a pseudopregnant foster mother and subsequently develops into a chimeric offspring (bottom panel). The contribution of the embryonic stem cells to the germ cells of the chimeric mouse results in germline transmission of the embryonic stem cell genome to offspring that are heterozygous for the mutated tumor suppressor allele. The heterozygotes are mated to produce mutant, cancer-prone mice that are homozygous for tumor suppressor deficiency. (Modified from Mazjoub JA, Muglia LJ: Knockout mice. N Engl J Med 1996;334:904–906.)

potential for study in clinical trials. Therapy involving gene or cell replacement can be also tested in genetically engineered disease models. Several caveats are important in considering the use of knockout technology. Most knockout mutations are loss-of-function (null) germline mutations. Inactivation of widely expressed genes with multiple functions may have complex phenotypes. Conversely, if the

functions of two genes overlap, a mutation in one of the genes might not produce an abnormal phenotype, owing to compensation by the unaltered partner. Perhaps the greatest drawback of conventional knockout technology derives from the disruption of gene function at the earliest stage of its expression. If the gene has a vital developmental role, the identification of functions later in development can be occluded. There-

Molecular Tools in Cancer Research • CHAPTER 1

fore, although the generation of a null mutation is an excellent starting point for analysis, it is far from being functionally exhaustive. For these reasons, conditional mutagenesis is the method of choice for the elucidation of the gene functions that exert pleiotropic effects in a variety of cell types and tissues throughout the life of the animal, which is particularly relevant for the generation of mouse models of adult-onset diseases such as cancer. By using recombinase-mediated gene mutation described previously for conditional transgenesis, conditional knockout mutations can be designed to disrupt the function of a target gene in a specific tissue (spatial control) and/or life stage (temporal control). Depending on the design of the experiment, recombinase action can delete

an entire gene, remove blocking sequences to induce gene expression, or rearrange chromosomal segments. With the advent of recent internationally coordinated systematic mutagenesis programs that aim to place a conditional inactivating mutation in each of the 20,000 genes in the mouse genome, the possibilities for modeling cancer are limited only by a researcher’s choice of the gene loci to test. The constantly evolving techniques for gene manipulation in vivo constitute a major advance in cancer research. They promise to provide integration of underlying molecular biological principles of malignancy with pathophysiologic consequences, generating an invaluable resource for understanding the complex genetics of tumor formation that holds great promise for improved treatment of human cancer.

RECOMMENDED TEXTS Alberts B, Johnson A, Lewis J, et al: Molecular Biology of the Cell, 4th ed. London, Taylor and Francis Group, 2002.

Pecorino L: Molecular Biology of Cancer: Mechanisms, Targets, and Therapeutics. New York, Oxford University Press, 2005.

Weinberg RA: Biology of Cancer. London, Garland Science, 2006.

Goh KI, Cusick ME, Valle D, et al: The human disease network. Proc Natl Acad Sci USA 2007;104:8685– 8690. Hunter K: Host genetics influence tumour metastasis. Nat Rev Cancer 2006;6:141–146.

Rosenthal N, Brown S: The mouse ascending: perspectives for human-disease models. Nat Cell Biol 2007;9:993–999. Wu J, Smith LT, Plass C, Huang TH: ChIP-chip comes of age for genome-wide functional analysis. Cancer Res 2006;66:6899–6902.

FURTHER SELECTED READING Chen C-J: MicroRNAs as oncogenes and tumor suppressors. N Engl J Med 2005;353:1768–1771. Feinberg AP: Phenotypic plasticity and the epigenetics of human disease. Nature 2007;447:433–440. Frese KK, Tuveson DA: Maximizing mouse cancer models. Nat Rev Cancer 2007;7:654–658.

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Intracellular Signaling Sara A. Courtneidge

S U M M ARY • Cell growth, metabolism, death, differentiation, movement, and invasion are all controlled by intracellular signaling pathways. These pathways are initiated by ligands binding to, and activating, their cognate receptors, which are usually plasma membrane proteins. • Receptor activation initiates cascades of signaling events, including activation of protein and/or lipid kinases, as well as the recruitment of adaptor proteins, the activation of transcription factors, and changes in the cytoskeleton. Together, these signaling cascades ultimately fashion the response of the cell to the ligand. Intracellular signaling

O F

K EY

P OI NT S

thus translates cues from the extracellular environment, such as peptide growth factors, extracellular matrix proteins, hormones, and cytokines, into appropriate cellular and organismal responses. • As normal cells make the transition to malignancy, alterations in key receptors and signaling pathways occur. Some of these alterations are a result of activating mutations in receptors and signaling pathway components. Other mutations inactivate negative regulators of these pathways. The net result is both enhanced proliferation and inappropriate survival of the cancer cells, as well as unregulated cell

INTRODUCTION Definition During embryonic development and in the adult organism, the fate of a cell is decided by the cues it receives from its surroundings. For example, growth factors instruct cells to divide, and extracellular matrix proteins provide survival signals. Other stimuli can cause cells to migrate, to differentiate, to undergo programmed cell death (apoptosis), or to enter a survival state that involves autophagy. Each of these outcomes is initiated by binding of diverse protein and nonprotein ligands to receptors, most of which are localized on the cell surface. Receptor activation results in the recruitment of adaptor molecules and enzymes, particularly protein and lipid kinases. These recruited proteins then relay signals to the nucleus, the cytoskeleton, and other subcellular compartments to affect the response. Each type of receptor initiates a discrete set of signaling pathways, such that different ligands binding to the same cell can have different effects. Furthermore, the same ligand can have different effects on different cell types because of innate differences in the signaling components present in the cells. Thus, the combinatorial action of several intracellular signaling pathways dynamically controls the responses of cells and organs to external cues from the environment.

Clinical Relevance The first clues that components of intracellular signaling pathways are important in causing cancer came from research on tumor viruses in model systems such as chickens and mice.1 Many RNA tumor

movement and invasive capacity. Together, then, alterations in signaling pathways underlie all aspects of the cancer phenotype. • In recent years, as the genetic alterations in cancer cells have begun to be characterized, new drugs have been developed that target these unregulated signaling pathways. In several cases, these drugs have been shown to be effective against tumors that harbor the appropriate mutations. Current research and development efforts are aimed at uncovering all aberrant intracellular signaling pathways in cancer cells and designing drugs to control them.

viruses (retroviruses) contain cancer-causing genes called oncogenes, which derive from host sequences. For example, the first retroviral oncogene to be discovered, src, the transforming gene of Rous sarcoma virus, derives from the cellular src gene, which encodes a protein tyrosine kinase. During the transduction event, 3′ sequences of the src gene were lost, resulting in the production of a protein that lacks the regulatory carboxy-terminus. Thus, while the cellular Src protein is normally tightly regulated in cells, the viral form of the protein is constitutively active and transforming. Other examples of oncogenes that are transduced by retroviruses include ras, myc, sis (encoding the growth factor PDGF), akt, raf, fos, and many more. Equally important lessons were learned from the study of DNA tumor viruses. The oncogenes of these viruses do not derive from the host genome. Rather, in this case, the oncogenes associate with and modify the functions of key signaling proteins. For example, most DNA tumor viruses encode proteins that are able to inactivate negative regulators of signaling, such as p53 and Rb. Many can also activate signaling by binding to proteins such as Src, the platelet-derived growth factor (PDGF) receptor, and cyclins. These oncogene studies therefore provided the early tools necessary to dissect the signaling pathways that control cell growth. Other important information, particularly on the control of cell survival, has come from the study of genetically tractable organisms such as fruit flies and the nematode Caenorhabditis elegans. More recently, whole genome sequencing projects have allowed the direct analysis of human clinical specimens for alterations in key signaling pathways. With the use of the systems and tools described previously, much progress has been made in the last decades in the characterization of intracellular signaling: Several classes of receptor have been defined,

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all protein kinases (the kinome) have been described,2 and some signaling pathways are now known in their entirety.3 Furthermore, there has been intense study of the perturbations that occur in intracellular signaling during cancer progression. This effort has resulted in the definition of new molecular targets for cancer drug discovery.4 Indeed, some new drugs that target cellular signaling have recently been approved, and many more are in clinical testing. While not yet fully realized, the promise is that defining and targeting signaling pathways responsible for all aspects of the cancer phenotype will result in more potent and less toxic chemotherapies. This chapter reviews the basic principles of intracellular signaling. Then some examples of receptors and their mechanisms of activation are given (antigen and other immune receptors are not discussed). This is followed by an overview of some common signal transduction intermediates and some selected examples of signal transduction pathways elicited by certain receptors. Finally there is a brief discussion of how these signaling pathways are dysregulated in cancer and how this might be exploited for targeted therapeutic intervention. This is not intended to be a comprehensive list of all receptors and signals; rather, examples have been chosen that have relevance to the cancer phenotype. The references that are provided are intended to point the reader to more detailed and thorough reviews of the topics covered; primary references are provided only for new discoveries that have not yet been the subject of reviews. Furthermore, many of the examples and themes that are briefly described in this chapter are explored in more detail in later chapters. For example, in Chapter 5, Craig Thompson and Rebecca Elstrom describe in some detail the control of cell death and in Chapter 4, Jacqueline Lees reviews how signaling pathways feed into the cell cycle. Furthermore, each of the chapters on specific malignancies contains descriptions of the dysregulated signaling pathways that are involved in each disease.

activation. Signaling specificity can be provided by the recruitment of distinct adaptor proteins by different receptors. A schematic of how signal transduction pathways can involve different components is outlined in Figure 2-1.

Receptor Activation by Ligand Most of the receptors that receive cues from the extracellular environment are found in the plasma membrane of the cell, where they initiate signaling for those peptide and protein ligands that cannot cross the lipid bilayer. In contrast, receptors for lipid soluble ligands are found in the cytoplasm and the nucleus. A broad overview of the molecular makeup and mechanism of activation of several different classes of receptor will be described later.

G Protein-Coupled Receptors The largest class of plasma membrane receptor is the so-called G protein-coupled receptor (GPCR) family, which has more than 1000 members.12 Ligands include agents that stimulate neurotransmission, light and taste perception, and cell division and differentiation as well as the chemokines, which are ligands that are involved in cell attraction.13 All GPCRs share a common architecture, with seven transmembrane α-helical domains, connected by both extracellular and intracellular loops. Because of this architecture, GPCRs are sometimes also referred to as serpentine or heptahelical receptors. The extracellular domain is responsible for ligand binding, which causes a conformational change such that the G protein binding to the intracellular domain becomes activated. G proteins are heterotrimeric proteins that consist of α, β, and γ subunits. Activation causes the dissociation of the α subunit from the βγ complex; both separated components then go on to mediate signaling events that are very similar to those initiated by receptor tyrosine kinases.14

FUNDAMENTAL SCIENCE

Receptor Tyrosine Kinases

General Principles of Intracellular Signaling

The next largest class of receptors is the receptor tyrosine kinases (RTKs).2,15,16 This class consists of approximately 90 members, most of which are involved in the control of cell growth, motility, and differentiation as well as metabolic control. Examples include PDGF receptors, epidermal growth factor (EGF) receptors, the ephrin receptors, hepatocyte growth factor receptors, fibroblast growth factor (FGF) receptors, insulin receptors, and many more. While the ligands for most classes of RTK are now known, some remain “orphans,” with their ligands yet to be discovered. At least one RTK, Her2 (also known as ErbB2), has no known ligand but instead signals by heterodimerization with other EGFr family members.17,18 Also, by sequence analysis of the catalytic domains, several RTKs are predicted to lack catalytic activity. In one of these cases, Her3, the receptor is transphosphorylated when heterodimerized with other EGFr family members (particularly Her2) and acts as an adaptor protein. The majority of the RTKs are single polypeptide chains that contain an extracellular ligand-binding domain, a short hydrophobic transmembrane domain, and a cytoplasmic region containing the kinase domain, as well as other sequences that regulate recycling and/or turnover of the receptor and interaction with signaling molecules. One exception to this rule is the insulin receptor family, which is composed of a disulfide-bonded tetramer, with two identical extracellular ligand binding subunits and two transmembrane subunits with catalytic activity. Another exception is the Met/Hepatocyte growth factor receptor family, in which a single polypeptide chain is cleaved to produce a stable dimer with ligand binding invested in one subunit and catalytic activity in the other. While it is generally thought that the single membrane pass RTKs are monomeric in the absence of ligand,15,19,20 there is some recent evidence that EGF receptors can exist in a dimeric but inactive state in the absence of ligand.15 Some RTK ligands are dimers (e.g., the PDGFs), while others are monomeric (the EGFs). The FGF receptors require both FGF ligands and heparin sulfate proteoglycans for full activation.21,22 Yet other

Intracellular signaling is the mechanism by which cues that are present in the extracellular environment are relayed and interpreted by the cell. These external cues can be growth factors that signal a cell to divide, extracellular matrix proteins that promote survival, hormones that change the metabolism of the cell, cytokines that instruct the cell to differentiate, or other signals that promote motility and invasive ability. Complex multicellular organisms have evolved to have a large array of receptors for these external cues, as well as an even larger number of intracellular signaling molecules. Both embryonic development and adult homeostasis require specificity as well as temporal and spatial control of these signaling pathways. Many disease states, including cancer, diabetes, and immune disorders, can arise if these signaling pathways are inadequately controlled. Intracellular signaling can at first glance seem overwhelmingly complicated. A given receptor can engage a number of different signaling pathways, each eliciting a distinct phenotype. One signaling pathway can affect the output of another, a phenomenon known as cross-talk. The same ligand can have different effects in different cell types. And the activation of a receptor frequently also elicits negative regulatory pathways that are designed to switch the system off after a defined period of signaling.5–7 But this complexity can be reduced somewhat. Experimental observation has shown that the members of any given family of receptors signal in approximately the same way. Furthermore, many receptors use similar components to signal; for example, cytokine, growth factor, and G protein-coupled receptors all activate the MAP kinase pathway and the PI 3-kinase pathway.8,9 Finally, most signaling pathways make use of adaptor or scaffolding proteins. These proteins lack catalytic activity and instead are made up of multiple protein-protein and sometimes also protein-lipid interaction domains.10,11 In this way, adaptor proteins orchestrate the simultaneous activation of a number of pathways following receptor

Intracellular Signaling • CHAPTER 2 Ligand

Extracellular space Receptor

Figure 2-1 • Schematic of intracellular signaling. A prototype receptor is shown. Dimerization of the receptor by ligand elicits multiple signaling pathways, whose outputs result in changes in gene expression, in the cytoskeleton, and in metabolism. These signaling pathways also elicit the production of negative regulators, which ultimately turn the signal off.

Cytoplasm

Negative regulators

Negative regulators Adaptor proteins

Cytoplasmic tyrosine kinases

Ras Cytoskeletal changes Serine/threonine kinases

PI 3-kinases Metabolic changes

Nucleus Modulation of gene expression

RTKs, notably Ret and MuSK, do not bind ligands directly, requiring a coreceptor to present the ligand.23,24 Despite these differences, RTKs generally share a common mechanism of activation on ligand binding, which involves dimerization and often further oligomerization to form higher-order structures. Recent crystallographic studies have revealed the molecular mechanisms behind ligand-induced activation and how monomer ligands can induce dimerization. In the case of the EGFr, the dimerization occurs at an interface between two receptor monomers, the ligand presumably serving to initiate the domain rearrangements that are necessary to make these contacts.19,20 In contrast, FGFs, while monomeric, have two receptor-binding sites. Dimerization is thus achieved by interactions between the receptor and the ligand. The heparin sulfate is required to organize and strengthen the interaction of ligand and receptor as well as to provide specificity to the interactions.21,22 Regardless of how the extracellular domains are brought into close apposition, the result is the juxtapositioning of the two catalytic domains and the subsequent transphosphorylation of tyrosine residues in the activation loop of the kinase domain, as well as elsewhere in the intracellular domain. These phosphorylations serve to initiate further conformational changes that stabilize the active form of the enzyme as well as to provide binding sites for the enzymes and adaptor proteins that are described later.

Serine and Threonine Kinase Receptors The proteins in this family act as receptors for transforming growth factor (TGF)-β, activins and inhibins, and bone morphogenetic protein.25 Members of this family of receptors, which together are often known as the TGF-β superfamily, play key roles during development and morphogenesis, as well as in cell cycle progression, motility and wound healing, and immune surveillance.26 The ligands can be homodimers or heterodimers, and they interact with two single-pass membrane proteins called type I and type II receptors. Both receptor subunits have intrinsic serine/threonine kinase activity in the cytoplasmic domain. Signaling is thought to be initiated by ligand-induced oligomerization of two type I receptors with two type II receptors.25 The type II receptor, which has constitutive kinase

activity, phosphorylates and activates the type I receptor, which then goes on to phosphorylate substrate proteins.

Integrin Receptors Integrins are heterodimers of an α and a β subunit, each of which is membrane-spanning.27 Mammals have 18 α subunits and 8 β subunits that can combine to form 24 distinct heterodimers. Integrins derive their name from their ability to bind to ligands outside the cell and cytoskeletal components inside the cell and so integrate the two environments.28 Their extracellular ligands can be either extracellular matrix proteins or cell surface proteins on neighboring cells. In their low-affinity state, the extracellular domains of the β subunits adopt a “bent” conformation. Ligand binding generates a higher-affinity, straightened structure of the β subunit, corresponding allosteric changes in the extracellular domain of the α subunit, followed by conformational changes in the transmembrane and cytoplasmic domains.29 Clustering of integrins also occurs because of the multivalent nature of the ligands. The net result is a separation of the cytoplasmic domains of the α and β subunits, which mutational studies have shown is required for this “outside-in” signaling. Integrins do not contain intrinsic kinase activity but instead signal through associated cytoplasmic kinases such as Src and FAK.30 Unlike any other class of membrane receptor, integrins can also signal from the cytoplasm to the extracellular space, so-called insideout signaling.31 In this form of signaling, cytoplasmic signal transduction pathways, for example, caused by activation of the EGFr or Frizzled receptors, generate a conformational change in the integrin extracellular domain, resulting in increased adhesion to extracellular ligands and subsequent outside-in signaling. Although the exact mechanism by which this conformational change is transmitted was unclear until very recently, it was known that the cytoskeletal protein talin was required. Talin binds to the cytoplasmic tails of most β integrins through its PTB-like domain. Reducing talin expression or preventing its association with the β subunit is sufficient to inhibit inside-out signaling. Talin adopts an autoinhibited structure in

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quiescent cells. Intracellular signaling pathways release this inhibition, allowing it to bind to and change the conformation of the integrin, thus promoting high-affinity interactions of the integrins with their ligands.32

Cytokine Receptors The cytokine receptor superfamily consists of the receptors for growth hormone, prolactin, erythropoietin, thrombopoetin, G-CSF, and the interleukins as well as the interferon receptors.33–35 They are grouped together because of their use of functionally related receptors. Most receptors use a common γ chain, together with variant β chains and sometimes α chains. Structural homologies among these chains include four conserved cysteine residues in the extracellular domain and a WSXWS motif located near the transmembrane domain. The cytoplasmic domains of these subunits lack any catalytic activity but instead have two regions of low homology, called “box 1” and “box 2.” Although the exact functions of these boxes are not known, it is clear from mutagenesis studies that they are necessary for receptor function. Activation of cytokine receptors occurs when ligand binding causes dimerization, perhaps by promoting a rotational switch near the WSXWS motif.36 Kinases of the JAK family, which are constitutively associated with the cytoplasmic domains, then transphosphorylate the receptors to initiate signaling. The most important substrates of the JAKs are the Stat proteins, which have an SH2 domain and a transcriptional activation domain. Signaling by this class of receptors thus involves tyrosine phosphorylation-induced dimerization of the Stats, which renders them transcriptionally competent.37 Another class of cytokine receptors is represented by the tumor necrosis factor (TNF) receptor superfamily, also known as death receptors, which control apoptosis in response to exogenous signals.38 Members of this family include the TNF receptors 1 and 2, Fas, CD40, and TRAIL and are simple single-pass transmembrane proteins, with cysteine-rich ligand-binding domains on the outside and either a death domain (R1 receptor) or a TRAF-binding domain (R2 domain) on the inside.38 They have no intrinsic catalytic activity. It was originally thought that TNF activated its receptor by inducing trimerization, but it is now known that this class of receptors breaks the ligand-induced oligomerization rule. Instead, the TNF receptors are found as preformed trimers on the cell surface.39 This is thought to be required for their function. For example, TNF-α signals through TNF-R1 to initiate cell death and through TNF-R2 to activate NFkB. If the ligand were to initiate the formation of trimers, mixed oligomers would form in any cell that expresses both receptors. Since only a trimer of death domain-containing receptors is functional, this would result in the suppression of death signals. Thus, preassembly of receptor complexes might promote the formation of homotypic receptors and confer more specificity in the response. As with other, more conventional receptors, ligand binding is thought to elicit conformational changes that render the cytoplasmic domains competent for signal transduction.

Frizzled Receptors Frizzled receptors are the binding partners for the Wnt family of ligands.40 This is a large class of peptides that control a wide array of developmental processes and have also been implicated in cancer. Of particular current interest is the role of Wnts in the control of stem cell fate.41,42 Frizzled receptors have a cysteine-rich extracellular Wnt-binding domain, followed by seven transmembrane domains and a short cytoplasmic tail. Frizzled alone is unable to transduce Wnt signals. Rather, members of the LRP family, which are singlepass transmembrane proteins, are required as coreceptors. The current model for the way in which Wnts activate their receptors is an interesting and novel one in which it is postulated that Wnts bring LRP and Frizzled into close proximity.40 In support of this model, coexpression of chimeric fusion proteins that cause the close apposition of Frizzled and LRP is sufficient to induce Wnt-independent signal transduction.

Notch Receptors There are four mammalian Notch receptors, which play important roles in development and tissue homeostasis, by specifying cell fates and creating boundaries between different cells.43–47 Each receptor is a single-pass transmembrane protein, with an EGF repeat domain that binds ligand in the extracellular domain, and a cytoplasmic domain that lacks catalytic activity but instead contains several ankyrin repeats and a C-terminal PEST domain. During transport through the Golgi, the receptor is cleaved into ligand-binding and transmembrane domains that remain associated with each other through noncovalent interactions. The ligands for Notch are called DSLs (for delta-serrate-lag2 family), and they are also single-pass transmembrane proteins with EGF-like repeats in their extracellular domains. Signaling between Notch and DSLs therefore takes place when one cell carrying the ligand and one cell expressing the receptor are in close apposition. Unlike the other receptors discussed here, in which ligand binding usually controls oligomerization of the receptor and phosphorylation of the cytoplasmic domains, activation of the Notch receptors is accomplished by regulated and specific proteolysis.48 The binding of ligand to Notch makes the receptor susceptible to cleavage first by an ADAMs family metalloprotease in the extracellular domain and then by a γ-secretase in the transmembrane domain. This frees the intracellular domain of Notch, which translocates to the nucleus, where, together with a DNA-binding protein called CSL and coactivators, it activates transcription. Uniquely among the plasma membrane receptors, then, the Notch receptor family does not use a series of signal transduction pathways to exert their effects but rather affects gene expression directly.47

Nuclear Hormone Receptors Some receptors, particularly those for estradiol, progesterone, androgen, glucocorticoids, polyunsaturated fatty acids, and the retinoids, predominantly exert their effects by acting as transcriptional activators and/or repressors.49 These nuclear hormone receptors are regulators of many aspects of homeostasis, as well as sexual development. Many have been implicated in disease, including cancer, lipid disorders, and diabetes.49,50 In addition, several are the targets of important drugs; for example, PPARγ is the target of the antidiabetes drug rosiglitazone. Most of the nuclear hormone receptors consist of a single polypeptide chain with a DNA-binding domain, a transactivation domain, a ligand-binding domain, and sequences that mediate interaction with coregulators. While some receptors can bind DNA with high affinity as monomers, most require dimerization.51 This requirement is particularly true of the retinoid receptor family, in which the exact nature of the heterodimers that are formed dictates the transcriptional output. The ligands for the nuclear hormone receptors either diffuse passively into the cell or are produced within the cell during normal metabolism. In some cases, association between receptor and ligand takes place in the nucleus. In other cases, particularly the sex hormone receptors, the receptor is cytoplasmic and bound to chaperone proteins in the absence of ligand. Ligand binding dissociates the chaperones and allows translocation to the nucleus. Once they are nuclear, ligand-dimer complexes interact with DNA and recruit transcriptional regulators to effect their responses. Thus, in this canonical mechanism of action, nuclear hormone receptors as a class do not require intracellular signal transduction pathways. However, there are a growing number of cases, particularly well studied in the case of the estrogen receptor, in which hormone can also bind a plasma membrane receptor.52,53 Although the identity of the membrane receptor is currently not established (some investigators think that it is the same as the nuclear form, whereas others have suggested it is a GPCR54), it is clear that this form of the receptor can elicit canonical signaling involving Src, MAP kinases, and lipid kinases in a similar way to RTKs.52 The roles of these two forms of receptor signaling, which are often called genomic and nongenomic, particularly in cancer causation, awaits further clarification.

Intracellular Signaling • CHAPTER 2

important for signal transduction being the Src family, the Abl family, the Tec family, the FAK family, and the JAK family. These kinases become activated by association with the receptor, usually through dimerization and transphosphorylation. In these cases, the CTKs can be considered to be noncovalently associated receptor subunits that provide the required catalytic activity. But it is interesting that even the RTKs recruit CTKs, which is required for their full function.69,70 An example of this, the recruitment of Src family kinases by the PDGFr, is given later. While these observations led some researchers to speculate that RTKs were able to phosphorylate only associated proteins and therefore required CTKs to phosphorylate all downstream signaling components, it now seems clear that different tyrosine kinase families have broadly different substrate specificities and that some signal transduction pathways are dependent on the substrates phosphorylated by the RTK and some on the CTK substrates. In keeping with this, cytokine receptors and GPCRs also recruit and/or activate more than one class of CTK.

Components of Intracellular Signaling Pathways With the exception of the nuclear hormone receptors and the Notch receptors, ligand-activated receptors do not have direct mechanisms of action. Rather, their activation results in the initiation of one or more signal transduction cascades that ultimately change the phenotype of the cell. Many general principles and some individual elements of these signal transduction cascades are shared by different classes of receptor. Some of the more commonly used strategies are described in overview here.

Adaptor Proteins Perhaps one of most common features shared by most receptor signal transduction cascades is the use of adaptor proteins,10 which are defined as proteins with protein-protein interaction and sometimes also protein-lipid interaction domains. Common protein interaction motifs include SH2 and some PTB domains, which bind to phosphotyrosine-containing peptides55; SH3 domains, which bind to proline-rich ligands56; WW and WD40 domains, which bind phosphorylated serine and threonine residues, respectively57,58; and PDZ domains, which bind carboxy-terminal sequences of proteins.59,60 In each case, neighboring amino acids provide specificity to the interaction. For example, the SH2 domain of Grb2 preferentially binds a phosphotyrosine followed by the sequence XN, where X is any amino acids and N is asparagine. The SH2 domain of Src, in contrast, prefers the sequence Y(p)EEI. PH, PX, and FYVE domains bind phosphoinositides, particularly those phosphorylated in the 3, 4, and/or 5 positions by phosphatidylinositol (PI) kinases.61–63 Since most adaptor proteins have multiple interaction domains, they act as scaffolds that cluster together distinct signaling molecules. Adaptor proteins can serve to give specificity to receptor signaling by clustering distinct sets of signaling proteins in a particular subcellular location. In some instances, the recruited proteins might modify each other’s activity, for example, when a kinase phosphorylates a coassociated protein. Some adaptor proteins are selective for certain receptor-signaling systems. For example, death domain-containing adaptors are used exclusively by the superfamily of TNF receptors,64,65 the insulin receptor substrate family of proteins is dedicated to the insulin receptor and cytokine receptor families,66,67 and FRS2 is involved in FGF and nerve growth factor signaling.68 Others, such as Grb2 and Shc and the Gab, Dok, and Vav families, are used by multiple receptor types.

Cytoplasmic Serine and Threonine Kinases There are 518 protein kinases in the human genome. Of these, 90 are tyrosine kinases, either RTKs or cytoplasmic kinases as described previously. All other members of the kinome phosphorylate serine and threonine residues.2 While many of these enzymes have housekeeping functions in the control of metabolism, DNA replication, and so on, many more are obligate proximal members of signal transduction cascades from receptors. The best studied of these kinases are the ones that make up what is called the MAP kinase (MAPK) pathway.8 MAPKs are small single-subunit serine/threonine kinases. They have a number of substrates, but chief among them are transcription factors, whose subcellular localization and activity are regulated by MAPK phosphorylation. MAPKs fall into three classes: the ERKs, the JNKs, and the p38 family. All of these enzymes are normally inactive in quiescent cells, but become activated upon growth and/or stress stimulation of the cells. This activation is accomplished by enzymes that are generally known as MAPK kinases (MKKs). These are dual-specificity enzymes; they phosphorylate MAPKs on both a tyrosine and a threonine in the activation loop phosphorylation, causing a large increase in catalytic activity of the MAPKs. While there is some cross-talk, different MKKs appear to be somewhat selective in activating the different classes of MAPKs (Fig. 2-2). The MKKs in turn need to be phosphorylated to be activated, which is accomplished by a broad range of serine/threonine kinases, generally called MKK kinases (MKKKs), from a number of different families. While the combinatorial complexity of these cascades of kinases could be staggering, recent evidence suggests that scaffold proteins exist that provide specificity and regulation to each cascade.71 The best studied of all the MAPK pathways, and one that is central to all mitogenic signaling from receptors, is the one known

Cytoplasmic Tyrosine Kinases Some receptors have intrinsic tyrosine kinase activity, while others, such as GPCRs and cytokine receptors, do not. Yet each of these receptor classes uses tyrosine phosphorylation as a signal. They do this by recruiting, or being stably associated with, cytoplasmic tyrosine kinases (CTKs). There are several subfamilies of CTKs,2 the most

Figure 2-2 • Mitogen-activated protein kinase (MAPK) cascades. Intracellular signaling from various receptors involves activation of members of the mitogen-activated kinases. The MAPKs consist of three families of related kinases (Erks, p38s, and Jnks). Activation of MAPKs is mediated by phosphorylation by a series of related kinases, MKKs (MAPK kinases), which are activated by a broad spectrum of kinases through phosphorylation. Activated MAPKs translocate to the nucleus and phosphorylate a variety of structurally unrelated transcription factors. The transcription factors and gene expression are thus activated.

MKKs

MKKs

MAP kinases

MEKK(1–4), PAK(1–3), ASK, Hpk1, GCK, MLK, Raf, etc.

MKK1, MKK2

MKK3, MKK6

MKK4, MKK7

Erk1, Erk2

p38α, p38β, p38γ, p38δ

Jnk1, Jnk2, Jnk3

Phosphorylation of a variety of transcription factors: Elk-1, Atf-2, c-Jun, etc.

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as the Ras-Raf-MAPK pathway.72 The Ras family consists of three small GTPases: H-, N-, and K-Ras. Of these, K-Ras has been implicated in many human cancers. For example, more that 90% of pancreatic cancers and approximately 50% of colon cancers have an activating mutation in K-Ras.73–77 In unstimulated cells, Ras is found in an inactive, GDP-bound form. Many ligands activate Ras, by stimulating the exchange of GDP for GTP, which is accomplished by activating proteins called guanine nucleotide exchange factors and inhibiting proteins called GTPase-activating proteins. In the case of growth factor signaling, the guanine nucleotide exchange factor that is involved is called Sos, which is usually associated in the cytoplasm with an adaptor protein called Grb2. The GTPase-activating protein that is involved is p120Gap. On receptor activation, two different types of adaptor protein, called Shc and Gab, become recruited to receptors in the membrane and phosphorylated on several tyrosine residues. Several of these sites are in YxN motifs, which are canonical binding sites for the Grb2 SH2 domain. Thus, receptor activation results in the following cascade of signaling: Shc (or Gab) > Grb2: Sos > Ras. Once Ras is in the GTP-bound state, it initiates a number of different signaling pathways, including PI 3-kinase activation, and cytoskeletal changes.78–81 But of particular importance, it recruits the serine/threonine kinase Raf to the plasma membrane, where other signals, including tyrosine phosphorylation, activate its intrinsic kinase activity.82 Raf then goes on to phosphorylate and activate MEK 1 and 2, which in turn activate ERK1 and 2. Space does not allow a full description of all serine/threonine kinases that are involved in signal transduction here. But there is one enzyme complex that has attracted much recent attention: mTor (mammalian target of rapamycin).83–88 mTor is a signal integrator, linking information about nutrients, energy status, and growth factor stimulation to outputs such as protein synthesis, ribosome biogenesis, metabolism, and cell survival and proliferation (Fig. 2-3). There are two structurally distinct mTor complexes, with proteins known as Raptor and Rictor. The mTor/Raptor complex is regulated by a small GTPase known as Rheb, which is normally kept in an inactive state by a GTPase-activating protein consisting of a complex of two tumor suppressors TSC1 and TSC2. A variety of signals, including signals from Akt, ERKs, and other serine/threonine kinases, inactivate the TSC1/2 complex, thus activating Rheb and in turn mTor/Raptor. The signaling outputs from this complex include mRNA translation, ribosome biogenesis, and autophagy through phosphorylation of substrates such as 4E-BP1 and ribosomal S6K. The mTor/Rictor complex is less well understood, but it is activated by RTKs and plays an important role in activating Akt.

Rac

Lipid Signaling As we saw earlier, many adaptor proteins have domains that interact with phosphorylated phosphoinositides, which frequently serves to recruit them to the plasma membrane or subcellular organelles such as endosomes. In other cases, phosphoinositide binding serves to activate the catalytic activity of the enzyme to which it is bound. The most important lipid modifiers in signal transduction pathways are a family of enzymes known as phosphatidylinositol 3-kinases (PI 3-Ks).9,89 These come in three classes, based on sequence alignment and substrate preference. Class I enzymes have two subunits, one with catalytic activity and one that specifies the association of the enzyme with other signaling molecules. They generally generate PI 3,4,5-P3 (PIP3) from PI 4,5-P2. Class II enzymes have a single subunit, which contains both catalytic activity and regulatory sequences. They predominantly generate PI 3-P and PI 3,4-P2. Class III has just one member, the Vps34 protein, which was originally identified as a gene required for vacuolar sorting in budding yeast and generates PI 3-P. Relatively little is known about the functions of the class II and class III enzymes in mammals, although Vps34 has recently been implicated in mTor regulation and autophagy, suggesting a role in the response of cells to nutrients. Class II enzymes bind clathrin, suggesting a possible role in receptor trafficking and/or endocytosis. But it is the class I family members that have been most intensively studied for their roles in intracellular signaling pathways and cancer.86,90–93 Class I enzymes can be further subdivided into Class IA and Class IB. All members have structurally related catalytic subunits. But the regulatory subunits of class IA members all contain 2 SH2 domains, and some also have an SH3 domain, whereas in class IB, the regulatory subunits are structurally distinct. Class IA members are activated by RTKs, whereas class IB members are activated by GPCRs. In all cases, activation occurs when the PI 3-K is recruited to the membrane by regulatory domain sequences, for example, the SH2 domains in the case of class IA. This brings the enzyme into close proximity with its substrate and allows catalysis to take place. PIP3 then acts as a lipid second messenger by binding to PH domains in a variety of proteins, including the serine/threonine kinases PDK1 and Akt (see Fig. 2-3). One important target of Akt is the FOXO family of transcription factors, which are sequestered in the cytoplasm and inactivated by phosphorylation, thus inhibiting gluconeogenesis.94 Other metabolic targets include the glucose transporter Glut4, glycogen synthase kinase 3, and ATP citrate lyase. Akt also has effects on the cell cycle via its inhibitory effects on FOXO and GSK3 as well as by directly phosphorylating proapoptotic proteins such as BAD. In

Class I PI 3-K

Class II PI 3-K

Class III PI 3-K

PI 3,4-P2 > PI 3,4,5P3

PI 4-P > PI 3,4P2

PI > PI 3P

Cdc42

PDK

Vesicle sorting

Actin rearrangement Protein synthesis

Rictor

Akt

mTor

Raptor

Cell survival Cell cycle

Metabolism Proliferation

Autophagy Ribosome biogenesis

mRNA translation

Figure 2-3 • mTor and PI 3-K signaling. The figure illustrates some of the key components of the mTor and PI 3-K signaling pathways and highlights the central role that the serine/threonine kinases Akt and mTor play in the control of several intracellular signaling events. The three classes of PI 3-K are activated by a variety of cell surface receptors and make different lipid products, which bind to lipid-binding domains in a variety of signaling molecules. Lipid binding activates signaling by recruiting the signaling molecules to intracellular or plasma membranes or by causing a conformational change. Once Akt is activated by PDK1 and other serine/threonine kinases, it phosphorylates a number of substrates to cause profound changes in cell homeostasis. mTor responds to changes in nutrients and growth factors and exerts its effects via forming stable complexes with either Rictor or Raptor.

Intracellular Signaling • CHAPTER 2

concert with the small GTPases Rac and Cdc42, class I enzymes also control actin dynamics and thus regulate cell polarity and motility.

PDGF

Negative Regulators of Signaling Given the power of signal transduction pathways to regulate all aspects of a cell’s phenotype, it is vital that these pathways be tightly controlled. Indeed, in the absence of such control, cancer often arises. We have seen that some control is provided in the recruitment and activation of defined signaling modulators. But there are also other layers of control that ensure that the signal is switched off in a timely manner. A number of different mechanisms are used. The first is at the level of the receptor itself. Following ligand binding, most receptors are internalized via mechanisms using clathrin-coated pits and endocytosis.95–98 In some cases, this results in degradation of both receptor and ligand in lysosomes; in other cases, the unoccupied receptor is recycled back to the plasma membrane. Both RTKs and some cytoplasmic tyrosine kinases are also regulated by ubiquitinmediated degradation at the proteasome.99,100 In the case of the EGFr and Src, it is the activation of these proteins that initiates the degradation by recruiting an E3 ubiquitin ligase called Cbl via SH2 domain interactions.101–104 This ensures that the proteasome selectivity degrades the active forms of the enzymes. Two other inhibitory mechanisms are also elicited by the very signaling pathways that they inactivate. For example, the activation of MAPK-signaling pathways results in the transcriptional activation of a class of enzymes called MAPK phosphatases (MPKs), which, as the name suggests, dephosphorylate and inactivate MAPKs.5,105 Also, the JAK-Stat signaling pathway results in the production of the SOCS (suppressor of cytokine signaling) proteins, which have an SH2 domain and a region known as a SOCS box. The SOCS proteins are thought to inhibit signaling by binding to specific phosphotyrosine-containing motifs on receptors and the JAK kinases and perhaps initiating ubiquitinmediated degradation.6,106 Finally, the action of both phosphoprotein and phospholipid phosphatases can also serve to regulate signaling. Of particular importance is the phosphoinositide phosphatase and tumor suppressor known as PTEN, whose loss has been associated with malignancy in many cancers, particularly glioblastomas and prostate cancer.93,107

Selected Examples of Signal Transduction Pathways Throughout this chapter, we have touched on the concept that many extracellular ligands engage multiple signaling pathways and that there are some common themes to the signaling outputs that emerge. For example, stimulation with most ligands results in profound changes in gene transcription by activating transcriptional regulators such as Fos, Myc, NFkB, Stats, Smads, hormone receptors, and so on. Many factors elicit changes in the cytoskeleton, polarity, and movement of cells. Alterations in metabolism, hormone responses, differentiation, and survival are other common outputs. In this section, a few examples of receptor-initiated signal transduction pathways will be given, illustrating the concepts that have been discussed throughout.

Receptor Tyrosine Kinase PDGFr There are five PDGF ligands encoded by four different genes (A, B, C and D), and two different receptors encoded by unique genes (α and β).108 Each ligand is a dimer, with known isoforms including AA, AB, BB, CC, and DD. PDGF AA can bind only to a homodimeric αα receptor, whereas AB, BB, and CC can bind both αα and αβ receptors. PDGF DD binds predominantly to ββ receptors. PDGF acts predominantly on cells on mesenchymal origin and promotes cell growth, migration, and survival. Alterations in PDGFr signaling play a role in sarcomas and gliomas.109,110 Furthermore, chromosomal translocations that cause the constitutive activation of the kinase domain of PDGFr are found in some leukemias and lymphomas.

Src

Y Y

Nck Y Y Grb7 Y Shc Y

Y PLC␥ Y

Y Y Stat

GAP Y Y Y

PI 3-K Grb2 Y

Y Y Shp-2

Figure 2-4 • Recruitment of SH2 domain-containing proteins by activated PDGF receptors. The figure illustrates how the ligand PDGF, which is a dimer, causes the dimerization and transphosphorylation of its receptor on a number of tyrosine residues, in the juxtamembrane region, in the kinase insert region, on the activation loop, and on the carboxy-terminal tail sequences. Each one of these phosphorylated tyrosines then binds selectively to SH2 domain-containing proteins, which then initiate a number of different intracellular signaling pathways. The selectivity of SH2 domain binding to each tyrosine is determined by the amino acid sequences immediately proximal to each tyrosine.

Once activated by dimerization and transphosphorylated on several tyrosine residues, the PDGFr recruits at least 10 different SH2 domain-containing signaling molecules to these sites (Fig. 2-4).69,70,111 One class of binding proteins are members of the Src family of tyrosine kinases (SFKs). These enzymes become activated by this association and then phosphorylate a number of other signaling molecules. While the mechanistic details have yet to be worked out, one important effect of SFK activation is the stabilization of myc mRNA and subsequent transcription.69 Another important signaling cassette to be recruited is the class IA PI 3-K, which associates directly with the receptor via the SH2 domains on its regulator subunit; the signaling cascades that were discussed earlier are then initiated. Recruitment and phosphorylation of the adaptor protein Shc, together with the subsequent binding of Grb2/Sos, allow the Ras/MAPK pathway to become activated. Other signaling effectors that are more selective for RTKs such as the PDGFr include phospholipase Cγ, the adaptor protein Nck, and the tyrosine phosphatase Shp2, as well as the negative regulators Ras-GAP and Cbl. It is important to note that the example of signaling that is illustrated here—the recruitment of multiple signaling cascades both to the receptor itself and to adaptor proteins—is a general theme that is seen throughout RTK signaling. Specificity is provided by the nature of the phosphorylation sites on the receptor and the domain makeup of the adaptor proteins that are recruited, as well as the

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complement of signaling proteins that are expressed in any given cell type. Furthermore, other receptors, such as integrins and GPCRs, use very similar strategies. In these cases, cytoplasmic tyrosine kinases (FAK and Src in the case of integrins and Src and Tec family members in the case of GPCRs) are responsible for generating the SH2 domain recruitment sites.

TGFb Receptor As we saw earlier, the receptors in this family are transmembrane serine/threonine kinases, with ligand stimulating the phosphorylation of the type I receptor by the type II subunit, which activates the intrinsic kinase activity of the type I subunit. The best-characterized intracellular effectors of TGFβ signaling are the Smad proteins.112–116 There are three types of Smads: R-Smads, a common Smad (in vertebrates this is known as Smad4), and inhibitory Smads. The RSmads and Smad4 have two conserved domains called MH1 and MH2, joined by linker sequences. In addition, the R-Smads have carboxy-terminal serine phosphorylation sites. On ligand binding, the receptors recruit and phosphorylate one or more R-Smads. The conformational changes that are induced by this phosphorylation release the R-Smads from the receptor and allow the formation of a trimeric complex of two R-Smads with Smad4. This complex translocates to the nucleus, where, along with coactivators such as CBP/ p300 and factors such as FOXO and Forkhead, transcription is activated. The specificity of the transcriptional response is provided by the R-Smads that are present in the complex and thus the coactivators and transcription factors that are recruited. In turn, recruitment of individual R-Smads is determined by the exact nature of the heterotetramer receptors that are formed in response to the diverse TGFβ family ligands.112 In the cases in which the ligand is TGFβ itself or activin, efficient recruitment of R-Smads to the receptor also requires an adaptor protein called SARA, which has a FYVE domain that interacts with PI 3-P, and a Smad binding site.116,117 The inhibitory Smads interfere with signaling by binding to type I receptors and interfering with R-Smad recruitment. Although the Smads are the only known mechanism by which TGFβ receptors modulate transcription, other signal transduction pathways also control Smad activation. For example, R-Smads are phosphorylated by MAPKs, which may prevent nuclear localization of the Smads and transcriptional responses. In contrast, phosphorylation by JNK enhances nuclear translocation and activity of Smad3. Other kinases that have been reported to phosphorylate Smads include cell cycle kinases such as Cdk2 and Cdk4, PKC, and CaMKII. In this way, signaling from other receptors can greatly influence the transcriptional responses to TGFβ receptor stimulation.112

TNF Receptor The death receptors are the means by which extracellular signals are linked to the apoptotic machinery of the cell, a process known as instructional apoptosis. One of the best characterized of these receptors is called Fas (also known as CD95 or Apo1); it will be used as illustration.65,118–120 Trimerization of Fas by its ligand clusters the receptor’s death domains, leading to the recruitment of the protein FADD by death domain interaction. The death effector domain in FADD then binds to the inactive, zymogen form of caspase-8. The oligomerization of caspase-8 that ensues causes the self-activation of caspase-8 by proteolysis, resulting in the subsequent cleavage and activation of effector caspases such as caspase-9 and commitment to apoptosis.

Wnt Signaling The last example to be considered is that of Wnt signaling through Frizzled and its coreceptors, the LRPs.40 One of the most important signaling components in Wnt signaling is the β-catenin protein, which accumulates in the cells in response to the ligand and activates transcription by associating with the DNA-binding protein TCF.121–123 In unstimulated cells, TCF is in a complex with the

negative regulator Groucho and is transcriptionally inactive. In these same cells, β-catenin turns over rapidly, a consequence of its recruitment by a so-called destruction complex. This complex contains Axin, APC, and GSK3, a serine/threonine kinase. Phosphorylation of β-catenin by GSK3 causes β-catenin to become ubiquitinated and targeted for destruction by the proteasome. The binding of Wnt to its ligand causes the phosphorylation of LRP by GSK3 and CK1 and the recruitment of the Axin/APC/GSK3 complex to the receptor (Fig. 2-5). This is presumably sufficient to prevent the phosphorylation of β-catenin, thus allowing its concentration to rise, enter the nucleus to associate with TCF, and activate transcription. Another key intermediate in Wnt signaling is Dsh, which is required upstream of Axin/APC/GSK3. Dsh is also a cytoplasmic protein that becomes recruited to the Frizzle/LRP complex in response to Wnts, but the exact mechanism by which it participates in signaling is unknown at this time. In an intriguing new twist to the story of Wnt signaling, it was recently found that Wnts can act as ligands for the atypical RTKs, Ryk, Ror1, and Ror2.124–126 Little is yet known about the signal transduction pathways that are elicited by Wnt2 binding to these RTKs or about the cellular outputs they specify.

CLINICAL RELEVANCE AND APPLICATIONS From research conducted over the last two decades, it is clear that many human cancers have their origins in dysregulated signaling pathways.1,127,128 Some of the earliest oncogenic events to be discovered in humans were mutated and activated K-Ras, chromosomal translocations that result in the overexpression of Myc and the production of the Bcr-Abl tyrosine kinase, and chromosomal amplification of the RTK Her2. Since then, many other activating mutations in signaling pathway components have been discovered in cancer cells. Examples include activating mutations in the EGFr in some non-small-cell lung cancers,129 in B-Raf in most melanomas as well as other tumor types,82 in Jak2 in myelodysplastic syndromes,130,131 and in PI 3-K, particularly in breast cancers.90,91 Furthermore, chromosomal translocations involving the RTKs Kit, Flt3, and PDGFr are detected in gastrointestinal stromal tumors and some lymphomas. Other mechanisms that cancer cells use to promote their growth and survival include the overexpression of the antiapoptotic protein Bcl2 in lymphoma,132,133 activating mutations in G proteins in pituitary tumors,13,134 the acquisition of insensitivity to the inhibitory growth effects of TGFβ while maintaining the positive signals in many carcinomas,135–137 and the overexpression of estrogen or androgen receptors in breast and prostate cancers, respectively.52,138–142 The discovery of each of these activated signaling pathways has been rapidly followed by attempts by both the academic community and the pharmaceutical industry to develop new therapeutics targeting these events.4,143–145 Some of these attempts have yet to be successful despite enormous effort; for example, the Ras GTPase has so far proved intractable to small molecule inhibition.72,146 But for other targets, there have been successes, the most notable being the development of imatinib (target: Abl, PDGFr) and later dasatinib (target: Abl, SFKs) for the treatment of chronic myelogenous leukemia147 (see Chapter 108). Other therapeutics that target signal transduction pathways include antiestrogens and antiandrogens for breast and prostate cancer, respectively148,149; trastuzumab for Her2-positive breast cancer150; gefitinib and erlotinib (target: EGFr) for lung cancer151; sorafenib (targets: VEGFr and Raf) for renal cell carcinoma152; and sunitinib (targets: VEGFr, PDGFr, Kit) for renal cell carcinoma and gastrointestinal stromal tumor.153,154 It can be anticipated that in the future, most cancers will be classified not just according to anatomic site of origin and histopathology, but also on the basis of the genetic alterations that are present in the tumor, including those in signaling pathways. New therapeutic modalities are likely to include strategies (small molecules, antibodies, microRNA, etc.) to target these activated signaling pathways.

Intracellular Signaling • CHAPTER 2

Wnt LRP5/6

Frizzled

LRP5/6

Frizzled

Dsh

Axin

Dsh APC Axin

GSK3

APC GSK3 ␤-catenin

P ␤-catenin

degradation

␤-catenin

␤-catenin

␤-catenin

␤-catenin

Groucho TCF

X

TCF

Figure 2-5 • Canonical intracellular signaling from Wnt receptors. In unstimulated cells, a complex of Axin, APC, and GSK (glycogen synthase kinase) phosphorylates β-catenin, which causes its degradation via the proteasome pathway. When Wnt ligand is present, Frizzled and Lrp associate and create binding site for axin, APC, and GSK3 in cooperation with disheveled (Dsh). Phosphorylation of β-catenin is thus prevented, which allows its accumulation and transport into the nucleus. Once in the nucleus, β-catenin displaces the association of Groucho with the transcriptional activator TCF, allowing transcription of several target genes. In some cancer cells, particularly from the colon, mutations in APC prevent the downregulation of β-catenin and thus allow constitutive TCF signaling.

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54. Wehling M, Losel R: Non-genomic steroid hormone effects: membrane or intracellular receptors? J Steroid Biochem Mol Biol 2006;102: 180–183. 55. Schlessinger J, Lemmon MA: SH2 and PTB domains in tyrosine kinase signaling. Sci STKE 2003;(191):RE12. 56. Mayer BJ: SH3 domains: Complexity in moderation. J Cell Sci 2001;114:1253–1263. 57. Ilsley JL, Sudol M, Winder SJ: The WW domain: linking cell signalling to the membrane cytoskeleton. Cell Signal 2002;14:183–189. 58. Chen S, Spiegelberg BD, Lin F, et al: Interaction of Gbetagamma with RACK1 and other WD40 repeat proteins. J Mol Cell Cardiol 2004;37:399– 406. 59. Kay BK, Kehoe JW: PDZ domains and their ligands. Chem Biol 2004;11:423–425. 60. Nourry C, Grant SG, Borg JP: PDZ domain proteins: plug and play! Sci STKE 2003;2003:RE7. 61. Ellson CD, Andrews S, Stephens LR, et al: The PX domain: a new phosphoinositide-binding module. J Cell Sci 2002;115:1099–1105. 62. Maffucci T, Falasca M: Specificity in pleckstrin homology (PH) domain membrane targeting: a role for a phosphoinositide-protein co-operative mechanism. FEBS Lett 2001;506:173–179. 63. Hayakawa A, Hayes S, Leonard D, et al: Evolutionarily conserved structural and functional roles of the FYVE domain. Biochem Soc Symp 2007;95–105. 64. Park HH, Lo YC, Lin SC, et al: The death domain superfamily in intracellular signaling of apoptosis and inflammation. Annu Rev Immunol 2007;25:561–586. 65. Tibbetts MD, Zheng L, Lenardo MJ: The death effector domain protein family: regulators of cellular homeostasis. Nat Immunol 2003;4:404– 409. 66. White MF: Regulating insulin signaling and betacell function through IRS proteins. Can J Physiol Pharmacol 2006;84:725–737. 67. Giovannone B, Scaldaferri ML, Federici M, et al: Insulin receptor substrate (IRS) transduction system: distinct and overlapping signaling potential. Diabetes Metab Res Rev 2000;16:434– 441. 68. Gotoh N, Laks S, Nakashima M, et al: FRS2 family docking proteins with overlapping roles in activation of MAP kinase have distinct spatialtemporal patterns of expression of their transcripts. FEBS Lett 2004;564:14–18. 69. Bromann PA, Korkaya H, Courtneidge SA: The interplay between Src family kinases and receptor tyrosine kinases. Oncogene 2004;23:7957–7968. 70. Abram CL, Courtneidge SA: Src family tyrosine kinases and growth factor signaling. Exp Cell Res 2000;254:1–13. 71. Morrison DK, Davis RJ: Regulation of MAP kinase signaling modules by scaffold proteins in mammals. Annu Rev Cell Dev Biol 2003;19:91– 118. 72. Rodriguez-Viciana P, Tetsu O, Oda K, et al: Cancer targets in the Ras pathway. Cold Spring Harb Symp Quant Biol 2005;70:461–467. 73. Aviel-Ronen S, Blackhall FH, Shepherd FA, et al: K-ras mutations in non-small-cell lung carcinoma: a review. Clin Lung Cancer 2006;8:30–38. 74. Friday BB, Adjei AA: K-ras as a target for cancer therapy. Biochim Biophys Acta 2005;1756:127– 144. 75. Smakman N, Borel Rinkes IH, Voest EE, et al: Control of colorectal metastasis formation by KRas. Biochim Biophys Acta 2005;1756:103–114. 76. Nakayama T, Morishita T, Kamiya T: K-ras as a genetic marker in pancreatic cancer. Acta Gastroenterol Latinoam 2003;33:43–46. 77. Ellis CA, Clark G: The importance of being KRas. Cell Signal 2000;12:425–434.

78. Zebisch A, Czernilofsky AP, Keri G, et al: Signaling through RAS-RAF-MEK-ERK: from basics to bedside. Curr Med Chem 2007;14:601– 623. 79. Plowman SJ, Hancock JF: Ras signaling from plasma membrane and endomembrane microdomains. Biochim Biophys Acta 2005;1746:274– 283. 80. Shapiro P: Ras-MAP kinase signaling pathways and control of cell proliferation: relevance to cancer therapy. Crit Rev Clin Lab Sci 2002;39: 285–330. 81. Stacey D, Kazlauskas A: Regulation of Ras signaling by the cell cycle. Curr Opin Genet Dev 2002;12:44–46. 82. Wellbrock C, Karasarides M, Marais R: The RAF proteins take centre stage. Nat Rev Mol Cell Biol 2004;5:875–885. 83. Sabatini DM: mTOR and cancer: insights into a complex relationship. Nat Rev Cancer 2006;6: 729–734. 84. Mamane Y, Petroulakis E, LeBacquer O, et al: mTOR, translation initiation and cancer. Oncogene 2006;25:6416–6422. 85. Petroulakis E, Mamane Y, Le Bacquer O, et al: mTOR signaling: implications for cancer and anticancer therapy. Br J Cancer 2006;94:195–199. 86. Shaw RJ, Cantley LC: Ras, PI3 and mTOR signalling controls tumour cell growth. Nature 2006;441:424–430. 87. Averous J, Proud CG: When translation meets transformation: the mTOR story. Oncogene 2006;25:6423–6435. 88. Sarbassov DD, Ali SM, Sabatini DM: Growing roles for the mTOR pathway. Curr Opin Cell Biol 2005;17:596–603. 89. Foster FM, Traer CJ, Abraham SM, et al: The phosphoinositide (PI) 3-kinase family. J Cell Sci 2003;116:3037–3040. 90. Dillon RL, White DE, Muller WJ: The phosphatidyl inositol 3-kinase signaling network: implications for human breast cancer. Oncogene 2007;26:1338–1345. 91. Liu Z, Roberts TM: Human tumor mutants in the p110alpha subunit of PI3K. Cell Cycle 2006;5: 675–677. 92. Bader AG, Kang S, Zhao L, et al: Oncogenic PI3K deregulates transcription and translation. Nat Rev Cancer 2005;5:921–929. 93. Parsons R: Human cancer, PTEN and the PI-3 kinase pathway. Semin Cell Dev Biol 2004;15: 171–176. 94. Manning BD, Cantley LC: AKT/PKB signaling: navigating downstream. Cell 2007;129:1261– 1274. 95. Le Borgne R, Bardin A, Schweisguth F: The roles of receptor and ligand endocytosis in regulating Notch signaling. Development 2005;132:1751– 1762. 96. Dikic I: Mechanisms controlling EGF receptor endocytosis and degradation. Biochem Soc Trans 2003;31:1178–1181. 97. Seachrist JL, Ferguson SS: Regulation of G protein–coupled receptor endocytosis and trafficking by Rab GTPases. Life Sci 2003;74:225– 235. 98. Waterman H, Yarden Y: Molecular mechanisms underlying endocytosis and sorting of ErbB receptor tyrosine kinases. FEBS Lett 2001;490: 142–152. 99. Marmor MD, Yarden Y: Role of protein ubiquitylation in regulating endocytosis of receptor tyrosine kinases. Oncogene 2004;23:2057–2070. 100. Holler D, Dikic I: Receptor endocytosis via ubiquitin-dependent and -independent pathways. Biochem Pharmacol 2004;67:1013–1017. 101. Swaminathan G, Tsygankov AY: The Cbl family proteins: ring leaders in regulation of cell signaling. J Cell Physiol 2006;209:21–43.

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The Cellular Microenvironment and Metastases Amato J. Giaccia and Janine T. Erler

S U M M ARY • Metastatic disease kills the majority of cancer patients. • Gene mutations, the tumor microenvironment, and host cells drive the metastatic spread of tumor cells. • Metastasis can be subdivided into four steps: invasion, intravasation, survival in circulation, and extravasation. • Colonization of metastatic tumor cells requires the ability to

O F

K EY

P OI NT S

proliferate in a foreign tissue and angiogenesis. • The formation of a premetastatic niche is essential for the growth of extravasating metastatic tumor cells. • Organ specificity of tumor metastases is determined both by blood flow and tissue-specific factors. • Primary tumors possess stem cells that can recapitulate the tumor from a

INTRODUCTION Tumors are described as benign or malignant. Malignant tumors can spread by invasion and metastasis, whereas benign tumors cannot and remain localized. One of the hallmarks of cancer cells is their ability to grow and divide without undergoing senescence, provided they have sufficient oxygen, nutrients, and space. As tumors grow, oxygen and nutrients can quickly become limiting in large part as a result of an inadequate vascular supply. Cancer cells will adapt to these growth-limiting environments and also seek out fresh terrain to take up residence, where neither space nor nutrients are (initially) limiting. The spread of cancer from its primary site to secondary sites in the body is defined as metastasis, which comes from the Greek word meaning “change of state.” These secondary sites may be located in a new organ or in a different region of the same organ. In reductionist terms, cancer cells metastasize by dislodging from the primary tumor, penetrating through lymphatic and blood vessels, and establishing new growth at a new site in normal tissue. Cancer cells must acquire the capability for invasion and metastasis to escape the primary tumor mass and colonize new terrain in the body where nutrients and space are not limiting. Acquisition of this capability for invasion and metastasis is another of the hallmarks of cancer1 and is significantly influenced by changes in gene expression and by microenvironmental factors. It is the ability to spread to other tissues and organs that makes cancer a potentially life-threatening disease, in that metastases are responsible for 90% of cancer patient deaths.2 Very few treatment options exist for patients with metastatic cancer, and furthering our understanding of the process of metastasis will aid in the development of new approaches to treat metastatic disease.3

MULTISTEP PROCESS OF METASTASIS Metastasis is a multistep process (Fig. 3-1) consisting of a series of discrete biological processes. These steps allow primary tumor cells

single cell, and a subset of these cancer stem cells may inherently possess altered gene expression changes with increased metastatic potential. • Antimetastatic therapy will probably require the targeted inhibition of many pathways that control proliferation, invasion, and angiogenesis.

to invade the surrounding tissue, intravasate through blood vessels to enter the circulatory or lymph system, acquire mutations to survive fluctuating environmental changes, extravasate from the circulatory or lymph system into new tissue, proliferate at secondary sites, and develop a vascular system to support growth of the metastases. In some cases, metastases can also give rise to new metastases. There is a propensity for certain tumors to seed in particular organs in part as a result of blood flow. However, blood flow alone cannot explain the different patterns of metastases found for different primary tumors. The most popular theory to explain the patterns of metastases is the “seed and soil” theory put forth by Stephen Paget over a century ago in 1889.4 Paget described tumor cells as “seeds” and the host environment as the “soil,” and proposed that their interaction determines metastatic outcome. Although the Paget theory is appealing, we know that metastasis is a very inefficient process, because very few cells that escape the primary tumor take up residence in new tissues. For this reason, the tumor cells that survive the process have been termed by Fidler as the “decathlon champions,” because they excel at all the steps in the metastatic process.5 Metastasis is strongly influenced by the interactions between tumor and host cells, and by both the immediate and extended tumor microenvironments. There is ever more evidence demonstrating that these interactions between tumor and host cells are key determinants for the success of metastatic growth and spread.

The Tumor Microenvironment Acts as a Selection Pressure for Metastatic Tumor Cells The first step in tumorigenesis is transformation, where cells accumulate mutations in proto-oncogenes that result in dysregulated cell growth and increased life span, and loss of tumor suppressor genes that normally act to limit cell growth and viability. In addition, alterations in DNA damage sensing and repair pathways result in

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Part I: Science of Clinical Oncology Tumor Normal epithelium BM ECM

(1) Primary tumor growth [benign]

(5) Extravasation at distant site

(2) Invasion of neighboring tissue [malignant]

(6) Proliferation at secondary organ

(3) Intravasation

(7) Angiogenesis to sustain growth

(4) Interaction with blood components and travel in bloodstream

(8) Metastasis of metastases

Figure 3-1 • Metastasis is a multistep process consisting of a series of discrete biological events. These allow primary tumor cells to invade the surrounding tissue, intravasate the circulatory system and survive this harsh environment, extravasate into new tissue, proliferate at secondary sites, and develop a vascular system to support growth of the metastases. See text for details. BM, basement membrane; ECM, extracellular matrix. (Adapted from Le QT, Denko NC, Giaccia AJ: Hypoxic gene expression and metastasis. Cancer Metastasis Rev 2004;23:293–310.)

decreased genomic stability and can promote tumor progression. In contrast to cellular transformation, tumor cells must overcome a different set of barriers to metastasize. These are external barriers created by the tumor microenvironment that limit tumor progression (Fig. 3-2). External forces include physical barriers such as extracellular matrix (ECM) components and basement membranes, as well as physiologic barriers such as limited oxygen (hypoxia) and nutrients, changes in pH, and immunologic barriers by the immune system.2 Cells respond to external microenvironmental influences by altering gene expression such that they are able to adapt and survive. The tumor microenvironment thus exerts a selection pressure for cells capable of overcoming these barriers, driving tumor progression. Tumor hypoxia is a potent microenvironmental influence and is associated with metastasis and poor survival in cancer patients.6–9 Hypoxia selects for cells with low apoptotic potential10–12 and increases genomic instability, allowing rapid mutational adaptations.13,14 Hypoxia additionally increases the expression of genes involved in glucose transportation, angiogenesis, anaerobic metabolism, cell survival, invasion, and metastasis (a list is given in Table 3-1).15,16 All of these changes allow cells to adapt to oxygen-deprived conditions and permit cells to escape these conditions by establishing new blood supplies or by physically moving from an oxygen-poor environment

Transformation

• Tumor suppressor function • DNA repair • Limited lifespan

Internal

to an oxygen-rich environment. A large number of gene expression changes are mediated by hypoxia-inducible factor (HIF)-1, a helixloop-helix transcription factor that is activated by oxygen-deprived conditions. HIF-1 is often found overexpressed in cancer cells as a result of the hypoxia microenvironment of solid tumors as well as oncogene and tumor suppressor gene mutations, and is associated with metastasis and poor survival.17 Several HIF-1 targets have been shown to be mediators of metastasis, such as CXCR4, which promotes organ-specific metastasis in renal cancer,18 and c-met, which increases tumor cell invasion (see section on Invasion).19,20 Hypoxia-regulated genes represent potentially specific therapeutic targets that should be highly tumor or metastases specific.9,21 Recent studies have suggested that lysyl oxidase (LOX ) is a hypoxia-induced gene that is a very promising target for metastatic disease. Research has demonstrated that inhibition of the secreted protein can prevent both invasion and metastatic growth.22 In addition, LOX and other HIF-1α targets such as CA-IX have been shown to be independent markers of prognosis.22–25 Animal imaging studies have revealed that tumor cells can move rapidly along collagen fibers in the ECM that act as “highways” for metastasis.26 This process is facilitated by host macrophage cells.27 Furthermore, increased fiber deposition enhances ECM stiffness,

Metastasis

• pH and hypoxia • Immune response • ECM components • Basement membrane External

Figure 3-2 • Barriers for tumor progression. The first step in tumorigenesis is cellular transformation where cells must overcome several internal barriers. To metastasize, cells must overcome external barriers put in place by the tumor microenvironment. (Adapted from Gupta GP, Massague J: Cancer metastasis: building a framework. Cell 2006;127:679–695.)

The Cellular Microenvironment and Metastases • CHAPTER 3

Table 3-1 Hypoxia-Regulated Genes METABOLISM

Lipocortin

Matrix metalloproteinase-7, 13

Aldolase A, C

Nuclear factor κB (NF-κB)

Vimentin

Enolase-1

NIX

Integrin 5a

Glucose transporter 1, 3

NR3C1 Glucocorticoid receptor-α

Plasminogen activator inhibitor-1

Glyceraldehyde-3-phosphate dehydrogenase

Nuclear factor IL-3 (NFIL-3)

Urokinase plasminogen activator receptor

Hexokinase-1, 2

GROWTH FACTORS/CYTOKINES

Tissue factor

Lactate dehydrogenase A. B

IGF-2

Mucin 1

Phosphoglycerate kinase

IL-6, 8

CXCR-4

6-Phosphofructo-2 kinase

Intestinal trefoil factor

Prolyl-4-hydroxylase

Fructose 2–6 bisphosphatase-3

Macrophage inhibitory factor (MIF-1)

Osteopontin

Pyruvate kinase-M

PDGF-B

Met tyrosine kinase (HGF receptor)

Transglutaminase-2

Stanniocalcin-2

APOPTOSIS

Acetoacetyl CoA thiolase

TGF-α

BNIP3, BNIP3L

Adenylate kinase-3

ANGIOGENESIS

IGFBP1, 3, 5

Aminopeptidase A

VEGF A, B, C, D

Bid

Triose phosphate isomerase

VEGF R1

Pim1, Pim2

Phosphoribosyl pyrophosphate synthetase

Placental growth factor

Bcl-w like

Spermidine N1-acetyltransferase

Angiopoietin 2

RTP801

Tyrosine dehydroxylase

Adrenomedullin

Glycogen branching enzyme

Endothelin 1, Endothelin 2

Hepatic fibrinogen/angiopoietin-related protein

Solute carrier family

Ephrin A1

STRESS RESPONSE

Carbonic anhydrase IX, XII

Nitric oxide synthase

GRP78, GRP94, ORP150

Ceruloplasmin

COX1, COX2

Gadd153

Erythropoietin

Thrombospondin 1, 2

HAP-1

Ferritin light chain

Fibroblast growth factor-3

Thioredoxin

Heme oxygenase

Hepatocyte growth factor

Heat shock factor

Transferrin & receptor

Transforming growth factor-α, β-1, 3

PROLIFERATION/DIFFERENTIATION

TRANSCRIPTIONAL FACTORS/GENE EXPRESSION

Tie-2

BTG1

Nitric oxide synthase

Cyclin G2

Early growth response 1

TISSUE REMODELING

DEC1/stra13

p35srj

Lysyl oxidase

Adipophilin

ETS-1

Lysyl hydroxylase-2 (PLOD2)

p21 CDKI

Mxi-1

Galectin-1

CDKN1b (p27, kip-1)

Annexin V

CD99

N-myc downstream reg-1 (Cap43)

BCL-interacting killer (BIK)

Collagen-5a

Cyclin G2

FOS

Ku 70

Mitogen-inducible gene-6 (MIG-6)

Jun

LDLR-related protein

ID-2 (DNA binding protein inhibitor)

Adapted from Le QT, Denko NC, Giaccia AJ: Hypoxic gene expression and metastasis. Cancer Metastasis Rev 2004;23:293–310.

which has been shown to increase cancer cell malignancy.28 These events occur through activation of ERK and Rho by integrin clustering (see section on Cell Motility).29 Production of reactive oxygen and nitrogen species by host immune cells and rapidly proliferating tumor cells not only increases genomic instability but has also been proposed to upregulate the expression of metastasis-promoting genes.30

Invasion Changes in Cell Adhesion The first step of metastasis is invasion. Cells must undergo changes in their cell-cell and cell-matrix adhesion interactions to dissociate

themselves from the tumor.31 Acquisition of an invasive phenotype requires changes in expression of genes that control cell-cell adhesion as well as proteolytic degradation of the ECM.32 Cell-cell adhesions are mediated primarily by E-cadherin proteins expressed at junctions between cells.31 Cadherins bind cells through protein-protein interactions at their extracellular domains, whereas their intracellular domains signal to catenins and the actin cytoskeleton. Changes in E-cadherin expression allow cells to detach from their neighbors and begin their migratory route toward the circulatory or lymphatic system to seek out new terrain. Reduced expression of E-cadherin is often observed in aggressive cancers through epigenetic silencing, proteosomal degradation, proteolytic cleavage, or mutation. In fact, inactivating mutations of E-cadherin have been shown to predispose

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patients to gastric cancer, implicating E-cadherin as a tumor suppressor gene.31 Loss of E-cadherin is highly associated with epithelial to mesenchymal transition (EMT), a program that is essential for numerous developmental processes.33 The acquisition of the invasive phenotype has many similarities to EMT, including loss of cell-cell adhesion mediated by E-cadherin repression and an increase in cell mobility. During EMT, there is a switch from E-cadherin (an epithelial cell marker) to N-cadherin expression (a mesenchymal cell marker), which promotes cell-matrix adhesion instead of cell-cell adhesion.33 Several signal transduction pathways, such as the Ras-MAPK and Wnt pathways, have been shown to regulate EMT (Fig. 3-3). In particular, the Ras-MAPK pathway activates two related transcription factors known as Snail and Slug.34,35 Both of these proteins act as transcriptional repressors of E-cadherin, and their expression induces EMT in cancer cells.36 Studies have indicated that Slug is an independent prognostic parameter for poor survival in colorectal carcinoma patients.37 Twist, another basic helix-loop-helix transcription factor that is necessary for proper embryonic development, has also been shown to induce EMT through the repression of E-cadherin.38 Both Twist and Snail expression levels are elevated in breast cancer patients, and are associated with poor prognosis.38,39 Dysregulation of Wnt signaling is common in many types of human cancers and regulates EMT in part through Snail activation, an important early step in metastasis.40

Cell Motility Cancer cells are able to take advantage of many mechanisms to migrate and invade, including both individual and collective cellHGF

migration strategies (Table 3-2).32 Most cancer cells of epithelial origin undergo EMT and acquire invasive migration capacity to enter the circulatory or lymphatic system. Invasive migration is a dynamic and complex process involving changes in cell-matrix adhesion and the cytoskeleton (Fig. 3-4). Changes in cell-matrix adhesion are necessary for the leading edge of the cell to grab onto the matrix surrounding it and pull itself forward in a movement similar to an inchworm. This invasive migration can be viewed as cycles of adhesion and detachment, allowing the cell to bind, then detach after pulling forward. Cell-matrix adhesions are in large part regulated by integrin proteins. Integrins are heterodimers of one of 18 alpha and 8 beta transmembrane proteins that bind to specific components of the ECM.41 They can transmit signals into or out of the cell and are important mediators of malignant transformation. Integrins are stimulated when they come into contact with specific ECM substrates, or through growth factor-stimulated signaling where they interact with receptor tyrosine kinases.42–44 For example, hepatocyte growth factor (HGF, also known as scatter factor) influences invasion by signaling through its receptor c-met.45 Integrin stimulation also promotes formation of focal adhesion contacts, focal adhesion kinase (FAK) activation through phosphorylation, and formation of FAK-Src complexes.44 It is noteworthy that Src mutations that have been implicated in tumor cell motility are often observed in human cancers such as adenocarcinoma of the colon.46 Intracellular signaling mediated by FAK activates Rac, RhoC, cdc42, and other guanosine triphosphatases (GTPases) that mediate cellular changes required for invasion.42 These include actin-myosin contraction that propels the cell forward, and recruitment of matrix metalloproteases (MMPs) to focal adhesion sites where they degrade the ECM,

α6β4 integrin E-cadherin

c-Met CD44

TGFβ

Wnt

IGF

TGFβRI IGF1R

TGFβRII Frizzled

Shp2 ERM

Src

α-catenin

Ras GRB2 SHC P13K G3K3β

PLCγ GRB1 Smad

β-catenin

α-catenin PLCγ IRS1 β-catenin Ras

β-catenin

P13K

MAPK

Nucleus

Snail Slug Twist

E-cadherin

DNA

Figure 3-3 • Signaling pathways involved in epithelial to mesenchymal transition (EMT). EMT is a program of development of biological cells essential for numerous developmental processes. Tumor cell invasion has many phenotypic similarities to EMT, including a loss of cell-cell adhesion mediated by E-cadherin repression and an increase in cell mobility. Several signal transduction pathways have been shown to be involved in regulation of EMT. These include Ras-MAPK and Wnt. These pathways are activated by the binding of ligands to transmembrane receptors. These include: TGF-β binding to TGF-βRI and TGF-βRII; HGF binding to c-Met; Wnt binding to Frizzled; and IGF binding to IGF-1R. Activation of these pathways results in transcriptional repression of E-cadherin, and transcriptional activation of Snail, Slug, and Twist. These transcription factors regulate expression of genes involved in EMT. An important repressor of E-cadherin is β-catenin, which is normally targeted for degradation by GSK-3β. Activation of the Wnt pathway inhibits GSK-3β activity, resulting in stabilization of the β-catenin and translocation to the nucleus. (Adapted from Lee JM, Dedhar S, Kalluri R, Thompson EW: The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol 2006;172:973–981.)

The Cellular Microenvironment and Metastases • CHAPTER 3

Table 3-2

Mechanisms of Cancer Cell Migration INDIVIDUAL

INTEGRINS

COLLECTIVE

Ameboid

Clusters

(e.g., lymphoma,

(e.g., epithelial cancer,

SCLC,

melanoma)

CADHERINS

leukemia)

+GAP

ADHESION

JUNCTIONS

INTERACTIONS

+PROTEASES Mesenchymal

Sheets

(e.g., fibrosarcoma,

(e.g., epithelial cancer,

glioblastoma,

vascular tumors)

anaplastic tumors)

Adapted from Friedl P, Wolf K: Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 2003;3:362–374.

Contraction

Cell protrusion

P

FAK signaling [Src/Rho/Rac] P

ECM fibers

(1) Increased adhesion and actin fiber assembly

(2) Focal adhesion formation

Forward movement

(3) FAK activation and cell contraction

Actin Integrin FAK P Phosphate

Protease

Key (4) Recruitment of proteases and ECM cleavage

(5) Detachment and complex disassembly

Figure 3-4 • Invasive migration. Invasive migration is a dynamic and complex process involving changes in cell-matrix adhesion and the cytoskeleton. It begins with pseudopod protrusion at the leading edge. This increases cell-matrix interaction, stimulating integrin receptors. Integrin stimulation promotes formation of focal adhesion contacts, focal adhesion kinase (FAK) activation through phosphorylation, and formation of FAK-Src complexes. Intracellular signaling mediated by FAK activates Rac, RhoC, cdc42, and other GTPases that mediate cellular changes required for invasion. These include actomycin contraction that propels the cell forward, and recruitment of proteases to focal adhesion sites where they degrade the ECM, allowing the cell to glide forward when focal adhesion complexes are disassembled after contraction. The remodeled matrix tracks left behind the cell have been shown to facilitate movement of subsequent cells. (Adapted from Friedl P, Wolf K: Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 2003;3:362– 374.)

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allowing the cell to glide forward when focal adhesion complexes are disassembled after contraction.32 In addition, the remodeled matrix tracks left behind the cell have been shown to facilitate movement of subsequent cells, similar to the generation of ski tracks by the first cross-country skier that allow following skiers to move more easily.26

Whether or not genes such as LOX that are required for cross-linking collagen are involved in this process is still unknown. Interestingly, the transcription factor Twist that is implicated in EMT (as described previously) has also been shown to increase the ability of tumor cells to intravasate.38 However, the underlying molecular mechanisms involved in promoting intravasation by Twist are as yet unknown.

Disruption of the Basement Membrane

Survival in the Circulatory System

The basement membrane provides a physical barrier between epithelial cells and the stroma. It is composed of numerous glycoproteins and proteoglycans that provide ligands for integrins permitting control of cell orientation and outside to inside signaling. Epithelial and stromal cells produce a mixture of these components that form a dense meshwork underlying the epithelial cells. The basement membrane is not normally permeable to cells. Tumor cells overcome this barrier by altering the expression of their cell surface receptors such that they can now adhere to basement membrane components.32,47,48 For example, tumor cells will increase expression of integrins that can bind laminin and collagen IV.49 Increased CD44 expression permits cell binding to hyaluronan, a basement membrane proteoglycan, and is observed in several types of human cancer including metastatic colon carcinomas.50 In addition, cancer cells modify the components of the basement membrane to ease penetration. For example, reduced laminin expression is observed in poorly differentiated human colon carcinomas.51 ECM protease activity is tightly regulated by proteins that inhibit their functions.45,52 Tumor cells proteolytically disrupt the basement membrane by altering the balance between ECM proteases and their inhibitory proteins. For example, elevated MMP expression and activity increases degradation of the basement membrane. MMP-1 (also known as collagenase or gelatinase) degrades collagen IV and is increased in highly metastatic cancer cells.53 MMP degradation of the ECM not only facilitates cell movement but additionally generates a large number of bioactive cleaved peptides, and releases growth factors and chemokines trapped within the ECM mesh.52 For example, MMP activity releases active forms of proteoglycans including heparin, hyaluronate, and chondroitin sulfate.54,55

Tumor cells that have successfully entered the bloodstream through intravasation theoretically have access to most organs in the body. However, before these tumor cells can extravasate into a target tissue they must first survive the environment of the circulatory system. Tumor cells in the circulatory system are subjected to immune attack, circulatory forces, and anoikis (apoptosis induced by loss of adhesion).2 Circulating tumor cells bind to platelets to protect themselves from these dangers, thus increasing their chance of survival.56–58 Tumor cells also bind to coagulation factors including thrombin, fibrinogen, tissue factor, and fibrin, creating emboli.59 These tumor cell emboli are more resilient to both circulatory forces and immune attack, and have been shown to have greater metastatic potential than single tumor cells.57 In the circulation, aggregates of tumor cells are termed homotypic clumps, because they are homogeneous in their cellular composition, whereas those associated with platelets are termed heterotypic clumps and may possess greater metastatic potential for the reasons described previously.56,58 The ability to resist apoptotic cell death is important at a variety of steps in the metastatic process. First, tumor cells must survive the lack of oxygen and nutrients in the primary tumor to be able to migrate and invade. This is particularly noteworthy, because hypoxia increases the metastatic potential of tumor cells. Apoptotic resistance in response to decreased oxygen and nutrients is achieved by loss of the p53 tumor suppressor gene, increased expression of antiapoptotic members of the Bcl-2 family and decreased expression of proapoptotic members, and increased activity of the HIF transcription factor. Hypoxic tumor cells that are resistant to apoptosis have a greater probability of surviving for sufficient periods of time to intravasate into the circulation. Apoptosis can also play a role in anoikis, death induced by loss of cell adhesion. Obviously, resistance to anoikis is important both in the early phases of invasion as well as during intravasation and circulation. Although a variety of receptor tyrosine kinases can impart resistance to anoikis,59 most probably the formation of homotypic and heterotypic cell aggregates promotes resistance to anoikis as well.

Intravasation The entry of tumor cells into the circulation (intravasation) and the exit of tumor cells from the circulation (extravasation) to host tissue represent critical steps in the metastatic process. One clear difference between intravasation and extravasation has to do with the composition of the blood vessels. Tumor blood vessels are malformed and irregular, often possessing breaks in their thin lining that permit the easy access of tumor cells into the circulation. In contrast, the vasculature of normal tissue where tumor cells extravasate do not have these same features. This very observation suggests that the processes of intravasation and extravasation are distinct and probably require different gene functions. The abnormal vasculature found in tumors is the result of the dysregulated expression of proangiogenic growth factors, inhibition of antiangiogenic genes and pathways, recruitment of vascular progenitor cells from the bone marrow, and, in some cases, vascular memory by tumor cells. Tumors typically do not possess abundant lymphatics and are under high interstitial pressure. Although tumors secrete lymphangiogenic factors such as vascular endothelial growth factor-C (VEGFC), the development of lymphatics in tumors is also abnormal. In fact, the intravasation of tumor cells into lymphatics is probably easier than through vasculature in that lymphatic vessels function as a collection point for interstitial fluids. Our knowledge of the genetic determinants involved in intravasation is limited. Gradients of chemo-attractant proteins such as chemokines have been proposed to guide cells toward the circulatory system.26 In addition, tumor cells move along collagen fibers produced by invading cells, a process facilitated by host macrophages.26

Arrest and Extravasation Much of our knowledge of tumor cell extravasation is patterned after leukocyte transmigration through endothelium. It is well known that leukocytes arrest before transmigration. Similarly, tumor cell arrest can occur passively through mechanical lodging or can be allowed by cell-surface molecules.60–62 Endothelial cells are constantly shed from the blood vessel walls, creating temporary gaps to which tumor cells can more easily attach because basement membrane components are exposed.63–65 Vessel wall damage also attracts platelets and tumor cells associated with platelets, which is enhanced by fibrinogen expression on the endothelial cell surface.66,67 Fibrin blood clots at the sites of tumor cell arrest can further damage vessels, attracting more platelets and circulating tumor cells.68 Increased blood coagulation is often observed in cancer patients as a result of elevated levels of thromboplastin, procoagulant A, and phosphatidylserine produced by tumor cells.69,70 The most severe manifestations of this hypercoagulation state were described by Trousseau many years ago. The induction of the enzymes involved in this state can also be enhanced by changes in the tumor microenvironment.71 Tumor cell arrest is allowed by endothelial cell P- and E-selectins that bind to the tumor cells72,73 and by tumor glycosylation patterns

The Cellular Microenvironment and Metastases • CHAPTER 3

and cell-cell adhesion molecules such as integrins and CD44.74–78 Increased cell-surface expression of mucin carbohydrate is associated with increased metastatic potential in human colon carcinoma.79 Tumor clump formation additionally facilitates tumor cell arrest by increasing the number of adhesive interactions. ECM components such as fibronectin and laminin enhance tumor cell arrest, and administration of targeting peptides to fibronectin and laminin can reduce metastatic formation.80 Tumor cells may reside and grow within the intravascular space until the metastatic lesion physically breaks through the vessel.81 Tumor cells may also extravasate by inducing endothelial cell retraction permitting cell attachment to the ECM.81 It is highly noteworthy that VEGF increases vascular permeability and may permit extravasation through Src activation.82,83 Thus, anti-VEGF therapy could potentially act to inhibit metastases by decreasing vascular permeability. In some cases, tumor cells direct their movement and invasion into new organ terrain by following migrating white blood cells and tissue motility factors.84

Proliferation The final steps of metastasis involve the resumption of cell proliferation at the secondary site and induction of angiogenesis to supply oxygen and nutrients. Studies have shown that the host tissue can influence tumor growth through autocrine, paracrine, and endocrine signals. However, it is the net balance of positive and negative signals that determines metastatic proliferation. This can partially explain organ-specific metastasis, because only certain cells will be able to respond to tissue-specific proliferation-stimulating signals and leave their dormant state.85 For example, insulin-like growth factor-1 (IGF1), HGF, and transforming growth factor α (TGFα) are highly expressed in the liver,86–88 and cancer cells from colon, breast, and bladder overexpress receptors for these ligands such as epidermal growth factor receptor (EGF-R)89–93 and c-met receptor,94 resulting in proliferation of metastatic cells in these tissues.

Angiogenesis The formation of a new blood supply from pre-existing vasculature is stimulated by an angiogenic “switch” that occurs when the ratio of inducers to inhibitors is increased. Inhibitors of angiogenesis include ECM proteins thrombospondin and endostatin.95–97 Angiogenic inducers include VEGF, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), TGFβ, and ephrin, and their family members.3 Of these, VEGF is the best characterized and has successfully been targeted through the use of monoclonal antibodies and soluble receptors.98 VEGF increases angiogenesis by stimulating endothelial cells, mobilizing endothelial progenitor cells, stimulating outgrowth of pericytes that line the walls of mature blood vessels, and increasing vascular permeability allowing macromolecules to traverse endothelium.99,100 Furthermore, VEGF is thought to be a key molecule for the homing of VEGFR-positive bone marrow-derived progenitor cells involved in premetastatic niche formation (see later discussion),101 and for homing of VEGFR-positive tumor cells to metastatic sites.102 Recruitment of bone marrow-derived circulating endothelial cells additionally increases angiogenesis.103 Thus, angiogenesis is important both for primary tumor and metastatic tumor growth, making it an attractive target.

Metastasis of Metastases Tumor cells that have successfully colonized secondary organs are capable of further metastasis and colonization of other organs. Cells within the metastatic tumors are subjected to similar microenvironmental stresses experienced by the primary tumor, and adapt to overcome these external barriers and seed new terrain. These cells from metastases may have an intrinsic colonization capability allowing them to constantly reseed both primary and secondary tumors.104

LYMPHATIC METASTASIS The vascular and lymphatic systems have numerous connections,105 and metastasizing tumor cells can easily pass from one system to another.106,107 Invading tumor cells can additionally enter small lymphatic vessels directly and be passively transported to the lymph. Cancer cells may spread to lymph nodes near the primary tumor, known as the regional lymph nodes (RLNs). This is often referred to as nodal involvement, positive nodes, or regional disease. Tumor cells may become trapped in the first lymph node, or form distal nodal metastases referred to as “skip metastases” because they have bypassed the first draining lymph nodes in the area.4 RLNs may become enlarged and are often removed to prevent cancer spread. Lymph node involvement and presence of micrometastases in the sentinel lymph node (the lymph node draining the tumor site) correlate with decreased survival.108 Localized spread to RLNs near the primary tumor is not normally considered as metastasis per se, although it is also a sign of worse prognosis.109,110 Some malignancies, such as sarcomas in contrast to breast carcinomas, do not spread to the RLNs before metastasizing to distant sites. Thus, node status does not always correlate with metastasis.2

COLONIZATION BY METASTATIC TUMOR CELLS The patterns of colonization cannot solely be explained by circulatory routes above. The propensity for certain tumors to seed in particular organs was first discussed as the “seed and soil” theory by Stephen Paget over a century ago in 1889.4 For example, prostate cancer often metastasizes to the bones, and colon cancer has a tendency to metastasize to the liver. Colonization is an extremely inefficient process that is heavily dependent on the interactions between “seeding” tumor cells and the “soil” microenvironment of the secondary site. Many factors including formation of a premetastatic niche and organ specificity determine these patterns of colonization.

Premetastatic Niche Recent in vivo data have suggested that the formation of a premetastatic niche is essential for the growth of extravasating metastatic tumor cells.101 Factors secreted by primary tumor cells stimulate mobilization of bone marrow-derived cells that enter circulation and reside in sites of future metastasis. These bone marrow-derived cells express VEGFR-1, c-kit, CD133, and CD134, and increase angiogenesis at the premetastatic sites. Targeted inhibition of VEGFR-1 prevented niche formation and subsequent metastatic progression. This tissue preconditioning may thus represent a key step that could be targeted therapeutically, although studies with anti-VEGF therapy fail to show significant benefit in preventing metastatic growth for long periods of time. The role of hematopoietic progenitor cells and other bone marrow-derived cells in tumor progression is reviewed by Kaplan and colleagues111 and shown in Figure 3-5. An additional function of the premetastatic niche is to guide metastases to specific organs. Kaplan and coworkers demonstrated that injection of secreted factors collected from cancer cells that metastasize to multiple organs could permit cancer cells that only metastasize to the lung when grown as subcutaneous tumors in mice, to display widespread metastasis through governing bone marrowderived cell accumulation.101 Elevated fibronectin expression by fibroblasts and fibroblast-like cells resident at premetastatic sites seems to be a critical factor in the development of the premetastatic niche. The key tumor-secreted factors that determine metastatic sites and mediate premetastatic niche formation have yet to be identified, although a role for tumor necrosis factor α (TNFα), TGF-β, and VEGF-α pathways has been demonstrated.112 MMPs may also play an important role in this process. For example, VEGF-R1 signaling has been shown to be required for

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Part I: Science of Clinical Oncology Bone marrow niche

Osteoblastic niche HPC

Stroma

ESC HSC MSC

Vascular niche

Circulatory system

Primary tumor niche

Premetastatic niche

Stromal cell

Osteoclast

Hematopoietic progenitor cell (HPC)

Osteoblast

Tumor cell Key

Figure 3-5 • The role of bone marrow-derived cells in tumor progression. Cells derived from the bone marrow niche are involved at many stages during tumor progression. Hematopoietic progenitor cells (HPCs), hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), and stromal cells including macrophages and fibroblasts, permit tumor growth and development at primary and metastatic niches. (Adapted from Wong SY, Hynes RO: Lymphatic or hematogenous dissemination: how does a metastatic tumor cell decide? Cell Cycle 2006;5:812–817.)

premetastatic induction of MMP-9 expression in endothelial cells and macrophages of the lungs by distant primary tumors.113 This is thought to make the lung microenvironment more compliant for invasion of metastasizing cells. This concept is supported by the finding that pericyte recruitment and angiogenesis are not observed in tumor-bearing mice with MMP-9 knockout bone marrow cells.114 Furthermore, stromal-derived MMP-2 and MMP-9 have also been shown to contribute to establishment and growth of metastases.115 Thus, whereas there is evidence that MMPs play multiple roles in metastases, clinical trials with MMP inhibitors have failed to show significant efficacy. In large part, this has been due to unexpected normal tissue toxicities and conflicting roles in metastases.

Organ Specificity The organ distribution of metastases from a primary is not random. Minn and colleagues used bioluminescence imaging to reveal patterns of metastasis formation by human breast cancer cells in mice.116 They also showed that individual cells from the pleural effusion of a breast cancer patient showed distinct patterns of organ-specific metastasis.117 Single-cell progenies derived from this population demonstrated different abilities to metastasize to the bone, lung, or adrenal medulla.

These studies indicate that there are particular requirements for metastasis to colonize specific organs. Some of the key molecules determining organ-specific metastasis have been identified and are briefly discussed in the following section.

Metastases to the Bone There are two types of bone metastases: osteoblastic and osteolytic.118,119 Osteoblastic metastases are observed in patients with advanced prostate cancer. Both the differentiation of osteoblastic precursors as well as the activity of osteoblast cells are stimulated by tumor and microenvironmental signals such as bone morphogenetic protein (BMP), FGFRs, and IGF-1R.120 Runx-2 is a key transcription factor that regulates the differentiation of osteoblasts and osteoblastic precursor cells,121 and represents a potential new target for inhibiting osteoblastic metastases by preventing osteoblastic precursor differentiation. In contrast, osteolytic metastases are observed in patients with breast cancer or multiple myeloma,122,123 and in these patients interactions between tumor cells and the bone microenvironment result in bone resorption and metastatic growth due to the unique interplay between osteoblasts and osteoclasts (Fig. 3-6).118,122 Parathyroid hormone-related protein (PTHrP) secreted by the tumor cells, stimulates osteoblasts to produce receptor activator of nuclear

The Cellular Microenvironment and Metastases • CHAPTER 3

PTHrP

RANKL RANKL

Figure 3-6 • The vicious cycle of osteoclastic bone metastasis. Interactions between the tumor cells and the bone microenvironment create a “vicious cycle” of osteolytic metastatic lesion development. Parathyroid hormone-related protein (PTHrP), secreted by the tumor cells, stimulates osteoblasts to produce RANK ligand (RANKL). Bone-resorbing osteoclast cells are activated by RANKL when it binds to the RANK receptor. The activated osteoclasts upregulate MMPs that degrade the bone matrix-releasing growth factors such as TGFβ, IGFs, PDGF, FGFs, and BMP. These factors stimulate tumor cells to release PTHrP, thus completing the vicious cycle. (Adapted from Steeg PS: Tumor metastasis: mechanistic insights and clinical challenges. Nat Med 2006;12:895–904.)

Tumor cell

factor–κB (RANK) ligand (RANKL). Consequently, bone-resorbing osteoclast cells are activated by RANKL when it binds to the RANK receptor. Activated osteoclasts upregulate MMPs, which degrade the bone matrix-releasing growth factors such as TGFβ, IGFs, PDGF, FGFs, and BMP.118,124,125 These factors stimulate tumor cells to release PTHrP, thus restarting this pathway of bone resorption. Gene profiling has identified other important mediators of osteoclastic bone metastases including CXCR4, MMP-1, CTGF, and osteopontin.126 Tumor cells additionally induce osteoclast formation by overexpressing interleukins such as IL-8 and IL-11, and by downregulating macrophage colony-stimulating factor.127,128 All of these latter factors represent new targets for metastases, although the importance of each factor in osteoclastic bone metastases requires further clarification.

Metastases to the Brain Brain metastases are most commonly observed in patients with breast cancer, lung cancer, and melanoma. Vascular access to the brain is strictly regulated by the blood-brain barrier, an endothelial layer surrounding the brain connected by tight junctions and further lined by a basement membrane, pericytes, and astrocytes.129 Macromolecules are not usually able to traverse the blood-brain barrier, and it remains unclear how tumor cells are able to penetrate the blood-brain barrier. However, once the tumor cells are within the brain parenchyma, glial cells permit establishment and growth of metastases by secreting chemokines, cytokines, and growth factors.130 Other neurotransmitter hormones in the brain such as norepinephrine have also been reported to increase tumor cell motility and metastatic spread.131 Little is known about the key factors that determine colonization of the brain, mostly because there is a lack of good in vivo models of brain metastasis. Overexpression of Stat3 increases melanoma metastasis to the brain and increases invasion of the melanoma cells and angiogenesis, although the pathways modulated by Stat3 signaling require elucidation.132 The dependence of brain metastases on VEGF has been demonstrated experimentally in animals through inhibition studies where VEGF neutralization reduces brain metastases.133,134 In general, patients with brain metastases have an extremely poor prognosis. It is of concern that there has been an increase in the incidence of brain metastases in patients whose systemic disease is well controlled.135–137 For example, patients with breast tumors that overexpress Her-2 and who are treated with Her-2 targeting trastuzumab (see later discussion) have an incidence of brain metastases twice that of breast cancer patients who are treated with other agents.135 This is thought to be because the brain offers a sanctuary when systemic disease is being controlled.3 The development of drugs

Osteoblast

RANK Osteoclast

TGFβ BMP IGFs PDGF FGFs

Bone marrow niche

MMPs

that can cross the blood-brain barrier and target brain metastases are of paramount importance in the development of new targeted therapies to tackle this problem. Currently, the best treatment for oligometastases to the brain is radiosurgery.

Metastases to the Lung Pulmonary metastases are frequently observed in patients with sarcoma, breast, melanoma, gastrointestinal, and kidney cancers. Because cardiac output from the pulmonary artery circulates through the lungs, a high incidence of pulmonary metastases in cancer patients can be expected on the basis of blood flow alone. Metastases therefore often initiate in pulmonary arterioles and later traverse the basement membrane into the lung parenchyma. TGFβ and NF-κB facilitate this process in breast cancer,138–140 as does osteopontin in hepatocellular cancer,141 and ezrin in osteosarcoma and breast cancer.142,143 In vivo studies have identified a gene expression signature for lung metastasis including several membrane-localized and secreted proteins that has been validated in breast cancer patients.116 Interestingly, this group of genes was able to induce lung metastasis when expressed together but not individually, suggesting essential cooperation between proteins. Increased expression of antiapoptotic proteins such as Bcl-2 and Bcl-xl is also observed in lung metastases, facilitating survival and providing resistance to therapy.144–148 These studies suggest that multiple targets must be inhibited with combination therapy to effectively inhibit lung metastases.

Metastases to the Liver Liver metastases are observed in patients with breast, lung, and pancreatic cancers. However, liver metastases are most commonly found in patients with metastatic colorectal cancer, because the liver is the first capillary bed encountered by the metastasizing cells. The circulatory system of the liver, in particular the liver sinusoids, does not have a barrier limiting macromolecule flux, and it is well perfused and highly permeable, permitting metastasizing cancer cells to establish themselves and grow. Thus, tumor cell invasion and survival are probably the key determinants in metastatic colonization of the liver.149 There are two types of liver metastases: a nonangiogenic “replacement” of liver cells with tumor cells that preserves the stroma,150 and a “pushing” type of metastasis3 whereby the liver stroma is not preserved and has higher levels of endothelial cell proliferation.151,152 In light of the “pushing” type of metastases that stimulate angiogenesis, targeting the VEGF pathway experimentally in vivo has been shown to prevent liver metastases,153–155 and is effective when combined with cytotoxic agents in patients with metastatic colon cancer.156,157 Other molecules thought to be important in

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colonization to the liver and that could be targeted therapeutically are COX-2,158,159 integrins,160 and Src.161

HOST-TUMOR CELL INTERACTIONS Tumor progression requires collaboration between tumor and host cells. Research has revealed much cross-talk between cancer cells and bone marrow-derived host immune and stromal cells.162 For example, disruption of TGFβ signaling in fibroblasts can induce stomach and prostate cancer in mice.163 Mutations commonly found in cancer cells, such as p53 and PTEN mutations, can also be found in cancerassociated stroma and have been hypothesized to be important during tumor progression.164,165 In fact, gene expression profiling of activated fibroblasts in vitro generated a signature that could predict primary tumor metastasis.166 For example, the chemokine CXCL12 is produced by breast cancer-associated fibroblasts167 and increases tumor cell migration and recruitment of endothelial progenitor cells expressing CXCR4,168 a CXCL12 receptor. Cells that respond to tissue injury (such as leukocytes and lymphocytes) are often associated with tumor cells and enhance their progression, for example by assisting travel in the bloodstream. The tumor-suppressing activities of cells in charge of immune attack (such as natural killer cells and antigen-presenting cells) can be dampened by overexpression of tumor-derived immunosuppressive cytokines such as TGFβ and interleukins,169–171 and by lack of necessary costimulatory signals from tumor cells.172 Thus, host cells can directly promote tumor progression by secreting growth factors and cytokines that stimulate tumor metastases and suppress tumor immune surveillance.

Inflammation and Metastases Ironically, cells involved in chronic inflammation facilitate tumor formation and progression, mostly mediated by NF-κB and COX2.173,174 In particular, infiltration of activated macrophages into tumors correlates with poor prognosis.27,175 The tumor-suppressing and tumor-promoting roles of these tumor-associated macrophages (TAMs) are shown in Table 3-3. TAMs are especially attracted to regions of hypoxia, where they secrete abundant angiogenic inducers and proteases, including VEGF and MMPs,175,176 that have been reported to enhance angiogenesis.27 TAMs express high levels of the HIF-2 transcription factor that has been found to be an independent prognostic factor of outcome.177 Interestingly, work by Cramer and associates178 has shown that in fact HIF-2 is needed for myeloid cell infiltration and activation. Furthermore, TAMs release growth factors such as PDGF, EGF, and HGF, which enhance proliferation, survival, and invasion.175 Mutation of the macrophage colony-stimulating factor-1 gene that affects macrophage differentiation has been shown to prevent metastasis in mice bearing aggressive breast cancer tumors.179 Targeting TAMs may be a viable mechanism for antimetastatic therapies.180

Table 3-3 Conflicting Roles of Tumor-Associated Macrophages Pro-Tumor

Anti-Tumor

Proangiogenic cytokines

Tumor cell lysis

Immunosuppressive cytokines

Immunostimulatory cytokines

Protumorigenic chemokines

Immunostimulatory chemokines

Reactive oxygen species (ROS)

ROS

Elevated MMPs, TF, and uPA MMPs, matrix metalloproteinases; TF, tissue factor; uPA, urokinase plasminogen activator. Adapted from Condeelis J, Pollard JW: Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell 2006;124:263–266.

DORMANT CELLS The prolonged survival of single cells or micrometastases with no apparent progression is referred to as dormancy.181 Dormant cells are frequently observed in patients with prostate, melanoma, and breast cancer,182–184 and are found to reside in the lungs, liver, and bone marrow. These micrometastases represent minimal residual disease that results from the inefficiency of metastasizing tumor cells to colonize organs properly following extravasation.185 Incompatibilities between tumor cells and their tissue soil, or inability of tumor cells to generate sufficient angiogenesis result in cell-cycle arrest and dormancy.181 What genes and pathways are important in controlling metastatic dormancy are unknown and are important to identify, because they represent a “metastatic tumor suppressor mechanism.” The presence of dormant tumor cells is associated with poor patient prognosis.186 Isolation and reimplantation of dormant cells can generate primary tumors, demonstrating that these cells are viable.185,187,188 In vivo experiments have indicated that growth of dormant metastatic cells can be activated by angiogenesis or removal of the primary tumor,189 suggesting that limited levels of growth factors or cytokines may induce this dormant state. The angiogenic switch required for dormant cells to grow into tumors may be detectable by markers in the blood, such as VEGF, and could thus be used to monitor undetectable and asymptomatic disease in patients.190 Interestingly, circulating tumor cells can be detected in breast cancer patients as long as 22 years after mastectomy.191

CANCER STEM CELLS AND METASTASIS Stem cells are primal cells that retain the ability to renew themselves through cell division and can differentiate into a wide range of specialized cell types. They give rise to all tissues during embryogenesis and control tissue homeostasis in the adult. Cells with stem cell properties have been identified in some cancer types.192–194 These cancer stem cells (CSCs) are able to self-renew and differentiate into multiple cell types and are believed to arise either by mutation of an adult stem cell or fusion of an adult stem cell with a CSC.195 It is hypothesized that tumors arise from CSCs and that these cells persist in tumors as a distinct population that is responsible for disease relapse and metastasis. Because CSCs are the only cells capable of giving rise to new tumors by themselves, targeting this subpopulation may eradicate cancer. The recent discovery of CSCs has revolutionized our way of thinking about cancer. Cancer is classically thought of as a disease of progression, facilitated through the accumulation of mutations and driven by microenvironmental signals. Whereas the stem cell microenvironment or niche is thought to be the key determinant for stem cell regulation, the role of CSCs in multistage tumor progression, particularly with respect to metastasis, is poorly understood. There may exist a subset of CSCs with the inherent property to metastasize (Fig. 3-7). Research into these metastatic CSCs is greatly anticipated.

ANTIMETASTATIC THERAPY The literature is replete with genes that have been implicated in the metastatic process.196,197 Because of the immense heterogeneity of metastatic cells, metastatic selective therapeutic targets have been difficult to identify and develop for targeted therapy. Clinically, there have been two success stories thus far: bevacizumab and trastuzumab. However, both agents affect both primary and metastatic tumor growth. Bevacizumab targets VEGF and has displayed activity in several metastatic cancer types, especially when administered in combination with cytotoxic compounds.157 Other small-molecule inhibitors to VEGF-R have also shown some antimetastatic effectiveness. Trastuzumab is a recombinant monoclonal antibody to Her-2 that is very effective against metastatic breast cancer, again, particularly in combination with cytotoxic agents.198 However, only 30% to 40%

The Cellular Microenvironment and Metastases • CHAPTER 3

Stem cell Stromal cell mCSC

Mutation/fusion with cancer cell

HPC Tumor cell

Key

Pool of cancer stem cells

Primary tumor

mCSC

CSC

Premetastatic niche

Microenvironmental signals

Homing and anchorage factors

Circulatory system

Metastatic growth

Figure 3-7 • The role of cancer stem cells (CSCs) in metastasis. CSCs are able to self-renew and differentiate into multiple cell types and are believed to arise either by mutation of an adult stem cell or fusion of an adult stem cell with a cancer stem cell. It is hypothesized that a pool of CSCs develops and gives rise to the primary tumor. A subpopulation of CSCs is believed to exist with the inherent property to metastasize. Microenvironmental signals stimulate primary tumor cells to secrete factors involved in premetastatic niche formation, and additionally stimulate invasion and dissemination of the metastatic CSCs (mCSCs). These mCSCs are attracted to homing and anchorage signals produced by bone marrow-derived cells at the premetastatic niche. (Adapted from Al-Hajj M, Clarke MF: Self-renewal and solid tumor stem cells. Oncogene 2004;23:7274–7282.)

of breast cancer patients overexpress Her-2 and are suitable for treatment.198 In addition, whereas MMP inhibitors demonstrated good antimetastatic effects in vivo, these compounds failed in clinical trials, and additional research has revealed their conflicting roles in metastasis.199 A recent study by Gupta and associates200 analyzed gene expression profiles of metastases from a breast cancer cell line, and identified four genes: epiregulin, which encodes a ligand that binds to the EGFR; cyclooxygenase, which encodes an enzyme that is involved in inflammatory responses and wound healing; and two MMPs that encode proteins involved in tissue remodeling and angiogenesis that affect both primary and metastatic tumor growth. The investigators found that inhibition of each gene individually through genetic knockdown resulted in a modest effect on lung metastases. In contrast, if combinations of these genes were inhibited, there was a significantly greater effect on the metastatic process, suggesting that combination therapy is more effective in controlling metastases. However, many of these reported genes, such as Her-2/Neu and EGFR, as described previously, affect both primary as well as metastatic tumor growth, somewhat obfuscating their roles as direct modulators of tumor metastases. What the field is desperately in need of is new candidate genes and proteins that specifically affect the metastatic process and have little effect on primary tumor growth. One very promising target is LOX, a hypoxia-induced secreted protein involved in multiple stages of metastasis (Fig. 3-8).201 LOX contributes to tumor cell invasion by cross-linking collagens in the ECM, which stimulates integrin-mediated cell-matrix adhesion and

activation of FAK, and additionally provides a route (“highway”) by which tumor cells may travel. Furthermore, LOX is involved in the formation and maintenance of the metastatic niche, allowing metastatic dissemination and growth. Targeting secreted LOX through antibody or small-molecule inhibition significantly reduced formation and growth of metastases to the lung, liver, bone, and brain, in several models of cancer. These data provide hope for the future, because there is an urgent need for new metastasis-targeting therapies.

CONCLUSION Metastatic disease, not the primary tumor, kills the majority of cancer patients. For such an important determinant of long-term survival, progress has been slow in understanding the crucial genes and pathways that drive metastatic progression. The reasons for this slow progress have been numerous, including inadequate animal models that reflect the metastatic process in humans, failure to identify genes that specifically affect metastatic tumor growth, the complexity of host and metastatic tumor interactions, and premature clinical trials focusing on “attractive” gene targets. The future for metastasis research looks very promising in large part because we understand the mistakes of the past and are using multiple genomic and proteomic approaches to target what has for so long seemed to be an untractable problem. It is only when we are able to attack the problem of metastases that we will make significant inroads in our war against cancer.

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Part I: Science of Clinical Oncology (a) Early stage (invasion)

(b) Premetastatic niche

Primary tumor

Hypoxia

ECM

Premetastatic niche

Pseudopod protrusion and cell matrix adhesion

LOX-enriched leading edge

c) Late stage (metastasis)

Metastatic growth

Stromal cell

Migration and intravasation

HPC LOX involvement

Tumor cell

Invasion Lox protein Key

Figure 3-8 • The role of lysyl oxidase in metastasis. Lysyl oxidase (LOX) is a hypoxia-induced secreted protein involved in many stages of metastasis. LOX contributes to early-stage metastasis by increasing tumor cell invasion through the cross-linking of collagens in the ECM, which stimulates integrinmediated cell-matrix adhesion and activation of focal adhesion kinase. LOX is expressed at the leading edge on invasive cells and extends along hairlike fibers in the ECM. Collagen cross-linking additionally provides a route (“highway”) by which tumor cells may travel. LOX secreted by hypoxic cells in the primary tumor is involved in premetastatic niche formation at distant sites. Furthermore, LOX is involved in later stages of metastasis where cell-matrix adhesion interactions are again required for arrest and extravasation, and invasive migration. LOX is further required for the formation of a mature ECM, which is essential for metastatic tumor cell growth.

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The Cellular Microenvironment and Metastases • CHAPTER 3

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163. Bhowmick NA, Chytil A, Plieth D, et al: TGFbeta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia. Science 2004;303:848–851. 164. Kurose K, Gilley K, Matsumoto S, et al: Frequent somatic mutations in PTEN and TP53 are mutually exclusive in the stroma of breast carcinomas. Nat Genet 2002;32:355–357. 165. Hu M, Yao J, Cai L, et al: Distinct epigenetic changes in the stromal cells of breast cancers. Nat Genet 2005;37:899–905. 166. Chang HY, Sneddon JB, Alizadeh AA, et al: Gene expression signature of fibroblast serum response predicts human cancer progression: similarities between tumors and wounds. PLoS Biol 2004;2: E7. 167. Allinen M, Beroukhim R, Cai L, et al: Molecular characterization of the tumor microenvironment in breast cancer. Cancer Cell 2004;6:17–32. 168. Orimo A, Gupta PB, Sgroi DC, et al: Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 2005;121:335–348. 169. Zou W: Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer 2005;5:263–274. 170. Gorelik L, Flavell RA: Transforming growth factor-beta in T-cell biology. Nat Rev Immunol 2002;2:46–53. 171. Langowski JL, Zhang X, Wu L, et al: IL-23 promotes tumour incidence and growth. Nature 2006;442:461–465. 172. Chambers CA, Kuhns MS, Egen JG, et al: CTLA4-mediated inhibition in regulation of T cell responses: mechanisms and manipulation in tumor immunotherapy. Annu Rev Immunol 2001;19:565–594. 173. Karin M: Nuclear factor-kappaB in cancer development and progression. Nature 2006;441:431–436. 174. Dannenberg AJ, Subbaramaiah K: Targeting cyclooxygenase-2 in human neoplasia: rationale and promise. Cancer Cell 2003;4:431–436. 175. Lewis CE, Pollard JW: Distinct role of macrophages in different tumor microenvironments. Cancer Res 2006;66:605–612. 176. Murdoch C, Lewis CE: Macrophage migration and gene expression in response to tumor hypoxia. Int J Cancer 2005;117:701–708. 177. Knowles H, Leek R, Harris AL: Macrophage infiltration and angiogenesis in human malignancy. Novartis Found Symp 2004;256:189–200; discussion 200–204, 259–269. 178. Cramer T, Yamanishi Y, Clausen BE, et al: HIF1alpha is essential for myeloid cell-mediated inflammation. Cell 2003;112:645–657. 179. Aharinejad S, Paulus P, Sioud M, et al: Colonystimulating factor-1 blockade by antisense oligonucleotides and small interfering RNAs suppresses growth of human mammary tumor xenografts in mice. Cancer Res 2004;64:5378– 5384. 180. Bingle L, Brown NJ, Lewis CE: The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. J Pathol 2002;196:254–265. 181. Townson JL. Chambers AF: Dormancy of solitary metastatic cells. Cell Cycle 2006;5:1744–1750. 182. Crowley NJ, Seigler HF: Relationship between disease-free interval and survival in patients with recurrent melanoma. Arch Surg 1992;127:1303– 1308.

183. Demicheli R, Abbattista A, Miceli R, et al: Time distribution of the recurrence risk for breast cancer patients undergoing mastectomy: further support about the concept of tumor dormancy. Breast Cancer Res Treat 1996;41:177–185. 184. van Moorselaar RJ, Voest EE: Angiogenesis in prostate cancer: its role in disease progression and possible therapeutic approaches. Mol Cell Endocrinol 2002;197:239–250. 185. Luzzi KJ, MacDonald IC, Schmidt EE, et al: Multistep nature of metastatic inefficiency: dormancy of solitary cells after successful extravasation and limited survival of early micrometastases. Am J Pathol 1998;153:865–873. 186. Pantel K, Brakenhoff RH: Dissecting the metastatic cascade. Nat Rev Cancer 2004;4:448– 456. 187. Naumov GN, MacDonald IC, Weinmeister PM, et al: Persistence of solitary mammary carcinoma cells in a secondary site: a possible contributor to dormancy. Cancer Res 2002;62:2162–2168. 188. Goodison S, Kawai K, Hihara J, et al: Prolonged dormancy and site-specific growth potential of cancer cells spontaneously disseminated from nonmetastatic breast tumors as revealed by labeling with green fluorescent protein. Clin Cancer Res 2003;9:3808–3814. 189. Holmgren L, O’Reilly MS, Folkman J: Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nat Med 1995;1:149–153. 190. Naumov GN, Akslen LA, Folkman J: Role of angiogenesis in human tumor dormancy: animal models of the angiogenic switch. Cell Cycle 2006;5:1779–1787. 191. Marches R, Scheuermann R, Uhr J: Cancer dormancy: from mice to man. Cell Cycle 2006;5:1772–1778. 192. Reya T, Morrison SJ, Clarke MF, et al: Stem cells, cancer, and cancer stem cells. Nature 2001;414:105–111. 193. Al-Hajj M, Wicha MS, Benito-Hernandez A, et al: Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003;100:3983–3988. 194. Al-Hajj M, Clarke MF: Self-renewal and solid tumor stem cells. Oncogene 2004;23: 7274–7282. 195. Li F, Tiede B, Massague J, et al: Beyond tumorigenesis: cancer stem cells in metastasis. Cell Res 2007;17:3–14. 196. Shevde LA, Welch DR: Metastasis suppressor pathways—an evolving paradigm. Cancer Lett 2003;198:1–20. 197. Steeg PS:, Metastasis suppressors alter the signal transduction of cancer cells. Nat Rev Cancer 2003;3:55–63. 198. Slamon DJ, Leyland-Jones B, Shak S, et al: Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344:783–792. 199. Coussens LM, Fingleton B, Matrisian LM: Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science 2002;295:2387– 2392. 200. Gupta GP, Nguyen DX, Chiang AC, et al: Mediators of vascular remodelling co-opted for sequential steps in lung metastasis. Nature 2007;446:765–770. 201. Erler JT, Giaccia AJ: Lysyl oxidase mediates hypoxic control of metastasis. Cancer Res 2006;66:10238–10241.

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4

Control of the Cell Cycle Jacqueline Lees

S U M M ARY • Cells in most postnatal tissues are quiescent. Exceptions include cells of the hematopoietic system, skin, and gastrointestinal mucosa. • The key challenges for proliferating cells are to make an accurate copy of the 3 billion bases of DNA (S phase) and to segregate the duplicated chromosomes equally into daughter cells (mitosis). • Progression through the cell cycle is dependent on both extrinsic and intrinsic factors. • Extrinsic factors include cell-to-cell contact, basement membrane attachments, and growth factor or cytokine exposure. • The internal cell cycle machinery is controlled largely by oscillating levels of cyclin proteins and by modulation of cyclin-dependent kinase activity. • One way in which growth factors regulate cell cycle progression is by

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affecting the levels of the D-type cyclins in the G1 phase of the cell cycle. The restriction point of the cell cycle occurs in late G1 and is the point beyond which the cell is committed to progress through the rest of the cell cycle. It is governed by a known tumor suppressor, the retinoblastoma protein. Cell cycle checkpoints are surveillance mechanisms that link the rate of cell cycle transitions to the timely and accurate completion of prior dependent events. Cells can arrest at cell cycle checkpoints temporarily to allow for (1) the repair of cellular damage; (2) the dissipation of an exogenous cellular stress signal; or (3) availability of essential growth factors, hormones, or nutrients. The major function of the p53 tumor suppressor protein is to induce cell cycle arrest, senescence, or death in response to cellular stress.

INTRODUCTION The majority of the cells in the adult body are arrested in a quiescent state, called the G0 state. Most of these cells are terminally differentiated and never divide. However, specific populations retain the ability to proliferate throughout the adult life span, and this is essential for viability. For example, cells of the hematopoietic compartment and the gut have a high rate of turnover, and high rates of proliferation are therefore essential for the maintenance of these tissues. On average, about 2 trillion cell divisions occur in an adult human every 24 hours (about 25 million per second). The decision to proliferate or not is very tightly regulated. It is influenced by a variety of exogenous signals, including nutrients, mitogenic (e.g., epidermal growth factor and platelet-derived growth factor) and inhibitory (e.g., transforming growth factor-β) growth factors, and the interaction of the cell with its neighbors and with the underlying extracellular matrix. Each of these factors stimulates intracellular signaling pathways that can either promote or suppress proliferation. The cell integrates all of these signals, and if the balance is favorable, the cell will initiate the proliferation process. Anything that disrupts this balance can lead to either the reduction or expansion of a particular cell population. It is now clear that such changes are a hallmark of tumor cells. They

• Activation of the G1, S, and G2 phase checkpoints after DNA damage minimizes replication of damaged DNA templates or their segregation to daughter cells. • Activation of the mitotic spindle checkpoint prevents defects in chromosome segregation and protects against aneuploidy. • Disruption of cell cycle controls is a hallmark of all malignant cells. Disruption can manifest as alterations of growth factor signaling pathways, dysregulation of the core cell cycle machinery, and/or disruption of cell cycle checkpoint controls. • Because cell cycle control is disrupted in virtually all tumor types, the cell cycle-related gene products that are mutated in tumors provide therapeutic targets that might preferentially affect tumor cells more than normal tissues.

carry mutations that impair signaling pathways that suppress proliferation and/or activate pathways that promote proliferation. It is essential that proliferating cells copy their genomes and segregate them to the daughter cells with high fidelity. Over the past three decades, extensive effort has been placed on unraveling the basic molecular events that control this process. Studies in a variety of organisms have identified evolutionarily conserved machinery that controls eukaryotic cell cycle transitions through the action of key enzymes called cyclin-dependent kinases (CDKs). Eukaryotic cells have also evolved a series of surveillance pathways, termed cell cycle checkpoints, that monitor for potential problems during the cell cycle process. Human cells are continuously exposed to external agents (e.g., reactive chemicals and ultraviolet light) and to internal agents (e.g., by-products of normal intracellular metabolism, such as reactive oxygen intermediates) that can induce DNA damage. The cell cycle checkpoints detect DNA damage and activate cell cycle arrest and DNA repair mechanisms, thereby maintaining genomic integrity. Most, if not all, human tumor cells have mutations within key components of both the cell cycle machinery and checkpoint pathways. This has important clinical implications, as the presence of these defects can modulate cellular sensitivity to chemotherapeutic regimens that induce DNA damage. This chapter focuses on the

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Cyclin B: cdk1

Mitosis Cyclin A: cdk1

G2

it becomes growth factor independent and is fully committed to undergoing cell division. Within an hour or two, the cell enters the synthesis phase, or S phase, in which each of the chromosomes is replicated once and only once. The cell then enters a second gap phase, called G2, which lasts 3 to 5 hours, and then initiates mitosis, or M phase, in which the chromosomes are segregated. On completion of mitosis, the daughter cells can enter quiescence or initiate a second round of cell division, depending on the milieu.

G0

Cyclin D: cdk4/6 G1 Cyclin E: cdk2

S-phase

Cyclin and Cyclin-Dependent Kinase Complexes

Restriction point

The CDKs constitute a large subfamily of highly conserved ser/thr kinases that are defined by their dependence on a regulatory subunit, called a cyclin.1,2 Only a subset of these CDKs are specifically involved in cell cycle regulation. Yeast use a single CDK for cell cycle control that is called cdc2 in Schizosaccharomyces pombe and cdc28 in Saccharomyces cerevisiae. In contrast, mammals employ multiple CDKs.3 The first identified human CDK, called CDK1 (originally cdc2), was cloned by virtue of its ability to complement a mutant cdc2 yeast strain.4 Subsequent studies identified additional human CDKs and determined that they regulate distinct cell cycle stages; CDK4 and CDK6 regulate passage through G1, CDK2 regulates the G1-to-S transition and S phase, and CDK1 controls G2 and mitosis. The activity of these kinases is controlled by multiple regulatory mechanisms.5,6 Most important, the CDKs act in association with a cyclin subunit that binds to the conserved PSTAIRE helix within the kinase.3,5 Cyclin binding causes a reorientation of residues within the active sites that is essential for kinase activity.3 The associated cyclin also determines the substrate specificity of the resulting cyclin-CDK complex. The cyclins are quite divergent, especially in their Nterminal sequences, but they all share a highly conserved 100-aminoacid sequence, called the cyclin box, that mediates CDK binding and activation. As their name implies, cyclins were originally identified as proteins whose expression was restricted to a particular stage of the cell cycle.7 This is due to cell cycle-dependent regulation of both cyclin gene transcription and protein degradation. Notably, there is frequently a delay between the formation of a particular cyclin/CDK complex and the appearance of kinase activity (Fig. 4-2). This reflects considerable post-translational regulation of the cyclin/CDK complex.8–11 First, kinase activation is absolutely dependent on phosphorylation of a threonine residue that is adjacent to the active site (thr 160 in CDK2). This is catalyzed by a kinase, called CDKactivating kinase (CAK).11–13 In mammalian cells, phosphorylation occurs after cyclin binding. Although there appear to be at least two

Cyclin A: cdk2

Figure 4-1 • The cell cycle. One round of cell division requires highfidelity duplication of DNA during the S phase of the cell cycle and proper segregation of duplicated chromosomes during mitosis, or M phase. Before and after the S phase and M phase, the cell transits through “gap” phases, termed G1 and G2. The appropriate transition through these stages is controlled by the action of specific cyclin/CDK complexes.

mechanics of the cell cycle and checkpoint-signaling pathways and discusses how this knowledge can lead to the efficient use of current anticancer therapies and to the development of novel agents.

CELL CYCLE MACHINERY Overview of Cell Cycle Phases Cell proliferation proceeds through a well-defined series of stages (Fig. 4-1). First, the cell moves from the quiescent G0 state into the first gap phase, or G1, in which the cell is essentially readying itself for the cell division process. This involves a dramatic upregulation of both transcriptional and translational programs not only to yield the proteins that are required to regulate cell division but also to essentially double the complement of macromolecules so that one cell can give rise to two cells without a loss of cell size. This protein synthesis phase is frequently referred to as cell growth. Not surprisingly, this takes a significant amount of time (anywhere from 8 to 30 hours) and energy. Studies with cultured cells show that mitogenic growth factors are essential for continued passage through G1. Specifically, if growth factors are withdrawn at any point during this phase, the cell will not divide. However, as it nears the end of G1, the cell passes through a key transition point, called the restriction point, at which

Restriction point E-type cyclins

A-type cyclins

Cyclin B-associated kinase activity

D-type cyclins B-type cyclins G1

S

G2

M

Figure 4-2 • The expression of the cyclin subunits in tightly linked to cell cycle phases. Extracellular stimuli, such as mitogenic growth factors and hormones, induce the expression of the D-type cyclins early in G1. Cyclin E expression occurs late in G1, synonymous with the restriction point, and its levels peak at the G1-to-S transition and then decline during S phase. Cyclin A is induced a little later than cyclin E and is degraded at the metaphase of mitosis. The B-type cyclins are expressed primarily in G2, and the levels remain high until the anaphase of mitosis. There is typically a delay between the expression of the cyclin protein and the appearance of cyclinassociated kinase activity that results from post-translational regulation. This is most pronounced in the case of cyclin B, for which activation of the cyclin B/CDK1-associated kinase activity does not occur until the G2-to-M transition.

Control of the Cell Cycle • CHAPTER 4

mammalian CAKs, the major CAK is a trimolecular complex composed of CDK7, cyclin H, and Mat1.12–14 This kinase is constitutively active, and to date, there is no evidence that it is cell cycle regulated. Indeed, the CDK7-cyclinH-Mat1 complex is also required for the control of basal transcription via regulation of RNA polymerase II function.12,15 Second, when it is first formed, the cyclin/CDK complex is frequently subject to inhibitory phosphorylation of Thr-14 and Tyr-15 residues within the CDK’s active site by the Wee1 (Tyr-15) and Myt1 (Thr-14 and Tyr-15) kinases.9 Activation of the cyclin/ CDK complex is now dependent on the action of a dual-specificity phosphatase called Cdc25.10 Mammalian cells have three different Cdc25 proteins, called Cdc25A, Cdc25B, and Cdc25C, which show some specificity for different cyclin/CDK complexes.10 The mammalian cyclins are divided into four distinct classes—Dtype cyclins, E-type cyclins, A-type cyclins, and B-type cyclins—on the basis of both their sequence homology and the stage of the cell cycle at which they act (see Figs. 4-1 and 4-2).16 Each of these classes has two or three paralogs (cyclin D1, D2, and D3; cyclin E1 and E2; cyclin A1 and A2; and cyclin B1 and B2). The relative roles of these paralogs are still unclear. In some cases, differences exist (e.g., in subcellular localization) that suggest that particular paralogs will have distinct activities or regulation. However, other studies (particularly the analysis of mouse models) show that there is considerable functional redundancy, or at least an ability to substitute for one another, between paralogs of a particular cyclin type.17,18 For simplicity, we will focus largely on the core properties of the cyclin types. The D-type cyclins represent an unusual class of cyclins.16,19 First, they do not participate the cell division process itself. Instead, they play a critical role in determining whether a cell will divide under the direction of external cues. Second, their expression is not really cell cycle regulated; D-type cyclins are present at very low levels in quiescent cells, in large part because they are phosphorylated by an abundant G0 kinase called GSK3β and then exported to the cytoplasm for degradation, but their expression is induced during G1, and it persists through all subsequent cell cycle stages.20 This G1 induction is a direct consequence of mitogenic signaling. This inhibits expression of GSK3β and activates transcription of the D-type cyclins. Notably, individual mitogenic signaling pathways induce different D-type cyclins. For example, signaling by EGF/ras (via the AP-1 transcription factor) and Wnt/β-catenin (via the Tcf/Lef transcrip-

Rbx1

tion factor) specifically induces cyclin D1, while c-myc specifically induces cyclin D2.21–26 This specificity helps to ensure that the presence of multiple proproliferative signals gives rise to more D-type cyclins than a single mitogen does. Importantly, the analysis of mouse models shows that cyclins D1, D2, and D3 are functionally redundant; the key factor appears to be the total level of D-type cyclin that is present in the cell.27 This promotes cell cycle entry through two distinct mechanisms. First, the D-type cells titrate inhibitory molecules, called CDK inhibitors, away from other CDK-kinase complexes and thereby promote their activation.5,28,29 Second, cyclins D1, D2, and D3 associate with CDK4 and CDK6, and the resulting complexes phosphorylate the retinoblastoma protein (pRB), a key gatekeeper for cell cycle reentry.30–34 We will discuss both the CDK inhibitors and pRB in more detail later because of their central importance in controlling cell cycle reentry and their frequent disruption in cancer. E-type cyclins are expressed during late in G1 under the control of the E2F transcription factors.35,36 Cyclin E binds specifically to CDK2, and the resulting complex is required for cells to move through the G1-to-S transition.37–39 To date, several cyclin E/CDK2 substrates have been identified. Some cyclin E/CDK2 substrates play a positive role in cell cycle progression. For example, cyclin E/CDK2 phosphorylates NPAT, a transcription factor that mediates transcriptional activation of histone gene clusters.40–42 The resulting increase in histone pools is essential for the appropriate packaging of newly replicated DNA in S phase cells. Cyclin E/CDK2 also induces the duplication of centrosomes that is required for formation of the mitotic spindle.43–46 Other cyclin E/CDK2 substrates are key cell cycle inhibitors. First, cyclin E/CDK2 phosphorylates pRB at completely different sites from those that have already been modified by the cyclin D/CDK4/6 kinases, and this is sufficient to inactivate pRB’s growth suppressive function.31,34,47 Second, cyclin E/CDK2 phosphorylates the CDK inhibitor p27 on Thr-187.48,49 This creates a high-affinity binding site for a ubiquitin ligase, called SCF, which plays a very important role in G1/S control.50–53 SCF has three core components: a RING finger protein, called Rbx1, which recruits the E2-ubiquitin conjugate; a cullin (Cul1); and Skp1 (Fig. 4-3).52–54 Skp1 acts to recruit a family of proteins, called F-box proteins, that determine the target specificity of the SCF complex (see Fig. 4-3). In the case of p27, the F-box

Apc11

E2

Ubiquitin

Ubiquitin Substrate P

Cul1

E2

Substrate

Apc2 P

Activator (cdc20 or cdh1)

F-box protein

skp2 SCF ubiquitin ligase

APC ubiquitin ligase

Figure 4-3 • Ubiquitin ligases. The SCF and APC ubiquitin ligases play a key role in enabling forward passage through key cell cycle transitions. These are both large complexes that include three core components: a scaffolding protein called a cullin, a protein that recruits the E2 and its associated ubiquitin molecule, and a specificity factor (called the F-box protein in SCF and the activator in APC) that recruits the substrate. SCF and APC catalyze polyubiquitination of their substrates, and this acts as a signal for substrate degradation by the 26S proteasome. SCF has numerous substrates whose degradation promotes passage through the early stages of the cell cycle, including p27Kip1 (the restriction point) and cyclin E, E2F-1, and Cdt1 (S-phase). APC is essential for completion of mitosis (by promoting degradation of securin and the mitotic cyclins) and to allow origin licensing (by promoting degradation of geminin and thereby allowing accumulation of cdt1 during G1).

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protein is Skp2, and the SCF complex is therefore designated as SCFSkp2.55,56 Once SCF binds its substrate, it transfers a ubiquitin molecule to lysine residues within the target protein and subsequently to lysine residues in the ubiquitin molecule to create a polyubiquitin chain.54 This polyubiquitin chain targets the substrate to the proteasome for degradation.54 Notably, SCF is also responsible for the decline in cyclin E/CDK2 levels that is activated during S phase (see Fig. 4-2). First, cyclin E/CDK2 actually phosphorylates itself on multiple sites, creating a recognition site for SCFFbw7/Cdc4 and thereby ensuring its own destruction.57,58 Second, cyclin A/CDK2 (the S phase kinase) phosphorylates E2F-1, the transcription factor that activates cyclin E transcription, and this is targeted for degradation by SCFSkp2.59–61 Together, these mechanisms restrict the action of cyclin E/CDK2 to a small window in the cell cycle. The A-type cyclins are first transcribed during late G1 under the control of the E2F transcription factors in a similar manner to that of cyclin E. However, in contrast to cyclin E, cyclin A associates with both CDK2 and CDK1 and it acts at two distinct cell cycle stages.62,63 First, cyclin A/CDK2 is absolutely required for S phase progression. This was established by showing that injection of either cyclin A antisense constructs or antibodies was sufficient to block S phase progression.62,64 Notably, cyclin A/CDK2 enters the nucleus at the start of S phase, and it is specifically localized at nuclear replication foci and therefore is thought to be actively involved in the firing of replication origins.65 As was described previously, cyclin A/CDK2 is also required to phosphorylate E2F-1 and mediate its degradation, and this is required to prevent E2F1 from triggering apoptosis.59–61 Second, cyclin A/CDK1 complexes act during G2 and at the beginning of mitosis.62 Here, they are thought to play a key role in initiating the condensation of chromatin and might also participate in the activation of the cyclin B/CDK1 complexes. Cyclin A/CDK2 is destroyed in the prometaphase of mitosis through the action of another ubiquitin ligase, called anaphase-promoting complex (APC).66 APC is a much larger complex that SCF, but it also contains a RING finger protein, called Apc11, to recruit the E2-ubiquitin conjugate, a core cullin subunit (Apc2), and it binds a variety of activators that are required for APC activity and, in a manner comparable to that of the F-box proteins of SCF, establish substrate specificity (see Fig. 4-3).54,66 In the case of cyclin A/CDK2, the activator is CDC20, and the APC complex is therefore designated as APCCdc20. There are two B-type cyclins, B1 and B2, which show significant differences in their subcellular localization. The analysis of mutant mouse models shows that cyclin B2 loss has no detectable effect on development, while cyclin B1 is absolutely required for embryogenesis.67 This indicates that cyclin B1 is the major B-type cyclin in vivo; therefore, we will restrict our discussion to this isoform. Cyclin B1 protein first appears at the beginning of G2. It accumulates steadily through G2 and associates specifically with CDK1.63 However, the resulting cyclin B1/CDK1 complex is mostly sequestered in the cytoplasm, and it is retained in an inactive form throughout G2 via the inhibitory phosphorylation of Thr-14 and Tyr-15 in CDK1’s active site by the Myt1 and, to a lesser extent, Wee1 kinases.68–70 Activation of cyclin B1/CDK1 occurs in a highly synchronous manner during the first stage (called prophase) of mitosis (see Fig. 4-2).71 This activation is mediated by two changes. First, the activities of myt1 and wee1 are dramatically downregulated at the transition between G2 and M. Second, there is a dramatic increase in the activity of the Cdc25A and C phosphatases that relieves the inhibitory phosphorylation of Thr-14 and Tyr-15.10 These activity changes are triggered by the phosphorylation of Myt1, Wee1, Cdc25A, and Cdc25C. Three different kinases are thought to contribute to this phosphorylation: polo-like kinase, cyclin A/CDK1, and cyclin B1/CDK1 itself. The involvement of cyclin B1/CDK1 creates a powerful feedforward loop; once a small amount of cyclin B1/CDK1 is activated, it simultaneously inactivates its own inhibitors and activates its activators, enabling a rapid transformation of the entire cyclin B1/CDK1 pool from the inactive state to the active state. Once active, cyclin B1/

CDK1 phosphorylates components of the centrosomes and initiates a process called centrosome separation, in which the centrosomes move to opposing poles of the nascent spindle, an event that is essential for formation of the mitotic spindle.72 Cyclin B1/CDK1 then translocates across the nuclear membrane (which is still intact at this point in the mitosis) to orchestrate mitotic events.73 Notably, cyclin B1/ CDK1 is degraded at the end of metaphase (see Fig. 4-2).74 This is triggered by the ubiquitination of cyclin B1 by the ubiquitin ligase APC and its subsequent recognition and degradation by the proteosome.66,75,76 This downregulation of mitotic CDKs is required for cytokinesis (the separation of the daughter cells) and reentry into G0/G1.74

Cyclin-Dependent Kinase Inhibitors The CDK inhibitors (CDKIs) play a key role in establishing the activity of the cyclin/CDK complexes in response to either external signals or internal stresses.5 The CDKIs can be divided into two distinct families based on their biological properties. The first CDKI family is named INK4, based on their roles as inhibitors of CDK4. The INK4 family has four members called p16INK4a, p15INK4b, p18INK4c, and p19INK4d. These INK4 proteins specifically target CDK4 and CDK6 and not other CDKs. They preferentially target the monomeric CDK and prevent cyclin binding. Consistent with their inhibitory role, the alterations in the INK4 genes are observed in human tumors.77 Ink4a appears to be most the most frequently affected; it was identified as a tumor suppressor that is associated with familial melanoma, and it is inactivated by point mutation, deletion, and/or promoter methylation in approximately 30% of all human tumors.78,79 In contrast, point mutations in p15INK4b, p18INK4c, and p19INK4d are rare, but promoter methylation of Ink4c has been detected in Hodgkin lymphomas and medulloblastomas, and reduced p18INK4c protein expression has been seen in a variety of tumor types.80–84 The second CDKI family is named CIP/KIP and includes three members: p21Cip1 (also called p21Waf1), p27Kip1, and p57Kip2.5 These CIP/KIP proteins have two major activities. First, they associate with, and inhibit the activity of, the G1/S and S phase kinases cyclin E/ CDK2 and cyclin A/CDK2. Second, p21Cip1 and p27Kip1 bind to the D-type cyclins outside of the CDK binding site and actually promote assembly of cyclin D/CDK4/6 complexes.85 These two activities are clearly paradoxical. However, they are critical in establishing how the cell decides whether or not to divide in response to external signals. In general, the expression and/or activity of CDKIs is promoted by growth suppressive signals and inhibited by pro-proliferative signals. For example, the inhibitory growth factor transforming growth factor-β induces transcription of p15INK4b, while several mitogenic signaling pathways cause Akt to phosphorylate p21Cip1 and p27Kip1 and induce their cytoplasmic sequestration.86–90 Importantly, signaling pathways have the opposite effect on the D-type cyclins: Growth suppressive signals inhibit their expression and activity while mitogens are activating. The opposing regulation of CDKIs and D-type cyclins controls cell cycle entry by creating a tipping point (Fig. 4-4). G0/G1 cells have low levels of D-type cyclins and high levels of CDKIs; thus, cell cycle entry is blocked. However, an increase in mitogenic signals boosts the levels of cyclin D, and this eventually exceeds the level of INK4 proteins, which are simultaneously declining. At this point, the D-type cyclins begin to bind to the CIP/KIP proteins. This helps the D-type cyclins to assemble into active cyclin D/CDK4/6 complexes and draws the CIP/KIP proteins away from the cyclin E/CDK2 complexes as they begin to accumulate. The cyclin D/CDK4/6 and cyclin E/CDK2 complexes cooperate in the phosphorylation and inactivation of the pRB protein. This appears to be the tipping point in commitment to cell cycle entry.

pRB Tumor Suppressor The retinoblastoma protein (pRB) was originally identified by virtue of its association with hereditary retinoblastoma protein.91 It behaves

Control of the Cell Cycle • CHAPTER 4

Inhibitory growth factors

Mitogens

INK4 family

Cip/Kip family

p15ink4b

p18ink4c

p21Cip1

p16ink4a

p19ink4d

p27Kip1 p57Kip2

cdk4/6

cycD

cdk2

cdk4/6 cycD

cycE

Figure 4-4 • The CDK inhibitors (CDKIs) play a key role in regulating the G1-to-S transition. The levels of nuclear INK4 and Cip/Kip CDKIs are typically elevated by growth inhibitory stimuli and reduced by mitogenic stimuli. The INK4 family members bind specifically to CDK4 and CDK6 and inhibit their association with cyclin D. The Cip/Kip CDKIs interact with both the cyclin and CDK components and have highest affinity with the intact cyclin–CDK complex. Cip/Kip binds in a different way to cyclin D versus cyclin E complexes, and this has opposing effects on their activity. Cip/Kip binding enables formation of active cyclin D–CDK4/6 complexes. In contrast, Cip/Kip associates with cyclin E–CDK complexes and blocks their activity. Cyclin D complexes have a higher affinity for Cip/Kip than do cyclin E complexes. The mitogen-induced accumulation of D-type cyclins during G1 titrates Cip/Kip from cyclin E–CDK2 and facilitates activation of both cyclin D–CDK4/6 and cyclin E–CDK2 kinases. Cyclin E–CDK2 can also phosphorylate p27 to promote its ubiquitin-mediated degradation.

as a classic tumor suppressor: Affected individuals inherit a germline mutation within one Rb-1 allele, and loss of heterozygosity is seen in all of the tumors. Subsequent studies showed that the transforming ability of small DNA tumor viruses, including human papilloma virus, adenovirus, and simian virus, was dependent on the ability of virally encoded oncoproteins (E7, E1A, and SV40, respectively) to bind and inhibit pRB.92 Moreover, the RB-1 gene was found to be inactivated in approximately one third of all sporadic human tumors.91 Thus, pRB is a major human tumor suppressor. To date, numerous pRB-associated proteins have been identified.93 However, studies in mouse models indicate that pRB’s tumor suppressive activity is largely dependent on its ability to prevent cell cycle entry through inhibition of the E2F transcription factors.94–96 The E2F proteins regulate the cell cycle-dependent transcription of numerous targets, including core components of the cell cycle control (e.g., cyclin E and cyclin A) and DNA replication (e.g., cdc6, CDT1, and the MCM proteins) machineries.47,97–99 pRB regulates E2F through two distinct mechanisms. First, its association with E2F is sufficient to block its transcriptional activity.100 Second, the pRB-E2F complex can recruit histone deacetylases (HDACs) to the promoters of E2F-responsive genes and thereby actively repress their transcription.101–103 Cell cycle entry requires the sequential phosphorylation of pRB by cyclin D/CDK4/6 and cyclin E/CDK2 complexes and the consequent dissociation of pRB from E2F.34,47,104 Importantly, tumors that retain wild-type pRB almost always carry activating mutations in cyclin D1 or CDK4 or inactivating mutations in the CDK4inhibitor, p16.104 This suggests that the functional inactivation of pRB, and the resulting deregulation of E2F, is an essential step in tumorigenesis.

Studies to date have identified eight E2f genes that encode nine different E2F proteins.99 pRB and its relatives p107 and p130 (collectively called the pocket proteins) regulate a subset of the E2Fs: E2F1, E2F2, E2F3a, E2F3b, E2F4, and E2F5. These E2F proteins associate with a dimerization partner, called DP, and the resulting complexes function primarily as either activators (E2F1, E2F2, and E2F3a) or repressors (E2F4 and E2F5) of transcription under the direction of the pocket proteins.47 Most classic E2F target genes are regulated by the coordinated action of these repressor and activator E2Fs (Fig. 4-5). In G0/G1 cells, the DP-E2F4 and DP-E2F5 complexes associate with the promoters of E2F-responsive genes and recruit p107 and p130, along with their associated HDACs, to actively repress their transcription.105,106 At the same time, the activating E2Fs are bound by pRB, inhibiting their potential to activate transcription. Whether complexes containing pRB and activating E2Fs contribute to the repression of E2F target genes is still unclear.107 In response to mitogenic signaling, CDK activity increases, and the phosphorylation of the pocket proteins causes them to release their associated DP-E2Fs. E2F4 and E2F5 dissociate from the DNA and translocate to the cytoplasm because they have potent nuclear export signals.108,109 The free E2F complexes—DP-E2F1, DP-E2F2, and DP-E2F3—now occupy the promoters and activate their transcription. Thus, in every cell cycle, there is a coordinated switch from the repressive to the activating E2Fs that enables the simultaneous activation of genes promoting cell cycle progression. Since cyclin E is itself an E2F-responsive gene, this regulation creates a strong feedforward loop: The appearance of a small amount of the cyclin E/CDK2 kinase promotes pRB inactivation and further cyclin E expression. This signal is further amplified by cyclin E/CDK2’s ability to phosphory-

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P

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P

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cdk4/6 cycD

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cdk2 cycE

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Transcriptional activation

Cell cycle and DNA synthesis regulators

Figure 4-5 • The retinoblastoma protein (pRB) and the restriction point. The pocket proteins—pRB, p107, and p130—regulate a subset of the E2F family of transcription factors. The pocket proteins bind to these E2Fs during G1 and suppress their activity through two mechanisms. First, pRB binds to E2F1, E2F2, and E2F3a (collectively called the activating E2Fs) and blocks their transcriptional activity. Second, p107 and p130 associate with E2F4 and E2F5 (together called the repressive E2Fs), and the resulting complexes recruit histone deacetylases (HDACs) to the promoters of E2F-responsive genes and actively repress their transcription. E2F-responsive genes encode core components of the cycle control and DNA replication machinery, and cell cycle entry is impossible without these products. Mitogenic signaling leads to the sequential activation of cyclin D–CDK4/6 and cyclin E/CDK2, and these phosphorylate the pocket proteins and release their associated E2Fs. This causes the repressive E2Fs to dissociate from E2F-responsive promoters and allows the activating E2Fs to bind and activate their transcription.

late p27 and signal its destruction. Importantly, pRB-inactivation is largely synonymous with the restriction point, defined as the point at which cells become committed to divide even in the absence of mitogenic stimuli.110,111 Consistent with this model, the exogenous expression of any individual activating E2F in cell culture is sufficient to stimulate DNA synthesis in the absence of growth signals.112–116

DNA Replication The DNA replication machinery is optimized to ensure that the genome is copied once—and only once—in each cell cycle.117–120 This is achieved through a two-step process that first establishes a prereplication complex (pre-RC) at each origin of replication, a process that is frequently referred to as origin licensing, and subsequently transforms pre-RCs into the preinitiation (pre-IC) complex that activates DNA replication (Fig. 4-6). These two steps occur at distinct stages of the cell cycle to ensure that origins are only licensed once per cell cycle, and rereplication cannot occur. Pre-RC formation takes place during G1. The first step in this process is the recruitment of the multiprotein complex called the origin recognition complex (ORC) to the origin DNA.121,122 Although ORC binds a subset of genomic sites, there is no evidence that ORC exhibits sequence-specific DNA binding, and it is still unclear how ORC is recruited to specific sequences. Once bound, ORC recruits additional proteins including Cdc6, Cdt1, and finally the MCM complex, a helicase that is required to unwind the DNA strands to form the pre-RC. Once cells enter S phase, the transformation of the pre-RC to the pre-IC requires the activity of two kinases: a CDK (likely, but not yet proven, to be cyclin A/CDK2) and the Ddf4-dependent kinase, which is composed of the Dbf4 regulatory subunit and the Cdc7 kinase.123,124 In mammals, the precise target(s) of these kinases is still unclear. However, the action of these kinases allows numerous additional proteins to associate with the pre-RC and form the pre-IC.125 Assembly of the pre-IC is thought to trigger DNA unwinding by the MCM complex, recruitment of the DNA polymerases, and initiation of the replication process, frequently called origin firing.

The transformation of the pre-RC to the pre-IC can occur at different time points in S phase, depending on whether the origin fires early or late.118–120 The system can tolerate this heterogeneity because the pre-RC is disassembled after firing and cannot reform until the subsequent cell cycle. This occurs through several mechanisms. The MCM complex travels with the replication fork in its role as the DNA helicase. There is also some evidence that phosphorylation of ORC1 reduces its ability to bind to origins. Finally, and most important, Cdt1 is prevented from participating in pre-RC formation outside of G1 phase in two distinct ways. First, Cdt1 is marked for destruction by ubiquitination.126 This is mediated by SCFSkp2 and particularly by an E4 ubiquitin ligase that includes Rbx1 (to recruit the E2-ubiquitin), a cullin (Cul4), Ddb1, and Dtl/Cdt2 (the substrate specificity factor).127–129 Importantly, this Cul4-Ddb1Dtl/Cdt2 complex functions independently of Cdt1 phosphorylation. Instead, Cdt1 is targeted only when proliferative cell nuclear antigen is present on the DNA, which occurs primarily as a consequence of the initiation of DNA replication.130 Second, cells possess a protein called geminin that sequesters Cdt1 and prevents it from participating in pre-RC formation. Geminin is present specifically in S, G2, and early M phase cells. However, the two major mitotic APC complexes, APCCdc20 and APCCdh1, ubiquitinate geminin and thereby trigger its destruction. This creates a window between anaphase of mitosis and late G1 (when APCCdh1 is inactivated) in which geminin is absent and therefore Cdt1 is free to participate in pre-RC formation. The importance of both the Cul4-Ddb1Dtl/Cdt2 complex and geminin is underscored by the finding that the loss of either one of these regulators is sufficient to trigger inappropriate Cdt1 accumulation and rereplication of the genome.128,129,131,132

Mitosis The mitotic machinery is optimized to ensure that the replicated chromosomes are faithfully segregated to the daughter cells. This is achieved through the use of a specialized microtubule-based structure, the mitotic spindle, on which the original chromosomes and

Control of the Cell Cycle • CHAPTER 4

MCM

MCM CDT1

CDC6 ORC

G1

CDC6 MCM

MCM CDT1

S

DDK + CDK dependent ORC

C

T 1 D

Figure 4-6 • Origin licensing and firing. The origin replication complex (ORC) associates with replication origins. During G1, Cdc6 and Cdt1 are loaded on chromatin, and they in turn load the MCM complex on chromatin, at which point licensing is considered complete, and the multiprotein complex is called the pre-RC. Once cells pass the G1-to-S transition, this complex is activated to form the pre-IC, and DNA replication is initiated. Activation requires both CDK and Ddf4-dependent kinase activity. It results in recruitment of numerous proteins and activation of the MCM complex, which unwinds the DNA. Subsequently, core components of the replication machinery, including DNA polymerase α and DNA polymerase ε, are recruited to initiation sites. The transition from pre-RC to pre-IC results in inhibition of cdt1 by ubiquitin-mediated degradation and geminin binding. Origin licensing cannot occur again until activation of APC at the end of mitosis allows accumulation of cdt1.

their newly replicated copies, called sister chromatids, align and are then partitioned to opposite poles of the cell. The appropriate sideby-side alignment of the sister chromatids, termed biorientation, is facilitated by the physical tethering of the sister chromatids to one another. This process, called cohesion, actually occurs in S phase in a manner that is coordinated with the replication process.133,134 Cohesin is mediated by four proteins that together make up the cohesin complex. Two of these proteins, Smc1 and Smc3, have a long coiledcoil structure with a dimerization domain at one end that allows them to heterodimerize to form a V-like structure. Importantly, the remaining ends of Smc1 and Smc3 can associate with each another to form a functional ATP domain. This acts in an ATP-dependent manner to recruit two additional proteins, Scc1 and Scc3, that form a closed ring structure.135 It is still unclear precisely how this ring links the sister chromatids; some investigators hypothesize that the cohesin complex encircles the chromosomes; others argue that the ring (which is known to be approximately 50 nM) is too small to surround complex chromatin structures. Regardless of the mechanism, the cohesin complex links the sister chromatids at the centromeres and at periodic intervals along the arms. The sister chromatids are essentially strung out and become entangled. Consequently, the chromosome structure must be modified before segregation. This occurs toward the end of G2 and the beginning of mitosis. Largely on the basis of morphologic features, mitosis is divided into five different stages—prophase, prometaphase, metaphase, anaphase, and telophase (Fig. 4-7)—prior to separation of the daughter cells or cytokinesis. Prophase is essentially a preparative stage. One of the major events is the modification of the DNA. In a process called resolution, the

sister chromatids are untangled via the action of topoisomerase II.133,134 Resolution requires removal of the chromosome arm cohesin through phosphorylation of Scc3 by polo-like kinase and histone H3 by the aurora B kinase. Importantly, the cohesin complex at the centromere is somehow protected from this modification by a protein called shugosin (Sgo).136 This is the glue that keeps the sister chromatids together until the appropriate point in mitosis. In addition to resolution, the sisters undergo condensation, essentially packaging into a more compact chromatin structure. This process involves two multimeric complexes, condensin I and II, which also contribute to sister chromatid resolution, and it requires phosphorylation by mitotic CDKs.133,134 During prophase, the nuclear envelope is still intact; consequently, differences in subcellular localization of the condensin and CDK complexes allow only condensin II and cyclin A/CDK1 (nuclear), and not condensin I and cyclin B/CDK1 (cytoplasmic), to initiate condensation. The second major event in prophase is the activation of the cytoplasmic cyclin B1/CDK1. This initiates formation of the mitotic spindle by triggering the centrosomes, which are located in the cytoplasm and are already nucleating microtubules, to segregate to the opposite poles of the nascent spindle. The active cyclin B1/CDK1 complex then translocates into the nucleus. Once there, it phosphorylates components of the nuclear envelope and triggers its breakdown.137,138 This defines the transition from prophase to prometaphase. During prometaphase, the condensation process is accelerated because condensin I and cyclin B/CDK1 now have access to the DNA. The sister chromatids become attached to spindle microtubules through a structure called the kinetochore, which is assembled onto centromeric DNA.139 Microtubules nucleated from the centrosomes attach to the kinetochore through a process called search and capture, in which individual microtubules grow and shrink until they contact and bind the kinteochore.140 Typically, one sister chromatid of the pair attaches first, and this attachment is further stabilized through the recruitment of additional microtubules from the same pole of the mitotic spindle to create a kinetochore fiber: highly bundled microtubules bound to the kinetochore. The sister chromatids oscillate in the cell until the second sister chromatid is captured by microtubules emanating from the other pole. These oscillations continue until all of the chromosomes are properly aligned on the metaphase plate during metaphase. Metaphase is defined as the point at which all of the chromosome pairs are fully condensed, attached to the mitotic spindle, and aligned at the center—termed the metaphase plate. The pulling of the kinetochore fibers toward the poles creates tension through the cohesin complex at the kinetochores that indicates that the sister chromatids have achieved appropriate biorientation. The cell constantly monitors the attachments of microtubules to the chromosomes, and the tension that is generated by microtubules on the kinetochores ensures that the sister chromatids are properly aligned at the metaphase plate.141 This is one of several cell cycle checkpoints, called the mitotic spindle checkpoint, that we will describe in more detail in the following sections. Anaphase is characterized by the segregation of the chromosomes. This event is controlled by the mitotic ligase APCCdc20.75,76,141–144 APCCdc20 ubiquitinates, and thereby triggers the degradation of, a protein called securin that exists to bind and inhibit a protease called separase. Once released, separase cleaves the Scc1 component of the cohesin complex. This opens the cohesin ring, unlinking the sister chromatids and allowing them to be pulled to opposite poles. The spindle poles then move farther apart to ensure that the chromosomes are fully segregated. APCCdc20 also activates the ubiquitination and degradation of geminin, allowing accumulation of Cdt1 for origin relicensing in the subsequent G1 phase, and the mitotic cyclins, allowing loss of CDK kinase activity. This latter event is critical to the completion of mitosis and cytokinesis. During telophase, the mitotic spindle disassembles, leaving a single centrosome and a single set of chromosomes with each nascent

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Interphase

Cyclin A:cdk1 activity Chromatin begins to condense Centrosomes move to poles and mitotic spindle starts to form

NE breakdown

Chromasomes attach to microtubules of spindle

Prophase

Prometaphase

Chromosomes align at metaphase plate

Sister chromatids separate, centromeres divide Chromotin expands Cytoplasm divides APC Metaphase

Anaphase

Telophase

Figure 4-7 • Key stages of mitosis. As the parent cell enters prophase, the chromosomes begin to condense, and proteins associate to form the kinetochores. The centrosomes segregate to the poles to begin formation of the mitotic spindle. Nuclear envelope (NE) breakdown denotes the start of prometaphase. In this phase, the sister chromatids continue to condense, and they attach to spindle microtubules via their kinetochores. During metaphase, the sister chromatids align at the metaphase plate and eventually achieve appropriate biorientation. At the onset of anaphase, the sister chromatids separate and move toward the poles of the spindle. During telophase, the parent cell is divided into two daughter cells by cytokinesis.

Control of the Cell Cycle • CHAPTER 4

daughter cell. As the DNA begins to decondense the nuclear envelope reforms around the segregated chromosomes to create two nuclei. These events are dependent on the loss of CDK kinase activity and the dephosphorylation of CDK substrates. Finally, the cell undergoes cytokinesis, or cytoplasmic division. This involves formation of an actin- and myosin-containing structure, called the contractile ring, on the inner face of the cell membrane. The position of the contractile ring is carefully controlled. For most mammalian cells (ones that are not undergoing asynchronous division), the ring begins to form in anaphase and its position is established by the position of the metaphase plate. As the membrane grows, the contractile ring contracts steadily to form a constriction, termed the cleavage furrow, which ultimately separates the two nuclei and forms the two daughter cells. These cells can adopt either a G0 or a G1 state, depending on the extrinsic signals that exist.

CELL CYCLE CHECKPOINTS At key transitions during eukaryotic cell cycle progression, signaling pathways monitor the successful completion of events in one phase of the cell cycle before proceeding to the next phase. These regulatory pathways are commonly referred to as cell cycle checkpoints.145–147 In a broader context, cell cycle checkpoints are signal transduction pathways that link the rate of cell cycle phase transitions to the timely and accurate completion of prior dependent events. Checkpoint surveillance functions are not confined to monitoring normal cell cycle progression; they are also activated by both external and internal stress signals. To minimize the possibility of errors, checkpoints exist at four different points in the cell cycle: G1/S, intra-S, G2/M, and at the metaphase to anaphase transition (called the spindle checkpoint).

The best-studied of the cell cycle checkpoints are those that monitor the status and structure of chromosomal DNA during cell cycle progression.147–149 In particular, cells scan the chromatin for partially replicated DNA as well as DNA strand breaks and other DNA lesions that can result from both extrinsic (e.g., chemicals, ionizing or ultraviolet radiation) and intrinsic (e.g., by-products of intracellular metabolism) DNA-damaging agents. The checkpoint pathways include sensor proteins that detect these DNA lesions and simultaneously trigger two processes: They recruit additional complexes to repair the DNA and activate signaling pathways that induce a temporary cell cycle arrest. In certain situations, which are determined by the cell type and the degree of damage, the checkpoint pathways can induce permanent cell cycle arrest (a process called senescence) or apoptosis. The central components of the DNA damage response (DDR) are two members of the phosphoinositide 3-kinase-related kinase family: ATM and ATR.147,148 ATM was original identified by virtue of its mutation in a hereditary syndrome, ataxia-telangiectasia, which is associated with radiation hypersensitivity and cancer predisposition.150 ATR is also associated with a hereditary syndrome called Seckel syndrome. Early studies suggested that ATM and ATR played distinct roles in the response to double-stranded DNA breaks (ATM) versus replicative defects and single-stranded breaks (ATR). However, we now know that the regulation is more complex; there is considerable cross-talk between ATM and ATR, and they share many mediators and effectors, but the precise composition and role of the DDR complexes vary depending on both the type of the damage and the stage of the cell cycle.151 In this chapter, we focus primarily on how the DDR activates cell cycle checkpoints (Fig. 4-8). However, it is important to note that many of the components of the core DDR machinery are affected in hereditary disease syndromes and/or

G2/M

Intra S

G1

Replicationassociated error

DNA damage

DNA damage

DSB

DSB SSDNA

MRN

RPA

MRN

ATM

ATR

ATM

␥H2AX

␥H2AX

ATM

Mediators repair machinery

chk2P

␥H2AX

ATR

Mediators repair machinery

Mediators repair machinery

chk2P

chk1P

chk1P

chk2P

Figure 4-8 • ATM/ATR signaling is activated by DNA damage and replication stress. The cell constantly monitors the chromatin for lesions, using complex signal transduction pathways that center on the ATM and ATR kinases. The precise mechanism of response varies according to the type of DNA damage and the cell cycle stage. Double-stranded breaks (DSBs) are the most deleterious form of DNA damage. DSBs are recognized by the MRN complex that consists of Mre11, Rad50, and Nbs1. This complex recruits ATM to the site of damage. ATM phosphorylates histone H2AX, to form γH2AX, and this creates a binding platform for additional proteins that propagate the DNA damage response and activate repair. For S and G2 phase cells, but not G1 cells, ATR is also recruited to the damage site. ATR and/or ATM signal to their effector kinases—CHK1 and CHK2—respectively, to influence cell cycle progression as described in Figure 4-9. Errors in DNA replication can also activate the DNA damage response machinery through the presence of single-stranded DNA (ssDNA) that is a hallmark of the replication fork. The ssDNA is coated with RPA and bound by ATR. Active ATR then recruits the DNA damage and repair machinery, including ATM, leading to the sequential activation of CHK1 and then CHK2.

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abrogated in human tumors (e.g., NBS1, BRCA1, BRCA2, and the Franconi’s anemia proteins).

G1/S Checkpoint In G1 cells, double-stranded DNA breaks (DSBs) are the most common and most deleterious type of DNA damage. These DSB breaks are recognized by the multifunctional Mre11-Rad50-Nbs1 (MRN) complex.147 This complex recruits ATM to the site of damage. It is still unclear whether ATM activation occurs before or in response to MRN binding. The active ATM then recruits proteins to modify the chromatin at the region of the break and activate repair and signaling. As a first step in this process, ATM phosphorylates histone H2AX, to form γH2AX. This helps to hold the damaged ends together and acts as a binding platform for additional factors, including Mdc1, 53BP1, and BRCA1, as well as more MRN and ATM. In contrast to the S and G2 response, there is no recruitment of ATR to DSB in G1 cells; therefore, ATM is solely responsible for checkpoint activation. The recruitment of additional ATM amplifies the signal, and ATM acts via phosphorylation and activation of the effector kinase CHK2.152,153 CHK2 influences G1 cell cycle arrest via two mechanisms (Fig. 4-9). First, it phosphorylates all three members of the Cdc25 family. Phospho-Cdc25A is ubiquitinated by the SCFTrcpβ and degraded, while phospho-Cdc25B and phospho-Cdc25C are bound and sequestered by a cytoplasmic protein called 14–3-3.10,154,155 This is a rapid response that can take effect within minutes after DNA damage, and it has a widespread effect on cell cycle progression by preventing activation of all CDK2 and CDK1 complexes. In the case of the G1/S checkpoint, cyclin E/CDK2 is the relevant target. Second, CHK2 phosphorylates p53, a critical regulator of cell cycle checkpoints.156 In normal, nonstressed cells, p53 protein is maintained at low steady-state levels because it has a very short half-life. This halflife is a result of rapid ubiquitination of p53 by HDM2 (the human ortholog of murine MDM2 protein) and its consequent degradation. The importance of MDM2 for maintenance of appropriate p53 levels in vivo is highlighted by the fact that absence of MDM2 in knockout

mice results in early embryonic lethality that is rescued by a dual knockout of MDM2 and p53.157 Phosphorylation of p53 by CHK2 is sufficient to prevent its association with HDM2/MDM2.158 This leads to an accumulation of p53, which functions as a transcriptional activator. p53 induces expression of many genes. One of the key targets for the G1/S (and also G2/M) checkpoint is the CDK inhibitor p21Cip1.159,160 This p53-mediated arrest takes longer to develop than Cdc25 response (because it requires transcription and protein synthesis) but appears to be much more robust. Moreover, in addition to inducing cell cycle arrest, p53 has the capacity to induce apoptosis through the transcriptional activation of proapoptotic regulators (e.g., the BH3-only proteins PUMA and NOXA).161 How p53 chooses to activate arrest versus apoptosis targets is not fully understood, but it is clearly influenced by both the cell type and the level of damage.161 Importantly, p53 is also activated by other stress signals (see Fig. 4-9). In particular, it is now well established that numerous oncogenes trigger a stress response (called oncogene-induced stress) that leads to the activation of p53.162–165 The emerging view is that this occurs through two distinct mechanisms. First, oncogene activation is thought to yield replicative stress that activates p53 via activation of CHK kinases and phosphorylation of HDM2/MDM2 as just described.166,167 Second, many oncogenes activate transcription of Arf.168 This gene is encoded by the INK4a/Arf locus, and it actually shares two coding exons, which are read in alternate reading frames (hence the name ARF), with the p16Ink4A tumor suppressor.169 The Arf protein product, called p14Arf in humans and p19Arf in mouse, binds to HDM2/MDM2 and prevents it from regulating p53.170–174 As with the DDR, this frees p53 to activate the transcription or proarrest or proapoptotic targets. The central importance of this p53 pathway is underscored by the finding that the majority of human tumors carry mutations in p53, have upregulated HDM2 (typically by gene amplification), or have inactivated p19Arf.169 This is very analogous to the selective pressure to deregulate pRB pathway components (pRB, cyclin D/CDK4, and p16Ink4A) that was discussed previously.104 Together, the pRB and p53 pathways are critical gatekeepers of G1to-S progression in normal cell cycle and stress response.

G1, Intra S, G2/M

Intra S

G1/S

DNA damage

Replicative stress

Oncogenic stress

chk1

P

chk2

and/or P

chk1

P

chk2

p14ARF

and P

hdm2

Phosphorylation of all three cdc25 proteins cdc25A

cdc25B cdc25C

P

P P

Ubiquitinated by SCFTrcpβ and degraded Bound and inhibited by 14-3-3

cdk activation

Ubiquitination Regradation

P p53

p53

Oligomerizes to form active transcription factor

P21Cip1 cdk activity

Pro-apoptotic genes

Figure 4-9 • DNA damage, replicative stress, and oncogenic stress induce cell cycle arrest. DNA damage and replication stress lead to the rapid phosphorylation and activation of the CHK1 and/or CHK2 kinases. These enforce cell cycle arrest through two mechanisms. CHK1 and CHK2 both phosphorylate the cdc25 phosphatases, and this triggers their ubiquitination and degradation (cdc25A) or binding and inhibition by 14-3-3 (cdc25B and cdc25C), thereby preventing activation of either cyclin/CDK2 or cyclin/ CDK1 kinases. CHK1 and CHK2 also phosphorylate p53 and prevent it from being targeted by HDM2 for ubiquitin-mediated degradation. As a result, p53 accumulates and activates transcription of p21Cip1, inhibiting CDK2 and CDK1 kinase complexes, or proapoptotic genes. Oncogenic stress also leads to cell cycle arrest by activating replicative stress and/or inducing transcription or the p14Arf tumor suppressor and suppressing HDM2-mediated inhibition of p53.

Control of the Cell Cycle • CHAPTER 4

As an additional DNA damage response in G1 cells, genotoxic agents also inhibit origin licensing by way of an ATM/ATR-independent process. This is achieved through regulation of Cdt1.127–129,175 As was described previously, Cdt1 is required for pre-RC formation. In an undamaged cell, Cdt1 is available during G1 but is inhibited after origin firing by degradation (mediated by the SCFskp2 and Cul4-Ddb1-Dtl/Cdt2 ubiquitin ligases) and geminin binding. As a key feature of this regulatory system, Cdt1 is completely resistant to Cul4-Ddb1-Dtl/Cdt2 in the G1 phase. However, DNA damage allows Cul4-Ddb1-Dtl/Cdt2 complex to ubiquitinate Cdt1 and induce its degradation. This process requires binding of Cdt1 to proliferative cell nuclear antigen, but the mechanism by which this induces Cdt1 ubiquitinylation is not understood. Importantly, the degradation of Cdt1 is extremely rapid, occurring within minutes of the DNA damage. As a result, origin licensing is completely blocked until the damage is repaired and Cdt1 is resynthesized.

Intra-S Phase Checkpoint One of the major goals of cell cycle checkpoints is to prevent the deleterious consequences of replicating damaged DNA. Therefore, S phase cells must respond virtually instantaneously to DNA damage to halt initiation of new replication forks throughout the S phase.149 The most deleterious damage is DSBs. These can occur through the action of DNA damaging agents (from either extrinsic or intrinsic sources) or as a consequence of the replication process itself, for example, if the replication fork passes through nicked DNA or replication stalls at sites of DNA damage. The cell senses the damage in different ways depending on whether or not the lesion is associated with replication. Ultimately, both ATM and ATR are recruited to the site of damage, but the order of binding is different.149,151 Replication-linked DSBs are distinguished by the presence of singlestranded DNA, a hallmark of the replication process. The single-stranded DNA is coated by RPA and bound by ATR and its regulator subunit ATRIP, even during the normal replication process. In response to DNA damage, the ATR kinase is activated, and it

then recruits a variety of complexes that mediate both repair and checkpoint activation, including ATM. In contrast, nonreplicationassociated DSBs initially recruit and activate ATM through the MRN-dependent process described previously for G1/S checkpoint. However, in S phase cells, DSB resection causes the formation of single-stranded DNA (through the action of the MRN endonuclease), and this is then bound by RPA and ATR/ATRIP.149,151 Thus, in S phase cells, ATR and ATM jointly orchestrate the DDR. ATR contributes to the checkpoint response in a similar manner to ATM: It activates an effector kinase, called CHK1, which can also phosphorylate the cdc25 proteins and p53.176–179

G2 Checkpoint The G2 checkpoint is required to prevent the passage of DNA lesions to two daughter cells during mitosis.147,180 DSBs are detected exactly as we described previously for the S phase nonreplication-associated DSBs. Similarly, the ATR/CHK1 and ATM/CHK2 pathways enforce arrest through inhibition of G2 and mitotic CDK complexes via the rapid removal of the cdc25 phosphates and the p53-dependent induction of the p21Cip1 CDKI.

Spindle Checkpoint The preceding sections focused on the steps the cell takes to prevent the propagation of DNA errors to the daughter cells. In contrast, the spindle checkpoint acts to ensure that there is appropriate partitioning of the chromosomes.181 We have already introduced the concept that chromosome segregation is prevented until all of the condensed sister chromatid pairs are aligned at the metaphase plate with the appropriate biorientation. This is actually controlled by a signaling network that constitutes the spindle checkpoint (Fig. 4-10). The core components of the spindle checkpoint—called MAD1, MAD2, BUBR1, and BUB1 in humans—were originally identified through screens in yeast for “mitotic arrest deficient” (MAD) and “budding uninhibited by benzimidazole” (BUB) mutants.181 These proteins become active in the prometaphase of mitosis (see Fig. 4-10). They

Prometaphase No tension

Figure 4-10 • The spindle checkpoint. Improper chromosome alignment on the mitotic spindle, disruption of microtubule dynamics, or unattached kinetochores can activate the spindle checkpoint. Spindle checkpoint signaling is mediated by the Bub1, Bub3, BubR1, and Mad2 proteins, which all localize to kinetochores. These core spindle checkpoint regulators prevent the activator protein Cdc20 from binding to APC and therefore protects securin, a major APCcdc20 target, from ubiquitin-mediated degradation. As a result, securin remains bound to separase, and this prevents cleavage of Scc1 and loss of centromeric cohesin. The spindle checkpoint is relieved at the end of the metaphase by the appropriate biorientation of the sister chromatids at the metaphase plate. The sensing mechanism involves detecting tension through the cohesin complex at the kinetochores that is created by the pulling of the spindle fibers toward the poles. Mad2 then dissociates from the attached kinetochore, and this allows cdc20 to activate APC and trigger sister chromatid segregation.

Pole “Wait” Unattached kinetochore Low MAD2

Cohesion Spindle checkpoint proteins

Metaphase

Cdc20

High MAD2

Tension

Securin ubiquitination + degradation

Active APC

Anaphase

Inactive APC

Securin Scc1 Active separase Cohesion (by scc1 cleavage)

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associate with the kinetochore and, in the absence of biorientation, prevent the CDC20 activator from binding to the APC. As a result, separase is sequestered by securin and unable to cleave the centromeric cohesin (see Fig. 4-10). It is still unclear precisely how the spindle checkpoint is inactivated by appropriate biorientation. However, it involves monitoring the tension through the cohesin complex at the kinetochores (created by the pulling of the spindle fibers toward the poles) and the dissociation of MAD2 from the attached kinetochore (see Fig. 4-10). Because aneuploidy is a shared feature of many cancer cells, there has been considerable speculation that disruption of the spindle checkpoint could occur during tumor progression.182–184 Notably, inactivating mutations in Bub1 have been identified in human colon carcinoma cell lines, which are known to have a high degree of aneuploidy.185 Moreover, haploinsufficiency of Mad2 has been shown to cause elevated rates of lung tumor development in Mad2+/− mice compared with age-matched wild-type mice.186 However, it is still an open question whether spindle checkpoint defects make a significant contribution to tumor development.

CELL CYCLE DEREGULATION IN HUMAN CANCERS Molecular analysis of human tumors demonstrates that alterations in components of the cell cycle machinery and checkpoint-signaling pathways occur in the majority of human tumors (Table 4-1). This finding underscores how important maintenance of cell cycle control is in the prevention of human cancer. The alterations in the cell cycle machinery that occur most frequently include loss or mutation of the

pRB tumor suppressor; overexpression of cyclins, CDKs, and Cdc25 phosphatases; and loss of expression of CDKIs. The most frequently altered cell cycle checkpoint-signaling molecule is the p53 tumor suppressor. Proteins that reside upstream of p53 (including ATM and CHK2) are also targeted for mutation in human tumors, and their discovery and analysis have greatly deepened our insight into DNA damage response-signaling pathways. Mutations that affect the pRB pathway have been identified in the majority of human cancers.91,187 The RB-1 gene was originally identified by virtue of its mutation in both familial and sporadic retinoblastoma, but it is defective in many other tumor types, especially osteosarcoma and lung cancer. Indeed, more than 90% of small-cell lung cancers have mutant RB-1, suggesting that disruption of the pRB pathway (through the genetic or epigenetic targeting of RB-1 or upstream signaling components) is a requirement for the genesis of lung cancer.188 It is important to note that inactivation of the parallel and interconnecting p14Arf-p53 axis is also essential in functionally pRB-deficient lung cells to bypass efficient apoptosis.169 In breast cancer, loss of normal pRB function due to RB-1 mutation is observed in 20% of tumors.189 In the 80% of breast carcinomas that lack RB-1 mutations, alterations in components of the signaling pathways that regulate pRB are frequently found, including cyclin D1 and cyclin E overexpression and cdk4 and cdk6 gene amplification.190–192 Nearly 50% of invasive breast cancers have elevated cyclin D expression compared with surrounding normal breast epithelium, while transgenic mice with overexpression of human cyclin D1 or cyclin E in mammary gland cells develop mammary adenocarcinomas.193–195 Similarly, cdk4 and cdk6 gene amplification occurs in breast cancers, sarcomas, gliomas, and melanomas.196

Table 4-1 Mutations of Cell Cycle Checkpoint Regulators in Human Tumors* Hereditary Syndromes Associated with Germline Mutations

Gene/Protein

Tumors Associated with Mutations or Altered Expression

ATM

Breast carcinomas, lymphomas, leukemias

Ataxia-telangiectasia

Bub1

Colorectal carcinomas

NR

BRCA1

Breast and ovarian carcinoma

Familial breast and ovarian cancer

Cdc25A

Carcinomas of breast, lung, head and neck, and lymphoma

NR

Cdc25B

Carcinomas of breast, lung, head and neck, and lymphoma

NR

Cdk4

Wide array of cancers

NR

Cdk6

Wide array of cancers

NR

Chk1

Colorectal and endometrial carcinomas

NR

Chk2

Carcinomas of breast, lung, colon, urogenital tract, and testis

Li-Fraumeni syndrome

Cyclin D1

Wide array of cancers

NR

Cyclin D2

Lymphoma and carcinomas of the colon, testis and ovary

NR

Cyclin D3

Lymphoma, pancreatic carcinoma

NR

Cyclin E

Wide array of cancers

NR

MDM2

Soft tissue tumors, osteosarcomas, esophageal carcinomas

NR

MRE11

Lymphoma

Ataxia-telangiectasia-like disorder

NBS

Lymphomas, leukemias

Nijmegen breakage syndrome

p15INK4b

Wide array of cancers

NR

p16INK4a

Wide array of cancers

Familial melanoma

p27KIP1

Wide array of cancers

NR

p53

Wide array of cancers

Li-Fraumeni syndrome

p57KIP2

Bladder carcinomas

NR

p130

Wide array of cancers

NR

pRB

Wide array of cancers

Familial retinoblastoma

NR, not reported. *Only alterations that are present in more than 10% of primary tumors are represented.

Control of the Cell Cycle • CHAPTER 4

Modifications of CDKIs that act upstream of pRB activity are also commonly found in human tumors. The CDK inhibitor p27Kip1 is often aberrantly expressed in human breast cancer, and reduced p27Kip1 protein levels are correlated with more aggressive breast tumors.197,198 Likewise, decreased expression of the CDK inhibitor p57Kip2 is found in human bladder cancers.199 Germline mutations in p16INK4a predispose individuals to melanoma, while deletion of p15INK4b and p16INK4a is linked to the pathogenesis of lymphomas, mesotheliomas, and pancreatic cancers.78,79,196,200 In tumor types in which p15INK4b and p16INK4a are not deleted, methylation of the gene locus leads to transcriptional repression and loss of gene expression. In some tumors, hypermethylation prevents expression of both p16INK4a and p14Arf, which are encoded by alternative reading frames of the Ink4a/Arf locus.199 Both Cdc25A and Cdc25B phosphatases are overexpressed in more than 30% of primary breast tumors, 40% to 60% of non-small-cell lung cancers, 50% of head and neck tumors, and a significant fraction of non-Hodgkin’s lymphomas.10,201,202 Elevation of these oncogenic phosphatases can result in increased activation of CDK and override of checkpoint arrest. p53 mutation is the most frequently observed mutation in the majority of human tumors. The importance of p53-dependent signaling in tumor suppression is underscored by the frequency of mutation in sporadic tumors and the finding that germline mutations of p53 result in Li-Fraumeni syndrome, a highly penetrant familial cancer syndrome that is associated with significantly increased rates of brain tumors, breast cancers, and sarcomas.203,204 In human tumors that lack p53 gene mutation, p53 function may be disrupted by alterations in cellular proteins that modulate the levels, localization, and biochemical activity of p53. For example, in some tumors with wild-type p53 alleles, MDM2 gene amplification occurs, resulting in MDM2 protein overexpression and subsequent p53 inactivation.205 In human papillomavirus-induced cervical carcinoma, p53 is typically not mutated; however, the human papillomavirus E6 protein binds p53 and targets it for degradation, abrogating p53-dependent signaling.206 Mutation in components of the DNA damage response pathway also leads to enhanced tumorigenesis, as was discussed previously. For example, ATM mutations occur in ataxia-telangiectasia, a disorder in which patients have increased sensitivity to radiation and an elevated incidence of leukemias, lymphomas, and breast cancer.150,207 ATMnull mice exhibit growth retardation, neurologic dysfunction, infertility, defective T lymphocyte maturation, and sensitivity to ionizing radiation.208,209 The majority of ATM-deficient animals develop malignant lymphomas by 4 months of age, while ATM −/− fibroblasts have abnormal radiation checkpoint function after exposure to ionizing radiation.208,209 The DNA double-strand break repair gene MRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder.210 Mutations of Chk2 and Chk1 also arise in human cancers. Chk2 mutations have been reported in several cancers, including lung, while Chk1 mutations have been observed in human colon and endometrial cancers.211,212 In addition, heterozygous alteration of Chk2 occurs in a subset of individuals with Li-Fraumeni syndrome who lack p53 gene mutations.213 These findings support the theory that in human tumors in which p53 is intact, the function of this tumor suppressor might be disrupted by alterations in cellular proteins that modulate the levels or activity of p53. In addition, the breast cancer susceptibility tumor suppressors BRCA1 and BRCA2 are known to participate in the DNA damage response and repair.214 Similarly, the Fanconi’s anemia proteins, which were originally identified by virtue of their association with a recessive development disorder called Fanconi’s anemia, which is associated with increased cancer predisposition (particularly acute myeloid leukemia), also function in the DNA damage response.214 The spindle checkpoint disruption has also been linked to the pathogenesis of several human tumors. BUB1 mutations have been identified in human colon carcinoma cells, and Bub1 mutation facilitates the transformation of cells that lack the breast cancer

susceptibility gene, BRCA2.185,215 Moreover, Michel and colleagues demonstrate that Mad2+/− mice have significantly higher rates of lung tumor development than do age-matched wild-type mice.186

THERAPEUTIC MANIPULATION OF CELL CYCLE CONTROLS Research over the past two decades has shown that alterations in cell cycle machinery and checkpoint signaling lead to tumorigenesis. These findings have important implications for the optimization of current therapeutic regimens and for the selection of novel cell cycle targets for the future development of anticancer agents. A leading goal of cancer-based research is to identify compounds that will target key cell cycle controls in a tumor-specific manner.

Targeting Cyclin-Dependent Kinase Activity There has been considerable debate about whether inhibition of CDK activity is a rational strategy for anticancer therapies. CDK activity is frequently elevated in human tumors, but it is also required to maintain specific cells populations in the adult (e.g., the hematopoietic compartment and gut) that are essential for viability. Thus, the key issue is whether there is sufficient difference in the CDK activity in tumor versus normal cells to create a therapeutic window. Over the last few years, the analysis of CDK and cyclin mouse models has yielded considerable insight into this question but has also raised additional questions.18 On the positive side, studies in mouse models clearly show that tumors can be more dependent on CDK activity, or at least a specific CDK activity, than normal tissues can. For example, loss of D-type cyclins has been shown to have little or no effect on the development and maintenance of many tissues, but loss of cyclin D-associated kinase activity can greatly suppress the development of certain tumor types, depending on the tissue and the identity of the initiating oncogenic lesions.22,29,216 On the negative side, the mouse models also show that the cell cycle machinery is extremely robust; it adapts easily to the loss of CDKs or cyclins by using other CDKs or cylins to substitute for the missing activity. For example, CDK2 knockout mice are fully viable because CDK4/6 and CDK1 now form novel cyclin/CDK complexes and assume roles that are normally specific to CDK2.217–219 This raises the possibility that tumor cells will rapidly develop resistance to CDK-inhibitory drugs by simply adapting their cell cycle machinery. In light of these complexities, efforts have been placed on generating pharmacologic inhibitors of CDKs that either are CDK-specific or have pan-CDK activities. Numerous small molecule inhibitors have been developed, and many are in clinical trials.220,221 One of the first compounds to be tested, flavopiridol, is a panCDK inhibitor that inhibits CDK4/6, CDK2, and CDK1 kinase activity. Consistent with this broad action, flavopiridol arrests cells at G1/S (in a pRB-dependent manner) and G2/M. This antiproliferative activity against a variety of human cancer cell lines produced favorable clinical responses in phase I and phase II studies of patients with renal, colorectal, gastric, lung, and esophageal carcinomas.222–224 Notably, it was also determined that if target cells are first induced to induced to enter S phase, then treatment with flavopiridol had significant cytotoxic effects.221 This arises through two mechanisms. First, flavopiridol inhibits the action of cyclin A/CDK2 and thereby prevents the phosphorylation of E2F1 and its subsequent degradation.59,61 The persistence of E2F1 in late stages of the cell cycle is known to trigger apoptosis, and this effect shows strong specificity for tumor cells versus normal cells, presumably because of higher E2F1 levels. Second, flavopiridol suppresses the activity of CDK7 (which functions both as a component of both CAK and as a RNA polymerase II CTD kinase that promotes transcriptional elongation) and CDK9 (which acts in association with cyclin T to form another CTD kinase called P-TEFb).225,226 Inhibition of CDK7 and CDK9 suppresses mRNA synthesis, and this leads to a rapid loss of

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transcripts that have short half-lives, including many cell cycle regulators (e.g., cyclins) and antiapoptosis regulators. As a result of these observations, clinical trails have been conducted using sequential treatment of an S phase chemotherapeutic agent, gemcitabine, and then flavopiridol.221 On a similar theme, sequential treatment with paclitaxel (which inhibits mitotic spindle function) and then flavopiridol also yields cytotoxic synergy.221 In this case, flavopiridol is acting by inhibiting cyclin B/CDK1 and thus prevents phosphorylation and stabilization of a protein, called survivin, that is required to maintain the spindle checkpoint. Thus, the cells proceed through cytokinesis and enter G1 without segregating their chromosomes, and this triggers apoptosis. Phase I and phase II trials have been conducted with paclitaxel and flavopiridol, and currents efforts are focused on optimizing the dosage and the time interval of administration.221 More selective CDK inhibitors are also being analyzed.220,221 These include small molecules that show a strong selectively for CDK2 and CDK1 or are highly specific for CDK4/6. Cell- and animal-based studies show that these drugs yield the anticipated affects. For example, the CDK4/6 inhibitor PD0332991 yields a G1/S arrest in an pRB-dependent manner, and it can yield regression of xenografts generated from pRB-positive cell lines.227 Many of these drugs have yet to be tested in clinical trials.

Targeting DNA Damage Response Proteins In the last decade, there has been a growing appreciation that many tumors cells carry mutations that disrupt their DNA damage response (DDR). This is a major factor in establishing the resistance of tumors to chemotherapeutic agents, many of which work by causing DNA damage and triggering apoptosis through induction of DNA damage pathways. Therefore, considerable attention has focused on designing cancer treatments that would be effective in cells with an impaired DDR. Since it is hard to restore the function of mutant or missing proteins, the prevailing strategy is to identify drugs that would synergize with the defective DDR to selectively kill the tumor cells and not the normal cells. For example, inhibitors of poly(ADP-ribose) polymerase selectively kill cells that lack either

BRCA1 or BRCA2.228–230 The rationale for this is that these proteins provide two alternative repair mechanisms in response to DNA damage: homologous recombination (BRCA1 and BRCA2) and base excision repair (poly(ADP-ribose) polymerase). Therefore, loss of one but not both of these pathways can be tolerated. As a second example, inhibition of CHK1 sensitizes p53 mutant cells to DNA damage.220 Since p53 is mutated in approximately half of all human tumors and the absence of p53 is a major predictor of poor response to classic chemotherapeutic agents, considerable efforts are being made to develop small molecular inhibitors of CHK1.

SUMMARY Over the past several decades, investigators have uncovered a wealth of information about the proteins that control cell growth and division in human cells. A key finding is that deregulation of the cell cycle machinery and/or checkpoints is a universal alteration that has been identified in human cancer.104,231 Although numerous genetic alterations can result in loss of normal checkpoints, the hope is that common strategies will be developed against a wide variety of cancers. Even though several of the currently used anticancer therapies target nonselective and non-mechanism-based targets, their effectiveness, albeit limited in many cases, is likely due to the fact that they ultimately target cell cycle regulatory or DDR-signaling pathways, the status of which is different in normal cells versus tumor cells. Identifying all the components of the cellular machinery that control the cell cycle both positively and negatively is vital to the continued development of anticancer agents that can preferentially eliminate cancer cells and minimize the toxicity to normal tissues. The information that is generated by the genomic and proteomic approaches using eukaryotic model systems will continue to reveal new cell cycle regulatory molecules. As our understanding of cell cycle regulation and checkpoint signaling improves, the goal is to use this knowledge in the design of mechanism-based therapeutics that will bring anticancer therapy to a new level. There can be little doubt of the value of targeting the cell cycle in drug discovery.

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Control of the Cell Cycle • CHAPTER 4 161. Vousden KH, Lu X: Live or let die: the cell’s response to p53. Nat Rev Cancer 2002;2:594– 604. 162. Serrano M, Lin AW, McCurrach ME, et al: Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 1997;88:593–602. 163. de Stanchina E, McCurrach ME, Zindy F, et al: E1A signaling to p53 involves the p19(ARF) tumor suppressor. Genes Dev 1998;2:2434–2442. 164. Dimri GP, Itahana K, Acosta M, Campisi J: Regulation of a senescence checkpoint response by the E2F1 transcription factor and p14(ARF) tumor suppressor. Mol Cell Biol 2000;20:273– 285. 165. Zindy F, Eischen CM, Randle DH, et al: Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization. Genes Dev 1998;12:2424–2433. 166. Bartkova J, Razaei N, Liontos M, et al: Oncogeneinduced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 2006;444:633–637. 167. Di Micco R, Fumagalli M, Cicalese A, et al: Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature 2006;444:638–642. 168. Zindy F, Williams RT, Baudino TA, et al: Arf tumor suppressor promoter monitors latent oncogenic signals in vivo. Proc Natl Acad Sci USA 2003;100:15930–15935. 169. Sherr CJ: The INK4a/ARF network in tumour suppression. Nat Rev Mol Cell Biol 2001;2:731– 737. 170. Kamijo T, Weber JD, Zambetti G, et al: Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc Natl Acad Sci USA 1998;95:8292–8297. 171. Honda R, Yasuda H: Association of p19(ARF) with Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumor suppressor p53. EMBO J 1999;18:22–27. 172. Llanos S, Clark A, Rowe J, Peters G: Stabilization of p53 by p14ARF without relocation of MDM2 to the nucleolus. Nat Cell Biol 2001;3:445– 452. 173. Pomerantz J, Schreiber-Agus N, Liégeois NJ, et al: The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2’s inhibition of p53. Cell 1998;92:713–723. 174. Weber JD, Taylor LJ, Roussel MF, et al: Nucleolar Arf sequesters Mdm2 and activates p53. Nat Cell Biol 1999;1:20–26. 175. Higa LA, Mihaylov IS, Banks DP, et al: Radiationmediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint. Nat Cell Biol 2003;5:1008–1015. 176. Sanchez Y, Wong C, Toma RS, et al: Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. Science 1997;277:1497–1501. 177. Furnari B, Blasina A, Boddy MN, et al: Cdc25 inhibited in vivo and in vitro by checkpoint kinases Cds1 and Chk1. Mol Biol Cell 1999;10: 833–845. 178. Liu Q, Guntuku S, Cui XS, et al: Chk1 is an essential kinase that is regulated by Atr and required for the G2M DNA damage checkpoint. Genes Dev 2000;14:1448–1459. 179. Jin J, Shirogane T, Xu L, et al: SCFbeta-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase. Genes Dev 2003;17:3062– 3074. 180. O’Connell MJ, Walworth NC, Carr AM: The G2phase DNA-damage checkpoint. Trends Cell Biol 2000;10:296–303. 181. Musacchio A, Salmon ED: The spindle-assembly checkpoint in space and time. Nat Rev Mol Cell Biol 2007;8:379–393.

182. Cimini D, Degrassi F: Aneuploidy: a matter of bad connections. Trends Cell Biol 2005;15:442– 451. 183. Kops GJ, Weaver BA, Cleveland DW: On the road to cancer: aneuploidy and the mitotic checkpoint. Nat Rev Cancer 2005;5:773–785. 184. Baker DJ, Chen J, van Deursen JM: The mitotic checkpoint in cancer and aging: what have mice taught us? Curr Opin Cell Biol 2005;17:583– 589. 185. Cahill DP, Lengaur C, Yu J, et al: Mutations of mitotic checkpoint genes in human cancers. Nature 1998;392:300–303. 186. Michel LS, Liberal V, Chatterjee A, et al: MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature 2001;409:355–359. 187. Sellers WR, KaelinWG Jr: Role of the retinoblastoma protein in the pathogenesis of human cancer. J Clin Oncol 1997;15:3301–3312. 188. Kaye FJ: RB and cyclin dependent kinase pathways: defining a distinction between RB and p16 loss in lung cancer. Oncogene 2002;21:6908– 6914. 189. Varley JM, Armour J, Swallow JE, et al: The retinoblastoma gene is frequently altered leading to loss of expression in primary breast tumours. Oncogene 1989;4:725–729. 190. Zheng L, Lee WH: The retinoblastoma gene: aprototypic and multifunctional tumor suppressor. Exp Cell Res 2001;264:2–18. 191. Nobori T, Miura K, Wu DJ, et al: Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 1994;368:753– 756. 192. Ravaioli A, Bagli L, Zucchini A, Monti F: Prognosis and prediction of response in breast cancer: the current role of the main biological markers. Cell Prolif 1998;31:113–126. 193. Weinstat-Saslow D, Merino MJ, Manrow RE, et al: Overexpression of cyclin D mRNA distinguishes invasive and in situ breast carcinomas from non-malignant lesions. Nat Med 1995;1:1257–1260. 194. Wang TC, Cardiff RD, Zukerberg L, et al: Mammary hyperplasia and carcinoma in MMTVcyclin D1 transgenic mice. Nature 1994;369:669– 671. 195. Bortner DM, Rosenberg MP: Induction of mammary gland hyperplasia and carcinomas in transgenic mice expressing human cyclin E. Mol Cell Biol 1997;17:453–459. 196. Elsayed YA, Sausville EA: Selected novel anticancer treatments targeting cell signaling proteins. Oncologist 2001;6:517–537. 197. Porter, PL, Malone KE, Heagerty PJ, et al: Expression of cell-cycle regulators p27Kip1 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients. Nat Med 1997;3:222–225. 198. Catzavelos C, Bhattacharya N, Ung YC, et al: Decreased levels of the cell-cycle inhibitor p27Kip1 protein: prognostic implications in primary breast cancer. Nat Med 1997;3:227–230. 199. Esteller M, Herman JG: Cancer as an epigenetic disease: DNA methylation and chromatin alterations in human tumours. J Pathol 2002;196:1–7. 200. Cannon-Albright LA, Goldgar DE, Meyer LJ, et al: Assignment of a locus for familial melanoma, MLM, to chromosome 9p13-p22. Science 1992;258:1148–1152. 201. Gasparotto D, Maestro R, Piccinin S, et al: Overexpression of CDC25A and CDC25B in head and neck cancers. Cancer Res 1997;57:2366– 2368. 202. Wu W, Fan YH, Kemp BL, et al: Overexpression of cdc25A and cdc25B is frequent in primary nonsmall cell lung cancer but is not associated with

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overexpression of c-myc. Cancer Res 1998;58: 4082–4085. Nigro JM, Baker SJ, Preisinger AC, et al: Mutations in the p53 gene occur in diverse human tumour types. Nature 1989;342:705–708. Ozbun MA, Butel JS: Tumor suppressor p53 mutations and breast cancer: a critical analysis. Adv Cancer Res 1995;66:71–141. Momand J, Jung D, Wilczynski S, Niland J: The MDM2 gene amplification database. Nucleic Acids Res 1998;26:3453–3459. Scheffner M, Werness BA, Huibregtse JM, et al: The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell,1990;63:1129–1136. Khanna KK: Cancer risk and the ATM gene: a continuing debate. J Natl Cancer Inst 2000;92: 795–802. Barlow C, et al: Atm-deficient mice: a paradigm of ataxia telangiectasia. Cell 1996;86:159–171. Xu Y, Ashley T, Brainerd EE, et al: Targeted disruption of ATM leads to growth retardation, chromosomal fragmentation during meiosis, immune defects, and thymic lymphoma. Genes Dev 1996;10:2411–2422. Petrini JH: The Mre11 complex and ATM: collaborating to navigate S phase. Curr Opin Cell Biol 2000;12:293–296. Matsuoka S, Nakagawa T, Masuda A, et al: Reduced expression and impaired kinase activity of a Chk2 mutant identified in human lung cancer. Cancer Res 2001;61:5362–5365. Bertoni F, Codegoni AM, Furlan D, et al: CHK1 frameshift mutations in genetically unstable colorectal and endometrial cancers. Genes Chromosomes Cancer 1999;26:176–180. Bell DW, Varley JM, Szydlo TE, et al: Heterozygous germ line hCHK2 mutations in LiFraumeni syndrome. Science 1999;286:2528– 2531. Wang W: Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins. Nat Rev Genet 2007;8:735– 748. Lee H, Trainer AH, Friedman LS, et al: Mitotic checkpoint inactivation fosters transformation in cells lacking the breast cancer susceptibility gene, Brca2. Mol Cell 1999;4:1–10. Yu Q, Geng Y, Sicinski P: Specific protection against breast cancers by cyclin D1 ablation. Nature 2001;411:1017–1021. Satyanarayana A, Hilton MB, Kaldis P: p21 inhibits Cdk1 in the absence of Cdk2 to maintain the G1/S phase DNA damage checkpoint. Mol Biol Cell 2007 [Epub ahead of print]. Berthet C, Aleem E, Coppola V, et al: Cdk2 knockout mice are viable. Curr Biol 2003;13: 1775–1785. Ortega S, Prieto I, Odajima J, et al: Cyclindependent kinase 2 is essential for meiosis but not for mitotic cell division in mice. Nat Genet 2003; 5:25–31. Collins I, Garrett MD: Targeting the cell division cycle in cancer: CDK and cell cycle checkpoint kinase inhibitors. Curr Opin Pharmacol 2005;5: 366–373. Shapiro GI: Cyclin-dependent kinase pathways as targets for cancer treatment. J Clin Oncol 2006; 24:1770–1783. Schwartz GK, Ilson D, Saltz L, et al: Phase II study of the cyclin-dependent kinase inhibitor flavopiridol administered to patients with advanced gastric carcinoma. J Clin Oncol 2001;19:1985– 1992. Senderowicz AM: Flavopiridol: the first cyclindependent kinase inhibitor in human clinical trials. Invest New Drugs 1999;17:313–320. Stadler WM, Vogelzang NJ, Amato R, et al: Flavopiridol, a novel cyclin-dependent kinase

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229. Farmer H, McCabe N, Lord CJ, et al: Targeting 227. Fry DW, Harvey PJ, Keller PR, et al: Specific the DNA repair defect in BRCA mutant cells as inhibition of cyclin-dependent kinase 4/6 by PD a therapeutic strategy. Nature 2005;434:917– 0332991 and associated antitumor activity in 921. human tumor xenografts. Mol Cancer Ther 230. Bryant HE, Schultz N, Thomas HD, et al: Specific 2004;3:1427–1438. killing of BRCA2-deficient tumours with inhibitors 228. Tutt AN, Lord CJ, McCabe N, et al: Exploiting of poly(ADP-ribose) polymerase. Nature the DNA repair defect in BRCA mutant cells in 2005;434:913–917. the design of new therapeutic strategies for cancer. Cold Spring Harb Symp Quant Biol 2005;70:139– 231. Hartwell LH, Kastan MB: Cell cycle control and cancer. Science 1994;266:1821–1828. 148.

5

Cell Life and Death Rebecca L. Elstrom and Craig B. Thompson

S U M M ARY • Apoptosis control mechanisms seem to be impaired in virtually all tumors, suggesting that a required step in carcinogenesis is to disengage the apoptotic machinery. • Two basic pathways of apoptosis have been described: the extrinsic or death receptor-mediated pathway and the intrinsic or mitochondrial pathway.

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• The fate of a cell is determined by the balance of proapoptotic and antiapoptotic factors within the cell. • Oncogenic transformation promotes proapoptotic pathways. Cancer cells must disable tumor suppressor molecules and/or activate survival signals to evade programmed cell death.

INTRODUCTION The evolution of a normal cell into cancer involves disruption and deregulation of several basic cellular processes. Multicellular organisms, in their evolution from simple, single cells, have developed redundant controls through which the homeostasis between different cell types is maintained. One of the safeguards that prevents excess cell accumulation is the presence of cell-intrinsic programs that can induce programmed cell death, the best studied being apoptosis. The growing understanding that transforming mutations can activate this intrinsic death response has emphasized the importance of this process in preventing cancer cell development. Apoptosis control mechanisms seem to be impaired in virtually all tumors, suggesting that a required step in carcinogenesis is to disengage the apoptotic machinery. The concept that genes regulating cell death could play a role in tumorigenesis arose in the mid-1980s, when investigators first discovered that a translocation commonly found in follicular lymphoma, between chromosomes 14 and 18, brings a region on chromosome 18 called breakpoint cluster region 2 (Bcl-2) into close proximity with the immunoglobulin heavy chain enhancer on chromosome 14, resulting in overexpression of the Bcl-2 gene.1 Later work showed that the Bcl-2 gene product promotes oncogenesis by a novel mechanism.2,3 Instead of inducing cell proliferation or invasion, the Bcl-2 protein inhibits the normal programmed death of B cells, resulting in the failure to eliminate the clonal B cells as they accumulate in excess. These findings demonstrated for the first time that disarming death pathways within a cell could predispose to development of malignancy. Since the description of Bcl-2, extensive progress has been made in understanding both the mechanisms of apoptosis and the ways in which this process contributes to tumorigenesis. The identification of a family of genes related to Bcl-2 that contribute to the balance between life and death, together with the discovery of the critical role of mitochondria in cellular homeostasis and apoptosis, have broadened our understanding of the dynamic interplay of forces determining the fate of cells.

• Therapeutic strategies aimed at restoring tumor suppressors and interfering with survival factors are playing increasingly important roles in antineoplastic treatments. • There is a growing understanding of the role of alternative forms of cell death, particularly necrosis, in the response of cancer cells to treatment.

In addition to progress in elucidating apoptotic mechanisms and deregulation, there has been an increasing understanding of the role of nonapoptotic mechanisms of cell death in both organismal homeostasis and pathogenic states such as cancer. Necrosis, a death process traditionally considered as unregulated and catastrophic, may instead represent a regulated event that contributes to organismal development and homeostasis. Autophagy, in which a cell “eats itself,” is also under investigation as a form of nonapoptotic cell death, although the function of this process, whether to induce death or, conversely, to maintain survival, is controversial. Harnessing these forces will improve our capability not only to understand the mechanisms by which normal cells become malignant but also to prevent and treat cancer in humans.

FUNDAMENTAL SCIENCE Cell death occurs by two general classes of mechanism: apoptosis, or programmed cell death, and necrosis. Autophagy has been proposed as a third potential mechanism of cell death, although the physiologic functions of this process remain under study. The process of apoptosis involves a cell-intrinsic suicide program that not only kills the cell but also stimulates the clearance and complete degradation of the corpse without inducing an inflammatory reaction. Apoptosis differs from other forms of cell death, such as necrosis, in that clearance of the cell is controlled through the activity of caspases—cysteine proteases with aspartate specificity that normally exist in an inactive, zymogen form. The initiator caspases 8 and 9 are activated by cellular signals and subsequently cleave downstream effector caspases, such as caspases 3, 6, and 7. These effector caspases set into motion the degradation of cellular components such as structural proteins, cell cycle machinery, and DNA. These processes result in the characteristic morphology of apoptotic cells, membrane blebbing, cell shrinkage, and DNA fragmentation. The end result is the disposal of the cell in a controlled manner, allowing turnover and phagocytosis without the inflammatory reaction to intracellular substances that accompanies death by necrosis.

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As noted in the preceding discussion, the caspase cascade is initiated through specific cellular signals. These signals come through one of two major pathways: the extrinsic, receptor-mediated pathway, or the intrinsic, mitochondrial pathway. Although these pathways often are considered separately, extensive cross-talk exists between them.

Cell Death by Murder In some cases, apoptosis is initiated through ligation of specific cellsurface receptors, the death receptors (Fig. 5-1). These molecules are members of the tumor necrosis factor receptor (TNFR) family. The best studied of these include Fas and TNFR1.4,5 These receptors exist as trimers at the cell surface that are activated on binding of ligand, Fas ligand, and TNF, respectively. The intracellular domains of these receptors contain death domains which, on activation of the receptor, can recruit a death-inducing signaling complex, which leads to activation of a caspase cascade. The Fas death domain binds the Fasassociated death domain (FADD) adapter protein, which directly recruits caspase 8 and allows its cleavage and activation. Activation of TNFR1, on the other hand, has multiple potential downstream effects.6 TNFR1 binds the TNFR-associated death domain (TRADD) adapter protein, which in turn might recruit FADD, resulting in caspase 8 activation and apoptosis, as with Fas. TRADD also, however, can bind TNFR-associated factor-2, which may recruit inhibitor of apoptosis proteins (IAPs), which bind the death-inducing signaling complex and inhibit activation of caspase 8. Alternatively, TNFR-associated factor-2 may recruit components of the mitogen-activated protein kinase (MAPK) cascade, leading to activation of Jun-N-terminal kinase (JNK) and c-jun. Although in some systems JNK seems to promote TNFR1-induced apoptosis,

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Figure 5-1 • Death receptors can initiate apoptosis or promote cell survival. On ligation of the death receptor, adaptors are recruited. For TNFR1, the outcome of ligand binding depends on the state of the cell. TRADD might bind the FADD molecule, which recruits caspase 8, resulting in its oligomerization and activation. Active caspase 8 in turn cleaves and activates effector caspases and the BH3-only molecule Bid, initiating an apoptotic cascade. This process is inhibited by IAPs. Alternatively, binding of TNFR1 might induce recruitment of RIP and IκB kinase (IKK), resulting in release of the NF-κB transcription factor from its inhibitor, IκB. NF-κB then enters the nucleus, activating transcription of survival genes such as IAPs, therefore antagonizing the proapoptotic factors.

these findings are inconsistent, and the role of JNK in receptormediated apoptosis is controversial. Finally, TRADD can bind receptor-interacting protein (RIP), resulting in activation of NF-κB, which antagonizes the apoptotic program. NF-κB is a transcription factor that activates expression of survival molecules such as IAPs and c-FLIP, a direct inhibitor of caspase 8 activation.7 IAPs were first described in baculoviruses, where they were shown to inhibit apoptosis of host cells following viral infection.8 Homologs such as XIAP and c-IAP-1 and -2 have since been identified in mammals; they seem to function by inhibiting caspase activation in both the death receptor and mitochondrial pathways.9–11 Another ligand-receptor pair that functions in both immune regulation and control of cancer is the TNF-related apoptosis-inducing ligand, TRAIL, and its receptors, TRAIL-R1/DR4 and TRAILR2/ DR5.12,13 TRAIL induces apoptosis in a variety of transformed cells but is much less toxic to normal cells than to abnormal ones. The intracellular signaling pathways induced by TRAIL are similar to those of the TNFR1. TRAIL is expressed as a cell-surface molecule on natural killer cells, and it can inhibit tumor growth.14 TRAIL also binds to decoy receptors TRAILR3/DcR1 and TRAIL-R4/DcR2, which sequester TRAIL from the signaling receptors, blocking TRAIL-induced apoptosis.15,16 Expression of these decoy receptors might provide the mechanism by which normal cells escape TRAIL-induced death, in that many tumor cells show lower expression of the decoy receptors.

Cell Death by Suicide The importance of mitochondria in apoptosis was demonstrated in 1996, when Liu and colleagues17 demonstrated that cytochrome c, a component of the electron transport chain normally contained in the mitochondrial intermembrane space, could initiate programmed cell death when present in the cytosol. This finding led to the demonstration that the intrinsic, or mitochondria-dependent, cell death program resulted from loss of mitochondrial integrity with release of intermembrane space contents (Fig. 5-2). Cytochrome c, on release into the cytosol, forms a complex with Apaf-1 and adenosine triphosphatase (ATP). This complex, known as the apoptosome, binds and activates procaspase 9.18 Other mitochondrial contents also participate in apoptosis. For example, Smac/DIABLO,19,20 Htra2,21 apoptosis-inducing factor (AIF), and endonuclease (endo) G also contribute to the cell death program.22–24 Inhibitory proteins such as XIAP and cIAPs-1 and -2 bind to the apoptosome and inhibit activation of caspases. Smac/DIABLO and Htra2 function by inhibiting the inhibitors, which allows apoptosis to proceed. The progression of the cell death program seems to depend on the relative ratios of apoptosis promoters and inhibitors. Evidence suggests that AIF, on the other hand, might function independently of caspase activity. Upon release from mitochondria, AIF translocates to the nucleus, where, in cooperation with endo G, it can initiate large-scale fragmentation of DNA. Controversy exists over the mechanism by which mitochondria lose integrity, resulting in release of their contents and initiation of the caspase cascade. General mechanisms proposed include mitochondrial dysfunction leading to matrix swelling and outer membrane rupture. Alternatively, loss of mitochondrial integrity could involve formation of specific pores large enough to release the intermembrane components. Another controversy exists over the importance of caspases in the actual death of cells following mitochondrial compromise as opposed to their role of simply orchestrating cellular disposal. Some investigators have shown that once mitochondria lose integrity, cells will die even in the absence of caspase activation.25 This finding is consistent with the idea that release of mitochondrial contents proceeds from large-scale catastrophe to the mitochondria, with cellular viability impossible in the absence of mitochondrial function. Others have suggested that, under some circumstances, cells can recover even after cytochrome c release if caspases are held in check.26 The function of IAPs in preventing apoptosome activity suggests that in some cases death can be prevented, but the role of caspases probably varies with different apoptotic stimuli.

Cell Life and Death • CHAPTER 5

might have largely redundant functions, presence of one or the other is critical to allowing apoptosis to proceed. The BH3-only molecules, in contrast, seem to have a signaling function. Various apoptotic stimuli induce expression and/or activation of specific BH3-only family members, which translocate to the mitochondria and initiate Bax/Bak-dependent apoptosis (Fig. 5-3). They could operate either by activating Bax and Bak or by inhibiting the antiapoptotic function of Bcl-2 and Bcl-Xl. It has been suggested that different members of this group could have distinct functions—some directly binding and activating Bax/Bak, and others indirectly activating these proapoptotic molecules by binding and antagonizing Bcl-2/Bcl-XL.31 The BH3-only protein Bid provides an example of cross-talk between the receptor-mediated and mitochondrial pathways of apoptosis. Bid is a target of active caspase 8.32,33 Once cleaved, this truncated form of the protein, tBid, can translocate to mitochondria, inducing cytochrome c release and amplification of the apoptotic signal. Although in some cell types death receptor engagement can kill cells independently of mitochondrial participation, in other cell types, this amplification step is critical to effect cell death.

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Figure 5-2 • Mitochondria-dependent apoptosis. Under conditions of cellular stress, the proapoptotic molecules Bax and/or Bak oligomerize at the mitochondria and induce release of mitochondrial contents, including cytochrome c. Cytochrome c then binds Apaf-1, and in combination with ATP forms the apoptosome. The apoptosome induces cleavage and activation of caspase 9, which activates a caspase cascade culminating in cell death. This process might be stimulated by DNA damage, which results in activation of p53, which, among several functions, promotes transcription of the BH3only molecules Noxa and PUMA. The apoptotic pathway is antagonized by Bcl-2, which can block cytochrome c release and mitochondrial dysfunction. Growth factor stimulation might also antagonize apoptosis, in part through activation of such survival factors as Akt, which can phosphorylate and inactivate proapoptotic molecules such as the BH3 protein, Bad.

Necrosis In contrast to apoptosis, necrosis has largely been considered an uncontrolled, default form of cell death. Morphologically, necrosis is characterized by swelling of organelles and loss of plasma membrane integrity, and it can be induced by exposure of cells to overtly pathologic conditions such as extreme temperature or pH, or mechanical force. Increasingly, however, it is being recognized that at least in many cases, necrosis can occur through a highly regulated process, dependent, as in apoptosis, on specific signaling pathways. The critical components of this process include effectors that induce irreversible bioenergetic compromise in the cell, and those that result in release of inflammatory mediators from the cell, inducing a host response.

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Bcl-2 Family Bcl-2 family proteins play a key role in regulation of mitochondrial integrity and programmed cell death. The Bcl-2 family includes proteins with both antiapoptotic and proapoptotic function. Bcl-2, the first identified member of this family, acts to prevent apoptosis, as do Bcl-XL and Mcl-1. Bax and Bak antagonize the function of the antiapoptotic family members and are critical in promoting apoptosis through the mitochondrial pathway. All these family members have multiple domains, termed Bcl-2 homology (BH) domains. A third type of Bcl-2 family member, exemplified by Bad, Bim, and others, consist of a single BH domain and are termed BH3-only molecules. The BH3-only family members also play a key role in promoting apoptosis. Extensive progress has been made in understanding the mechanisms by which these Bcl-2 family members regulate mitochondrial integrity and apoptosis, although many questions remain. The antiapoptotic molecules Bcl-2 and Bcl-XL localize to the mitochondria and maintain mitochondrial integrity. This could occur through promotion of exchange of metabolic substrates across the mitochondrial membrane, allowing maintenance of respiration.27 Alternatively, antiapoptotic Bcl-2 family members might bind and inhibit the function of proapoptotic family members.28 The multidomain proapoptotic molecules, Bax and Bak, are required for mitochondrial apoptosis initiated through most stimuli.29 Mice with targeted knockouts of either of these molecules show largely normal apoptotic function, but loss of both Bax and Bak results in a severe defect in apoptosis.30 This finding suggests that although these two molecules

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Figure 5-3 • BH3-only proteins initiate apoptosis. Several BH3-only proteins exist and could function in various ways to promote apoptosis. Bid is cleaved by caspase 8 on death receptor stimulation and subsequently translocates to mitochondria, where it can activate oligomerization of Bax and Bak. Other proteins, such as Bad and Noxa, function by inhibiting antiapoptotic Bcl-2 molecules. Noxa is transcriptionally regulated by p53, and Bad is regulated by inhibitory phosphorylation, which is growth factor dependent.

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Necrosis can be activated both by death receptor signaling and by cell-intrinsic events. The absence of specific caspase activities, such as caspase 8, or the presence of apoptosis inhibitors, such as may be present in viral infection, may block apoptotic death in response to TNFR or Fas ligand stimulation, but induce a necrotic program. The regulated nature of this necrotic stimulus is illustrated by the fact that blocking the activity of the RIP kinase blocks necrotic cell death.34 Programmed necrosis may also be an important way in which organisms dispose of cells that have accumulated DNA damage. Although cells sustaining DNA damage in many cases die an apoptotic death, a functioning apoptotic pathway is not required for DNA damage–induced death. Zong and colleagues have shown that the activation of poly(ADP-ribose) polymerase (PARP) in response to DNA damage plays a critical role in the death of actively growing cells sustaining such damage.35 PARP binds to DNA strand breaks, catalyzing the synthesis of poly(ADP-ribose) polymers on histones, promoting recognition of strand breaks by DNA repair enzymes. β-nicotinamide adenine dinucleotide (NAD) is a critical substrate of this reaction, and therefore PARP activation leads to depletion of NAD from the cytosol. Because cytosolic NAD is required for glycolytic metabolism, this consumption of NAD in response to DNA damage compromises the production of ATP in cells that are dependent on glycolysis, rather than mitochondrial metabolism, for energy production, such as rapidly proliferating cells and most cancer cells. In contrast, vegetative cells, deriving the bulk of their ATP from oxidative phosphorylation, remain bioenergetically intact. This necrotic death via PARP activation does not depend on apoptotic mediators such as Bak and Bax. The activation of programmed necrosis might act as a warning system to the organism, resulting in release of proinflammatory mediators and activation of the immune system. This could be of particular benefit in the cases discussed previously, viral infection and cancer, in allowing the organism to mount an effective, global response to the insult.

Autophagy Autophagy is a cellular process in which cytoplasmic components, including proteins and organelles, are sequestered into acidic vacuoles for degradation. The degradation products then become available as a source to support biosynthesis and energy production. Autophagy is promoted by metabolic stress and forms a response to metabolic emergencies within the cell. The role of autophagy in cell death is not clear and is under active investigation. Some have hypothesized that autophagy is in and of itself a mechanism of cell death. Other investigators, however, have suggested that autophagy is instead a protective mechanism, allowing the cell to maintain bioenergetic integrity in the face of an inability to access nutrients. In this scenario, death would occur as a result of the depletion of intracellular resources, despite, not because of, autophagy. Support for both these hypotheses can be found in the experimental literature. Lum and colleagues demonstrated that cells deprived of growth factors, leading to a defect in nutrient uptake, could maintain survival through activation of autophagy.36 Inhibition of autophagy induction in this system led to rapid cell death. On the other hand, the loss of beclin-1, a gene product necessary for the induction of autophagy in mammals, predisposes to cancer in animal models,37,38 and loss of beclin-1 is seen in some tumor types. The mechanism by which beclin-1 functions as a tumor suppressor is unclear, however. Beclin-1 associates with Bcl-2 and therefore might have a more direct role in apoptosis. Further investigation will be needed to clarify the role of autophagy in cancer.

APOPTOSIS IN CANCER The events that can lead to mitochondrial apoptosis are varied. These include loss of normal survival-promoting extrinsic signals, DNA

damage, metabolic stress such as hypoxia and nutrient limitation, oncogenic stresses, and toxins. Under normal circumstances, cells require extrinsic signals to promote cellular homeostasis and survival. These signals include growth factors and cell-cell or cell-matrix contact. Survival signals demonstrate to the cell that it is in an appropriate location and that cells like it are present in an appropriate number. Loss of these signals can occur if the cell finds itself in an ectopic position (loss of cell-cell or cell-matrix contact) or when specific cell types are in excess numbers (causing competition for growth factors), resulting in apoptosis.

Oncogenes as Triggers of Apoptosis Oncogenic stresses, such as activation of Myc or loss of Rb with subsequent uncontrolled activation of the cell cycle machinery, can induce apoptosis. The mechanisms by which this occurs are not clear, but it is well demonstrated that oncogenesis through these pathways requires the additional step of inhibition of programmed cell death. In that Myc promotes activity of biosynthetic pathways, one possibility is that it promotes metabolic stress.39,40 Other oncogenic stresses, such as loss of the Rb tumor suppressor or DNA damage, promote activity of the tumor suppressor p53.41 Research performed in recent years has demonstrated clearly the importance of apoptotic pathways in control of tumorigenesis. Cancer cells, through inappropriate growth and proliferation, outstripping of resources, and translocation to environments to which they are not adapted, subject themselves to death triggers and therefore must disable the apoptotic response to survive. A critical point in understanding the role of apoptosis in cancer is that lack of death alone does not suffice to make a cancer cell. Rather, tumors must activate proliferative, growth, and invasion programs—the targets of traditional oncogenes. It is these programs and their tendency to overwhelm the cell’s survival signals that place the cell under apoptotic stress. Disabling of apoptotic pathways makes the cancer cells intrinsically defective in initiation of programmed cell death; such disabling promotes resistance to antineoplastic therapy but also suggests that many cancer cells live constantly “on the edge” of death. It is possible that restoration of apoptotic function could suffice for, or at least contribute to, the elimination of a tumor.

Tumor Suppressors Promote Apoptosis p53 p53 is one of the best studied tumor suppressors, and its function is lost in at least half of human solid tumors. p53 activity is induced through stabilization of the protein in response to various oncogenic signals (including DNA damage), resulting in inhibition of cell growth through either cell cycle arrest or induction of apoptosis.42 The specific mechanisms by which one or the other response occurs are not completely clear but could include duration of activity or the prevailing state of the cell. The activity of p53 seems to be mediated largely through its ability to act as a transcription factor, playing the roles of both transcriptional activator and repressor for different targets. p53 activity and levels are controlled in large part by its upstream regulator, MDM2. MDM2 protein binds p53 and exports it from the nucleus, blocking its ability to act as a transcriptional regulator. MDM2 also targets p53 for proteasome-dependent degradation through its activity as a ubiquitin ligase. MDM2, in turn, is inhibited in its inhibition by p14ARF. In the absence of loss of p53 itself, overexpression of MDM2 can act as an oncogene, functionally suppressing p53 activity. MDM2 is overexpressed in multiple human tumor types, including lung cancer, brain cancers, and breast cancers.43,44 p14ARF, conversely, has been shown to be lost in various tumors, including colon cancers.45 Abnormalities of these upstream regulators tend to occur in tumors that retain wild-type p53, dem-

Cell Life and Death • CHAPTER 5

onstrating that dismantling of the p53/MDM2/p14ARF pathway plays a key role in tumorigenesis. In addition to regulation by MDM2, post-translational modifications of p53 also influence its biologic activity. Phosphorylation and acetylation affect p53 stabilization and DNA-binding activity, and it seems that both these modifications promote p53 function. Mutant p53 proteins found in cancer cells are in some cases highly acetylated and phosphorylated, leading to stabilization and accumulation of the mutant protein.46 This stabilized mutant protein can then act as a dominant negative, forming inactive complexes with residual wildtype protein. The antitumor activity of p53 is mediated largely through its transcriptional effects. p53-dependent genes play multiple roles in apoptosis. For example, several proapoptotic Bcl-2 family members are transcriptionally activated by p53, including Bax and the BH3only proteins Noxa and PUMA.47,48 Expression of Apaf-1, another important element in the mitochondrial pathway, is induced through p53 activity. p53 promotes death receptor pathways through activation of Fas transcription, and it inhibits survival signaling through induction of PTEN (see later discussion).42 Although this list of antitumor effects of p53 is far from exhaustive, the foregoing examples provide insight into the importance of this pathway in blocking tumorigenesis. Nontranscriptional roles for p53 in apoptotic regulation have also been proposed, including direct binding and inhibition of the activity of Bcl-XL and Bcl-2.49–51

Proapoptotic Bcl-2 Family Members In contrast to the oncogenic effects of the antiapoptotic Bcl-2 family members, proapoptotic family members, particularly Bax, have been implicated as tumor suppressors. Bax and its functional homolog, Bak, are critical in mediating apoptosis through the mitochondrial pathway induced by many cellular stresses.30,46 Experimental models have suggested that Bax and Bak have p53-independent function in suppression of tumorigenesis and act as tumor suppressors.52 In one study, murine cells expressing adenoviral E1A, a proliferative factor, and dominant negative p53 were unable to form tumors in mice. Additional loss of Bax and Bak, however, resulted in the formation of highly invasive tumors, emphasizing the capability of these molecules to inhibit carcinogenesis. Furthermore, mutations in Bax and Bak have been identified in many colon and gastric cancers.53,54 BH3-only proteins could also be important in preventing tumorigenesis. Although evidence implicating them as bona fide tumor suppressors is scant, BH3-only proteins play a role in the response to apoptotic stimuli of various death pathways, including p53 and death receptors.

PTEN The activity of the survival pathway mediated through PI3K and Akt, discussed subsequently, is antagonized by phosphatase and tensin homolog on chromosome 10 (PTEN), a dual-specificity (protein and lipid) phosphatase that degrades phosphatidylinositol-3,4,5triphosphate [Ptd(3,4,5)P3] back to the bisphosphate form, terminating the signal of PI3K. PTEN was discovered in the search for a tumor suppressor on chromosome 10 that is frequently lost in glioblastoma and prostate cancer. Since its discovery, researchers have shown that PTEN acts as a negative regulator of Akt.55 Tumorigenesis in response to loss of PTEN depends in part on deregulation of Akt activity.56 The frequency of PTEN loss in human tumors is exceeded only by that of p53. PTEN function is abnormal in the majority of glioblastomas and prostate cancers and has been described in many other human cancers, including breast cancer and endometrial cancer. Furthermore, mice bearing an inactive allele of PTEN develop tumors in multiple organ systems.57,58 The loss of a single allele of PTEN seems to be sufficient to promote tumorigenesis, as haploinsufficient mice frequently do not lose the second allele upon development of tumors, and loss of the second allele is a late event in many tumors.

Survival Factors Prevent Apoptosis in Cancer Cells Antiapoptotic Bcl-2 Family Members The central role of Bcl-2 family members in control of apoptosis suggests that these proteins may be appropriate targets of dysregulation in tumorigenesis, and, as predicted, many tumors show alterations in these proteins. The earliest description of antiapoptotic activity in cancer was that of overexpression of Bcl-2 in follicular lymphoma. This overexpression is brought about by the t(14;18) translocation, which brings the Bcl-2 gene locus into juxtaposition with the immunoglobulin heavy-chain enhancer, an abnormality found in at least 85% of follicular lymphomas. Since this discovery, Bcl-2 overexpression has been found in a multitude of different cancers, including other types of lymphoma and solid tumors such as breast cancer.59 The importance of Bcl-2 in the pathogenesis of cancer has also been demonstrated in experimental models. For example, mice expressing transgenic c-Myc in B cells develop lymphoma with a long latency period, suggesting the need for other transforming mutations for tumorigenesis. Coexpression of Bcl-2 markedly shortens the latency period, demonstrating synergy of these two molecules in lymphomagenesis.2 Myc activation in cell lines induces apoptosis, in part through activation of p53. These experiments imply that a critical step in Myc-induced transformation is inhibition of apoptosis, and that Bcl-2 can provide this function. Bcl-Xl, another antiapoptotic family member with function similar to Bcl-2, also seems to play a role in both experimental and naturally occurring human tumors.60–62 Mice expressing transgenic Bcl-2 and Bcl-XL illustrate the important concept that inhibition of apoptosis alone does not induce tumorigenesis. Enforced expression of these molecules in B lymphocytes of mice leads to accumulation of lymphocytes, but lymphoma develops only rarely.63 Instead, Bcl-2 and Bcl-XL facilitate lymphomagenesis by inhibiting the death that normally accompanies oncogenic activation. Likewise, lymphocytes bearing the t(14;18) can be detected in some healthy people with no evidence of lymphoma.58 Taken together, these points of evidence emphasize that although suppression of apoptosis is an important step in transformation, it is not sufficient to drive carcinogenesis.

NF-κB Another pathway through which tumor cells might suppress apoptosis is the nuclear factor-κB (NF-κB) pathway. As discussed previously, activation of NF-kB during death receptor stimulation sets into motion a transcriptional program that inhibits apoptosis and promotes survival. NF-κB, under normal circumstances, is held in check by binding of the inhibitor of NF-κB, IκB. NF-κB is released on phosphorylation of IκB through activity of two IκB kinases, IKKα and IKK-β.64 Phosphorylation targets IκB for ubiquitination and degradation by the 26S proteasome, releasing NF-κB to translocate to the nucleus and activate its target genes (Fig. 5-4). Oncogenic stimuli, as well as survival signals, promote NF-κB activation. For example, Ras-mediated transformation stimulates transcriptional activity of NF-κB.65 Furthermore, the Bcr-Abl fusion protein, a causative mutation in chronic myeloid leukemia, activates NF-κB by promoting nuclear translocation.66 Virally induced transformation by human leukemia/lymphoma virus type 1 is dependent in part on Tax-mediated activation of IKKs.67 The proto-oncogene Bcl-3 was identified on one arm of a translocation found in some lymphoid malignancies, t(14;19). This translocation brings the Bcl-3 gene in proximity to the immunoglobulin heavy-chain enhancer, resulting in its overexpression. The Bcl-3 gene encodes a member of the IκB family, but this gene product seems to function differently from other IκBs.68 Specifically, Bcl-3 localizes to the nucleus and modifies the function of NF-κB subunits.69 When overexpressed in normal T cells, Bcl-3 promotes survival following cellular activation, and expression seems to be induced on treatment

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PI3K/Akt

IKKbeta IKKalpha

IkB NFkB

P Ubiquitination

IkB

Phosphorylation

P IkB

NFkB Ubiquitin

Degradation

26S Proteasome

Inhibitors of apoptosis

Figure 5-4 • NF-κB is a transcriptional activator of multiple antiapoptotic genes. Under resting conditions, NF-κB is bound by its inhibitor, IκB, which prevents its transcriptional activity by maintaining it in the cytoplasm. Activation signals stimulate activity of IκB kinases (IKK), which phosphorylate IκB. Phosphorylated IκB can then be ubiquitinated and targeted to the proteasome for degradation. This process releases NF-κB to enter the nucleus, where it activates transcription from its target promoters.

with immunologic adjuvants, suggesting a physiologic survival role for it in the immune system.70 Mice expressing a Bcl-3 transgene in B lymphocytes do not develop lymphoma, but they do show accumulation of B cells and hyper-responsiveness of the immune system, similar to the findings in Bcl-2 transgenic mice.

Many tumors lose extracellularly derived survival signals during pathogenesis, either by outstripping limited growth factors or by translocating to inappropriate environments. One signaling pathway that has been implicated in provision of survival signals is the phosphatidylinositol-3-kinase (PI3K) pathway.71,72 Many growth factors, including interleukin-2, interleukin-3, platelet-derived growth factor, and insulin-like growth factor, signal in part through PI3K. PI3K phosphorylates phosphatidylinositide-4,5-bisphosphate (Ptd(4,5)P2) to Ptd(3,4,5)P3, which acts as a second messenger, activating downstream effectors such as the serine/threonine kinase Akt, also known as protein kinase B (PKB; Fig. 5-5). Akt seems to be a critical survival factor in many cell types, and its activity might promote survival through multiple functions, such as phosphorylation and inactivation of the BH3-only protein Bad, and through the Forkhead family transcription factor FKHRL1.73 In addition, Akt acts in the insulin signaling pathway to promote glucose uptake, and it seems to play a similar role in non-insulin-responsive cells, promoting glucose uptake and glycolysis upon growth factor stimulation. It has been proposed that this metabolism-promoting effect of Akt might protect mitochondrial integrity through maintenance of substrate availability, thereby preventing apoptosis (Box 5-1).74,75 Another important mediator in the PI3K/Akt pathway is the mammalian target of rapamycin (mTOR). Akt indirectly activates mTOR through inhibitory phosphorylation of the TSC1/TSC2 complex,76 leading to a complex interplay of proteins culminating in mTOR activation. Once active, mTOR promotes protein translation, leading to cell growth and proliferation. The complexity of this pathway, however, including multiple interacting protein partners and feedback mechanisms, makes it clear that the exact effects of mTOR activity on cell fate and cancer development require further elucidation. Multiple studies have demonstrated the importance of Akt activity in tumorigenesis, either through amplification of one of the three AKT genes, or through loss of PTEN function. Amplification of Akt results in a similar phenotype to PTEN loss and has been found in gastric cancers, breast cancers, and other tumor types.77–79 Furthermore, animal models also have demonstrated the role of Akt in tumorigenesis. Mice expressing constitutively active Akt in T cells develop thymic lymphoma at a high rate.80 Furthermore, inhibition of mTOR activity diminishes tumor formation in mice heterozygous for PTEN, demonstrating the importance of mTOR in tumorigenesis mediated by this pathway.81

P

P

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?

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PTEN

Akt

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Akt

72

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ras

P

PP2A Mdm2 phosphatidylinositol phosphatidylinositol 4–phosphate

p53 NF–κB

FoxO3a

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Figure 5-5 • The PI3K-Akt pathway is activated by multiple growth factor receptors and oncogenes and plays a critical role in promoting cell survival. PI3K is activated by growth factor stimulation or intracellular signals such as activated Ras or the oncogene BCR-Abl. Active PI3K phosphorylates phosphatidylinositols to phosphatidylinositol3,4,5-triphosphate (PIP3) at the plasma membrane. PIP3 recruits Akt and its activating kinases, PDK1 and an uncharacterized PDK2, to the membrane, where Akt is phosphorylated and activated. Akt then promotes survival functions such as Bad and Forkhead inactivation, activation of NF-κB and MDM2, and glucose metabolism. PI3K activity is antagonized by the phosphatase PTEN, which degrades PIP3.

Cell Life and Death • CHAPTER 5 Box 5-1.

METABOLIC DEREGULATION IN CANCER

Cells that lose survival signals fail to maintain themselves and undergo progressive atrophy. Loss of survival-inducing signal transduction leads to downregulation of cell-surface nutrient transporters (e.g., glucose transporters) and to a decreased rate of glucose metabolism, as indicated by decreased levels of hexokinase and phosphofructokinase, two key regulatory enzymes in the glycolytic pathway.69,100–102 The loss of glycolytic products reduces delivery of substrate to the mitochondria, resulting in mitochondrial damage. Nutrient limitation might have similar metabolic effects when cells accumulate in excess of the existing vascular supply. In the 1920s, Warburg observed that cancer cells metabolize glucose at a higher rate than their normal counterparts. Furthermore, he found that the malignant cells relied on glycolysis for a disproportionate amount of their ATP production, with comparatively little energy produced by oxidative phosphorylation. Warburg hypothesized that this shift to aerobic glycolysis resulted from defects in mitochondrial function in the cancer cells. Other researchers have subsequently confirmed Warburg’s findings of increased aerobic glycolysis in cancer cells. The high rate of glucose uptake in tumors has formed the basis of a novel imaging modality, positron emission tomography (PET), using a fluorine-18-labeled glucose analog. A study evaluating PET scanning in lymphoma showed that more than 90% of lymphomas—including very indolent tumors— metabolize glucose at an abnormally high rate.103 Although several research groups have found mutations in genes that encode mitochondrial enzymes in cancer cells, the fact that normal lymphocytes are unable to maintain glucose uptake and glycolysis in the face of dropping ATP levels suggests an alternative hypothesis. In this scenario, normal cells lack the ability to take up sufficient glucose to maintain themselves and instead are dependent on extrinsic signal

transduction to maintain the expression and function of nutrient transporters. As a corollary, mutations that activate such signaling pathways could permit the cell to take up glucose in excess of that needed for bioenergetic or synthetic activities. Under such conditions, cells would secrete the excess glucose as lactate and have sufficient bioenergetic reserves to support entry into and progression through the cell cycle. These findings raise the possibility that cancer cells, in the process of transformation, turn on signaling pathways that allow autonomous access to nutrients and metabolic pathways, rendering the cells independent of the extracellular signals normally required to maintain nutrient uptake. This facilitated access to nutrients would provide substrate to mitochondria, allowing maintenance of mitochondrial function and suppression of apoptosis even in the absence of growth factor signaling. If this hypothesis is true, autonomous access to nutrients is probably accomplished in multiple ways by different tumors. One potential contributor to this goal is Akt. Akt is critical in the insulin signaling pathway to activate glucose uptake in insulinresponsive tissues, and it seems to play a similar role in non-insulinresponsive cells on growth factor stimulation. Although the role of Akt as an oncogene might include several functions, it clearly has the potential to promote glucose transporter expression and activity of glycolytic enzymes. The role of metabolic control in tumorigenesis is poorly understood. Elucidation of this fundamental process in cancer might offer greater appreciation for the mechanisms of carcinogenesis. Furthermore, the recognition of the importance of metabolic control in cancer could offer new therapeutic targets, improving our chances of defeating cancer in the future.

Epigenetic Gene Silencing Inhibition of expression of tumor suppressor genes through epigenetic mechanisms is emerging as an important mechanism by which tumor cells might disable proapoptotic pathways. Promoter methylation at CpG islands could repress transcription of genes in the absence of mutation (Fig. 5-6). In addition, histone deacetylation might also turn off gene expression, possibly by inhibiting access of transcription factors (TF). For example, Soengas and associates82 have shown that many melanoma cells (both primary tumors and cell lines) suppress Apaf-1 expression. This results in inhibition of p53dependent apoptosis in these cells and renders them resistant to chemotherapy. Apaf-1 suppression is mediated not by mutation of Apaf-1 but instead through methylation, because treatment with the methylation inhibitor 5-azacytidine restores both Apaf-1 expression and chemosensitivity. Methylation seems to play an important role in suppression of tumor suppressors in other tumors as well. For example, childhood neuroblastomas show loss of expression of caspase 8 through methylation of its promoter at high frequency.83 Finally, promoter methylation of the p14ARF locus might suppress its expression in multiple tumor types.84 Acetylation of histones can also repress transcription of genes important in regulation of cell death. Global changes in histone acetylation have been found in cancer cells and are hypothesized to contribute to aberrant gene expression promoting survival and malignancy.85

MANIPULATING CELL DEATH IN CANCER TREATMENT As discussed previously, cancer cells must dismantle or inhibit apoptotic pathways to maintain their transformed phenotype. Many trans-

TF

Methyl

Methyl

CpG

CpG

Promoter

Target gene

Methylation inhibitor

Methyl TF Promoter

Target gene

Figure 5-6 • Epigenetic gene silencing is a mechanism by which cancer cells turn off tumor suppressor gene expression. In the process of development or oncogenesis, genes might be silenced through promoter methylation. In tumors, promoters of tumor suppressor genes such as Apaf-1 and caspase 8 are frequent targets of methylation. Methylation inhibitors such as 5azacytidine reactivate expression of these tumor suppressor genes, potentially contributing to anticancer therapy.

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formation-inducing mutations also have the effect of promoting programmed cell death, and cells are unable to pass through these initial changes to become cancer unless apoptosis is inhibited. This fact has two correlates. First, most traditional cancer chemotherapies act through induction of apoptosis, and the intrinsic apoptotic defects of these cells could make them inherently resistant to chemotherapy. On the other hand, cancer cells are constantly living “on the edge,” pushed beyond the normal limits of cell viability. This fact might make tumor cells profoundly susceptible to apoptosis if either the defect can be corrected or another death pathway can be activated. This reasoning is the basis of the many attempts currently in progress to design therapies that will attain one of these objectives.

Restoration of Apoptotic Capability Restoration of lost apoptotic pathways can be accomplished by several methods. First, if a proapoptotic gene, such as p53, is mutated, gene therapy provides a direct way in which to restore expression of the missing protein. One approach has been to attempt to deliver the gene in question via an adenoviral vector, for example with p53 in tumors of the head and neck, and in lung tumors86,87; some success has been seen with intratumoral injections. Systemic therapy poses an additional challenge, however, in terms of both feasibility and safety. The effect of gene therapy with p53 would be expected to be seen only in cells in which the transgene is expressed, requiring that every cell be infected by the vector. Furthermore, the safety of adenovirus vectors remains at issue. Another, similar approach has been to target cancer cells lacking p53 by taking advantage of the fact that adenovirus must inactivate p53 to replicate in cells. Usually this is accomplished through the activity of the virus’s E1Bp55 protein, which binds and inactivates p53. A virus that lacks E1Bp55 is unable to replicate in normal cells. Cancer cells that lack p53 present a viable target, however, allowing the virus to accomplish its lytic life cycle and killing the cell. This approach is being used clinically with the drug ONYX-015, which, similar to p53 gene therapy, has shown success with intratumoral injection in combination with chemotherapy in head and neck cancers.88 Once again, systemic delivery seems more problematic. In a strategy that is growing in importance in cancer therapy, small molecules targeting the p53 pathway have been designed to restore p53 function. One approach takes advantage of the fact that, in most cancers, p53 is inactivated not by deletion but rather through a point mutation that results in accumulation of inactive protein. CP-31398 is a drug found in a screen for therapeutic agents that restore wild-type conformation to mutant p53 in tumor cells.89 Since its identification, researchers have found conflicting data regarding its ability to restore p53 function in tumor cells, with some studies finding evidence of restoration of p53 function but others reporting nonspecific toxicity.90,91 Another approach has been to target the interaction of p53 with the inhibitory molecule MDM-2. Small-molecule inhibitors have been identified and have shown promise in preclinical studies.92 Early-phase clinical studies using this approach are expected to be underway shortly. The realization that many cancer cells turn off expression of proapoptotic genes by epigenetic mechanisms has provided another approach to the restoration of tumor suppressor function. Histone deacetylase inhibitors have entered clinical trials and have shown evidence of activity.93,94 One of these, suberoylanilide hydroxamic acid, has shown activity in several tumor types and has been approved for use in cutaneous T-cell lymphomas. DNA methylation inhibitors are also in development.95 The lack of specificity of these treatments raises theoretical concerns that genes which have been silenced in differentiation (e.g., hTERT, the human telomerase gene that might play a role in tumor promotion) or other tumor-promoting genes could be turned on through demethylation or deacetylation. Silencing of TRAIL decoy receptors through methylation has been demonstrated in cancer cells.96 The end result of these therapies

could depend on the balance of genes silenced through epigenetic mechanisms in each cancer, but early clinical trials are promising.

Inhibition of Survival Factors Small-molecule inhibitors might show promise in the inhibition of survival factors expressed in cancer cells. The 26S proteasome is important in myriad cellular pathways, but its importance in activation of NF-κB, through degradation of IκB, has raised the possibility that inhibition of proteasomal degradation could have proapoptotic effects in cancer cells. Akt has also been reported to phosphorylate the tumor suppressor genes TSC1 and TSC2 and target them for proteasomal degradation.97 Bortezomib, a proteasomal inhibitor, has shown activity in several hematologic malignancies, notably multiple myeloma,98 in which it has been approved by the FDA for use, and mantle cell lymphoma. The activity of bortezomib as a sensitizing agent, lowering the threshold for apoptosis in response to other cytotoxic agents, is also under active investigation. Small-molecule inhibitors targeting several other components of survival pathways are under development. Inhibitors of mTOR and Akt are currently in clinical trials, and a small-molecule inhibitor of Bcl-2 is undergoing preclinical testing at the time of this writing.99 Inhibitors of IAP molecules are also drawing interest and under preclinical development. Antisense strategies have been explored to target antiapoptotic molecules. Antisense oligonucleotides act by binding to specific messenger RNAs, forming double-stranded RNA complexes.These complexes might inhibit expression of the target messenger RNAs, either by blocking translation or through targeting them for destruction by the cell through recognition of abnormal double-stranded RNA. The most developed of these antisense oligonucleotides is one targeted against Bcl-2,100–102 oblimersen, also known as Genasense. Trials in hematologic malignancies and in several solid tumors have shown some activity and it might be particularly useful as a sensitizing agent when used in combination with other cytotoxic therapies.

Death Receptor Activation As discussed in previous sections, death receptor signaling seems to play an important role in some tumors. Initial studies examined the use of Fas and TNF as death-inducing ligands. Yet, although these molecules demonstrated antitumor activity, their utility as therapeutic agents has been compromised by toxicity, with both normal cells and malignant cells targeted for death. Investigations of TRAIL have raised the possibility that this ligand might be more selective for tumor cells. Most normal cells express decoy receptors that sequester TRAIL, preventing it from sending an intracellular death signal. Tumor cells seem to be uniquely sensitive to the apoptotic stimulus of TRAIL; one reason might be downregulation of decoy receptor expression, possibly by promoter methylation.87 Studies in mice and nonhuman primates have demonstrated minimal toxicity to normal cells with administration of TRAIL.103,104 When researchers examined the effects of TRAIL on human hepatocytes in vitro, however, it was found to cause significant cell death, raising the question whether TRAIL, like its counterparts Fas and TNF, might be too toxic for use in humans at therapeutic doses.105 Early-phase clinical trials of TRAIL are underway.

Induction of Necrosis The recognition of necrosis as a regulated, physiologic process raises the possibility that induction of necrotic cell death might present advantages in targeting cancer cells. Some currently used therapeutics might act in large part through inducing necrosis, and a strategy priming cells for necrotic cell death could contribute to the effectiveness of these treatments. As discussed previously, the metabolic state of a cancer cell seems to predispose it to necrosis in response to DNA damage through loss of the ability to produce ATP through glycolysis. Cancer cells might be sensitized to the effects of DNA-damaging

Cell Life and Death • CHAPTER 5

agents if also treated with agents that interfere with glycolysis. Whereas all cells depend to some extent on glycolytic metabolism, a modest inhibition of glycolysis in cells that are highly dependent on this pathway could potentially enhance the effects of DNA damagebased treatments. Other strategies to induce necrosis, such as induction of reactive oxygen species and promotion of RIP activity, might also be optimized as our understanding of these pathways increases. A potential advantage of necrosis as a treatment strategy lies in the potential to promote an immune reaction, and therefore possibly recruit the immune system to assist in fighting the tumor.

SUMMARY Understanding of mechanisms of cell death and their importance in tumorigenesis is evolving rapidly. Our interpretation of these findings

will certainly undergo revision as more information constantly becomes available. The current appreciation for these processes, however, already has provided opportunities for advancement of patient care. In the future, research further clarifying the basic mechanisms of programmed cell death—including events mediating mitochondrial demise along with a better understanding of the nature of death signals—will continue to improve our arsenal of potential weapons against cancer. As has been found with traditional antineoplastic therapies, success will most likely be found with a combination of therapeutic approaches. These might include the addition of apoptosis-based therapies to traditional agents, combining proapoptotic with antisurvival approaches, along with strategies to manipulate both forms of cell death that await development as knowledge of these complex processes evolves.

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68. Kerr LD, Duckett CS, Wamsley P, et al: The proto-oncogene bcl-3 encodes an I kappa B protein. Genes Dev 1992;6:2352–2363. 69. Zhang Q, Didonato JA, Karin M, McKeithan TW: Bcl3 encodes a nuclear protein which can alter the subcellular location of NF-kappa B proteins. Mol Cell Biol 1994;14:3915–3926. 70. Mitchell TC, Hildeman D, Kedl RM, et al: Immunological adjuvants promote activated T cell survival via induction of Bcl-3. Nat Immunol 2001;2:397–402. 71. Kauffmann-Zeh A, Rodriguez-Viciana P, Ulrich E: Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB. Nature 1997;385:544–548. 72. Kennedy SG, Wagner AJ, Conzen SD, et al: The PI 3-kinase/Akt signaling pathway delivers an antiapoptotic signal. Genes Dev 1997;11:701–713. 73. Dudek H, Datta SR, Franke TF, et al: Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 1997;275:661–665. 74. Vander Heiden MG, Plas DR, Rathmell JC, et al: Growth factors can influence cell growth and survival through effects on glucose metabolism. Mol Cell Biol 2001;21:5899–5912. 75. Plas DR, Talapatra S, Edinger AL, et al: Akt and Bcl-xL promote growth factor-independent survival through distinct effects on mitochondrial physiology. J Biol Chem 2001;276:12041–12048. 76. Manning BD, Tee AR, Logsdon MN, et al: Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/Akt pathway. Mol Cell 2002;10:151–162. 77. Cheng JQ, Godwin AK, Bellacosa A, et al: AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas. Proc Natl Acad Sci USA 1992;89:9267–9271. 78. Cheng JQ, Ruggeri B, Klein WM, et al: Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. Proc Natl Acad Sci USA 1996;93:3636–3641. 79. Staal SP: Molecular cloning of the Akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma. Proc Natl Acad Sci USA 1987;84:5034–5037. 80. Malstrom S, Tili E, Kappes D, et al: Tumor induction by an Lck-MyrAkt transgene is delayed by mechanisms controlling the size of the thymus. Proc Natl Acad Sci USA 2001;98:14967–14972. 81. Podsypanina K, Lee RT, Politis C, et al: An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/− mice. Proc Natl Acad Sci USA 2001;98:10320–10325. 82. Soengas MS, Capodieci P, Polsky D, et al: Inactivation of the apoptosis effector Apaf-1 in malignant melanoma. Nature 2001;409:207–211. 83. Teitz T, Wei T, Valentine MB, et al: Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN. Nat Med 2000;6:529–535. 84. Esteller M, Cordon-Cardo C, Corn PG, et al: p14ARF silencing by promoter hypermethylation mediates abnormal intracellular localization of MDM2. Cancer Res 2001;61:2816–2821. 85. Fraga MF, Ballestar E, Villar-Garea A, et al: Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 2005;37:391–400. 86. Merritt JA, Roth JA, Logothetis CJ: Clinical evaluation of adenoviral-mediated p53 gene transfer: review of INGN 201 studies. Semin Oncol 2001;28:105–114. 87. Shimada H, Matsubara H, Shiratori T, et al: Phase I/II adenoviral p53 gene therapy for chemoradiation resistant advanced esophageal

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6

Cancer Immunology Drew M. Pardoll

S U M M ARY • Cancer cells develop, grow, invade, and metastasize in the context of an organized microenvironment. This is reflected by the fact that for many cancers the majority of cells within the tumor mass are nontransformed. • The relationship between the tumor cells and the nontransformed cells composing the tumor microenvironment is dynamic and active. • The immune system is a major component of the tumor microenvironment, and therefore, the tumor must actively organize the immunologic component of its microenvironment.

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• Because the immune response— particularly that mediated by killer T cells and cells of the innate immune system—can be a potent enemy to the tumor, a successful cancer must develop mechanisms to instruct the immune system to “tolerate” its existence, particularly when it is invading through tissue barriers and metastasizing to many organ sites. • If a tumor fails to develop these tolerance induction and immunologic resistance mechanisms, the immune system will eliminate it. • Oncogenic pathways in the tumor not only mediate cell growth, metabolic

OVERVIEW Historically, interest in cancer immunology stemmed from the perceived potential activity of the immune system as a weapon against cancer cells. In fact, the term “magic bullet,” commonly used to describe many visions of cancer therapy, was coined by Paul Ehrlich in the late 1800s in reference to antibodies targeting both microbes and tumors. Central to the concept of successful cancer immunotherapy are the dual tenets that tumor cells express an antigenic profile distinct from their normal cellular counterparts and that the immune system is capable of recognizing these antigenic differences. Support for this notion originally came from animal models of carcinogen-induced cancer in which it was demonstrated that a significant number of experimentally induced tumors could be rejected upon transplantation into syngeneic immunocompetent animals.1–4 Extensive studies by Prehn on the phenomenon of tumor rejection suggested that the most potent tumor rejection antigens were unique to the individual tumor.5 As cancer genetics and genomics has exploded over the past decade, it is now quite clear that altered genetic and epigenetic features of tumor cells indeed result in a distinct tumor antigen profile. Overexpression of “oncogenic” growth factor receptor tyrosine kinases such as HER2/Neu and epidermal growth factor receptor (EGFR) via epigenetic mechanisms has provided clinically relevant targets for one arm of the immune system—antibodies.6,7 Indeed, monoclonal antibodies are the largest growing single class of cancer therapeutics based on successful new U.S. Food and Drug Administration approvals. In striking contrast, cellular immunotherapy of cancer has been quite disappointing in establishing therapeutic success in clinical trials thus far. Emerging insights about the nature of the interaction between the

activity, and antiapoptotic activity, they also mediate interactions with the immune system. • Ultimately, tumors do more than merely inhibit effecter functions of the immune system that can be detrimental to them. Tumors can in fact alter immunologic activity to promote tumor growth and development. • We are now beginning to understand the molecular and cellular basis for tumor–immune system interactions, providing specific molecular targets for immunologic intervention.

cancer and the immune system have led us to understand why cellbased cancer immunotherapy approaches such as therapeutic vaccines have been less potent against established cancer than originally imagined. In general, we have learned that tumors use mechanisms of tolerance induction to turn off T cells specific for tumor-associated antigens. Oncogenic pathways in tumors result in the elaboration of factors that organize the tumor microenvironment in ways that are quite hostile to antitumor immune responses. Not only is the cancer capable of inducing potent tolerance among tumor-specific T cells, we now know that there are distinct forms of inflammatory and immune responses that are procarcinogenic. Thus, two frontiers in cancer immunology are the elucidation of how the tumor organizes its immune-microenvironment as well as the nature of immune responses that are anticarcinogenic versus procarcinogenic. As the receptors, ligands, and signaling pathways that mediate immune tolerance and immune-induced procarcinogenic events are elucidated, these factors and pathways can be selectively inhibited by both antibodies and drugs in a way to shift the balance to antitumor immune responses. This chapter will outline the major features of tumor–immune system interactions and set the stage for molecularly based approaches to manipulate immune responses for successful cancer therapy.

HOW DO TUMORS DIFFER FROM SELF TISSUES? Tumors differ fundamentally from their normal cell counterparts in both antigenic composition and biologic behavior. Genetic instability, a basic hallmark of cancer, is a primary generator of tumor-specific antigens. The most common genetic alteration in cancer is mutation,

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which arises from defects in DNA damage repair systems of the tumor cell.8–15 Recent estimates from genome-wide sequencing efforts suggest that every tumor contains a few hundred mutations in coding regions.16 Additionally, deletions, amplifications, and chromosomal rearrangements can result in new genetic sequences resulting from juxtaposition of coding sequences not normally contiguous in untransformed cells. The vast majority of these mutations occur in intracellular proteins, and thus, the “neoantigens” they encode would not be readily targeted by antibodies. However, the major histocompatibility complex (MHC) presentation system for T-cell recognition makes peptides derived from all cellular proteins available on the cell surface as peptide-MHC complexes capable of being recognized by T cells. Based on analysis of sequence motifs, it is estimated that roughly onethird of the mutations identified from genome sequencing of 22 breast and colon cancers16 were capable of binding to common human lymphocyte antigen (HLA) alleles based on analysis of sequence motifs (J.P. Allison, personal communication). In accordance with the original findings of Prehn,5 the vast majority of tumor-specific antigens derived from mutation as a consequence of genetic instability are unique to individual tumors. The consequence of this is that antigen-specific immunotherapies targeted at most truly tumor-specific antigens would by necessity be patient specific. However, there are a growing number of examples of tumor-specific mutations that are shared. The three best-studied examples are the Kras codon 12 G→A (found in roughly 40% of colon cancers and >75% of pancreas cancers), the BRAF V599E (found in roughly 70% of melanomas) and the p53 codon 249 G→T mutation (found in ∼50% of hepatocellular carcinomas).17–20 As with nonshared mutations, these common tumorspecific mutations all occur in intracellular proteins, and therefore require T-cell recognition of MHC-presented peptides for immune recognition. Indeed, both the Kras codon 12 G→A and the BRAF V599E mutations result in “neopeptides” capable of being recognized by HLA class 1- and class II-restricted T cells.21–24 The other major difference between tumor cells and their normal counterparts derives from epigenetics.25 Global alterations in DNA methylation as well as chromatin structure in tumor cells results in dramatic shifts in gene expression. All tumors overexpress hundreds of genes relative to their normal counterparts, and in many cases, turn on genes that are normally completely silent in their normal cellular counterparts. Overexpressed genes in tumor cells represent the most commonly targeted tumor antigens by both antibodies and cellular immunotherapies. This is because, in contrast to most antigens derived from mutation, overexpressed genes are shared among many tumors of a given tissue origin or sometime multiple tumor types. For example, mesothelin, which is targeted by T cells from vaccinated pancreatic cancer patients,26 is highly expressed in virtually all pancreatic cancers, mesotheliomas, and most ovarian cancers.27,28 Whereas mesothelin is expressed at low to moderate levels in the pleural mesothelium, it is not expressed at all in normal pancreatic or ovarian ductal epithelial cells. The most dramatic examples of tumor-selective expression of epigenetically altered gene are the so-called cancer-testis antigens.29 These genes seem to be highly restricted in their expression in the adult. Many are expressed selectively in the testis of males and are not expressed at all in females. Expression in the testis seems to be restricted to germ cells, and some of these genes actually seem to encode proteins associated with meiosis.30–32 Cancer-testis antigens therefore represent examples of widely shared tumor-selective antigens whose expression is highly restricted to tumors. Many cancertestis antigens have been shown to be recognized by T cells from nonvaccinated and vaccinated cancer patients.29 From the standpoint of immunotherapeutic targeting, a major drawback of the cancertestis antigens is that none appear to be necessary for the tumors’ growth or survival. Therefore, their expression seems to be purely the consequence of epigenetic instability rather than selection, and antigen-negative variants are easily selected out in the face of immunotherapeutic targeting.

A final category of tumor antigen that has received much attention encompasses tissue-specific antigens shared by tumors of similar histologic origin. Interest in this class of antigen as a tumor-selective antigen arose when melanoma-reactive T cells derived from melanoma patients were found to recognize tyrosinase, a melanocytespecific protein required for melanin synthesis.33,34 In fact, the most commonly generated melanoma-reactive T cells from melanoma patients recognize melanocyte antigens.35 Although one cannot formally call tissue-specific antigens tumor specific, they are nonetheless potentially viable targets for therapeutic T-cell responses when the tissue is dispensable (e.g., prostate cancer or melanoma). From the standpoint of T-cell targeting, tumor antigens upregulated as a consequence of epigenetic alterations represent “selfantigens” and are therefore likely to induce some level of immune tolerance. However, it is now clear that the stringencies of immune tolerance against different self-antigens differ according to tissue distribution and normal expression level within normal cells. The mesothelin antigen described previously is such an example. In a recent set of clinical pancreatic cancer vaccine studies, mesothelin-specific T-cell responses were induced by vaccination with genetically modified pancreatic tumor cell vaccines and induction of mesothelinspecific T cells correlated with ultimate disease outcome. Given that the immune system is capable of differential responsiveness determined by antigen levels, it is quite possible to imagine generating tumor-selective immune responses against antigens whose expression level in the tumor is significantly greater within normal cells in the tumor-bearing host. Additionally, upregulated antigens that provide physiologically relevant growth or survival advantages to the tumor are preferred targets for any form of therapy, because they are not so readily selected out. Beyond the antigenic differences between tumor cells and normal cells, there are important immunologic consequences to the distinct biologic behavior of tumor cells relative to their normal counterparts. Whereas uncontrolled growth is certainly a common biologic feature of all tumors, the major pathophysiologic characteristics of malignant cancer responsible for morbidity and mortality are their ability to invade through natural tissue barriers and ultimately to metastasize. Both of these characteristics, never observed in nontransformed cells, are associated with dramatic disruption and remodeling of tissue architecture. Indeed, the tumor microenvironment is quite distinct from the microenvironment of normal tissue counterparts. One of the important consequences of tissue disruption, even when caused by noninfectious mechanisms, is the elaboration of proinflammatory signals. These signals, generally in the form of cytokines and chemokines, are potentially capable of naturally initiating innate and adaptive immune responses. Indeed, the level of leukocyte infiltration into the microenvironment of tumors tends to be significantly greater than the leukocyte component of their normal tissue counterparts. Cancers are therefore constantly confronted with inflammatory responses as they invade tissues and metastasize. In some circumstances these inflammatory and immune responses can potentially eliminate a tumor—so called immune surveillance. However, as will be discussed, oncogenic pathways in the tumor seem to organize the immunologic component of the microenvironment in a fashion that not only protects itself from antitumor immune responses, they can qualitatively shift immune responses to those that actually support and promote tumor growth. Thus, just as with Annekin Skywalker, the tumors can entice the immune system to the dark side. It is these elements of the cancer–immune system interaction that will be the central targets of future immunotherapeutic strategies.

EVIDENCE PRO AND CON FOR IMMUNE SURVEILLANCE OF CANCER The fundamental tenet of the immune surveillance hypothesis, first conceived nearly a half-century ago,36,37 is that a fundamental role of

Cancer Immunology • CHAPTER 6 Box 6-1.

HOW IMPORTANT IS IMMUNE SURVEILLANCE OF CANCER?

Few questions in cancer immunology have been more controversial than the immune surveillance hypothesis. First put forward by Lewis Thomas over a half century ago, the original immune surveillance hypothesis proposed that a natural role for the immune system was to survey the body for tumors similarly to the way the immune system surveys the body for invading foreign pathogens. Tumors were proposed to be distinguished from self-tissues by virtue of expression of tumor-specific antigens (TSAs). One aspect of this hypothesis that has held up to experimental testing has been the existence of TSAs. We now know that the genetic instability of tumor cells generates genetic and epigenetic changes that translate to antigens capable of being recognized by the immune system. However, because tumors seem to have developed mechanisms to subvert immunogenicity and tolerize the immune system, it is generally no longer believed that immune surveillance represents a major endogenous defense against tumorigenesis. It is likely that guardians of the genome that sense DNA damage, such as the ATR/ATM/p53 system, are much more fundamental mechanisms to protect against transformation. However, certain specialized components of the immune system, such as interepithelial lymphocytes activated by stress-induced ligands, may indeed play a complementary role in immune surveillance among specific epithelial tissues that are frequently exposed to carcinogenic stress. The best example is the intraepithelial lymphocytes within cutaneous epithelium that is constantly exposed to carcinogenic ultraviolet irradiation. A more moderate view of immune surveillance has been summarized by the hypothesis of Schreiber and colleagues that tumors “edit” themselves to either become resistant to immunologic surveillance or upregulate pathways (such as the STAT3 pathway) that actively induce tolerance among components of the immune system capable of recognizing TSAs.57–59

the immune system is to survey the body for tumors as it does for infection with pathogens, recognizing and eliminating them based on their expression of tumor-associated antigens (Box 6-1). In animal models, carcinogen-induced tumors can be divided into those that grow progressively (termed progresser tumors) and those that are rejected after an initial period of growth (termed regresser tumors).1,2 The phenomenon of regresser tumors was thought to represent an example of the ongoing process of immune surveillance of cancer. A corollary to the original immune surveillance hypothesis is that pro-

gresser tumors in animals (presumed to represent clinically progressing cancers in humans) fail to be eliminated because they develop active mechanisms of either immune escape or resistance (Fig. 6-1). A fundamental prediction of the immune surveillance hypothesis is that immunodeficient individuals would display a dramatic increase in tumor incidence. After an extensive analysis of spontaneous tumor formation in immunodeficient nude mice, which have atrophic thymi and therefore significantly reduced numbers of T cells and T-cell-dependent immune responses, no increased incidence of tumors was observed.38–42 These studies were taken as a major blow to the immune surveillance hypothesis. However, a caveat to the interpretation of these results is that nude mice still produce diminished numbers of T cells via thymus-independent pathways and therefore can mediate some degree of T-cell-dependent immunity. In addition, nude mice frequently display compensatory increases in innate immunity that, as in the following discussion, may represent a potent form of antitumor immunity and could contribute to immune surveillance of cancer. Epidemiologic studies of patients with heritable immunodeficiencies revealed a significantly increased risk of certain cancers that are distinct from the epithelial cancers commonly observed in normal immunocompetent adults.43–45 Many of these cancers are also observed in transplant patients on chronic pharmacologic immune suppression as well as in human immunodeficiency virus/acquired immunodeficiency syndrome patients whose immune system is depressed. The most common cancers in these individuals include lymphoplastic lymphomas as well as Kaposi’s sarcoma; however, certain epithelial cancers, such as stomach cancer, were also observed at increased frequency. A unifying theme for the majority of cancers observed in immunodeficient individuals is their microbial origin. The majority of lymphoplastic lymphomas are Epstein-Barr virus-associated lymphomas,46 and Kaposi’s sarcoma is a result of infection with the herpesvirus KSHV (Kaposi’s sarcoma herpesvirus).47 Other virusassociated cancers such as cervical cancer (from human papillomavirus [HPV])48,49 are also observed at increased frequency. It is now appreciated that stomach cancer is associated with ulcer disease related to infection with the bacterium Helicobacter pylori.50,51 From these studies, the notion emerged that immune surveillance indeed protects individuals against certain pathogen-associated cancers by either preventing infection or altering chronic infection by viruses and other microbes that can eventually induce cancer. These studies were taken to represent evidence that the common non-pathogenassociated cancers most commonly seen in adults in developed countries (e.g., prostate cancer, colon cancer, lung cancer) are not subject to immune surveillance.

Immune surveillance Normal cell

Genetic alterations Transformation progression

Tumor cell x x

x x

ELIMINATION Resistance mechanisms Tolerance induction

SURVIVAL SURVIVAL

Figure 6-1 • The balance among immune surveillance, resistance, and tolerance. Transformation of normal cells to cancer cells involves the creation of true neoantigens resulting from mutation as well as upregulation of self-antigens resulting from epigenetic forces. Successful immune surveillance of tumors based on recognition of these tumor-specific antigens would lead to tumor elimination at early stages. Clinically relevant tumor survival and progression requires that tumors develop resistance mechanisms that inhibit tumor-specific immune responses to kill tumor cells. Alternatively, if the tumor develops mechanisms to induce immune tolerance to its antigens, antitumor effector responses do not develop. Evidence is accumulating that tumors actively develop immune resistance mechanisms as well as immune tolerance mechanisms to survive despite displaying antigens capable of recognition by the immune system.

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Two caveats to this interpretation must be noted, however. First, detailed epidemiologic analyses of immunodeficient individuals were performed at a time when these patients rarely lived beyond their 20s and 30s, when cancer incidence normally increases most significantly. It is therefore possible that a more subtle cumulative increased incidence of common non-pathogen-associated cancers would have been observed had these individuals lived further into adulthood. Indeed, more recent analyses definitively demonstrate an increased incidence of some non-pathogen-associated cancers, in immunodeficient individuals, particularly melanoma.52 In addition to epidemiologic data, dramatic anecdotal examples are difficult to ignore. There have been reports that patients receiving kidneys from a cadaver donor that had been in complete remission from a melanoma before organ donation each rapidly developed metastatic melanoma of donor origin after the transplant.53–55 These results indicate that at least for some non-pathogen-associated tumors, the immune system can play a significant role in maintaining the micrometastatic disease in a dormant state. Whether this principle applies to other non-pathogen-associated human tumors besides melanoma remains to be demonstrated. Several recent studies reevaluating tumor immune surveillance in genetically manipulated mice have revealed clear-cut evidence that various components of the immune system can at least modify, if not eliminate, both carcinogen-induced and spontaneously arising cancers. In a series of studies by Schreiber and colleagues reexamining cancer incidence in mice rendered immunodeficient via genetic knockout of either the RAG2 gene (deficient in both B and T cells), the γ-interferon receptor gene, STAT 1 gene, or the type 1 interferon receptor gene.56–59 When these knockout mice are either treated with carcinogens or crossed onto a cancer-prone p53 knockout background, the incidence of cancers was modestly but significantly increased relative to nonimmunodeficient counterparts when observed over an extended period (longer than 1 year). Transplantation studies demonstrated that direct γ-interferon insensitivity by the developing tumors played a significant role in the defect in immune surveillance. Interestingly, in contrast to γ-interferon receptor knockout mice, the mechanism for increased tumor incidence in tumors in type 1 interferon receptor knockout mice did not involve sensitivity by the tumor to type 1 interferons but rather reflected the role of the type 1 interferons in induction of innate and adaptive immunity. Even animals not crossed onto a cancer-prone genetic background or treated with carcinogens developed an increased incidence of invasive adenocarcinomas when observed over their entire life span. Furthermore, γ-interferon, RAG2 double-knockout mice developed a broader spectrum of tumors than RAG2 knockout mice. All of the tumors that arise in these genetically manipulated immunodeficient animals behave as regresser tumors when transplanted into immunocompetent animals. These findings indeed suggest that tumors that arise in immunodeficient animals would have been eliminated had they arisen in immunocompetent animals. The relatively subtle effects on tumorigenesis, requiring observation over the life span of the animal, suggest that the original concept of immune surveillance of tumors arising on a daily basis is in fact not correct. Instead, it is clear that the presence of a competent immune system “sculpts” the tumor through a process that has been termed immunoediting. One of the caveats in the interpretation of these studies comes from the work of Enzler and Dranoff, who studied mechanisms of increased tumorigenesis in granulocyte-macrophage colony-stimulating factor (GM-CSF), γ-interferon double-knockout mice.60 Although they observed an increase in gastrointestinal and pulmonary tumors, they noted that such animals harbored infection with a particular bacterium not normally observed in immunocompetent animals. Maintenance of these double-knockout mice on antibiotics essentially eliminated the increased rate of tumor formation. Thus, it is possible that some of the increased tumor rates in genetically immunodeficient animals could be related to unappreciated chronic infections that develop in these animals, which are not housed

under germ-free conditions. Nonetheless, although the classic concepts of immune surveillance of cancer remain unsupported by experimental evidence, studies on tumorigenesis in genetically manipulated immunodeficient mice indeed suggest that developing tumors must actively adapt themselves to their immune microenvironment to exist within the context of a competent immune system.

INNATE IMMUNITY, EPITHELIAL IMMUNITY, AND TUMOR IMMUNE SURVEILLANCE Although much emphasis has been placed on the role of adaptive immunity, particularly of conventional T cells in immune surveillance of cancer, a confluence of more recent findings points to innate immunity and epithelial immunity in the immunologic sensing of carcinogenic events in the skin, gut, and possibly other sites. Much of the evidence focuses on the NKG2D receptor. NKG2D was originally defined as an activating natural killer (NK) receptor.61–63 Most NK receptors seem to be inhibitory when engaged; this inhibition is often associated with ITIM (immunoreceptor tyrosine kinasebased inhibitory motif) domains in the cytoplasmic tails. ITIMs provide docking sites for phosphatases that oppose the activity of tyrosine kinases involved in lymphocyte activation. NK activation status is a balance between engagement of activating and inhibitory receptors. NKG2D, the best-studied activating receptor on NK cells, is somewhat unusual in that it does not contain an ITAM (immunoreceptor tyrosine kinase-activating motif) and is associated with an adaptor molecule, DAP 10, which contains neither conventional ITIMs nor ITAMs.64 Instead, DAP 10 contains a KYXXM motif that seems to bind to phosphatidyl inositol (PI) 3 kinase upon phosphorylation of the tyrosine in this motif. NKG2D is expressed on all NK cells as well as on some αβ and γδ T cells. Beyond NK cells, NKG2D is expressed at high levels on a number of subsets of interepithelial lymphocytes (IELs). IELs represent a distinct population of lymphocytes residing in epithelial tissues that display features of both adaptive and innate immune responses.65–69 They are thought to represent a major first line of defense against pathogens attempting to invade across epithelial linings exposed to the environment (i.e., skin, gut, respiratory tract). Fifty percent of the IELs of the gut express the γδ T-cell receptor (TCR), which is normally expressed by less than 3% of circulating T cells, whereas the other 50% express the common αβ TCR. γδ TCR-expressing IELs in different compartments express a very restricted repertoire and are thought to recognize certain types of microbial antigens or potentially self-antigens associated with stress or inflammatory responses to microbial infection. Even the αβ TCRexpressing IELs have an extremely restricted TCR repertoire similar to invariant NK T cells. A significant subset of gut IELs express a particular VαVβ and are thought to recognize a limited subset of microbial or self nonpeptide antigens presented by nonclassical class 1 MHC molecules. Thus, NKG2D expression marks diverse subsets of lymphocytes that, though expressing different families of recognition receptors, act as components of innate immunity in that they recognize a stereotypical set of antigens associated with infection or stress (see later discussion). The first evidence that the NKG2D receptor might play a role in tumor immune surveillance came from the finding that normal colonic epithelium as well as a significant proportion of tumors could express the two defined human ligands for NKG2D: MICA and MICB. MICA and MICB, which represent nonclassical MHC class I-type molecules whose structure demonstrates no antigen-binding groove characteristic of most MHC molecules, are stress-induced proteins whose genes contain stress response elements in their promoters.70,71 Raulet and colleagues have demonstrated that upregulation of MICA/B is induced through the ATM/ATR/Chk1 pathway of DNA damage recognition.72 An analysis in human cancer suggested a correlation between expression of MICA/B and infiltration of certain subsets of γδ T cells that express NKG2D. Initially it was proposed

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that MICA and MICB were direct ligands for specific γδ receptors themselves as well as NKG2D,73,74 but this idea is controversial. MICA and MICB do not have any murine orthologs, but murine NKG2D does bind to products of the retinoic acid-inducible gene family, RAE-1α-RAE-1ε, as well as the product of the H60 gene. ULBP3 is an additional NKG2D ligand to be described.75,76 These NKG2D ligands seem to be involved in immune recognition and possibly tumor surveillance in mice.77–79 Recognition and killing of murine skin keratinocytes or intestinal epithelial cells by γδ IELs require expression of NKG2D ligands and are blocked by antiNKG2D antibodies. Trasnfection of murine tumors with genes encoding NKG2D ligands renders them susceptible to NKG2Ddependent killing by NK cells. Emerging data on NKG2D function on IELs together with the potentially stress-induced nature of its ligands suggests that the IEL system of immune surveillance may indeed be relevant to carcinogenesis as well as infectious challenges.80 The major initiating event of carcinogenesis in the skin—ultravioleet light—is a potent source of DNA damage that, as mentioned previously, has been shown to induce NKG2D ligands via the ATM pathway. Thus, in addition to endogenous killers of genome-damaged cells, such as p53, IELs and NK cells may represent an extrinsic sensor of DNA damage and genotoxic stress via recognition of cells that have upregulated NKG2D ligands (Fig. 6-2). As with the case of classic immune surveillance mediated by classical T cells, the emergence of a clinically evident cancer implies that

Genotoxic event/ Transformation

the tumor has developed a mechanism to circumvent or evade any innate immune surveillance systems. In the case of the NKG2D system, Spies and colleagues have provided suggestive evidence that tumors can shed MICA/B in a soluble form as a means of evading NKG2D-dependent recognition. They demonstrated that certain tumors are associated with high levels of shed MICA/B and that soluble MICA/B binds to and downmodulates NKG2D on NK cells, thereby acting as an antagonist to NKG2D activation via cell surfacebound MICA/B.81 Although this mechanism remains to be proven as a true evasion system for NKG2D-dependent tumor recognition, it points out the diversity of mechanisms that tumors utilize to evade immune recognition. It also points out straightforward approaches to block these evasion systems. If indeed soluble MICA/B does represent a mechanism for tumor immune evasion of innate immune recognition, antibodies that would bind to and clear soluble MICA/ B but not block the interaction between cell membrane MICA/B and NKG2D on NK cells could potentially restore the capacity of NK cells to recognize MICA/B-expressing tumors.

IMMUNE TOLERANCE AND IMMUNE EVASION—THE HALLMARK OF A SUCCESSFUL TUMOR Although controversy over the ultimate role of immune surveillance in natural modulation of cancer development and progression will

Cytolysis

NKG2D

␣␤ IEL

Basement membrane ␣␤ TCR Genotoxic event/ Transformation

Cytolysis

NKG2D

␥␦ IEL

Basement membrane ␥␦ TCR

Figure 6-2 • Epithelial linings contain intraepithelial lymphocytes that can recognize epithelial cells undergoing genotoxic stress. Intraepithelial lymphocytes (IELs) fall into two categories: those that express the classical αβ T-cell receptor (TCR) that recognizes peptide-MHC complexes on the cell surface and those that express the γδ TCR, whose ligands are less well characterized. IELs also express the NKG2D receptor, which serves as a costimulatory receptor for activation of IELs. The ligands for NKG2D, MICA, and MICB in humans and RAE1α–RAE1ε, H60, and ULBP-3 in mice, are induced by genotoxic stress via the ATM/ATR pathways. This is a mechanism by which IELs can survey for damaged epithelial cells due to irradiation (skin) or mutagens that can cause transformation.

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undoubtedly continue into the future, one can summarize the current state of knowledge as supporting the notion that natural immune surveillance plays a much smaller role than originally envisioned by Thomas and Burnet. However, developing tumors need to adapt to their immunologic milieu in a manner that either turns off potentially harmful (to the tumor) immune responses or creates a local microenvironment inhibitory to the tumoricidal activity of immune cells that could inadvertently become activated in the context of inflammatory responses associated with tissue invasion by the tumor. These processes—tolerance induction and immune evasion—have become a central focus of cancer immunology efforts and will undoubtedly provide the critical information necessary for development of successful immunotherapies that break tolerance to tumor antigens and break down the resistance mechanisms operative within the tumor microenvironment (see Fig. 6-1). Evidence from both murine tumor systems as well as human tumors strongly demonstrates the capacity of tumors to induce tolerance to their antigens. This capacity to induce immune tolerance may very well be the single most important strategy that tumors use to protect themselves from elimination by the host’s immune system. Tolerance to tumors seems to operate predominantly at the level of T cells; B-cell tolerance to tumors is less certain, because there is ample evidence for the induction of antibody responses in animals bearing tumors as well as human patients with tumors. However, with the exception of antibodies against members of the EGFR family, there is little evidence that the natural humoral response to tumors provides significant or relevant antitumor immunity. In contrast, numerous adoptive transfer studies have demonstrated the potent capacity of T cells to kill growing tumors, either directly through cytotoxic T-lymphocyte (CTL) activity, or indirectly through multiple CD4-dependent effector mechanisms. It is thus likely that induction of antigen-specific tolerance among T cells is of paramount importance for tumor survival. The first direct evidence for induction of T-cell tolerance by tumors was provided by Bogen and colleagues, who examined the response of TCR-transgenic T cells specific for the idiotypic immunoglobulin expressed by a murine myeloma tumor.82,83 They first demonstrated induction of central tolerance to the myeloma protein followed by peripheral tolerance. Using influenza hemagglutinin as a model tumor antigen, Levitsky and colleagues demonstrated that adoptively transferred hemagglutinin-specific TCR-transgenic T cells were rapidly rendered anergic by hemagglutinin-expressing lymphomas and hemagglutinin-expressing renal carcinomas.84,85 Tolerance induction has been demonstrated in both the CD4 and CD8 compartment. In general, initial activation of tumor-specific T cells is commonly observed; however, the activated state of T cells is typically not sustained with failure of tumor elimination as a frequent consequence. Tolerance induction among tumor antigen-specific T cells is an active process involving direct antigen recognition, although in some murine systems, tolerance to tumors seems to be associated with failure of antigen recognition by T cells—that is, the immune system “ignores” the tumor.86,87 Beyond studies on transplantable tumors, more recent analyses of immune responses to tumor antigens in tumor-transgenic mice developing spontaneous cancer have further emphasized the capacity of spontaneously arising tumors to induce tolerance among antigen-specific T lymphocytes. In a model of prostate tumorigenesis, Drake and associates evaluated CD4 responses to hemagglutinin in double-transgenic animals expressing hemagglutinin and simian virus 40 (SV40) T antigen under control of the prostate-specific probasin promoter.88 Development and progression of prostate tumors did not result in enhanced activation of adoptively transferred hemagglutinin-specific T cells. Tolerance to hemagglutinin as a normal prostate antigen occurred largely through ignorance, because there was no evidence for antigen recognition by hemagglutinin-specific T cells. However, increased recognition was observed upon either androgen ablation (which causes massive apoptosis within the prostate) or development of prostate cancer. Nonetheless,

enhanced antigen recognition was not accompanied by activation of effector functions such as γ-interferon production. Analysis of the consequences of transformation in additional tumor-transgenic mouse systems has also been performed. Willimsky and Blankenstein evaluated T-cell responses and rejection in a model of sporadic induction of tumors associated with expression of a tumor-specific antigen only at the time of transformation.89 They found that preimmunization of mice against the tumorassociated antigen prevented the development of tumors. However, nonimmunized mice developed spontaneous tumors without any significant evidence of natural immune surveillance in the absence of preimmunization. They further demonstrated that an initial antigendependent activation of tumor-specific T cells could be observed at the time of spontaneous tumor induction but that this recognition ultimately resulted in an anergic form of T-cell tolerance similar to that observed by Drake and colleagues in the prostate system. The capacity of spontaneously arising tumors to tolerize T cells has not been uniformly observed. A contrasting result by Ohashi and colleagues was observed when lymphocytic choriomeningitis virus (LCMV) GP33-specific TCR-transgenic CD8 T cells were adoptively transferred into double-transgenic mice expressing both SV40 T antigen and LCMV GP33 under control of the rat insulin promoter.90 These animals develop pancreatic islet cell tumors that express GP33. These investigators found that as tumors progressed in the mice, enhanced T-cell activation occurred. CD8 T-cell activation was demonstrated through bone marrow chimera experiments to occur exclusively via cross-presentation in the draining lymph nodes. Despite the activation of tumor-specific T cells, the tumors grew progressively, indicating that the degree of immune activation induced by tumor growth was insufficient to ultimately eliminate the tumors. These results suggest that developing tumors can induce immune responses but may titrate their level of immune activation to one that ultimately does not “keep up” with tumor progression. Such a circumstance is one that is highly susceptible to the immunoediting concept put forward by Schreiber and colleagues, in which the tumor edits itself genetically to maintain a sufficient level of resistance to induced immune responses. In the case of the LCMV GP33 T antigen-transgenic mice, because neither anergic nor deletional tolerance was observed, animals treated with the dendritic cell (DC) stimulatory anti-CD40 antibody demonstrated significant slowing of tumor growth. Thus, it may be possible under some circumstances to shift the balance between tumor immune evasion and tumor immune recognition by agents that affect the overall activation state of either antigen-presenting cells (APCs) or T cells (see later discussion). It has been more difficult to obtain definitive evidence that human cancers tolerize tumor-specific T cells, because humans cannot be manipulated the way mice are. However, the T cells that are grown out from patients with cancer tend to be either of low affinity for their cognate antigen or recognize antigens that bind poorly to their presenting HLA (human MHC) molecule, resulting in inefficient recognition by T cells. Recently, the first crystal structure of the TCR-peptide-MHC trimolecular complex has been solved for an MHC class II-restricted human tumor antigen.91 Interestingly, the orientation of the TCR, which is of low affinity for the peptide-MHC complex, is distinct from trimolecular complexes for viral (foreign) antigens and is partially similar to trimolecular complexes for a selfantigen. Thus, there may be fundamental structural features of tumor antigen recognition that lie between those of foreign-antigen and self-antigen recognition. As will be discussed later, one of the features of the tumor microenvironment that is probably central to the capability of tumors to tolerize tumor-specific T cells is the immature or inactive state of tumor-infiltrating DCs. DCs are the major APCs that present peptides to T cells to initiate adaptive immune responses. In the context of infection, microbial ligands or endogenous “danger signals” associated with tissue destruction activate DCs to a state whereby they

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Figure 6-3 • Dendritic cells (DCs) can either activate adaptive immunity or tolerize T cells depending on their state of maturation. DC progenitors develop from hematopoietic (bone marrowderived) progenitors under the influence of various cytokines, particularly GM-CSF. Under circumstances of microbial infection, specific pathogenassociated molecular patterns (termed PAMPs) engage pattern recognition receptors (PRRs), leading to release of proinflammatory danger signals that induce DC maturation. DC maturation leads to upregulation of costimulatory molecules, MHC, and chemokines that result in activation of T cells to effector cells (right). In the absence of these “danger signals,” DCs follow a default pathway (left) in which they become “tolerizing DCs” that present antigen (Ag) to T cells in the absence of costimulatory signals. This represents a steady-state pathway for continuous presentation of self-antigens. The consequence is that these T cells are turned off (anergy), inducing tolerance.

GM-CSF IL-4,FLT-3L Bone Bon e marrow marrow progenit prog enitor enit or

Dendrit Den dritic drit ic cell cell progenit prog enitor enit or Ag upta upta ptake/Pr ke/Proce ke/Pr ocessing oce ssing No dange dangerr signals sig nals

“Tolerizi “To lerizing ng DC” DC”

Microbial infe Microbial infection ction dangerr signal dange signals s Exogenous LPS CpG

Endogenous TNF␣ CD40L

Activat Acti vated ed DC DC

Modera Mod erate era te MHC MHC II Chemokin Che mokines mokin es Adhesion Adh esion molec molec olecules ules Costimu Cos timulatory timu latory molecule molecules s

present antigens to T cells together with costimulatory signals that induce T-cell activation and development of effector function. However, in the absence of microbial products or danger signals, DCs remain in an immature state in which they can still present antigens to T cells but without costimulatory signals. These immature DCs function as “toleragenic” DCs, inducing a state of antigenspecific T-cell unresponsiveness (termed anergy; Fig. 6-3). It is thought that steady-state presentation of self-antigens by immature DCs is an important mechanism of peripheral self-tolerance. Thus, if a tumor is able to produce factors that inhibit local DCs from becoming activated in response to the endogenous danger signals associated with tissue invasion, it could shift tumor-specific T cells from a state of activation (Fig. 6-4A) to one of tumor-specific tolerance (Fig. 6-4B).

High MHC II II Chemokin Che mokines mokin es Adhesion Adh esion molec molec olecules ules Costimu Cos timulatory timu latory molecule molecules s

REGULATORY T CELLS AND CANCER Over the past 10 years, regulatory T (Treg) cells have emerged as a central player in maintenance of the tolerant state as well as general downregulation of immune responses to pathogens.92,93 Not surprisingly, they seem to play a role in tolerance to tumor antigens as well as in the resistance of tumors to immune-mediated elimination.94,95 In contrast to the ephemeral CD8 suppressor cells of the 1970s that failed to withstand experimental scrutiny, the more recently defined CD4+ regulatory T cells are characterized by expression of a central master regulatory transcription factor—FoxP3—whose role in the gene expression programs of regulatory T cells is being actively studied.96 Although CD4+ regulatory T cells selectively (but not specifically) express several cell membrane molecules, including

LN Activated DCs

Activation of tumorspecific T cells

Danger

Figure 6-4 • Inhibition of DC activation in the tumor microenvironment can shift tumor-specific immune responses from activation to tolerance. Based on the scenario presented in Figure 6-3, if a tumor is able to produce factors that inhibit local DCs from becoming activated in response to the endogenous danger signals associated with tissue invasion, it could shift tumor-specific T cells from a state of activation (A) to one of tumor-specific tolerance (B). LN, lymph nodes.

A Inhibition of danger signals Unactivated DCs Danger

B

LN

Anergy/ Deletion of tumorspecific T cells

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CD25, neuropilin, GITR, and LAG3,92,97–99 their overall genetic program and inhibitory capacity is absolutely dependent on sustained expression of FoxP3.100,101 Mechanisms of immune suppression by regulatory T cells vary and include production of inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β).102–104 In keeping with the emerging appreciation that tumors are by nature highly toleragenic, numerous murine studies have demonstrated that Treg cells expand in animals with cancer and significantly limit the potency of antitumor immune responses—either natural or vaccine induced. For example, in a study by Sutmuller and coworkers a combination of GM-CSF–transduced tumor vaccine plus anti-CTLA4 antibodies was much more effective at eliminating established tumors when animals were treated with anti-IL-2 receptor α antibodies to eliminate CD4+ Treg cells.105 It is now appreciated that treatment with low-dose cytoxan is a relatively simple and reasonably effective way to temporarily eliminate cycling Treg cells.106–109 This seems to be a major mechanism by which pretreatment with low-dose cytoxan before vaccination can significantly enhance the capacity of vaccines to break tolerance. As new cell membrane molecules that define Treg cells are identified, the capacity to block regulatory T-cell activity with antibodies to these molecules presents new opportunities for immunotherapeutic strategies to break tolerance to tumor antigens.

ONCOGENIC PATHWAYS ACTIVELY MEDIATE TUMOR–IMMUNE SYSTEM INTERACTIONS The previous sections outline the complex interplay between tumor and host immune system and describe the experimental evidence that the immune system is in general tolerant to tumors and their antigens under circumstances in which a tumor has established and is expanding within the host. Is this tolerance to tumor antigens a passive default pathway, or does the tumor actively manipulate its immune microenvironment in a way to render the immune system tolerant to its antigens. Indeed, evidence is accumulating that activation of oncogenic pathways in the tumor as well as inactivation of tumor suppressor genes have immunologic consequences far beyond the more commonly studied roles in growth regulation and antiapoptosis. Critical signaling pathways whose role has been studied in this context include STAT3, NF-κB, BRAF, and PTEN. Although each of these pathways (either activation or inactivation) has been well studied for its role in “classic” tumor biology such as dysregulated growth, regulation of apoptosis, and resistance to DNA-damaging agents, additional roles in the organization of the immune microenvironment of the tumor have also been elucidated recently. The best-studied oncogenic pathway to play a role in tumor immune evasion is the STAT3 pathway. STAT3 is one of two STATs (the other being STAT5A) to be constitutively activated in many diverse tumor types.110–113 Activation of STAT3 involves tyrosine phosphorylation resulting in homodimerization in the cytosol that leads to nuclear transport where it participates in transcriptional activation (and in some cases repression) of diverse genes. Although synthetic mutations in STAT3 can confer upon it oncogenic activity, constitutive activation of STAT3 in tumors is not a consequence of mutation. Instead, STAT3 is downstream of several important oncogenic tyrosine kinases, both receptor tyrosine kinases and src family tyrosine kinases. Several receptor tyrosine kinases that play important roles in human cancer, including EGFR, HER2/Neu, and cMet, signal in part through STAT3.114–116 In addition, src and potentially other src family tyrosine kinases can activate STAT3.117 In fact, the original association of STAT3 with oncogenesis came from the demonstration that src-dependent transformation required STAT3.118 Activated STAT3 in tumors participates in transcriptional activation of several genes associated with common cell-autonomous and noncell-autonomous mechanisms of carcinogenesis and cancer promo-

tion. These include cell cycle regulation (e.g., cyclin D1), antiapoptosis (e.g., BCL-Xl and survivin), and angiogenesis (e.g., vascular endothelial growth factor [VEGF]).119,120 In addition, STAT3 activation in tumors has been shown to repress the production of proinflammatory cytokines and chemokines that could enhance antitumor immune responses.121 These include proinflammatory cytokines such as type 1 interferons and tumor necrosis factor as well as proinflammatory chemokines such as RANTES and IP-10. Thus, blockade of STAT3 signaling in tumor cells results in the release of multiple proinflammatory mediators and consequent infiltration with cells of both the innate and adaptive immune system that ultimately inhibit tumor growth. Beyond simply repressing the production and release of molecules that could promote antitumor immune responses, STAT3 signaling also induces the release of factors that inhibit activation of multiple immune cell types in the tumor microenvironment. These include DCs, NK cells, and granulocytes, which, though present in significant numbers within tumors, are generally found in an unactivated state. Some of the STAT3-regulated factors that induce this “quiescent microenvironment” include IL-10, VEGF, IL-6, and possibly IL-23. As will be described later, some of these cytokines promote distinct forms of immune responses that promote rather than inhibit tumor growth. The receptors for each of these factors are expressed on cells of the hematopoietic system and signal through STAT3. Thus, infiltrating hematopoietic cells within the tumor microenvironment are found to also express constitutively activated STAT3. Blockade of STAT3 in the hematopoietic system (for example, via hematopoieticspecific STAT3 knockout) results in dramatically enhanced activation of DCs and cells in the innate immune system (such as NK cells and granulocytes) and leads to antitumor immune responses. In fact, even aggressive tumors fail to grow when transplanted into animals with hematopoietic STAT3 knockout.122 Thus, STAT3 seems to be an important global signaling pathway that restrains antitumor immunity. Another immunologically relevant pathway that is commonly constituently activated in cancer is the NF-κB pathway.123,124 Normally, NF-κB is activated in a highly stimulus-dependent fashion, but is constitutively activated in many types of tumors. Multiple NF-κB family members participate in either a canonical or noncanonical NF-κB activation pathway. Common to both pathways is the activation of IκB kinase (IKK), which phosphorylates IκB leading to ubiquitin-dependent degradation and release of NF-κB to traffic from the cytosol to the nucleus and activate gene transcription programs.125 Alternatively, IKK phosphorylation can result in cleavage of a precursor protein for the activation of the noncanonical NF-κB pathway. The mechanism for constitutive NF-κB activation in tumors is not currently known. Normally, NF-κB plays a central role in the activation of virtually all cells in the immune system—both innate and adaptive. In the case of innate immunity, Toll-like receptors (TLRs) on the surface of cells or intracellular sensors of viral RNA or DNA (the RIGI or MDA5 pathway) result in a signaling cascade that activates NF-κB via TRAF6.126,127 Paradoxically, constitutive activation of NF-κB in tumors is associated predominantly with activation of antiapoptotic genes, whereas many of the typical NF-κB-responsive proinflammatory/proimmunity genes are not activated in tumors. Recently, it was demonstrated that the selective NF-κB gene activation program in tumors is dependent on its association with STAT3. Indeed, coactivation of STAT3 and NF-κB is commonly observed in tumors. This coactivation seems in part to be due to a newly defined role for STAT3 in enhancing acetylation of NF-κB p50 subunit, resulting in enhanced retention of active NF-κB in the nucleus of tumor cells. This retention seems to be through the p300 acetyl transferase. The result is a shift in equilibrium toward nuclear retention of NF-κB. In addition, STAT3–NF-κB complexes fail to bind promoters of proinflammatory/proimmunity genes that are typically repressed in tumor cells, whereas STAT3–NF-κB dimers

Cancer Immunology • CHAPTER 6

are found associated with promoters driving antiapoptotic genes such as BCL-Xl and survivin. These findings highlight the interactivity between key signaling pathways of tumor cells as well as the interplay between gene expression programs mediating tumor immunity versus tumor survival. An additional oncogenic pathway that seems to play a role in tumor immune evasion is the BRAF pathway.128 BRAF is constitutively activated in the majority of human melanomas as a result of a single activating mutation. Kawakami and colleagues128 have demonstrated that factors produced by melanoma cells that inhibit DC activation are in part driven by Braf. Knockdown of Braf with short interfering RNA abrogates the production by melanoma cells of factors that inhibit DC activation. This inhibition seems to be independent of but complementary to that provided by STAT3 activation in melanoma cells. Thus, it seems that multiple oncogenic pathways active in tumor cells may contribute to the release of factors that inhibit DCs and other components of innate immunity, shifting the balance of immune responses toward tolerance. In addition to oncogenic pathways, inactivation of tumor suppressor pathways may also play a role in immune evasion by tumors. In one example, Parsa and colleauges demonstrated that expression of a T-cell-inhibitory molecule by tumors, B7-H1 (see later discussion), is linked to inactivation of PTEN. PTEN, an inhibitor of the oncogenic AKT pathway, is emerging as one of the most important tumor suppressor pathways in cancer.129 More recently, Lowe and colleagues provided evidence that the p53 pathway may play a role in inhibiting innate immune responses to tumors. In a transgenic system in which inactivated p53 is conditionally reexpressed in tumors, they found that the inhibition of tumor growth induced by reactivation of p53 might be in part dependent on induction of innate immune responses mediated by NK cells.130 Taken together, these findings strongly suggest that oncogene and tumor suppressor gene pathways in tumors play important roles in orchestrating the interaction between the tumor cell and its immune microenvironment such that immune responses induced by the invasion and metastasis process do not eliminate the tumor cell itself. Whereas most of the focus on the function of oncogenic and tumor suppressor pathways has been on cell-autonomous functions within

the tumor such as growth regulation, there is growing appreciation that these pathways additionally affect the tumor microenvironment via nontransformed cells. As an integral part of the tumor microenvironment, the immune system is clearly subject to regulation by these pathways. Understanding of the immunologic consequences of these pathways ultimately provides direct opportunities to develop therapeutic approaches that integrate inhibitors of oncogenic pathways, activators of tumor suppressor pathways, and other immunotherapeutic approaches to cancer.

IMMUNOLOGIC CHARACTERISTICS OF THE TUMOR MICROENVIRONMENT Ultimate understanding of the relationship between the tumor and the host immune system requires elucidation of local cross-talk at the level of the tumor microenvironment. As mentioned at the outset, the hematopoietic/immune system is a major component of the tumor microenvironment. The systemic tolerance to tumor antigens begins with events that occur in this microenvironment. Beyond mechanisms that skew tumor-specific T cells toward immune tolerance, the tumor microenvironment is replete with mechanisms that dampen antitumor immune responses locally (Fig. 6-5). This represents an important barrier to successful immunotherapy even when activated effector responses can be generated with vaccines. As the specific cells and molecules within the tumor microenvironment that mediate this hostile immune environment are elucidated, inhibitors are being developed and tested to use as adjuncts to vaccination that will allow activated immune cells to function more effectively within the tumor microenvironment. The previous section described how oncogenic pathways in the tumor cell directly affect the immune microenvironment of the tumor. In addition to its role in inhibiting the activation and effector function of DCs, granulocytes, and NK cells in the tumor microenvironment, STAT3 signaling has also been reported to play a role in guiding immature myeloid cells (iMCs) in the tumor microenvironment to differentiate into myeloid suppressor cells (MSCs) rather than DCs with APC activity. iMCs131,132 and MSCs133–136 represent a cadre of myeloid cell types, including tumor-associated

Lytic activity

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X IL-10 XX TGF-␤ X NO B7-H4

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Figure 6-5 • The hostile immune microenvironment of the tumor. Activation of oncogenic pathways and inactivation of tumor suppressor pathways in the tumor lead to a cascade of molecular and cellular processes in the tumor microenviroment that block the killing function of innate immune effectors such as NK cells and granulocytes and block DC maturation (see Figs. 6-3 and 6-4). In addition, multiple cell membrane molecules such as IL-10, TGF-β, B7-H1, and B7-H4 are upregulated. These molecules bind to receptors that inhibit T-cell effector function. Immature myeloid cells (iMC) produce NO, which inhibits T cells, and immature plasmacytoid DCs (iPDC) produce indoleamine dioxygenase (IDO), which depletes tryptophan. Regulatory T cells also accumulate in the tumor microenvironment, further blunting antitumor T-cell responses.

Tumorspecific CTL

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macrophages, that share the common feature of inhibiting both the priming and effector function of tumor-reactive T cells. It is still not clear whether these myeloid cell types represent distinct lineages or different states of the same general immune-inhibitory cell subset. In mice, iMCs and MSCs are characterized by coexpression of CD11b (considered a macrophage marker) and Gr1 (considered a granulocyte marker) while expressing low or no MHC class II or the CD86 costimulatory molecule. In humans, they are defined as CD33+ but lack markers of mature macrophages, DCs, or granulocytes and are HLA-DR–. Several molecular species produced by tumors tend to drive iMC/MSC accumulation. These include IL-6, CSF-1, IL-10, and gangliosides. IL-6 and IL-10 are potent inducers of STAT3 signaling. Another cytokine reported to induce iMC/MSC accumulation is GM-CSF.137 This finding is somewhat paradoxical, in that GM-CSF is a critical inducer of DC differentiation and GM-CSF– transduced tumor vaccines enhance antitumor T-cell immunity via accumulation of DCs at the vaccine site followed by increased DC numbers in vaccine draining lymph nodes. It seems that the paradox is solved based on levels of GM-CSF. High local levels drive DC differentiation at the vaccine site, whereas chronic production of low levels of GM-CSF can promote iMC/MSC accumulation. GMCSF–transduced vaccines that produce extremely high GM-CSF levels can induce iMC/MSC accumulation at distant sites (i.e., spleen and lymph nodes), because they release enough GM-CSF systemically to drive iMC/MSC accumulation. Several mechanisms have been proposed to explain how iMC/ MSC inhibit T-cell responses within the tumor microenvironment. Most include the production of reactive oxygen species (ROS) and/or reactive nitrogen species. Nitric oxide production by iMC/MSC as a result of arginase activity, which is high in these cells, has been well documented, and inhibition of this pathway with several drugs can mitigate the inhibitory effects of iMC/MSC. ROS, including hydrogen peroxide, have been reported to block T-cell function associated with the downmodulation of the ζ chain of the TCR signaling complex,138 a phenomenon well recognized in T cells from cancer patients and associated with generalized T-cell unresponsiveness. Another mediator of T-cell unresponsiveness associated with cancer is the production of indolamine-2,3 dioxygenase (IDO).139 IDO seems to be produced by DCs either within tumors or in tumordraining lymph nodes. Interestingly, IDO in DCs has been reported to be induced via backward signaling by B7-1/2 upon ligation with CTLA-4.140,141 Apparently, the major IDO-producing DC subset is either a plasmacytoid DC (PDC) or a PDC-related cell that is B220+.142 IDO seems to inhibit T-cell responses through catabolism of tryptophan. Activated T cells are highly dependent on tryptophan and are therefore sensitive to tryptophan depletion. Thus, Munn and Mellor have proposed a bystander mechanism, whereby DCs in the local environment deplete tryptophan via IDO upregulation, thereby inducing metabolic apoptosis in locally activated T cells.139 Another inhibitory molecule produced by many cell types that has been implicated in blunting antitumor immune responses is TGF-β, which is produced by a variety of cell types, including tumor cells, and which has pleiotropic physiologic effects. For most normal epithelial cells, TGF-β is a potent inhibitor of cell proliferation, causing cell cycle arrest in the G1 stage.143 In many cancer cells, however, mutations in the TGF-β pathway confer resistance to cell cycle inhibition, allowing uncontrolled proliferation. Additionally, in cancer cells the production of TGF-β is increased and may contribute to invasion by promoting the activity of matrix metalloproteinases. In vivo, TGF-β directly stimulates angiogenesis; this stimulation can be blocked by anti-TGF-β antibodies.144 A bimodal role of TGF-β in cancer has been verified in a transgenic animal model using a keratinocyte-targeted overexpression.145 Initially, these animals are resistant to the development of early-stage or benign skin tumors. However, once tumors form, they progress rapidly to a more aggressive spindlecell phenotype. Although this clear bimodal pattern of activity is more difficult to identify in a clinical setting, it should be noted that

elevated serum TGF-β levels are associated with poor prognosis in several malignancies, including prostate cancer,146 lung cancer,147 gastric cancer,148 and bladder cancer.149 From an immunologic perspective, TGF-β possesses broadly immunosuppressive properties and TGF-β knockout mice develop widespread inflammatory pathology and corresponding accelerated mortality.150 Interestingly, a majority of these effects seem to be T-cell mediated, in that targeted disruption of T-cell TGF-β signaling also results in a similar autoimmune phenotype.151 Recent experiments by Chen and associates rather convincingly demonstrated a role for TGF-β in Treg-mediated suppression of CD8 T-cell antitumor responses.152 In these experiments adoptive transfer of CD4+ CD25+ regulatory T cells inhibited an antitumor CD8 T-cell effector response, and this inhibition was ameliorated when the CD8 T cells came from animals with a dominant negative TGF-β1 receptor. One of the unresolved issues in the study of tumor immune evasion relates to the mechanisms by which tumors induce antigenspecific T-cell tolerance. Whereas the many mechanisms described previously, including STAT3 signaling-dependent mechanisms, IDO, ROS, reactive nitrogen species, TGF-β, and others, clearly inhibit priming of T-cell responses and/or tumor killing by activated effector T cells, it remains to be definitively determined which processes actively induce antigen-specific T-cell tolerance that has been documented in transgenic models. Self-tolerance induction for peripheral tissue antigens is now thought to involve specific presentation of tissue-specific antigens to mature T cells in the absence of appropriate costimulatory signals. Similar mechanisms are probably operative in the case of tumor-induced tolerance. Originally, the relevant costimulatory signals were envisioned to be provided by B7 family costimulatory molecules expressed by DCs.153 It is now becoming clear that additional proinflammatory cytokines such as interferons, IL-12, tumor necrosis factor, and others are critical in the distinction between effector T-cell induction and tolerance induction. An emerging concept is that immature or not fully matured DCs are critical in presenting self-antigens to induce T-cell tolerance in the absence of TLR-mediated danger signals associated with infection.154,155 Unquestionably, DCs found within the tumor microenvironment have a relatively immature, unactivated phenotype characterized by low levels of proinflammatory cytokine production, and CD86 and surface MHC class II expression. As described previously, a major inhibitory signaling pathway induced in tumor-infiltrating DCs is the STAT3 pathway, which, when activated, strongly antagonizes TLR- and CD40-mediated DC activation. As mentioned, tumor-derived factors such as IL-10, IL-6, and VEGF (in part induced by STAT3 signaling in the tumor cell) can induce STAT3 activation in DCs. As described in the previous section, constitutive BRAF signaling in melanoma cells has additionally been shown to induce release of factors that inhibit DC activation.128 These immature “activation-inhibited” DCs clearly represent a prime candidate for the induction of tumor-specific T-cell tolerance. It remains an open question whether iMC/MSC represent a distinct intertumoral cell subset capable of presenting antigens to T cells in a toleragenic fashion.156 A recent report indeed suggested that iMCs loaded with antigen and adoptively transferred into mice can induce antigen-specific T-cell tolerance. Finally, it has been suggested that IDO-expressing DCs can induce antigen-specific T-cell tolerance, because IDO-mediated tryptophan selectively kills or inhibits proliferation of activated T cells.157 According to this model, IDOexpressing DCs would present antigen to T cells inducing activation followed by activation-associated cell death mediated by depletion of local tryptophan stores by the IDO in the presenting DCs. As described later, regulatory T cells play an additional important role in induction of or maintenance of tumor antigen-specific T-cell tolerance. Whether Treg cells mediate T-cell tolerance independently from immature or toleragenic APCs, or whether the two mechanisms are completely interrelated (i.e., toleragenic DCs inducing a Treg

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phenotype among antigen-specific T cells and antigen-specific Treg cells acting upon DCs to enhance their toleragenic capacity), remains to be definitely determined. One of the most important classes of immune-inhibitory molecules shown to be expressed by both tumors and myeloid cells in the tumor microenvironment are members of the growing class of B7 molecules. Originally, B7.1 (also called CD80), and then B7.2 (CD86), were identified as critical costimulatory molecules expressed by APCs (first found on B cells, then macrophages, then DCs). Costimulation of T cells, defined as amplification of activation signals delivered through engagement of the TCR by antigen, was shown to be mediated by binding of B7.1 and B7.2 to CD28, expressed on all naive T cells. Subsequently, feedback inhibition of T-cell activation was shown to be mediated by inhibitory signals delivered by a second receptor for B7.1 and B7.2, termed CTLA-4. CTLA-4 is not expressed on the surface of naive T cells but is rapidly induced after T-cell activation, and, as is the case for TGF-β, CTLA-4 knockout mice develop lymphoproliferative autoimmunity, indicating a tonic role for CTLA-4–B7-1/B7-2 interactions in the prevention of autoimmunity.158 According to these data, CTLA-4 blockade would be expected to function mostly during T-cell priming events, facilitating or enhancing an immune response. In several murine systems, CTLA4 blockade exerts a pronounced antitumor effect,159 generating enthusiasm for translating these observations to the clinic.160 A second coinhibitory molecule on T cells is PD1 (programmed death 1), a T-cell surface molecule originally discovered in a T-cell hybridoma undergoing apoptosis.161 Further studies of PD1 identified expression on activated, but not naive T and B cells, in addition to potential overexpression in anergized CD4 T cells.162 Recent data show that PD1 is also expressed on the surface of certain CD8 T cells, where it serves as a marker for T cells that have been “exhausted” by exposure to persistent viral antigen in vivo. As is the case for CTLA-4 and TGF-β, PD1 knockout mice develop strain-dependent autoimmune disease.163 In murine models of experimental autoimmune encephalomyelitis and diabetes, anti-PD1 antagonist antibodies enhance disease progression.164–166 There are currently two known ligands for PD1: B7-H1 (also known as PD-L1) and B7-DC (also known as PD-L2). B7-H1 and B7-DC represent two of the five additional B7 family members identified over the past 10 years. These ligands have very different tissue distribution patterns, with B7-DC expression primarily confined to DCs and macrophages.167 B7-H1 messenger RNA is widely expressed, but cell surface protein is not detectable in normal tissues other than a subset of macrophages.168 Interestingly, B7-H1 expression can be detected in several tumor types,169 and engagement of PD1 by tumor-associated B7-H1 promotes CD8 T-cell apoptosis. In addition, B7-H1 has been reported to be upregulated on both DCs and macrophages within the tumor microenvironment. Clinically, it has been reported that B7-H1 expression is correlated with poor prognosis in renal cell carcinoma.170 Interestingly, whereas B7-H1 expression on tumor cells was correlated with poor clinical prognosis, combined expression on tumor cells together with hematopoietic cells within the tumor sections was even more highly correlated with poor clinical outcome. Thus, it seems that PD1/B7-H1 interactions mediate a potent and specific immunoregulatory effect, preventing activated and trafficking CD8 T cells from lysing their targets in vivo. In recent data, this observation has been confirmed in murine tumor models, where blockade of either PD1 or of the PD1 ligand B7-H1 potentiates an antitumor immune response.171 In contrast, the molecular role of B7-DC ligation in an immune response is complex,172 and under some circumstances ligation of B7-DC on APCs seems to potentiate a costimulatory interaction with T cells.173 Another more recently identified inhibitory B7 family member, B7-H4, also seems to play an important role in the tumor microenvironment.174 The receptor for B7-H4 has not yet been identified, but this molecule has been definitively shown to play an inhibitory

role because treatment of mice with blocking antibodies and gene knockout resulted in increased immune responses. As with B7-H1, B7-H4 is expressed by several tumors and also by macrophages in the tumor microenvironment.175 B7-H4 is regulated differently than B7H1 and there seems to be a distinct pattern of tumor-selective expression for the two molecules, with some overlap. Recently, B7-H4 expression in human renal cancer has been shown to correlate with poor clinical prognosis, similarly to B7-H1. Patients whose tumors expressed high levels of both molecules displayed the worst clinical outcome, with almost all developing distant metastases.176

PROCARCINOGENIC VERSUS ANTICARCINOGENIC ROLES OF THE IMMUNE RESPONSE Much to the chagrin of the immunotherapy community, skepticism among the oncology community regarding the capacity to induce therapeutically meaningful antitumor immune responses has been accompanied by increasing focus on the capacity of immune responses to induce cancer and potentially enhance cancer progression. Understanding the paradox between the potential procarcinogenic and anticarcinogenic immunity is arguably the most important frontier in cancer immunology (Fig. 6-6). The capacity of inflammatory (i.e., innate) immune responses to enhance carcinogenesis has become well appreciated on the basis of clinical observations that chronic infections that induce chronic inflammation can lead to cancer. One of the best examples is hepatitis C virus (HCV) infection.177 HCV infection leads to a chronic persistent state in the majority of infected individuals associated with chronic hepatitis. This chronic hepatitis is associated with development of hepatocellular carcinoma at the rate of roughly 1% per year. In contrast to other procarcinogenic chronic infections with viruses such as HPV that carry their own oncogenes, the HCV genome contains no oncogenes or genes encoding proteins that inactivate tumor suppressor genes. Thus, the evidence is quite strong that the chronic inflammatory response to HCV is responsible for the genesis of hepatocellular carcinoma. Similarly, the inflammatory responses associated with chronic H. pylori infection of the stomach are thought to be central to the genesis of stomach cancer.178 Further evidence for the procarcinogenic effects of inflammation come from the findings that anti-inflammatory drugs, such as COX-2 inhibitors, can decrease the incidence of colon cancer.179 Additional evidence includes the propensity of patients with certain forms of chronic colitis (e.g., ulcerative colitis) to develop colon cancer and the association of microinflammatory foci in the prostate with prostate intraepithelial neoplastic lesions.180 In animal models, experimental induction of inflammation in both the colon and the liver are associated with increased incidences of cancer. Recently, Karin and colleagues have used conditional knockouts of IKK to demonstrate an important role for NF-κB signaling in experimental models of colonic carcinogenesis.181 Interestingly, they found that tissue-specific knockout of IKKβ in both colonic epithelial cells as well as in myloid cells resulted in a diminished incidence of dextran sulfate (DSS)-induced colon cancers. Epithelium-specific knockout of IKKβ resulted in a decreased incidence, whereas myeloid-specific knockout of IKKβ resulted in both decreased incidence and decreased progression rate. The effects of myeloidspecific IKK knockout were taken as evidence for an NF-κB-dependent proinflammatory role in carcinogenesis. More recently, carcinogen-induced liver cancer and colon tumor development in mice bearing heterozygous adenomatous polyposis coli gene mutations (Min mice) was shown to be dependent on MyD88, an adapter for TLR signaling that is necessary for TLR-dependent NF-κB activation.182,183 In one case, IL-6 production was found to be an important downstream cytokine for liver carcinogenesis. Although the majority of evidence linking immunity to cancer involves the innate immune system, Coussens and colleagues have

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Evidence for the immune system as PROCARCINOGENIC ⵧ Chronic inflammation enhances cancer risk (HBV, HCV→ liver cancer; H. pylori→ gastric cancer) ⵧ Anti-inflammatory drugs reduce cancer risk ⵧ Mouse models • IKK KO in macrophages and PMNs → increased colon cancer incidence/faster progression • MyD88 KO decreases incidence of colon cancer in Min mice and carcinogen-induced liver cancer • Elimination of humoral immunity→ decreased skin carcinogenesis in keratin-E6/E7 transgenic mice

Evidence for the immune system as ANTICARCINOGENIC ⵧ Lymphocytic infiltrates correlate with favorable clinical prognosis (ovarian cancer, colon cancer) ⵧ Activation of antitumor adaptive immune responses suppress tumor growth ⵧ Increased incidence of spontaneous oncogene- and carcinogen-induced cancer in immunodeficient mice

provided evidence that components of the adaptive immune response could contribute to carcinogenesis in a positive fashion. In a transgenic model of HPV E6/E7-induced skin carcinogenesis, they demonstrated that elimination of B cells resulted in a decreased incidence of tumorigenesis.184 Surprisingly, T-cell knockout did not alter the incidence of tumorigenesis. The ultimate mechanism by which B cells contribute to carcinogenesis in this system is not fully defined, although antibody production seems to be involved. Understanding the paradox between procarcinogenic versus anticarcinogenic effects of the immune response will be critical to ultimately defining successful immunotherapies. Recent studies suggest a solution to this paradox and provide insight into the notion that different forms of immune responsiveness can respectively be anticarcinogenic or procarcinogenic. Much evidence has been accumulated that a certain type of T-cell response, termed Th1, can be potently anticarcinogenic. Th1 responses are characterized by production of γ-interferon by CD4 T cells as well as induction of CTL responses by CD8 T cells. γ-interferon not only can enhance the activity of CD8 CTLs, it can also activate components of innate immunity such as macrophages that can kill tumors. Th1 responses are induced by STAT1 signaling and are significantly enhanced by IL-12 that can be produced by macrophages or DCs. Indeed, IL-12 activates not only Th1 responses but also innate immune responses by NK cells that can additionally kill tumor cells.185,186 Recently, a distinct IL-12 family cytokine, termed IL-23, has been discovered.187,188 IL-23 shares the same β chain as IL-12 but has a distinct α chain, termed IL-23 p19. Likewise, the IL-23 receptor shares a common β chain with the IL-12 receptor but also has a distinct IL-23 receptor-specific α chain. Several immunopathologic states related to autoimmune disease that had been attributed to IL12 and linked Th1 responses have now been shown to be instead attributable to IL-23.189 Analogous to IL-12 driving both NK-dependent innate as well as Th1-type adaptive immune responses, IL-23 drives distinct innate immune responses from IL-12 that are just now being elucidated (characterized by granulocyte recruitment). In addition, IL-23 promotes the growth of a distinct type of helper T cell, termed Th17.190–193 Th17 is applied to this helper T-cell pathway, because it is characterized by production of cytokine IL-17a rather than γ-interferon. Recently Langowski and coworkers evaluated skin carcinogenesis and tumor growth in mice with either an IL-23 p19 gene knockout or an IL-12 specific p35 gene knockout.194 As predicted from previous studies on the role of IL-12 in promoting both innate and Th1-dependent antitumor immunity, tumor formation in a

Figure 6-6 • Evidence for procarcinogenic and anticarcinogenic roles of the immune system. Ample evidence exists in both animal models and human disease settings that immune responses can promote or inhibit cancer development.

carcinogen-induced skin cancer model as well as growth of transplanted tumors was increased in IL-12 p35 knockout mice. In striking contrast, carcinogenesis and tumor growth was decreased in the IL-23 p19 knockout mice. Carcinogenesis and tumor growth was also reduced in knockout mice for p40, the common subunit for IL-12 and IL-23. This result suggests that the procarcinogenic effects of IL-23 production dominate over the anticarcinogenic effects of IL-12 production. Although these initial findings will require extensive follow-up, they support the notion that qualitatively distinct types of immune responses, characterized by distinct cytokines that mediate distinct functions, can be procarcinogenic or anticarcinogenic. Analysis of signaling pathways involved in the IL-12–Th1 axis and the IL-23–Th17 axis further suggest a model of “competition” between these two immune pathways. Th1 responses depend on signaling through STAT1, which is essential for commitment to the Th1 lineage, and STAT4, which is the major signal transducer for the IL-12 receptor. In contrast, both IL-23 transcription as well as IL-23 receptor transcription and signal transduction require STAT3 signaling. IL-17 production also requires STAT3 signaling, which is generated by IL-6, which, together with TGF-β, is a critical cytokine for Th17 development from naive T cells.195,196 This may explain the role of IL-6 in the MyD88-dependent carcinogenesis described previously.182 At several levels, STAT3 signaling and STAT1 signaling are mutually antagonistic such that increases in STAT3 signaling inhibit STAT1-induced gene expression programs whereas increases in STAT1 signaling inhibit STAT3-dependent gene expression programs.197 This finding suggests that therapeutic manipulations of STAT signaling could potentially convert procarcinogenic to anticarcinogenic pathways of immune responsiveness. Ultimately it will be critical to evaluate the qualitative nature of immune responses in chronic infections that lead to carcinogenesis to determine whether this “yin-yang” paradigm of procarcinogenic IL-23–Th17 immunity versus anticarcinogenic IL-12–Th1 immunity represents a general principle translatable to human cancer initiation or promotion (Fig. 6-7).

CLINICAL IMPLICATIONS FOR MANIPULATION OF THE IMMUNE RESPONSE TO TUMOR CELLS Fundamentally, we now have clear-cut evidence that antibodies and T cells can selectively recognize and kill cancer cells in patients. Cancer genetics, epigenetics, and genomics have provided us with a

Cancer Immunology • CHAPTER 6

DC1

Figure 6-7 • Two mutually inhibitory pathways of immunity may inhibit or promote cancer. The Th1 pathway is promoted by STAT1 and STAT4 signaling, is initiated by type I interferons, and depends on IL-12. Th1 cells are characterized by production of γ-IFN but also produce many other cytokines. The IL-12/Th1 pathway is typically anticarcinogenic. The Th17 pathway is promoted by Stat3 signaling, is initiated by IL-6 and TGF-β, and depends on IL-23. Th1 cells are characterized by production of IL-17a but also produce many other cytokines. Evidence exists that the IL-23-Th17 pathway can promote carcinogenesis. These pathways are mutually inhibitory in a yinyang fashion.

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Box 6-2.

CAN THE IMMUNE SYSTEM BE MANIPULATED TO OVERCOME TUMOR-INDUCED TOLERANCE AND IMMUNE EVASION?

One of the most consistent therapeutic failures thus far has come from attempts to treat cancer with tumor vaccines. Evidence has now mounted that a major reason for the lack of efficacy of tumor vaccines and other immunotherapeutic interventions is the barrier of immune tolerance to tumor antigens as well as the upregulation of molecules in the tumor microenvironment that inhibit effector immune responses. The bad news from these insights is that therapeutic efficacy of tumor vaccines used as single agents will probably always be limited, no matter how effective the vaccine is in inducing immunity in a naive host. It is likely that the only viable therapeutic application of cancer vaccines used as single agents will be in the setting of minimal residual disease. However, as the specific molecular pathways of immune tolerance and immune evasion are defined, antibodies as well as small molecule agonists and antagonists are being developed that can either enhance costimulatory pathways that amplify immune responses or specifically block receptors and pathways that inhibit immune effectors such as cytotoxic T cells. Preclinical experiments as well as very earlystage clinical experience suggest that combinations of vaccines that direct immune responses to particular tumor antigens together with agents that either enhance or costimulate immunity, as well as agents that block immune checkpoints, produce strong synergy in enhancing antitumor immune responses. It is likely that these combinatorial approaches will be the most fruitful for developing clinically relevant immune therapies of cancer.

far better understanding of the nature and specificity or selectivity of tumor antigens, providing new opportunities for targeted antigen-specific immunotherapy. We know much more about the details of antigen recognition by T cells allowing for the opportunity to modify antigens at critical residues to provide for enhanced immunestimulatory capacity. Finally, we are learning much about the ligands, receptors, and signaling pathways that regulate immune responses and how they are expressed within the tumor microenvironment. Elucidation of these regulatory pathways has demonstrated that the outcome of antigen recognition is in large part determined by the balance between costimulatory signals and inhibitory signals. These relatively recent insights into the molecular basis of immune regulation are demonstrating profound significance for the development of more potent combinatorial immunotherapy approaches to cancer (Box 6-2). Several consensus have emerged from both preclinical immunotherapy models as well as analysis of cancer patients. First and foremost, the natural state of endogenous tumor-reactive T cells is characterized by general hyporesponsiveness or anergy. This is probably due to several mechanisms that tumors utilize to induce tolerance as they develop. Whereas a number of the newer generation vaccines such as recombinant viral vaccines or GM-CSF genemodified vaccines can effectively transfer antigen to and activate DCs, T-cell tolerance remains a major barrier that is difficult to overcome by vaccination alone. Preclinical models demonstrate that for poorly immunogenic tumors, once tolerance has been established, therapeutic vaccines alone are ineffective at curing animals with a significant established tumor burden. However, strategies combining vaccination with inhibitors of immunologic checkpoints and agonists for costimulatory pathways are proving capable of overcoming tolerance and generating significant antitumor responses even in cases of established metastatic cancer.

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4. Old LJ, Boyse EA, Clark DA, Carswell EA: Antigenic properties of chemically-induced tumors. Ann NY Acad Sci 1962;101:80–106. 5. Prehn RT: Immunity to methylcholanthreneinduced sarcomas. J Natl Cancer Inst 1957;18: 769–778. 6. Slamon DJ, Leyland-Jones B, Shak S, et al: Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344:783–792.

7. Ciardiello F, Bianco R, Damiano V, et al: Antitumor activity of sequential treatment with topotecan and anti-epidermal growth factor receptor monoclonal antibody C225. Clin Cancer Res 1999;5:909–916. 8. Fearon ER, Vogelstein B: A genetic model for colorectal tumorogenesis. Cell 1990;61:759–767. 9. Lu X, Lane DP: Differential induction of transcriptionally active p53 following UV or ionizing radiation: defects in chromosome instability syndromes? Cell 1993;75:765–778.

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169. Dong H, Strome SE, Salomao DR, et al: Tumorassociated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 2002;8:793–800. 170. Thompson RH, Gillett MD, Cheville JC, et al: Costimulatory B7-H1 in renal cell carcinoma patients: indicator of tumor aggressiveness and potential therapeutic target. Proc Natl Acad Sci USA 2004;101:17174–17179. 171. Hirano F, Kaneko K, Tamura H, et al: Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity. Cancer Res 2005;65:1089–1096. 172. Shin T, Kennedy G, Gorski K, et al: Cooperative B7-1/2 (CD80/CD86) and B7-DC costimulation of CD4+ T cells independent of the PD-1 receptor. J Exp Med 2003;198:31–38. 173. Nguyen LT, Radhakrishna S, Ciric B, et al: Crosslinking the B7 family molecule B7-DC directly activates immune functions of dendritic cells. J Exp Med 2002;196:1393–1398. 174. Sica GL, Choi IH, Zhu G, et al: B7-H4, a molecule of the B7 family, negatively regulates T cell immunity. Immunity 2003;18:849–861. 175. Kryczek I, Zou L, Rodriguez P, et al: B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma. J Exp Med 2006;203:871–881. 176. Krambeck AE, Thompson RH, Dong H, et al: B7H4 expression in renal cell carcinoma and tumor vasculature: associations with cancer progression and survival. Proc Natl Acad Sci USA 2006;103: 10391–10396. 177. Levrero M: Viral hepatitis and liver cancer: the case of hepatitis C. Oncogene 2006;25:3834– 3847. 178. Fox JG, Wang TC: Inflammation, atrophy, and gastric cancer. J Clin Invest 2007;117:60–69. 179. Koehne CH, Dubois RN: COX-2 inhibition and colorectal cancer. Semin Oncol 2004;31(Suppl 7):12–21. 180. De Marzo AM, Platz EA, Sutcliffe S, et al: Inflammation in prostate carcinogenesis. Nat Rev Cancer 2007;7:256–269. 181. Greten FR, Eckmann L, Greten TF, et al: IKKbeta links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell 2004;118:285–296. 182. Naugler WE, Sakurai T, Kim S, et al: Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science 2007;317:121–124. 183. Rakoff-Nahoum S, Medzhitov R: Regulation of spontaneous intestinal tumorigenesis through the adaptor protein MyD88. Science 2007;317:124– 127. 184. de Visser KE, Korets LV, Coussens LM: De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent. Cancer Cell 2005;7:411–423. 185. Colombo MP, Trinchieri G: Interleukin-12 in anti-tumor immunity and immunotherapy. Cytokine Growth Factor Rev 2002;13:155–168. 186. Trinchieri G: Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003;3:133–146. 187. Oppmann B, Lesley R, Blom B, et al: Novel p19 protein engages IL-12p40 to form a cytokine, IL23, with biological activities similar as well as distinct from IL-12. Immunity 2000;13:715– 725. 188. Kastelein RA, Hunter CA, Cua DJ: Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation. Annu Rev Immunol 2007;25:221–242. 189. Cua DJ, Sherlock J, Chen Y, et al: Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 2003;421:744–748.

Cancer Immunology • CHAPTER 6 190. Langrish CL, Chen Y, Blumenschein WM, et al: IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 2005;201:233–240. 191. Bettelli E, Oukka M, Kuchroo VK: T(H)-17 cells in the circle of immunity and autoimmunity. Nat Immunol 2007;8:345–350. 192. Aggarwal S, Ghilardi N, Xie MH, et al: Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production

of interleukin-17. J Biol Chem 2003;278:1910– 1914. 193. Dong C: Diversification of T-helper-cell lineages: finding the family root of IL-17-producing cells. Nat Rev Immunol 2006;6:329–333. 194. Langowski JL, Zhang X, Wu L, et al: IL-23 promotes tumour incidence and growth. Nature 2006;442:461–465. 195. Veldhoen M, Hocking RJ, Atkins CJ, et al: TGFbeta in the context of an inflammatory

cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 2006;24:179– 189. 196. Bettelli E, Carrier Y, Gao W, et al: Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006;441:235–238. 197. Stephanou A, Latchman DS:Opposing actions of STAT-1 and STAT-3. Growth Factors 2005;23: 177–182.

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Stem Cells, Cell Differentiation, and Cancer Michael F. Clarke and Irving L. Weissman

S U M M ARY • Most cancers arise in tissues (e.g., the gut, breast, prostate, lungs, and bone marrow) that contain a stem cell population. • Stem cells have three fundamental properties: the ability to divide and give rise to a new stem cell in a process called self-renewal, the ability to give rise to the differentiated cells of an organ, and genetic constraints on expansion. • The fact that stem cells are the only long-lived cells in most tissues in which cancers arise suggests that early mutations that lead to cancer accumulate in stem cells.

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• In addition to classes of oncogenes that affect cell survival and proliferation, there is a class of oncogenes that permits cells to self-renew. Thus, in some cancers, the target cells for neoplastic transformation may be progenitor cells that have acquired the ability to self-renew as a result of one or more mutations. • New data suggest that in both leukemia and solid tumors, including tumors of the breast, colon, head and neck, pancreas, and prostate, a small, phenotypically distinct subset of cancer cells has the exclusive ability to form tumors.

INTRODUCTION Common cancers arise in tissues that contain a large subpopulation of proliferating cells that are responsible for replenishing the shortlived mature cells. In such organs, cell maturation is arranged in a hierarchy in which a rare population of stem cells, which perpetuate themselves through a process called self-renewal, gives rise to intermediate progenitors and then mature cells, neither of which self-renew.1–11 Because of their rarity, stem cells must be isolated prospectively to study their biologic, molecular, and biochemical properties. Although it is likely that each tissue regenerates from tissue-specific stem cells, stem cells have been rigorously identified and purified in only a few. The stem cells that give rise to the lymphohematopoietic system, called hematopoietic stem cells (HSCs), have been isolated from mice and humans and are the best-characterized stem cells. The utility of tissue containing HSCs has been demonstrated in cancer therapy with its extensive use for bone marrow transplantation to regenerate the hematolymphoid system after myeloablative protocols.12 The prospective isolation of HSCs from patients can result in a population of cancer-free cells for autologous transplantation.13–17 Understanding the cellular biology of the tissues in which cancers arise, and specifically that of the stem cells that reside in those tissues, could provide new insights into cancer biology. Several aspects of stem cell biology are relevant to cancer. First, both normal stem cells and cancer stem cells undergo self-renewal, and emerging evidence suggests that similar molecular mechanisms regulate self-renewal in normal stem cells and their malignant counterparts. Next, it is quite likely that mutations that lead to cancer accumulate in normal stem

• At present, therapeutic targets are selected on the basis of the proposition that all of the cancer cells within a particular tumor are capable of driving tumor formation and metastasis. However, because the bulk of the cancer cells in the tumor are unable to form tumors and most cancer agents are selected to reduce the bulk, these cells are the targets of many therapies. • To be effective, therapies must target the critical tumorigenic cancer cell population. • The ability to prospectively identify tumorigenic cancer cells should allow the identification of new diagnostic markers and therapeutic targets.

cells.18 Finally, as was stated previously, it is likely that tumors contain a minority “cancer stem cell” population with indefinite proliferative potential that drives the growth and metastasis of tumors.19–28

PROPERTIES OF NORMAL STEM CELLS HSCs are the most studied and best understood somatic stem cell population and serve as a model for stem cells from other tissues.1,9,27,29,30 Hematopoiesis is a tightly regulated process in which a pool of HSCs eventually gives rise via oligo lineage intermediates1,31–33 to the lymphohematopoietic system consisting of the formed blood elements (e.g., red blood cells, platelets, granulocytes, macrophages, and B and T lymphocytes). These cells are important for oxygenation, prevention of bleeding, immunity, and fighting infections. In the adult, HSCs have three fundamental properties. First, HSCs need to selfrenew to maintain the stem cell pool. Self-renewal is not synonymous with proliferation. Self-renewal is a cell division in which one or both of the daughter cells remain undifferentiated and have the ability to give rise to another stem cell as well as to the spectrum of more differentiated progenitors. Second, HSCs must undergo differentiation to maintain a constant pool of mature cells in normal conditions and to produce increased numbers of a particular lineage in response to stresses such as bleeding or infection. Third, the total number of HSCs is under strict genetic regulation.34 In the mouse hematopoietic system, multipotent cells constitute 0.05% of bone marrow cells and are heterogeneous with respect to their ability to self-renew. There are three different populations of multipotent cells: long-term self-renewing HSCs, short-term

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Granulocytes Common myeloid progenitor

GMP Monocytes

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self-renewing HSCs, and multipotent progenitors without detectable self-renewal potential.7,35 These populations form a hierarchy in which the long-term HSCs give rise to short-term HSCs, which in turn give rise to multipotent progenitors7 (Fig. 7-1). As HSCs mature from the long-term self-renewing pool to multipotent progenitors, they become more mitotically active but lose the ability to self-renew. Only longterm HSCs can give rise to mature hematopoietic cells for the lifetime of the animal, whereas short-term HSCs and multipotent progenitors reconstitute in lethally irradiated mice for fewer than 8 weeks.7 Despite the fact that the phenotypic and functional properties of mouse and human HSCs have been extensively characterized,2 understanding of the fundamental stem cell property, self-renewal, is minimal.27,29,36 In most cases, HSCs differentiate when exposed to combinations of growth factors that can induce extensive proliferation in long-term cultures.37 Although recent progress has been made in identifying culture conditions that maintain HSC activity in culture for a limited period,38 it has proven to be exceedingly difficult to identify tissue culture conditions that promote a significant and prolonged expansion of progenitors with transplantable HSC activity.

GENETIC REGULATION OF SELF-RENEWAL IN NORMAL STEM CELLS AND CANCER CELLS Maintenance of a tissue or a tumor is determined by a balance of cell proliferation and cell death.39 As would be expected, many of the mutations that drive tumor expansion regulate either cell proliferation or survival. For example, the prevention of apoptosis by enforced expression of the oncogene Bcl-2 promotes the development of lymphoma and also results in increased numbers of HSCs in vivo, suggesting that cell death plays a role in regulating the homeostasis of HSCs; enforced expression of bcl2 does not endow short-term HSCs or multipotent progenitors with self-renewal properties.40,41 In fact, the progression to experimental acute myelogenous leukemia (AML) in mice requires at least four independent events to block the several intrinsically triggered and extrinsically induced programmed cell death pathways of myeloid cells.42 Proto-oncogenes such as c-myb and c-myc that drive proliferation of tumor cells are also essential for HSC development43–46 as well as increased expression of the telomere regenerating enzyme and RNA.47

Figure 7-1 • Blood development hierarchy. All of the diverse mature blood cells arise from the hematopoietic stem cells (HSCs). The cells that are capable of multilineage reconstitution of a lethally irradiated mouse are contained within two identifiable and separate populations of cells: the longterm hematopoietic stem cells HSCs (LT-HSCs) and the short-term hematopoietic stem cells HSCs (ST-HSCs). Only the LT-HSCs are capable of selfrenewal for the lifetime of the animal. In contrast, other cells, even the ST-HSCs that can give rise to large numbers of mature blood cells, have very limited life spans (measured in hours to 1 or 2 months). GMP, granulocyte-macrophage progenitors; MEP, myeloid-erythroid progenitors.

Self-renewal is critical for both normal stem cells and cancer stem cells. In a normal tissue, stem cell numbers are under tight genetic regulation, resulting in the maintenance of a constant number of stem cells in the organ.34,48,49 In contrast, cancer stem cells have escaped this homeostatic regulation, and the number of cells within a tumor with the ability to self-renew is constantly expanding, resulting in the inevitable growth of the tumor. Because cancer cells and normal stem cells share the ability to self-renew, it is not surprising that a number of genes classically associated with cancer may also regulate normal stem cell development.27,50 In combination with other growth factors, sonic hedgehog (Shh) signaling has also been implicated in the regulation of self-renewal by the finding that cells that are highly enriched for human HSCs (CD34+Lin−CD38−) exhibit increased self-renewal in response to Shh stimulation in vitro.51 Several other genes related to oncogenesis have been shown to be important for stem cell function. For example, mice that are deficient for tal-1/SCL, which is involved in some cases of human acute myeloid leukemia, lack embryonic hematopoiesis,52 suggesting that tal-1/SCL is required for intrinsic or extrinsic events necessary to initiate hematopoiesis, for maintenance of the earliest definitive blood cells, or for the formation of blood cells downstream of embryonic HSCs.52,53 Members of the Hox family have also been implicated in human leukemia, and enforced expression of HoxB4 can affect stem cell functions.54,55 One of the major targets of the p53 tumor suppressor gene is p21cip1. Bone marrow from p21cip1-deficient mice has a reduced ability to serially reconstitute lethally irradiated recipients. Failure at serial transfer could result from exhaustion of the stem cell pool, loss of telomeres, or loss of transplantability.18,56,57 Thus, many genes that are involved in decisions about stem cell fate are also involved in malignant transformation. The notion that the function of certain oncogenes is to regulate self-renewal is perhaps best illustrated by studies of the oncogene bmi-1. In mice, bmi-1 cooperates with c-myc to induce lymphoma.58,59 The number of HSCs is markedly reduced in postnatal bmi-1−/− mice, and transplanted bmi-1−/− fetal liver and bone marrow cells are able to contribute only transiently to hematopoiesis, indicating a cell autonomous defect of HSC self-renewal in bmi-1−/− mice.60 The expression of stem cell-associated genes,3 cell survival genes, transcription factors, and genes that modulate proliferation, including p16 Ink4a and p19 Arf, is altered in bone marrow cells of bmi-1−/− mice. This suggests that the function of bmi-1 is to regulate a cascade of genes

Stem Cells, Cell Differentiation, and Cancer • CHAPTER 7

that modulate stem cell self-renewal. In a mouse model of leukemia, leukemic cells that lack expression of bmi-1 eventually undergo proliferation arrest associated with signs of differentiation and apoptosis when they are transplanted in syngenic hosts. Infection of the cells with a bmi-1 retrovirus completely rescues the proliferative defect of the bmi-1−/− leukemic stem cells.61 Along a similar line, the regulated postnatal deletion of junB, an AP1 transcription factor, results in HSC expansion and then the development of a chronic myeloproliferative disorder that resembles human chronic myelogenous leukemia (CML) in its chronic phase.62 Transduced overexpression of junB in HSCs inhibits or prevents their self-renewing proliferations. In the junB knockout HSCs, p16 ink4a and p19 Arf levels are lowered, and the antiapoptotic proteins bcl2 and bclx are increased, while the reverse is true in HSCs that have junB overexpression.55 Only HSCs transplant this chronic phase of the disease, but later, in blast crisis, cells at a more differentiated stage emerge that transplant the blast crisis disease.62,63 These studies conclusively demonstrate that malignant transformation requires not only activation of proliferation pathways, as well as inactivation of cell death and cell cycle arrest pathways, but also activation of self-renewal pathways. Two other signaling pathways that are implicated in oncogenesis in both mice and humans, the Wnt/β-catenin and Notch pathways, may play central roles in the self-renewal of both normal and cancer stem cells. The Notch family of receptors was first identified in Drosophila species and has been implicated in development and differentiation.64 In Caenorhabditis elegans, Notch plays a role in germ cell self-renewal.65 In neural development, transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by embryonic neural crest stem cells.10 Notch activation of HSCs in culture with either of the Notch ligands Jagged-1 or Delta transiently increases the primitive progenitor activity both in vitro and in vivo, suggesting that Notch activation promotes either the maintenance of progenitor cell multipotentiality or HSC self-renewal.66,67 Although the Notch pathway plays a central role in development and the mouse oncogene int-3 is a truncated Notch 4,68 the role of Notch in de novo human cancer is complex and less well understood. Various members of the Notch signaling pathway are expressed in cancers of epithelial origin, and activation of the Notch pathway by chromosomal translocation is involved in some cases of leukemia.69–73 Microarray analysis has shown that members of the Notch pathway are often overexpressed by tumor cells.70,71 A truncated Notch 4 messenger RNA is expressed by some breast cancer cell lines.74 Overexpression of Notch1 leads to growth arrest of a small cell lung cancer cell line, whereas inhibition of Notch1 signals can induce leukemia cell lines to undergo apoptosis.64,66,75 Elegant work by Weizen and colleagues76 showed that activation of Notch1 signaling maintains the neoplastic phenotype in Ras-transformed human cells. They also found that in de novo cancers, cells with an activating Ras mutation also demonstrate increased expression of Notch1 and Notch4. Wnt/β-catenin signaling also plays a pivotal role in the selfrenewal of normal stem cells and malignant transformation.77–79 The Wnt pathway was first implicated in mouse mammary tumor virusinduced breast cancer in which deregulated expression of Wnt-1 caused by proviral insertion resulted in mammary tumors.80,81 Subsequently, it has been shown that Wnt proteins play a central role in pattern formation. Wnt-1 belongs to a large family of highly hydrophobic secreted proteins that function by binding to their cognate receptors, members of the Frizzled and low-density lipoprotein receptor-related protein families, resulting in activation of βcatenin.50,70,77,82,83 In the absence of receptor activation, β-catenin is marked for degradation by a complex consisting of the adenomatous polyposis coli, Axin, and glycogen synthase kinase-3β proteins.78,79,84–87 Wnt proteins are expressed in the bone marrow, and activation of Wnt/β-catenin signaling by Wnt proteins in vitro or by expression of a constitutively active β-catenin expands the pool of early progenitor cells and enriched normal transplantable HSCs in tissue culture and in vivo.27,79,84 Inhibition of Wnt/β-catenin by ectopic

expression of Axin, an inhibitor of β-catenin signaling, leads to inhibition of stem cell proliferation both in vitro and in vivo. Addition of Wnt3a to purified HSC leads to their expansion, presumably selflimited usually by the expression of the β-catenin-induced transcription of axinII; transduction of purified HSC with axin prevents their expansion in vitro or in vivo.88,89 Other studies suggest that the Wnt/ β-catenin pathway mediates stem or progenitor cell self-renewal in other tissues.85,86,90,91 The level of β-catenin in a particular keratinocyte directly correlates with its proliferative capacity.85,86,91 As in their normal HSC counterparts, enforced expression of an activated βcatenin in epidermal stem cells increases their ability to self-renew and decreases their ability to differentiate. Mice that fail to express TCF-4, one of the transcription factors that is activated when bound to β-catenin, soon exhaust their undifferentiated crypt epithelial progenitor cells, further suggesting that Wnt signaling is involved in the self-renewal of epithelial stem cells.50,90 Activation of β-catenin in colon cancer by inactivation of the protein degradation pathway, most frequently by mutation of adenomatous polyposis coli, is common.50,70,78,87 Expression of certain Wnt genes is increased in some other epithelial cancers, suggesting that activation of β-catenin might be secondary to ligand activation in such cancers.77,92–97 There is evidence that constitutive activation of the Wnt/β-catenin pathway might confer a stem/progenitor cell phenotype to cancer cells. Inhibition of β-catenin/TCF-4 in a colon cancer cell line induced the expression of the cell cycle inhibitor p21cip-1 and induced the cells to stop proliferating and to acquire a more differentiated phenotype.97 Enforced expression of the protooncogene c-myc, which is transcriptionally activated by β-catenin/ TCF-4, inhibited the expression of p21cip-1 and allowed the colon cancer cells to proliferate when β-catenin/TCF-4 signaling was blocked, linking Wnt signaling to c-myc in the regulation of cell proliferation and differentiation.97 The implication of roles for genes such as Notch, Wnt, c-myc, and Shh in the regulation of self-renewal of HSCs, and perhaps of stem cells, from multiple tissues suggests that there might be at least some common self-renewal pathways in many types of normal somatic stem cells and cancer stem cells. It will be important to identify the molecular mechanisms by which these pathways work and to determine whether the pathways interact to regulate the selfrenewal of normal stem cells and cancer stem cells.

TARGET CELLS FOR MALIGNANT TRANSFORMATION If oncogenic mutations often target signaling pathways that regulate proliferation and self-renewal, then are stem cells, highly proliferative progenitor cells, or both the target of neoplastic transformation? Several lines of evidence suggest that stem cells might be involved in the evolution of a cancer. First, the fact that multiple mutations are necessary for a cell to become cancerous98,99 suggests that in many cases, mutations accumulate in a stem cell. Progenitor cells have a very limited life span, making it less likely that all of the mutations occur during the life of these relatively short-lived cells.1,7,8,27,29,31,32,100 Second, the regulation of stem cell expansion and self-renewal is under strict genetic regulation by multiple genes, and unregulated expansion of stem cells could, in essence, result in a cancer.34,48,49 Third, most cancers arise in tissues that contain stem cells that have the intrinsic ability to self-renew. Because cancer cells must undergo self-renewal, this suggests that stem cells might more easily undergo steps in the progression to malignant transformation than will progenitor cells that lack this fundamental property and must therefore activate these self-renewal pathways to become malignant. In the hematopoietic system, the only cells that have the ability to self-renew are HSCs and mature lymphocytes. The common blood cancers, acute leukemias and lymphomas, may arise from the HSCs or lymphocytes, respectively, via constitutive activation of mitogenic pathways associated with the proliferation of normal cells.27,39,101,102 Although stem cells

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may undergo steps toward malignant transformation, it is possible, if not likely, that in many cases, their progenitor cells that inherit the changes in stem cells, and in addition add the ultimate transforming event, give rise to cancer. For example, the initial mutations that occur in the stem cell could permit a single mutation to transform a progenitor cell, or perhaps events that shut down self-renewal could occur in stem cells, but only progenitors outside of the stem cell regulatory niche expand as cancer stem cells.18 It is also possible that certain oncogenic mutations such as bmi-1 or its downstream targets could confer the property of self-renewal on a progenitor cell. The target cells for transformation are best understood in hematopoietic malignancies because the developmental hierarchy of the blood is well established.1,7,31,32 One of the most frequent mutations in AML in elderly patients is the t(8;21) translocation, which results in the expression of a chimeric AML-ETO transcript in the leukemic cells.103–105 Marrow samples from patients with early onset 8;21 leukemia in Hiroshima Hospital had CD34+Thy1(CD90)+CD38−Lin− HSCs, which, when isolated from patients in clinical remission, had up to 90% incidence of the chimeric AML-1-ETO transcript.105 When these HSCs were analyzed by means of in vitro differentiation assays, the HSCs gave rise to normal myeloerythroid progeny, demonstrating that the mutation was present in the otherwise normal stem cells. In these patients, the CD90 neg subset of CD34+CD38− Lin− cells gave rise to leukemic colonies in vitro; this could represent HSCs that have lost Thy1 expression or downstream multipotent progenitors106 that have gained self-renewal capacity.105 Taken together, these observations support the notion that mutations accumulate in stem cells and that subsequent mutations in either the stem cells or their progeny result in overt leukemia. Although stem cells are frequently the target of mutations that are on the path to malignant transformation, it is likely that their clonal progenitor cells may be transformed by subsequent genetic events that confer immortality, self-renewal potential, or both to these normally non-self-renewing cells (Fig. 7-2).18 In patients with CML, the BCR-ABL mutation is present in both normal and leukemic stem

Normal stem cell

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Mature cells

Figure 7-2 • Target cells for neoplastic transformation. In many tissues in which cancers arise, the stem cells are the only long-lived cells and are the only cells capable of self-renewal. Because they are already capable of extensive self-renewal, they are good targets for neoplastic transformation. Dysregulation of the self-renewal process may be simpler in these cells than in progenitor cells that lack this ability. For progenitor cells to undergo malignant transformation, they must acquire the ability to undergo extensive selfrenewal as a result of oncogenic mutations.

cells. In otherwise normal hematopoietic cells, the BCR-ABL mRNA is expressed solely by the progenitor cells.2,107,108 In a mouse model of CML, BCR-ABL expression targeted to myeloid progenitor cells by the hMRP-8 promoter resulted in CML-like disease in a subset of the mice. Furthermore, when hMRP8p210BCR/ABL mice were crossed with hMRP8bcl-2 mice, a proportion of the mutant mice developed a disease resembling AML or myeloid blast crisis.109 Although the expression of transforming genes was targeted to early progenitor cells, the appearance of the leukemia cells and clinical course resembled human CML and AML in the hMRP8p210BCR/ABL mice and the hMRP8p210BCR/ABL/hMRP8bcl-2 mice, respectively. In the chronic phase of human CML, the CML phenotype of the CML stem cell is identical to that of a normal HSC. β-Catenin signaling is active in both the normal HSCs and the CML stem cells but not in the progenitor cells. When CML progresses to “myeloid blast crisis,” the patients develop an acute leukemia-like disease. The leukemic stem cell in CML blast crisis phenotypically is at the developmental stage of a granulocyte-macrophage progenitor cell that transplants the disease to immunodeficient mice and that self-renews replating potential in vitro.110 Furthermore, the CML blast crisis stem cells appear to have activated the β-catenin signaling pathway and are inhibited in their in vitro self-renewal by transduced axin. Thus, disease progression in human CML appears to result from activation of self-renewal pathways in a progenitor cell population or, more likely, failure to shut down this pathway in the HSC to granulocytemacrophage transition. These studies strongly suggest that the leukemia stem cell in CML blast crisis is derived from a progenitor cell, not from a normal stem cell, although the initial event and likely several progression events are in successive subclones of the initial bcr-abl HSC stage cells. In a mouse model of high-grade glioblastoma, enforced expression of the epidermal growth factor receptor-enriched populations in either Ink4a/Arf null neuronal stem cells or Ink4a/Arf null astrocytes led to malignant glioblastomas when the cells were injected orthotopically into mice.111 Notably, in the majority of the cases, the transformed astrocytes appeared to acquire an immature phenotype in the brains of the mice, suggesting to some researchers that there was “dedifferentiation,”111 although we have never documented a case of dedifferentiation with purified normal hematopoietic progenitor cells. There are two other possible explanations for these results. First, the astrocyte tissue culture cells could have contained a rare population of neuronal stem cells that were transformed, and these stem cells were responsible for generating the tumors. Second, it is possible that the tissue culture conditions could have caused the dedifferentiation of the astrocytes and that unless the astrocytes are grown in tissue culture, they cannot give rise to glioblastomas in an animal. These observations in humans and mice support the notion that oncogenic mutations accumulate in the stem cells, but expression of the mutated gene by progenitors downstream of the stem cells can lead to their neoplastic transformation of progenitor cells. These observations have implications for targeted therapies. It is possible that only a minority of the mice whose progenitors express BCR-ABL develop leukemia because the progenitors must acquire an additional mutation or epigenetic change that causes deregulated self-renewal. Two lines of evidence support this notion. First, expression of bmi-1 is necessary for the self-renewal of adult HSCs, and the blast cells of patients with AML express large amounts of this protein.101 Although expression of HoxA9 and Meis1 induces transplantable AML in normal mice, expression of these genes in the absence of bmi-1 does not.60,61 This suggests that both normal HSCs and leukemic stem cells require bmi-1 to self-renew. Second, deregulated β-catenin signaling occurs in many de novo human cancers and causes cancer in transgenic mouse models. Because expression of a constitutively active β-catenin can promote the self-renewal of normal HSCs, as well as stem cells from other tissues, it is quite plausible that activation of this pathway promotes self-renewal of the cancer cells.2,27,90,91,97,102,107,109,112,113 From these results, it is evident that

Stem Cells, Cell Differentiation, and Cancer • CHAPTER 7

future studies focusing on the molecular regulation of the self-renewal of normal stem cells and cancer cells will likely lead to more effective therapies for cancer.

EVIDENCE FOR CANCER STEM CELLS It has long been known that cancers consist of phenotypically heterogeneous populations of cancer cells.23–26,114–117 These phenotypically distinct cell populations could arise in part from sequential mutations caused by genetic instability, environmental factors, or both (Fig. 7-3A). Alternatively, a tumor can be viewed as an aberrant organ containing a tumorigenic (stem cell) population that drives tumor growth. These tumorigenic cells would have acquired oncogenic mutations and epigenetic changes that result in unregulated self-renewal, extended cell survival, and avoidance of innate and adaptive immune surveillance and would also give rise to phenotypically diverse populations of tumor cells that lack the ability to self-renew (Fig. 7-3B). Several lines of evidence suggest that the latter model accounts for some of the cellular heterogeneity that is seen in tumors, although genetic instability and environmental factors could also contribute to the variability in phenotypes.118,119 It is well documented that many types of cancer contain heterogeneous populations of cells that variably express differentiation markers that reflect the tissues from which the tumors originate, as well as cancer cells that have an immature appearance.115,120,121 Examples of this include the variable expression of milk proteins by some breast cancers and the variable expression of myeloid markers, lymphoid markers, or both in CML and AML. Perhaps the most striking example of abnormal differentiation in cancer is the variable expression of diverse tissues in some germ cell tumors. Mature tissues such as teeth, skin, and hair are present in some cases of teratocarcinomas (Fig. 7-4). In contrast, in some tumors, only a minority of the cancer cells express immature cell markers such as α-fetoprotein (see Fig. 7-4). Because the terminally differentiated cells that form the teeth and hair in the tumors are unlikely to be able to proliferate and form new tumors, these data suggest that the minority population of α-fetoprotein-expressing cancer cells has the exclusive ability to form new tumors consisting of more tumorigenic cells, as well as the phenotypically diverse populations of non-self-renewing abnormally differentiated cells. If this is true, these cells can thus be considered cancer stem cells. If a tumor is viewed as an abnormal organ, then the principles of stem cell biology can be applied to better understand the biology of

A

these diseases.1,8,9 It was first shown in hematopoietic malignancies and subsequently in solid cancers that only a subset of cancer cells were clonogenic when placed in tissue culture or injected into immunodeficient mice.21,23–25,115,117,122,123 For example, only 1 in 100 to 1 in 10,000 mouse myeloma cells obtained from ascites fluid formed in in vitro colony-forming assays. Similarly, only 1% to 4% of leukemic cells formed spleen colonies when transplanted into mice. In solid cancers, only 1 in 1000 to 1 in 5000 ovarian cancer or lung cancer cells formed colonies in soft agar. Because only a minority of normal bone marrow cells was also clonogenic, the clonogenic cancer cells were described as cancer stem cells, implying that only a distinct population of cancer cells was able to proliferate extensively in these assays. However, an alternative explanation is that all the cancer cells had an intrinsic ability to proliferate extensively but only a minority of cells did so in a particular assay.117 Variable plating efficiency in a variety of in vitro cells and cell lines could have accounted for the poor performance of such cells, and in none of the assays did the investigators prospectively purify the plating cell to show that it was, or was not, a cancer stem cell. To prove that a phenotypically distinct population of cancer cells is solely responsible for perpetuating the disease, it is necessary to isolate different populations of cancer cells and demonstrate that one or more groups are enriched for the ability to initiate disease and other populations lack this ability. This was done in the case of AML, when it was shown that in most cases of human AML, a leukemic tumor initiating subpopulations of cells could be identified prospectively and enriched from the bone marrow of multiple patients. In most cases of AML, the minority population of CD34+CD38− cells was the only group of cells that was capable of establishing human AML in the bone marrow of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice.124,125 Remarkably, within the CD34+CD38− population are Thy1+CD34+CD38− Lineage− normal HSCs2,100,105,124–127 as well as Thy1−CD34+38−Lineage− progenitors.106 Because normal HSCs, but not their leukemic initiating cell counterparts, express Thy1, it is likely that the early mutations occurred in the HSCs and the final transforming mutations occurred either in early downstream progenitors or in HSCs if, as a consequence of neoplastic transformation, Thy1 expression was lost.105 Recently, tumorigenic and nontumorigenic subsets of cancer cells have been isolated from human breast cancer tumors. When a similar model for human breast cancer was used in which isolated

B

Figure 7-3 • The two most likely models of heterogeneity of the cancer cells, shown as different colored cells within a tumor. A, Heterogeneity is due to environmental factors (gold, red, green, and blue cells) or due to ongoing mutations in the cancer cells (magenta cells). In this model, all of the cancer cells have the intrinsic ability to form tumors. B, Cancer stem cells (yellow cells) have the exclusive ability to self-renew. As in normal tissues, the stem cells would give rise to more stem cells with the capacity to form new tumors, as well as the other heterogeneous populations of cancer cells that lack the ability to form new tumors. To date, therapeutic and diagnostic strategies have been based on model A, but these strategies might be limited because they might not target the rare population of cancer stem cells depicted in model B.

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A Post–therapy mature teratoma

B

C Figure 7-4 • Clinical evidence for the stem cell model. The clinical and radiographic information for a patient with metastatic teratocarcinoma of the testis is shown. A, In the original testicular biopsy specimen, expression of α-fetoprotein by only rare cancer cells (brown cells) was detected by immunohistochemistry of the original testicular tumor. The original histologic finding in this patient was believed to represent a teratoma. B, Computed tomographic scans before treatment (upper panel ) demonstrated large retroperitoneal masses that were still present after four courses of platinum-based chemotherapy (lower panel ). C, Biopsy specimen of the residual mass revealed only mature teratoma. No cells expressed the immature marker α-fetoprotein. The patient has survived for more than 10 years without recurrence of his germ cell cancer. This suggests that in some patients, therapies that selectively eliminate the rare stem cell population while sparing the “nontumorigenic” cancer cells could be curative.

cells were grown in immunocompromised mice, a minority population of breast cancer cells that had the ability to form new tumors was identified.19 Tumorigenic cells could be distinguished from nontumorigenic cancer cells on the basis of surface marker expression. In eight of nine patients, tumorigenic cells could be prospectively identified and isolated as CD44+CD24−/lowLineage− cells.19 As few as 100 CD44+CD24−/lowLineage− cells were able to form tumors, whereas tens of thousands of cells from other populations of cells within the tumor failed to form tumors in NOD/SCID mice. These tumorigenic cells could be serially passaged in mice, and each time, cells within this population generated new tumors containing additional CD44+ CD24−/lowLineage− tumorigenic cells, as well as phenotypically mixed populations of other nontumorigenic cancer cells. These data demonstrate the presence of a hierarchy of cells within a breast cancer tumor in which only a fraction of the cells have the ability to proliferate extensively and other cells have only a limited proliferative potential, suggesting that the tumorigenic cells can both self-renew and differentiate. The phenotype of the tumorigenic breast cancer cells may be similar to that of normal breast epithelial stem or progenitor cells, because early multipotent epithelial progenitor cells have been reported to express epithelial cell antigen and CD44.128–130 The CD44+CD24−/lowLineage− tumorigenic breast cancer cell and the CD34+CD38− CD90− leukemia-initiating cells share with normal

stem cells the abilities to proliferate extensively and to give rise to diverse cell types with reduced developmental or proliferative potential.5,19 The extensive proliferative potential of the tumorigenic breast cancer cell population was demonstrated by the ability of as few as 200 tumorigenic breast cancer cells or several thousand leukemiainitiating cells to give rise to tumors that could be serially transplanted in NOD/SCID mice. This extensive proliferative potential contrasts with the bulk of the breast cancer cells that lack the ability to form detectable tumors. Not only was the CD44+CD24−/lowLineage− population of cells able to give rise to additional tumorigenic CD44+CD24−/low Lineage− cells, it was also able to give rise to phenotypically diverse nontumorigenic cells that made up the bulk of the tumors. Thus, both tumorigenic breast cancer cells and leukemia-initiating cells from most tumors appear to exhibit properties of cancer stem cells. However, before these cells can definitively be called cancer stem cells, new assays are needed to demonstrate that a single transplanted cell gives rise to all of the diverse populations of cancer cells within a tumor. Cancer stem cells have since been identified in multiple tumors, including cancers arising in the brain, head and neck, pancreas, colon, and prostate.131–135 Interestingly, CD44 seems to be useful as a marker for isolation of cancer stem cells from multiple types of tumors of epithelial origin, including head and neck cancer. Importantly, in histology sections, the cancer stem cells in well-differentiated or mod-

Stem Cells, Cell Differentiation, and Cancer • CHAPTER 7

erately differentiated tumors showed that the cancer stem cells, but not the nontumorigenic cancer cells, expressed CD44 and bmi-1, which had previously been shown to be involved in self-renewal in some types of stem cells.133 However, the nontumorigenic cells expressed mature cell markers, while the cancer stem cells did not.133 These studies demonstrate that the differential expression of the stem cell markers was not an artifact of flow cytometry.

IMPLICATIONS OF CANCER STEM CELLS FOR THE DIAGNOSIS AND TREATMENT OF CANCER Although the immunocompromised mouse model provides compelling evidence in support of the stem cell model of cancer, the ultimate confirmation of the hypothesis requires proof in humans. If the growth of solid cancers is driven by cancer stem cells, this would have profound implications for the diagnosis and treatment of cancer. At present, all of the phenotypically diverse cancer cells are treated as if they possess the ability to form tumors and the ability to metastasize. However, if in most tumors, only a small population of cancer cells has the ability to self-renew and other populations of cancer cells have only limited ability to proliferate, then this would explain several conundrums of cancer biology. For example, for many years, it has been recognized that disseminated cytokeratin-positive breast cancer cells can be detected in the bone marrow of patients who never experience relapse, even without adjuvant therapy.136–143 One possibility is that the cancer cells lie dormant until some unknown event triggers them to proliferate. Another explanation is that the cancer cells in the bone marrow in this group of patients arose from the spread of nontumorigenic cancer cells, and only when the cancer stem cells metastasize and subsequently self-renew will frank tumors form. Thus, the development of diagnostic reagents that allow cancer stem cells to be identified may have prognostic significance for patients with breast cancer. The ability to prospectively isolate cancer stem cells and nontumorigenic cancer cells makes it possible to do molecular analyses of each population of cancer cells in a tumor. A 186-gene signature, called the invasiveness gene signature (IGS), was derived by identifying genes that are differentially expressed by breast cancer stem cells and normal breast epithelial cells.144 The IGS was used to stratify patients with early stage breast cancer on the basis of the similarity of the gene expression of the whole tumor to the cancer stem cell-derived signature. Remarkably, the IGS was associated with both survival and the risk of developing metastasis. The prognostic power of the IGS was greater than gene signatures derived from the nontumorigenic cancer cells, indicating that the IGS contained both cancer-specific and cancer stem cell-specific elements. The prognostic power of the IGS was even greater when combined with a wound repair signature145 derived from serum-stimulated fibroblasts. This suggests that cancer stem cells interact with normal tumor stromal elements. Such an interaction is further supported by the observation that the cancer stem cells are adjacent to normal stromal elements in well-differentiated and moderately differentiated head and neck tumors.133 Another example of the implications of cancer stem cells is the observation that in most solid cancers, such as breast cancers, chemotherapy can frequently shrink tumors, but in most patients, the tumors rapidly recur, and there is only a small impact on patient survival.13,146,147 Most of the cancer therapeutic agents that are in current use have been developed largely for their ability to shrink a tumor. If only a minority of the cancer cells are tumorigenic and are responsible for driving tumor growth and metastasis, then tumor shrinkage must reflect primarily the elimination of the bulk population of nontumorigenic cells. If a substantial number of tumor stem cells were spared, then the tumors would regenerate from these cells. In support of this model, many patients who are treated with chemotherapy experience an initial shrinkage of their tumors, but tumors recur in sites of prior disease.

The similarities of the AML tumor-initiating cells and normal HSCs suggest that the AML tumor-initiating cells may be more resistant to chemotherapy than the bulk population of leukemic blasts. Compared with their differentiated progeny, normal HSCs express high levels of genes that make them more resistant to cytotoxic agents including antiapoptotic members of the bcl-2 family, as well as members of the ABC transporters that pump many drugs out of the cell.30,41,148–151 If the same is true for their cancer stem cell counterparts, then these cells may be significantly more resistant to cytotoxic agents than their nontumorigenic progeny. In support of this possibility, although the chemotherapeutic agent cytosine arabinoside very efficiently killed leukemic blast cells isolated from many patients, the leukemia-initiating cells were selectively spared.152 In glioblastoma, the cancer stem cells are more resistant to radiation than are their nontumorigenic cancer cell counterparts.153 These observations suggests that the effect of a particular therapeutic agent on the cancer stem cell population must be taken into account when its curative potential is evaluated.154 Because therapeutic agents are selected on the basis of their ability to shrink tumors rapidly, agents that selectively target the cancer stem cells could be overlooked in screens to identify potential therapeutic agents. Initially, such agents would be expected to slow the growth of a tumor only modestly. However, the elimination of the cancer stem cells would eventually halt the spread of the tumor. Perhaps the best clinical evidence of this model occurs in patients with teratocarcinoma. Platinum-based chemotherapy is curative in the majority of these patients;155 however, many patients are left with residual masses (see Fig. 7-4). After surgical resection, the immature cancer cells have been eliminated, leaving only differentiated cancer cells in a mature teratoma (see Fig. 7-4). Patients with mature teratomas only occasionally have metastases, and most are cured, demonstrating that the elimination of the presumed stem cell population by the chemotherapy is sufficient for curing this solid cancer. Box 7-1 discusses alternative models for cancer cell heterogeneity. Box 7-1.

ALTERNATIVE CANCER CELL HETEROGENEITY MODELS

An alternative explanation for the ability of a single, phenotypically unique population of AML cells or breast cancer cells to engraft in NOD/SCID mice is that all cancer cells are tumorigenic in humans but that only the CD34+CD38-Thy1−Lineage− AML cells or the CD44+CD24−/low Lineage− breast cancer cells are able to proliferate in mice. However, there are several reasons that this appears unlikely. First, NOD/SCID mice have previously been validated as in vivo models for the growth of normal human HSCs and human neural stem cells.2,9,126,156–158 Second, tumors that are passaged in mice contain heterogeneous cancer cells that are phenotypically similar to the cancer cells that were present in the original tumors from patients, including both tumorigenic and nontumorigenic fractions.19,20,125,126 This demonstrates that the mouse environment is not incompatible with the survival of the nontumorigenic cell fractions. Third, in the case of breast cancers, the tumorigenic and nontumorigenic fractions of cancer cells exhibit a similar cell cycle distribution in mouse tumors, demonstrating that the nontumorigenic cells are able to divide in mice.19 Thus, on the basis of these data and the data obtained for other types of normal and malignant human stem cells, the NOD/SCID mouse model reliably supports the engraftment of clonogenic human progenitors. However, human data are required to completely exclude the possibility that different populations of cancer cells are clonogenic in mice than are clonogenic in humans. Because ethical issues preclude the injection of cancer cells into humans, unequivocal proof of the stem cell model will require clinical studies that confirm that therapeutic agents that effectively target cancer stem cells in the immunodeficient mice also eliminate cancer stem cells in patients and result in clinical cures.

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FUTURE IMPLICATIONS OF CANCER STEM CELLS The ability to prospectively identify cancer stem cells should have a major impact on the development of new diagnostic and therapeutic agents. At present, all of the cancer cells within a tumor are treated as if they had the ability to drive tumor growth, invasion, and metastasis. The ability to identify these crucial cells will allow efforts to develop new diagnostic markers and therapies to be focused on the cells that are responsible for the maintenance of the malignancy—the cancer stem cells. For example, in efforts to identify the genes and proteins expressed by cancer cells, either whole tumors or all of the phenotypically diverse cancer cells within a tumor are currently used. Because the cancer stem cells represent only a minority of the cancer cells in most tumors, it is nearly impossible to identify diagnostic markers or therapies that target these cells. However, directing expression analyses to enriched populations of cancer stem cells should allow the identification of novel diagnostic markers and novel therapeutic targets that can be exploited to more effectively diagnose and

treat cancer. This principle is illustrated by the observation that BCR/ABL oncogene mRNA is not expressed by HSCs that carry the mutation in their DNA;159,160 such an approach may have implications even when oncogenic mutations are targeted. The ability to prospectively identify the cancer stem cells should also improve the ability to evaluate the curative potential of new therapeutic agents. Although cancer cell lines are useful for evaluating particular biologic pathways, they have proven to be somewhat unreliable when used in attempts to predict the clinical efficacy of a particular therapeutic agent in patients.159,160 Because the tumors that arise in immunodeficient mouse models of human cancer appear to more closely recapitulate the phenotypic diversity of patients’ original tumors, including the generation of tumorigenic and nontumorigenic cells, these models might more effectively predict the potential usefulness of a particular drug. New agents could be tested for their ability to eliminate the tumorigenic (cancer stem cell) component of tumors from multiple patients, allowing the agents that have the greatest curative potential to proceed to human clinical trials.

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62. Passegue E, Wagner EF, Weissman IL: JunB deficiency leads to a myeloproliferative disorder arising from hematopoietic stem cells. Cell 2004; 119:431–443. 63. Passegue E, Weissman I: Unpublished data, 2008. 64. Artavanis-Tsakonas S, Rand MD, Lake RJ: Notch signaling: cell fate control and signal integration in development. Science 1999;284:770–776. 65. Berry L, Westlund B, Schedl T: Germ-line tumor formation caused by activation of glp-1, a Caenorhabditis elegans member of the Notch family of receptors. Development 1997;124:925– 936. 66. Shelly LL, Fuchs C, Miele L: Notch-1 inhibits apoptosis in murine erythroleukemia cells and is necessary for differentiation induced by hybrid polar compounds. J Cell Biochem 1999;73:164– 175. 67. Varnum-Finney B, Xu L, Brashem-Stein C, et al: Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat Med 2000;6:1278–1281. 68. Gallahan D, Callahan R: The mouse mammary tumor associated gene INT3 is a unique member of the NOTCH gene family (NOTCH4). Oncogene 1997;14:1883–1890. 69. Zagouras P, Stifani S, Blaumueller C, et al: Alterations in Notch signaling in neoplastic lesions of the human cervix. Proc Natl Acad Sci USA 1995; 92:6414–6418. 70. Leethanakul C, Patel V, Gillespie J, et al: Distinct pattern of expression of differentiation and growthrelated genes in squamous cell carcinomas of the head and neck revealed by the use of laser capture microdissection and cDNA arrays. Oncogene 2000;19:3220–3224. 71. Liu Y, Dehni G, Purcell KJ, et al: Epithelial expression and chromosomal location of human TLE genes: implications for notch signaling and neoplasia. Genomics 1996;31:58–64. 72. Capobianco AJ, Zagouras P, Blaumueller CM, et al: Neoplastic transformation by truncated alleles of human NOTCH1/TAN1 and NOTCH2. Mol Cell Biol 1997;17: 6265–6273. 73. Ellisen LW, Bird J, West DC, et al: TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 1991;66:649–661. 74. Imatani A, Callahan R: Identification of a novel NOTCH-4/INT-3 RNA species encoding an activated gene product in certain human tumor cell lines. Oncogene 2000;19:223–231. 75. Jehn BM, Bielke W, Pear WS, Osborne BA: Cutting edge: protective effects of notch-1 on TCR-induced apoptosis. J Immunol 1999;162: 635–638. 76. Weizen S, Rizzo P, Braid M, et al: Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells. Nat Med 2002;8:979–986. 77. Cadigan KM, Nusse R: Wnt signaling: a common theme in animal development. Genes Dev 1997; 11:3286–3305. 78. Spink KE, Polakis P, Weis WI: Structural basis of the Axin-adenomatous polyposis coli interaction. EMBO J 2000;19:2270–2279. 79. Austin TW, Solar GP, Ziegler FC, et al: A role for the Wnt gene family in hematopoiesis: expansion of multilineage progenitor cells. Blood 1997;89: 3624–3635. 80. Tsukamoto AS, Grosschedl R, Guzman RC, et al: Expression of the int-1 gene in transgenic mice is associated with mammary gland hyperplasia and adenocarcinomas in male and female mice. Cell 1988;55:619–625. 81. Nusse R, Brown A, Papkoff J, et al: A new nomenclature for int-1 and related genes: the Wnt gene family. Cell 1991;64:231.

82. Reya T, O’Riordan M, Okamura R, et al: Wnt signaling regulates B lymphocyte proliferation through a LEF-1 dependent mechanism. Immunity 2000;13:15–24. 83. Wu C, Zeng Q, Blumer KJ, Muslin AJ: RGS proteins inhibit Xwnt-8 signaling in Xenopus embryonic development. Development 2000;127: 2773–2784. 84. Van Den Berg DJ, Sharma AK, Bruno E, Hoffman R: Role of members of the Wnt gene family in human hematopoiesis. Blood 1998;92: 3189–3202. 85. Gat U, DasGupta R, Degenstein L, Fuchs E: De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin. Cell 1998;95:605–614. 86. Chan EF, Gat U, McNiff JM, Fuchs E: A common human skin tumour is caused by activating mutations in beta-catenin. Nat Genet 1999;21:410–413. 87. Hedgepeth CM, Deardorff MA, Rankin K, Klein PS: Regulation of glycogen synthase kinase 3beta and downstream Wnt signaling by Axin. Mol Cell Biol 1999;19:7147–7157. 88. Reya T, Duncan AW, Ailles L, et al: A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 2003;423:409–414. 89. Jamieson CH, Ailles LE, Dylla SJ, et al: Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML. N Engl J Med 2004;351:657–667. 90. Korinek V, Barker N, Moerer P, et al: Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nat Genet 1998; 19:379–383. 91. Zhu AJ, Watt FM: Beta-catenin signalling modulates proliferative potential of human epidermal keratinocytes independently of intercellular adhesion. Development 1999;126:2285– 2298. 92. Nusse R: The Wnt gene family in tumorigenesis and in normal development. J Steroid Biochem Mol Biol 1992;43:9–12. 93. Weeraratna AT, Jiang Y, Hostetter G, et al: Wnt5 signaling directly affects cell motility and invasion of metastatic melanoma. Cancer Cell 2002;1:279– 288. 94. Saitoh T, Mine T, Katoh M: Up-regulation of WNT8B mRNA in human gastric cancer. Int J Oncol 2002;20:343–348. 95. Saitoh T, Mine T, Katoh M: Frequent upregulation of WNT5A mRNA in primary gastric cancer. Int J Mol Med 2002;9:515–519. 96. Kirikoshi H, Inoue S, Sekihara H, Katoh M: Expression of WNT10A in human cancer. Int J Oncol 2001;19:997–1001. 97. van de Wetering M, Sancho E, Verweij C, et al: The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 2002;111:241–250. 98. Knudson AG Jr, Strong LC, Anderson DE: Heredity and cancer in man. Prog Med Genet 1973;9:113–158. 99. Fearon ER, Vogelstein B: A genetic model for colorectal tumorigenesis. Cell 1990;61:759–767. 100. Uchida N, Weissman IL: Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57bL/ Ka-Thy1.1 bone marrow. J Exp Med 1992;175: 175–184. 101. Park IK, Qian D, Kiel M, et al: Bmi-1 is required for the maintenance of self-renewing adult hematopoietic stem cells. Nature 2002;423:302– 305. 102. Wechsler-Reya R, Scott MP: The developmental biology of brain tumors. Annu Rev Neurosci 2001;24:385–428. 103. Kowenz-Leutz E, Twamley G, Ansieau S, Leutz A: Novel mechanism of C/EBP beta (NF-M)

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143. Ingle JN, Tormey DC, Bull JM, Simon RM: Bone marrow involvement in breast cancer: effect on response and tolerance to combination chemotherapy. Cancer 1977;39:104–111. 144. Liu R, Wang X, Chen GY, et al: The prognostic role of a gene signature from tumorigenic breastcancer cells. N Engl J Med 2007;356:217–226. 145. Chang HY, Nuyten DS, Sneddon JB, et al: Robustness, scalability, and integration of a woundresponse gene expression signature in predicting breast cancer survival. Proc Natl Acad Sci USA 2005;102:3738–3743. 146. Schultz LB, Weber BL: Recent advances in breast cancer biology. Curr Opin Oncol 1999;11:429– 434. 147. Lippman ME: High-dose chemotherapy plus autologous bone marrow transplantation for metastatic breast cancer. N Engl J Med 2000;342:1119–1120. 148. Harrison DE, Lerner CP: Most primitive hematopoietic stem cells are stimulated to cycle rapidly after treatment with 5-fluorouracil. Blood 1991;78: 1237–1240. 149. Peters R, Leyvraz S, Perey L: Apoptotic regulation in primitive hematopoietic precursors. Blood 1998;92:2041–2052. 150. Feuerhake F, Sigg W, Hofter EA, et al: Immunohistochemical analysis of Bcl-2 and Bax expression in relation to cell turnover and epithelial differentiation markers in the non-lactating human mammary gland epithelium. Cell Tissue Res 2000;299:47–58. 151. Zhou S, Schuetz JD, Bunting KD, et al: The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001; 7:1028–1034. 152. Guzman ML, Neering S, Upchurch D, et al: Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells. Blood 2001;98:2301–2307. 153. Bao S, Wu Q, McLendon RE, et al: Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006;444:756–760. 154. Guzman ML, Swiderski CF, Howard DS, et al: Preferential induction of apoptosis for primary human leukemic stem cells. Proc Natl Acad Sci USA 2002;99:16220–16225. 155. Williams SD, Birch R, Einhorn LH, et al: Treatment of disseminated germ-cell tumors with cisplatin, bleomycin, and either vinblastine or etoposide. N Engl J Med 1987;316: 1435–1440. 156. Bhatia M, Bonnet D, Murdoch B, et al: A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat Med 1998;4: 1038–1045. 157. Larochelle A, Vormoor J, Hanenberg H, et al: Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med 1996;2:1329–1337. 158. Rosu-Myles M, Gallacher L, Murdoch B, et al: The human hematopoietic stem cell compartment is heterogeneous for CXCR4 expression. Proc Natl Acad Sci USA 2000;97:14626–14631. 159. Hoffman RM: Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic. Invest New Drugs 1999;17:343–359. 160. Brown JM: NCI’s anticancer drug screening program may not be selecting for clinically active compounds. Oncol Res 1997;9:213–215.

8

Vascular and Interstitial Biology of Tumors Rakesh K. Jain and Dan G. Duda

S U M M ARY • A solid tumor is an organ composed of neoplastic cells and host stromal cells nourished by the vasculature made of endothelial cells—all embedded in an extracellular matrix. The interactions among these cells and between these cells, their surrounding matrix, and their local microenvironment control the expression of various genes. The products encoded by these genes, in turn, control the pathophysiologic characteristics of the tumor. The tumor pathophysiology governs not only tumor growth, invasion, and metastasis but also the response to various therapies. • Tumor vasculature is made of host vessels co-opted by tumor cells and by new vessels formed by the processes of vasculogenesis and angiogenesis. A constellation of positive and negative regulators of

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angiogenesis governs the process of neovascularization. Tumor vessels are abnormal in terms of their organization, structure, and function. These abnormalities contribute to heterogeneous vascular permeability, blood flow, and microenvironment. Tumor interstitial matrix is formed by proteins secreted by host and tumor cells and by those leaked from the nascent blood vessels. Tumor interstitium is heterogeneous, with some regions fairly permeable and others difficult to penetrate. Modification of the collagen matrix can improve penetration of large-molecular-weight therapeutics. Interstitial hypertension is a hallmark of solid tumors and results from vessel leakiness, lack of functional lymphatics, and compression of vessels by proliferating cancer cells.

INTRODUCTION A solid tumor is an organ composed of neoplastic cells and host stromal cells nourished by the vasculature made of endothelial cells— all embedded in an extracellular matrix (Fig. 8-1). The interactions among these cells and between these cells, their surrounding matrix, and their local microenvironment, control the expression of various genes. The products encoded by these genes, in turn, control the pathophysiologic characteristics of the tumor. The tumor pathophysiology governs not only the tumor growth, invasion, and metastasis but also the response to various therapies. In this chapter we will discuss various pathophysiologic parameters that characterize the vascular and extravascular compartments of a tumor and the mechanisms governing the formation and function of these compartments.

VASCULAR COMPARTMENT Neoplastic cells, like normal cells, need oxygen and other nutrients for their survival and growth. Every normal cell in our body is located within 100 to 200 µm from a blood capillary so that it can receive oxygen and other nutrients by the process of diffusion. Likewise, cells

• Judicious application of angiogenic therapy can normalize the tumor vessels and make them more effcient for delivery of oxygen (a known radiosensitizer) and drugs. Antiangiogenic agents can prune tumor vessels, induce cancer cell apoptosis, reduce the number of blood circulating endothelial cells and progenitor cells, and lower interstitial hypertension in tumors. • Thus far, three antiangiogenic agents have been approved for patients with certain types of cancer. Based on these successes, antiangiogenic therapy is expected to make a difference in many other tumor types. Two main hurdles to further development of antiangiogenic agents are the better understanding of the mechanisms of action of these agents and developing biomarkers to monitor their effects.

undergoing neoplastic transformation depend on nearby capillaries for growth. These preneoplastic (i.e., hyperplastic or dysplastic) cells can grow as a spherical or ellipsoidal cellular aggregate. Once the size of the cellular aggregate reaches the diffusion limit for critical nutrients and oxygen, however, the aggregate as a whole can become dormant. Indeed, human tumors can remain dormant for many years because of a balance between neoplastic cell proliferation and apoptosis. However, once they have access to new blood vessels they may grow and metastasize. What triggers the growth of new vessels? What molecular and cellular mechanisms are involved? How do these vessels compare with normal vessels with respect to structure and function? Can we prevent or delay tumor progression only by interfering with the neovascularization process?

New Vessel Formation It has been known for nearly a century that the vascular system is associated with tumor growth in animals and humans.1 Powerful insights into the neovascularization of transplanted tumors using the transparent window techniques were developed in the 1940s.2–5 The possibility that tumors produce an “angiogenic” substance was suggested in 1968.6,7 The hypothesis that blocking angiogenesis should

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antiangiogenic factors.14,15 This balance is spatially and temporally regulated under physiologic conditions, so that the “angiogenic switch” is “on” when needed (e.g., during embryonic development, wound healing, formation of the corpus luteum) and “off ” at other times. During neoplastic transformation and tumor progression, this regulation is deranged, and blood vessels form ectopically to support a growing tumor mass.

Blood vessel Basement membrane

Cellular Mechanisms

Interstitial matrix and fluid

Cancer cells

Host cells

Figure 8-1 • Schematic representation of a solid tumor. The key components include cancer cells, host cells, and vasculature made of endothelial cells—all embedded in a matrix bathed in interstitial fluid. Arrows indicate interactions between the components. (Adapted from Jain RK: Angiogenesis and lymphangiogenesis in tumor: insights from intravital microscopy. Cold Spring Harbor Symp Quant Biol [The Cardiovascular System] 2002;67:239– 248.)

block tumor growth and metastasis was proposed shortly thereafter in 1971.8 The concept that a tissue acquires angiogenic capacity during neoplastic transformation—and, by extension, that antiangiogenesis could be used to prevent cancer—was put forward in 1976.9 The first antiangiogenic agent approved for cancer patients was bevacizumab, an antibody specific to vascular endothelial growth factor (VEGF), on the basis of the increased survival seen in metastatic colorectal cancer patients with the combination of bevacizumab with standard chemotherapy in a pivotal randomized placebo-controlled phase III trial.10 At present, various anti- and proangiogenesis strategies are being evaluated clinically to prevent or treat a large number of diseases, including cancer.11–13 Both normal and pathologic angiogenic processes are governed by the net balance between pro- and

At least four cellular mechanisms are involved in the vascularization of tumors: co-option, intussusception, sprouting (angiogenesis), and vasculogenesis (Fig. 8-2).11 Tumor cells can co-opt and grow around existing vessels to form “perivascular” cuffs. However, as stated earlier, these cuffs cannot grow beyond the diffusion limit of critical nutrients and may actually cause the collapse of the vessels due to the growth pressure (referred to as “solid stress”). Alternatively, an existing vessel may enlarge in response to the growth factors released by tumors, and an interstitial tissue column may grow in the enlarged lumen and partition the lumen to form an expanded vascular network. This mode of intussusceptive microvascular growth has been observed during tumor growth, wound healing, and gene therapy.16–19 “Sprouting” angiogenesis is perhaps the most widely studied mechanism of vessel formation. During sprouting angiogenesis, the existing vessels become leaky in response to growth factors released by normal cells or cancer cells; the basement membrane and the interstitial matrix dissolve; pericytes dissociate from the vessel; endothelial cells (ECs) migrate and proliferate to form an array/sprout; a lumen is formed in the sprout (a process referred to as canalization); branches and loops are formed by confluence and anastomoses of sprouts to permit blood flow; and finally, these immature vessels are invested in basement membrane and pericytes. During physiologic angiogenesis, these vessels differentiate into mature arterioles, capillaries, and venules, whereas in tumors they remain largely immature.5,11,12,20 During embryonic development, a primitive vascular plexus is formed from endothelial precursor cells (EPCs, also known as angioblasts) by a process referred to as vasculogenesis. In adults, EPCs—mobilized from bone marrow niches into the peripheral blood circulation—can also contribute to neovascularization (process referred to as “postnatal” vasculogenesis) in tumors and other tissues.21–23 The current challenge is to discern the relative contribution of each of the four

Endothelial precursor

Intussusceptive growth Angiogenic sprouting

Figure 8-2 • Cellular mechanisms of vascularization in tumors. At least four mechanisms are involved: (1) intussusception, where tumor vessels enlarge and an interstitial tissue column grows in the enlarged lumen, expanding the network; (2) vasculogenesis, where endothelial precursor cells mobilized from the bone marrow or peripheral blood contribute to the endothelial lining of tumor vessels; (3) “sprouting” angiogenesis, where the existing vascular network expands by forming sprouts or bridges; and (4) co-option (not shown), where tumor cells grow around existing vessels to form “perivascular” cuffs. (Adapted from Jain RK, Carmeliet PF: Angiogenesis in cancer and other diseases. Nature 2000;407:249– 257.)

Vascular and Interstitial Biology of Tumors • CHAPTER 8

mechanisms of neovascularization during the growth and/or during treatment of tumors.24

Molecular Mechanisms Various pro- and antiangiogenic molecules that orchestrate different steps in vessel formation, along with their functions, are listed in Table 8-1. VEGF is currently considered the most critical proangiogenic molecule. Originally discovered in 1983 as the vascular permeability factor and cloned in 1989, VEGF increases vascular permeability, promotes migration and proliferation of ECs, serves as an EC survival factor, can mobilize EPC populations from the bone marrow, and is known to upregulate leukocyte adhesion molecules on ECs.16,22,25–27 During tumor progression, or with treatment, the number of distinct angiogenic molecules produced by a tumor can increase.28–30 Thus, after VEGF signaling is blocked, a tumor might rely on other, alternative angiogenic molecules (e.g., basic fibroblast growth factor [bFGF], stromal-derived factor 1α [SDF1α], placental-derived growth factor [PlGF], or interleukin-8 [IL-8]).31 Other

positive regulators of angiogenesis include the angiopoietins that are involved in stabilizing vessels and controlling vascular permeability; various proteases involved in dissolving/remodeling matrix and releasing growth factors; and recently discovered organ-specific angiogenic stimulators (e.g., endocrine gland VEGF).20,32,33 Angiogenesis inhibitors include endogenous soluble receptors of various proangiogenic ligands (e.g., sVEGFR1) and molecules that downregulate the expression of stimulators (e.g., interferons) or that interfere with the release of the stimulators or binding with their receptors (e.g., platelet factor 4). Thrombospondins are among the first and most well-characterized endogenous inhibitors that interfere with the growth, adhesion, migration, and survival of ECs.14 Other endogenous inhibitors include fragments of various plasma or matrix proteins (e.g., angiostatin, a fragment of plasminogen; endostatin, a fragment of collagen XVIII; tumstatin, a fragment of collagen IV).34–36 Neither the mechanisms of action of the matrix-derived inhibitors nor their physiologic role are well understood.37 The generation of proand antiangiogenic molecules can be triggered by metabolic stress

Table 8-1 Angiogenesis Activators and Inhibitors* Activators

Function

Inhibitors

Function

VEGF family members†‡

Stimulate angio/vasculogenesis, permeability, leukocyte adhesion

VEGFR-1; soluble VEGFR-1; soluble neuropilin-1 (NRP-1)

Sink for VEGF, VEGF-B, PlGF

VEGFR‡, NRP-1, NRP-2

Integrate angiogenic and survival signals

Ang 2†‡

Antagonist of Ang 1

EG-VEGF

Stimulate growth of endothelial cells derived from endocrine glands

TSP-1,2

Inhibit endothelial migration, growth, adhesion, and survival

Ang 1 and Tie 2†‡

Stabilize vessels

Angiostatin and related plasminogen kringles

Inhibit endothelial migration and survival

PDGF-BB and receptors

Recruit smooth muscle cells

Endostatin (collagen XVIII fragment)

Inhibit endothelial survival and migration

TGF-β1§, endoglin, TGF-β receptors

Stimulate extracellular matrix production

Tumstatin (collagen IV fragment)

Inhibit endothelial protein synthesis

FGF, HGF, MCP-1

Stimulate angio/arteriogenesis

Vasostatin; calreticulin

Inhibit endothelial growth

Integrins αvβ3, αvβ5, α5β1

Receptors for matrix macromolecules and proteinases

Platelet factor-4

Inhibit binding of bFGF and VEGF

VE-cadherin; PECAM (CD31)

Endothelial junctional molecules

Tissue-inhibitors of MMP (TIMPs); MMP-inhibitors; PEX

Suppress pathologic angiogenesis

Ephrins‡

Regulate arterial/venous specification

Meth-1; Meth-2

Inhibitors containing MMP-, TSP-, and disintegrin domains

Plasminogen activators, MMPs

Remodel matrix, release growth factor

IFN-α, -β, -γ; IP-10, IL-4, IL-12, IL-18

Inhibit endothelial migration; downregulate bFGF

PAI-1

Stabilize nascent vessels

Prothrombin kringle-2; antithrombin III fragment

Suppress endothelial growth

NOS; COX-2

Stimulate angiogenesis and vasodilation

16 kD-prolactin

Inhibit bFGF/VEGF

AC133

Regulate angioblast differentiation

VEGI

Modulate cell growth

Chemokines§

Pleiotropic role in angiogenesis

Fragment of SPARC

Inhibit endothelial binding and activity of VEGF

Id1/Id3

Inhibit differentiation

Osteopontin fragment

Interfere with integrin signaling

Maspin

Protease inhibitor

Canstatin, proliferin-related protein, restin

Mechanisms unknown

See text for explanation of abbreviations. *Selected list updated from ref. 11; for complete function and references, see supplementary information (http://steele.mgh.harvard.edu). † Also present in or affecting nonendothelial cells. ‡ See ref. 20. § Opposite effect in some contexts.

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A

Figure 8-3 • Tumor induction of host promoter activity in stromal cells. The expression of VEGF in host cells can be examined using transgenic mice expressing a green fluorescent protein (GFP) under the control of the VEGF promoter. A, A murine mammary carcinoma xenograft shows host cell VEGF expression mainly at the periphery of the tumor after 1 week. B, After 2 weeks, the VEGF-expressing host cells have infiltrated the tumor. C, A GFPexpressing layer of host cells can be seen at the tumor-host interface. D and E, The VEGF-expressing host cells colocalize with the angiogenic tumor vessels. (A and B, From Fukumura D, Xavier R, Sugiura T, et al: Tumor induction of VEGF promoter activity in stromal cells. Cell 1998;94:715–725. C–E, From Brown EB, Campbell RB, Tsuzuki Y, et al: In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy. Nat Med 2001;7:1069.)

B

C

E

D 100 µm

100 µm

(e.g., low pO2, low pH, or hypoglycemia), mechanical stress (e.g., shear stress, solid stress), immune/inflammatory cells that have infiltrated the tissue, and genetic mutations (e.g., activation of oncogenes or deletion of suppressor genes that control the production of angiogenesis regulators).14,15,38–41 These molecules can emanate from cancer cells, endothelial cells, stromal cells, blood, and extracellular matrix (Fig. 8-3).42–45 Because the normal host cells differ among organs, the detailed mechanisms of angiogenesis might depend on the specific host-tumor interactions operating within a given tissue.46–53 Furthermore, because the tumor microenvironment is likely to change during tumor growth, regression, and relapse, profiles of pro- and antiangiogenic molecules are likely to change with time and space.54,55 The challenge currently is to develop a unified conceptual framework to describe the temporal and spatial profiles of this increasingly diverse array of angiogenesis regulators with the aim of developing effective therapeutic strategies.56,57

50 µm

tributor.20,65 In mice, “normalization” of the tumor vasculature observed during therapies that reduce VEGF (e.g., hormone withdrawal from a hormone-dependent tumor), interfere with VEGF signaling (e.g., treatment with anti-VEGF or anti-VEGFR2 antibody; Fig. 8-4), or mimic an antiangiogenic cocktail (e.g., trastuzumab [Herceptin] treatment of a HER2-overexpressing tumor) is in concert with this molecular hypothesis.12,54–56,66 Mechanical stress generated by proliferating tumor cells also can lead to the partially compressed or totally collapsed vessels often found in tumors.67,68 The decompression of blood vessels observed after induction of apoptosis in perivascular cells supports this mechanical hypothesis.69,70 Perhaps the combination of both molecular and mechanical factors renders the tumor vasculature abnormal, and, thus, both types of factors must be taken into account when designing novel strategies for cancer treatment.

Blood Flow and Microcirculation Vascular Architecture In a normal tissue, blood flows from an artery to arterioles to capillaries to venules to a vein. Although the tumor vasculature originates from these host vessels and the mechanisms of angiogenesis are similar, its organization may differ dramatically, depending on the tumor type, its location, and whether it is growing, regressing, or relapsing.16,58–61 In general, tumor vessels are dilated, saccular, tortuous, and chaotic in their patterns of interconnection.62 For example, whereas normal vasculature is characterized by dichotomous branching, tumor vasculature has many trifurcations and branches with uneven diameters.63,64 The fractal dimensions and minimum path lengths of tumor vasculature are different from those of normal host vasculature.58–60 The molecular mechanisms of this abnormal vascular architecture are not understood, but it seems reasonable to hypothesize that the imbalance of VEGF and angiopoietins is a key con-

Blood flow in a vascular network, whether normal or abnormal, is governed by the arterio-venous pressure difference and flow resistance. Flow resistance is a function of the vascular architecture (referred to as geometric resistance) and of the blood viscosity (rheology, referred to as viscous resistance).62 Abnormalities in both vasculature and viscosity increase the resistance to blood flow in tumors.64,71–73 As a result, overall perfusion rates (blood flow rate per unit volume) in tumors are lower than in many normal tissues.74–76 Both macroscopically and microscopically, tumor blood flow is temporally and spatially chaotic. Macroscopically, four spatial regions can be recognized in a tumor (Fig. 8-5): 1. 2. 3. 4.

An avascular necrotic region A semi-necrotic region A stabilized microcirculation region An advancing front77,78

Vascular and Interstitial Biology of Tumors • CHAPTER 8

Tumor

Normalized

Normal

tumor, blood flow fluctuates with time and can reverse its direction.77,79,80 In addition to the elevated geometric and viscous (rheologic) resistance, other molecular and mechanical factors contribute to this spatial and temporal heterogeneity. These include imbalance between pro- and antiangiogenic molecules, “solid stress” generated by proliferating cancer cells, vascular remodeling by intussusception, and coupling between luminal and interstitial fluid pressure via hyperpermeability of tumor vessels.17,57,58,67–69,81–83 As we will learn later, this heterogeneity contributes to both acute and chronic hypoxia in tumors—a major cause of resistance to radiation and other therapies. Considerable effort has gone into increasing tumor blood flow for improving radiation therapy, or decreasing tumor perfusion in the case of hyperthermia. This has been difficult to achieve reproducibly, because tumor vasculature consists of both vessels co-opted from the pre-existing host vasculature and vessels resulting from the angiogenic response of host vessels to cancer cells. The former are invested in normal contractile perivascular cells, whereas the latter lack these perivascular cells or these cells are abnormal.42,62,84 Presumably as a result, efforts to increase the tumor blood flow by pharmacologic or physical agents have not always been reproducible or successful.62,75 On the other hand, the strategy of decreasing or shutting down the tumor blood flow—by “stealing” blood away from the “passive component” of the tumor vasculature by vasodilators, by vascular targeting, or by intravascular coagulation—has shown promise in experimental systems.75,76,85–87 It also appears that judiciously applied antiangiogenic therapy could “normalize” the abnormal tumor microcirculation by pruning the immature vessels (see Fig. 8-4), thus rendering the remaining vasculature more responsive to vasoactive agents.88

Inadequate

VASCULAR PERMEABILITY

y n de liv er

ra tio

ru g

ol

ife

llu tra ce

2

pO

la r

lp re ia

n

tit

si o

te rs

++

+

++

80% of infants with ALL

Hilden et ald

t(12;21)(p13;q22)

ETV6/RUNX1

Low risk disease

Pui et ale; Al-Sweedan et alf; Stams et alg

t(1;19)(q23;)(p13.3)

TCF3/PBX1

Favorable with intensified therapy

Pui et alh

54–65 chr with +4, +10, +17

Chromosome enumeration

Low risk

Sutcliffe et ali

> 1, but the P is greater than 0.05 (the typical cut-point for statistical significance), is there an association or not? It depends: if the hypothesis were motivated by existing scientific evidence, then the conclusion from the study might be that the findings suggest an association, rather than that no association exists. Interpretation of association magnitudes and significance is an art; it is not dictated by simple rules. After considering alternative explanations for an epidemiologic finding, investigators still cannot conclude that the observed association reflects causation. In epidemiology, any given research question must be evaluated many times using different designs and study populations, and usually by independent investigators. Each epidemiologic study design has different potentials for each type of bias. If findings from studies that used different designs are similar, then this may provide evidence in favor of a causal association. If findings differ, then more weight should be given to studies that used designs that maintained temporality and were the least susceptible to bias. In general, prospective cohort studies and their derivative designs provide stronger evidence for a causal association than retrospective cohort studies and case-control studies, because they maintain the correct temporal sequence between the factor and the endpoint and because they are less susceptible to selection and observation biases. Although both prospective cohort studies and randomized trials are likely to have the correct temporal sequence, evidence from randomized trials usually trumps evidence from cohort studies because randomization reduces the likelihood of selection bias and confounding. Different study populations (defined, for example, by country, race/ethnicity, or socioeconomic status) may have different characteristics that influence the likelihood or extent of confounding. If findings are the same across study populations, then this may provide evidence in favor of a causal association. If findings differ, reasons may include differences in unmeasured confounders or differences between study populations in the presence and prevalence of factors that might modify the association. At this point, investigators should think about possible reasons for differences across study populations and investigate these reasons in subsequent studies. Chance variability is possible in any study. When the results from independent epidemiologic studies are not too heterogeneous, metaanalyses can be performed to generate a more stable (i.e., less variable) summary estimate of the association. When considering whether an observed association between a factor and endpoint is causal, some investigators find it useful to think about the nine “aspects” of an association that Sir Austin Bradford Hill described in 19653: 1. Strength: How big is the measure of association? 2. Consistency: Are findings from studies conducted using different designs in different populations similar? 3. Specificity: Is the factor only associated with the endpoint of interest, and is this the only factor that has been found to be associated with the endpoint of interest? 4. Temporality: Was the factor experienced before the endpoint occurred? 5. Biological gradient: Does the magnitude of the measure of association increase (or decrease) with increasing extent of the factor? 6. Plausibility and 7. Coherence: Is the association supported or refuted by the contemporary biologic literature? 8. Experiment: Does the magnitude of the measure of association decrease (or increase) after changing the factor status? 9. Analogy: Have associations between similar factors and similar endpoints been observed?

Temporality has been highlighted in this chapter because it is the only aspect of an association necessary for causality. The other aspects neither rule in nor rule out the potential for an association to be causal. In light of all these considerations, the original therapeutic effectiveness example is now reexamined. The investigators observed that patients with 2 copies of the haplotype had 0.5 times the risk of death, and patients with 1 copy had 0.7 times the risk of death compared with patients with no copies of the haplotype. These results were statistically significant (P < 0.05). What should investigators infer? They conducted a prospective cohort study in which haplotyping was performed using samples collected at the start of the study; hence, the correct temporal sequence was obtained. All individuals from one arm of the trial were included in the study; therefore, selection bias is unlikely to have been introduced into the study. All trial participants were followed actively and in the same manner for survival. Therefore, it is unlikely that the ability to observe the endpoint differed by haplotype (i.e., no observation bias). The investigators observed that the prevalence of the haplotype did not vary by any of the participant characteristics measured at the start of the study, including race or ethnicity; therefore, confounding also is unlikely. The strength of the association between the haplotype and survival is relatively strong, suggesting that confounding alone is unlikely to explain the association. Although the findings were statistically significant, chance cannot be ruled out as an explanation for the findings. This is the first study of this hypothesis, so there are no other studies to which to compare the findings. The RR of death decreased with increasing number of copies of the haplotype, demonstrating a biological gradient. The hypothesis was well motivated by scientific evidence that the P450 enzyme enhances the action of the chemotherapeutic agent, and that the haplotype of interest encodes a more active form of the enzyme. Findings from this study are analogous to those from a study that investigated genetic variation in a different metabolic enzyme with cancer survival after treatment with a different chemotherapeutic agent. After contemplating all of these aspects of the study and its findings, the investigators might conclude that the findings are promising, but because the study is observational and the first to investigate this hypothesis, additional studies should be conducted before the findings can be translated into clinical practice. As already mentioned, a randomized trial of this research question is not possible because haplotype is an inherent characteristic of an individual. However, if, in the future, findings from this study are upheld in other studies, the next step might be to conduct a trial in which patients without the beneficial haplotype are randomized to either a drug that induces the P450 enzyme or placebo, concurrent with administration of the new chemotherapeutic agent.

FINAL THOUGHTS ON THE USE OF EPIDEMIOLOGIC METHODS IN ONCOLOGIC RESEARCH This chapter has described epidemiologic methods for formally testing hypotheses pertinent to oncology and discussed their rationale. Epidemiology is a flexible method that can be applied to many different types of translational and clinical studies that do not necessarily have an etiologic or population-level focus. For instance, in the hypothetical prognosis example, the focus of the study appears to be on the measurement of a newly discovered tissue marker using an innovative technique. However, use of this innovative technique does not ensure correct inferences about whether or not the tissue marker predicts prognosis in the study population, nor its applicability to other populations, even if the technique is highly accurate. Obtaining a valid answer also hinges on choosing the

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appropriate study population, design, and analysis, all of which require epidemiologic thinking. For this reason, a multidisciplinary research team that includes collaborators from clinical science, basic

science, biostatistics, and epidemiology is optimal for addressing the complex translational and clinical questions posed in modern oncology.

REFERENCES 1. Gordis L: Epidemiology, 3rd ed. Philadelphia: Elsevier Saunders, 2004.

2. Rothman K, Greenland S: Modern Epidemiology, 2nd 3. Hill AB: The environment and disease: association or ed. Philadelphia: Lippincott Williams & Wilkins, 1998. causation? Proc R Soc Med 1965;58:295–300.

26

Cancer Prevention, Screening, and Early Detection Jason A. Zell and Frank L. Meyskens

S U M M ARY

Etiology and Pathogenesis • Prevention of cancer is based on an understanding of the etiology and pathogenesis of the individual organ malignancies. The identification of atrisk individuals is based on familial/ genetic and environmental influences. • Smoking tobacco remains the number one cause of malignancy and accounts for about 30% of the mortality from cancer. The role of diet in cancer risk is substantial. • Infections are an important component of cancer risk, and major etiologic agents for different organs include viruses (hepatitis B and C [hepatocellular], human papillomavirus [cervix and some oral cancers], Epstein-Barr virus [nasopharyngeal carcinoma]), bacteria (Helicobacter pylori [stomach]), and parasites (Schistosoma haematobium [bladder], Clonorchis sinensis [cholangiocarcinoma]).

Screening and Early Detection • Effective screening and early detection techniques for cancer include visual examination (skin, cervical, and oral

O F

K EY

P OI NT S

cancers), cytology (cervical cancer), mammography (breast cancer), and fecal occult blood, sigmoidoscopy, and colonoscopy (colorectal cancers). • Screening for prostate cancer by serum prostate-specific antigen (PSA) measurement has been widely adopted, although its impact on overall survival remains uncertain. No successful method has been established for lung cancer screening.

Chemoprevention • “Proof of principle” for the prevention of primary cancers has been established convincingly for breast cancer (tamoxifen and raloxifene), hepatocellular carcinoma (vaccination against hepatitis virus B), and cervical cancer (vaccination against human papillomavirus). • Prevention or regression of various intraepithelial neoplasias has been demonstrated: actinic keratoses (diclofenac), oral leukoplakia (retinoids), cervical intraepithelial neoplasms (topical retinoic acid), adenomatous polyps (calcium, aspirin,

INTRODUCTION The guiding principles of this chapter and oncology should be that the best treatment of malignant disease is its prevention, and that the disease to be prevented is carcinogenesis, not cancer (Fig. 26-1).1 By the time a cancer is diagnosed, even with the advanced techniques now available, more than 90% of the biologic life of the tumor is over, and the best chance to control the malignant process has been missed. The extensive advances in our understanding of carcinogenesis at the molecular level in the past decade, the rediscovery of intraepithelial neoplasia as an early, recognizable precursor of many solid tumors that can be managed simply, and the well-defined successes of screening and early detection in reducing the morbidity and mortality from several major cancers, need to be brought to bear on the problem of malignancy in a concerted and widespread fashion, with clinical oncologists working closely with primary care physicians and subspecialists. Because fewer than 50% of cancers are cured, once

•

•

•

•

•

celecoxib), and gastric dysplasia (anti-H. pylori therapy, antioxidants). Secondary aerodigestive cancers can be prevented with high-dose 13-cisretinoic acid but at the price of unacceptable toxicity. An increased incidence of secondary lung cancers in smokers supplemented with β-carotene or 13-cis-retinoic acid demands particular caution in the development of chemoprevention agents. Unexpected cardiovascular toxicity demonstrated by selective cyclooxygenase-2 inhibitors demonstrated in colon polyp trials has led to a major concern about risk-risk in the evaluation of risk-benefit. Attempts to develop less toxic or low-dose combination interventions for all of the major cancers are being investigated. Useful resources for those interested in research in cancer prevention, screening, and early detection are provided.

established, and because gains in treatment effectiveness have been increasingly incremental and expensive, early detection and prevention of cancer should be pursued aggressively as a means to reduce morbidity and mortality. Many major diseases of humankind have been controlled by the systematic application of prevention strategies, including morbidity and mortality from nutritional and infectious diseases and vehicular trauma.1 Among chronic diseases, the incidence of cardiovascular disease has decreased markedly as smoking has declined, cholesterol and blood pressure lowered, and exercise encouraged. It is likely that these simple approaches have led to a greater overall benefit to health for the population than the effect of all intensive care units, but such direct comparisons are difficult to make. In general, appreciation of the role of prevention strategies in the overall management of cancer has been neglected by clinical oncologists, although health care planners and society as a whole are intensely interested in this topic.2 Cancer prevention strategies can be considered at three different

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Part I: Science of Clinical Oncology Biology of carcinogenesis Pathway* Classical

Initiation

Promotion

Progression

Invasion

Immortalization

Genetic Event

Molecular

Markers

Biochemical

Histologic Immunologic

Epigenetic influence Clinical appearance

Intraepithelial neoplasia†

Normal

Neoplasia

Prevention strategies

Figure 26-1 • Integration of the biology of carcinogenesis and prevention. *Hereditary alteration(s) or baseline polygenic representation provides the constitutive “set point” on which postzygote changes occur. †Precancer, premalignant lesion in general clinical parlance.

Smoking cessation Screening Early detection Chemoprevention

major levels: primary, secondary, and tertiary. This chapter will deal primarily with primary and secondary prevention and with tertiary prevention as represented by chemoprevention of second malignancies. Normal, asymptomatic individuals are the population at which primary prevention is addressed. Major strategies for risk reduction include changes in diet, increased physical activity, tobacco awareness, decreased exposure to the sun, and reduced intake of alcohol. With the increasing identification of constitutive genetic alterations that predispose individuals to cancer, this group has been targeted for primary interventions such as prophylactic surgery.3 Annual screening mammography in women older than 50 years of age and smoking cessation or chemoprevention in a group of asymptomatic smoking individuals are also examples of targeted primary prevention. Secondary prevention is directed toward individuals with evidence of preneoplastic, clinically identifiable progression, but without frank

Table 26-1 Common Clinical Precursors (Intraepithelial Neoplasia) of Cancer

malignancy. The phenomenon of intraepithelial neoplasia, also called preneoplasia or precancer, has become of widespread interest, and management of these lesions has the potential to abrogate the disease process early. Many organ sites have preneoplastic counterparts that should be amenable to early intervention (Table 26-1). Representative examples of this type of secondary prevention include suppression or reversal of oral leukoplakia, cervical intraepithelial neoplasia (CIN) or Barrett’s esophagus and inhibition of polyp formation or progression (Fig. 26-2). Tertiary prevention involves decreasing the morbidity of established disease. Chemoprevention of second malignancies is a good example of tertiary prevention. The distinction between primary, secondary, and tertiary prevention can sometimes become blurred. Further, tertiary prevention and adjuvant therapies can share many of the same goals. From the viewpoint of the clinical oncologist, probably the best way to look at prevention is as one more therapeutic modality for the management of cancer, directed at its control in the earliest stages. The observation that the addition of a retinoid after bone marrow transplantation markedly enhances the survival of children with refractory neuroblastoma represents an informative synthesis of a quaternary treatment approach and a tertiary prevention modality.4

Organ Site

Precursor

Method of Detection*

Oropharynx

Leukoplakia

Visual†

Avoidable Causes

Skin

Actinic keratoses/moles

Visual†

Esophagus

Barrett’s esophagus

Endoscopy

Colon

Adenoma (polyp)

Sigmoidoscopy, colonoscopy

Breast

LCIS, DCIS‡

Mammography, ultrasound, MRI

Cervix

Intraepithelial neoplasia

Colposcopy

An extensive analysis of the topic of avoidable causes of cancer was performed over 2 decades ago by Doll and Peto5; these investigators concluded that 50% to 70% of all human cancers were preventable. No new data have emerged that would alter that overall estimate, although some of the specifics have changed.6 The major avoidable risk factors can be broadly separated into four areas: tobacco, infectious, chemical (including hormonal), and diet. Tobacco smoke is far and away the most important carcinogen to which humans are exposed on a routine basis. The morbidity and mortality from tobacco smoke is huge and represents the major preventable cause of all diseases, not just cancer, in modern and many undeveloped societies. It is estimated that more than 500 million smokers now living will die of tobacco-related illnesses.7,8 What is generally not appreciated is the wide carcinogenic range of molecular

*Cytology and/or biopsy is required in almost all cases before definitive therapy can be initiated. † Elegant in situ optical spectroscopic methods are being developed to detect early preneoplastic changes, including enhancing the signals with fluorescent molecules. ‡ Lobular and ductal carcinoma in situ.

Cancer Prevention, Screening, and Early Detection • CHAPTER 26

A

B

C

D

Figure 26-2 • Examples of premalignant lesions. A, Leukoplakia. Whitish lesion on side of tongue. B, Erythroplakia. Reddish (dark area) lesion in otherwise normal-looking buccal cavity. C, Barrett’s esophagus (pale area) with high-grade lesion (dark portion). D, Adenomatous polyp in proximal sigmoid colon. (Courtesy of Bill Armstrong and Ken Chang.)

damage and the numerous organ sites affected by cigarette smoke.9,10 In addition to the lungs, cigarette smoking contributes significant attributable risk to the development of cancers of the oropharynx (75%), bladder (50%), esophagus (50%), pancreas (25%), cervix (20%), kidney (15%), and bone marrow (10%). Cigarette smoke facilitates chromosomal instability and enhances transformation at all levels of cancer formation (initiation, promotion, progression), including adversely affecting the natural history of successfully resected early-stage lung cancer.11 Although the incidence of cigarette smoking has fallen among males, in 1994 lung cancer surpassed breast cancer as the most common cause of death from cancer in females. Lung cancer incidence among females has increased from 1975 through 2003, although the rate of increase has declined since 1991.12 Well-tested modules have been developed to assist health care

workers, including physicians, in applying smoking prevention and cessation strategies.7 Various forms of nicotine (gum, patch, inhalers, nasal sprays) and behavioral modulators (bupropion) have been effective in increasing the quit rate significantly at very low cost.8 The evidence for infectious involvement in human cancer has increased dramatically in the last 15 years (Table 26-2). Hepatitis and human papillomavirus (HPV) clearly play major roles in the development and evolution of hepatocellular and cervical carcinoma, respectively. Vaccines using various viral components as the target have recently been tested; results from these trials strongly suggest that liver cancer and cervical cancer are preventable.13,14 In addition to the classical and long-recognized associations of the parasites C. sinensis to cholangiocarcinoma and S. haematobium to squamous cell carcinoma of the bladder, the bacterium H. pylori has now been accepted

Table 26-2 Cancers with an Infectious Etiology Cancer

Agent

Major Mode of Transmission

Hepatocellular carcinoma

Hepatitis virus

Maternal, oral

Vaccine

Gastric

Helicobacter pylori

Oral

Antibiotics

Cervix

Papillomavirus

Sexual

Vaccine

Several major cancers are of infectious etiology and can be eradicated by preventive intervention.

Intervention

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as an etiologic agent associated with gastric dysplasia, stomach cancer, and a rare type of lymphoma.15 Early on, these diseases can be treated with antibiotics and the process reversed. Finally, it seems that viruses have a role in the evolution of some lymphomas (human T-lymphotropic virus-1, Epstein-Barr virus [EBV]). These findings all offer new approaches for primary and secondary prevention using standard and new microbiologic and immunologic approaches. A number of chemicals are known to play a role in cancer causation, perhaps the most widespread being aniline dyes (bladder cancer), asbestos (lung, mesothelioma), and hormones (breast, prostate). The role of endogenous and exogenous hormones in cancer causation is complex and is considered in detail in the sections on breast, prostate, and gynecologic organ sites.16 Some of the most intensely debated issues in medicine relate to this area. For example, is the overall health benefit of hormone replacement therapy (HRT) worthwhile? Results from the Women’s Health Initiative (WHI) trial suggest not,17 and long-term use of HRT is not recommended. The question of the specific role of dietary components in cancer prevention remains largely unanswered. Many comprehensive reviews on the topic are available, and the overall recommendation to eat an abundant amount of fruits and vegetables has not changed in 20 years.18 Ambitious campaigns such as the well-known “5-a-day for better health” campaign to encourage large-scale dietary changes continue.19 The specific components responsible for the protective effects against cardiovascular disease and cancer remain unclear. The roles of macronutrients, fat, and fiber in prevention have been topics of much discussion. The general recommendation to reduce total calories and fat consumption and to increase fiber is a good one with regard to cardiovascular disease prophylaxis, but whether such a strategy affects cancer outcome remains unproven. Increasing epidemiologic data suggests that physical activity and basal metabolic index play critical roles, and several trials are underway to address these issues.20 There is growing evidence that changes in the insulin-like growth factor pathways play an important role in many aspects of lifestyle changes represented by a high basal metabolic index and its control.20 With increasingly positive protective effects of physical exercise on cardiovascular disease being shown, there has been a renewed interest in the influence of physical exercise on malignant transformation and progression.21 There has been a great deal of interest in micronutrients as preventive agents, but the results emanating from supplementation in well-done randomized clinical trials thus far have been disappointing.22 Notable exceptions have included the report that supplementation with a modest dose of vitamin A (25,000 IU per day) can decrease the appearance of cutaneous squamous cell cancer of the skin in individuals with prior actinic keratoses and several trials showing that supplementation with a modest dose of calcium can decrease the subsequent prevalence of cancer polyps by 20%.23–25 Probably the most exciting diet-related development has been the identification of a wide range of potentially new and active chemoprevention compounds in food, such as protease inhibitors (soybeans), monoterpenes (citrus fruit oils), polyphenols (nuts), dithiolethiones (cruciferous vegetables), alliums (onion/garlic family), and many others.26 The opportunity to genetically engineer foods to reduce the risk of heart disease and cancer is a topic of much scientific interest and commercial activity.27 Nature created these molecules to deal with a hostile toxic environment, and figuring out how to use them for the prevention of cancer should be both scientifically interesting and clinically rewarding.

Screening and Early Detection Strictly speaking, screening is limited to normal individuals. The science of screening identifies many pitfalls in the design, analysis, and interpretation of such trials, including length and lead-time biases and many others.28,29 Beyond the technical issues involved in study design, three other requirements addressing implementation,

analysis, and interpretation, must be met to demonstrate that a screening test is useful: 1. A test must be available that will detect cancer earlier than routine methods (e.g., clinical or self-examination). 2. There must be evidence that treatment at an earlier stage of disease will result in an improved outcome (decreased cause-specific morbidity or mortality). 3. There must be evidence of a total health benefit. For example, the benefits of early detection via screening that meets the aforementioned criteria must also outweigh the adverse risks of subsequent diagnostic and therapeutic interventions. In current screening trials, disease-specific vs. all-cause mortality, and risk-benefit have become increasingly important issues—particularly among older individuals.30,31 Fulfilling these requirements is difficult, and the issues specifically related to screening of different organ sites for precancers or cancers are discussed in those sections. Some generic comments are worthwhile. Enough evidence exists for a specific test for some organ sites that has been proven effective to recommend the routine adoption of screening (Table 26-3). Although the availability of cancer screening is generally increasing, usage is relatively low for some organ sites (e.g., colon) and among groups that lack health insurance or a usual source of care.32 Many screening tests, however, are ineffective (e.g., routine chest roentgenograms in smokers being the most notable). Also, a positive screening test may lead to aggressive intervention that could allow “cure” of the organ site disease but result in an overall increased morbidity or mortality that is not efficacious for a person’s general health (e.g., radical prostatectomy for older individuals with a minimally increased PSA). Finally, screening for currently incurable malignancies (e.g., pancreatic cancer) offers new ethical dilemmas. If we have little to offer therapeutically, do we want to know the risk? Maybe, maybe not. Perhaps earlier prophylactic surgery might be able to affect the outcome in a few patients—for example, in families with early-onset pancreatic cancer. Over the past few years, there have been rapid developments applying new imaging modalities for the purpose of cancer screenings. The United States Preventive Services Task Force now recommends screening with imaging techniques for breast cancer (mammography for women aged 40 to 70 years) and colorectal cancer (barium enema, fecal occult blood, endoscopy).33 Cancer screening trials are ongoing for breast cancer (ultrasound, magnetic resonance imaging [MRI]), colorectal cancer (computed tomography [CT] colonography), liver cancer (ultrasound, CT), and pancreas (endoscopic ultrasound, CT, endoscopic retrograde cholangiopancreatography).34 Major trials have completed accrual, including the Prostate, Lung, Colorectal and Ovarian (PLCO) trial and the National Lung Screening Trial. Mortality results from the PLCO are expected in 2015 and will assess the role of chest radiography (i.e., x-ray) in lung cancer screening, and transvaginal ultrasound for ovarian cancer screening. Mortality data from the National Lung Screening Trial are expected in 2009, to evaluate low-dose CT for lung cancer screening among high-risk adults. The age of molecular diagnosis in screening is upon us and holds both promise and peril.35 Novel functional and molecular imaging techniques for cancer diagnosis currently include measurements of tumor angiogenesis via dynamic contrast-enhanced (DCE)-MRI, DCE-CT with ultrasound, positron emission tomography (PET), and combinations of these techniques.36 Diffuse optical imaging is another functional imaging technique that has emerged as an adjunct for cancer diagnosis. This noninvasive imaging method uses near-infrared light intensity to address tissue-specific changes between normal and tumor tissue (i.e., tissue hemoglobin concentration, tissue oxygen saturation) in vivo.37 DCE-MRI, DCE-CT with ultrasound, and PET imaging have varying capabilities to profile microvessel density—a hallmark of angiogenesis—which is essential for tumor growth and invasion. However, even in the setting of cancer diagnosis, these methods are not

Cancer Prevention, Screening, and Early Detection • CHAPTER 26

Table 26-3 Effectiveness of Major Screening Approaches for Cancer* Test

Positive Level of Evidence†

Over age 50

Mammography

Strong

Yes

Age 40–50

Mammography

Fairly strong

Yes

Papanicolaou‡

Strong

Yes

Occult fecal blood

Strong

Yes

Sigmoidoscopy

Strong

Yes

Organ Site

Recommended

Breast

Cervix Colorectal Over age 50

§

Lung

Colonoscopy

Fairly strong

Yes

Chest roentgenogram

None||

No

Melanoma

Skin examination

Moderate

Yes

Prostate

Prostate-specific antigen

Moderate

Yes¶

*Listed here are organ sites for which sufficient data exist to make a judgment. Although no specific trial evidence exists, routine physical examination of the skin, oral cavity, testicles, and ovary/uterus is worthwhile, because treatment success is closely related to stage at diagnosis and effective treatment is available in most cases. † The concept of level of evidence is a valuable approach—a quantitative approach that is presented in detail in the PDQ section of the NCI Web site (http://cancernet.nci: nih.gov/clinpolq/screening). A randomized trial with survival as an endpoint is at the top of the hierarchy, whereas anecdotal evidence by experts is at the lowest. ‡ Screening should begin with the onset of sexual activity. § Evidence also exists that colonoscopy with excisional biopsy is an effective therapeutic maneuver, but the cost of the procedure has precluded its general usage. || Several randomized trials of screening chest roentgenograms showed no effect on outcome. Spiral CT is currently being tested in a large national trial. ¶ With careful follow-up and appropriate testing.

yet standardized.36 Appropriate methods for incorporating these functional and molecular imaging techniques into screening trials must be considered, particularly in light of their associated high costs. Thus, the potential impact of such molecular and functional imaging techniques on cancer screening is great, though not yet realized. Although early detection is, formally speaking, the evaluation of a symptomatic individual for cancer and is therefore different from screening, many of the caveats regarding evaluation of this approach are the same. The increasing ability to identify high-risk populations, either by phenotypic criteria or by genetic analysis, also tends to lead to a blurring of the classic division between screening and early detection. With the rapid advances in molecular diagnostics, routine genetic typing of individuals at risk for major tumor types should not be too distant in the future, and quantitation of that risk (a concept we proposed quite some time ago)38 and its evaluation at the time of detection leads to real-world angst. Identification and referral of families at high risk for cancer susceptibility should be an increasing emphasis of clinical oncologists, but thus far, participation by oncologists has been low.2,39 At the very least, the ability to downshift the stage of a disease at the time of detection should eventually lead to improved survival, because new treatment approaches emanate from causative understanding of a particular cancer. Proving this point, though, has been difficult for cancers of many organ sites. The effectiveness of screening for the major types of cancer is summarized in Table 26-3 and is discussed in detail in the individual sections of this chapter. There are effective screening modalities for breast cancer, colorectal cancers, melanoma, and cervical cancers, whereas no compelling evidence exists for the value of screening for lung cancer. The effectiveness of PSA in screening for prostate cancer has been a subject of intense debate; we feel that the tide of evidence has turned and that the evidence supports the routine use of PSA screening over age 50 with thoughtful management and appropriate follow-up of abnormal values.

early detection and provided a guide to thinking about risk assessment and chemoprevention.40 Figure 26-1 serves as a useful general roadmap to reflect on these issues for all tumor types (see Shureiqi and colleagues).41 The classical model of carcinogenesis divides cancer evolution into three major epochs: initiation, promotion, and progression. This classification has served as a useful heuristic model for which considerable experimental evidence has been developed. In the past 15 years, the genetic paradigm for the development of cancer has been elegantly articulated and experimentally confirmed for some organ sites. A series of steps in response to separate molecular events at the genetic level is a useful platform from which to understand carcinogenesis in human epithelial tumors. Almost all human cancers examined in any detail have shown evidence of several acquired molecular abnormalities. Although the “pathway” is different for each cancer, the tumor suppressor genes p53 and p16 seem to play a central role in many malignancies.42,43 The expression of these abnormalities has allowed the development of markers that could serve as indicators of cancer risk or disease progression or possibly as surrogate endpoints for chemoprevention agent testing.44 Use of markers of carcinogenesis to assess the status of the disease relative to diagnosis and treatment and for assessment of chemoprevention effect is an important and complex issue.45 The continued development and validation of markers will be critical to the intelligent management of early-stage cancer.46 What also has become clear is that environmental phenomena (e.g., hormones, diet, carcinogens) can influence the expression of genetic changes. At one extreme of the paradigm is retinoblastoma, in which loss of a single gene inevitably results in an ocular tumor at a young age; however, most common solid tumors in adults seem to have underlying polygenic contributions, which can be affected by a large range of exogenous factors, even when a deleterious mutation such as BRCA1 or BRCA2 is present.

Carcinogenesis and Chemoprevention

The idea of the chemoprevention of human cancer has been with us for nearly 3 decades, but only in the recent years have positive clinical trials validated the results of preclinical data and their potential for use in human beings.47–50 Retinoids are a major group of compounds that have provided convincing “proof of principle” of

Carcinogenesis Advances in our understanding of the biology of carcinogenesis (cancer formation) have sharpened our thoughts about screening and

Chemoprevention

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chemoprevention in humans, but in general they have been too toxic for widespread use and have not been adopted widely.51 The overall results of some of the key randomized chemoprevention trials are summarized in Table 26-4 and are discussed in more detail in the individual organ site sections. Major problems in developing chemoprevention as a modality for cancer management have been the length and size of the trials required to show changes in a definitive endpoint.52,53 Consequently, only the National Cancer Institute and a few large research groups have been able to marshal the resources to develop broad-based chemoprevention efforts. Another significant issue in the design of early trials was that many large studies evolved primarily from epidemiologic observations, with little experimental data available. Because the implementation of large phase III or IV chemoprevention trials is a 10- to 100-million-dollar exercise, political influences on the funding process have also been substantial. An attempt to develop chemoprevention agents logically has been outlined, and systematic preclinical testing and evolution of sequential clinical trials are likely to avoid some of the mistakes of the past.53,54 Several key elements are featured in this decision analysis process: • Preclinical in vitro and in vivo testing against a battery of molecular targets and cellular and animal models • Accurate identification of side effects and assessment of their importance • Evidence of modulation of anticipated biochemical or molecular markers in the relevant tissue in short-term human trials • A randomized 6- to 12-month study of multiple low doses of the candidate agent in a relevant patient/participant population, with careful identification of side effects and assessment of their importance, and evaluation of their biochemical, molecular, and/or histologic effects

In this regard we have performed a particularly informative series of studies in assessing topical all-trans-retinoic acid in cervical cancer prevention and difluoromethylornithine (DFMO) in colon cancer prevention,55–57 whereas the M.D. Anderson group58 has performed a series of important trials in aerodigestive cancers and the Arizona group has done the same for skin cancers.59 Following this logical pathway of chemoprevention agent development assures that the probability of conducting a definitive phase III or IV study will be high and maximizes the chance for a successful outcome. Although the term “chemoprevention” has been widely used to describe chemical or dietary intervention to prevent or reverse cancer, it is a misnomer that is out of sync with the nosology used in other areas of medicine. Therapeutic prevention might be a better term—for example, cholesterol-lowering agents and antihypertensives to prevent cardiovascular diseases, prophylactic antibiotics to prevent infections, and many more. Risk-benefit of a drug in a prevention setting has always been a key issue, particularly of individuals at low risk. The unexpected increase of lung cancers and adverse cardiovascular events in two large trials in which β-carotene was administered called this issue into sharp focus in the mid-1990s.60,61 Recently, a different level of risk has been appreciated, designated risk-risk. In several large trials, selective/specific COX-2 inhibitors markedly reduced the incidence of colonic polyps but at the price of increased serious cardiovascular events.62,63 A careful analysis indicated that overall morbidity would be increased by using selective COX-2 inhibitors to reduce adenomatous colon polyps in individuals at low risk.64 These important observations do not necessarily preclude using other nonsteroidal anti-inflammatory drugs (NSAIDs) that have more complex effects on the prostaglandin pathways (e.g., aspirin, sulindac); the results of other trials will help in this decision making, but the unexpected outcomes in the COX-2 inhibitor trials has clouded the field of chemoprevention for reduction of cancer incidence, particularly in

Table 26-4 Current Overall Status of Chemoprevention in Preventing Human Cancers* Organ Site

Pretrial Level of Evidence

Agent

Status of Chemoprevention

Breast

Strong

Tamoxifen

Effective

One large trial, very positive; two smaller trials showed no effect. Overall long-term health benefits must be determined.

Cervix†

Strong

Multiple

Ineffective to marginal

Numerous phase III trials of several compounds have not substantiated phase II trials except for topical all-trans-retinoic acid.

Colon

Strong

Multiple

Mixed

Slight decrease (25%) in colon polyp recurrence by calcium or aspirin

Head and neck (secondary)

Moderately strong

13-cis retinoic acid

Effective but toxic

Follow-up trial at lower dose ineffective

Leukoplakia

Moderately strong

β-carotene

Promising

Single randomized trial needs confirmation.

Tertiary

Strong

13-cis- retinoic acid

Effective but toxic

Follow-up studies at lower doses in progress

Primary

Strong

β-carotene

Ineffective

More lung cancers and increased higher overall mortality

Secondary (metaplasia)

Strong

Retinoids

Ineffective

Impressively negative

Moderately strong

Finasteride

Accrual complete

Results indicate positive effects but are preliminary.

Comment

Lung

Prostate

SELECT Skin

Strong

‡

Ongoing

Results in 2010

Retinoids

Effective in some cases

Seems to depend on stage of cancer development and strength of agent

Diclofenac

Effective

Causes regression of actinic keratoses

*Details are discussed in individual sections. † Cervix, effects on regression of cervical intraepithelial neoplasia. ‡ Selenium and vitamin E. Epidemiologic data from secondary analysis; experimental data moderate.

Cancer Prevention, Screening, and Early Detection • CHAPTER 26

individuals at low risk. It is important to recall that the development of cardiovascular risk reduction trials met similar problems in the early days until effective and nontoxic agents were developed, and 20 to 30 years ago the regulatory burdens and the ethical bars were considerably lower.65 Although thus far most agents have been developed based on epidemiologic observations or carcinogen models in animals, increasing knowledge about the molecular basis of cancer progression in human tumors should result in the discovery and synthesis of highly specific drugs based on altered biochemical and signaling pathways.66,67 The number of specific tumors for which prevention strategies could be reviewed is large. In this chapter we review the major organ sites (aerodigestive, colon, breast, prostate) and those sites (skin, ovary, cervix) in which sufficient evidence exists to suggest that preventive strategies currently have a role in clinical oncology. We also offer a few comments on less studied cancers (stomach, liver) that are extremely common outside the United States and Europe. With the rapidly increasing scientific understanding of the biologic basis for many tumor types and the recognition that screening, early detection, and chemoprevention should play a large role in the management of the carcinogenic process, we can anticipate that the list of therapeutic prevention strategies for intraepithelial neoplasia and possibly earlier manifestations of cancer will grow rapidly over the next few years.

AERODIGESTIVE MALIGNANCIES Risk Reduction Aerodigestive malignancies encompass a subset of cancers including those that arise from the oral cavity, pharynx, esophagus, and lung.58 These organ sites have been grouped together, because they share a mucosal epithelial field that is directly subject to malignant transformation by the common toxin (tobacco), the major underlying etiologic agent for these malignancies. In addition to cigarette smoke, smokeless tobacco has also played an increasing contributory role in oral carcinogenesis, and oral cancer in the young adult has become increasingly common.68 Alcohol also clearly plays a synergistic role with tobacco carcinogens in the development of oral and esophageal cancers, including second cancers.69,70 Polymorphisms in the alcohol dehydrogenase gene involved with tobacco carcinogen metabolism may also play an important role in determining risk for head and neck and tobacco-related malignancies.71 The identification of HPV in more than 50% of oropharyngeal and nearly 100% of laryngeal tumors has led to acceptance of the role of HPV in head and neck squamous cell carcinomas (HNSCCs) pathogenesis.72,73 Compared with HPV-negative HNSCC tumors, which harbor tobacco-induced p53 mutations, HPV-positive HNSCC tumors have wild-type p53, which is inactivated by the viral oncoprotein.73,74 Interestingly, HPVpositive HNSCC patients have a different clinical profile compared with HPV-negative patients: they are younger, less likely to ingest alcohol or use tobacco, and show equal gender distribution; the tumors show poorly differentiated and basaloid histology, but such patients have improved survival characteristics.73,75 Progress in understanding the biology of tobacco-associated carcinogenesis in the past few years has been rapid, and molecular models of head and neck and lung cancers have been characterized to a substantial degree.76 What is clear from cytogenetic genomic hybridization and other studies is that, although aerodigestive cancers share many similar changes early on (e.g., loss and gain of 3p and cyclin alterations), discrete subsets exist.77 Because different genes are involved, this information should have a practical effect on the development of chemoprevention and other interventions. In 2006 more than 230,000 cancers are expected to develop in aerodigestive sites, and 188,000 related deaths are anticipated in the United States.78 Because it is estimated that the etiology of more than 80% of aerodigestive cancers is tobacco related, the cost to society of

this legal carcinogen is extraordinarily high. The application of prevention strategies should have a favorable impact on decreasing morbidity and mortality from aerodigestive cancers; particularly important for the medical profession should be the adoption of proven, physician-facilitated smoking cessation methods.7,8

Screening and Early Detection Oropharyngeal cancer occurs in a region of the body that is easily accessible to examination by a health care worker. The morbidity and mortality from oropharyngeal cancer is directly related to the stage at diagnosis, so effective screening and early detection should be worthwhile.79 However, no definitive trial has demonstrated that an early detection program can downshift stage at diagnosis of oropharyngeal cancer or reduce mortality in a screened population. Nevertheless, an inspection of the oral cavity should be part of every examination in high-risk patients (smokers) and can be made efficacious by careful inspection of the soft palate, tongue, and floor of the mouth, where 90% of all squamous cell cancers occur.80 The preneoplastic lesions, leukoplakia and particularly erythroplakia, should be identified and biopsy specimens obtained if necessary, because they represent early observable signs of squamous cell carcinomas with different prognoses (see Fig. 26-2A and B). In appropriately screened populations of high-risk smokers and drinkers over age 40, a detection rate of oral cancers as high as 1 cancer in every 200 individuals examined has been achieved.81 A successful screening program can be mounted using health caseworkers; for example, in Sri Lanka, where oral cancer is a common malignancy, a sensitivity of 58% was obtained for 660 patients with suspected cancers.82 Recent advances in optical biology also suggest that screening using autofluorescent techniques may allow detection of premalignant changes before the clinical appearance of disease.83 The strong association of HPV with oral cancers in young, nonsmoking individuals72 and the success of screening techniques in detecting cervical intraepithelial neoplasia (see the discussion that follows) suggest that oral screening for HPV should be adopted for those who are sexually active. Established risk factors for HNSCC related to sexual behavior (i.e., history of genital warts, young age at onset of sexual activity, and a high number of sexual partners) indicate that this group is the same population at risk for genital HPV. Routine screening of esophageal cancer in the United States has not been attempted to a significant degree, because it is relatively uncommon and therapeutic options are poor. In China and other Asian countries, however, the disease is much more common and found at high frequency in certain geographic locales. In these areas, screening and early detection using esophageal cytology are widely used, although the efficacy of these approaches has yet to be firmly established.84 Although lung cancer incidence among men has declined since the 1980s, the incidence for females has increased from 1975 through 2003 in the United States.12 This trend may be changing, in that recent data indicate that the rate of increase among women has declined since 1991.12 The issue of screening for lung cancer has been a long and complicated one, without a strong supporting evidence base. In the Mayo Lung Project (a large controlled trial designed to assess lung cancer screening with chest roentgenograms and sputum cytology among high-risk adults conducted in the 1970s and 1980s), chest x-ray imaging was noted to detect early-stage lung cancers, without leading to any difference in lung cancer mortality.85,86 Furthermore, excess cases were noted in the intervention arm, leading to “overdiagnosis.”87 Four large randomized trials of screening chest xrays in smokers have demonstrated no difference in survival between the randomized groups.88 In subsequent chest CT screening trials at The Mayo Clinic, CT was shown to detect early-stage tumors, but with a high rate of false-positives (i.e., benign nodule detection). Recent support for lung cancer CT screening among high-risk individuals comes from the singular outcome analysis of a large trial in

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which 85% of the screen-detected lung tumors were stage I at diagnosis, and 10-year overall survival was 88% for these stage I patients.89 Although these observational findings are impressive, we must await the results of confirmatory randomized controlled prospective screening trials before making broad screening recommendations for lung cancer. Thus, the National Lung Screening Trial (CT imaging) and PLCO (chest x-ray) trials for lung cancer screening will serve as important validation studies to evaluate the efficacy of lung cancer screening among high-risk individuals.

Chemoprevention Epithelial cancers of the upper aerodigestive tract and lungs are the most extensively studied system for chemoprevention in humans, and the results are the most negative. The natural history of the disease process has been studied extensively and provides a rich platform from which to conduct chemoprevention trials. Field carcinogenesis by tobacco carcinogens with its associated epidemiologic risk and characterized molecular changes is a straightforward concept that has guided the development of chemoprevention studies in this area.46 The recent identification of molecular (e.g., DNA repair, telomerase), metabolic (e.g., cytochrome P450, alcohol dehydrogenase), and mutagen sensitivity profiles that predispose to aerodigestive cancers, acquired chromosomal abnormalities in the field and in the cancers, and alterations of several molecular parameters that predict responsiveness and unresponsiveness, have recently provided useful detail from which to consider the next generation of rational chemoprevention trials.90 The identification of a variety of molecular changes during head and neck cancer progression, in addition to readily identifiable histologic precursors, has provided a biologic base for understanding the interaction of carcinogenesis and chemoprevention of this disease. Recent studies of the molecular changes that accompany the progression of lung cancer also provide a useful paradigm and platform from which to develop well-considered chemoprevention approaches. Thus far, however, the results of primary, secondary, and tertiary chemoprevention trials of the lung have been disappointing (see Goodman91). Two large placebo-controlled, multiagent randomized trials in heavy smokers (more than 47,000 participants) have been negative and showed no beneficial effect of retinol (vitamin A) or α-tocopherol (vitamin E).60,61 More disturbingly, these two large, randomized trials indicate that current smokers supplemented with oral β-carotene developed lung cancers at a rate 25% greater than the placebo group and also showed an increased overall mortality. Although these findings remain unexplained, possibilities that might help explain the adverse effect of β-carotene in these studies include the following: • The formation of cyclic epoxides in the setting of tobacco carcinogens, inflammation, and high β-carotene concentrations • The suppression of RAR-β, a major transcription factor important for differentiation in epithelial tissues • Lowering of the concentration of other micronutrients that might be protective • Stimulation of preneoplastic clones by enhancement of growth factor production • Complex genetic polymorphisms that lead to alteration of tobacco carcinogen metabolism Definitive secondary (metaplasia, atypia) and tertiary (second malignancy) chemoprevention trials that use a number of different retinoids and other compounds (folic acid, N-acetylcysteine) have also yielded negative results.91,92 Treatment with anethole dithiolethione (an organo-sulfur compound) was shown in a randomized trial to reduce development of new bronchial dysplasia lesions and to slow progression of pre-existing disease in current or former smokers.93 However, a randomized trial of inhaled budesonide in smokers with bronchial dysplasia indicated no effect of the active agent in the

regression of bronchial dysplastic lesions or prevention of new lesions.94 A modest decrease in p53 and BCL-2 protein expression in bronchial biopsy specimens was seen, as was a slightly higher rate of resolution of CT-detected lung nodules. Whether budesonide or anethole dithiolthione will be useful in managing preneoplastic lesions of the lung will require assessment in larger and longer trials. Studies of secondary and tertiary chemoprevention of head and neck cancers have led to somewhat more encouraging results. Randomized trials have shown that isotretinoin causes regression of oral leukoplakia, though accompanied by substantial side effects.95 Two randomized trials confirmed activity of β-carotene, although results from the later study were less convincing.96,97 Several other, less toxic agents (retinol, 4-[hydroxyphenyl]-retinamide, and α-tocopherol) and selenium have also produced responses of premalignant lesions in phase II trials.98 A randomized trial of the cyclooxygenase inhibitor ketorolac as an oral rinse was negative in patients with oropharyngeal leukoplakia.99 A recently reported phase II trial of the complex retinoid fenretinide suggested activity for this compound in patients with retinoic acid–resistant oral leukoplakia.100 We have demonstrated substantial potential activity of Bowman-Birk inhibitor (a soybeanderived compound) against oral leukoplakia.101 The results of randomized studies for these latter two compounds have not yet been reported. In a randomized phase III adjuvant trial of patients treated for head and neck cancer by local therapy, the synthetic retinoid 13cis-retinoic acid (isotretinoin, Accutane) at a high daily oral dose (50 to 100 mg/m2) led to a reduction in the incidence of second primary tumors, a difference that was maintained for more than 5 years.102 The rate of second primary tumors was affected greatly by tobacco smoking status, with the efficacy of chemoprevention decreasing sequentially in current and former smokers as compared with nonsmokers.103 The side effects in the 13-cis-retinoic acid trial were substantial at this dosage level, however. These results with 13-cisretinoic acid were particularly significant in that a similarly designed randomized trial using another retinoid (etretinate) at a high dose showed no reduction of second primary tumors.104 Therefore, the efficacy of low-dose isotretinoin (30 mg/day) to prevent second primary tumors after treatment of early-stage (I and II) head and neck cancer was tested in a randomized trial, with the hope that side effects could be decreased without losing efficacy. This strategy was unsuccessful, and no difference in the appearance of second malignancies in the placebo and treatment groups could be demonstrated; it is noteworthy that patients who continued to smoke had an increased rate of second primary tumors and death.105 The incidence of adenocarcinoma of the esophagus has been increasing over the past 2 decades. Gastroesophageal reflux has been identified as a risk factor leading to the development of a columnarlined esophagus, called Barrett’s esophagus, which progresses to adenocarcinoma via a metaplasia-dysplasia sequence (Fig. 26-2C). Both endoscopic resection and thermal or photodynamic ablation have been used to treat this condition,106 but long-term benefit is unknown. Epidemiologic and clinical observations also suggest that NSAIDs and aspirin, by inhibiting COX enzymes, and proton pump inhibitors by decreasing gastric reflux107 should be effective as chemopreventive agents, but randomized controlled trials proving this supposition have not yet been reported. Attempts to reverse or suppress these lesions with 13-cis-retinoic acid have been unsuccessful.108 Overall, these trials suggest that oral leukoplakia, but not bronchial metaplasia, can be reversed or suppressed by currently available chemopreventive agents. Thus far, “proof of principle” of chemoprevention in head and neck cancers has been achieved. However, largescale phase III trials will have to show efficacy with a favorable risk-benefit profile before the strategy of chemoprevention can be adopted into standard medical practice for the management of aerodigestive cancers.

Cancer Prevention, Screening, and Early Detection • CHAPTER 26

COLORECTAL CANCER Screening for and early detection of colorectal cancers results in 5year survival rates of 90% for colon cancer and 80% for rectal cancer, providing that diagnosis and treatment occur before the lesions have spread beyond the bowel to regional lymph nodes or distant metastatic sites. Unfortunately, more than 60% of patients still present with higher staged disease, leading to a lower overall 5-year survival rate of 65%.78 In the United States, colorectal cancer affects 148,000 individuals annually and is responsible for more than 55,000 deaths per year, which is surpassed only by cancer deaths secondary to lung cancer.78 These statistics highlight the critical importance of identifying individuals at risk for the development of colorectal cancer and of screening and early detection in its management. This section will discuss various risk factors, both modifiable (i.e., extrinsic risk factors) and not modifiable (i.e., intrinsic risk factors), that increase susceptibility for the development of colorectal cancer and will review current screening guidelines. This information can be used to design more effective preventive strategies, which could make use of genetic testing for individuals at risk and apply behavioral modification and chemoprevention.

Pathogenesis Numerous epidemiologic, international, and experimental studies have evaluated various hereditary and environmental factors that lead directly or indirectly to the development of colorectal cancers. It is believed that colon cancer is the result of a complex series of genetic and epigenetic events that occur when environmental factors interact with an individual’s inherited or acquired susceptibility.42,109 This interaction produces somatic mutations that accumulate over time and lead to neoplastic transformation of normal colonic epithelium into premalignant adenomatous polyps (see Fig. 26-2D) and ultimately into invasive disease. The natural history preceding the development of cancer can progress through several decades. Adenomatous polyps, especially the villous subtype, are the premalignant lesions in more than 90% of colorectal cancers. The risk of malignant degeneration depends on the size of the polyp (increasing greatly in those greater than 1 to 2 cm in size), duration of its presence, number present at the time of the initial examination, and the histologic type.110 Only adenomatous polyps seem to carry a premalignant risk; however, hyperplastic polyps are diagnosed more commonly in individuals with a smoking or drinking history, two predisposing factors for adenomatous polyp development.111 The presence of hyperplastic

Genetic changes

Figure 26-3 • Colorectal cancer results from inherited genetic predisposition and acquired molecular alterations interacting with environmental and endogenous toxins that are themselves modified by gene products. TSG, tumor suppressor gene; ONC, oncogene; ROS/RNS, reactive oxygen and reactive nitrogen species form from normal metabolic processes (endogenous) and from exposure to carcinogens and toxins (external).

polyps may warrant increased screening and prevention counseling, although further study of this issue is needed before definitive conclusions can be made.

Etiology Heredity Our understanding of the genetic and molecular alterations that precede the development of colorectal cancers has broadened and deepened over the past 10 to 15 years (Fig. 26-3).112 This information has facilitated the identification of individuals who might benefit from early interventions with more vigilant screening, chemoprevention, or treatment. Based on studies of family histories, it is estimated that 20% to 30% of colorectal cancers have a significant hereditary component.112 Thus far, however, genes associated with only two major syndromes—familial adenomatous polyposis (FAP) and hereditary nonpolyposis colon cancer (HNPCC)—have been identified clearly. Allelic deletions have been identified in patients diagnosed with these two autosomal dominant syndromes, FAP and HNPCC.113–115 FAP accounts for only 1% of colon cancer cases per year and is associated with a deletion of the APC gene on chromosome 5 (band q21). These patients develop thousands of adenomatous polyps that tend to be evenly distributed throughout the colon and rectum by the second or third decades of life. If surgical treatment by complete colectomy is not done, affected individuals are at high risk to develop colon cancer by the age of 40. A highly specific mutation (T to A at nucleotide 3920) has been found in 6% of Ashkenazi Jews, and about 28% of Ashkenazim have a family history of colorectal cancer.116 This mutation created a small hypermutable region of the gene, thereby indirectly causing predisposition. The incidence of HNPCC was determined to be less than 1% of annual colon cancer cases in a large population-based study, although estimates of 5% are typically reported from nonpopulation-based studies.117 Diagnosis requires that three or more relatives be diagnosed with colorectal cancer, representing at least two successive generations, and at least one relative must have been diagnosed before the age of 50.115 One relative must be a first-degree relative of the proband patient. Patients tend to have cancers that arise in the proximal colon, and they also develop ovarian and endometrial cancers at a higher rate than the population at large. This syndrome is associated with defective DNA repair mechanisms, which lead to aberrant cell growth and tumor formation. These mutations occur on chromosomes 3 (hMLH1, 3p21) and 2 (hMSH2, 2p).118,119 Based

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on extensive experimental and clinical data, Vogelstein and colleagues42,113 proposed that it is the progressive accumulation of mutations that ultimately lead to invasive disease. This proposal has been substantiated extensively, and mutations associated with colorectal cancers have been identified involving proto-oncogenes, tumor suppressor genes, and certain key regulatory enzymes, such as cytochrome P450 and acetyltransferase.120–122 It has been suggested that colorectal cancers in adults develop through one of three different pathways (chromosomal instability, microsatellite instability, and CpG island methylator phenotype) and have different biologic behaviors.109 The role of polymorphisms in metabolizing key molecules (including those present in the diet) is being examined closely and should provide a platform from which to understand geneenvironment interactions. For example, polymorphisms in hepatic cytochrome P450 and acetyltransferase enzymes lead to rapid oxidation and acetylation of genotoxic compounds such as heterocyclic amines, which are present in processed foods.122 Accelerated metabolism of these compounds increases an individual’s risk of developing colorectal cancers threefold.

Diet Although diet seems to play a significant role in colon carcinogenesis, the degree to which individual macronutrients and micronutrients contribute to the development of colorectal cancer has been elusive. In part, this difficulty stems from differences in design and methodology in studies that have been performed to evaluate this subject, including the type of dietary questionnaire administered, differences in cohorts such as age and ethnicity, confounding effects of other dietary components, selection and recall biases, sample size, and length of follow-up. The majority of past evidence has demonstrated an increase in incidence and mortality rates from colorectal cancers in groups of people who consume a more “westernized” diet that is high in animal fat, total calories, and red meat but low in fiber and fruit and vegetable intake.123 International and migrant studies have supported this observation. Recent studies, however, have indicated that the older evidence should be reconsidered. Large prospective cohort studies and several large randomized trials indicate that fiber does not seem to be protective nor fat contributory to colon cancer development.124–126 In contrast, mechanistic considerations, metabolic studies, and epidemiologic studies suggest a strong protective effect of folate and an important role of insulin and insulin-like growth factors in colon cancer pathogenesis. Potter109 and other researchers have done a particularly nice job in attempting to relate genetic changes, risk factors (including diet), and downstream molecules and pathogenesis.

Other Factors (Alcohol, Smoking, Exercise, Body Mass Index) Primary prevention of colorectal cancer also requires that we understand factors other than diet that increase risk for colorectal carcinoma by initiating or promoting carcinogenesis. These include use of alcohol and tobacco, sedentary lifestyle, and the metabolic changes that proceed from these.18,127 Many studies have demonstrated a relationship between alcohol use and colorectal cancer and adenoma formation.128,129 It is still uncertain whether alcohol directly initiates DNA damage or acts as a promoter on cells that already have undergone preneoplastic changes. A low-methionine or low-folate diet might contribute to a situation leading to adenomas and colorectal cancer, in that both methionine and folate are cofactors for DNA synthesis; lowered concentrations of these compounds leads to hypomethylation of DNA, which is a precursor to aneuploidy and loss of heterozygosity.130 Various forms of a key enzyme, 5,10-methylenetetrahydrofolate reductase, which catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, have also been identified.131 Some mutations of this enzyme increase its activity, whereas others decrease it. Low activity leads to decreased methionine synthesis and antagonizes methyl group metabolism in DNA synthesis.131 This

theory gained some support in the U.S. Male Health Professionals Study.111 An association was found between high alcohol intake and methionine-deficient diets, after controlling for intakes of fat, red meat, fiber, level of physical activity, body mass index (BMI), and multivitamin and aspirin supplementation. Alcohol might be particularly important for progression of large adenomas to tumors.128 Avoiding excess alcohol, while increasing dietary folate and methionine, seems like a reasonable approach to decreasing risk for colon cancer. Several large studies are underway to test whether supplementation with folate can reduce adenomata development. Cigarette smoking has been consistently associated with adenoma formation but less so with colon tumors, an observation that could be explained by the molecular nature of a subset of colon tumors in which microsatellite instability and/or p53-negative status is a prominent feature.132 The Health Professionals Follow-up Cohort Study and the Nurse’s Health Study observed more adenomas in individuals with a history of smoking than in those who did not smoke.133 Analysis of the very large American Cancer Society Cancer Prevention II study indicates that 20% of colorectal cancers and 12% of deaths are associated with long-term cigarette smoking134 (Fig. 26-4). Physical inactivity and high BMI also increase one’s risk for colorectal cancers. A prospective study found a significant inverse association between leisure-time physical activity and incidence of colon cancer in participants of the Nurse’s Health Study.135 An inverse association was also observed between physical activity and the development of large (>1 cm) adenomas in the distal colon. In this same study, more adenomas were observed in individuals with a high BMI. Obesity, and in particular abdominal adiposity, has also been associated with an elevated risk for adenomatous polyps and colon cancer.136 Recently, two separate cohort studies have demonstrated that physical activity is associated with improved outcomes among resected colon cancer patients.137,138 Increasing physical activity and maintaining lean body weight for the prevention of colorectal cancer probably has considerable merit for decreasing the incidence of polyps and colon cancer, as well as of other chronic diseases. The mechanisms underlying these proposed effects are not clear, but a unifying hypothesis has been proposed recently and involves the sequential steps of consumption of excess dietary energy, development of insulin resistance, and increased circulating levels of insulin, triglycerides, and nonesterified fatty acids, which results in secondary colonic epithelial damage.139 Thus, the beneficial effects of physical activity on colon cancer risk and outcomes represents the sum total of numerous cellular and molecular events. A goal of prevention research is to determine which molecular pathways are being affected by physical activity and to evaluate potential biomarkers in this process. However, validated mechanisms have been lacking. Epidemiologic evidence associating insulin, insulin-like growth factor-1, and insulin-like growth factor–binding protein levels with colorectal cancer incidence provides considerable support for the proposal that changes in diet and physical activity may affect key molecular events.140

Screening and Early Detection Population-Based Data A definitive amount of data has accumulated indicating that colorectal cancer screening for persons at average risk is effective and reduces colorectal morbidity; agreement on the best screening modality remains unsettled, however.141 Cost-saving analysis even supports the use of universal colonoscopy over the long term, notwithstanding the considerable practical challenges and total cost in achieving this goal. Probably everyone over 50 years of age should be screened. Yet surprisingly, fewer than 50% of individuals who should be screened are evaluated by fecal occult blood tests (FOBT), digital rectal exams (DRE), and/or sigmoidoscopy.32 Conflicting recommendations from government and private agencies contribute to the confusion. In general, all groups advocate

Cancer Prevention, Screening, and Early Detection • CHAPTER 26

Dietary energy

Insulin resistance

Insulin

Triglycerides

NEFA

ROS

Proliferation

Enhances epithelial damage

Focal inflammatory response

Cox-2

Accumulation of genetic damage ROS

APC

DNA mismatch repair Aberrant crypt K-Ras Adenomas P53 Carcinoma

Figure 26-4 • The potential effect of diet on risk of colon cancer. NEFA, nonesterified fatty acids; ROS, reactive oxygen species.

screening in men and women beginning at the age of 50, because the incidence of colon cancer rises sharply between the ages of 50 and 55 and continues to double with each succeeding decade, reaching a peak by the age of 75. The American Cancer Society and the American College of Obstetricians and Gynecologists support yearly DREs beginning at age 40 and FOBTs performed yearly, once a person reaches 50 years of age. Sigmoidoscopy should occur every 3 to 5 years beginning at 50 years of age. Flexible sigmoidoscopy is superior to rigid sigmoidoscopy, because it allows the examiner to visualize up to 60 cm of bowel mucosa and is easier on the patient. There are numerous studies validating the use of DRE, FOBT, and sigmoidoscopy as effective screening tools, providing regular screening is performed (to detect lesions in this disease with a long preinvasive phase).142–144 This is particularly important with the FOBT, because reported sensitivities are low and range between 22% and 92%. Sensitivity is higher when at least three tests are performed on different days and the samples are rehydrated with hydrogen peroxide. To decrease false-negative results, which also increases the sensitivity of the test, patients should be instructed to avoid vitamin C and to eat a high-residue diet for several days before the test. The incidence of false positives is lowered when gastrointestinal irritants (e.g., aspirin, oral iron, and meat products) are not consumed a few

days before the FOBT. In general, screened, asymptomatic patients have a positive test 4% to 6% of the time. Only 5% to 10% of these individuals have colorectal cancers, and an additional 30% have benign polyps.145,146 The positive predictive value is only about 20%, so a positive test can be costly, because follow-up requires evaluation by sigmoidoscopy or colonoscopy. Randomized clinical trials have demonstrated a decrease in mortality by 15% to 33% with the regular use of serial FOBT, however.143 Patients who received FOBT in conjunction with sigmoidoscopy in a Memorial Sloan-Kettering colon cancer trial had a significantly higher survival probability when compared with those who received sigmoidoscopy alone (70% vs. 48%, respectively).143 This is an impressive result. Regular screening with sigmoidoscopy among patients over 50 years of age both reduces mortality from colorectal cancer and prolongs survival.147,148 Studies must be performed to clarify the optimal interval between screening and to develop recommendations for individuals at higher risk for adenomas or colorectal cancers. The early results of a “once-only” sigmoidoscopy at age 60, in which a high yield of adenomas was obtained, suggests that a less intense approach to screening could be a cost-effective strategy to prevent colon cancers.149 Follow-up colonoscopy suggested that a significant number of proximal adenomas were present. The efficacy of screening

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colonoscopy for adenoma detection was directly related to prolonged endoscope withdrawal time in a recent study, a finding that may influence patient management in the near future.150 There is little doubt that screening colonoscopy is effective at identifying silent, large adenomas and colon cancers, but its cost effectiveness must be demonstrated; so must the optimal usage of fecal occult blood, sigmoidoscopy, and colonoscopy with regard to efficacy and cost. A particularly exciting approach is the identification of colon cancerspecific mutations in fecal DNA.151 A study of the adenomatous polyposis coli (APC ) gene, the initiating abnormality in most sporadic colon cancers, in the fecal DNA of normal individuals and patients with polyps shows considerable promise.152 Although the specificity of this test was high (100%), the sensitivity was only 57%. However, integration of the test into the screening paradigm (perhaps with FOBT) as a low-cost, general screen is an important goal. The DRE should be included in all examinations, because it permits evaluation of the distal rectum and prostate. Its sensitivity has decreased, however, with the temporal shift to more proximal lesions in the colon.149 Other tests, including double-contrast barium enema, have not been evaluated completely as screening tests and are more expensive than DRE, FOBT, and sigmoidoscopy. We feel that these modalities should be reserved for those patients with positive screening tests.

High-Risk Individuals More frequent screening is recommended for certain postpolypectomy individuals and other individuals at increased risk for the development of adenomatous polyps or colorectal cancers.153 One particularly important group includes those who have had previous treatment for a primary colorectal cancer. Recent analysis indicates that evaluation of this high-risk group has been inadequate. Proctosigmoidoscopy or colonoscopy should begin at age 10 for first-degree relatives of individuals with FAP and at age 25 for individuals with a strong family history suggestive of HNPCC.154,155 Genetic testing for the mutated APC gene in peripheral mononuclear cells of patients with early onset of adenomatous polyps or in first-degree relatives of FAP-affected individuals will help to determine who needs closer surveillance.156 Counseling about diet, chemoprevention, and lifestyle issues can be provided for individuals who test positive for the mutated APC gene. In addition, a colectomy should be considered if polyps are found. Genetic testing for mutations in DNA repair genes should be performed on anyone whose family history is suggestive of HNPCC; counseling can then be provided for individuals who test positive.157 A positive family history of colorectal cancer that does not meet criteria for FAP or HNPCC probably also warrants earlier screening, but definitive guidelines have not been established. A prospective study of approximately 120,000 men and women who underwent colonoscopy or sigmoidoscopy surveillance concluded that the ageadjusted relative risk for cancer was 1.72 with one first-degree relative with the disease, 2.75 with two first-degree relatives, and 5.37 with one first-degree relative who was under age 45 at the time of diagnosis.158 It is estimated that as many as 25% of individuals with colon cancer have positive family histories. Baseline colonoscopy before age 50 seems to be a reasonable consideration among this group of patients with a positive family history. Other high-risk conditions requiring close surveillance include individuals with a long-standing history of inflammatory bowel disease, a prior history of polypectomy or ureterosigmoidostomy, a personal history of ovarian, endometrial, or colon cancer, and finally anyone who has been treated for Streptococcus bovis bacteremia.154,159–161

Other Health Factors In considering health care resources, other issues that affect the development of colon cancer should be considered. The role of estrogen replacement in the development of colon cancer and in other health parameters in postmenopausal women is of great importance. The

results of the Nurse’s Health Study suggested that current estrogen replacement in postmenopausal women can reduce the risk of colon cancer (relative risk = 0.65); this effect disappeared 5 years after discontinuation of estrogen replacement.162 However, a detailed analysis of the group of postmenopausal women in the WHI trial who received estrogen plus progestin indicated that the colorectal cancers were diagnosed in a more advanced stage even though the number of colorectal cancers was reduced by 45%.163 When reproductive factors were examined among women who were diagnosed with colon cancer in this study, oral contraceptive pill use and later age of menarche were also associated with a decreased risk. In another study, women who delivered more than five children, especially if they had a positive family history of adenomas, were at increased risk for the development of adenomas.164 In this study, however, no association was found with age at menarche, menopause, first birth, or oral contraceptive pill use. Although estrogen plus progestin lowered the risk for colorectal cancer in the WHI trial,163 these favorable results will have to be balanced against the increased risk for breast cancer and cardiovascular disease (see later discussion).

Chemoprevention Despite the enormous amount of epidemiologic observations and experimental data that support a protective role of many dietary constituents against the development of adenomatous polyps and colorectal polyps, the results from definitive randomized trials have been modest, at best. Many micronutrients and dietary constituents have been studied. Trials using vitamins C, D, and E, and β-carotene as well as supplementation with fiber or lowering of dietary fat have been uniformly negative. Trials in which calcium has been supplemented have been modestly positive with a 25% to 35% reduction of adenomatous polyps demonstrated.24,25 However, daily supplementation of calcium with vitamin D for 7 years had no effect on the incidence of colorectal cancer among postmenopausal women in the WHI.165 These results suggest that calcium inhibits colon carcinogenesis at early but not late stages. Additionally, in a double-blind, randomized, placebo-controlled trial of selenium supplementation for skin cancer prevention, a secondary analysis showed that the numbers of colon, prostate, and lung cancers were found to be reduced by 50% in the treatment arm.166 The results of additional randomized trials are pending and should become available in the next 3 to 5 years. Study of the effects of micronutrient supplementation on the formation of polyps and colorectal cancers is difficult, given the inherent complexity of carcinogenesis. Understanding how various dietary components inhibit carcinogenesis will be instrumental to the development of novel dietary-derived chemopreventive agents in the future. Factors such as type of micronutrient, dose, and duration of treatment, as well as cohort demographics (age and geographic location), and endpoints (polyp formation, or changes in the incidence and mortality of invasive cancer) are all important variables that will affect trial results. However, despite these difficulties in study design, analysis, and interpretation, we should not dismiss the large amount of epidemiologic evidence and supportive experimental data demonstrating that the consumption of diets rich in fruits and vegetables, but low in fat, have a lower incidence of bowel cancer and cancer in general.167 Observational and animal studies suggest that reduction of caloric intake and moderate physical exercise should also decrease the risk of colorectal cancer.168 Meat consumption—particularly red meat that has been processed, has been associated with increased risk of colorectal cancer in numerous epidemiologic studies. Substances in meat that have been implicated in colon carcinogenesis include the known carcinogens: heterocyclic amines (formed in meat during cooking), polycyclic aromatic hydrocarbons and N-nitroso compounds (both found in processed meats), and the amino acid arginine (i.e., a key substrate of the polyamine synthetic pathway).169–172 Research from clinical trials should provide us with invaluable data

Cancer Prevention, Screening, and Early Detection • CHAPTER 26

on which to base dietary and lifestyle recommendations for the prevention of colorectal cancer. There are also abundant scientific opportunities to explore the role of nondietary chemoprevention compounds in controlling colorectal cancer based on substantial studies of colon carcinogenesis (see review173). Some of the more active nondietary compounds being studied include NSAIDs and DFMO. Mechanistic studies indicate that epithelial regeneration and focal inflammation may be important early changes in the pathogenic process,139,173 so that the use of nontoxic antiproliferative and anti-inflammatory agents as chemoprevention agents has a strong rationale. These biologic features of colon carcinogenesis are also used to support a role for probiotics and prebiotics for the prevention of colorectal cancer.174 A considerable amount of experimental and epidemiologic evidence exists to support the use of NSAIDs to decrease the risk of colon cancer.173 These compounds exert their antiproliferative effects on colonic cells through inhibiting prostaglandin synthesis by reversibly binding to cyclooxgenase as well as through several other newly discovered mechanisms. Laboratory studies have consistently demonstrated that NSAIDs can inhibit chemically induced and transplanted tumors in rodents. The interpretation of epidemiologic trials involving NSAIDs is challenging because of differences in design and methodology, including the particular agent chosen, the dose, frequency, and duration of use, and variable follow-up periods. Nevertheless, most case-control and cohort studies have demonstrated an association of a reduced risk of colon cancer with increased consumption of NSAIDs. Analyses of subgroups of patients who routinely take these drugs—such as patients with rheumatoid arthritis (aspirin), inflammatory bowel disease (sulfasalazine), and FAP (sulindac)—have reported a decrease in either adenomatous polyp formation or the development of colorectal cancer. A particularly important study was the Nurse’s Health Study, which used three consecutive questionnaires to determine the rate of colorectal cancer among women who consumed aspirin and compared these rates to women who reported no aspirin use.175 After at least a decade of regular aspirin use, at doses similar to those recommended for the prevention of cardiovascular disease, aspirin consumption was found to reduce the risk of colorectal cancer substantially. Both celecoxib and sulindac have been shown to cause regression of polyps in patients with FAP.176,177 Sulindac was not effective in preventing the development of new polyps in these patients, however.178 The results of several large randomized trials using NSAIDs have been reported.168,179,180 Aspirin has uniformly reduced the recurrence of adenomatous polyps by 25% to 35% in patients at moderate risk with acceptable toxicity. Studies with COX-2 selective inhibitors have produced an unusual conundrum. Several randomized trials have demonstrated substantial benefit with a 40% to 50% reduction in recurrence of adenomas; however, cardiovascular events were markedly increased in the treatment arm.62,63 These results have put a significant damper on the development of chemoprevention agents for cancer. Whether similar adverse results will be seen with less selective NSAIDs (e.g., sulindac) is unknown and awaits the results of ongoing trials. DFMO has been found to be a potent inhibitor of carcinogenesis in experimental animal models by reducing the number and size of adenomas and carcinomas. This drug exerts its effects by irreversibly inhibiting ornithine decarboxylase, the first enzyme in the polyamine synthesis pathway. Suppressing intracellular pools of polyamines decreases cell growth and interferes with the process of carcinogenesis in essentially all animal models. We have reported the results of a long-term clinical trial, which serially measured the effects of different doses of DMFO on rectal mucosal polyamines over a 12month time period and demonstrated consistent suppression without side effects.57 Demonstration of a dose of a chemopreventive agent that has a substantial biochemical effect without producing clinical side effects is an important goal. Currently, we are studying the

Fecal occult blood, digital rectal exam and flexible sigmoidoscopy* at age 50

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Repeat yearly !2 yrs then less frequently if negative

;

:

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Prevention Do not smoke Lowfat, vegetablebased diet Lower alcohol intake Increase physical exercise

Figure 26-5 • Routine screening and early detection for colorectal cancer and its prevention. *There is a growing consensus that a colonoscopy should be performed at age 50 and all polyps removed. If negative, repeat about every 5 years. If positive, repeat in 3 years. If there is a familial tendency, start 5 years before age of youngest family member with disease.

prevention of adenomas in patients that have undergone polypectomies for adenomatous polyps. DMFO will be given in conjunction with sulindac over a 3-year period, and its effect in reducing polyp recurrence is being studied in a randomized, placebo-controlled trial. The role of tertiary prevention in colorectal cancer has been little explored. As effective chemoprevention agents are developed, the group of patients who have been “cured” by standard therapy should become a focus of investigation, because the incidence of second primary colon cancers is high (about 25%).181

Integration of Prevention Activities Several approaches exist to prevent the development of colorectal cancers. Successful primary prevention depends on public education and counseling about behavioral and dietary modifications that can be made to decrease an individual’s risk, including increased physical activity and reduction of total calorie intake. In patients with adenomatous polyps, polypectomy is a useful preventive measure. Patients with FAP or HNPCC might require colectomies at a younger age to prevent development of cancer. New advances in genetic testing will help to select people who not only need closer surveillance but also might benefit from surgical treatment before cancer or premalignant polyps develop. More vigilant screening in individuals at increased risk for the development of colorectal cancer is reasonable, but the appropriate screening tests, and the optimal interval between tests, require further clarification. More important, given that the majority of cancers occur in patients without a family history, everyone probably should be screened beginning at 50 years of age. Clinical applications of chemoprevention with DFMO, NSAIDs, and various micronutrients are still under development but may offer important alternatives for the future or as part of an overall approach to prevention (Fig. 26-5).

BREAST CANCER The morbidity and mortality from breast cancer remains high despite significant advances in our understanding and management over the

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last several decades. Therefore, prevention and early detection have become important challenges for the medical community. In addition to an enormous health benefit, several billion dollars would be saved annually if breast cancer were prevented and/or the disease were detected at an earlier stage. The widespread use of screening mammography, the increasing recognition that breast density is a major risk factor, the identification of high-risk individuals based on family history, the detection of deleterious mutations, and the “proof of principle” that tamoxifen and raloxifene can reduce the risk for breast cancer all anticipate more effective early management of this disease.

Etiology Heredity Primary prevention depends on our ability to identify individuals who are at increased risk for the development of breast cancer.182 Although many risk factors cannot be changed, knowledge of their presence can be used to identify high-risk individuals. Age, socioeconomic class, geographic location, race, and ages of menopause, menarche, and first birth are examples of risk factors that are difficult to change but important to recognize. The incidence of breast cancer, like that of most cancers, increases with age. The majority of cases are diagnosed in women older than 40 years of age, with only 10% to 15% occurring in women younger than 40 years old and fewer than 5% occurring among women younger than 35 years of age. Affluent women and individuals born in colder climates or in the Western hemisphere also tend to have a higher incidence of breast cancer. White women have more breast cancers than black, Asian, Hispanic, or Native American women. It is of considerable interest that Hispanic and Native American women have many of the same demographic variables (obesity, high fat, and low vegetable diet) that are associated with a high incidence of breast cancer in whites, yet their incidence of breast cancer is less than half that of whites.183 Determining whether this difference is genetically based or if it reflects some other protective dietary or environmental factor is an important issue to address. By identifying who is at higher risk for breast cancer, health professionals can then counsel this subgroup of women and their families about the risks for breast disease and various ways to modify these risks, and they can encourage enrollment into clinical trials aimed at studying novel approaches for breast cancer risk reduction. The most important step in trying to discern who is at risk is to take a detailed personal and family history extending back at least three generations.184 Nearly 25% of women diagnosed with breast cancer have a family history of the disease.184 Recent advances in our understanding of the molecular biology of breast cancer have led to the identification of specific mutations that might help identify women with a hereditary predisposition to developing breast cancer and might help predict who will respond to adjuvant therapy. Medical records, including pathology reports, should be obtained whenever possible to help complete an accurate pedigree. Recall bias is a significant problem when constructing pedigrees and can profoundly influence how we counsel patients; therefore, it is important to collect documentation whenever possible. Family histories must be gathered from the maternal and paternal sides of the family. This latter step is often neglected and makes it impossible to counsel anyone in a meaningful way. Although most cases of breast cancer are sporadic and a product of many genetic insults, approximately 5% are due to specific inherited germline mutations in the BRCA1 and BRCA2 tumor suppressor genes.185 The estimated lifetime risk for breast cancer in BRCA1 and BRCA2 mutation carriers ranges from 55% to 85%, in comparison with the 13% lifetime risk for the general population.185–187 Women with these mutations are also at increased risk for the development of a second breast cancer; BRCA1 mutation carriers carry up to a 65% lifetime risk, and BRCA2 carriers might share a similar risk.187

Therefore, carriers of mutations in the BRCA genes and women with a personal or family history might benefit from prevention strategies and genetic counseling. Genetic testing should be offered to individuals with a strong family history (breast or ovarian cancer in two or more generations), a history of multiple primaries (ovarian or breast, colon, endometrial), early age of onset of breast cancer (60 g/day) were not associated with additional increased risk. Low dietary levels of selenium and antioxidants such as vitamins C and E, and β-carotene have been associated with breast cancer development and differences in survival.213 β-carotene is the major provitamin A carotenoid and has differentiating and antiproliferative effects on a variety of cells, including mammary carcinomas. Levels of β-carotene have been analyzed in numerous studies and are lower in women with higher staged breast cancer and breast cancer in general. A case-control study from Europe, however, observed no differences in vitamin A or β-carotene levels between cases and control subjects.214 Studies of soy intake in Singapore Chinese women are of great interest.210,215 Soy intake was significantly associated with lowered plasma estrone and with more favorable mammographic patterns. Recently, there has been a great interest in gene-environment interactions and studies of micronutrients in relationship to metabolic pathways, and genetic polymorphisms are likely to be informative. Among women at high risk for breast cancer for other reasons (e.g., heredity), reducing alcohol consumption should be a straightforward way to reduce breast cancer risk.

Screening and Early Detection Secondary prevention is aimed at detecting preinvasive lesions such as ductal carcinoma in situ, lobular carcinoma in situ, or early-staged breast cancers that have the potential to be cured with limited treatment. Screening tests include the breast self-examination, the clinical breast examination administered by health care professionals, and mammography. Successful implementation of wide-scale screening

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programs that incorporate these techniques, followed by treatment of detected lesions, is probably responsible for most of the decline in the overall death rate from breast cancer that occurred among American women from 1989 through 1993.216 This decline continues and probably represents both the increased use of mammography and the effectiveness of systemic adjuvant therapy. Currently, 5-year survival rates for localized breast cancers have increased to more than 98%.78 Although we encourage women to perform monthly breast examinations, a randomized trial indicated that this practice does not decrease overall mortality rates.217 The most effective combination for decreasing the incidence of invasive disease is the clinical breast examination and mammography. The Breast Cancer Detection Demonstration Project showed that the sensitivity of the clinical breast examination and mammogram together was 70% to 80%, with sensitivity increased for older patients.218 Although it is standard practice for a clinical breast examination to be performed annually, there has been a great deal of controversy surrounding the appropriate time to begin routine screening with mammograms. Randomized controlled trials of a large number of women on trials from several countries have unequivocally demonstrated a 40% decrease in mortality from breast cancer in women who have annual mammograms beginning at the age of 50.219 Throughout the years, controversies have erupted regarding the magnitude of benefit from screening mammography. We continue to recommend the practice to women beginning at the age of 50 but encourage discussion of the risks and benefits associated with this procedure. The opinion on routine screening in women between the ages of 40 and 49, however, is mixed.220 Eight randomized controlled trials performed between 1963 and 1982 do not demonstrate a statistically significant difference in breast cancer mortality within 7 years after screening was initiated in women randomized to receive or not receive screening mammograms. A majority in a recent consensus panel used this information to state that there currently was not sufficient evidence to advocate routine screening mammography in women ages 40 to 49. Five of these trials, however, demonstrated a 16% decrease in mortality if follow-up continued for 10 years, prompting release of a minority report advocating routine screening in the 40 to 49 age group.220 As the minority report highlighted, the goal of mammography is to detect preinvasive lesions, and 15% to 20% of breast cancers are now diagnosed as ductal carcinoma in situ or lobular carcinoma in situ in younger women. The minority report correctly pointed out that the risk from radiation during mammography screening was overemphasized. The U.S. Preventive Task Force has considered the issues carefully and recommends a screening mammography every 1 or 2 years for women aged 40 and older,221 a position we favor. An important recent observation is that mammographic density is strongly associated with risk and is heritable.222 This information should further target the premenopausal woman at higher risk for breast cancer; determination of the underlying genetic basis for breast density has now become of great interest, particularly in that the use of a postmenopausal hormone therapy was strongly associated with an increase in mammographic density in the Postmenopausal Estrogen/Progestin Interventions and WHI trials.195,196 New areas that are currently being evaluated for the enhancement of primary and secondary prevention strategies include digital mammography, DCE-MRI, DCE-CT with ultrasound, PET, optical scanning, ductal lavage for cytologies and molecular testing, nipple aspirates, and blood and urine assays for growth factors and autoantibodies to oncoproteins and to tumor DNA. Validated biologic markers of breast cancer risk and/or more sophisticated screening modalities might well increase our ability to detect lesions earlier in high-risk populations.

Chemoprevention Successful therapeutic prevention for breast cancer has progressed faster than for any other malignancy.223 Two compounds, tamoxifen

and raloxifene, which are estrogen receptor antagonists, have been shown to reduce the incidence of primary breast cancer in women at high risk.224,225 In a head-to-head randomized trial of tamoxifen and raloxifene, efficacy was comparable with a 50% reduction of breast cancers; the toxicity of raloxifene was considerably less than that of tamoxifen.226 Based on the positive activity of anastrozole (an inhibitor of estrogen synthesis) in the adjuvant setting and its minimal toxicity,227 a randomized trial of this compound versus raloxifene in women at high risk for breast cancer is now underway. Preclinical studies at our institution using a transgenic mouse model suggest that antiprogestational agents may be of particular benefit in BCRA1positive breast cancers.228 The pros and cons of chemoprevention for breast cancer were recently reviewed, and the overall conclusion was that it cannot yet be recommended for general usage but should be useful for reduction of risk among high-risk individuals.229 There are several other options for a woman who is at very high risk for breast cancer, including bilateral mastectomy or oophorectomy and lifestyle modification. Prophylactic bilateral mastectomies have been performed on some mutation carriers, but cases of breast cancer developing in the remaining breast tissue after subcutaneous and total mastectomies have been reported. In addition, such surgeries are dramatic procedures for a woman who has only a “probability” of developing breast cancer, and a decision analysis paradigm for these interventions is available.230 No long-term data exist on the effect of these surgeries in increasing the life expectancy of mutation carriers, yet one meta-analysis reveals considerable global variation in the utility of prophylactic surgery among unaffected BRCA1 and BRCA2 mutation carriers.231 Bilateral oophorectomy has been proposed as another option for premenopausal women who have completed their childbearing. Although castration has been shown to decrease the risk of breast cancer in young, nulliparous women, especially when it is performed before the age of 35, this remains a very controversial area in the management of breast disease. Secondary prevention can be accomplished by instructing these high-risk women about the importance of clinical breast examinations by a physician and screening mammograms, which should begin at a younger age, preferably at least 5 years earlier than the age at which the relative developed breast cancer. Observational studies and initial clinical trials suggest that moderate physical exercise and control of obesity may decrease the risk for breast cancer,232–234 as has been suggested for colorectal cancer. Recently, several randomized interventional trials have been initiated to definitively address these issues.

PROSTATE CANCER Etiology The age-adjusted incidence of prostate cancer rose slowly from 1965 to 1985 for unclear reasons. Parallel with aging of the baby-boomer population, the prevalence has also markedly increased since the general recommendation in the late 1980s by the American Cancer Society and the American Urological Association of yearly screening for PSA after age 50. This recommendation led to widespread screening and a rapid increase in the incidence of prostate cancer that peaked in the early 1990s.235 In the most recent national estimates using SEER data from 1999 through 2003, the age-adjusted incidence of prostate cancer was 165 cases per 100,000 individuals—the highest rate of any cancer type among men.12 Major differences in the incidence of prostate cancer are observed across the major U.S. ethnic groups, with blacks having the highest incidence rates (243 per 100,000) and Asian-Pacific Islanders having the lowest incidence rates (104 per 100,000). Overall, 234,460 cases of prostate cancer are expected in the United States in 2006, along with 27,350 deaths—making prostate cancer the third deadliest cancer in men after cancer of the lung and colorectal cancer.78 Several factors—age, familial/genetic, environmental, and hormonal—seem to contribute to the development of prostate cancer.236

Cancer Prevention, Screening, and Early Detection • CHAPTER 26

Prostate cancer shows a familial tendency that is currently not well defined, but at least one study suggests that 10% to 15% of cases could have a strong genetic component.237 The loss of heterozygosity in certain chromosomes in prostate cancer suggests that a gene related to some prostate cancers will be found.238 The existence of a locus in chromosome 1 (band q 24) that predisposes men to develop earlyonset prostate cancer has been verified, but a gene has not yet been isolated.239 The androgen dependence of prostate cancer led to the interesting hypothesis that variations in transcriptional activity by the androgen receptor regulated by CAG repeats could determine risk. Subsequent findings, however, argue against such an association, although there may be specific underlying situations in which other genotype influences lead to such an effect.240 Because these studies concentrate on identifying prostate cancer risk and most prostate cancers are not clinically significant, others have argued for identifying genotypes that are associated with clinically aggressive cancers that predict outcome (mortality). In this regard, a report documenting extensive mitochondrial mutations in primary prostate cancers might provide new insights into progression.241 Both epidemiologic and experimental data suggest that hormones, particularly testosterone, play a definitive role in the development of prostate cancer. In the rat model, testosterone induces prostate cancer, and in humans prostate cancer rarely occurs in castrated men.242,243 Also, black men have a higher incidence of prostate cancer at all ages than white men, and Japanese men have the lowest incidence.244,245 Whether this racial-ethnic variation in prostate cancer risk has a hormonal basis is still unclear, but a substantive amount of data supports this viewpoint.245 A high-fat diet and obesity may be associated with an increased risk of prostate cancer, but the studies to date have yielded inconsistent results.246 One investigation suggests that the preadult hormonal milieu, as reflected in attained height and childhood obesity, could have a strong influence on prostate carcinogenesis.247 Epidemiologic, animal model, and in vitro studies indicate that n-3 polyunsaturated fatty acids, lycopene, and selenium might also be important in the pathogenesis of prostate cancer.248 Additionally, GSTP1 has been proposed as a caretaker gene that serves to detoxify carcinogens associated with various lifestyle habits.249

Screening and Early Detection The relative benefits and costs of screening for prostate cancer are currently among the most contentious issues in the medical community.250–252 There are several major reasons why this controversy continues: • All available first-line techniques (DRE and serum PSA) have high rates of false-positive results. This leads to a relatively low positive predictive value and the unnecessary workup of many normal individuals. • The natural history of prostatic intraepithelial neoplasia (PIN), the probable precursor of prostate cancer, is highly variable, and the natural history of the disease cannot currently be predicted reliably in any one particular case or by any specific biologic or pathologic marker. • The workup of abnormal screening tests is invasive, requiring several biopsies of the prostate. • The treatment of prostate cancer produces significant morbidity and measurable mortality. • The rate of false-negative results is also high, which can produce a false level of assurance about the reliability of the screening tests (Fig. 26-6). These same five concerns regarding the use of PSA and DRE for screening also exist for their use for early detection purposes, but the consequences are mitigated somewhat, because individuals are by definition symptomatic on presentation.

At age 50 digital rectal exam and prostatic specific antigen ;

:

Ultrasound

Repeat every two years

Biopsy :

;

Yearly complete surveillance, PSA more frequently

Surgery or radiation

Figure 26-6 • Screening and early detection for prostate cancer.

The two most commonly used screening tests for prostate cancer are PSA and DRE, with transrectal ultrasound (TRUSP) reserved for patients with a positive PSA and/or DRE. Before the 1990s, yearly DRE after age 50 was the standard test used both for detection of prostate cancer and for screening. Although many primary care physicians use the DRE as part of a routine physical examination, assessment of its routine use indicates that DRE is performed in less than 50% of primary care encounters in which one would expect it to be done.253 A summary of the data indicates that the positive predictive value of DRE is relatively low (11% to 26%), whereas the negative predictive value (85% to 96%) is relatively high.254 The most complete assessment thus far evaluated 811 unselected serial patients from 50 to 80 years of age who underwent DREs; 43 patients had a palpable nodule, and the positive predictive value of the 38 patients who underwent biopsy was 25%.254 It is of great interest that 68% of the detected tumors were clinically localized, but only 30% were pathologically localized after radical prostatectomy. These data and other studies suggest that only about 20% to 25% of cases are localized at the time of a positive DRE; on the other hand, more than 25% of cases of prostate cancer are metastatic by the time a detectable palpable lump is detected on DRE.254,255 Although the effectiveness of DRE is probably also significantly influenced by the skill of the examiner, the proper technique can easily be taught to health workers, is inexpensive, and is relatively noninvasive. Its usage as a primary screening tool, however, has not been widely adopted, probably because of its inconvenience. Whether routine screening by DRE alone can reduce mortality from prostate cancer is unknown. With the emergence of serum PSA as the screening test of choice, it is unlikely that the specific value of DRE in reducing prostate cancer morbidity and mortality per se will ever be demonstrated conclusively. Annual measurement of serum PSA as a screening test for prostate cancer has been adopted widely following the initial 1993 recommendation of the American Cancer Society. At the current time, the professional community remains split over the question whether serum PSA should be recommended for routine screening in men older than age 50. The issues have been presented and analyzed extensively, and the same arguments that are used to discourage routine screening are used by others to recommend its widespread use. Estimates of overdiagnosis of clinically insignificant lesions have ranged from 15% to 84% in well-done investigations.251,252 Several studies have demonstrated that PSA screening results in a stage downshift and increases the detection rate of early-stage cancers.256,257 The false-positive rate (25% to 50%) is high, however, resulting in a positive predictive value of PSA in screening studies of

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about 30%. Because most of the studies have been done on symptomatic men, the positive predictive values in a true screening effort are likely to be lower (i.e., due to an expected lower prevalence of disease). In practical terms, this observation means that less than one-third of men with an elevated PSA will have biopsy-proven prostate cancer, and two-thirds will have a biopsy result that is negative for prostate cancer. Even if biologically aggressive tumors (and this is unlikely in most cases) were being identified (see the discussion later in this chapter), a large number of men would undergo unnecessary prostate biopsies with the attendant fiscal cost and morbidity. Because the value of a “normal” level of PSA (4 ng/mL) or DRE were identified.260 Among the 2717 men with elevated PSA, 64% received biopsy within 3 years, and among men with positive DRE and negative PSA only 27% underwent biopsy within 3 years. As might be expected, higher biopsy rates were noted for those with PSA above 7 ng/mL as compared with those whose PSA level was 4 to 7 ng/mL. Follow-up data regarding mortality, and also the positive predictive value for PSA, DRE, or both among different demographic strata are not yet available but are expected to assist greatly in our understanding of the utility of these commonly used screening tests. The second major obstacle to the successful use of any screening modality for prostate cancer is that the biologic aggressiveness of PIN and early prostate cancer is not identified with high reliability by serum PSA. Progressive prostate cancer is a serious disease with high morbidity and mortality; however, not all prostate cancers are serious, and indolent behavior is more common than not. For example, about 30% of men over age 50 have histologic evidence of prostate cancer at routine autopsy, suggesting a prevalence of prostate cancer of about 9 million.261 About 1.2 to 1.5 million of these 9 million men, or about 15%, will eventually die of their disease. Thus, most prostate cancers in the population are latent and do not progress to clinical adversity; therefore, an aggressive workup of an elevated PSA should not be a reflex action. A third major consideration in evaluating PSA as a useful screening test is that the subsequent workup and treatment have a significant complication rate. Follow-up testing of an elevated PSA requires a repeat PSA, DRE, TRUSP, and biopsy. These are relatively safe procedures, but about 0.1% to 0.4% of the 20% of screened men who undergo biopsy experience infection or bleeding, and almost all experience considerable anxiety while waiting for the results.262,263 The potential complications of treatment can be quite serious and

include impotence, incontinence, and death from radical prostatectomy. Adverse outcomes of radical prostatectomy have been compared with watchful waiting in a randomized clinical trial. In this study, erectile dysfunction (80% vs. 45%) and urinary leakage (49% vs. 21%) were more common among patients who underwent radical prostatectomy; however, symptoms of urinary obstruction were improved (28% vs. 44%).264 Radiation therapy is no less benign, but it carries a lower incidence of incontinence, a higher incidence of acute gastrointestinal complications, and a similar incidence of impotence. A fourth major issue in PSA screening is the high rate of falsenegative results. Numerous studies have demonstrated that many individuals (25%) with a normal PSA level have disease beyond the prostate.265 Such results can lead to false reassurances and decreased follow-up when other factors suggest that a more aggressive workup might be reasonable. Several studies suggest that a rising PSA, even in the normal range, is a cause for concern and reason enough to biopsy.266,267 Although serum measurement of PSA has been widely adopted in men over age 50 as a primary screening tool for prostate cancer, its value in improving the overall health of men has not been shown to date. The equally important issue of whether screening does more harm than good also remains unanswered, because the natural history of prostate cancer is so variable.265 Decision analysis has been used to determine the benefits and risks of age- and quality-adjusted survival, but the results remain inconclusive.268,269 Other studies suggest that screening might have the potential to decrease survival, particularly in the older individual.269 There is, of course, no lack of critics of this viewpoint.270 What information is needed to resolve this difficult and important issue? Perhaps only a series of randomized trials can lay this question to rest, and to this aim, the prostate component of the PLCO Cancer Screening Trial may provide answers to these important questions. The results of a randomized trial involving more than 40,000 men in the city of Quebec have been reported, and those subjects with a regular PSA screening had a 60% decrease in mortality from prostate cancer after 7 years.271 The conduct and interpretation of this type of trial is complex, and the results of several other large screening studies will have to be available before definitive recommendations about the value of routine screening PSA can be made.272 These targeted trials, however, will provide only a general guide regarding population-based screening using serum PSA in men over age 50 as an approach to identify potential prostate cancers. The current consensus by the U.S. Preventive Services Task Force is that the evidence is insufficient to recommend for or against routine screening for prostate cancer using PSA.272 An equally important issue is this: How do we identify and distinguish a biologically aggressive tumor in any one individual from those that will remain latent for the life of the individual? This is a very difficult problem to study. Although the earliest features of prostate cancer pathogenesis remain obscure, recent studies of the biologic features of intraepithelial hyperplasia of the prostate and of the “normal” prostate in individuals with a strong family history could shed some light on this issue.272–274 Recent studies of the cytogenetic and molecular alterations in high-grade PIN have indicated that loss of heterozygosity is prominent and that certain oncogenes are expressed.274 Defining the biologic features of the preclinical phase of prostate cancer is critical to answer for innumerable reasons, not the least of which is to increase the effectiveness of PSA screening. In this regard, a large, randomized trial of men with T1b, T1c, or T2 prostate cancer demonstrated that radical prostatectomy was superior to “watchful waiting” in terms of disease-free survival but not in terms of overall survival.275

Chemoprevention Although the development of rat prostate tumors has been studied for some time, this model system has been regarded as a poor one

Cancer Prevention, Screening, and Early Detection • CHAPTER 26

for carcinogenesis of the human prostate. The development of transgenic models that simulate the human disease represents an improvement in this regard.276 Epithelial changes, including PIN, were identified in the human prostate long ago, although only recently have the biologic (and clinical) implications of these changes been recognized. The importance of these alterations, the recognition of the analogous evolution of the process to other epithelial cancers (e.g., cervical, oral), and its association with a wide spectrum of biologic abnormalities has moved PIN into the forefront as the probable, but clinically uncommon, preneoplastic precursor of prostate cancer.276 An impressive array of studies measuring various biologic and molecular parameters in PIN have been done, and various biologic changes associated with the progression of prostate cancer have been identified.277 What should be done now is to relate these biologic findings to the clinical aggressiveness of PIN and/or the eventual outcome of clinically relevant (nonindolent) prostate cancer. To be able to do so will help guide the difficult decisions after detection of an elevated serum PSA in biopsy samples during the screening and/or identification of PIN and during the early detection process. Three major categories of chemoprevention agents are being currently considered: inhibitors of proliferation, hormonal modulators, and stimulators of differentiation. Thus far, two definitive randomized trials have been launched. The results of a phase III study using the 5α-reductase inhibitor finasteride (thereby lowering levels of dihydrotestosterone, the active metabolite of testosterone) in a high-risk population has been reported. The incidence of prostate cancer was decreased about 25%, and the side effects were minimal.278 However, conclusions about the overall benefit were compromised by the finding that the risk for higher grade tumors was increased in the treatment arm. Although a large number of explanations have been offered for these contradictory findings, the future of finasteride as a therapeutic agent for prostate prevention is uncertain. A second trial uses selenium, vitamin E, or both. The rationale for the study was based on secondary analyses of several large intervention trials in which prostate cancer was not the target, although recently some supportive experimental data also have become available.279,280 Accrual to this 2 × 2 factorial randomized trial (vitamin E, selenium) is ongoing, and results are anticipated in 2013. A large number of compounds are being investigated at the preclinical level, and a few have advanced to the phase I/II clinical level. Studies of fenretinide failed to show an effect on relevant surrogate markers, whereas DFMO was more successful.273,281 Two other relatively unexplored areas of chemoprevention research in prostate cancer should also be mentioned: PIN and familial risk. Just as understanding the biology of PIN will affect our screening and early detection decisions, PIN should also serve as a useful marker in chemoprevention studies. Although the heterogeneity of lesions will make interpretation of effect of an intervention a challenge, PIN represents an important parameter for advancing our knowledge of early prostate cancer carcinogenesis and its modulation by candidate chemoprevention agents. Several studies are in progress to use PIN as a screening tool for new chemoprevention agents including studies of biologic markers to determine risk.282 The roles of family studies and genetics in identifying individuals at high risk for prostate cancer are in their infancy, but epidemiologic studies support the notion that genetic risk plays a role, and clinical studies support the observation that early prostate cancer in some individuals is highly aggressive, whereas in others it is indolent. Linking these two parameters should identify a population of individuals in whom screening, early detection, and chemoprevention agents should be intensively directed. Advances in the systemic therapy of advanced prostate cancer have been slow in coming. In a real sense, advances in the management of prostate cancer have been minimal since the introduction of hormonal therapy more than 50 years ago. It is likely that the widespread use of screening and early detection with an appropriate follow-up will reduce the morbidity and mortality from prostate cancer in a

substantial way and that effective chemoprevention will be developed, because the major biologic enhancer (androgens) of prostate cancer carcinogenesis is known.

SKIN CANCERS Each year, more than $2 billion is spent to treat patients diagnosed with skin cancers, the majority of which are malignant melanoma and basal and squamous cell carcinomas. These figures underestimate the true cost, because many of these cancers are treated in physicians’ offices, and nonmelanoma skin cancers are not routinely reported to tumor registries. An aging population, depletion of the stratospheric ozone layer, and increased recreational exposure to ultraviolet radiation (UVR) represent some of the factors that contribute to the development of over 1 million cases of nonmelanoma (basal and squamous cell carcinomas), 49,000 cases of melanoma in situ, and 62,000 cases of invasive melanoma diagnosed annually in the United States.78 Understanding how these and other risk factors lead to alterations in key cellular processes like DNA synthesis and repair, oncogene activation, cell-cycle control, and apoptosis is the focus of intense research efforts aimed at designing novel preventive, diagnostic, and treatment strategies.59 This section details the various primary, secondary, and tertiary preventive approaches for melanoma and nonmelanoma skin cancers. Incorporating these strategies into medical practice should decrease the incidence and mortality from skin cancer and should also decrease health care costs.

Etiology and Primary Prevention Environmental Successful primary prevention depends on the ability to identify individuals at risk for skin cancer and to use this information to educate both high-risk groups and the general population about various ways to reduce risk (Table 26-5). Many factors have been identified that increase an individual’s risk for the development of melanoma and nonmelanoma skin cancers. Exposure to UVR is a major risk factor.283 Not only is cumulative UVR exposure important in the development of skin cancers, but it is apparent that acute, intermittent exposure to UVR is carcinogenic. The electromagnetic spectrum is composed of infrared, visible, and UV light; the latter is responsible for causing the cellular and architectural changes in the epidermis and dermis that lead to photoaging and skin cancer.283 Although the UVR spectrum is broad, UVR-B (290–320 nm) and UVR-A (320–400 nm) are the only wavelengths that routinely reach the earth’s surface, in that shorter wavelengths (UVR-C) are absorbed by the ozone layer. UVR-B is more potent than UVR-A in inducing neoplastic transformation in epidermal keratinocytes and melanocytes, which give rise to basal and squamous cell cancers, and melanoma, respectively. UVR-A, however, has been found to penetrate the skin more deeply and is the predominant wavelength emitted from artificial lamps found in tanning salons.283 More than a million adolescent and young women frequent these facilities daily and expose themselves to up to five times the amount of UVR that is emitted from the sun at any given time. The role of UVR-A radiation in the development of skin cancer will increase as this industry continues to grow. The mechanism of action of UVR on the skin has been studied extensively. Once photons penetrate through the stratum corneum, they are absorbed by cellular DNA and produce base substitutions in pyrimidines.284 The substitution of thymidine for cytosine is pathognomonic for UVB-induced skin damage and is found in the tumor suppressor gene p53 in more than 90% of squamous cell skin cancers.285,286 Basal cell cancers also contain p53 mutations.287 Although UVR is regarded as contributing to the pathogenesis of melanoma, these types of mutations are uncommon, therefore raising the likelihood that the role of UVR is associative or complementary to the process. Normally, p53 acts to protect damaged cells by either

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Table 26-5 Predisposition and Risk Factors for Skin Cancer NONMELANOMA Ultraviolet light (sun) exposure (cumulative) Genetic Xeroderma pigmentosum Nevoid basal cell syndrome Phenotypic Skin complexion Sunburn/tanning response Degree of freckling Premalignant dermatoses Actinic (solar) keratoses Leukoplakia Chemical, thermal, and scar keratoses Chronic inflammation Immunosuppression Prior history of skin cancer

MELANOMA Ultraviolet light exposure (intermittent) Genetic Melanocortin receptor variants Atypical or dysplastic nevi Dysplastic nevus syndrome Phenotypic Less cutaneous pigmentation

inducing cell-cycle arrest (so that mutated DNA can be repaired or excised) or by inducing apoptosis.288 UVR-induced p53 mutations disturb the cell cycle by inhibiting cyclin-dependent kinases, leading to uncontrolled cell proliferation. Cells with one mutated p53 allele can undergo clonal expansion, and, if the other p53 allele is mutated, neoplastic transformation occurs. Therefore, UVR could both initiate and promote carcinogenesis.288 UV light might also have immunosuppressive effects by interfering with the ability of Langerhans cells to process antigens.289 Other risk factors that increase susceptibility for the development of skin cancers include skin complexion and response to sunlight, degree of freckling, ethnicity, gender, age, geographic location, presence of premalignant skin lesions, medical history of exposure to ionizing radiation or psoralen and UV therapy, chronic skin irritation (ulcers, inflammation, or trauma), or a personal history of a germatodermatoses (xeroderma pigmentosum, nevoid basal cell carcinoma syndrome, and familial dysplastic nevus syndrome), lymphoreticular malignancy, granulomatous diseases, or other immunosuppressed states such as organ transplantation in which development of multiple cutaneous squamous cell carcinomas is a major problem. Health care providers should be aware of several premalignant dermatoses for the purpose of identifying individuals who are at increased risk for the development of skin cancers. The most common lesion, actinic keratoses (solar keratoses), has been reported to undergo malignant transformation to squamous cell cancer in 12% of patients.290 Histologic evaluation of white patches occurring on mucous membranes, known as leukoplakia, is also important, because up to 20% could be dysplastic, with 3% to 6% becoming invasive cancers. Atypical and dysplastic nevi, large congenital nevi (>9 cm),

and an increased number of moles are common precursor lesions of melanoma.291 Chronic skin irritation from radiation (radiation dermatitis), chemicals (tar and arsenical keratoses), infrared light (thermal keratoses), and scars (scar keratoses) might also lead to malignant transformation. Any patient who is immunosuppressed (human immunodeficiency virus [HIV] diagnosis or transplant recipient) or who has a history of epidermodysplasia veruciformis or Bowen’s disease (an intradermal carcinoma that often occurs on sun-exposed areas) should be considered for prevention protocols. Anyone who has a prior history of skin cancer is also at risk for a second primary cutaneous malignancy.292,293 Patients diagnosed with thin melanomas (2000/3500

Platelet Count (/mm3) >100,000 50,000–90,000 50%) with acceptable treatment-related mortality rates.

Non-Hodgkin’s Lymphoma Allogeneic, syngeneic, and autologous HSCT have been reported to yield long-term disease-free survival and an apparent cure for patients with intermediate and high-grade non-Hodgkin’s lymphomas (NHL).118–120 Due to its relative sensitivity to chemotherapy, there is substantial evidence that autologous HSCT is efficacious for patients with primary refractory or chemotherapy-sensitive recurrent NHL of specific histologies including “intermediate-grade” (e.g., disease-diffuse, large B-cell) NHL.121 It is now apparent that patients who fail to achieve an initial complete remission, but who do not have other adverse prognostic factors such as poor performance status or bulky disease, also can achieve long-term disease-free survival.122 Because of the superior results achieved in patients treated earlier in the course of the disease, a number of investigators have incorporated high-dose therapy and autologous HSCT into the primary treatment of patients with intermediate and high-grade non-Hodgkin’s lymphoma.123,124 Autologous HSCT also has been used to treat patients with indolent (“low-grade”) NHL (e.g., follicular center cell) with either purged bone marrow or peripheral blood stem cells, resulting in disease-free survival rates as high as 60%.125,126 However, the late relapses seen in this illness and long overall survival observed with conventional therapy make very long follow-up necessary to document the efficacy of this approach. The demonstration of a potent graft-versus-leukemia effect against NHL is less clear, and the efficacy of donor lymphocyte infusion in lymphoma is anecdotal at best.126–128 Consequently, the specific role of allogeneic HSCT has not been defined. However, there are data that myeloablative allogeneic HSCT can result in long-term survival for patients with recurrent or refractory NHL,120,129 and evidence exists that nonmyeloablative allogeneic HSCT may provide benefit for patients with recurrent follicular non-Hodgkin’s lymphoma. Some evidence, however, indicates that this approach requires that the disease remains chemotherapy-sesitive.130

Hodgkin’s Disease High-dose therapy with autologous or allogeneic HSCT has been widely used in patients with recurrent Hodgkin’s disease (HD; also known as Hodgkin’s lymphoma).131–133 As in NHL, patients with HD whose disease fails to respond to front-line therapy can derive benefit from high-dose therapy and autologous HSCT.134 Allogeneic HSCT has had a limited role in the treatment of HD due to the efficacy of autologous HSCT, the treatment-related toxicities associated with myeloablative allogeneic HSCT, and lack of evidence of a graft-versus-leukemia effect against HD. However, recent data indicate that reduced-intensity allogeneic HSCT may benefit patients with recurrent HD, and a graft-versus-leukemia effect against HD may exist.135

Hematopoietic Stem Cell Transplantation • CHAPTER 32

Soft Tissue Sarcomas and Neuroblastoma High-dose therapy and autologous HSCT have been used as either consolidation of primary therapy or treatment of metastatic or recurrent soft tissue sarcomas, such as Ewing’s sarcoma, rhabdomyosarcoma, and osteosarcoma in children.136,137 Burdach and colleagues reported a 45% relapse-free survival for patients with poor-risk and recurrent Ewing’s sarcoma who received high-dose therapy and autologous HSCT, as compared with 2% for a historical control group.136 The European BMT Solid Tumor Registry reported the results from 21 European transplant centers on 50 patients with Ewing’s sarcoma in first or second complete remission consolidated with high-dose chemotherapy and autologous HSCT.137 Thirty-two patients with high-risk or metastatic disease in first complete remission achieved an actuarial event-free survival of 21% at 5 years. Results of prospective studies of high-dose therapy and autologous HSCT in soft tissue sarcomas, primarily in children, suggest a possible improvement in remission duration and possibly on overall survival.138,139 Autologous HSCT remains investigational in adults with sarcomas. Autologous HSCT has been found to be beneficial for both newly diagnosed and recurrent neuroblastoma.140–143 The Children’s Cancer Group assessed whether high-dose therapy and autologous bone marrow transplantation improved event-free survival as compared with chemotherapy alone.140 All patients were treated with the same initial regimen of chemotherapy, and those without disease progression were then randomly assigned to receive continued treatment with high-dose therapy and purged autologous bone marrow transplantation or to receive three cycles of intensive chemotherapy alone. The mean event-free survival rate was significantly better among the patients who were assigned to undergo transplantation.

Germ Cell Tumors In patients with germ cell tumors for whom platinum-based chemotherapy regimens fail to effect a cure, the use of high-dose chemotherapy and autologous BMT has resulted in prolonged disease-free survival, including patients with refractory disease.144,145 Evidence exists suggesting that tandem transplant may result in improved results; however, no direct comparison of single versus tandem transplants has been performed.146

Other Solid Tumors Several nonrandomized trials and retrospective analyses had suggested that high-dose chemotherapy and autologous stem cell transplantation were beneficial in regard to prolongation of survival in stage II/III breast cancer.147,148 Subsequently, several randomized trials addressed the role of high-dose therapy and autologous stem cell transplantation in patients with stage II/III breast cancer.149–153 Most of these trials found no evidence of a survival advantage for patients randomized to receive high-dose therapy, although some of the trials suggested an improvement in event-free survival in the high-dose arm. Evidence also is lacking that high-dose chemotherapy and autologous HSCT improve survival in metastatic breast cancer.154 There has been considerable interest in investigating the presence of a “graft-versus-tumor” effect in a variety of solid tumors, including renal cell carcinoma and breast cancer.155–157 Childs and colleagues155 reported on a series of 19 patients with metastatic renal cell carcinoma who underwent nonmyeloablative allogeneic stem cell transplantation. Nine patients had responsive disease (47%), of which three were complete responses.

COMPLICATIONS AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION In addition to the acute toxicities associated with prolonged cytopenia, other organ toxicities can be associated with transplantation. A simple index, based on pretransplant comorbidities, has been devel-

oped that reliably predicts nonrelapse mortality and survival.158 This comorbidity index is useful for patient counseling prior to HSCT. The late toxicities always must be kept in mind when choosing therapies for patients.159

Graft Rejection Graft rejection occurs when immunologically competent cells of host origin destroy the transplanted cells of donor origin.160 This complication occurs more commonly in patients who receive transplants from alternative or HLA-mismatched donors, in T cell-depleted transplants, and in patients with aplastic anemia who receive a non– total body irradiation (TBI)-containing regimen. Graft rejection is less likely to occur in nontransfused patients with aplastic anemia.

Cardiac Toxicity Most transplant centers screen potential patients for underlying cardiac abnormalities that would place them at potential increased risk during the procedure.161 Despite this screening, however, a small number of patients experience cardiotoxicity, either acutely during the transplant or at a later time, manifested as a cardiac arrhythmia, congestive heart failure, or cardiac ischemia due to the large volumes of fluids administered during the procedure or from the added physiologic stress.162 Complications associated with a pericardial effusion can be seen in some patients during or after transplant and are more common in patients with disease near that area and those receiving radiation therapy in that field. An idiosyncratic cardiomyopathy associated with the administration of high doses of cyclophosphamide can be demonstrated in a small number of patients. Viral cardiomyopathies also can be seen as a late transplant complication.

Engraftment Syndrome Engraftment syndrome occurs during neutrophil recovery following both autologous and allogeneic HSCT.163 It consists of a constellation of symptoms and signs that may include fever, erythrodermatous skin rash, and noncardiogenic pulmonary edema, and, in its most extreme forms, acute renal failure and diffuse alveolar hemorrhage. These clinical findings reflect the manifestations of increased capillary permeability. Making a distinction from hyper-acute GVHD in the allogeneic setting has been difficult, however. Corticosteroid therapy often is dramatically effective for engraftment syndrome, particularly for the treatment of the pulmonary manifestations.

Pulmonary Toxicities Pulmonary toxicities are common during and after transplantation. Patients who receive certain chemotherapeutic agents, such as 1,3-bis (2- chloroethyl)-1-nitrosourea (BCNU; carmustine) have an increased incidence of chemotherapy-induced lung tissue damage after transplant, which usually can be treated successfully with the prompt initiation of corticosteroid therapy.164 In addition to these complications, patients who are undergoing allogeneic transplant are at increased risk for pneumonitis caused by cytomegalovirus, fungal infections due to the patient’s increased immunosuppression, and adult respiratory distress syndrome or interstitial pneumonia of unknown etiology. Chronic GVHD also can manifest as bronchiolitis obliterans in the lung.165

Liver Toxicity The most common liver complication associated with transplantation is veno-occlusive disease (VOD [sinusoidal obstruction syndrome of the liver]).166,167 Symptoms associated with VOD include jaundice, tender hepatomegaly, ascites, and weight gain. Progressive hepatic failure and multiorgan system failure can develop in the most severe cases. Predisposing factors appear to be previous hepatic injury, use of estrogens, and high-dose intensity conditioning.167

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Table 32-2 Graft-versus-Host Disease Categories PRESENCE OF GVHD FEATURES

Time of Manifestation after HSCT or DLI

Acute

Chronic

Classic acute GVHD

≤100 d

Yes

No

Persistent, recurrent, or late-onset acute GVHD

>100 d

Yes

No

Classic chronic

No time limit

No

Yes

Overlap syndrome

No time limit

Yes

Yes

Category ACUTE GVHD

CHRONIC GVHD

DLI, donor lymphocyte infusion; GVHD, graft-versus-host disease; HSCT, hematopoietic stem cell transplantation.

Renal Toxicity Acute renal failure requiring dialysis during the transplant occurs infrequently.168 However, patients with underlying renal dysfunction are at risk for this complication. The judicious use of nephrotoxic agents can decrease its incidence. The need for dialysis typically is a short-term complication, because the patient’s underlying problem (e.g., a septic event) either improves with time or becomes lifethreatening, sometimes leading to death. An idiopathic or cyclosporineinduced hemolytic-uremic syndrome can be a serious complication after allogeneic stem cell transplantation, posing a high mortality risk or resulting in end-stage renal disease. Recently, nephrotic syndrome and membranous nephropathy have been described in long-term survivors; these complications seem to be associated more commonly with chronic GVHD and nonmyeloablative conditioning.169

Graft-versus-Host Disease In the setting of allogeneic HSCT, complications associated with acute and chronic GVHD also are of concern. Previously, acute and chronic GVHD were distinguished chronologically by whether GVHD occurred before or after the fist 100 days after transplant,

respectively. However, acute or chronic GVHD is a clinical diagnosis, based on the characteristic clinical manifestations and context, and neither is specifically defined by the “day 100” dichotomy any longer (Table 32-2). In the evaluation of both acute and chronic GVHD, which usually includes a tissue biopsy, it is important to exclude other potential diagnoses such as infection, drug reaction, or second malignancies, which can mimic GVHD. Acute GVHD is manifested by symptoms in several organ systems, including the skin, gastrointestinal tract, and liver (Table 32-3).170 This complication typically occurs within the first 100 days after transplantation. The skin manifestations range from a maculopapular rash up to generalized erythroderma or desquamation. The severity of liver GVHD is scored on the basis of the bilirubin and the gastrointestinal severity on the quantity of diarrhea per day. Patients who receive transplants from unrelated donors are at much higher risk for GVHD, the incidence and severity of which rise with the age of the patient. Other risk factors for the development of GVHD include a female donor (particularly a multiparous donor), more advanced age, and cytomegalovirus seropositivity of the donor or patient. Patients receive prophylaxis for GVHD prevention most commonly with cyclosporine, with or without methotrexate and corticosteroids.171 Treatment for acute GVHD includes high-dose corticosteroids, antithymocyte globulin, or various monoclonal antibodies.172–174 Chronic GVHD occurs most commonly between 100 days and 2 years from the transplant and has polymorphic features similar to a number of autoimmune diseases. It is most likely to develop in older patients who also had acute GVHD or received peripheral blood rather than bone marrow grafts.175 Symptoms associated with chronic GVHD include sicca syndrome, rashes or skin thickening, diarrhea, wasting syndrome, bronchiolitis obliterans, or liver function abnormalities.176–178 Patients also are at greatly increased risk for infectious complications, due to either the GVHD itself or the treatment administered. Adverse prognostic factors include thrombocytopenia, a progressive clinical presentation, extensive skin involvement, and an elevated bilirubin. Treatment for the chronic form of the disease includes corticosteroids, cyclosporine, thalidomide, ultraviolet light treatments, or other immunosuppressive agents.179,180

Infertility Many of the preparative regimens used for transplant are associated with a high incidence of permanent sterility. The use of TBI almost

Table 32-3 Classification of Patients with Acute Graft-versus-Host Disease CLINICAL STAGING Stage

Skin

Liver

Gut

+

Rash 1500 mL/day

++++

Desquamation and bullae

Total bilirubin >15 mg/dL

Pain, with or without ileus

CLINICAL GRADING STAGE Grade

Skin

Liver

Gut

PS

0 (none)

0

0

0

0

I

+ to ++

0

0

0

II

+ to +++

+

+

+

III

++ to +++

++ to +++

++ to +++

++

IV

++ to ++++

++ to ++++

++ to ++++

+++

BSA, body surface area.

Hematopoietic Stem Cell Transplantation • CHAPTER 32

always is associated with sterility. However, successful pregnancies have occurred after the use of non–TBI-containing regimens.181 This is most likely to be the case in patients who were less heavily retreated before the transplant and were under the age of 25 years at the time of transplant.

Secondary Malignancies With the increasing number of long-term survivors following HSCT, complications that develop years later are beginning to be recognized. One complication of the chemo- plus radiotherapy that is used to treat a malignancy is the development of a secondary malignancy.182,183 Several reports have now been published of the development of secondary AML or MDS after autologous transplantation. Some studies have suggested that the use of TBI may increase the risk of these complications.183 It is unclear to what degree the transplant itself contributed to the development of the AML/MDS, because all

patients received chemotherapy or radiotherapy, or both, before the transplant and, in some cases, after the transplant.

CONCLUSION There has been tremendous success since the 1980s in the increased safety of hematopoietic stem cell transplantation and in the expanding application of this treatment to more patient populations. Areas currently under development that may further improve the use and efficacy of transplantation include continuous improvements in supportive care for transplant patients, broadened use of alternative donors, more refined graft manipulations, and further improvements in the nonmyeloablative transplantation techniques and GVHD prevention. Future progress depends on our ability to identify safer and better-targeted antitumor therapies that can be incorporated in the transplantation regimens without attenuating the graft-versus-tumor responses. This remains a challenge for future clinical research.

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responses from graft-versus-host disease. Blood 1995;86:1261–1268. Hughes TP, Kaeda J, Branford S, et al: International Randomised Study of Interferon versus STI571 (IRIS) Study Group. Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med 2003; 349:1423–1432. Bhatia R, McGlave PB: Autologous hematopoietic cell transplantation for chronic myelogenous leukemia. Hematol Oncol Clin North Am 2004; 18:715–732. Tefferi A, Solberg LA, Silverstein MN: A clinical update in polycythemia vera and essential thrombocythemia. Am J Med 2000;109:141–149. Tefferi A: Myelofibrosis with myeloid metaplasia. N Engl J Med 2000;342:1255–1265. Guardiola P, Anderson JE, Bandini G, et al: Allogeneic stem cell transplantation for agnogenic myeloid metaplasia: a European Group for Blood and Marrow Transplantation, Société Francaise de Greffe de Moelle, Gruppo Italiano per il Trapianto del Midollo Osseo, and Fred Hutchinson Cancer Research Center Collaborative Study. Blood 1999;93:2831–2838. Jurado M, Deeg H, Gooley T, et al: Haemopoietic stem cell transplantation for advanced polycythaemia vera or essential thrombocythaemia. Br J Haematol 2001;112:392. Rondelli D, Barosi G, Bacigalupo A, et al: Myeloproliferative Diseases-Research Consortium. Allogeneic hematopoietic stem-cell transplantation with reduced-intensity conditioning in intermediate- or high-risk patients with myelofibrosis with myeloid metaplasia. Blood 2005;105:4115–4119. Pavletic SZ, Khouri IF, Haagenson M, et al: Unrelated donor marrow transplantation for B-cell chronic lymphocytic leukemia after using myeloablative conditioning: results from the Center for International Blood and Marrow Transplant research. J Clin Oncol 2005;23:5788– 5794. Khouri IF, Keating MJ, Vriesendorp HM, et al: Autologous and allogeneic bone marrow transplantation for chronic lymphocytic leukemia: preliminary results. J Clin Oncol 1994;12:748– 758. Khouri IF, Przepiorka D, van Besien K, et al: Allogeneic blood or marrow transplantation for chronic lymphocytic leukaemia: timing of transplantation and potential effect of fludarabine on acute graft-versus-host disease. Br J Haematol 1997;97:466–473. Dreger P, Brand R, Hansz J, et al: Treatmentrelated mortality and graft-versus-leukemia activity after allogeneic stem cell transplantation for chronic lymphocytic leukemia using intensityreduced conditioning. Leukemia 2003;17:841– 848. Schetelig J, Thiede C, Bornhauser M, et al: Evidence of a graft-versus-leukemia effect in chronic lymphocytic leukemia after reducedintensity conditioning and allogeneic stem-cell transplantation: the Cooperative German Transplant Study Group. J Clin Oncol 2003;21:2747–2753. Delgado J, Thomson K, Russell N, et al: Results of alemtuzumab-based reduced-intensity allogeneic transplantation for chronic lymphocytic leukemia: a British Society of Blood and Marrow Transplantation Study. Blood 2006;107:1724– 1730. Phillips GL, Herzig RH, Lazarus HM, et al: Treatment of resistant malignant lymphoma with cyclophosphamide, total body irradiation, and transplantation of cryopreserved autologous marrow. N Engl J Med 1984;310:1557–1561.

119. Philip T, Armitage JO, Spitzer G, et al: High-dose therapy and autologous bone marrow transplantation after failure of conventional chemotherapy in adults with intermediate-grade of high-grade nonHodgkin’s lymphoma. N Engl J Med 1987;316: 1493–1498. 120. Appelbaum FR, Sullivan KM, Buckner CD, et al: Treatment of malignant lymphoma in 100 patients with chemotherapy, total body irradiation, and marrow transplantation. J Clin Oncol 1987;5: 1340–1347. 121. Philip T, Guglielmi C, Hagenbeek A, et al: Autologous bone marrow transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive non-Hodgkin’s lymphoma. N Engl J Med 1995;333:1540–1545. 122. Kewalramani T, Zelenetz AD, Nimer SD, et al: Rituximab and ICE as second-line therapy before autologous stem cell transplantation for relapsed or primary refractory diffuse large B-cell lymphoma. Blood 2004;103:3684–3688. 123. Haioun C, Lepage E, Gisselbrecht C, et al: Survival benefit of high-dose therapy in poor risk aggressive non-Hodgkin’s lymphoma: final analysis of the prospective LNH87–2 Protocol-A Groupe d’Etude des Lymphomes de l’Adulte Study. J Clin Oncol 2000;18:3025–3030. 124. Milpied N, Deconinck E, Gaillard F, et al; Groupe Ouest-Est des Leucemies et des Autres Maladies du Sang: Initial treatment of aggressive lymphoma with high-dose chemotherapy and autologous stem-cell support. N Engl J Med 2004;350:1287–1295. 125. Freedman AS, Ritz J, Neuberg D, et al: Autologous bone marrow transplantation in 69 patients with a history of low-grade B-cell nonHodgkin’s lymphoma. Blood 1991;77:2524–2529. 126. Rohatiner AZ, Johnson PW, Price CG, et al: Myeloablative therapy with autologous bone marrow transplantation as consolidation therapy for recurrent follicular lymphoma. J Clin Oncol 1994;12:1177–1184. 127. Bierman PJ, Sweetenham JW, Loberiza FR Jr, et al: Syngeneic hematopoietic stem-cell transplantation for non-Hodgkin’s lymphoma: a comparison with allogeneic and autologous transplantation—The Lymphoma Working Committee of the International Bone Marrow Transplant Registry and the European Group for Blood and Marrow Transplantation. J Clin Oncol 2003;21:3744–3753. 128. Grigg A, Ritchie D: Graft-versus-lymphoma effects: clinical review, policy proposals, and immunobiology. Biol Blood Marrow Transplant 2004;10:579–590. 129. Ratanatharathorn V, Uberti J, Karanes C, et al: Prospective comparative trial of autologous versus allogeneic bone marrow transplantation in patients with non-Hodgkin’s lymphoma. Blood 1994;84: 1050–1055. 130. Robinson SP, Goldstone AH, Mackinnon S, et al: Chemoresistant or aggressive lymphoma predicts for a poor outcome following reduced-intensity allogeneic progenitor cell transplantation: an analysis from the Lymphoma Working Party of the European Group for Blood and Bone Marrow Transplantation. Blood 2002;100:4310–4316. 131. Carella AM, Congiu AM, Gaozza E, et al: Highdose chemotherapy with autologous bone marrow transplantation in 50 advanced resistant Hodgkin’s disease patients: an Italian study group report. J Clin Oncol 1988;6:1411–1416. 132. Armitage JO, Bierman PJ, Vose JM, et al: Autologous bone marrow transplantation for patients with relapsed Hodgkin’s disease. Am J Med 1991;91:605–611. 133. Linch DC, Winfield D, Goldstone AH, et al: Dose intensification with autologous bone-marrow transplantation in relapsed and resistant Hodgkin’s disease: results of a BNLI randomized trial. Lancet 1993;341:1051–1054.

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Part I: Science of Clinical Oncology 134. Moskowitz CH, Nimer SD, Zelenetz AD, et al: A 2-step comprehensive high-dose chemoradiotherapy second-line program for relapsed and refractory Hodgkin disease: analysis by intent to treat and development of a prognostic model. Blood 2001;97:616–623. 135. Peggs KS, Hunter A, Chopra R, et al: Clinical evidence of a graft-versus-Hodgkin’s-lymphoma effect after reduced-intensity allogeneic transplantation. Lancet 2005;365:1934–1941. 136. Burdach S, Jurgens H, Peters C, et al: Myeloablative radiochemotherapy and hematopoietic stemcell rescue in poor-prognosis Ewing’s sarcoma. J Clin Oncol 1993;11:1482–1488. 137. Ladenstein R, Lasset C, Pinkerton R, et al: Impact of megatherapy in children with high-risk Ewing’s tumours in complete remission: a report from the EBMT Solid Tumour Registry. Bone Marrow Transplant 1995;15:697–705. 138. Craft A, Cotterill S, Malcolm A, et al: Ifosfamidecontaining chemotherapy in Ewing’s sarcoma: The Second United Kingdom Children’s Cancer Study Group and the Medical Research Council Ewing’s Tumor Study. J Clin Oncol 1998;16:3628–3633. 139. Arpaci F, Ataergin S, Ozet A, et al: The feasibility of neoadjuvant high-dose chemotherapy and autologous peripheral blood stem cell transplantation in patients with nonmetastatic high grade localized osteosarcoma: results of a phase II study. Cancer 2005;104:1058–1065. 140. Matthay KK, Villablanca JG, Seeger RC, et al: Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children’s Cancer Group. N Engl J Med 1999;341:1165–1173. 141. Sung KW, Yoo KH, Chung EH, et al: Double high-dose chemotherapy with autologous stem cell transplantation in patients with high-risk neuroblastoma: a pilot study in a single center. J Korean Med Sci 2002;17:537–543. 142. Luksch R, Podda M, Gandola L, et al: Stage 4 neuroblastoma: sequential hemi-body irradiation or high-dose chemotherapy plus autologous haemopoietic stem cell transplantation to consolidate primary treatment. Br J Cancer 2005;92:1984–1988. 143. Matthay KK, Tan JC, Villablanca JG, et al: Phase I dose escalation of iodine-131metaiodobenzylguanidine with myeloablative chemotherapy and autologous stem-cell transplantation in refractory neuroblastoma: a New Approaches to Neuroblastoma Therapy Consortium Study. J Clin Oncol 2006;24:500–506. 144. Broun ER, Nichols CR, Kneebone P, et al: Longterm outcome of patients with relapsed and refractory germ cell tumors treated with high-dose chemotherapy and autologous bone marrow rescue. Ann Intern Med 1992;117:124–128. 145. Bhatia S, Abonour R, Porcu P, et al: High-dose chemotherapy as initial salvage chemotherapy in patients with relapsed testicular cancer. J Clin Oncol 2000;18:3346–3351. 146. Schmoll HJ, Kollmannsberger C, Metzner B, et al: Long-term results of first-line sequential high-dose etoposide, ifosfamide, and cisplatin chemotherapy plus autologous stem cell support for patients with advanced metastatic germ cell cancer: an extended phase I/II study of the German Testicular Cancer Study Group. J Clin Oncol 2003;21:4083–4091. 147. Antman K, Ayash L, Elias A, et al: A phase II study of high-dose cyclophosphamide, thiotepa, and carboplatin with autologous marrow support in women with measurable advanced breast cancer responding to standard-dose therapy. J Clin Oncol 1992;10:102–110. 148. Antman KA, Rowlings PA, Vaughn WP, et al: Highdose chemotherapy with autologous hematopoietic stem-cell support for breast cancer in North America. J Clin Oncol 1997;15:1870–1879.

149. Rodenhuis S, Richel DJ, van der Wall E, et al: Randomised trial of high-dose chemotherapy and haemopoietic progenitor-cell support in operable breast cancer with extensive axillary lymph-node involvement. Lancet 1998;352:515–521. 150. Hortobagyi GN, Buzdar AU, Theriault RL, et al: Randomized trial of high-dose chemotherapy and blood cell autografts for high-risk primary breast carcinoma. J Natl Cancer Inst 2000;92:225–233. 151. Rodenhuis S, Bontenbal M, Beex LV, et al: Highdose chemotherapy with hematopoietic stem-cell rescue for high-risk breast cancer. N Engl J Med 2003;349:7–16. 152. Tallman MS, Gray R, Robert NJ, et al: Conventional adjuvant chemotherapy with or without high-dose chemotherapy and autologous stem-cell transplantation in high-risk breast cancer. N Engl J Med 2003;349:17–26. 153. Zander AR, Kroger N, Schmoor C, et al: Highdose chemotherapy with autologous hematopoietic stem-cell support compared with standard-dose chemotherapy in breast cancer patients with 10 or more positive lymph nodes: first results of a randomized trial. J Clin Oncol 2004;22:2273– 2283. 154. Stadtmauer EA, O’Neill A, Goldstein LJ, et al: Conventional-dose chemotherapy compared with high-dose chemotherapy plus autologous hematopoietic stem-cell transplantation for metastatic breast cancer. Philadelphia Bone Marrow Transplant Group. N Engl J Med 2000;342:1069–1076. 155. Childs R, Chernoff A, Contentin N, et al: Regression of metastatic renal-cell carcinoma after nonmyeloablative allogeneic peripheral-blood stemcell transplantation. N Engl J Med 2000;343:750– 758. 156. Bishop MR, Fowler DH, Marchigiani D, et al: Allogeneic lymphocytes induce tumor regression of advanced metastatic breast cancer. J Clin Oncol 2004;22:3886–3892. 157. Carella AM, Beltrami G, Corsetti MT, et al: Reduced intensity conditioning for allograft after cytoreductive autograft in metastatic breast cancer. Lancet 2005;366:318–320. 158. Sorror ML, Maris MB, Storb R, et al: Hematopoietic cell transplantation (HCT)-specific comorbidity index: a new tool for risk assessment before allogeneic HCT. Blood 2005;106:2912– 2919. 159. Antin JH: Clinical practice. Long-term care after hematopoietic-cell transplantation in adults. N Engl J Med 2002;347:36–42. 160. Champlin RE, Horowitz MM, van Bekkum DW, et al: Graft failure following bone marrow transplantation for severe aplastic anemia: risk factors and treatment results. Blood 1989;73:606– 613. 161. Cazin B, Gorin NC, Laporte JP, et al: Cardiac complications after bone marrow transplantation. A report on a series of 63 consecutive transplantations. Cancer 1986;57:2061–2069. 162. Hertenstein B, Stefanic M, Schmeiser T, et al: Cardiac toxicity of bone marrow transplantation: predictive value of cardiologic evaluation before transplant. J Clin Oncol 1994;12:998–1004. 163. Spitzer TR: Engraftment syndrome following hematopoietic stem cell transplantation. Bone Marrow Transplant 2001;27:893–898. 164. Chao NJ, Duncan SR, Long GD, et al: Corticosteroid therapy for diffuse alveolar hemorrhage in autologous bone marrow transplant recipients. Ann Intern Med 1991;114:145–146. 165. Dudek AZ, Mahaseth H, DeFor TE, Weisdorf DJ: Bronchiolitis obliterans in chronic graft-versus-host disease: analysis of risk factors and treatment outcomes. Biol Blood Marrow Transplant 2003;9: 657–666. 166. Bearman SI, Anderson GL, Mori M, et al: Venoocclusive disease of the liver: development of

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174. 175. 176.

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179. 180.

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a model for predicting fatal outcome after marrow transplantation. J Clin Oncol 1993;11:1729–1736. DeLeve LD, Shulman HM, McDonald GB: Toxic injury to hepatic sinusoids: sinusoidal obstruction syndrome (veno-occlusive disease). Semin Liver Dis 2002;22:27–42. Gruss E, Bernis C, Tomas JF, et al: Acute renal failure in patients following bone marrow transplantation: prevalence, risk factors and outcome. Am J Nephrol 1995;15:473–479. Srinivasan R, Balow JE, Sabnis S, et al: Nephrotic syndrome: an under-recognised immune-mediated complication of non-myeloablative allogeneic haematopoietic cell transplantation. Br J Haematol 2005;131:74–79. Przepiorka D, Weisdorf D, Martin P, et al: 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant 1995;15:825–828. Storb R, Deeg HJ, Pepe M, et al: Methotrexate and cyclosporine versus cyclosporine alone for prophylaxis of graft-versus-host disease in patients given HLA-identical marrow grafts for leukemia: long-term follow-up of a controlled trial. Blood 1989;73:1729–1734. Martin PJ, Schoch G, Fisher L, et al: A retrospective analysis of therapy for acute graft-versus-host disease: initial treatment. Blood 1990;76:1464–1472. Kennedy MS, Deeg HJ, Storb R, et al: Treatment of acute graft-versus-host disease after allogeneic marrow transplantation: randomized study comparing corticosteroids and cyclosporine. Am J Med 1985;78:978–983. Jacobsohn DA, Vogelsang GB: Novel pharmacotherapeutic approaches to prevention and treatment of GVHD. Drugs 2002;62:879–889. Lee SJ, Vogelsang G, Flowers ME: Chronic graftversus-host disease. Biol Blood Marrow Transplant 2003;9:215–233. Shulman HM, Sullivan KM, Weiden PL, et al: Chronic graft-versus-host syndrome in man: a long-term clinicopathologic study of 20 Seattle patients. Am J Med 1980;69:204–217. Pavletic SZ, Carter SL, Kernan NA, et al: Influence of T-cell depletion on chronic graftversus-host disease: results of a multicenter randomized trial in unrelated marrow donor transplantation. Blood 2005;106:3308–3313. Filipovich AH, Weisdorf D, Pavletic S, et al: National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and staging working group report. Biol Blood Marrow Transplant 2005;11:945–956. Vogelsang GB: How I treat chronic graft-versushost disease. Blood 2001;97:1196–1201. Couriel D, Carpenter PA, Cutler C, et al: Ancillary therapy and supportive care of chronic graft-versushost disease: national institutes of health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: V. Ancillary Therapy and Supportive Care Working Group Report. Biol Blood Marrow Transplant 2006;12:375–396. Salooja N, Szydlo RM, Socie G, et al: Pregnancy outcomes after peripheral blood or bone marrow transplantation: a retrospective survey. Lancet 2001;358:271–276. Curtis RE, Metayer C, Rizzo JD, et al: Impact of chronic GVHD therapy on the development of squamous-cell cancers after hematopoietic stem-cell transplantation: an international case-control study. Blood 2005;105:3802–3811. Darrington DL, Vose JM, Anderson JR, et al: Incidence and characterization of secondary myelodysplastic syndrome and acute myelogenous leukemia following high-dose chemoradiotherapy and autologous stem-cell transplantation for lymphoid malignancies. J Clin Oncol 1994;12: 2527–2534.

33

Gene Therapy in Oncology James E. Talmadge and Kenneth H. Cowan

S U M M ARY

O F

K EY

P OI NT S

Recent Major Improvements in Gene Therapy

Current Concerns Regarding Gene Therapy

• • • •

• Gene therapy has been implicated in the death of at least one patient, resulting in the temporary suspension of clinical trials (2000) with adenovirus (Adv) vectors in the United States. The first gene therapeutic to be approved (China, 2003) was an Adv vector with a p53 transgene used in combination with chemotherapy for the treatment of head and neck squamous cell carcinoma (HNSCC). A second, conditionally replicative Adv vector, H101 (ONYX-015), was approved in China in December of 2005. • Leukemic transformation by insertional mutagenesis occurred in several

Vector development Vector targeting Regulated transgene expression Clinical development strategies

Ideal Vector Attributes • Can be targeted either physically or via promoter expression, and is nontoxic, noninflammatory, and nonimmunogenic • Should have the potential to incorporate a large transgene and result in high levels of both transduction and transgene expression • Duration of transgene expression and/ or genomic integration ought to be regulatable

INTRODUCTION The development of nucleic acid technologies has provided insight into the molecular basis of neoplasia. The resultant detection of phenotypic and genotypic alterations in neoplastic diseases has increased optimism that molecular intervention might lead to improved clinical care for patients with cancer. One such approach is gene therapy, which is the introduction of a nucleic acid sequence into a target cell. The objectives are the delivery of a transgene to an adequate number of cells and at an effective level of expression sufficient to result in therapeutic outcomes. Both criteria require the use of a vector and potentially, a formulation such that these objectives can be met. Although this approach is both simple and attractive, thus far gene therapy has promised much and delivered little because of the technical hurdles. Several preclinical studies and clinical trials have been undertaken to improve gene transfer systems. In 2003 the first gene therapeutic was approved in China. This was an adenovirus (Adv) serotype 5 vector engineered to express TP53 (Gendicine) for treatment of patients with head and neck squamous cell carcinoma (HNSCC).1 A second gene therapy product, H101 (ONYX-015), an Adv vector modified to replicate in and kill cancer cells with TP53 mutations, was approved in December 2005.2 Regardless of these approvals, the primary challenges in gene therapy remain improvements in the targeting of our existing vectors and increasing gene transduction efficiency. Overcoming these obstacles will facilitate the development of targetable vectors and, given the systemic nature of

patients treated with retroviral vectors. However, because retroviral vectors have been successful in the treatment of patients with severe combined immune deficiency (SCID-X1) for whom no other therapy is available, their use continues to be approved on a case by case basis.

Future Directions of Gene Therapy • The choice of disease, clinical implementation, and vector are critically important to the future development of successful gene therapy. • Because of deficiencies in gene delivery and targeting, as well as expression levels, it is critical to pair protocols with specific vector attributes.

most malignancies, help in the development of vectors that can be administered intravenously (IV). This chapter focuses on strategies to improve efficacy, as well as ongoing gene therapeutic strategies. We also will examine and discuss recent advances and indicate areas that require further development for clinical gene therapy to become a widely used treatment modality.

VECTORS Viral Gene Transfer Vectors Viral gene delivery has developed from a virus’ innate ability to infect cells, which offers many intrinsic advantages:3,4 • Specific cell-binding and cell-entry properties • Efficient targeting of the transgene to the nucleus of the cell • Ability to avoid intracellular degradation Most viral vectors are based on the principle that an intact wildtype (wt) virus can be modified for safe and effective gene transfer. In general, the more severely attenuated the viral vector is from the wt, the safer the virus is for use in gene therapy protocols, yet the poorer the yield obtained after propagation. Typically, two (or preferably, three) noncontiguous partial or complete gene sequences are deleted to reduce the potential for homologous recombination. Specific genes critical to viral replication are then modified or deleted, resulting in a recombinant viral vector that is “replication defective.”

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The transgene to be delivered by the virus is then inserted into the viral genome at the site created by the removal of the viral replication genes. The transgene must be a smaller size to fit within the available space, which is a critical characteristic because the transgene cannot be packaged into an infectious particle if the new viral genome is too large. Many of the viruses that are used as vectors lack genes for replication in normal cells; therefore, the recombinant virus and its transgene must be grown in a packaging cell line that provides the complementary genes required for viral replication. The recombinant viral particles are purified as live infectious viruses and are replication incompetent in the absence of the packaging cell line. Alternatively, the packaging cell line can be used to infect (transduce) cells or tissues in vitro.

Retroviridae—Retrovirus The Retroviridae is a large family of ribonucleic acid (RNA) viruses including Moloney-murine-lentivirus-related viruses (e.g., Moloney murine leukemia virus [MMLV]) and lentiviruses (e.g., human immunodeficiency viruses 1 and 2 [HIV-1 and HIV-2]).5 Their genomes consist of two identical positive-sense, single-stranded RNA molecules (∼3.5 kb), and are encased in a capsid along with integrase and reverse transcriptase enzymes. Initially, retroviral vectors were the most widely used viral vectors, a distinction that has been replaced by Adv vectors in recent years. Retroviruses can transduce only those cells that are actively undergoing mitosis, limiting their utility with certain cell populations, especially hematopoietic stem cells. Retroviral vectors provide good gene expression and are technically easy to produce, although the titers obtained are suboptimal. In addition, the production of retroviral vectors requires careful monitoring because of the potential for helper virus contamination.

RECOMBINANT MOLONEY MURINE LEUKEMIA VIRUS. Most of the retroviral vectors that are used for gene therapy are based on MMLV. Vector replication is prevented by the deletion of the gag, pol, and env gene regions. The gag region encodes the capsid proteins, the pol region encodes reverse transcriptase and integrase, and the env region encodes proteins required for receptor recognition and envelope anchoring (Fig. 33-1). The genome includes long terminal repeats at either end that play a vital role in initiating

gag Pr55gag • Poly-protein (p55) processed by Pro • MA Matrix (p17) • CA Capsid (p24) • NC Nucleocapsid (p7) • p6

5'

vif Viral infectivity factor (p23) Overcomes inhibition by host factors and results in a more stable RT complex.

deoxynucleic acid (DNA) synthesis and regulating transcription of the viral genes. The gag, pol, and env gene products are supplied by a complementary packaging cell line. When a retroviral vector plasmid is introduced into a packaging cell line, viral RNA is produced, packaged into virions, and secreted into the medium. Each resultant viral particle is able to integrate itself into the genome of the host cell, but is unable to produce additional viral particles because it lacks the gag, pol, and env genes. The transduced DNA sequences are stably integrated into the chromosomal DNA of the target cells and in this way are transferred to cellular progeny of transduced cells. Highlights of results obtained to date with retroviral vectors include the therapeutic studies in children with severe combined immune deficiency (SCID-X1), which will be discussed later in this review, as well as the gene-marking studies of Malcolm Brenner and others.6,7 In the latter studies, it was shown that tumor cells within autologous stem cell transplant products could be responsible for tumor relapse, at least in leukemia patients.

RECOMBINANT LENTIVIRUS. The most recently discovered members of the retrovirus family are the human and simian immunodeficiency viruses (HIVs and SIVs, respectively), which belong to a subclass of retroviruses known as lentiviruses.8,9 The development of HIV gene therapy vectors has several potential advantages based on the following characteristics: • Transduce actively dividing and nondividing cells. • Long-term, stable transgene expression occurs due to genetic integration. • Inherent tropism for CD4 T cells, macrophages, and hematopoietic stem cells. Genetic modifications, such as the introduction of vesicular stomatitis virus G protein into the lentiviral envelope, can widen the tropism of this vector. Until it has been demonstrated that HIVbased vectors are safe, however, the use of these vectors for therapies targeting diseases other than HIV could be difficult to initiate clinically.10 The in vitro efficiency of lentiviral vectors is at an acceptable level; however, in vivo expression has not been demonstrated at an acceptable level for clinical utility. In addition, there is a need to find protocols and/or procedures that can elevate the expression levels of the HIV virus in nondividing cells.11

vpu Viral protein U env Envelope protein (gp160) Promotes CD4 degradation • Cleaved in ER to gp120 and gp41 and facilitates virion release. • gp120 mediates CD4 and chemokine binding • Contains RNA response element (RRE) that binds to Rev

nef Negative effector (p24) • Promotes downregulation of membrane CD4 and class I expression • Blocks apoptosis • Enhances infectivity • Alters cellular activation

U3 R U5

LTR Long terminal repeat • With regions that bind host transcription factors • Required for initiation of transcription • With RNA trans-acting response element (TAR) that binds tat.

U3 R U5

pol Polymerase Encodes numerous viral proteins: • Pro protease (p10) • RT reverse transcriptase • RNase H (p66/51) • IN integrase H (p32)

vpr Viral protein R (p15) • Promotes G2 cell cycle arrest • Aids HIV infection of Macs

rev Regulator of viral gene expression (gp19) • Binds RRE • Inhibits viral RNA splicing and promotes nuclear export of incompletely spliced viral RNAs

3'

tat Transcriptional activator (p14) • Binds TAR • Can enhance RNA Pol II elongation

Genes that can be deleted and replaced with a transgene

Figure 33-1 • Retrovirus proviral genome and gene product functions. Overview of the 9-kb genome of the HIV provirus and a brief summary of the functions for the 9 genes encoding 15 proteins.

Gene Therapy in Oncology • CHAPTER 33

The first clinical study using a lentivirus vector was undertaken in HIV-infected patients.12 This study investigated the safety of infusing autologous T cells modified with an HIV type 1 (HIV-1)–based lentiviral vector expressing an antisense gene against the HIV envelope. Five patients with HIV infections that were resistant to antiviral therapy and had viral loads of more than 5000 copies/mL and CD4+ T cells counts between 200 and 500 cells/mm3 were treated. The primary endpoints included adverse events, viral load, CD4+ counts, and the emergence of replication-competent lentivirus derived from the vector. In this phase I study, one subject was reported to have a sustained decrease in viral load. The CD4 counts remained steady or increased in the other four subjects, and sustained gene expression was observed. These preliminary studies support the safety of lentivirus vectors.

Recombinant Adenovirus Recombinant Adv is a nonenveloped, icosahedral, double-stranded DNA virus with a capsid containing 252 capsomeres (240 hexons and 12 pentons).13 The large genome of Adv (36 kilobases, kb) allows large genes to be inserted into an Adv-based vector. Transgenes in Adv vectors are not incorporated into the genome of transduced cells but instead remain as an extrachromosomal entity in the nucleus. First isolated from U.S. army recruits who had acute respiratory symptoms, Adv vectors have been found to be common human pathogens. To date, 49 serotypes have been characterized and associated with a variety of symptoms, ranging from a mild cold to acute febrile pharyngitis.14 Replication-defective recombinant Adv vectors are currently the most commonly used viral vectors in clinical trials. Ad2 and Ad5 are used primarily for gene therapy applications. Recently, however, the Ad11 and Ad35 serotypes were shown to exhibit a unique tropism that includes hematopoietic stem cells, a finding that potentially widens their utility.15–17 The Adv vector’s genome (Fig. 33-2) can be divided into two main regions: early (E) and late (L) according to the time at which their genes are expressed during virus replication. There are four regions of early genes that are termed E1, E2, E3, and E4, and one region of late genes comprising the five coding units termed L1, L2, L3, L4, and L5. The E1 region is essential for viral replication; therefore, recombinant Advs without the E1 region are considered replication defective. In a replication-defective Adv vector, the E1 region can be replaced with a transgene for expression. Moreover, removal of genetic material from the vector, such as the E3 and/or the E4 region(s), allows for larger genes to be inserted and reduces the viral immunogenicity.18 Viruses without the E3 and E4 regions are referred to as “gutless” and have decreased antigenicity.19 The E1 region of Adv vectors is subdivided into E1A and E1B. The E1A gene product is a viral transcription unit that activates the expression of other Adv transcription units by binding to viral pro-

moters. The E1B region codes for a 55-kd protein that interacts with the cellular p53 tumor suppressor protein and regulates the host cells’ cycle progression supporting viral replication. E1B also binds to viral E4 proteins and to p53, which together act to depress host protein synthesis. The E2 region codes for viral DNA polymerase and the Adv single-stranded DNA-binding protein. The E3 region is not required for in vitro replication; however, it does offer the virus some protection against host defense mechanisms. The E4 region codes for proteins involved in • Regulation of viral and cellular protein expression • Replication of viral DNA • Switching off of host protein synthesis The late genes (L1–L5) are expressed at the onset of viral DNA replication and encode structural polypeptides that are needed for virion assembly. This understanding of viral replication has allowed the development of extremely elaborate, conditionally replicative Adv vectors capable of replication only in cancer cells.20 The transduction efficiency of Adv vectors is high as compared with that of most other viral vectors. Because of the structural stability of the capsid polypeptides of Adv, viral particles can be purified and concentrated to a very high titer of ∼1 × 1012 plaque-forming units (pfu)/mL. This is in contrast to retroviral titers that achieve much lower titers (∼1 × 107 pfu/mL) because of their envelopes’ instability. Another distinguishing characteristic of Adv vectors is their lack of integration into the human genome. The Adv genome remains in the nucleus of the target cells as a nonreplicating extrachromosomal entity, thereby avoiding any potential for mutagenic effects caused by random integration into the host. However, Adv vectors have potential shortcomings, including • Transient expression because the viral DNA does not integrate into the host • Viral protein expression by the Adv vector after administration into a host • A common pathogen and in vivo delivery could be hampered by the prior induction of host immunity.3 The period of Adv transgene expression is relatively short; therefore, this is a suboptimal vector if expression is desired for longer than 10 to 14 days. This short expression time is due primarily to the induction of a cytotoxic T lymphocyte (CTL) response to viral polypeptides, as well as potentially to the transgene itself, especially if it is not expressed normally. Because the Adv genome does not integrate into the target cell, only one of the daughter cells (if the target cells are dividing) will contain the transgene. Manipulation of the immune response can result in longer expression; however, Adv gene delivery is ideally suited to those situations that require only a single period of transgene expression in which transient expression is desired, for

Leader 5' ITR

E1A

E1B

Seq

L1/VA

L2

L3

L4

E3 L5

E2B

E4

ITR

5'

E2A/E2B Hatched genes are deleted in vectors. E1a and E1b were deleted in the initial vectors, E3 and/or E4 are also deleted in the second-generation gutless vectors.

Figure 33-2 • Adenovirus genome. The Adv genome is composed of early and late genes. The E1A gene encodes the initial viral transcription unit and must be deleted to prevent the recombinant virus from replicating. In most of the original Adv vectors, E1A and E1B are deleted. The second-generation vectors (known as gutless vectors) typically also have the E3 and/or E4 genes deleted. This deletion allows larger transgenes to be inserted into the Adv vector, and the deletion of E4 significantly reduces vector immunogenicity with the potential for a more prolonged transgene expression.

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ADENOVIRAL VECTORS

Adenoviral (Adv) vectors have a number of positive and negative attributes. The positive attributes include the transduction of a wide profile of cellular phenotypes such as epithelial, carcinoma, and hematopoietic cells. Further, the use of Adv vectors results in a high frequency of transduction and high levels of transgene expression. A negative attribute of Adv vectors is transient expression, although for appropriate targets such transitory infection is a positive attribute. The transient expression is due, in part, to the high level of innate vector immunogenicity, which can limit multiple cycles of transduction and chronic transgene expression. The resulting Adv profile of activity is ideal for the transduction of dendritic cells (DCs) as vaccines, the purging of tumor cells from stem cell products, and intralesional injection of carcinomas. Further, the ability to develop Adv vectors that are conditionally replicative has great potential for the treatment of neoplastic disease. It is noted that two Adv vectors have received regulatory approval in China. These controversial studies have resulted in the treatment of several thousand patients, supporting the safety of these vectors.

example, growth factor therapy. A second major disadvantage of Adv vectors used in vivo is the immune response (CTL and antibody [Ab]), both endogenous and induced, which can preclude infection and cause the destruction of transduced cells, resulting in local tissue damage and inflammation. This shortcoming was demonstrated in the initial studies with intrabronchial delivery of Adv for the treatment of cystic fibrosis.21 Host cells presenting peptides from Advencoded transgene products target the host cell for CTL-mediated destruction. A third major disadvantage of Adv vectors is that most humans are primed against at least one serotype, because Adv is a naturally occurring virus. Using the same serotype in a gene therapy context will probably result in a rapid and vigorous immune response such that high levels of Adv-specific Abs occur in the sera within days of Adv vector administration. Another similar problem is the potential secondary immune response induced by the readministration of a vector. It must be stressed that transgene expression can occur during a boost, although a shortened duration is observed. The augmentation of a CTL response by an Adv vector suggests the utility of Adv vectors as vaccine adjuvants (Box 33-1).

Recombinant Adeno-associated Virus Adeno-associated virus (AAV) vectors offer many of the same advantages as Adv vectors, including a wide host-cell range and a relatively high transduction efficiency.22,23 AAV vectors stably integrate at specific sites in the host genome, resulting in a longer lasting transgene expression. In addition, these stable vectors can infect a variety of dividing and nondividing cells without inducing an immune response. AAV vectors cause little damage to target cells—unlike Adv vectors that can cause a high degree of cytopathogenicity. There is evidence, however, to suggest that AAV vectors are significantly less efficient than retroviral vectors at transducing primary cells, because most of their DNA remains extrachromosomal and does not integrate into the host genome. Furthermore, they cannot incorporate genes larger than 5 kb and must be screened closely for Adv contamination.

Recombinant Herpes Simplex Virus Herpes simplex virus (HSV) vectors are developed primarily for protocols that target neuronal tissue.24 Similar to Adv vectors, HSV vectors are maintained as an extrachromosomal DNA element in the nucleus of host cells, but can establish long-lived asymptomatic infections in the sensory neurons of the peripheral and central nervous tissue.25 HSV vectors also have a wide host range and are similar to Adv vectors in that they allow large gene inserts of up to 20 kb. These

vectors are infective even with multiple deletions of immediate-early (IE) genes that are essential for replication, resulting in less cytotoxic vectors, thereby reducing safety concerns.26 HSV vectors can be produced at high titers and express transgenes for a long period of time in the central nervous system.27 The major concern associated with HSV is the potential for wt virus to replicate lytically in the human brain, resulting in encephalitis. Other significant disadvantages with HSV vectors include • Requirement for additional engineering to increase efficiency26 • Transient expression associated with lytic infection and viral protein expression • Relatively low transduction efficiency

Recombinant Pox Vectors The origin of vaccinia virus (VV), the virus used for vaccination against smallpox, is not known, but it was probably derived from cowpox virus, variola virus, or a hybrid of the two.28,29 Percutaneous VV vaccine administration results in protective cellular and humoral immune responses in greater than 95% of primary vaccinees. Recombinant VV vectors are highly attenuated, host-restricted, and non- or poorly replicating poxvirus strains (including the modified vaccinia Ankra [MVA] and canarypox or avipox vector [Alvac]) and thus do not create productive infections.30,31 MVA is avirulent in normal and immunosuppressed animals, and safe in humans.32 Recent studies using transgenic mice provided a comparison of VV immunogenicity, including MVA and Western Reserve (WR). These studies demonstrated that MVA vaccines elicited CD8+ T-cell responses that are comparable to those induced by the replication-competent WR strain. Furthermore, MVA vaccination was shown to be protective against a lethal respiratory challenge with the virulent WR strain.33 The most frequent adverse complication of VV vaccination is inadvertent inoculation (usually autoinoculation) at other sites. Serious complications, which are more common among primary vaccinees and infants than among revaccinees and adults, include the following: • Generalized vaccinia in otherwise healthy individuals, which is generally self-limiting • Eczema vaccinatum, which consists of disseminated cutaneous lesions in highly susceptible patients with eczema or other chronic skin diseases, which can be severe or even fatal • Progressive vaccinia (vaccinia necrosum), which is a severe, potentially fatal illness seen in patients with immunodeficiency, whether congenital, acquired (e.g., via leukemia or lymphoma), iatrogenic (e.g., via chemotherapy or glucocorticoid treatment), or HIV induced • Postinfectious encephalitis, which is rare (three cases per million primary vaccinees), but can be fatal in 15% to 25% of cases and can leave 25% of patients with permanent neurologic sequelae Similar to Adv vectors, VV vectors are used for immune manipulation and as a vector for vaccines.34 VV vectors have been used worldwide to eradicate smallpox and as discussed earlier, provide a relatively safe live vaccine. Vaccinia vectors do not integrate into the genome of the host cell; however, they can accommodate large transgenes and are extremely immunogenic. VV vectors are used to immunize patients against tumor antigens (Ags) by cloning Ags and/or genes encoding proteins with adjuvant activity (e.g., cytokine or costimulating factor genes) into the viral genome. Most transgenes are expressed at high levels in vivo, eliciting an Ag-specific response. Vector-induced immunity, however, can limit the ability of the vaccinia transgenes to boost an immune response, which is an observation similar to that seen with Adv vectors. The current emphasis is on VV infection of dendritic cells (DCs) using a vector with an antigenic transgene.34,35 In association with the immunogenicity of VV vectors and their ability to deliver an antigenic transgene, they have been used clinically

Gene Therapy in Oncology • CHAPTER 33

as a melanoma vaccine. In clinical studies by Wallack and colleagues,36 a phase III trial of a vaccinia melanoma oncolysate, delivered as an active specific immunotherapy, was found to increase the disease-free or overall survival of patients with stage III melanoma in a surgical adjuvant setting. Other studies have used VV mutants that are conditionally replicative and can lyse cancer cells after viral replication. These vectors have been used in a strategy whereby insertional inactivation of the VV thymidine kinase (tk) gene was used to limit viral replication in cells with large intracellular nucleotide pools, such as tumor cells. In a similar approach, Mastrangelo and coworkers37 inserted the gene for granulocyte-macrophage colony-stimulating factor into the VV tk gene locus as a strategy to generate an oncolytic virus that induced antitumor immunity after infection of malignant melanoma. This vector is currently in a clinical trial of intralesional administration to patients with refractory recurrent melanoma. In the first seven patients studied, two patients had a complete response and three other patients had partial responses. Other oncolytic VV vectors have been engineered with complementary DNAs for cytokines such as interleukin 2 or with prodrug-activating enzymes such as cytosine deaminase to augment antineoplastic efficacy.38,39 The role of VV vectors as vaccines has focused predominantly on carcinoembryonic Ag (CEA) as the vaccine Ag. CEA is a glycoprotein self-Ag found on breast, lung, gastric, colon, and ovarian tumors. One such vector is a recombinant VV containing the CEA gene (rV-CEA).40,41 In a phase I clinical trial, the safety of rV-CEA was demonstrated; however, no significant antineoplastic effects were observed.42 Possible reasons for the lack of clinical efficacy in these trials include • Prior exposure to the VV, leading to the development of antivaccinia immune responses after repeated vaccinations • Advanced state of the patients’ tumors • Potentially compromised immune status of the patients Another phase I rV-CEA vaccine study demonstrated that CEAspecific T-cell responses could be generated in humans after vaccination.42 A second recombinant anti-CEA vaccine, Alvac-CEA, has been developed.43,44 Similar to rV-CEA, Alvac-CEA contains the CEA gene; however, unlike rV-CEA, it cannot replicate in mammalian cells. The safety of Alvac-CEA has been documented in phase I trials in patients with advanced carcinomas.45 A moderate but statistically significant increase in the number of CEA-specific CTL precursors was observed in seven of nine HLA-A2+ patients treated with Alvac-CEA, although objective anticancer effects were not observed. Preclinical studies have suggested that the combination of rV-CEA and Alvac-CEA in a prime and boost protocol can induce a more vigorous T-cell response than either vaccine alone.44 In a clinical prime-and-boost study, 18 patients with advanced tumors expressing CEA were randomized to receive either rV-CEA followed by three Alvac-CEA vaccinations, or Alvac-CEA (three times) followed by one rV-CEA vaccination. In this study, vaccination with rV-CEA followed by Alvac-CEA resulted in an increased frequency of Ag-specific interferon γ cells by enzyme-linked immunospot assay (ELISPOT) relative to the reverse order of vaccination.46 Another method to enhance the responses to a vaccine is to incorporate a costimulatory signal. In the absence of a costimulatory signal, presentation of an Ag to T cells can result in anergy.47 B7.1, which binds to CD28 on T cells, is one such costimulatory signal that results in the production of interleukin 2 and interferon γ by T cells. In a vaccine study using VV vectors, 39 patients were treated with AlvacCEA B7.1.48 In one study using the Alvac-CEA-B7.1 vaccine, patients with metastatic CEA-expressing adenocarcinomas received vaccine intradermally every 2 weeks for a total of four injections. In this phase I trial, 27% of the patients had disease stabilization after four vaccinations. Six of 31 patients with elevated serum CEA levels had a temporary decline in CEA. In addition, HLA-A2+ patients demonstrated increased CEA-specific T-cell frequencies after three vaccinations. Based on these studies and additional phase II data, a phase III

Box 33-2.

VACCINIA VIRAL VECTORS

Vaccinia viral (VV) vectors have a profile of activity analogous to that of adenoviral (Adv) vectors. That is, they can easily transduce a wide range of cells, resulting in transient expression, and have as a negative attribute a brief transgene expression due to the innate antigenicity of the vector. In contrast to Adv vectors, VV vectors almost inevitably lyse the transduced cell, rendering it potentially less attractive as a vector (especially for dendritic cell [DC] transduction) due to the shorter halflife of the transduced cell. Significant experience with the administration of both VV and Adv vectors as vaccines has provided a strong safety profile for both. In theory, the concomitant use of VV and Adv vectors makes possible cycles of vaccine delivery via transduced DCs, allowing a prime and boost immunization with DCs, which have a high frequency of transduction and levels of transgene expression, while reducing the concerns associated with the innate antigenicity of the viral vectors.

trial was initiated in 255 patients with advanced pancreatic cancer at approximately 60 medical centers.49 The protocol was powered to detect a 2-month improvement over control chemotherapy based on a median overall survival of 6 months. Unfortunately, this study did not meet its primary endpoint of improving overall survival compared with palliative chemotherapy or best supportive care. However, this outcome is not unexpected, because patients with advanced pancreatic cancer have a rapid disease progression and are poorly responsive to intervention in general (Box 33-2).

Recombinant Alphavirus Vectors (Sindbis) High-titer alphavirus vectors can provide efficient gene delivery both in vitro and in vivo. In addition, efficient central nervous system infections via intranasal and vascular injections with virulent and avirulent replication-competent Semliki Forest virus (SFV) strains have been shown in animal models.50–52 Replication-deficient alphavirus particles have a high local and transient transgene expression in rodent brains. Furthermore, repeated SFV injections are possible in the absence of an immunogenic response against SFV, which is in contrast to Adv and VV vectors. Modifications to the envelope structure of Sindbis virus are possible with resultant changes in host range and targeting. The favorable characteristics of alphavirus vectors include • • • •

Rapid production of high-titer virus Broad host range High RNA replication rate in the cytoplasm High transgene expression levels Negative attributes include

• Short-term expression • Strong cytotoxic effects on host cells Nonetheless, both these properties are advantageous for certain indicators, particularly vaccine production.

Nonviral Gene Transfer Vectors Nonessential genes can be removed from viral vectors to allow room for transgene(s) to reduce inflammatory responses and to increase safety.53,54 This process involves simplifying the virus, sometimes to an extreme. After undergoing such a process, a virus vector can be an artificial “vector shell” allowing the gene of interest to be expressed at high levels, in a highly regulated manner, and for a controlled period of time. Another approach to achieve the same result is to produce a vector that can introduce genetic material to the nucleus of cells.53,55,56 This strategy has resulted in the development of several

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nonviral vector systems; however, the efficiency of “naked DNA” as a therapeutic is suboptimal without some form of carrier or formulation.

Direct DNA Injection and Transduction One form of nonviral gene delivery is the use of purified DNA plasmids.55 The transgene expression is low following intramuscular or intratumoral injection; however, high levels are observed if hydrodynamic injection is used.57,58 The approach of naked DNA injection is typically done as an intramuscular or intratumoral injection. Despite the simplicity of this approach, transfection efficiency is low and results in limited expression. Various formulations, including lipid or pluronic formulations, and incorporation into nanoparticles or liposomes, have been used to improve transduction efficacy and gene expression (Box 33-3).59–61 Nonviral liposomal delivery systems can be IV injected with limited vector-associated toxicity, but with transgene expression, especially in the lungs.62 Tumor targeting using tumor-specific promoters, ligandation of receptors to the liposome surface, and pegylation of liposomes have all been studied.63–69 Although some degree of tumor targeting has been observed using these delivery systems, the level of transgene expression is generally low. Studies have revealed that liposome-DNA complexes can also elicit an inflammatory response when injected systemically, resulting in suppression of transgene expression.70–72 Furthermore, failure to achieve increased or sustained gene expression after repeated injections has been a major obstacle in the development of liposomes.70,73 Recently, it was shown that cationic liposome (DOTAP : cholesterol or DOTAP : Chol)–DNA complexes can achieve effective levels of transgene expression in tumor-bearing lungs and when injected IV can achieve levels sufficient to cure immunocompetent mice with disseminated experimental metastases.74 Furthermore, repeated daily injections can result in a dose-dependent increase in transgene expression in tumorbearing lungs.75

Hydrodynamic Gene Delivery Hydrodynamic tail-vein plasmid delivery results in high levels of transgene expression in the livers of rodents.76 Lower levels of transgene expression (100- to 1000-fold) are found in the spleen, heart, kidneys, and lungs. This simple nonviral gene transfer procedure entails the rapid delivery of naked plasmid DNA in a relatively large volume of physiologic saline.57 In a typical mouse, weighing 20 g, the plasmid is delivered in a total volume of 2.0 mL over a period of 5 to 7 sec. Although there are toxicity issues, clinical studies are under

Box 33-3.

PLASMID AND RETROVIRAL VECTORS

The transduction efficiency of plasmid vectors is low, even with the use of formulations to improve transfection efficiency and increase transgene expression. Further, this approach appears to work better in vitro than in vivo. In contrast to viral vectors, plasmid vectors offer little innate antigenicity, although there have been reports of immune responses to bacterial genes. Positive attributes of plasmid vectors include the low level of innate immunogenicity and the potential for genomic integration. The use of hydrodynamic delivery in rodents has provided a powerful preclinical tool. However, clinical translation is problematic with the potential for utilization in an isolated limb. In contrast, retroviral and lentiviral vectors provide the same characteristics with higher levels of transgene expression and improved transduction efficiency relative to plasmids. However, the improved transgene expression and transduction levels of retroviral and lentiviral vectors remain significantly lower than those of adenoviral and vaccinia vectors.

discussion for the delivery of dystrophin into the arms of Duchenne muscular dystrophy patients.77

Liposomes and Virosomes In their most basic form, liposomes consist of two lipid species: a cationic amphiphile and a neutral phospholipid.75,78 Liposomes spontaneously bind to and condense DNA to form complexes that have a high affinity for the plasma membranes of cells, resulting in the uptake of liposomes to the cytoplasm by endocytosis. Many variations of this approach are used, resulting in varying levels of gene expression. Unfortunately, liposome-facilitated gene delivery is relatively ineffectual in vivo. More recently, some of the advantages of viral delivery vectors have been combined with the safety and “simplicity” of the liposome to produce fusigenic virosomes.78 Virosomes are engineered by forming complexes of the membrane fusion proteins with liposomes that have already-encapsulated plasmid DNA. The inherent ability of the viral proteins in virosomes to fuse with cell membranes results in the efficient introduction of DNA to the target cell, providing improved gene expression. Viral vectors have limitations based on the size of transgene that can be incorporated; in contrast, no such limit exists for virosome or liposome technology (at least in theory).

Ballistic Delivery (Gene Gun) This physical method of gene delivery involves microcarriers (usually gold particles) coated with DNA and “fired” at high velocity using an explosive or gas-powered ballistic device called a “gene gun.”79–81 Once the particles are inside the target cell, the DNA is slowly released from the microcarriers, resulting in gene transcription and translation. This application has been used extensively in vivo, but its clinical use is restricted to exposable surfaces or ex vivo transduction because the fired particles do not penetrate tissues deeply.82

Nanoparticles Novel polymeric delivery systems (e.g., nanospheres) that can be administered in novel ways are being developed.83–85 These particles are potentially useful because the smaller the condensed DNA particles are, the better will be their in vivo diffusion toward target cells and the trafficking within the cell. Individual plasmid molecules can be collapsed into a nanoparticle using detergents. For example, nanoparticle-based gene delivery was targeted to the neovasculature of mice using an integrin-targeting ligand, resulting in tumor regression.86 Nonetheless, the size of the transgene that can be delivered by nanoparticles is limiting, and the primary focus has been on small interfering RNA (siRNA) delivery, which will be discussed in the following section.

Nucleic Acid-Based Therapeutics DNA Transduction High-molecular-weight, double-stranded DNA constructs containing transgenes, which encode specific proteins, are classically used in gene therapy to introduce transgenes into cells that inherently lack the ability to produce a protein of interest. In addition to being used to treat congenital diseases, DNA vectors can be used as vaccines for genetic immunization.87 Suicide gene therapy is another rapidly emerging strategy for the induction of transgenes.88,89 In this approach, chemosensitization genes are delivered to tumor cells, which upon gene expression convert a separately administered, nontoxic prodrug into a chemotoxic drug. Because only the transfected tumor cells can convert the prodrug, the susceptibility to the chemotoxic entity is limited to the tumor cells—hence the term suicide gene therapy.

RNA Transduction Thus far the primary transgene source for DC transduction is DNA, although other Ag sources are also used with DCs, including peptides, recombinant or purified proteins, cellular extracts from tumor

Gene Therapy in Oncology • CHAPTER 33

cells, apoptotic bodies, and RNA or DNA plasmid vectors. Nevertheless, the carrier of choice for loading DCs with tumor Ags is DNA or RNA.90 Nucleic acid transfection leads to the display of multiple antigenic epitopes by both class I and II major histocompatibility complex via the Ag-processing machinery of the patients’ DCs, resulting in the display of the “most appropriate” peptides. This is in contrast to vaccine strategies based on synthetic peptides, which require the knowledge of the patient’s unique peptide epitopes. Thus, nucleic acid transfection of DCs offers several advantages for both immunologic and practical considerations. The bias for the use of DNA vectors includes an increased stability as compared with RNA, the ability to produce plasmids in large quantities, and the ease with which the sequence can be modified to regulate expression.91 In several respects, however, RNA vectors are also advantageous when compared with DNA transfection. RNA vector advantages include the ability to use total messenger RNA (mRNA) isolated from tumors to transfect DCs with no intervening cloning steps and the ability to express several or potentially all tumor-derived genes within DCs. Transfected RNA need only reach the cytoplasm of DCs, whereas DNA requires entry into the nucleus and subsequent transcription. Furthermore, it has been suggested that the low level of antigenic epitope expression that occurs with RNA-transfected DCs could be advantageous, provided that expression levels are sufficient to generate a T-cell response.90,91 When low levels of antigenic peptides are presented by DCs, only those T cells with high-affinity recognition are activated, thus skewing the response toward T cells that can better recognize the tumor cells. Conversely, when DCs present high levels of antigenic peptides, T cells of low affinity may be activated, thus masking or even preventing the activation of high-affinity T cells. This could result in T cells that kill cells with high Ag expression but cannot kill tumor cells, which typically express low Ag levels. Thus, RNA-transfected DCs might have greater efficacy for the activation of high-affinity T cells.91

Oligonucleotides Oligonucleotides are short single-stranded segments of DNA or RNA that upon cellular internalization can selectively inhibit the expression of a single protein.92 Multiple forms of oligonucleotides are used in gene therapy including antisense, siRNA, and ribozymes. Most of these constructs form a duplex with the mRNA or the pre-mRNA and inhibit their translation or processing, consequently inhibiting protein biosynthesis. This occurs by multiple mechanisms, as discussed in the following section.

Small Interfering RNA RNA interference is a recently discovered mechanism for silencing the transcription of mRNA. siRNA is generated by dicer, an endonuclease that cleaves long double-stranded RNA molecules into fragments of 21 to 23 base pairs (bp) and is highly specific for the nucleotide sequence of its target mRNA siRNAs.93–96 These siRNAs associate with helicase and nuclease molecules and form a large complex, which is termed RNA-induced silencing complex that unwinds siRNA and directs precise, sequence-specific degradation of mRNA. Although RNA interference was discovered only recently, the field has exploded.97 It is now apparent that RNA interference is a highly conserved molecular mechanism that is used by eukaryotic organisms to control gene expression during development and to defend their genomes against invaders, such as transposons and RNA viruses. Recently, it was shown that siRNA is active in vivo with resultant therapeutic activity.98 In one study, siRNA knockdown of the mutant K-ras oncogene had pronounced antitumor activity.99 In this study, siRNA was delivered as a nonreplicative retroviral transgene and was shown to inhibit the relevant mutant K-ras and prevent anchor-independent growth and tumorigenicity. Antitumor activities can also be induced in vivo through siRNA knockdown of other critical components of tumor growth, metastasis, angiogenesis, and chemoresistance.100 Stable transfection and

expression of siRNA is obtained with nonreplicating viruses101,102; however, oncolytic virus vectors provide a potential method to extend bioactivity. The tumor-selective infectivity has the potential to restrict transgene expression to the cancer microenvironment, potentially reducing toxicity and extending transgene expression via viral replication and multiple cycles of the infection of permissive cancer cells.103,104 Furthermore, viral oncolysis has the potential to augment antitumor outcomes by siRNA-mediated therapeutic activity. In a recent study,105 the replication-competent, oncolytic adenovirus, ONYX411, was used to deliver a mutant K-ras siRNA transgene. In this study, additive tumor growth-inhibitory responses via siRNAmediated K-ras knockdown and ONYX-411–mediated oncolysis were observed. Therapy with ONYX alone or ONXY-411 with green fluorescent protein siRNA as controls had significantly lower therapeutic activity.

Antisense The principles of antisense technology are conceptually simple. Oligonucleotides are designed to hybridize to a defined target mRNA and to inhibit its translation into protein.106,107 This approach was first employed in 1978 by Stephenson and Zamecnik108 to inhibit the Rous sarcoma virus expression in chicken fibroblasts. Several antisense oligonucleotides are in clinical trials, and one has received Food and Drug Administration (FDA) approval for the treatment of cytomegalovirus retinitis. Currently, an antisense to Bcl2 has been submitted for licensing by the Food and Drug Administration for the treatment of leukemia.109 Although it is relatively easy to synthesize phosphodiester oligonucleotides, they cannot be used as drugs because of their sensitivity to nuclease degradation. To improve their resistance to nuclease digestion, different chemical modifications are used, including phosphorothioates, methylphosphonates, and phosphoramidates.110 These modifications increase the stability of oligonucleotides, but they also alter the capacity to hybridize with RNA and reduce cellular internalization.

Ribozymes Ribozymes are RNA molecules capable of sequence-specific cleaving of mRNA molecules.111 They selectively bind to target mRNAs and form a duplex that is easily hydrolyzed, suppressing specific genes.54 Two types of ribozymes, the hammerhead and hairpin, have been extensively studied.112 However, the RNA backbone in ribozymes is an easy target for RNases, so they are biologically unstable in vivo.54 Ribozymes have been used primarily for gene suppression, the induction of apoptosis, and antiproliferative effects. Phase I clinical trials using ribozyme gene therapy to treat AIDS patients are ongoing.113

GENE TARGETING Targeted gene therapy of cancer can be achieved through • Targeted gene expression • Vector targeting13,114,115 Although it is less important during ex vivo or intratumoral gene delivery, targeted gene therapy becomes crucial with systemic gene transfer. Impediments to gene therapy include the poor selectivity of existing vectors and the low efficiency of gene transfer. Overcoming these hurdles is critical to achieving vectors that can be targeted and injected IV—an important goal given the systemic nature of cancer.

Conditional Gene Targeting Vector targeting is a goal for both viral and nonviral vectors13,114; however, the current emphasis is on tissue- or target-specific promoters. Transcriptional regulatory sequences are used because they are responsible for protein production in carcinoma cells such as oncogene products. One example is the use of tissue-specific promoters to

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facilitate tumor-specific killing via expression of a suicide gene (such as the HSV-tk) followed by exposure to ganciclovir, or the expression of the cytosine deaminase gene and exposure to 5-fluorocytosine. In addition, transcriptional targeting is used to achieve conditionally targeted transgene expression.

Tissue-Specific Promoters The production of proteins within a cell requires that the appropriate gene be transcribed into mRNA and then translated to protein.116 This process is under multiple levels of control, with the regulation of transcription mediated by interactions between the enhancer/promoter region of the appropriate piece of DNA and the specific proteins or transcription factors that bind to this region. Activation or repression of promoters is achieved through interactions with specific transcription factors. Thus, some tissues might express specific proteins because the promoter for that gene is activated in that tissue alone. The success of transcriptional targeting is dependent on achieving a differential gene expression in cancer cells as compared with normal cells. Transcriptional control of gene therapy is an important goal for two reasons: 1. Current gene transfer vectors can be inefficient in gaining entry into the types of cells needing treatment. 2. Many therapeutic genes can be toxic if delivered to an unintended cellular target. Criteria for selecting a promoter for use in a gene therapy protocol include consideration of the promoter’s strength, tissue specificity, and size. Promoter candidates include regulatory elements that are already expressed by the malignant cell, tissue-specific promoters, or externally inducible sequences. Unfortunately, any of these candidate promoters can lack sufficient activity, specificity, or both. To address promoter potency, promoters and enhancers that retain cellspecific function are often linked to transactivators. Additional strategies to enhance promoter activity in malignant tissues include the use of cell-cycle elements, normal or abnormal tissue differentiation factors, hormones, cytokines, chemicals, or physical stimuli. A convenient classification of candidate promoters for cancer gene therapy (Table 33-1) includes tumor-associated, tissue-specific, and inducible promoters. These are further discussed in the ensuing sections, as is the role of transcriptional regulation of replicationcompetent viruses. Specific examples are provided that are the most mature developmentally but should be viewed as representative. The most focused reviews and articles are referenced.

Tumor-Associated Promoters Selective delivery of a transgene to a tumor is not currently an achievable clinical goal. Consequently, tissue and tumor promoters are used to regulate transgene expression in a given tumor tissue with the goal of reducing nonspecific transgene expression. However, this approach retains challenges, including the infection of only a small fraction of tumor cells within the target tissue. As such, tumor-specific and associated promoters are also extensively utilized to target transgene expression, especially ones associated with the tumor vasculature.117–119

TELOMERASE. Telomerase, an RNA-dependent DNA polymerase that synthesizes new telomeric repeats at the end of chromosomes, is expressed in high levels in malignant tumors, stem cells, and germ cells, but not in normal tissues. It is thought to be essential for the maintenance of the proliferative capacity of tumor cells and, for this reason it represents an attractive target for gene therapy. The human telomerase reverse transcriptase is regulated primarily at the transcriptional level, and its promoter has the potential for targeted cancer gene therapy.120,121 TUMOR VASCULATURE. Another target for gene therapy is provided by the tumor’s vasculature. The tumor vasculature has excellent accessibility to systemic delivery across all solid tumor

types.122 Indeed, high levels of vascular endothelial growth factor (VEGF), a growth stimulus for endothelial cells, have been correlated with a poor prognosis for specific tumor histotypes. VEGF activity is mediated by two high-affinity receptors: the tyrosine kinases VEGFR-1/fms-like tyrosine kinase (Flt-) 1 and VEGFR-2/flk-1. These ligand-stimulated tyrosine kinases are induced in a tumor stage-dependent manner during cancer progression and are expressed exclusively in tumor vascular endothelial cells.123 This suggests that VEGF receptors are promising targets for tumor endothelial cellspecific therapy.122,124 Thus, the 939-bp Flk-1 promoter fragment and an enhancer element located in a 2.3-kb fragment upstream have been used to induce tumor endothelium-specific reporter gene expression in transgenic mice.125 Targeting of the VEGF receptor/ligand system has been shown to be a useful approach with which to inhibit tumor growth and prolong survival in colon cancer.124 The human preproendothelin-1 promoter has also been shown to have specificity for breast microvascular endothelial cells using a recombinant retroviral vector.126

Tumor-Specific Promoters PROSTATE-SPECIFIC ANTIGEN. Prostate-specific Ag (PSA) is expressed at a high level in the luminal epithelial cells of the prostate and is absent or expressed at low levels in other tissues. The PSA promoter is usually regulated by androgens, but it might retain its activity in an androgen-free environment. The minimal PSA promoter, however, is weak in both PSA+ and PSA− cells and does not respond to androgenic stimuli. Nonetheless, the PSA promoter has been used to target the delivery of therapeutic genes to prostate tumors.127,128

TYROSINASE. Specificity for malignant melanoma may be con-

ferred by the human tyrosinase promoter.129,130 Driven by this promoter, in vitro and in vivo melanoma transduction by constructs results in selective transgene with the potential to induce tumor regression. Similarly, a construct consisting of the human tyrosinase promoter linked to two enhancer elements causes high-level, melanoma-specific expression of a reporter gene in transient transfection assays. The murine tyrosinase promoter-enhancer expression cassette expressed by an Adv vector maintains transcriptional specificity for pigment cell lineages, especially human melanoma cell lines.

Conditional Replication and Inducible Promoters During evolution, various stress response genes developed, and their promoters are now considered as gene therapy transcriptional regulators. Heat, hypoxia, glucose deprivation, irradiation, and chemotherapeutic agents upregulate stress response genes. Because of the relative weakness of tissue- and tumor-specific promoters, these inducible promoters are attractive as mediators of transient transgene activation. Promoters of these genes are also attractive for cancer gene therapy because they depend to a large extent on the biology of the tumor or are already induced by various therapeutic modalities.131,132

STRESS-ASSOCIATED GENES. Genes that are upregulated during stress include ABCB1, human heat-shock protein (HSP), VEGF, irradiation-inducible EGR1 (early growth response gene), and the tissue plasminogen activator (tpa) promoters. Irradiation-responsive promoter sequences have been identified for the tpa and EGR1 genes.133 The first irradiation-inducible promoter system used in combination with gene therapy involved the EGR1 promoter driving either the radiosensitizing cytokine tumor necrosis factor α (TNF-α) or tk. The HSP family is induced by a variety of environmental conditions, including heat, irradiation, photobeam irradiation, hypoxia, acidosis, hypoglycemia, and osmotic changes. These conditions can exist in poorly vascularized tumors and can trigger anticancer gene expression linked to the HSP70 promoter.134 It is significant that HSP70 expression is upregulated in p53-deficient tumor cells, thereby providing transcriptional targeting.

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Table 33-1 Transcriptional Regulation for Cancer Gene Therapy Transcriptional Mechanism

Promoter

Target Tumor

TISSUE SPECIFICITY

PSA, Kallikrein

Prostate

ABERRANT TUMOR BIOLOGY

INDUCIBLE PROMOTER

Tyrosinase

Melanoma

CEA

Hepatocellular carcinomas (HCC): breast, lung, and pancreas cancers

α-fetal protein (AFP)

HCC

c-erb B2

Pancreas

Amylase

Pancreas

SP-B

Lung cancer

Grp

Small cell lung carcinoma

AVP

Small cell lung cancer

Immunogloblin heavy chain

B lymphomas

AP-2

Breast cancer

α-lactalbumin

Breast cancer

Osteocalcin

Osteosarcoma

Prolactin

Prolactinoma

Insulin

β-islet cells

Whey acidic protein

Breast cancer

Cirulatory leukoprotease inhibitor (CLPI)

Lung, colon, breast, bladder, oropharyngeal, ovarian, and endometrial carcinomas

Glial fibrillary acidic protein

Brain astrocytes, glioma cells

Albumin

Liver

T-cell receptor

T lymphocytes

Her 2/neu

Breast, pancreatic, and gastric carcinomas

Myc-Max responsive element

Lung cancer

MUC-1

Adenocarcinomas

Telomerase

Urinary bladder and HCC

FLK-1

Melanoma, fibrosarcoma and breast tumor vessels

E-selectin

Tumor vasculature

VEGF

Lung cancer

Hexokinase II

Lung cancer

c-erb B2

Breast and pancreas tumors

c-Myc

Small cell lung cancer

L-plastin

Ovarian carcinoma

SLPI

Lung and ovary tumors

EGR-1

Glioma

Hsp70

Prostate, breast, and melanomas

Grp78

Fibrosarcoma

ABCB1

Breast

MULTIDRUG RESISTANCE GENES. ABCB1 encodes a membrane effluxing glycoprotein, whose expression is induced by vincristine, actinomycin D, and doxorubicin. Its promoter is indirectly transactivated by these compounds and induces transcription and expression of therapeutic genes, such as TNF-α in tumors exposed to chemotherapy.135 Chemotherapy can also induce another mechanism of drug resistance, namely, activation of the glutathione detoxification system and apoptosis-controlling gene alterations (especially p53 and bcl-2). Because the ABCB1 promoter contains heat-responsive elements, it is also activated by HSP. In addition to its promoter activity, ABCB1 can be transduced into hematopoietic

stem cells to reduce the myelosuppressive effects of chemotherapy and radiotherapy.136

DEXAMETHASONE. Several

drug-related gene expression systems are available to control target gene transcription through the use of small-molecule-inducing compounds.137,138 Although the utility of such systems has been demonstrated in vitro and in transgenic mice, they are also targeting use in a therapeutic context.139,140 Dexamethasone, a synthetic glucocorticoid, can selectively activate the p21 promoter in rat hepatoma cells via a glucocorticoid-responsive region between nucleotides 21481 and 21184.141 This region

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does not contain a canonical glucocorticoid response element, but it confers specific dexamethasone responsiveness to heterologous prostate promoters.

TETRACYCLINE RESPONSE ELEMENTS. The Tetcontrolled transcription system is made up of Tet-off and Tet-on transcriptional regulation, derived from the Escherichia coli Tet-resistance operon.142 The Tet-R system can be used to suppress or induce cytotoxic and reporter gene expression.143,144 The latter selects gene expression to p53-deficient tumor cells. Similar to Tet-R, mifepristone is an orally bioavailable antiprogestin that can switch on gene expression in allosteric systems, whereby a chimeric transactivator activates a target gene.145 This system can circumvent constitutive expression of transgenes in normal tissues by drug-specific and temporal regulation of the target gene. In addition, the replacement of the activation domain of the chimeric transactivator with a transcriptional repressor domain results in inducible repression of the transgene.146 Conditionally Replicative Viruses Toxic or tumor suppressor gene expression from nonreplicative vectors, as a single therapeutic, is inadequate to control solid-tumor growth in humans.147,148 Thus, replication-competent viruses have been developed and tested as therapeutic agents in cancer. Adv vectors are the most commonly used agents in this context, although retrovirus, reovirus, HSV, and vesicular stomatitis virus are all used for the treatment of malignancies. The criteria governing the utility of replication-competent viruses include infection efficacy, replication selectivity, viral dispersion from the injection site, and evasion of the host immune response. Augmented gene transfer efficiency has been reported for Adv vectors based on the coxsackievirus Adv receptor (CAR)-independent cellular entry pathways.149 Propagation of these vectors within tumor tissue remains a challenge, however. Recent improvements in our understanding of cancer biology have made possible the development of viral vectors with improved tumor-selective replication and the restriction of lytic effects to cancer cells. Dysregulation of the normal control over cell cycle and circumvention of physiologic apoptotic signals might allow tumor-selective replication of an engineered virus and, subsequently, direct oncolysis by viral cell killing.150

Conditionally Replicative Adenoviruses Conditionally replicative Ads (CRAds) are designed by the deletion of Adv natural genes encoding cell-cycle regulatory proteins and/or by placing a tissue-specific promoter to control a viral gene essential for viral replication. An example of a vector with a deleted Adv gene is the deletion of CRAd E1A (Table 33-2). This results in a loss of its conserved region 2, which precludes binding to the retinoblastoma gene (Rb) and eliminates the inhibitory effect of Rb on E2F. Consequently, the engineered Adv replicates selectively within cells in which the G1-S phase checkpoint is impaired (i.e., tumor cells).151,152

Deletion of the Adv E1B 55-kd protein was initially suggested to be selective for replication in p53-mutant cells, but this hypothesis has since been questioned.153,154 Despite the mechanistic uncertainty, the E1B-deleted ONYX-015 virus selectively infects head and neck tumor cells and could show a clinical benefit in patients with recurrent carcinomas.155 Although ONYX-015 does not have a therapeutic transgene and relies on its lytic effect, this is the first clinical utility of a CRAd for cancer therapy. It should be remarked that development of ONYX-015 by the American biotechnology company, ONYX Pharmaceuticals, was halted primarily because of financial concerns. Shanghai Sunway Biotech Co. licensed world rights to ONYX-015 and obtained regulatory approval in China in December 2005.2 This gene therapy product, H101 (ONYX-015), is a recombinant Adv modified to selectively replicate in and kill tumor cells with TP53 mutations. This involves a loss-of-function mutation at the E1B locus. The E1B locus product is a 55-kd protein that binds to and inactivates the p53 tumor suppressor protein. Thus, the vector is crippled in its ability to grow in cells with wt TP53, and replicates and causes the death of cells with mutant TP53.156 Phase I, II, and III clinical trials undertaken in China have been reported to show the safety of H101 with efficacy demonstrated in patients with HNSCC. Approval was based on a study that combined H101 and chemotherapy, which was reported to be effective in 78.8% of patients with this disease.157 Recently, mutants of human Adv 5 (Ad5) with enhanced oncolytic activity have been isolated using a procedure termed bioselection. In this process, Ad5 is mutagenized and repeatedly passaged in a human colorectal cancer cell line. From such a cell line, mutants can be found that replicate more rapidly than wt Ad5 and that lyse cells up to a thousand-fold more efficiently.158 Another strategy for designing CRAds uses tissue-specific promoters to drive expression of E1A, thereby restricting viral replication to specific tissues or tumors.159 The application of heterologous promoters in Adv vectors is difficult because their activity and specificity are often affected by viral enhancers and promoters. The E1A gene expressed from the alpha fetoprotein (AFP) gene promoter induces relatively selective replication in hepatocellular carcinoma cells.159 Control of Adv E1A expression under the minimal PSA enhancer/promoter has also been shown to confer prostate-specific oncolytic viral replication.160 Recently, a reengineered Adv vector with enhanced oncolytic efficacy was developed. This vector contained a novel regulatory circuit in which p53-dependent expression of an antagonist of the E2F transcription factor inhibits viral replication in normal cells. In tumor cells, however, the combination of the p53 pathway defects and deregulated E2F allows replication at near-wt levels. This Adv vector also has significantly enhanced efficacy for the treatment of human xenograft tumor models compared with the extensively studied E1Bdeleted Adv vectors.20 CRAds for breast tumors have been created using the DF3/MUC1 promoter (which is abnormally activated in breast tumors) and are

Table 33-2 Transcriptional Regulation of Adv Replication Genetic Modification

Biologic Result

Deletion of E1A (AA 121–127)

Transformation deficiency

Deletion of E1B 55 K protein

Susceptibility to apoptosis

E1A control by the αFP promoter

E1A transcription limited to αFP+ cells

E1A control of the PSA promoter

E1A transcription limited to PSA+ cells

E1A control by the DF3/MUC1 promoter

E1A transcription limited to DF3/MUC1+ cells

E1A control by the pS2 promoter

E1A transcription limited to estrogen receptor+ cells

E1A control by the Sp-B promoter

E1A transcription limited to surfactant producing cells

E1 deletion

Selective DNA replication of Adv vectors in trans-complementing tumor cells

Gene Therapy in Oncology • CHAPTER 33

used to drive the expression of E1A. This CRAd selectively replicates in MUC1+ cells and can inhibit the growth of human breast cancer xenografts.161 Another approach is to target CRAd replication within estrogen receptor (ER)-positive tumors based on replacing the E1A and E4 promoters with a portion of the pS2 promoter containing two estrogen-responsive elements.162 This promoter induces transcriptional activation of the E1A and E4 in response to estrogen in cells that express an ER. This CRAd is able to lyse ER+ human breast cancer cell lines as efficiently as Adv, with decreased capacity to affect ER− cells. Another strategy that has been reported recently makes use of the generation of a functional promoter/gene constellation only on Adv DNA replication, thereby providing selective transcriptional activation.163 These strategies to discriminate between tumor and normal tissue are based on selective DNA replication of Adv vectors with the entire E1 gene in tumor cells deleted. An E1 deletion is considered to abolish Adv replication; however, human tumor cell lines apparently can support DNA replication of Ad with an E1 deletion. Inverted repeats insert into the E1 region of AdE1 vectors can mediate genomic rearrangements, and bring a transgene into control of a promoter. Thus, formation of a functional expression cassette depends on viral DNA replication, which is expected to occur specifically in tumor cells.

Vector Targeting Targeted in vivo gene transfer is becoming a reality as a result of an improved understanding of influences that govern gene delivery.13,14,114 Viral-based vectors are designed to avoid gene transfer through their native receptors and are redirected to tissue- and tumorspecific receptors. In most therapeutic applications, the vector is introduced into a mixed population of cells with the goal of delivering the therapeutic transgene to specific cells. Transduced stem cells can also be targeted to treat certain genetic diseases, improve tolerance to chemotherapy, or assist in tissue repair and remodeling. DCs can also be targeted for the development of improved vaccines. Finally, a systemically administered, targeted vector can potentially reach systemic disease. Nevertheless, these vectors require additional development, including clinical testing, reduced liabilities (including innate and acquired immune augmentation), and an improved understanding of the mechanisms that govern biodistribution and pharmacokinetics. Ligand-directed targeting of gene vectors allows control of the site at which genes are expressed by imparting the capacity to distinguish between target and nontarget tissue(s). These ligand-directed targeting vectors achieve this capability through the addition of ligands to the vector that recognize receptors specific for a tissue or disease. This approach has met two goals: 1. Improved efficiency for the current gene transfer vectors in transducing the targeted cells that need treatment 2. Reduction in the toxicity due to delivery of therapeutic genes to unintended target cells Thus, ligand-directed targeting can potentially improve both the safety and the efficacy of gene transfer and make possible therapies that could not be envisioned with standard gene transfer vehicles. Although targeting gene transfer to specific cells and tissues holds promise, it is a challenge for vector design. Regardless of the vector, three variables are critical to ligand-directed targeting. These include cellular specificity, physical barriers, and the host innate or acquired response, which could eliminate the vector from the circulation. Cellular specificity can be achieved by the use of ligands that recognize cell-specific receptors. For viral-based vectors, specificity requires a targeting element plus modification of the vector so that it no longer binds to its native cellular receptors. In the case of nonviral vectors, targeting requires modification of the vector to avoid nonspecific uptake. Apart from cellular specificity, physical barriers (e.g., the

cellular matrix) can limit access of the vector to the target cell. Finally, avoiding elimination and neutralization of vectors by innate and acquired immunity is critical to gene transfer. This is critical, because Abs and serum proteins can directly inactivate the vector or direct it to the liver for rapid clearance, if the vector is given systemically.

Adenoviral Vectors Our improved understanding of the attachment and entry processes of Adv vectors has facilitated the development of Adv-targeting vectors.13,34,114 The nonenveloped subgroup C Adv vectors use at least two coat proteins to gain entry into cells. The knob portion of the fiber coat protein binds to the cellular receptor, CAR, and mediates virus attachment.164,165 At the base of the fiber protein, the penton base coat protein contains an Arg-Gly-Asp (RGD) motif that binds to integrins and facilitates vector uptake into the cell.166 Compared with a vector with native receptor binding interactions intact, gene expression in the liver and other organs is substantially reduced after systemic administration of a vector containing mutations that ablate CAR and integrin binding.167 This observation suggests that these receptor interactions are important for in vivo gene transfer. The loss of CAR and integrin binding also reduces gene transfer after direct injection. Furthermore, it allows Adv vectors to be retargeted genetically.17,168 Ablation of CAR binding alone does not significantly reduce liver gene transfer, which suggests that the standard two-step model of attachment via the CAR and entry by means of integrins does not apply to in vivo gene transfer to the liver, which instead probably involves Kupffer cells. Adv vectors have been retargeted by both genetic and nongenetic means. Peptides (including the fiber, penton base, and hexon) have been functionally incorporated into coat proteins, although few functional peptide ligands have been identified thus far.166,169–173 In addition to genetic modifications for retargeting viral vectors, ligation approaches have also been used with Adv vectors. Such approaches involve a bifunctional adaptor or bridging molecule that binds to the vector and to a target receptor. Such systems have demonstrated the feasibility of targeting conventional Adv vectors to more than 20 different receptors, including αv integrins, endoglin, E-selectin, EpCAM, and folate receptors.114 Specific targeting to the lung vasculature has also been demonstrated through a combination of receptor-based targeting via lung endothelial-specific receptor, angiotensin-converting enzyme, and promoter-based targeting through the endothelial-specific promoter, Flt-1.

Structural Modification of the Fiber Protein One approach to Adv retargeting involves engineering of the knob domain of the fiber protein. In this domain, the introduction of heterologous cell targeting peptides requires consideration of the structural limitations of the fiber three-dimensional configuration. The fiber is synthesized as a monomer, which undergoes trimerization before its attachment to the penton base. Thus, any modification of the knob domain of the fiber must not impair trimer formation. In addition, the final quaternary configuration of the new fiber must make the incorporated ligand accessible to target cell receptor recognition and binding. Recombinant Adv vectors have been constructed with a heparin/ heparan sulfate-binding domain, consisting of polylysine residues added to the C terminus of the fiber. Gene transfer to different mammalian cells has been obtained with a level of efficiency 10- to 300fold higher as compared with unmodified vector.174 The main drawback with this approach is the lack of specificity, because most mammalian cells express heparin-containing cellular receptors. Genetic modification of the Adv fiber C terminus is limited, because the addition of more than 25 to 30 amino acid residues renders the fiber trimer unstable and limits function.175 The modification of Adv vectors by placing an RGD peptide in the HI loop rather than in the C terminus of the fiber knob domain was reported recently.176 This

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modification resulted in an increase in gene transfer to ovarian cancer cell lines (30- to 600-fold) and ovarian cancer cells (twofold to threefold).

Modification of the Penton Base Retargeting of Adv vectors has also focused on the modification of the penton base, which mediates the second step of Adv infection (i.e., internalization). Recombinant Adv vectors have been generated in which the RGD motif in the penton base has been replaced by the FLAG peptide. A complex of this vector with a bispecific Ab— consisting of a monoclonal Ab to the FLAG epitope and a monoclonal Ab to integrins—was shown to target cells lacking the Adv fiber receptor, such as endothelial cells or human intestinal smooth muscle cells. Thus, the first two steps of Adv infection binding and internalization are both mediated by α integrins.177 In addition, recombinant Adv vectors can be constructed of chimeric penton base proteins that recognize tissue-specific integrin receptors.178

Retroviral Vectors Retroviral vectors were the first viral vectors to be targeted and the first to demonstrate the promise of vector targeting.34,114 Since that time, the challenge has been to incorporate targeting ligands without compromising vector entry into target cells. There are now several approaches used to address this problem. One approach exploits pseudotyping, classically with the G-glycoprotein from the vesicular stomatitis virus, in which entry events are mediated through common membrane phospholipids.179 Pseudotyped lentiviral and retroviral vectors have also been generated with glycoproteins from a variety of enveloped viruses, including Ebola virus, Marburg virus, rabies virus, lymphocytic choriomeningitis virus (LCMV), Mokola virus, human foamy virus, gibbon ape leukemia virus, murine leukemia virus, influenza virus, avian leukosis-sarcoma virus, and respiratory syncytial virus.180–184 Although these pseudotyped vectors vary in terms of degree of envelope shedding, efficiency of packaging, titer, and stability, they can be concentrated to high titers for in vivo comparisons of cellular tropisms. Another vector modification involves the ligation of polypeptides at the N terminal of env to extend the host range of ecotropic murine leukemia virus (MLV). Examples include erythropoietin, heregulin, and CD4.185–187 It should be stressed that coexpression of the wt env protein is necessary for infection to occur, possibly because incorporation of the engineered env protein in the virons can be facilitated by the oligomerization of both wt and chimeric env proteins. Another approach for engineering of the ecotropic MLV env protein involves the display of different polypeptide binding domains to the N terminus of the ecotropic MMLV surface protein. Examples include the N-terminal moiety of the amphotropic MLV env, single-chain Abs recognizing different cell surface receptors, heregulin, and epidermal growth factor.188–191 In some of these studies, infection specificity was redefined, though with lower efficiencies than those obtained with viruses expressing wt amphotropic envelopes.192,193 Bifunctional bridging agents that recognize both the retrovirus and the targeted cell surface molecule provide evidence that retroviruses can enter cells via cell surface molecules that are not viral receptors. Such bridging agents have been used to infect human cells that are naturally resistant to ecotropic MLV-based vectors. The agents used usually consisted of an Ab to the MLV envelope protein connected to either another Ab or a growth factor that would bind to the appropriate receptor such as the epidermal growth factor receptor, the insulin receptor, or major histocompatibility complex class I and class II molecules. Unfortunately, infection efficiencies for these agents are extremely low, emphasizing that binding of a retrovirus to a target other than the natural receptor cannot guarantee success.194,195

Nonviral Vectors Nonviral vectors have no native receptor binding, yet do have a high incidence of nonspecific gene transfer from the high positive charge

on many nonviral vectors.54–56 Many nonviral vectors transduce lung vasculature, potentially as a result of vector-mediated red blood cell aggregation and arrest in the lung subsequent to IV administration. The solution to the problem of nonspecific delivery is to shield the vectors, either with hydrophilic polymers such as polyethylene glycol (PEG) or with a ligand to reduce the surface charge. This type of shielding is applied successfully to lipid-based systems and to cationic polymer-based systems (polyplex). Ligand-directed liposomes have shown some success in targeting tumors. Coupling of a synthetic αvβ3-integrin ligand to cationic liposomes permits the selective delivery of a mutant Raf gene that causes apoptosis in angiogenic blood vessels within tumors. Systemic injection of the αvβ3-targeted liposome results in apoptosis of tumorassociated endothelium and the regression of primary and metastatic tumor(s). This accomplishment highlights the extension of this approach from in vitro to in vivo efficacy.86 Another promising advance is to coat polyethylenimine (PEI)DNA polyplexes with a ligand, such as transferrin or transferrin plus PEG. Shielding by PEG or transferrin prevents nonspecific interactions with plasma proteins and erythrocytes but does not interfere with target cell interactions. When systemically administered in a subcutaneous tumor mouse model, the shielded complexes were shown to selectively transduce a well-vascularized, rapidly growing tumor. Although the specificity of this approach is high, the overall level of transduction is low.196 In all the studies to date that use nonviral approaches, the need for relatively high dosing levels (∼100 mg/mouse) suggest that innate clearance mechanisms might have to be saturated before substantial gene transduction can occur. Other potential issues that remain include determining whether the high doses used in the animal studies can be manufactured and delivered successfully for human studies. Attaining further improvements in the efficiency of gene transfer and better defining the toxicity profiles associated with these vectors are also critical steps.

CLINICAL TRIAL STRATEGIES The development of gene therapy over the last decade has been on a roller-coaster ride that has yet to fulfill the promise of this exciting new research and therapeutic tool. Retroviral gene therapy has inarguably been shown to reverse congenic diseases. This was the first success for gene therapy, whereby a retroviral-based treatment was undertaken for infants suffering from X chromosome-linked SCID-X1. These studies provided the first demonstration of the potential for long-term treatment of hereditary diseases.197 The success of this approach is due not only to gene therapy but also to improvements in our understanding of hematology and the availability of clinical-grade cytokines to support the transduction of adequate numbers of stem cells. These studies have resulted, however, in the concept of retroviral insertional carcinogenesis moving from a theoretic to a real concern in recent months. Two of the initial 11 children who received retroviral gene therapy for the treatment of SCID developed a leukemia-like condition.198 Both of these cases, as well as a third in which leukemia has not yet developed, seem to be due to the insertion of the corrective gene near another gene called Lmo2, which helps to control cell growth and can contribute to cancer if turned on at the wrong time.198 Nonetheless, the unique nature of this therapeutic strategy for patients who have no other viable therapeutic modality, and the responsiveness of the resultant leukemias to chemotherapy, suggest that it remains a justifiable therapeutic strategy for those patients who have no matching allotransplant donor. Therapeutic activity has also been demonstrated199 for retroviral vector transduced stem cells in infants with a defective gene for adenosine deaminase (ADA-SCID). In contrast to children with SCID-X1, enzyme replacement therapy has been available for children with ADA-SCID using pegylated ADA (PEG-ADA). Initially,

Gene Therapy in Oncology • CHAPTER 33

ethical concerns required that gene therapy studies in ADA-SCID patients be undertaken concomitant with PEG-ADA therapy. A generalized schema for these gene therapy protocols is shown in Figure 33-3. However, the SCID-X1 studies by Cavazzana-Calvo revealed that a strong selective pressure was needed to expand the transduced cells following infusion.197 This revelation provided the impetus for studies in ADA-SCID whereby PEG-ADA was discontinued, allowing the selection of transfected stem cells and more importantly differentiated T cells. In addition, nonmyeloablative conditioning with busulfan was used to provide space in the marrow for the infused stem cells. Although insertional mutagenesis remains a concern, no clonal expansion has been reported to date. A recent follow-up at a median of 3 years was presented at the 2006 American Society of Hematology (ASH) annual conference and reported that seven of eight children responded to gene therapy, with approximately 95% of T cells expressing ADA. The one patient who did not show improvement was treated at an older age, suggesting a higher level of disease damage. The second vector type that has shown significant clinical potential is Adv vectors. In contrast to retroviral vectors, Adv vectors induce a transient gene expression and demonstrate both high transduction efficiency and high transgene expression; in addition, these vectors can be grown to high titer for virus stocks. Furthermore, the activity profile of these vectors (particularly transient gene expression) provides an attractive characteristic for many current clinical development strategies. Most notably, these protocols involve the induction of tumor apoptosis via systemic or intralesional injection of vectors

with transgenes that induce apoptosis or result in the activation of cytotoxic drug precursors, such as TK or cytosine deaminase. In addition, Adv vectors are being used to deliver Ags to Ag-presenting cells (e.g., DCs), resulting in the induction of an Ag-specific immune response and (theoretically at least) therapeutic activity. In a recent phase I/II trial of extensive stage, small cell lung cancer,200 29 patients received standard first-line chemotherapy and three vaccinations with Adv-transfected DCs as a vaccine. Patients with stable disease to first-line chemotherapy had DCs prepared from enriched monocytes, which were then transfected with Adv-p53 and the resultant p53-DC injected three times. Following disease progression, patients received second-line chemotherapy. The objective response rate to vaccine alone was low, although it was reported that following p53-DC vaccination one patient had lymph node metastasis regress with the remainder of patients rapidly progressing. In contrast, 60% of patients with progressive disease following vaccination had objective clinical responses to second-line chemotherapy, resulting in an overall survival of greater than 11 months after immunotherapy. Indeed, the major response rate to second-line chemotherapy (complete and partial responses) was 90% in those patients who developed an immune response to p53 as compared with 40% for those patients who did not develop an immune response. In this disease, aggressive combination chemotherapy regimens can extend the median survival time to 9 to 10 months from diagnosis, as compared with 2 to 3 months if untreated. Similar to retroviral vectors, Adv vectors have experienced “growing pains.” Although the majority of the vectors used in current

5. Patient’s immunodefiency provides a strong selective pressure, enriching for transduced T cells and resulting in a functional immunity and protection against infection

1. Patient diagnosed with a congenital disease and then bone marrow cells removed

Begin treatment

Stylized gene therapy protocol utilizing a retrovirus in the treatment of congenital diseases

2. Stem cells enriched by antibody magnetic bead isolation Magnetic field

Retroviral vectors

4. Optimization of gene therapy protocols may incorporate nonmyeloablative chemotherapy prior to intravenous infusion of transduced stem cells

3. Stem cells are cultured with a cytokine cocktail in a closed bag system and infected with a retroviral vector expressing the transgene of interest

Figure 33-3 • Characteristic protocol for retroviral treatment of congenital diseases. An improved understanding of stem cell culture has facilitated therapeutic efficacy for the treatment of children with common γ-chain, severe combined deficiency; SCIDX1; and ADA-SCID. Protocols for other congenital diseases are similar. In general, children have a bone marrow harvest either in utero or more commonly in the first few years of life. The harvested cells are briefly cultured, typically with a mixture of cytokines active on primitive hematopoietic stem cells; transfected with a retroviral vector; and then cultured again to allow transgene insertion into the chromosome. The patient may also undergo nonmyeloablative conditioning, typically with busulfan, before receiving the transfected cells. Several studies have shown that supportive therapy must be withdrawn to allow selection of the transfected cells. Typically within a few weeks post-transplant, a high percentage of the circulating T cells are transduced with retroviral vector, resulting in normalization of T-cell numbers and a significant decrease in infections.

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practice have been replication incompetent, the routes of administration and therapeutic targets have been variable and—in part because of this—various toxicity issues have developed. Studies using Advp53 vectors have shown clearly that these can be injected at doses up to approximately 7.5 × 1013 for intraperitoneal administration, and 2.5 × 1013 viral particles has been identified as the maximum total dosage.201 This same particle number, administered via the hepatic artery for the treatment of hepatic metastasis, has also been identified as the maximum total dosage.202 Initially, Adv vectors were delivered based on plaque-forming units, a strategy found to be less rigorous quantitatively when compared with a particle number strategy.203 Yet, despite the known biodistribution and toxicity profile of Adv vectors—including Adv vectors delivered by vascular injection—one child with a non-life-threatening disease, ornithine transcarbamylase deficiency, was dosed with a high number of viral particles, resulting in his untimely demise.204 The incident resulted in a regulatory hold on Adv for a period of time, which limited the use of Adv vectors. This toxicity problem has largely been overcome with increased clinical conservatism. Similarly, the immunologic reaction to the Adv vectors can be reduced as shown with some second- or third-generation vectors (gutless), providing a significant impact on safety, transgene expression, and duration of expression.205 Indeed, Adv vectors have been used to deliver receptors for retroviral vectors to improve their transduction frequency.206 Thus, in addition to “naked DNA” vectors, retroviral and Adv vectors have been used predominantly in the clinic thus far. Clearly, other agents are used, including AAV, α viruses, and herpes vectors, but to a lesser extent. Several factors directly related to vectors have hampered the clinical progression of gene therapy. These include • Inefficient gene delivery, which is associated predominantly with nonviral and retroviral vectors that have reasonable gene delivery efficacy in vitro but disappointing efficacy in vivo • Poor ability to target transgene expression to either cells or tissues of interest to avoid expression of toxic gene products in healthy or unintended target tissue • Short duration of expression due to poor replication and/or stability of episomal vectors and to inefficient or inappropriate integration of vectors into the host genome • Poor production of vectors at high titer, which is developmentally limiting in the cases of retroviral and gutless Adv vectors • Safety, which is a prerequisite for clinical gene therapy trials. Safety includes not only issues of direct toxicity but also the potential for homologous recombination, which has to be maintained at theoretically acceptable levels. Furthermore, targeted genomic integration has also recently been shown to be potentially critical. Because of the challenges associated with targeting and transfection efficiency, the therapeutic strategies currently in use take advantage of the positive aspects of the vectors and limit their deficiencies. There are four overall approaches that reduce the challenges associated with the targeting and delivery of the transgene: 1. Hepatic arterial delivery of Adv-p53 for the treatment of hepatic metastasis202,207,208 2. Intratumoral administration of Adv vectors for head and neck tumors155,209,210 3. Intralesional injection for the treatment of bladder cancer with Adv vectors211 4. Intralesional injection for the treatment of lung cancer The use of Adv vectors to purge hematopoietic stem cell products is also an exciting strategy that initially targeted breast cancer.212–216 It has become a historical approach, however, with the reduction in transplantation for the treatment of metastatic breast cancer. These are the types of approaches that are needed for the successful development of gene therapeutics. The future of gutless vectors or vectors with improved targeting is bright, but at present such

vectors introduce additional deficiencies such as low manufacturing titers. In 2004, Adv-p53 vectors were approved in China for the treatment of patients with HNSCC.1 This approval was based on a single clinical study using an Adv serotype 5 vector engineered to express p53 (Gendicine). In this multicenter, randomized clinical trial, 135 patients were randomized and entered to receive Gendicine in combination with radiotherapy (GTRT) or radiotherapy alone (RT). It was reported that the response rate in the GTRT group was 93%, with 64% showing complete regressions and 29% partial regressions. This contrasts with a phase I study in the United States by Introgen that entered 106 patients, which were reported to have only a 10% tumor response rate, defined as a 30% reduction in tumor size, in patients who received gene therapy alone.217 This response increased to 26.5% for the clinical biomarker defined population, resulting in a progression-free interval of greater than 12 months from initial treatment of patients who had prior chemotherapy. In the overall treatment population, tumor response was associated with a significant increase in survival. The median survival of responders was 16.9 months, a significant increase as compared with 5.4 months for nonresponders. Thus, there is some controversy regarding the Chinese studies; however, the resulting population of patients that have received Gendicine (≥3,000) has provided valuable information. One such observation is that Gendicine has a better response if injected directly into the tumor. This potentially minimizes any immune reaction against the Adv vector and improves the efficiency of p53 delivery. The majority of gene therapy trials are focused on cancer, and to date, approximately 66% of the over 1000 gene therapy trials in the United States have been initiated for this indication. This represents approximately 63% of all clinical trials. The predominance of clinical vectors used is retroviral vectors, although Adv vectors are also being used extensively. The majority of therapeutic strategies are focused on immunotherapy, with the predominance of transgenes used being either cytokine or Ag. When one considers the timeline for most drug development, gene therapy is on target. Although there was considerable initial optimism, the reality is that a period of time and appropriate attention to toxicities, adverse events, and pharmacologic issues (including biodistribution and cell targeting) are required before success can be achieved. Great strides have been made as vector biology begins to catch up with improvements in vectors. It is our expectation that future successes (such as those found with SCID and retroviral vectors) can be expected, although future frustrations are also to be expected and appropriate conservatism must be maintained. One area that has a high potential for success is the utility of vectors such as VV and Adv to deliver Ags to DCs as a vaccine for the treatment of infectious diseases or tumors. Clearly, Adv and VV vectors have innate vector antigenicity, which is limiting these approaches and providing opportunities for “naked DNA” and formulated “naked DNA” for either vaccine priming or boosts. A successful clinical protocol will be achieved only if these liabilities are considered carefully, with appropriate attention to well-designed protocols that take advantage of the positive attributes of vectors and minimize their negative attributes. Within this review, we have attempted to stress the great strides have been made recently with targeting and to illustrate that the future is clearly bright.

ACKNOWLEDGMENTS The authors wish to thank Ms. Kirsten Stites for her assistance with the preparation of the manuscript. This research was supported in part by the Nebraska Research Initiative Programs in Molecular Therapeutics (J.E.T.) and in Gene Therapy (J.E.T.), as well as the Avon-NCI Progress for Patients (PFP) Award Program (P30 CA036727-AV-93P-A1)(K.H.C. and J.E.T.).

Gene Therapy in Oncology • CHAPTER 33

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(ARCA) CN706: a selective cytotoxic for prostatespecific antigen-positive prostate cancer cells. Cancer Res 1997;57:2559–2563. Kurihara T, Brough DE, Kovesdi I, et al: Selectivity of a replication-competent adenovirus for human breast carcinoma cells expressing the MUC1 antigen. J Clin Invest 2000;106:763–771. Hernandez-Alcoceba R, Pihalja M, et al: A novel, conditionally replicative adenovirus for the treatment of breast cancer that allows controlled replication of E1a-deleted adenoviral vectors. Hum Gene Ther 2000;11:2009–2024. Steinwaerder DS, Carlson CA, Otto DL, et al: Tumor-specific gene expression in hepatic metastases by a replication-activated adenovirus vector. Nat Med 2001;7:240–243. Bergelson JM, Cunningham JA, Droguett G, et al: Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science 1997;275:1320–1323. Tomko RP, Xu R, Philipson L: HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc Natl Acad Sci USA 1997;94: 3352–3356. Wickham TJ, Tzeng E, Shears LL, et al: Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins. J Virol 1997;71:8221–8229. Stecher H, Carlson CA, Shayakhmetov DM, et al: Generation of adenovirus vectors devoid of all viral genes by recombination between inverted repeats. Methods Mol Med 2003;76:135–152. Dmitriev I, Krasnykh V, Miller CR, et al: An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Virol 1998;72:9706–9713. Yoshida Y, Sadata A, Zhang W, et al: Generation of fiber-mutant recombinant adenoviruses for gene therapy of malignant glioma. Hum Gene Ther 1998;9:2503–2515. Bouri K, Feero WG, Myerburg MM, et al: Polylysine modification of adenoviral fiber protein enhances muscle cell transduction. Hum Gene Ther 1999;10:1633–1640. Mizuguchi H, Kay MA: Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method. Hum Gene Ther 1998;9: 2577–2583. Mizuguchi H, Xu Z, Ishii-Watabe A, et al: IRESdependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol Ther 2000;1:376–382. Mizuguchi H, Koizumi N, Hosono T, et al: A simplified system for constructing recombinant adenoviral vectors containing heterologous peptides in the HI loop of their fiber knob. Gene Ther 2001;8:730–735. Wickham TJ, Roelvink PW, Brough DE, et al: Adenovirus targeted to heparan-containing receptors increases its gene delivery efficiency to multiple cell types. Nat Biotechnol 1996;14:1570– 1573. Krasnykh V, Dmitriev I, Mikheeva G, et al: Characterization of an adenovirus vector containing a heterologous peptide epitope in the HI loop of the fiber knob. J Virol 1998;72:1844– 1852. Hong JS, Engler JA: Domains required for assembly of adenovirus type 2 fiber trimers. J Virol 1996;70:7071–7078. Wickham TJ, Segal DM, Roelvink PW, et al: Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies. J Virol 1996;70:6831–6838. Bilbao G, Gomez-Navarro J, Curiel DT: Targeted adenoviral vectors for cancer gene therapy. Adv Exp Med Biol 1998;451:365–374.

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Part I: Science of Clinical Oncology 180. Burns JC, Friedmann T, Driever W, et al: Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci USA 1993;90:8033–8037. 181. Reiser J, Harmison G, Kluepfel-Stahl S, et al: Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles. Proc Natl Acad Sci USA 1996;93:15266– 15271. 182. Mitrophanous K, Yoon S, Rohll J, et al: Stable gene transfer to the nervous system using a nonprimate lentiviral vector. Gene Ther 1999;6:1808– 1818. 183. Lewis BC, Chinnasamy N, Morgan RA, et al: Development of an avian leukosis-sarcoma virus subgroup A pseudotyped lentiviral vector. J Virol 2001;75:9339–9344. 184. Kobinger GP, Weiner DJ, Yu QC, et al: Filoviruspseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo. Nat Biotechnol 2001;19:225–230. 185. Beyer WR, Westphal M, Ostertag W, et al: Oncoretrovirus and lentivirus vectors pseudotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concentration, and broad host range. J Virol 2002;76:1488–1495. 186. Kasahara N, Dozy AM, Kan YW: Tissue-specific targeting of retroviral vectors through ligandreceptor interactions. Science 1994;266:1373–1376. 187. Han X, Kasahara N, Kan YW: Ligand-directed retroviral targeting of human breast cancer cells. Proc Natl Acad Sci USA 1995;92:9747–9751. 188. Cosset FL, Morling FJ, Takeuchi Y, et al: Retroviral retargeting by envelopes expressing an Nterminal binding domain. J Virol 1995;69: 6314–6322. 189. Russell SJ, Hawkins RE, Winter G: Retroviral vectors displaying functional antibody fragments. Nucleic Acids Res 1993;21:1081–1085. 190. Somia NV, Zoppe M, Verma IM: Generation of targeted retroviral vectors by using single-chain variable fragment: an approach to in vivo gene delivery. Proc Natl Acad Sci USA 1995;92:7570– 7574. 191. Ager S, Nilson BH, Morling FJ, et al: Retroviral display of antibody fragments; interdomain spacing strongly influences vector infectivity. Hum Gene Ther 1996;7:2157–2164. 192. Schnierle BS, Moritz D, Jeschke M, et al: Expression of chimeric envelope proteins in helper cell lines

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and integration into Moloney murine leukemia virus particles. Gene Ther 1996;3:334–342. Valsesia-Wittmann S, Drynda A, Deleage G, et al: Modifications in the binding domain of avian retrovirus envelope protein to redirect the host range of retroviral vectors. J Virol 1994;68:4609–4619. Nilson BH, Morling FJ, Cosset FL, et al: Targeting of retroviral vectors through proteasesubstrate interactions. Gene Ther 1996;3:280–286. Roux P, Jeanteur P, Piechaczyk M: A versatile and potentially general approach to the targeting of specific cell types by retroviruses: application to the infection of human cells by means of major histocompatibility complex class I and class II antigens by mouse ecotropic murine leukemia virus-derived viruses. Proc Natl Acad Sci USA 1989;86:9079–9083. Etienne-Julan M, Roux P, Carillo S, et al: The efficiency of cell targeting by recombinant retroviruses depends on the nature of the receptor and the composition of the artificial cell-virus linker. J Gen Virol 1992;73 (Pt 12):3251–3255. Cavazzana-Calvo M, Hacein-Bey S, de Saint BG, et al: Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000;288:669–672. Check E: A tragic setback. Nature 2002;420:116– 118. Aiuti A, Slavin S, Aker M, et al: Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 2002;296:2410–2413. Antonia SJ, Mirza N, Fricke I, et al: Combination of p53 cancer vaccine with chemotherapy in patients with extensive stage small cell lung cancer. Clin Cancer Res 2006;12:878–887. Bowles C: Cancer risk clouds gene cures. New Scientist 2003;25:12. Buller RE, Runnebaum IB, Karlan BY, et al: A phase I/II trial of rAd/p53 (SCH 58500) gene replacement in recurrent ovarian cancer. Cancer Gene Ther 2002;9:553–566. Reid T, Warren R, Kirn D: Intravascular adenoviral agents in cancer patients: lessons from clinical trials. Cancer Gene Ther 2002;9:979–986. Hutchins B, Sajjadi N, Seaver S, et al: Working toward an adenoviral vector testing standard. Mol Ther 2000;2:532–534. Raper SE, Yudkoff M, Chirmule N, et al: A pilot study of in vivo liver-directed gene transfer with an adenoviral vector in partial ornithine transcarbamylase deficiency. Hum Gene Ther 2002;13:163–175.

206. Schiedner G, Morral N, Parks RJ, et al: Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity. Nat Genet 1998;18:180– 183. 207. Nathwani AC, Persons DA, Stevenson SC, et al: Adenovirus-mediated expresssion of the murine ecotropic receptor facilitates transduction of human hematopoietic cells with an ecotropic retroviral vector. Gene Ther 1999;6:1456–1468. 208. Sung MW, Yeh HC, Thung SN, et al: Intratumoral adenovirus-mediated suicide gene transfer for hepatic metastases from colorectal adenocarcinoma: results of a phase I clinical trial. Mol Ther 2001;4:182–191. 209. Warren RS, Kirn DH: Liver-directed viral therapy for cancer p53-targeted adenoviruses and beyond. Surg Oncol Clin N Am 2002;11:571–588, vi. 210. Nemunaitis J, Khuri F, Ganly I, et al: Phase II trial of intratumoral administration of ONYX-015, a replication-selective adenovirus, in patients with refractory head and neck cancer. J Clin Oncol 2001;19:289–298. 211. Villaret D, Glisson B, Kenady D, et al: A multicenter phase II study of tgDCC-E1A for the intratumoral treatment of patients with recurrent head and neck squamous cell carcinoma. Head Neck 2002;24:661–669. 212. Pagliaro LC: Gene therapy for bladder cancer. World J Urol 2000;18:148–151. 213. Kim M, Wright M, Deshane J, et al: A novel gene therapy strategy for elimination of prostate carcinoma cells from human bone marrow. Hum Gene Ther 1997;8:157–170. 214. Hirai M, Kelsey LS, Vaillancourt M, et al: Purging of human breast cancer cells from stem cell products with an adenovirus containing p53. Cancer Gene Ther 2000;7:197–206. 215. Hirai M, Kelsey L, Maneval DC, et al: Adenovirus p53 purging for human breast cancer stem cell products. Acta Haematol 1999;101:97–105. 216. Watanabe T, Kuszynski C, Ino K, et al: Gene transfer into human bone marrow hematopoietic cells mediated by adenovirus vectors. Blood 1996;87:5032–5039. 217. Nemunaitis J, Bier-Laning C, Clayman GL, et al: Predictive biomarkers associated with efficacy of adenoviral p53 gene therapy in patients with recurrent squamous cell carcinoma of the head and neck. MCMRC: The 14th International Conference on Gene Therapy of Cancer. Dallas, TX, 2006, p 42.

34

Therapeutic Antibodies and Immunologic Conjugates Nai-Kong V. Cheung

S U M M ARY • Because of their tumor selectivity, monoclonal antibodies offer exceptional opportunities for targeted therapy. • As naked antibodies, they kill tumors by receptor blockade and by actively inducing apoptosis. • Tumor cytotoxicity is mediated in the presence of white cells by activating antibody-dependent cell-mediated cytotoxicity; and in the presence of serum, it is mediated by complement. • The effector functions of antibodies can be greatly enhanced as immunoconjugates, which include radioimmunoconjugates, immunocytokines, immunotoxins, immunoenzymes, immunoliposomes, and cellular immunoconjugates. • Naked antibodies can, on occasion, have overlapping toxicity profiles

O F

K EY

P OI NT S

with chemotherapy and radiation therapies. • Dose-limiting toxicities of immunoconjugates depend on the cytotoxic moiety (e.g., myelosuppression in radioimmunoconjugates) being used. • Antibodies are likely to be most beneficial at the time of minimal residual disease, especially when used in conjunction with standard therapy. • The following antibodies have been licensed by the FDA for specific cancers: Alemtuzumab (Campath): B-chronic lymphocytic leukemia (CD52) Bevacizumab (Avastin): colorectal cancer (VEGF) Cetuximab (Erbitux): colorectal cancer, head and neck cancer (EGFR)

INTRODUCTION The clinical development of antibody therapy was accelerated by the introduction of the hybridoma technique in 1975 and the emergence of recombinant technology.1 Through these innovations, individual plasma cells can be immortalized, and cloning of heavy and light chain repertoires from animals and humans is now possible. In the last three decades, monoclonal antibodies (MAb) have evolved from research tools to inclusion in a rapidly increasing list of licensed pharmaceuticals. They have generated excitement on many fronts and will likely play a pivotal role in the history of cancer medicine (Box 34-1). The clinical utility of MAb for in vitro diagnosis and ex vivo manipulation of blood or stem cells is well recognized. Their role in the treatment and prophylaxis of graft versus host disease is detailed in Chapter 32. The use of B-cell idiotype and anti-idiotypic antibodies as tumor vaccines is described in Chapter 6. This chapter summarizes the application of therapeutic antitumor MAb and immunologic conjugates in cancer therapy.

EFFECTOR MECHANISMS OF MONOCLONAL ANTIBODIES Antitumor MAb can mediate highly effective tumoricidal functions both in vitro and in vivo (Fig. 34-1). These include signaling through

Gemtuzumab ozogamicin (Mylotarg): acute myelogenous leukemia (calicheamicin, CD33) Ibritumomab (Zevalin): non-Hodgkin’s lymphoma (90Y, CD20) Rituximab (Rituxan): non-Hodgkin’s lymphoma (CD20) Tositumomab (Bexxar): non-Hodgkin’s lymphoma (131I, CD20) Trastuzumab (Herceptin): breast cancer (HER2) • In the coming decade, other monoclonal antibodies that are currently in various phases of clinical trial as well as those approved for nononcologic indications could be added to the list. The prospects for further innovation in this maturing modality are highly favorable.

receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC).

Signaling by Receptor Cross-Linking and Receptor Blockade When the antigen is a cell surface receptor, its clustering by multivalent MAb can induce apoptosis.2 Apoptosis increases with hypercross-linking (e.g., CD20 target on lymphoma cells).3–5 Both caspase dependent and independent programmed cell death pathways appear to be involved.6 In AIDS-related lymphoma, anti-CD20 MAb diminishes p38MAPK signaling and Bcl-2 expression, while in non-AIDSrelated lymphoma, signaling through CD20 inhibits AP-1 in addition to NF-κB, leading to downregulation of Bcl-XL, sensitizing lymphoma cells to chemotherapy.7 Direct receptor blockade by MAb has also been reported for EGF-R18 and HER-2 (EFG-R2),9 leading to upregulation of the BH3-only protein Bnip3L, thereby sensitizing tumor cells to chemotherapy.10 MAb inhibition of VEGF-R111 or VEGF-R212 can also enhance the efficacy of chemotherapy.

Cytophilic MAb and ADCC The Fc region of IgG MAb interacts with both activating and inhibitory Fc receptors (FcγR).13 In humans, there are four activating FcγRs: FcγRI (CD64) is a high-affinity FcγR, whereas FcγRIIA

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Table 34-1 Properties of IgG Fc Receptors13

ANTIBODY THERAPY OF CANCER: HISTORICAL PERSPECTIVE

Fc Receptor

1901: Nobel prize awarded to Emil von Behring for work on serum therapy in collaboration with Shibasaburo Kitasato 1908: Nobel prize awarded to Paul Ehrlich for his work on passive immunization 1927: Serotherapy of chronic myelogenous leukemia 1975: Hybridoma technique of Hans Kohler and Caesar Milstein (winners of 1986 Nobel prize) 1980: MAb therapy of lymphoma 1986: FDA approval of MAb as standard pharmaceuticals 1992: Murine 111In-anti-B72.3 for imaging colon and ovarian cancer 1997: Chimeric anti-CD20 (rituximab) for B-cell lymphoma 1998: Humanized anti-HER2 (trastuzumab) for breast cancer 1999: Humanized anti-CD33 immunotoxin for acute myelogenous leukemia 2001: Humanized anti-CD52 (alemtuzumab) for B-chronic lymphocytic leukemia 2002: 90Y-anti-CD20 (Ibritumomab) for B-cell lymphoma 2003: Murine 131I-anti-CD20 (tositumomab) for B-cell lymphoma 2004: Chimeric anti-EGFR (cetuximab) for colorectal cancer and head and neck cancer 2004: Humanized anti-VEGF (Bevacizumab) for colorectal cancer

FcγR1

Distribution on WBC

A

High

PMN, MONO, MΦ, DC

CD32 FcγRIIA

A

Low*

PMN, MONO, MΦ, DC, NK

FcγRIIB

I

Low*

PMN, MONO, MΦ, B-cell

FcγRIIC

A

Low*

PMN, MONO, MΦ

FcγRIIIA

A

Intermediate

MONO, MΦ, NK, DC

FcγRIIIB†

A

Low*

PMN

CD16

A, activating; DC, dendritic cells; hIgG, human IgG; I, inhibiting; MONO, monocytes; MΦ, macrophages; PMN, neutrophils; WBC, white blood cells. *Prefers antibody-antigen complex. † Glycosylphosphatidylinositol-anchored.

kinase C activation and sustained calcium elevation.13 These biochemical cascades trigger phagocytosis, degranulation, cytokine release, and antibody-dependent cell-mediated cytotoxicity. In sharp contrast to activating FcγRs, FcγRIIB is a single-chain receptor that carries the immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic domain. Engagement of this inhibitory receptor downregulates both biochemical and cellular functions. The ratio of activating to inhibitory FcγRs on immune cells, such as dendritic cells, macrophages, and neutrophils, can greatly influence the antitumor properties of MAb. Inflammatory mediators (interferon-γ or C5a) increase activating FcγRs and downregulate inhibitory FcγRIIB, while IL-4, IL-10, and TGF-β upregulate FcγRIIB, thereby raising the thresholds for cell activation. Removing the inhibitory signals by FcγRIIB-blocking antibodies have shown efficacy in preclinical models.13 This is particularly relevant for cross-presentation of antigens that are acquired

3 Multistep targeting

Biotinylated radioactive ligand

ADCC CDC

Affinity for hIgG

CD64

(CD32A), FcγRIIIA (CD16A), and FcγRIIIB (CD16B) are lowaffinity FcγRs. FcγRIIB (CD32B) is the only known inhibitory FcγR (Table 34-1). All FcγRs (except FcγRIIIB) are transmembrane glycoproteins that are anchored on neutrophils by glycosylphosphatidylinositol. Activating FcγRs (with the exception of FcγRIIA) require the accessory γ chain, which carries a cytoplasmic immunoreceptor tyrosine-based activation motif for activation. Immunoreceptor tyrosine-based activation motif becomes tyrosine phosphorylated by members of the Src-kinase family with subsequent recruitment of SH2-containing kinases. These events lead to the activation of phosphatidylinositol 3-kinase and phospholipase-Cγ, followed by protein

1 Naked MAb

Function

Streptavidin

Radionuclide Radioimmunoconjugate Bispecific MAb Tumor cell Cytokine Killer cell

Immunocytokine

Cellular immunoconjugates Immunotoxin Liposome scFv-enzyme 2 Immunoconjugates

scFv

ADEPT Prodrug

Drug

Immunoliposome

Figure 34-1 • Effector mechanisms of monoclonal antibodies. ADEPT, antibody-directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement-dependent cytotoxicity; MAb, monoclonal antibody; scFv, singlechain variable fragment. (Modified from Carter P, Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer 2001;1:118–129.)

Therapeutic Antibodies and Immunologic Conjugates • CHAPTER 34

endocytically through Fc receptors on dendritic cells during the induction of tumor-specific T-cell responses.14 In addition to these FcγRs, a unique class of Fc receptor called FcRB (Brambell)/FcRn (neonatal) is found on endothelial cells and regulates antibody catabolism.15 Although most therapeutic antibodies have been primarily IgGs, both IgA1 and IgA2 can also mediate efficient ADCC by binding to FcαRI (CD89) on human neutrophils and monocytes/ macrophages.16 Certain cancer cells, such as colon carcinoma, lymphoma, leukemia, neuroblastoma, and melanoma, are effectively killed by natural killer (NK) lymphocytes, granulocytes, and activated monocytes in vitro in the presence of specific MAb. Depending on the affinity of the MAb for the individual FcγR, both NK cells (carrying FcγRII and FcγRIII) and neutrophils (bearing all three FcγRs) can mediate efficient ADCC. Because of the high affinity, FcγRI is generally occupied by monomeric IgG in human plasma. Human IgG subclasses (IgG1, IgG2, IgG3, and IgG4) have differential affinity for FcγRII and FcγRIII. Chimeric or humanized IgG1 antibodies (e.g., Lym-1 specific for HLA-DR and ch14.18 for GD2) exploit FcγRIII for lymphocyte ADCC while using FcγRII for myeloid ADCC.17,18 Among the four IgG subclasses, IgG2 has the lowest affinity for the inhibitory receptor FcγRIIB.13 Mouse IgG3 (e.g., 3F8 specific for GD2) can engage both FcγRII and FcγRIII in ADCC,19 despite its low affinity for human FcγRs. The correlation of patient FCGR2A20,21 and FCGR3A polymorphism21,22 with clinical responses to MAb suggests that affinity for Fc receptor can influence antitumor responses in patients. In addition to FcγRs, adhesion molecules are critical for MAb-mediated ADCC. These molecules include CR3 (CD11b/ Cd18),17–19 plus CD66b17 for neutrophil ADCC and LFA-1 (CD11a/ CD18) for lymphocyte ADCC.23 Because cytokines can increase the expression of adhesion molecules, GM-CSF or interferon-γ has been used to activate granulocyte ADCC,18,24–26 and IL-2 has been used similarly for lymphocyte ADCC.27,28 Furthermore, because both GM-CSF and IL-2 expand the effector cell pools, they can have additional benefits in tumor therapy. Optimal combinations of MAb and cytokines in the appropriate clinical setting are being explored.29–31

Complement Activation IgG initiates the classical complement cascade by binding C1q to its CH2 domain. C1q is more avid for human IgG1 and IgG3 than for IgG2 and has no affinity for IgG4.32 CDC potency of individual MAbs is also correlated with its slow off-rate.33 Although some tumor cell lines (e.g., lymphoma and neuroblastoma) are sensitive to CDC, many are resistant to complement because of anticomplement surface proteins such as decay-accelerating factor (DAF, CD55),34–36 homol-

ogous restriction factor (CD59),34,37,38 and membrane cofactor protein (CD46).35–37,39 The effect of complement activation extends beyond direct tumor lysis. Following complement activation, tumor-bound C3b is cleaved rapidly by plasma protease factor I to iC3b. Through CR3 (Mac-1 or αMβ2-integrin) and CR4 (CD11c/CD18, αXβ2integrin) receptors on leukocytes, tumor cells are opsonized.40 C3a and C5a, by-products of complement activation, are also potent mediators of inflammation41 and are chemotactic for phagocytic leukocytes, drawing them to the tumor sites. C5a can also downregulate the inhibitory receptor FcγRIIB13 or induce secondary cytokines to increase vascular permeability for both MAb and effector cells.

CLINICAL APPLICATION OF NAKED MAb DIRECTED AT CANCER CELLS Lymphoma and Leukemia In 1997, the anti-CD20 chimeric antibody rituximab became the first MAb to be approved by the U.S. Food and Drug Administration (FDA) for the treatment of cancer (Table 34-2). In a single-arm multicenter study of 166 patients with relapsed or refractory, lowgrade, or follicular non-Hodgkin’s lymphoma (NHL), rituximab at a dose of 375 mg/m2 four times weekly produced an overall response (OR) rate of 48%, a complete response (CR) rate of 6%, and a partial response (PR) rate of 42%. Median time to progression in responders was 13.1 months.42 In this study, rituximab demonstrated activity in chemoresistant disease (29%) and in patients relapsing after anthracycline therapy (51%). FDA approval was later expanded to include patients with bulky disease, retreatment of responders, and an extended treatment schedule of eight infusions. For most patients, rituximab was well tolerated.43 Severe adverse events that were thought to be secondary to complement activation often occurred with the first infusion,44 especially if there were high numbers of circulating tumor cells. These infusion-related reactions usually appeared 30 to 120 minutes after MAb injection and typically were associated with severe cardiopulmonary events, with deaths (1 year of age) with newly diagnosed stage 4 neuroblastoma.31 Extramedullary toxicities were limited to hypothyroidism, which occurred despite aggressive thyroid protection using potassium iodide, liothyronine (T3), and potassium perchlorate. Intrathecal and intraventricular administration for leptomeningeal carcinomatosis and intratumoral therapy of malignant brain tumors using 131I-81C6 (anti-tenascin MAb) have produced objective responses and prolonged patient survival.146,147 211At-81C6 is an example of α-particle therapy for minimal residual disease in malignant glioma.148 Intraventricular 131I-3F8149 and 131I-8H9150 are also being tested in RIT for leptomeningeal cancers in both children and

Therapeutic Antibodies and Immunologic Conjugates • CHAPTER 34

adults, with highly favorable CSF to blood radiation dose ratios; among children with recurrent neuroblastoma metastasized to the CNS, long-term remissions have been achieved.151

Multistep Targeting or Pretargeting To improve tumor uptake and reduce systemic toxicity, a multistep procedure that pretargets the antibody before the binding of the cytotoxic ligand to the tumor has been employed successfully.152 Generally, a tumor-specific antibody is conjugated to a ligand binder, such as streptavidin or avidin (with high affinity for biotin), or a ligandspecific antibody (binding to metal chelators such as diethylenetriamine pentaacetic acid [DTPA] or 1,4,7,10-tetraacetic acid [DOTA]).98,153 In the first step, these antibody-streptavidin or F(ab′)2streptavidin conjugates (172 to 200 kd) are allowed to localize to tumors in vivo, and any excess is cleared from the blood. A small radiolabeled ligand (or its biotinylated form) is then injected intravenously. The ligand penetrates tissues rapidly and, by virtue of the high affinity interaction, binds tightly to the antibody-conjugate at the tumor site. Unbound ligand is quickly excreted through the kidneys. Because of the short transit time of the toxic ligand (radionuclides or toxins), a substantial improvement in the therapeutic ratio is achievable without sacrificing the percent injected dose per gram in tumor. Antibody pretargeting has improved tumor imaging for colorectal, lung, and medullary thyroid cancers, especially when positron emission tomography radioisotopes are used. Multistep targeting of 110 mCi/m2 of 90Y-DOTA was well tolerated except for doselimiting gastrointestinal toxicity that was thought to be related to MAb NT-LU-10 cross-reactivity with the gut.154 Delayed renal toxicity was also observed. A similar approach that was applied to MAb CC49 in GI cancer155 and anti-CD20 MAb in NHL156,157 achieved tumor doses of 0.289 Gy/mCi and 0.26 Gy/mCi, respectively, although the tumor-to-kidney dose ratio was less than 2.5. A threestep approach, which used biotinylated MAb, followed by avidin/ streptavidin and then by biotinylated radiometal-chelate, was also applied to glioma with encouraging results.100,158 The bispecific antibody pretargeting system takes advantage of a bivalent hapten that binds to the two arms of a tumor-localizing bispecific antibody.92 When anti-CEA bispecific antibody was tested in patients with SCLC159 and in patients with medullary thyroid carcinoma,160,161 an average tumor dose of 0.192 Gy/mCi was achieved, accompanied by tumor stabilization in 45% of patients. Building on these early results, clinical studies combining with chemotherapy are under way.162 Furthermore, pretargeting concepts may be potentially useful in targeting small ligands in addition to radioisotopes.

Immunocytokines Cell-mediated cytotoxicity has been highly effective against tumors in vitro and in animal models. Immunocytokines93,163 have shown remarkable success in activating and redirecting effectors to human tumors. Most of these studies have focused on NK, natural killer T, or T cells93 and granulocytes.18 Antibody-IL-2 immunocytokine can eradicate metastatic murine neuroblastoma while inducing long-term antitumor immunity.93,163 Following initial successes with IL-2 immunocytokine, constructs containing other cytokines also have been tested with encouraging results.93 These include IL-12, tumor necrosis factor, and lymphotoxin. This emerging technology has been successfully applied to a number of antigens and tumor models, including GD2, human epithelial cell adhesion molecule (hEpCAM), CEA, EGF-R, HER2, folate receptor, and B-cell idiotype. More recently, the combination of a plasmid DNA vaccine and IL-2 immunocytokine in the mouse model was shown to be more effective than when either one was administered alone.164 KS-IL-2 (anticolorectal CA) and 14.18-IL-2 (anti-GD2) are both in clinical trials; their toxicity profiles are generally acceptable, but clinical efficacy has yet to be established.

Immunotoxins Ribosome-inactivating toxins can be potent cancer drugs. One major limitation is the lack of tumor selectivity.165 Two-chain toxins (e.g., ricin and diphtheria toxin [DT]) utilize their B chain for cell-binding and their A chain for inhibition of protein synthesis; other toxins (e.g., Pseudomonas exotoxin [PE], Pokeweed antiviral protein, and gelonin) have a built-in receptor for cell attachment. When conjugated to MAb, they become immunotoxins. These toxins can be genetically modified for MAb conjugation and for improved safety profile.94 In recombinant toxins (e.g., PE40, PE38, or diphtheria toxin DAB486), the cell-binding domains are replaced by single-chain variable fragments (scFv).94,165 Various monoclonal MAb have been conjugated to different toxins for clinical trials:165 ricin toxin A chain (RTA conjugated to anti-CD7, anti-CD22, and anti-CD25), DT (anti-IL-2R), and PE (anti-CD25, anti-CD22,166 anti-Lewis Y,167 and anti-HER-2). A common toxicity is the vascular leak syndrome, characterized by marked fluid overload, dyspnea, and sensorimotor neuropathies.168 Deglycosylated RTA devoid of mannose and fucose has reduced hepatic sequestration, allowing longer serum half-life. An OR of 31% (2.6% CR, 29% PR) was achieved in patients with NHL following anti-CD22-deglycosylated RTA treatment.169 Among 16 patients with cladribine-resistant hairy cell leukemia, anti-CD22-dsFv-PE (RFB4[dsFv]-PE38, BL22) induced 11 CR and 2 PR.170 In addition to transient hypoalbuminemia and elevated aminotransferase levels, 2 patients had serious but reversible hemolytic-uremic syndrome. Other highly toxic natural compounds have also been explored recently, such as calicheamicins171 and maytansinoids.172 Gemtuzumab ozogamicin (Mylotarg) is an anti-CD33 antibody that is conjugated to calicheamicin. Acting like a prodrug, calicheamicin is released from the antibody following internalization, forming a diradical that induces double-strand DNA breaks. Gemtuzumab was active in childhood refractory AML173 and achieved a 30% response rate among refractory AML patients 60 years of age or older.174 In contrast, antimucin MAb-calicheamicin conjugate has not been successful to date in solid tumors.175 With most immunotoxins, immunogenicity has been a major constraint, although pegylation may reduce immunogenicity.176

Immunoenzymes for ADEPT and Drug-Antibody Conjugates To enhance the effector functions of MAbs, drugs have been conjugated to MAb for selective tumor delivery. Doxorubicin, melphalan, methotrexate, and vinca alkaloids conjugated to MAb have limited clinical success. BR96-doxorubicin directed at Lewis Y antigen has shown no clinical benefit in phase II trials in breast cancer177 or gastric cancer.178 Another novel approach (ADEPT) uses MAb to deliver a covalently conjugated enzyme to the tumor, which can then activate a nontoxic prodrug.95,179 Despite preclinical successes, ADEPT has been difficult to translate into clinical benefit. Significant impediments to broaden their clinical implementation include immunogenicity of antibody-enzyme conjugate, as well as the presence of endogenous enzymes or endogenous substrates and endogenous inhibitors of these enzymes within the tumors.

Immunoliposomes With advances in liposome technology, several liposomal agents have been licensed for use in cancer patients.180,181 When coated with polyethylene-glycol, uptake by the reticuloendothelial system is inhibited, thereby prolonging residence time in the blood. Concurrent developments in drug-loading technology have improved the efficiency and stability of drug entrapment in liposomes. Although there is passive accumulation of liposomes in tumors through enhanced permeability and retention, their uptake can be greatly enhanced when engrafted with surface antibodies or their derivatives.

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For example, scFv or Fab can target liposomes for uptake into tumors bearing CD19,182 HER-2,183 EGFR,184 and GD2.185 When liposomes fuse with the tumor targets, their contents can be efficiently delivered intracellularly. While their potential is high, the clinical benefit of MAb-targeted liposomes remains to be proven.

Cellular Immunoconjugates with Bisepecific Antibodies Tumor-selective MAb can be rendered cytophilic by conjugation with MAb that are specific for trigger molecules on T-lymphocytes, NK cells, and granulocytes.186–188 These molecules include CD3, CD28, Fc receptors (CD64, CD16), and FcαRI (CD89).97 One binding site of the bispecific antibody engages CD3 on T-cells; the other binding site determines tumor specificity, for example, B-NHL (CD19),189 breast cancer (HER-2),190 and Hodgkin’s lymphoma (CD30).191 Similar successes have been reported for the trigger molecule CD28 for acute lymphoblastic leukemia (CD19 and CD20)192,193 and Hodgkin’s disease.194 A phase I trial of the bispecific (HER-2, CD3) and trifunctional (metastatic breast cancer, T cells and FcγRI/III) antibody at low doses (100 µg per injection) was tolerable, tumor responses being noted in 5 of 15 patients.195 Bispecific MAb targeted at FcγRI can redirect ADCC to specific tumors, including epithelial cancer (EGF-R)196 and breast cancer (HER-2),197 while those directed at FcγRIII have been successful against Hodgkin’s disease (CD30)198 and breast cancer (HER-2).197 Because serum IgG competes for FcγR, bispecific MAb that is made to recognize the FcγR outside its Fc-binding domain is also being tested. Although bispecific MAb can induce generalized cytokine release from leukocytes and trafficking of effector cells into tumors is limited,186 this treatment modality is being actively explored in clinic trials.

IMPROVING THE EFFICACY OF ANTIBODY-BASED CANCER THERAPIES Measures have been taken to improve the efficacy of antibody-based cancer therapies.199 To reduce immunogenicity, MAb have been chimerized and humanized, cloned from phage display libraries,200 or produced in human IgG-transgenic or human transchromosomal

mice (Fig. 34-2). Chimeric MAb are made by joining the antigencombining variable domains of a mouse MAb to human constant domains: mouse VL to human CL and mouse VH to human CH1CH2-CH3.87 In humanized MAb, the antigen-binding loops, known as complementarity-determining regions from a mouse MAb are grafted into a human IgG.201 Human antibodies can also be derived from scFv or Fab phage display libraries,202 which are particularly useful for self-antigens.203 Alternatively, human MAb can be made from hIgG-transgenic mice.204 Because Fc is necessary for antitumor effect, chimerizing mouse MAb with the human IgG1 or IgG3 Fc regions can improve ADCC and CDC functions. Similarly, removing FcγRIIB inhibitory receptor recognition also can enhance antitumor activity.205 Point mutations in the Fc region have increased its affinity for activation receptors or decrease its affinity for the inhibitory receptor.206 Glycosylation of IgG at Asn297 stabilizes the tertiary structure of the CH2 domain, which is critical for effector function.207 Glycosylation depends on the producer line, and increasing the bisected complex oligosaccharides in the Fc region89 or defucosylation has greatly improved ADCC properties of MAbs.90 Complement-dependent cytotoxicity can also be improved by Fc region mutations to increase C1q binding.208 The antigen-binding affinity, molecular architecture, and oligomerization states of MAb can be reengineered to enhance tumor delivery and therapy.209 For example, affinity can be increased by using phage display libraries,210 ribosome display,211 DNA shuffling,212 or yeast display combined with DNA shuffling.213 However, because the binding-site barrier can impede tumor penetration if the MAb has high affinity,214 the optimal MAb may indeed be a lowaffinity IgG binding to a surface antigen that is expressed at high density. In addition, the size of the MAb is critical. ScFv are small (25 kd) and rapidly cleared by the kidney. On the other hand, oligomers with molecular weights in the range of 100 to 200 kd should be ideal for tumor targeting. Besides increasing avidity, oligomerization can increase antitumor activity through a multitude of mechanisms, including CDC/ADCC, induction of apoptosis, growth arrest, and synergy with chemotherapy or immunotoxins.4 While scFv are a powerful building block for polymeric forms or novel fusion proteins,215,216 single-domain antibodies may further expand the possibilities of antibody-based cancer therapies.217

= Complementarity determining regions (CDR)

Human

VH VL

VH

Primatized

scFv VL Decreasing immunogenicity

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Humanized

VH CH1

VL

VH

Chimeric

CL

VH

CH2

VL

CH3 Murine

scFv

VL scFv-fusion protein

Figure 34-2 • Immunogenicity of MAb. CH1, CH2, and CH3, constant region domains of an IgG heavy chain; scFv, single chain variable fragment; VH, variable region of the heavy chain; VL, variable region of the light chain. Red, mouse; blue, human; green, recombinant protein to which scFv is genetically fused.

Therapeutic Antibodies and Immunologic Conjugates • CHAPTER 34

ALTERNATIVE TARGETS FOR ANTICANCER ANTIBODIES Besides the ability to block receptors from interaction with their natural ligand, MAb can inhibit receptor dimerization or receptor interaction with coreceptors.218 HER-2 is a ligand-less member of the ErbB receptor family that functions as a coreceptor with HER-1/ EGFR, HER-3, and HER-4. MAb 2C4 sterically hinders the recruitment of HER-2 into HER ligand complexes and inhibits in vitro and in vivo growth of breast and prostate tumors. The humanized antibody Omnitarg is currently in clinical trial. Most of the MAb targeting effort has been focused on individual tumor cells, but alternative strategies directed at tumor neovasculature,219 tumor stroma,220 or tumor infiltrating T cells221 are promising approaches. Bevacizumab (Avastin), a humanized IgG1 that is specific for vascular endothelial growth factor (VEGF) was effective for metastatic renal cancer222,223 and, when combined with chemotherapy, for non-small-cell lung cancer,224 metastatic CRC,225 and metastatic breast cancer.226 Furthermore, MAb can be made to inhibit homing of angiogenic progenitors (e.g., anti-VLA4 [Natalizumab]227 and anti-VEGF-R1228) or to block the VEGF-R2/KDR (e.g., IMC-1C11, chimeric anti-KDR).229,230 Targeting tumor vasculature may have significant advantages over direct tumor targeting,231 in that endothelial cells, unlike tumor cells, are less likely to acquire resistance. Another angiogenesis target is αVβ3 integrin, which initiates endothelial proliferation, migration, and matrix remodeling.232 In a phase I trial, chimeric IgG1 (MEDI522) that is specific for αVβ3233 was well tolerated, and tumor perfusion was possibly modified. Ipilimumab (also known as MDX-010) is a fully human antibody against human CTLA-4, a molecule on T cells that attenuates their immunocompetence. Ipilimumab is currently being tested in metastatic melanoma as monotherapy or in combination with melanoma-peptide vaccine.221

WHAT IS THE FUTURE ROLE OF MAb AS A TREATMENT MODALITY? Can One Size Fit All? Human tumors and their response to MAb-based therapies are heterogeneous. Although MAb share common structures and properties, the successful translation of their antitumor activity into survival benefit in patients requires a much better appreciation of the clinical biology of each individual tumor type as well as an understanding of the fundamental biology of the antigens being targeted.

Is There an Optimal Time to Use MAb Therapy? It is likely that MAb therapy is most beneficial at the time of minimal residual disease (MRD). Accurate and sensitive measures of MRD will provide objective indicators of tumor response to help guide clinicians to apply this modality more effectively.

What It the Future Role of Antibody Therapy in Treating Cancer? As a rapidly expanding class of pharmaceuticals, MAb are now an important modality for cancer treatment. They have demonstrated antitumor activity in a broad spectrum of malignancies in the last two decades. The successful integration of MAb and immunoconjugates with other treatment modalities has the potential for achieving further improvements in symptom control and patient survival.

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210. Schier R, McCall A, Adams GP, et al: Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site. J Mol Biol 1996;263:551–567. 211. Hanes J, Schaffitzel C, Knappik A, et al: Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat Biotechnol 2000;18:1287–1292. 212. Jermutus L, Honegger A, Schwesinger F, et al: Tailoring in vitro evolution for protein affinity or stability. Proc Natl Acad Sci USA 2001;98: 75–80. 213. Boder ET, Midelfort KS, Wittrup KD: Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc Natl Acad Sci USA 2000;97:10701–10705. 214. Fujimori K, Covell DG, Fletcher JE, et al: A modeling analysis of monoclonal antibody percolation through tumors: a binding-site barrier. J Nucl Med 1990;31:1191–1198. 215. Schultz J, Lin Y, Sanderson J, et al: A tetravalent single-chain antibody-streptavidin fusion protein for pretargeted lymphoma therapy. Cancer Res 2000;60:6663–6669. 216. Kortt AA, Dolezal O, Power BE, et al: Dimeric and trimeric antibodies: high avidity scFvs for cancer targeting. Biomol Eng 2001;18:95–108. 217. Holliger P, Hudson PJ: Engineered antibody fragments and the rise of single domains. Nat Biotechnol 2005;23:1126–1136. 218. Agus DB, Akita RW, Fox WD, et al: Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth. Cancer Cell 2002;2:127–137. 219. Halin C, Neri D: Antibody-based targeting of angiogenesis. Crit Rev Ther Drug Carrier Syst 2001;18:299–339. 220. Hofheinz RD, Al-Batran SE, Hartmann F, et al: Stromal antigen targeting by a humanised monoclonal antibody: an early phase II trial of sibrotuzumab in patients with metastatic colorectal cancer. Onkologie 2003;26:44–48. 221. Peggs KS, Quezada SA, Korman AJ, et al: Principles and use of anti-CTLA4 antibody in human cancer immunotherapy. Curr Opin Immunol 2006;18:206–213. 222. Yang JC, Haworth L, Sherry RM, et al: A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 2003;349: 427–434. 223. Rini BI, Halabi S, Taylor J, et al: Cancer and Leukemia Group B 90206: a randomized phase III trial of interferon-alpha or interferon-alpha plus anti-vascular endothelial growth factor antibody (bevacizumab) in metastatic renal cell carcinoma. Clin Cancer Res 2004;10: 2584–2586. 224. Johnson DH, Fehrenbacher L, Novotny WF, et al: Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small-cell lung cancer. J Clin Oncol 2004;22:2184–2191. 225. Hurwitz H, Fehrenbacher L, Novotny W, et al: Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004;350:2335–2342. 226. Miller KD, Chap LI, Holmes FA, et al: Randomized phase III trial of capecitabine compared with bevacizumab plus capecitabine in patients with previously treated metastatic breast cancer. J Clin Oncol 2005;23:792–799. 227. Polman CH, O’Connor PW, Havrdova E, et al: A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N Engl J Med 2006;354:899–910.

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230. Posey JA, Ng TC, Yang B, et al: A phase I study of anti-kinase insert domain-containing receptor antibody, IMC-1C11, in patients with liver metastases from colorectal carcinoma. Clin Cancer Res 2003;9: 1323–1332. 231. Huang X, Molema G, King S, et al: Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature. Science 1997;275:547–550.

232. Brooks PC, Clark RA, Cheresh DA: Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science 1994;264:569– 571. 233. McNeel DG, Eickhoff J, Lee FT, et al: Phase I trial of a monoclonal antibody specific for alphavbeta3 integrin (MEDI-522) in patients with advanced malignancies, including an assessment of effect on tumor perfusion. Clin Cancer Res 2005;11:7851–7860.

35

Complementary and Alternative Medicine James M. Metz and Heather Jones

S U M M ARY • Complementary and alternative medicine (CAM) therapies are used by a significant number of cancer patients worldwide. • Providing a nonthreatening environment for discussion of CAM will facilitate communication on this topic between physicians and patients. • Many types of CAM may interact with conventional medications or cancer

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treatments to increase the toxicity or decrease the efficacy of the drug or therapy. • Some CAM therapies have been shown to have side effects that mimic those of conventional cancer treatments. • Physicians must warn patients about potential problems with CAM. They also should support those patients using those types of CAM that do not

INTRODUCTION Complementary and alternative medicine (CAM) has infiltrated mainstream medical practices in recent years, driven mainly by patient desires to obtain these treatments. These therapies have gained increased exposure through television, radio, magazines, books, and the Internet. Most health care professionals have limited formal education on the subject and are unable to provide informed responses to questions about complementary and alternative methods. Patients’ expectations of their health care team have expanded to include open discussions of CAM. They no longer accept labeling all of these CAM treatments by health care providers as ludicrous and unfounded. Numerous interactions with conventional medical therapies have been described in the scientific literature, so there are important medical reasons for physicians to understand the utilization of CAM. Medical professionals must be able to converse intelligently with the patient about CAM practices and to provide information about relevant dangers and hoaxes when appropriate. Health care providers also must learn to accept patients’ use of alternative and complementary techniques so long as such modalities are safe. Physicians must ask all patients under their care specifically about alternative and complementary medical practices that may be in use. Patients are more likely to discuss their adoption of these techniques openly when the physician provides a nonthreatening environment for discussion. Patients who have cancer are prime consumers and targets for alternative medical therapies. Many feel that they are in a desperate and hopeless situation. Many simply want to regain control over their lives, and use of CAM is an expression of this desire. This chapter provides a general introduction to the topic of CAM and considers some of the more commonly used CAM therapies. Not every CAM modality in use is covered, because this would be beyond the scope of the discussion; rather, relevant information on commonly used therapies is presented to promote optimal physicianpatient interactions whenever CAM treatments are discussed.

adversely affect conventional cancer treatment. • A number of complementary therapies may be effective for stress reduction and combating pain or nausea. • Health care providers must familiarize themselves with the most common CAM treatments used by patients with cancer so that informed discussions can occur.

DEFINITION OF COMPLEMENTARY AND ALTERNATIVE MEDICINE The terms alternative medicine and alternative therapy have become popular in recent years but do not accurately reflect or encompass the practices for which they are used. Alternative is the generally accepted term that refers to a diverse assortment of philosophies, theories, diagnostic, preventive, and therapeutic practices not generally viewed as arising from or belonging to the modern Western medical paradigm.1 Other popular terms are complementary, unconventional, and integrative medicine. Each of these terms attempts to encompass the practice modalities not common to Western medicine. The terms complementary medicine and integrative medicine often are used to acknowledge the combination of these nontraditional treatment modalities with more conventional views and therapeutic approaches. Although some authors prefer to define alternative medicine as treatment approaches not amenable to combination with conventional therapy,2,3 several other terms have been used to define this subject: unconventional, unorthodox, nontraditional, holistic, and non-Western.1 The National Institutes of Health (NIH) Office of Alternative Medicine established a Panel on Definition and Description, charging it “to establish a definition of the field of complementary and alternative medicine for purposes of identification and research; and to identify factors critical to thorough and unbiased description of CAM systems and practices that would be applicable to both quantitative and qualitative research.”4 The panel defined complementary and alternative medicine as follows: Complementary and alternative medicine is a broad domain of healing resources that encompasses all health systems, modalities, and practices and their accompanying theories and beliefs, other than those intrinsic to the politically dominant health system of a particular society or culture in a given historical period. CAM includes all such practices and ideas self-defined by their users as preventing or treating illness or promoting health and well-being. Boundaries within CAM and between the CAM domain and the domain of the dominant system are not always sharp

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COMPLEMENTARY AND ALTERNATIVE MEDICINE AS DEFINED BY THE NATIONAL INSTITUTES OF HEALTH

In 1992, the National Institutes of Health (NIH) convened a meeting to discuss the major areas of alternative medicine and to direct future research activities.1 As part of this meeting, the group defined the following seven fields of alternative therapy: 1. Alternative systems of medical practice. This field includes “folk” medicine and organized health care systems based on alternative practice. Examples include acupuncture, homeopathy, and naturopathy. 2. Bioelectromagnetics. Researchers in this field study how living organisms interact with electromagnetic fields. Magnetic field therapy is one example of bioelectromagnetics practice. It most often is used to treat osteoarthritis and nonunion of bone fractures. 3. Diet and nutrition. This field includes the use of special diets to improve health. Examples include the macrobiotic diet and orthomolecular medicine. 4. Herbal remedies. This field includes the use of herbs and plants to promote and improve health. Herbal therapy is considered to be the most popular alternative therapy used in the United States. It is used for many conditions. 5. Manual healing methods. Practitioners use touch and manipulation to promote and improve health. Examples include chiropractic therapy, massage therapy, and therapeutic touch. 6. Mind-body interventions. This form of therapy uses the interconnectedness of mind and body to improve health. Examples include psychotherapy, meditation, guided imagery, hypnosis, biofeedback, and prayer. Such interventions most commonly are used to treat nausea and vomiting (particularly for anesthesia- or chemotherapy-induced hyperemesis gravidarum) and postoperative dental pain. 7. Pharmacologic and biologic treatments. This field includes treatment with drugs and vaccines not accepted by mainstream medicine. Examples include the use of shark cartilage, EDTA for chelation therapy (for coronary artery disease), and apiotherapy. EDTA, ethylenediaminetetra-acetic acid.

or fixed. Box 35-1 presents a list of CAM modalities defined by the NIH.

UTILIZATION OF COMPLEMENTARY AND ALTERNATIVE MEDICINE Many studies have been performed to identify the utilization patterns of CAM throughout the world. This section addresses the worldwide use of CAM, which can be influenced by demographic factures such as culture, religion, race, geographic location, and gender of the patient. A number of questionnaire studies have suggested that a significant percentage of cancer patients are using CAM. Estimates range from 9% to 64%, depending on the definition of CAM and the cancer patient population studied.5–10 Some evidence indicates that the use of these treatments within the general U.S. population increased during the past decade.11 Also, studies suggest that many patients do not discuss their use of these treatments with their physicians.8 Risberg and colleagues evaluated 252 cancer patients in Norway and found a 45% likelihood (i.e., cumulative risk) of using CAM among patients under observation or cancer treatment.5 Females were much more likely to use these therapies than males. Liu and associates evaluated 100 patients with advanced cancer receiving conventional cancer treatment in China; 64% were found to be using some form

of CAM, mainly herbal therapies.6 Begbie and colleagues evaluated 319 cancer patients in Australia and found that 22% used CAM treatments; 40% of the users did not discuss them with their physicians.7 Downer and coworkers found that the 16% of 415 patients with cancer surveyed in England used CAM. The typical user of CAM tended to be younger, of higher socioeconomic status, and female.8 In the United States, the use of CAM also is prevalent. In a telephone survey of patients with cancer, 452 of 5047 (9%) admitted to using CAM techniques.9 Mind-body interventions and diet therapies were the most common. A study from the University of Pennsylvania showed that 40% of patients with cancer undergoing radiation therapy were using CAM. However, only 7% admitted to using these therapies during the standard history and physical examination.11 Only after the addition of a few directed questions regarding use of CAM did a majority of these patients reveal that they were using these therapies. Exercise and prayer were specifically excluded as CAM practices in this study. A recent study of patients with stage I and II breast cancer found that a complementary or alternative medical system was used by 57.3% of the patient population.10 When exercise therapy was excluded from the analysis, 40% were using CAM. Younger age and increasing income and educational level were predictors of such use. The definition of CAM can vary significantly between studies, which affects the percentage utilization reported, as noted. Clearly, it is important to understand the precise definitions used in each study when comparisons are made. Very limited information is available concerning the use of CAM therapies in the pediatric oncology population. An interview of the parents of 84 pediatric patients with cancer in The Netherlands found that 26 of 84 children (31%) had used or were using CAM.12 Among these children, 19 of 26 (73%) had suffered a relapse before using these techniques. Fernandez and associates performed a study of pediatric oncology patients in British Columbia and found that 42% of the 366 respondents used some form of CAM treatment.13

CANCER PREVENTION AND COMPLEMENTARY AND ALTERNATIVE MEDICINE A growing body of literature relating cancer prevention and CAM is available to the public. More than 50% of U.S. adults use some type of vitamin, mineral, or other micronutrient supplement.14 Several studies indicate that users of dietary supplements believe that supplements can prevent or treat chronic diseases, such as cancer and cardiovascular disease, despite limited scientific support for the efficacy of such use.15–18 Information about micronutrient supplements is becoming more common in the popular medical literature and is creating increased curiosity and a broader awareness. The explosion of the micronutrient supplement market is compelling physicians to become aware of dietary supplements. Whether or not they are used in clinical practice is a decision for the individual physician. In view of the increasing number of patients who are using micronutrient supplements, however, it is imperative that physicians have a good understanding of this topic (Box 35-2).

Antioxidants Fruits and vegetables appear to be protective against the major cancers.19 People who eat more fruits and vegetables that are rich in carotenoids or who have higher serum beta-carotene levels have a lower risk of cancer, according to randomized trials in human populations.20 The antioxidant vitamins—vitamin A and related compounds such as beta-carotene, as well as vitamins C and E—are prominent components of many fruits and vegetables. Conjecture regarding the micronutrients responsible for this beneficial effect has been extensive because the antioxidant vitamins function as scavengers for DNA-damaging, mutagenic oxygen free radicals.

Complementary and Alternative Medicine • CHAPTER 35 Box 35-2.

WHY PATIENTS USE COMPLEMENTARY AND ALTERNATIVE MEDICINE

The complex trend of public awareness of and use of complementary and alternative medicine (CAM) has grown extraordinarily in the past decade. This seemingly insatiable desire for ancient philosophies and approaches to medical care by the general public seems particularly odd because it comes at a time of extraordinary technological and therapeutic advances. The physician should strive to understand what motivates a particular patient to seek CAM therapies before entering into a discussion with a patient—reaching this understanding can be quite challenging. The clinical literature for the most part has done an excellent job in documenting the incidence and patterns of CAM use but often has overlooked the more important question of why patients choose alternative modes of care. This motivation stems from a complex combination of social, cultural, philosophical, and personal factors that often differ among ethnic groups and disease types. One reason for this phenomenon, no doubt, is the enormous increase in public access to worldwide information available on the Internet and from extensive media coverage. Commercial advertising and continuous exposure through the lay press, ranging from tabloid publications to magazines, medical journals, and books, have vigorously promoted the concepts of disease prevention and healing by unconventional means, striking a responsive (and highly lucrative) chord in a truly global population. Another reason for the popularity of CAM is the escalating cost of modern allopathic medical care. New technologies have been developed at a record pace, producing many medical, surgical, and diagnostic innovations, most of which are unquestionably

improvements but which also are very costly. The expense and the resulting rationing of these new modalities by managed care programs in an attempt to reduce the costs of medical care have placed them out of reach for a considerable segment of the population. The outcome appears to be the creation of a strong public desire for a wide range of CAM modalities to prevent and treat the full scope of human illness. Other reasons sited for CAM use have included an affinity for a holistic or natural approach to healing, the need to manage side effects, dissatisfaction with the mindset of physicians, and an overall failure of conventional therapies to meet patient needs. Additional insight can be gained by reviewing patient assessments of CAM providers. Patients often praise CAM providers for the ability to define an illness, the amount of time provided for patient visits, continued involvement of the same provider over the course of treatment, and the attention to personality and personal experience. The satisfaction with treatments received often is not contingent on clinical improvement with regard to the presenting complaint. Many patients are more informed about complementary and alternative therapies than their physicians are, a situation that, in itself, should encourage physicians to learn more about CAM. CAM therapies offer patients “a participatory experience of empowerment, and authenticity, when illness threatens their sense of intactness and relationship to their world.”* Understanding what motivates patients may better enable physicians to enter into a dialogue with them and encourage a positive physician-patient relationship.

*Pappus S, Perlman A: Complementary and alternative medicine: the importance of doctor-patient communication. Med Clin North Am 2002;86:1–10.

Accumulating epidemiologic evidence suggests that a number of micronutrients may decrease the incidence of cancers of epithelial cell origin. These include vitamin A, vitamin C, vitamin E, and betacarotene. Dietary deficiency of vitamins A, C, and E has been implicated in the development of cancers of the lung, breast, oropharynx, stomach, bladder, prostate, and colon.21–26 Squamous tissues deficient in vitamin A exhibit metaplastic differentiation that can be reversed by administration of vitamin A and related compounds.27 Additional evidence suggests that diets rich in vitamin A and related compounds not only diminish the risk but also are protective against the development of certain cancers. Whereas vitamin C appears to inhibit the formation of carcinogenic nitrosamines that have been associated with the development of gastric cancer, vitamin E inhibits mutagenesis and cell transformation mainly through its antioxidant function.28 Nonetheless, the role of vitamins C and E in neoplastic development remains particularly unclear. Although vitamins C and E function as antioxidants, little evidence is available to support any direct role for these vitamins in the inhibition or reversal of neoplastic growth and development. Several recent studies of dietary supplementation highlight the importance of clinical trials in defining benefit.29 Both the CARET (beta-carotene and retinol) and the alpha-tocopherol–beta-carotene trials suggest that pharmacologic doses of beta-carotene increase the risk of lung cancer among smokers or those with asbestos exposure.30,31 A large, four-arm clinical trial of multiple dietary supplements in 30,000 subjects in Linxian, China, demonstrated no significant effect on cancer incidence. However, those subjects who received a combination of selenium, beta-carotene, and alphatocopherol enjoyed statistically significant lower total and gastric cancer–specific mortality rates.32

Vitamin D, Calcium, and Selenium Among the many minerals required for normal tissue development, calcium and selenium have received the most attention with regard

to carcinogenesis. Laboratory data, as well as preliminary clinical data, suggest a role for calcium deficiency in the development of colon cancer. In epidemiologic studies, an inverse relationship also has been reported between vitamin E, vitamin D, and calcium supplementation and prostate cancer.33 In addition, laboratory studies have shown that the active metabolite of vitamin D, 1,25-dihydroxyvitamin D (calcitriol), inhibits growth of both primary cultures of human prostate cancer cells and cancer cell lines; however, the mechanism by which cellular growth is inhibited has not been clearly defined.34 Recently, a large randomized trial of more than 1000 patients with a history of polyps showed that daily supplementation with 1.5 g of calcium reduced new adenomatous polyp formation by 20%.35 The Nutritional Prevention of Cancer Study by Clark and associates randomized 1312 patients to receive placebo or 200 µg of selenium per day and was designed to evaluate the effect of this supplement on risk of developing new basal cell and squamous cell skin cancers. In the analysis of the primary outcomes, selenium supplementation had no effect on reducing the incidence of these skin cancers. After preliminary analyses showed a reduction in total carcinoma, however, the protocol was modified in 1990 to add total and cancer mortality, as well as the incidence of lung, colon, rectal, and prostate cancers, as secondary endpoints. Analysis of these endpoints revealed a preventive effect for cancers of the lung, prostate, colon, and rectum, but no reduction in risk of breast or bladder cancer.36

Soybeans Soy products are the primary food source for the isoflavone glycosides genistin and daidzin, which are metabolized by colonic microflora to the biologically active aglycones genistein and daidzein. These compounds, along with lignans, are generically named phytoestrogens and have structural similarities to estradiol. Evidence suggests that the consumption of diets rich in soybean products is associated with lower cancer mortality rates, particularly for cancers of the colon, breast, and prostate.37–40

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The soy protein Bowman-Birk trypsin inhibitor (BBI), also found in other beans and peas, now in clinical trials, has been shown to suppress carcinogenesis in laboratory animals and in in vitro transformation systems.40 It is difficult to study the effects of micronutrient supplementation on the formation of cancers, in view of the inherent complexity of carcinogenesis. Information about the mechanisms of chemopreventive agents that inhibit carcinogenesis is still imperfect. Elucidation of how various dietary components inhibit carcinogenesis will be instrumental in the development of novel dietary chemopreventive agents in the future. Results from large trials such as the Women’s Health Initiative Trial and the Selenium and Vitamin E Cancer Prevention Trial (SELECT) will provide a great deal of information regarding the effects of dietary modification and calcium, selenium, vitamin D, and vitamin E supplementation on breast, prostate, and colon cancer.

COMPLEMENTARY AND ALTERNATIVE MEDICINE MODALITIES AND THEIR LEGISLATION AND REGULATION National Center for Complementary and Alternative Medicine In 1990, Congress became aware of the rapidly developing interest in CAM in the United States. In 1991, the Office of Alternative Medicine (OAM) was established at the NIH but did not have formal status as a section or subsection. Initially, it was given an annual budget of approximately $2 million. The budget was increased to $20 million by 1997, and the OAM has since been given increased status by being renamed the National Center for Complementary and Alternative Medicine (NCCAM), with an annual budget of $50 million.41 The charge of the NCCAM is to begin to appraise various CAM products as scientifically as possible through controlled studies. The NCCAM has thus far been slow in reporting results, no doubt at least in part because of the complex nature of conducting meaningful double-blind, clinical trials with CAM products. Numerous studies, however, are now in various stages of completion through the NCCAM and many other facilities throughout the world. At present, 13 institution-affiliated centers of research on CAM exist in the United States, and 75 medical schools in the United States are now teaching integrative medicine courses to their medical students.41

Herbal Medicine Herbs have been used for medicinal purposes for thousands of years. Ancient Egyptians used herbs for the treatment of disease as early as 3000 bc. Almost one fourth of the current pharmacopoeia is derived from botanicals. Digoxin is derived from foxglove, aspirin is derived from willow bark, narcotics are derived from opium poppy, and birth control pills were developed from the Mexican yam. With the advent of modern medical science, people came to believe that synthetic ingredients were more effective than those found in nature, and the use of herbal remedies quickly diminished, especially in the United States. Currently available herbal preparations are sold mainly as nutritional products.42 Today, herbs are widely used in Europe and are again gaining popularity in the United States.43 The most informative longitudinal data on use of herbal medicine derive from two Harvard surveys.43,44 The results indicated that between 1990 and 1997, the use of selfprescribed herbal medicines within the U.S. general population increased from 2.5% in 1990 to 12.1% in 1997. During the same period, the proportion of persons consulting practitioners of herbal medicine rose from 10.2% to 15.1%. These survey studies estimated that in 1997, the entire U.S. population spent approximately $5

billion on herbal medicines. Most of this was out-of-pocket expenditure.43–45

Regulation of Herbals The over-the-counter availability of herbal medicines fosters the notion that these medications are safe, and many casual users have inadequate knowledge about the use of these medications. Users often avoid discussing the use of these medications with their conventional care providers unless specifically asked.46 These factors may set the stage for potential adverse drug reactions and interactions. Consumers in the United States are accustomed to products that have been tested and approved before sale. The U.S. Food and Drug Administration (FDA) functions to oversee the safety of foods, drugs, and medical devices sold in this country. Most herbal products in the United States are considered dietary supplements and thus are not regulated as medicines and are not required to meet the standards for drugs specified in the Federal Food, Drug, and Cosmetic Act. In 1994 the Dietary Supplement and Health Education Act (DSHEA) was passed. This legislation had profound effects on the regulation and marketing of herbal products in the United States. Herbs or other botanicals could be sold as dietary supplements and were not subjected to the rigorous regulations that applied to medicines.47 Herbal products may be produced without the assurance of compliance standards for good manufacturing practice (although such standards are being developed), and they are marketed without previous approval of their efficacy and safety by the FDA. According to the DSHEA, the manufacturer of a herbal preparation is responsible for the truthfulness of claims made on the label and must have evidence that the claims are supported, yet the DSHEA neither provides a standard for the evidence needed nor requires submission of the evidence to the FDA. Under the DSHEA, the manufacturer is permitted to claim that the product affects the structure or function of the body, so long as no claim is made of efficacy for the prevention or treatment of a specific disease, and provided that a disclaimer is supplied informing the user that the FDA has not evaluated the agent. Some of the claims on the labels of herbal products suggest that they can be used to treat disease, and supplementary materials, produced by persons other than the manufacturer, that overtly promote such use may be available where the herbal remedies are sold. According to the DSHEA, the manufacturer is responsible for controlling quality and safety, but if a concern about safety arises, the burden of proof lies not with the manufacturer but with the FDA, which has to prove that the product is unsafe.48,49 Several European countries have implemented guidelines for licensing herbal remedies. In Germany, such products can be registered as medicines on the basis of information in approximately 300 monographs on herbs (“positive” monographs with concise information about terminology, composition, uses, contraindications, side effects, drug interactions, dosage, mode of administration, and actions, and “negative” monographs explaining insufficient benefits or unacceptable risks).50 The European Commission (which governs the European Union) has recently publicized a draft directive on the licensing of traditional herbal preparations. If accepted, this proposal will require all members of the European Union to introduce a simplified procedure for these preparations so that they can receive a “traditional use” registration without the need to present data on efficacy from randomized trials.49,51 The simplified licensing approach allows a premarketing assessment of the quality and safety of a product and facilitates postmarketing surveillance and product recalls.49 Generally, herbal products are not evaluated by the strict preclinical toxicology and pharmacology guidelines that are in place for conventional drugs. Instead, the focus of the clinical trials is on the efficacy of the products. A number of herbal formulations currently are undergoing clinical trials. The FDA has established a hotline for information about herbal products and one for reporting adverse effects.47

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Acupuncture Acupuncture has been practiced in China for more than 5000 years. Around the same time, acupressure, which uses similar points without needles, was developed in Japan. Acupuncture may be recommended by some practitioners for a variety of conditions, but abatement of pain or of nausea and vomiting is of greatest interest for cancer patients. Acupuncturists use fine needles that range in length from 0.5 cm to several centimeters. The needles, usually made of stainless steel or copper, are placed approximately 5 mm deep and are gently manipulated by hand. The needles may be stimulated with a weak electrical current or by heat. Many patients describe a tingling sensation and feel a sense of heaviness in the area where the needles are placed. The classic acupuncture teaching states that a life force called qi (pronounced “chi,” to rhyme with “eye”) dominates every organism and flows along interconnected meridians through the body and crosses at specific points. The meridians surface at various locations denoting the acupuncture points (these points are very similar for both acupuncture and acupressure). The opposing forces of yin and yang must be in balance before qi can get the body’s vital functions to work normally; imbalance causes an accumulation of lactic acid in the muscles. Stimulating the acupoints dissipates the lactic acid and restores the yin-yang balance and the flow of qi. To date, scientists in the Western world have found no evidence to support the existence of qi, yin, or yang. Bing and colleagues observed that stimulation of acupoints with needles activates the subnucleus reticularis dorsalis neurons that send projections to the dorsal horn of the spinal cord at all levels.52 These investigators suggest that this anatomic structure may be involved in the modulation of pain. Grossman and Clement-Jones postulated that the release of endorphins within the nervous system may reduce the perception of pain.53 Of interest, it also has been shown that the acupoints have a similarity in location to some of the anatomic sites used for local and regional anesthesia.54

Cancer Pain and Acupuncture Owing to the widespread use of acupuncture and evolving scientific studies, in 1997, an NIH panel of experts issued a consensus statement for the use of acupuncture. According to this statement, clear evidence supports the effectiveness of acupuncture for the treatment of postoperative and chemotherapy-induced nausea and vomiting, nausea associated with pregnancy, and postoperative dental pain.55 In the palliative care setting, acupuncture is increasingly being used alongside conventional medical treatment for pain and symptom management.56 Select evidence indicates that acupuncture effectively reduces acute perioperative and postoperative pain.57–60 Despite numerous observational studies showing the effectiveness of the technique for chronic cancer pain relief, randomized controlled trials (RCTs) of acupuncture remain scarce. A recent review of the published data revealed only a single high-quality RCT for pain control in cancer,61 by Alimi and colleagues.62 This study showed statistically significant pain relief with auriculoacupuncture in patients with cancer compared with those in two control groups: a “nonpoint” needling control group and a noninvasive control group. Numerous observational and case series suggest that acupuncture may help relieve a variety of pain symptoms, including postsurgical and treatment-induced breast pain, often resulting in reduction in analgesic requirements and an improvement in mobility and circulation.63–70

Non-Pain Syndromes and Acupuncture Nausea and vomiting are harrowing symptoms that commonly accompany cancer treatments. Numerous RCTs and reviews have been performed using the traditional point and have shown efficacy for postoperative nausea and vomiting and chemotherapy-related nausea and vomiting.71,72 Fatigue is a commonly encountered debilitating symptom in cancer and may persist after successful cancer

treatment.73,74 A recent study concluded that acupuncture was worthy of further study in the treatment of fatigue following chemotherapy.75 Studies indicate that pilocarpine-resistant xerostomia can be ameliorated by acupuncture.76 Vasomotor symptoms and hot flashes secondary to cancer treatment and hormone manipulation also have been ameliorated by acupuncture.5,77,78

Regulation of Acupuncture Acupuncturists can be certified in either of two ways: They can complete a formal, full-time educational program that includes both classroom and clinical hours, or they can participate in an apprenticeship program. Acupuncturists also must complete a “clean needle technique” approved course. Medical doctors with training in acupuncture also can obtain board certification. Certification for formally trained acupuncturists is through the National Certification Commission for Acupuncture and Oriental Medicine (NCCAOM). Medical doctors are certified through the American Academy of Medical Acupuncture and must possess a valid medical license. Currently, 37 of the 40 states that regulate acupuncturists require NCCAOM certification. In response to petitions submitted by the acupuncture community, the FDA has reclassified acupuncture needles for general use from class III, the category in which clinical studies are required to establish safety and efficacy, to class II, a category that involves less stringent control by the FDA but does require good manufacturing and proper labeling. Manufacturers are required to label FDA needles for single use only. Use of acupuncture needles for clinical practice would be restricted to qualified practitioners as determined by state practice laws.47

Homeopathy From the Greek words homoios (“like”) and pathos (“suffering”), homeopathy is a system of medicine whose first tenet is the “principle of similars”: A substance that can cause symptoms in a healthy person possibly can encourage self-healing in a person with an illness presenting with similar symptoms. This principle was developed into a practice of medicine in the nineteenth century by the renowned German physician Hahnemann. The theory of homeopathy is rooted in three of Hahnemann’s principles: (1) the “law of similars,” which states that a substance that can cause disease in a well person can cure similar symptoms in the diseased; (2) the “principle of the minimum dose,” which states that by diluting a substance, its curative properties are enhanced and its side effects minimized; and (3) prescribing for the individual, which advocates basing treatment not only on the medical diagnosis but also the patient’s temperament, personality, and emotional and physical responses.58 The principles of homeopathy are not well understood by the public or the medical profession, yet medical consumers are using homeopathic treatments in increasing numbers. In the United States and Europe, the sale of homeopathic medicines increased by 20% to 30% per year in the 1980s and 1990s.80 A recent survey study revealed that many women who have breast cancer turn to alternative treatments in the hope of minimizing adverse reactions to treatment, rather than in the hope of a cure,81 and use homeopathy for the reduction of treatment-related side effects. Two reviews identified 11 additional RCTs of homeopathy between 1997 and 2001.82,83 These trials did not show any strong evidence that homeopathy was effective for any specific condition, and some of the better-designed trials had the least positive results. Even more recently, a comparative study of 100 placebo-controlled trials of homeopathy and conventional medicine found no specific effect for homeopathy when sources of bias were removed.84 Two meta-analyses have been published suggesting that homeopathic remedies are more effective than is placebo alone. However, both studies conclude that the current research and literature in the field do not meet the rigorous, scientific proof needed to establish

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efficacy of homeopathy for specific clinical conditions.85,86 More research is needed before homeopathy can be declared clinically useful for any one condition.

Table 35-1 Six Basic Principles of Naturopathic Medicine

Regulation of Homeopathy

Belief

Resultant Principle

Although in other countries, homeopathic training and certification have been available for decades, the United States has not had fulltime homeopathic schools or accredited professional education for more than 40 years. Only three states—Arizona, Connecticut, and Nevada—license homeopaths. The scope of practice varies but includes the use of substances of animal, vegetable, or mineral origin given in microdoses and prepared according to homeopathic pharmacology. All three states use licensure as a means to authorize practice. These states require a DO or MD degree, as well as certification in the study of homeopathy. Arizona and Nevada have independent examining boards. In Delaware and New Hampshire, the practice of homeopathy is regulated by the state, although under no specific board. The Council of Homeopathic Education has implemented a voluntary certification process that includes a written multiple-choice examination, an oral examination, a videotaped interview, and 10 case reports. A person can be admitted to examination only after completion of a required curriculum and clinical supervision. Thus far, this certification process has not been recognized by the U.S. (or other) medical boards. The FDA currently is attempting to establish guidelines for the regulation of homeopathic products. The FDA takes the position that homeopathic remedies, which are used in the treatment of disease, are by definition drugs and should be regulated. In recent years, the FDA has exempted homeopathic products from the regular drugreviewing process if such drugs have been reviewed and approved by the Homeopathic Pharmacopeia of the United States.47

The belief that the body has the inherent nature to heal itself

The healing power of nature underlies the ability to recover from illness or injury.

Massage Massage therapy has had a long and well-known history, having been known to the ancient Chinese and Japanese and the Greeks, Romans, and Egyptians. The “laying on of hands” was the primary form of healing throughout history in places such as ancient Greece, where Hippocrates wrote that the “physician must be experienced in many things, most especially in rubbing.”87 Massage therapy is considered a form of medical treatment in several countries where it is covered by national health insurance, including China, Japan, Russia, and West Germany. In the United States, massage therapy still is considered a CAM modality. The popularity of massage therapy is growing. National and international massage therapy associations increased their membership by thousands of therapists during the 1990s.88

Regulation and Training for Massage To become certified, massage therapists must complete a formal therapeutic massage bodywork program. They also may be considered for certification if they have training in anatomy, physiology, and kinesiology, as well as formal education and professional experience in bodywork or massage, or both. Massage provider practice acts for the regulation of massage exist in 22 states. Most statutes include directives for the treatment of soft-tissue or muscle, or both. Techniques may include, but are not limited to, friction, beating, and percussion. Types of health conditions treated, depending on the practice act, include maintaining good health, improving muscle tone, and reducing stress. Board certification is through the National Certification Board for Therapeutic Massage and Bodywork and is required in 20 of the 29 states that regulate massage therapists.47,88

Naturopathic Medicine Benedict Lust, a German physician, introduced naturopathy to the United States. He used the term naturopathy (from natur, to indicate

The belief that health and disease result from the interaction of a person’s physical, mental, emotional, genetic, environmental, and social components The belief that the cause of disease, not merely the symptoms, should be treated

Treat the whole person. First, do no harm. Identify and treat the cause. Prevention is the best cure. The physician is a teacher.

The belief that a physician’s major role is to educate, empower, and motivate patients to take responsibility for their own health Shealy CN, Thomas R (eds): The Complete Family Guide to Alternative Medicine. Rockport, Mass., Element Books, 1996.

nature, and pathy, from homeopathy) to encompass all natural approaches to healing. Several healing modalities have been added to the healing module to arrive at modern naturopathy. Naturopathic medicine is far from being a single scientific discipline. The basic principle is that healing comes from within more than from without, and that medicine depends on the healing power of nature to cure (Table 35-1). Naturopathy employs various natural means to empower the patient to reach the ability to self-heal. The tools include lifestyle modifications, nutrition, dietetics, herbs, breathing, education, and hydrotherapy. In addition, naturopaths may elect to use a variety of healing modalities, including acupuncture, botanicals, homeopathy, massage, and Oriental medicine. Naturopaths are the “generalists” of the alternative medicine world. The emphasis of their practice is on prevention, education, and health maintenance.89

Regulation and Training for Naturopathy Naturopathic practitioners undergo a 4-year training program that includes therapies such as homeopathy, clinical nutrition, manipulation, herbal medicine, and hydrotherapy. Naturopaths often may have additional training in Chinese medicine (acupuncture and herbs). Naturopaths are licensed in 12 states. The naturopathic certification examination is administered by the North American Board of Naturopathic Examiners. Each state defines the scope of practice differently, using several adjunctive therapies including acupuncture, biofeedback, and nonprescription medications.47,89

Chiropractic The term chiropractic is derived from two Greek words meaning “done by hand” and is defined as “the diagnosis, treatment and rehabilitation of conditions that affect the neuromuscular system.”89 Chiropractic care origins are in the manipulative health care modalities. It became an organized discipline approximately 100 years ago when Canadian Daniel David Palmer introduced it in the United States. The chiropractic system of health is based on two principles: a testable principle, that the structure and condition of the body influence how it functions and heals, and the untestable principle that the mind-body relationship is instrumental in maintaining health and affects the healing processes. Hence, the focus is on the body’s ability to self-heal, on the nervous system’s role in overall health, and on the interaction between body structure and the functioning of the nervous system. In the past decade, chiropractic care

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gained measured acceptance and has developed into a treatment and wellness modality that is practiced by 55,000 licensed practitioners and used by roughly 10% of the U.S. population. A majority of visits to chiropractors in the United States are for back pain. According to some studies, chiropractic treatment is as beneficial for low back pain as treatment given by primary care providers, orthopedists, and physical therapists.90,91 The evidence for the use of chiropractic for other conditions is less compelling.

national standardization of training and credentialing of ayurvedic providers. A wide variance of training and experience exists among providers.

Regulation and Training for Chiropractic Care

The tendency of CAM modalities to change and shift in popularity does not arise in response to new developments or randomized clinical trials; rather, particular therapies simply go in and out of vogue. Typically, a CAM therapy remains popular for a limited period of time, after which it is replaced by a new and usually nonvalidated CAM therapy. Popular CAM modalities that currently are used by cancer patients are discussed in this section under the categories developed by the NIH (see Box 35-1).

Chiropractic care is licensed in all 50 states, with 45 requiring insurers to include it in their plans. A large variation exists in the scope of practice; certain states restrict the practice to spinal manipulation, whereas others permit different procedures to be performed, such as acupuncture, electromyography, and laboratory diagnosis. The Council on Chiropractic Education (CCE) (www.cce-usa.org) has accredited 17 colleges of chiropractic medicine in the United States. Since 1974, chiropractic education has been established with a 4-year curriculum monitored by the CCE. Admission requirements differ from school to school, although a minimum of 2 years of college education and specific science courses are required by all.47,92 Chiropractors must pass either a state licensing examination or an examination given by the National Board of Chiropractic Examiners.

Ayurveda The word Ayurveda is derived from the Sanskrit ayur, meaning “long life,” and veda, “knowledge.” One of the world’s oldest traditional healing systems, Ayurveda has been documented and practiced in India for thousands of years. Ayurveda is a holistic system that deals with all aspects of life: mind, body, and spirit. The ayurvedic practice is founded on the pooled wisdom of ancient Hindu saints and healers. Ancient Ayurveda was meant essentially to promote health, rather than fight disease. A basic theory of Ayurveda states that everything in the material world is a sign of the unseen universe of energy or life force. The world was created from the unseen universe when the primordial sound created the five fundamental elements responsible for the material world: space, air, fire, water, and earth. These five elements manifest in the human physiology as three life energies called doshas. The three doshas are vata (space and air), pitta (fire and water), and kapha (water and earth). Each dosha, its subdivision, and underlying structures confer a particular characteristic and quality to each person. Health is a state of balance among the mind, body, and consciousness. Several factors can disturb this balance, including congenital and genetic factors, natural tendencies, habits, seasonal factors, and internal and external traumas. The imbalance produced in the doshas disturbs the life force, producing the disease state. Diagnosis is based on identifying the exact quality and nature of the imbalance and correcting it. This is accomplished through a detailed history, inspection, and examination. Radial pulses (three superficial and three deep pulses, bilaterally) and tongue, nail, and eye examinations, among others, are important parts of the ayurvedic diagnostic examination. Treatment consists of amplifying or reestablishing the body’s balance through a combination of interventions, including lifestyle changes, diet modifications, meditation, yoga, breathing exercises, massage, aromatherapy, herbs, and detoxification.93 Studies have documented the favorable effects of regular meditation on reducing cardiovascular risk factors and stress. Further studies investigating the effects of the ayurvedic herbal products on a wide variety of conditions including cancer, aging, and health promotion are ongoing.94

Regulation and Training for Ayurveda Ayurvedic medicine is the progenitor of several CAM disciplines. These include aromatherapy, homeopathy, and massage. There is no

THE CANCER PATIENT AND POPULAR COMPLEMENTARY AND ALTERNATIVE MEDICINE THERAPIES

Mind-Body Techniques The effectiveness of meditation, biofeedback, and yoga in stress reduction and in the control of particular physiologic reactions is well supported by accepted research. The belief that patients can use mental attributes and mind-body work to prevent or cure cancer has not been demonstrated in clinical studies.

Biofeedback Biofeedback manipulates the body’s physiologic responses that are normally controlled by the autonomic nervous system. A biofeedback therapist, of which there are over 10,000 in the United States, can teach a patient how to control many involuntary functions. Some patients learn to control their heart rate, blood pressure, muscle tension, and emotions. Monitoring electrodes are placed on the body or scalp by the biofeedback therapist. The electrodes then are connected to a computer or polygraph, which will emit a noise or signal indicating the intensity or level of the process to be controlled. The patient is instructed to concentrate on influencing the signal. Specific mental exercises are carried out under the direction of the therapist. The patient is asked to visualize certain images that affect mood and in time may become able to identify which mental exercises change the signals. After a number of sessions (usually 8 to 10), the patient may be able to affect certain of the autonomic processes. Researchers at Vanderbilt University performed a randomized study to evaluate the effectiveness of a combination of biofeedback and relaxation training for the reduction of side effects of chemotherapy.95 Biofeedback reduced some indices of physiologic arousal but did not modify the side effects of chemotherapy. However, relaxation training was found to produce a decrease in nausea and anxiety during chemotherapy and a decrease in physiologic arousal after chemotherapy. The researchers concluded that the major benefit of biofeedback was the relaxation training that accompanies the instruction and not the biofeedback alone. The potential benefit from biofeedback therapy for the cancer patient is relaxation and reduction of stress. This can undoubtedly improve quality of life and allows the cancer patient to take an active role in overall management. Biofeedback is a noninvasive procedure. A group of 10 sessions with a biofeedback therapist costs approximately $500. No specific reports in the medical literature have described side effects attributed to the use of biofeedback.

Guided Imagery Guided imagery is a technique that relies heavily on the power of suggestion to create relaxing mental images. It is particularly useful for relieving stress and promoting serenity. Some patients find that it helps them cope more effectively with the impact of the diagnosis and the side effects of treatments.

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In this technique, the therapist instructs participants to visualize a specific image. Sometimes the participant is asked to visualize a mass of cancerous cells being attacked by the immune system, chemotherapy, or radiation therapy. Many patients use guided imagery audiotapes that provide instruction on meditation exercises, guided relaxation, and visualization techniques. Some patients use these tapes while they are undergoing chemotherapy or radiation therapy, or en route to receive treatment. Syrjala and colleagues evaluated relaxation and imagery training along with cognitive-behavioral coping skills for control of oral mucositis pain in patients undergoing bone marrow transplantation.96 These investigators found that patients who received relaxation and imagery training reported less pain than the control groups. No benefit was obtained with the addition of cognitive-behavioral skills, however. The goal of guided imagery is total relaxation. Patients learn breathing exercises to help them attain an “inner calm,” or they try to modify their experience of anxiety or pain by imagining a pleasurable scene or situation. Some patients with cancer find the method effective in promoting relaxation and relieving anxiety. It must be emphasized, however, that no reliable evidence indicates any effect of this technique on disease progression or survival. Guided imagery is a noninvasive therapy. Relaxation and guided imagery audiotapes cost approximately $10 to $20 and are available in local bookstores. Some patients prefer to visit a therapist for individualized training, which may be more expensive. No reports in the medical literature describe any side effects related to guided imagery.

Herbal Medicine and Biological Supplements PC-SPES PC-SPES (PC stands for “prostate cancer,” and SPES is the Latin word for “hope”) is a patented preparation of eight herbs97 (Box 35-3). PC-SPES is the only herbal medicine for prostate cancer that has been subjected to clinical trials. Four clinical trials of PC-SPES have been carried out in the United States and Germany.98–101 These trials have been single-arm, phase I and II studies in patients with prostate cancer and have demonstrated prolonged decreases in prostate-specific antigen (PSA) levels in most of the men in the study. The side effect profile of PCSPES has been suggestive of an estrogenic effect (i.e., breast tenderness, decreased libido, impotence, venous thromboses), and components of PC-SPES contain known phytoestrogens.99 The largest and most recently reported study was a phase II trial in 70 patients with prostate cancer.101 Each patient received 320 mg of dried PC-SPES extract orally, three times a day. All androgendependent patients experienced PSA declines of 80% or more, and 26 patients (81%) experienced PSA decreases to undetectable levels. At 15 months, only one patient exhibited biochemical or objective progression. More than half of the patients with androgenindependent disease had a PSA response, with a median duration of

Box 35-3.

COMPONENTS OF THE HERBAL PREPARATION PC-SPES

Dendranthema morifolium Tzvel. (chrysanthemum) Isatis indigotica Glycyrrhiza glabra L. Ganoderma lucidum Panax pseudo-ginseng Rabdosia rubescens Saw palmetto Scutellaria baicalensis Georgi (skullcap)

response of 18 weeks. In the two androgen-dependent patients with positive findings on bone scans at study entry, one patient’s follow-up scan revealed complete resolution of the osseous lesions, and the second patient’s follow-up scan showed improvement but did not yield normal results. One patient had measurable disease and experienced complete resolution of a bladder mass seen on pelvic CT scan, accompanied by a decline in PSA from 8.9 ng/mL to an undetectable level. PC-SPES generally was well tolerated but was associated with a number of endocrine side effects, including decreased libido, erectile dysfunction, gynecomastia or mastodynia, and hot flashes. Despite these encouraging results, a survival benefit for PC-SPES thus far has not been demonstrated. A worrisome possibility is that PC-SPES may decrease PSA levels while masking increases in tumor growth. PC-SPES also is associated with an increased risk of thromboembolic events.102–104 Of enormous concern, PC-SPES was found to contain warfarin (and SPES, a more generic version for all cancers, to contain alprazolam), prompting the U.S. Food and Drug Administration (FDA) to issue a recall of both products in February 2002.105

Hydrazine Sulfate Cachexia remains a major problem in patients with advanced disease undergoing cancer treatment. There has been interest for a number of years in hydrazine sulfate for combating cachexia seen in cancer patients. Gold evaluated 84 patients with disseminated cancer and found that 59 of the 84 patients (70%) improved subjectively and 14 of the 84 (17%) improved objectively when receiving hydrazine sulfate. It was concluded that the compound may favorably influence nutritional status and clinical outcome in patients with disseminated cancer.106 Enthusiasm has been dampened by three prospective trials that showed no benefit when hydrazine sulfate was added to standard treatment regimens. Loprinzi and coworkers randomized 243 patients with non-small-cell lung cancer and 127 patients with advanced colorectal cancer to receive hydrazine sulfate or a placebo in addition to conventional anticancer regimems and showed no benefit with use of hydrazine sulfate.107,108 Kosty and colleagues randomized 291 patients with advanced non-small-cell lung cancer to receive chemotherapy with or without hydrazine sulfate.109 No benefit was found for pain control, cachexia, or survival in any of these studies. Hydrazine sulfate is not recommended for the treatment of any cancerrelated symptoms, although it remains widely promoted on the Internet.

Shark Cartilage Shark cartilage has gained increased popularity as the basis for an unconventional medical therapy (UMT) for the treatment and prevention of cancer. Shark cartilage was initially promoted by William Lane, PhD, in his book Sharks Don’t Get Cancer and the follow-up book, Sharks Still Don’t Get Cancer. In fact, however, tumors, including malignant tumors, do develop in sharks, in which thyroid and central nervous system neoplasms,110,111 papillomas,110–112 oral cavity cancers, adenomas of the liver, chondromas, and odontomas110 have been observed. Shark cartilage is purported to contain angiogenesis inhibitors. In fact, a modest antiangiogenic effect has been seen in vitro.113 Shark cartilage is supplied in powder and capsule forms. It usually is taken orally but sometimes as an enema. The television news program 60 Minutes gave shark cartilage a huge boost a few years ago. The program reported a Cuban study of 29 patients with “terminal” cancer who were placed on shark cartilage; most “felt better” several weeks thereafter. “Feeling better” is not a reliable endpoint in a scientific study. The National Cancer Institute (NCI) performed a review of the study and found the data to be “incomplete and unimpressive.”114 The 60 Minutes program allegedly refused to broadcast the findings of the NCI.

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A small study on shark cartilage was reported at the American Society of Clinical Oncology in 1997.115 Of the 58 patients with advanced cancer who were given shark cartilage for 12 weeks, not one objective complete response or partial response to shark cartilage was obtained. Only two patients reported significant improvement in the quality of life. Rigorous studies of shark cartilage are ongoing at a number of institutions, but no positive results have yet been published. It has been reported that shark cartilage can cause an elevation of values on liver function tests (LFTs) and frank hepatitis.116 Patients on chemotherapy should be urged not to use shark cartilage enemas because of the risk of infection associated with chemotherapy-associated neutropenia. Shark cartilage is relatively expensive. If it is taken as described by William Lane, the cost of the 16-week program is approximately $3000.

Mistletoe Mistletoe (Viscum album L.) is one of the most commonly used CAM herbal medicines in Europe.5,117–118 The active compounds identified in mistletoe are lectins (glycoproteins) and viscotoxins (proteins). The lectin component has in vitro immunostimulant activity and has been shown to increase the number of peripheral blood lymphocytes, as well as lymphocyte activity, in patients with gliomas.119–121 The viscotoxins have been shown to have direct cytotoxic activity against certain cancer cell lines.122 Mistletoe has been used primarily as an adjuvant to conventional cancer therapies to manage micrometastatic disease. Various mistletoe products have been evaluated in several randomized, controlled clinical trials. An analysis of 11 of the randomized trials of mistletoe published in 1994 found that although 10 of these studies reported improved survival in the mistletoe treatment group, many of these trials had deficiencies in 5 or more of the 10 criteria of good methodology. These methodologic issues have led reviewers to question the validity of these studies and promoted demands for further welldefined studies.123 Recently, two well-designed prospective randomized trials have reported negative results. The effect of adjuvant treatment with mistletoe lectin-1, a standardized mistletoe preparation (Eurixor), was tested in a prospective, randomized clinical trial involving 477 patients with head and neck squamous cell carcinoma.124 The European Organization for Research and Treatment of Cancer (EORTC) has completed a phase III randomized trial of adjuvant treatment with low-dose interferon-α versus interferon-γ versus mistletoe extract (Iscador M) versus no further treatment after curative resection of high-risk stage I or IIB malignant melanoma. No benefit was seen in disease-free survival or overall survival for patients receiving either Eurixor or Iscador.124,125

Ginseng (Panax Ginseng) Ginseng has been touted as enhancing mental and physical strength. It may affect nitric oxide synthesis in the endothelial tissue of the lung, heart, and kidney.126 In addition, effects on serotonin and dopamine may be responsible for its actions. Survey data indicate that breast cancer survivors seeking treatment for fatigue often turn to this drug for amelioration of fatigue.127 A recent randomized, double-blind, placebo-controlled pilot trial was carried out by Kim and colleagues. Fifty-three patients were randomly assigned to receive sung ginseng 3000 mg a day or placebo. Quality of life was assessed using the World Health Organization Quality of Life Assessment and the General Health Questionnaire-12. The investigators concluded that ginseng did improve the quality of life scores of patients and that this intervention warranted further investigation.128 Adverse effects may include irritability, insomnia, and gastrointestinal disturbance. Ginseng may interact with oral anticoagulants, antiplatelet agents, corticosteroids, and hypoglycemic agents.129

Ginger Several studies have demonstrated the efficacy of ginger for the treatment of nausea and vomiting. Most of the investigations involved pregnant women.130–134 A randomized study of ginger for the amelioration of nausea caused by platinum-based chemotherapy showed that ginger is at least equivalent to the commonly used medication metoclopiramide and has less toxicity.135

Hoxsey Regimen and Essiac The Hoxsey regimen, a herbal compound comprising pokeroot, burdock root, barberry root, buckthorn bark, and stillingia root, was used first in 1924 by Harry Hoxsey. The recipe was passed down to him by his grandfather, a farmer who observed a horse cure itself of cancer by eating certain plants. Despite decades during which no supporting data have been forthcoming, the Hoxsey formula remains popular and in use among patients with cancer.80 Essiac is one of the most popular herbal medicines in North America and is a mixture of four herbs given by a Native American healer to nurse Renee Caisse. (“Essiac” is Caisse spelled backward.) Despite a lack of systematic research or documentation of its value, Essiac is promoted and purchased for all forms of cancer.135

Diet and Nutrition Macrobiotic Diet Various dietary regimens have been promoted for both prevention and treatment of cancer. The macrobiotic diet was first described by George Ohsawa (1893–1966). He developed a diet consisting of 10 stages, with each stage more restrictive than the previous one. The final stage consisted of only rice and water. The American Medical Association and various governmental agencies have opposed the macrobiotic diet owing to its restrictive nature. In fact, a number of health problems and even deaths have been reported among persons who have followed the diet.137 The macrobiotic diet subsequently has been modified and is regaining popularity in the United States and generally consists of 50% to 60% whole grains, 20% to 25% vegetables, 5% to 10% beans and sea vegetables, and 5% soups. Some variations of the diet allow small amounts of fish. There may be alterations of the diet depending on the disease process. The Kushi Institute in Massachusetts is a strong proponent of the macrobiotic diet. This institute teaches the macrobiotic diet and lifestyle. Specific foods for the individual cancer patient are recommended. Numerous testimonials supporting the effectiveness of the macrobiotic diet are provided, but no controlled studies have been performed to evaluate the Kushi Institute methods. A number of nutritional deficiencies have been reported in association with the macrobiotic diet. Breast milk from mothers who follow the macrobiotic diet contains less vitamin B12, calcium, magnesium, and saturated fatty acids than in the milk of mothers following “regular” diets.138 Infants of mothers on the macrobiotic diet were found to have retarded growth, fat and muscle wasting, and slowed psychomotor development. Bone mineral content was evaluated in a study of adolescents who had followed a macrobiotic diet and compared with that in control patients without dietary restrictions.139 The bone mineral content was found to be significantly lower in both boys and girls who had followed the macrobiotic diet. The study investigators suggest that this decreased bone density may hold important implications for fracture risk in later life. Machiels and associates reported a rare case of nutritional rickets in a young child due to the macrobiotic diet.140

Megadose Vitamin C: A Closer Look The use of vitamin C for the treatment of cancer has been publicized for many years. Many continue to claim efficacy without strong scientific data to back these claims. Linus Pauling, PhD, and Ewan

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Cameron, MD, claimed that high doses of vitamin C could significantly improve survival in cancer patients. The claim was based on the known antioxidant properties of vitamin C and some epidemiologic evidence that populations with high dietary intake of the vitamin have a decreased risk for development of some types of cancer. These scientists believed that much higher doses than the recommended daily intake of 60 mg/day of vitamin C were needed to prevent free radical damage within the body. Pauling and Cameron reported a study of 100 patients in the terminal stages of cancer treated with megadose vitamin C who had significantly improved survival when compared with historical controls.141 It was recommended that patients with cancer take 10,000 mg of vitamin C daily on the basis of their research. The study was plagued by significant design issues. The “terminal” patients in the vitamin C treatment group all came from Dr. Cameron’s practice, whereas the historical controls were “terminal” patients who came from other sources in the geograhic region. It is conceivable that significant selection bias occurred between Dr. Cameron’s patients given vitamin C and the patients of other physicians who were not offered any additional treatments. Owing to the exceptional reputation of Nobel Laureate Dr. Pauling, investigators at the Mayo Clinic performed a prospective randomized study to evaluate vitamin C. Creagan and colleagues randomized 150 patients with advanced cancer to receive 10 g of vitamin C or a placebo.142 No difference in symptoms, performance status, appetite or survival was found between the two groups. These investigators concluded that high-dose vitamin C had no therapeutic benefit. Dr. Pauling criticized the design of the Mayo Clinic study, claiming the patients had poor performance status and too much prior treatment with chemotherapy. Based on his criticisms, a new trial was launched. Moertel and coworkers randomized 100 patients with advanced colorectal cancer in a double-blind study to receive highdose vitamin C (10 g daily) or a placebo.143 No patient received previous cytotoxic therapy, and all had good performance status. Again, vitamin C showed no advantage over placebo therapy with regard to disease progression, objective improvement in measurable disease, or survival. The researchers concluded that high-dose vitamin C therapy is not effective against malignant disease regardless of whether the patient has had any prior chemotherapy. In a recent trial of oral supplementation with ascorbic acid (vitamin C, 6100 mg/d), dl-alpha-tocopherol (a form of vitamin E, 1050 mg/d), and beta-carotene (vitamin A precursor, 60 mg/d), patients who had stage IIIB or IV non-small-cell lung cancer were randomly assigned to chemotherapy with paclitaxel and carboplatin (72 patients) or to this chemotherapy regimen plus the supplements (64 patients). No significant survival differences were reported, with 1-year survival rates of 33% and 39% and 2-year survival rates of 11% and 16% in the chemotherapy and the combination treatment groups, respectively.144 The argument has been made that the inability to confirm Pauling’s results may reflect the use of oral rather than intravenous vitamin C.9,145 A 1.25-g dose of vitamin C given orally results in a mean plasma level of 135 µmol/L, whereas the same dose given intravenously results in a level of 885 µmol/L. The same study showed that the maximum tolerated oral dose of 3 g every 4 hours increased the plasma level to only 220 µmol/L, whereas the maximum tolerated intravenous dose of 50 g resulted in a plasma level of 13,000 µmol/L. The form of oral vitamin C also affects its bioavailability. A solution of vitamin C has an approximately 40% lower bioavailability than that of a slow-release formula.146 Vitamin C intake also has been examined for the amelioration of toxicity associated with chemotherapy administration. Weijl and colleagues147 investigated the use of oral vitamin C, vitamin E, and selenium for the prevention of cisplatin-induced renal toxicity and ototoxicity. No significant overall effect was demonstrated, but a correlation with plasma levels of the vitamins and lower

toxicity was observed. These investigators, however, concluded that poor compliance or inadequate supplementation may have confounded the results. Song and coworkers148 investigated the use of an intravenous administration of 10 g vitamin C twice with a 3-day interval and an oral intake of 4 g vitamin C daily for a week. They then evaluated demographic data and assessed changes in patients’ reported quality of life after the administration of vitamin C. Quality of life was assessed with the EORTC QLQ-C30 questionnaire. These researchers concluded that high-dose vitamin C therapy did indeed significantly improve quality of life scores. Side effects of megadose vitamin C include diarrhea, formation of renal stones, iron overload, and gastrointestinal discomfort. Ardent supporters of megadose vitamin C remain, however, despite the strong scientific evidence refuting its use in the treatment of cancer. Based on the current scientific literature, megadose vitamin C is not recommended for the prevention or treatment of cancer.

COMPLEMENTARY AND ALTERNATIVE MEDICINE AND TOXICITIES A multitude of potential interactions are possible between conventional cancer treatments and UMTs. Many such interactions are just beginning to be recognized by the medical establishment and reported in reputable scientific journals.149,150 Both renal and hepatic function can be impaired by various UMTs.151–156 Multiple biochemical pathways can be affected, including the lipoxygenase, cyclooxygenase, and cytochrome P-450 pathways.112 Such effects may have an impact on drug concentrations in the body, resulting in increased toxicity or changes in effectiveness of chemotherapy and radiation therapy. Antioxidants may decrease the effectiveness of radiation therapy as a result of the scavenging of free radicals, which can damage DNA, leading to cell death.157 Moreover, many of these therapies have their own side effects that can mimic those of conventional cancer treatments158–177 (Tables 35-2 and 35-3). If the oncologist is not aware

Table 35-2 Potential Adverse Effects of Some Common Herbal and Other Alternative Medicines Preparation/ Medication

Adverse Effect(s)

Ephedra species

Hypertension, tachycardia, stroke, seizures

St. John’s wort

Depression, nausea, hypersensitivity reactions

Amygdalin (Laetrile)

Emesis, headache, dizziness, obtundation, dermatitis

Antineoplastons

Somnolence, confusion

Ginseng

Sedative, diarrhea, headache, hypertension, insomnia, nausea

Echinacea

Hypersensitivity reactions

Kelp

Hyperthyroidism

Saw palmetto

Urinary retention, headache, diarrhea, constipation, hypertension, nausea

Mistletoe

Local irritation, allergic reactions

Shark cartilage

Hepatitis, emesis, constipation

Ginkgo

Emesis, headache

Green tea

Insomnia, emesis, diarrhea, confusion

Hydrazine sulfate

Hepatorenal failure

Goldenseal (Hydrastis canadensis)

Uterine contractions

Data from references 163 to 179.

Complementary and Alternative Medicine • CHAPTER 35

Table 35-3 Potential Interactions between Herbal Medicines and Conventional Drugs Herb(s)

Drug(s)

Potential Interaction/Effect(s)

St. John’s wort

Irinotecan, protease inhibitors, other drugs metabolized by cytochrome P-450

Reduced drug levels

Cyclosporine Oral contraceptives Digoxin Hawthorn flower, devil’s claw, licorice

Digoxin

Alters pharmacodynamics; drug levelmonitoring is prudent

Licorice

Potassium-sparing diuretics

Affects potassium levels

Kelp

Thyroxine

Iodine content of herb may interfere with thyroid replacement

Alprazolam

Additive sedative effects, coma

Kava

Terazosin Evening primrose oil

Anticonvulsants

Lowered seizure threshold

Feverfew, garlic, ginseng, gingko, ginger, dong quai

Warfarin

Altered bleeding time

Yohimbe bark

Centrally active antihypertensive agents

Yohimbine may antagonize guanabenz and methyldopa through its α2-adrenoceptor–antagonistic properties

Phenelzine sulfate

Headache, tremulousness, manic episodes

Estrogens, corticosteroids

Additive effects

Ginseng

Data from references 163 to 176.

that a patient is using a particular UMT, signs or symptoms developing as a result of that UMT may be erroneously attributed to a conventional cancer treatment with established efficacy, which may then may be unnecessarily altered or discontinued. Many forms of CAM are associated with no or minimal risk to a cancer patient; however, this is not true for all such therapies. It is well established that a variety of herbal medications may produce serious side effects. Quality control of these preparations can be a major concern. Issues include variability in biologic potency in different crops, the very realistic possibility of contamination (e.g., by fungal or bacterial organisms), and use of wrong plant species.158 Herbal remedies may contain lead, arsenic, mercury, tin, or zinc, each of which can itself be toxic.159 Immunoaugmentative therapy (IAT) of Burton is based on balancing four protein components in the blood while strengthening the patient’s immune system. The use of various organ extracts from cows and pigs is claimed to selectively suppress tumors and stimulate the immune defense cells.160,161 No studies have shown clinical effectiveness of immunoaugmentative therapy; however, samples of infected material from patients who received IAT revealed evidence of hepatitis virus.161 New toxic effects of a variety of herbal preparations continue to be reported. Kava, for example, a widely publicized natural sleep medication, has been associated with severe liver dysfunction, leading to at least one case of hepatic failure and the requirement for a liver transplant.167 Other herbal medications also have been shown to be associated with hepatotoxicity.168 Laetrile (amygdalin), derived from apricot and other fruit pits, one of the oldest CAM medications, continues to be marketed to the public.178 Amygdalin had been used for centuries, but, in the 1950s it was elevated to new heights under the trade name Laetrile. Proponents of Laetrile claim that proper use of this substance can eradicate cancer entirely (www.worldwithoutcancer.com and www.sumeria. net/health/laetrile.html). Moertel and colleagues carried out a phase

II clinical trial of Laetrile, along with vitamins A, C, E, and B complex and various minerals and pancreatic enzymes, in 178 patients with cancer not previously subjected to conventional cancer treatment and with good performance status. Only one patient, who had gastric carcinoma with cervical lymph node metastases, had a possible short-lived partial 10-week response. All others showed no signs of response. No evidence of disease stabilization was noted. Evaluation of toxicity revealed blood cyanide levels in the ranges known to kill animals and humans in several patients.171 Studies have demonstrated that this drug can produce signs and symptoms of nausea, vomiting, headache, dizziness, and obtundation.170,171 To date, one of the gravest examples of the potential for harm associated with herbal medications is that of the development of renal failure and urothelial carcinoma in persons who used the Chinese herb Aristolochia fangchi.179,180 As a result of a manufacturing error, this herb replaced another preparation (Stephania tetrandra) used in a weight-reducing pill. More than 40 people who took this pill experienced progressive renal failure, and almost 50% subsequently were found to have a urothelial cancer.179 Vitamin toxicities are uncommon but well defined. Megadoses of vitamin A can cause increased intracranial pressure and vomiting in children, and its chronic use in adults can lead to hypercalcemia.181 Vitamin B complex overdose can lead to cardiovascular toxicity including arrhythmias, edema, vasodilation, and allergic reactions. Megadoses of niacin can cause cardiac toxicity with arrhythmia, as well as liver toxicity and peptic ulceration. Long-term high-dose toxicities include gouty arthritis, hyperglycemia, dry skin, and rashes. Vitamin B6 in megadoses can cause peripheral neuropathies, with resulting numbness lasting for weeks. Vitamin C toxicities include the formation of renal stones.182 High-dose vitamin E therapy can interfere with blood coagulation by antagonizing vitamin K and inhibiting prothrombin production. A recent study of vitamin E demonstrated an increased number of strokes in the vitamin E treatment group as compared with the control group.183

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Both acupuncture and chiropractic medicine generally are quite safe; however, they too can be associated with irritating and more serious side effects.184,185 Reported toxic effects of acupuncture include transmission of infectious agents through needle insertion; broken, forgotten, or misapplied needles; pneumothorax; transient hypotension; minor bleeding; contact dermatitis; and pain.184 A small but finite risk of a cerebrovascular accident is always associated with cervical spinal manipulations.185

THE INTERNET AND COMPLEMENTARY AND ALTERNATIVE MEDICINE The Internet has become a hotbed of CAM offerings and information over the past decade. However, patients must be advised to be extremely careful about where the information is being derived and what is being marketed over the Internet. Since the public introduction of the Internet in 1994, this computer resource has experienced exponential growth. In August 2002 it was estimated 64% of the U.S. population (177.6 million people) had access to the Internet.186 This was increased from 56.5% of the population in October 2000. Studies have been performed to specifically evaluate the use of the Internet by patients with cancer to obtain information about CAM. In a study of 921 patients presenting to radiation oncology centers in the United States, it was found 42% of those presenting to an academic medical center and 25% of those presenting to a community medical center were using the Internet to find cancer-specific information.156 A questionnaire study from England showed that 24% of prostate cancer patients were using the Internet to obtain further health information.187 A Canadian questionnaire study of patients with prostate cancer revealed that 35% of patients had used the Internet to obtain cancer-related information.188 Another recent Canadian questionnaire study evaluated 191 cancer patients on the reliance on the news media and the Internet as sources of medical information.189 This study showed that 50% used the Internet to obtain information and 7% used the Internet as their primary source of information. A study reporting on use of the Internet by 295 patients with prostate cancer in the United States showed that 32% were using the Internet to gather information.190 Of interest, 58% of these patients used the Internet to search for information on CAM. Many more patients probably are obtaining Internet-derived information from other people. As indicated by questionnaires placed on OncoLink (http://www.oncolink.upenn.edu), the cancer information resource from the University of Pennsylvania, many patients’ friends and family members are using the Internet to obtain cancerrelated information. Vordermark and colleagues used a questionnaire study to identify where patients were obtaining information.191 Of 139 German radiation oncology patients, 12% had used the Internet to obtain information about their cancer, but an additional 15% received Internet-derived information about their cancer from friends or family members. Of note, only 24% discussed the information obtained from the Internet with their physicians. Yakren and coworkers used a patient survey to analyze the use of media information, including that from the Internet, among cancer patients and their companions at Memorial Sloan-Kettering Cancer Center.192 As indicated by the responses of the 443 patients who returned the completed surveys, 44% of the patients and 60% of the companions reported use of the Internet to obtain cancer-related information. This is very similar to the utilization rate of 41% for patients at the University of Pennsylvania Cancer Center.11 The identification of good Internet sites can be difficult for the nonmedical person searching for information on complementary and alternative medicine. The general public requested 30% of all PubMed searches performed in 1999. A study by Bernstam and associates evaluated the ability of a computer-literate lay user to

perform multiple searches for various question types related to cancer on MEDLINE.193 A blinded investigator then rated each search’s relevancy. The computer user was then given a custom interface to help with the searches through MEDLINE. Overall, significantly higher precision was observed with use of the MEDLINE interface than with unaided novice searching. This finding emphasizes the need for the medical community to help guide patients’ search for medically relevant information on the Internet. Although this study does not specifically evaluate Web searches, these results may be generalizable to patients looking for information on the Internet regarding CAM. It can be a daunting task for the nonmedical person to objectively evaluate the quality of Internet sites, particularly those offering CAM. A recent study by RAND Health reported on the quality of health information on the Internet.194 A variety of health Web sites were evaluated, including 20 major Web sites for breast cancer information. It was found that on average, two to four websites needed to be visited to find more than minimal coverage for at least 75% of the indicators for a topic. Although experts may be able to quickly evaluate the quality and appropriate coverage of a topic, as in the RAND study, this can be very difficult for the patient. Meric and colleagues found that popularity and traffic of breast cancer Websites do not always correlate with quality.195 This finding again emphasizes the need for professionals to help guide the lay public to appropriate medical material on the Internet. Most health care providers have experienced a visit from a patient entering the office with pages of CAM information printed from the Internet. Only a limited number of studies, however, have documented the use of the Internet to find information on CAM. A study from the University of Pennsylvania showed that 53% of cancer patients using the Internet were interested in finding information about CAM.196 Most of these patients were doing so without their health care providers’ knowledge. CAM therapies were purchased over the Internet by 12% of Internet users in this study. However, this study did not specifically evaluate the type of therapy purchased. Health care providers need to familiarize themselves with the therapies offered over the Internet in order to have an informed discussion with their patients regarding these treatments. Particular discussions should emphasize the potential side effects and interactions with conventional cancer treatments.11 A typical search on Yahoo, one of the Internet search engines (http://www.yahoo.com), for “alternative and complementary medicine” reveals greater than 6.14 million different Web site matches. This is increased from 432,000 in 2004. Some of these Web sites provide credible information. Unfortunately, many are designed only to sell a specific product and give false or misleading information. It can be overwhelming for the average person without medical knowledge to sift through and understand the claims presented on many of these sites. Some patients will ask their health care providers for recommendations on evaluating Web sites offering CAM information. It generally is recommended to start with sites managed by major academic centers and the government. These institutions maintain a level of quality outside of the Internet that typically is upheld on these Web sites in keeping with the general philosophies of these organizations. Patients also should be warned to be wary of sites designed to sell a specific product or treatment. Many of these sites do not give unbiased information and are designed strictly to generate revenue. Box 35-4 provides some suggestions to guide patients in evaluating Internet sites. Table 35-4 recommends selected Web sites with reliable information on CAM for the cancer patient and health care provider.

ALTERNATIVE MEDICINE CANCER CLINICS Numerous cancer clinics promoting alternative medicine techniques have formed throughout the world. Many centers are transient, but

Complementary and Alternative Medicine • CHAPTER 35 Box 35-4.

EVALUATING MEDICAL WEBSITES

• Accuracy of information: Websites that post information that is not referenced, or articles that do not state authors and dates for content, should be avoided. • Availability of editorial staff: An online resource should list the names of its editorial staff, the credentials of the people behind the resource. An address and email contact information also should be provided. • Qualifications of editorial staff: Many online resources are run by people who are not qualified to provide medical advice. Fundamentally, there is nothing wrong with this, provided that it is clearly stated. In general, however, the best information is provided by health care professionals who are health care providers themselves. Much of what physicians, nurses, and other professionals are trained to do involves interacting with patients and providing information in the clearest, most appropriate manner. • Freshness of content: Sites with content that is updated regularly are likely to be ones that are best managed and most up to date. • Disclosure of conflicts of interest: Conflicts of interest should either be obvious or clearly disclosed to the users. • Price of information: So far, very few online medical resources are charging for information. Although this may change in the future, open access to information with fees that are minimal should be the rule. If you are being charged for the information, make sure it is not information others are providing for free. • Confidentiality: Most online medical resources will not respond to direct medical inquiries by users. This is due in part to concerns about patient confidentiality, and about accuracy of information either sent to or received by the patient. It is important to ensure that sites that require registration are not releasing contact data without permission. • Reputation: Resources known to be run by reputable institutions are more likely to be providing more timely, accurate, and unbiased information. • Look and “feel”: Resources must balance between having an attractive resource and being able to provide the best possible information to users. Certainly, content rich in graphics is attractive, but if it is poorly organized, or if downloading it takes an inordinate amount of time, it may not be serving its primary purpose. • Navigation and searching: Make sure a site is well organized and easy to navigate and has a good search engine. Reproduced, with slight modifications, with permission from the editors at http:// www.oncolink.org.

some have been established for quite some time and have a large following and have marketed their facilities to the general public. A complete enumeration is beyond the scope of this chapter, but none of the treatments offered by these facilities are known to have shown verifiable beneficial results when compared with conventional medical treatments. Nevertheless, case reports and testimonials abound. It is important that health care providers recognize that these treatments are being promoted to their patients through the Internet and print publications, and by word of mouth. For example, Tijuana, Mexico, has long been a destination for patients seeking alternative medical therapies. The government has acted to close down a number of these centers at various times, but many continue to thrive. At any one time, some 50 to 70 alternative medicine clinics are in operation. It has been estimated that roughly 40,000 people travel to Tijuana in search of alternative treatments each year. Of these patients, 95% are from the United States. Many of these clinics operate in a hospital-type atmosphere, and patients stay for a number of days or weeks for their therapy. A few such clinics are strictly outpatient or day treatment centers. The therapies offered in these centers range from simple dietary management to complex operative treatments. Some of these treatments are innocuous, whereas others may be quite dangerous. For instance, some patients are offered insulin-induced hypoglycemia therapy (IHT). Patients are given insulin to drop the blood glucose level to less than 40 mg/dL and then are infused with a glucose solution incorporating a diluted chemotherapy preparation. The rationale for this treatment is that “starving” cancer cells need more glucose and thus more readily take up the chemotherapy drugs, so no side effects develop. Claims of benefit by these alternative medicine centers have not been substantiated by the outside medical establishment. As confirmed by our own observations in such centers, however, some patients with hormone-responsive tumors such as breast cancer and prostate cancer are receiving hormonal therapies. Also, as mentioned earlier, chemotherapy sometimes is offered along with the alternative treatments. This may account for some for the claims of response to these treatments. These treatments generally constitute out-of-pocket medical expenditures, because these are not covered by medical insurance programs. Table 35-5 shows the estimated cost at some of the more popular centers offering alternative cancer treatments. Some of the prices do not include room and board. Many have added charges that may not become specified until the patient is seen and evaluated at the clinic. All require cash payment before treatment. Travel expenses are not included in the estimates.

Table 35-4 Selected Web Sites with Reliable Information on Complementary and Alternative Medicine Organization

Web Address

American Botanical Council

http://www.herbalgram.org

American Cancer Society

http://www.cancer.org

M.D. Anderson Cancer Center

http://www.mdanderson.org/departments/cimer

Memorial Sloan-Kettering Cancer Center

http://www.mskcc.org/mskcc/html/11571.cfm

National Cancer Institute

http://www.cancer.gov/cancertopics/pdq/cam/cam-cancer-treatment/Patient

National Center for Complementary and Alternative Medicine

http://nccam.nih.gov

Office of Complementary and Alternative Medicine

http://www.cancer.gov/cam

OncoLink (University of Pennsylvania Cancer Center)

http://www.oncolink.org

Quackwatch

http://www.quackwatch.com

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Table 35-5 Popular Unconventional Cancer Therapy Clinics Clinic

Location

Type of Treatment

Duration of Therapy

Kushi Institute

Brookline, Massachusetts

Macrobiotic diet

1 week

Estimated Cost $1,500*

Burzynski Clinic

Houston, Texas

Antineoplastins

6 months

$50,000†

Immuno-Augmentive Centre

Bahamas

Immuno-Augmentive

3 months

$13,100‡

Hosp de Baja California del Sol

Tijuana

Gerson Method

1 month

$20,000§

Bio Medical Center

Tijuana

Hoxsey Herbal

Variable

$3,800||

Center for Cell Specific Therapy

Santo Domingo

Magnet Therapy

Variable

$20,000¶

*Based on published advertising on the Internet of $1,495 for 1 week. Private counseling sessions are an additional $225 each. † Based on telephone quotation of intravenous therapy of $14,000 for the first month and $7,200 for each additional month, which averages 6 months in duration. Cost of the oral formula is $6,000 for the first month and $2,000 for each additional month. ‡ Published advertising fee schedule from the Immuno-Augmentive Centre of $7,500 for the first 4 weeks and $700 per week thereafter, up to 8 additional weeks. Cost of supplies for home maintenance is $50 per week indefinitely. § Based on published advertising on the Internet of $3,990 per week for basic charges, with a recommended stay of 1 month; “. . . actual costs for any individual will become evident only during the course of treatment.” || Based on telephone quotation of $3,500 lifetime supply of Hoxsey Tonic, $300 to $400 for blood work, and $25 consultation fee. ¶ Based on brochure from the Center for Cell Specific Therapy. All patients are charged $20,000 regardless of the length of treatment.

CONCLUSIONS Many patients with cancer, as well as their friends and and family members, are searching for information regarding CAM. Health care providers must become educated on this topic so that they can appropriately guide these patients. CAM is here to stay, and patients need

to be supported in their decision to use complementary therapies that are safe. Health care providers must warn patients of potential or known interactions with conventional medications and treatments when appropriate. Clinical trials evaluating CAM should be developed and encouraged. CAM needs to be held to the same stringent criteria as those in place for conventional treatment modalities.

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Cancer Pain Stuart A. Grossman and Suzanne Nesbit

S U M M ARY

Incidence Major Presenting Symptom of Malignancies • Affects more than 30% of patients undergoing antineoplastic therapy • Moderate to severe pain occurs in over 70% of patients during the later phases of their illness • Significantly affects quality of life • Frequently managed poorly

Etiology of Complication • Can be of nociceptive, neuropathic, or sympathetically maintained origin • Due to direct tumor involvement (70%), evaluation or therapy (20%), or

O F

K EY

P OI NT S

illness unrelated to the malignancy (12 weeks) are principally hypertension and edema resulting from water retention, as well as thromboembolic events. In a study of elderly men treated with MA (800 mg/day), there was an antianabolic effect on muscle with a significant reduction in thigh muscle cross-sectional area despite a significant increase in body weight.52 Thus, administration of MA might actually accentuate the loss of lean body mass in cachectic cancer patients. It is surprising, therefore, that MA and MPA are the only agents that are routinely used for the treatment of weight loss in cancer anorexia and cachexia.

Corticosteroids These include dexamethasone, prednisolone, and methylprednisolone. They induce a temporary effect on symptoms such as appetite, food intake, sensation of well-being, and performance status but

show no beneficial effect on body weight. They tend to be used in patients in the end stages of cancer in an attempt to improve the quality of life.

Agents Undergoing Clinical Evaluation Omega-3 Fatty Acids Cold water fish such as mackerel, sardines, and salmon contain an oil under their skin containing omega-3 polyunsaturated fatty acids, the major components being eicosapentaenoic acid (EPA:20:5:) and docosahexaenoic acid (DHA; 22:6, ω3). Of the two fatty acids, only EPA has been shown to be effective in attenuating weight loss in a murine cachexia model.53 Muscle mass is preserved and protein degradation suppressed through inhibition of the ubiquitin-proteasome pathway. EPA has been shown to attenuate the induction of the ubiquitin-proteasome pathway by PIF in murine myotubes by preventing activation of NF-κB.54 While most of the clinical studies have been carried out with fish oil, two studies have been carried out with EPA either as free acid55 or as the propane diol diester.56 Patients with pancreatic cancer and a median rate of weight loss of 2 kg/month who were administered EPA (6 g/day) showed weight stabilization over the 12-week study period.55 However, in a placebo-controlled randomized study with EPA ester (2 or 4 g daily), patients with advanced gastrointestinal or lung cancer and with a mean weight loss of 18% showed no statistically significant improvement in survival, weight, or other nutritional variables.56 A further uncontrolled study of fish oil capsules (supplying 4.7 g EPA/day) in patients with advanced malignancy and weight loss showed weight stabilization over a 1.2-month period.57 When fish oil was combined with an energy- and protein-dense nutritional supplement, initial studies showed that patients gained 2 kg over a 7-week period and that this represented lean body mass.58 However, in a randomized double-blind trial, intention to treat group comparisons indicated that omega-3 fatty acids did not provide a therapeutic advantage, mainly because of problems with compliance.59 Post hoc analysis, however, showed a net gain of weight and lean tissue and improved quality of life in patients with measured increases in plasma EPA. In addition, total energy expenditure and physical activity level increased.14 The increased physical activity level might reflect an improved quality of life. Interestingly, in a further randomized study, although fewer patients taking the EPA supplement gained 10% or more of baseline weight over a 3-month period than did patients taking MA, the percentage of patients with appetite improvement was similar in the two groups.60 This suggests that EPA could also be used as an appetite stimulant. Further studies are required to confirm the anticachectic activity of EPA.

Thalidomide Thalidomide is being evaluated for the treatment of cancer cachexia owing to its ability to promote weight gain in HIV-infected patients. Thalidomide has been shown to block NF-κB regulated genes through suppression of IκB kinase activity.61 A small study with 10 patients with nonobstructing and inoperable esophageal cancer showed thalidomide (200 mg daily) to increase both body weight (1.29 kg) and lean body mass (1.75 kg), while an isocaloric diet caused a loss of both body weight and lean body mass.62 A further study in 50 patients with advanced pancreatic cancer who had lost at least 10% of their body weight substantiated these results.63 Thus, after 4 weeks, patients receiving thalidomide (200 mg daily) had gained an average of 0.37 kg in body weight and 1 cm3 in arm muscle mass compared with a weight loss of 2.2 kg and arm mass of 4.46 cm3 in the placebo group. After 8 weeks, patients on thalidomide had lost 0.06 kg in weight and 0.5 cm3 in arm mass, while the placebo group had lost 3.62 kg and 8.4 cm3, respectively. These results suggest that thalidomide has the potential to attenuate muscle wasting in cachexia.

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b-Hydroxy-b-methylbutyrate β-Hydroxy-β-methylbutyrate (HMB) is a metabolite of leucine formed by transamination to α-ketoisocaproate in muscle, followed by oxidation of the α-ketoisocaproate in the cytosol of the liver and possibly other tissues. HMB has been shown to attenuate the loss of body weight and skeletal muscle mass in mice bearing a cachexia-inducing tumor by attenuating the increased expression of the ubiquitin-proteasome pathway.64 HMB acts like EPA to attenuate PIF-induced signaling pathways in muscle, leading to an increased proteasome expression by preventing activation of NF-κB.65 Only a single clinical study has been reported on the effect of HMB in cancer cachexia.66 This showed that HMB (3 g/day) in combination with l-arginine (14 g/day) and l-glutamine (14 g/day) produced a 0.95 kg gain in body weight in 4 weeks, whereas control subjects lost 0.26 kg. This gain was the result of a significant increase in fat free mass, and this was maintained over a 24-week study period. Further studies are required to confirm the clinical efficacy of HMB.

Combination Treatment Evidence has been presented to indicate that oxidative stress plays an important role in age-dependent skeletal muscle atrophy.67 Since an increase in oxidative stress has been observed in cancer patients, Mantovani and colleagues68 administered vitamins A, E, and C (antioxidants), polyphenols, omega-3 fatty acids, α-lipoic acid, carboxycysteine, MPA, and a selective COX-2 inhibitor, with or without anti-TNF-α antibodies, to 25 cachectic cancer patients. Over a 4-month period, body weight was significantly increased from 51.3 to 58.1 kg together with lean body mass, appetite, grip strength, and quality of life. It is not certain which components of the treatment were responsible for the beneficial effect, since some have not been tested individually, but combination of agents attacking various components of the cachexia syndrome might be the way forward for improved clinical treatment of cachectic cancer patients. In addition to these treatments there are a number of experimental agents that require clinical investigation: • Adenosine 5′-triphosphate69 • Cyclooxygenase inhibitors (COX-1 and COX-2)70

• • • • • • • • •

Interleukin-471 Interleukin-1572 Ghrelin73 Reseveratrol35 Antibodies to parathyroid hormone–related protein74 Melanocortin-4-receptor antagonists75 Lipoxygenase inhibitors76 Erythropoietin77 β2-adrenergic receptor agonists (e.g., formoterol)78

SUMMARY Progress in basic science has improved our understanding of the cellular mechanisms underlying tissue wasting in cachexia and has provided new molecular targets for therapeutic drug development. Further studies on tumor and host factors involved in the cachectic process, together with their cellular receptors, will aid in drug design and provide rational drug combinations for therapy. Attention in clinical trials is moving away from appetite and total body weight to measurements of lean body mass and physical functioning, which are important factors in maintaining the quality of life. This is not to say that anorexia is not an important factor that needs attention. Feeding is an important social interaction and something in which the family can help in the treatment of severely malnourished patients. Advances in our understanding of neuropeptides and their relationship to cancer anorexia will lead to new developments in this field, although few have received rigorous clinical evaluation. Nutritional therapy could prove to be synergistic with other types of agents. Thus, inhibitors of the activation of NF-κB, such as EPA, HMB, and thalidomide, might be expected to attenuate muscle protein degradation but not improve protein synthesis. This is borne out by clinical trials, which show attenuation of the loss of muscle mass but no marked increase in size. Certain amino acids, principally the branched-chain amino acids, are known to stimulate protein synthesis at the translational level and might be expected to be synergistic with agents targeting muscle protein degradation. Cure of the cancer would of course abolish the need to treat cachexia. However, until this happens, there will be a need for palliative therapy to improve the quality of life of the cancer patient.

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15. Collins P, Bing C, McCullock P, et al: Muscle UCP-3 mRNA levels are elevated in weight loss associated with gastrointestinal adenocarcinoma in humans. Br J Cancer 2002;86:372–375. 16. Clapham JC, Arch JRS, Chapman H, et al: Mice overexpressing human uncoupling protein-3 in skeletal muscle are hyperphagic and thin. Nature 2000;406:415–418. 17. Bing C, Bao Y, Jenkins J, et al: Zinc-α2glycoprotein, a lipid mobilising factor, is expressed in adipocytes and is up-regulated in mice with cancer cachexia. Proc Natl Acad Sci USA 2004;101:2500–2505. 18. Russell ST, Zimmerman TP, Domin BA, et al: Induction of lipolysis in vitro and loss of body fat in vivo by zinc-α2-glycoprotein. Biochim Biophys Acta 2004;1636:59–68. 19. Russell ST, Tisdale MJ: Effect of a tumour-derived lipid-mobilising factor on glucose and lipid metabolism in vivo. Br J Cancer 2002;87:580– 584. 20. Russell ST, Tisdale MJ: The role of glucocorticoids in the induction of zinc-α2-glycoprotein expression in adipose tissue in cancer cachexia. Br J Cancer 2005;92:876–881. 21. Zhang HH, Halbeib M, Ahmad F, et al: Tumor necrosis factor-α stimulates lipolysis in differentiated

Cachexia • CHAPTER 38

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41. Costellli P, Bossola M, Muscaritoli M, et al: Anticytokine treatment prevents the increase in the activity of ATP-ubiquitin and Ca2+dependent proteolytic systems in the muscle of tumour-bearing rats. Cytokine 2002;19:1–5. 42. Li Y-P, Reid MB: NF-κB mediates the protein loss induced by TNF-α in differentiated skeletal muscle myotubes. Am J Physiol 2000;279: R1165–R1170. 43. Guttridge DC, Mayo MW, Madrid LV, et al: NFκB-induced loss of MyoD messenger RNA: possible role in muscle decay and cachexia. Science 2000; 289:2363–2366. 44. Anker SD, Sharma R: The syndrome of cardiac cachexia. Int J Cardiol 2002;85:51–66. 45. Falconer JS, Fearon KC, Ross JA, et al: Acute-phase protein response and survival duration of patients with pancreatic cancer. Cancer 1995;75:2077– 2082. 46. Watchorn TM, Waddell ID, Dowidar N, et al: Proteolysis-inducing factor regulates hepatic gene expression via the transcription factors NF-κB and STAT3. FASEB J 2001;15:562–564. 47. Wigmore SJ, Fearon KCH, Sangster K, et al: Cytokine regulation of constitutive production of interleukin-8 and -6 by human pancreatic cell lines and serum cytokine concentrations in patients with pancreatic cancer. Int J Oncol 2002;21: 881–886. 48. Zoico E, Roubenoff R: The role of cytokines in regulating protein metabolism and muscle function. Nutr Rev 2002;60:39–51. 49. Acharyya S, Butchbach MER, Sahenk Z, et al: Dystrophin glycoprotein complex dysfunction: a regulatory link between muscular dystrophy and cancer cachexia. Cancer Cell 2005;8:421–432. 50. Maltoni M, Nanni O, Scarpi E, et al: High-dose progestins for the treatment of cancer anorexiacachexia syndrome: a systematic review of randomised clinical trials. Ann Oncol 2001;12:289– 300. 51. Mantovani G, Maccio A, Massa E, et al: Managing cancer-related anorexia-cachexia. Drugs 2001;61: 499–514. 52. Lambert CP, Sullivan DH, Freeling SA, et al: Effects of testosterone replacement and/or resistance exercise on the composition of megestrol acetate stimulated weight gain in elderly men: a randomized controlled trial. J Clin Endocinol 2002;87:2100– 2106. 53. Whitehouse AS, Smith HJ, Drake JL, et al: Mechanism of attenuation of skeletal muscle protein catabolism in cancer cachexia by eicosapentaenoic acid. Cancer Res 2001;61:3604–3609. 54. Whitehouse AS, Tisdale MJ: Increase expression of the ubiquitin-proteasome pathway in murine myotubes by proteolysis-inducing factor (PIF) is associated with activation of the transcription factor NF-κB. Br J Cancer 2003;89:1116–1122. 55. Wigmore SJ, Barber MD, Ross JA, et al: Effect of oral eicosapentaenoic acid on weight loss in patients with pancreatic cancer. Nutr Cancer 2000;36:177– 184. 56. Fearon KCH, Barber MD, Moses AG, et al: Double-blind, placebo-controlled, randomised study of eicosapentaenoic acid diester in patients with cancer cachexia. J Clin Oncol 2006;24:3401– 3407. 57. Burns CP, Halabi S, Clamon G, et al: Phase II study of high-dose fish oil capsules for patients with cancer-related cachexia. Cancer 2004;101: 370–378. 58. Barber MD, Ross JA, Voss AC, et al: The effect of an oral nutritional supplement enriched with fish oil on weight-loss in patients with pancreatic cancer. Br J Cancer 1999;81:80–86. 59. Fearon KCH, von Meyenfeldt MF, Moses AGW, et al: Effect of a protein and energy dense n-3 fatty

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acid enriched oral supplement on loss of weight and lean tissue in cancer cachexia: a randomised double blind trial. Gut 2003;52:1479–1486. Jatoi A, Rowland K, Loprinzi CL, et al: An eicosapentaenoic acid supplement versus megestrol acetate versus both for patients with cancerassociated wasting: a North Central Cancer Treatment Group and National Institute of Canada collaborative effort. J Clin Oncol 2004;22:2469– 2476. Kiefer JA, Guttridge DC, Ashburner BP, et al: Inhibition of NF-κB activity by thalidomide through suppression of IκB kinase activity. J Biol Chem 2001;276:22382–22387. Khan ZH, Simpson EJ, Cole AT, et al: Oesophageal cancer and cachexia. The effect of short-term treatment with thalidomide on weight loss and lean body mass. Aliment Pharmacol Ther 2003;17:677– 682. Gordon JN, Trebble TM, Ellis RD, et al: Thalidomide in the treatment of cancer cachexia: a randomised placebo controlled trial. Gut 2005;54: 540–545. Smith HJ, Mukherji P, Tisdale MJ: Attenuation of proteasome-induced proteolysis in skeletal muscle by β-hydroxy-β-methylbutyrate in cancer-induced muscle loss. Cancer Res 2005;65:277–283. Smith HJ, Wyke SM, Tisdale MJ: Mechanism of the attenuation of proteolysis-inducing factor stimulated protein degradation in muscle by β-hydroxy-β-methylbutyrate. Cancer Res 2004;64: 8731–8735. May PE, Barber A, D’Olimpio JT, et al: Reversal of cancer-related wasting using oral supplementation with a combination of β-hydroxy-β-methylbutyrate, arginine and glutamine. Am J Surg 2002;183:471– 479. Muller FL, Song W, Liu Y, et al: Absence of CuZn superoxide dismutase leads to elevated oxidative stress and acceleration of age-dependent skeletal muscle atrophy. Free Rad Biol Med 2006;40:1993– 2004. Mantovani G, Madeddu C, Maccio A, et al: Cancer-related anorexia/cachexia syndrome and oxidative stress: an innovative approach beyond current treatment. Cancer Epidemiol Biomarkers Prev 2004;13:1651–1659. Agteresch HJ, Dagnelie PC, van der Geast A, et al: Randomised clinical trial of adenosine 5′triphosphate in patients with advanced non-smallcell lung cancer. J Natl Cancer Inst 2000;92: 321–328. Lundholm K, Daneryd P, Korner U, et al: Evidence that long-term COX-treatment improves energy homeostatis and body composition in cancer patients with progressive cachexia. Int J Oncol 2004;24:505–512. Sturlan C, Beinhauer BG, Oberhuber G, et al: In vivo gene transfer of murine interleukin-4 inhibits colon-26-mediated cancer cachexia in mice. Anticancer Res 2002;22:2547–2554. Quinn LS, Anderson BG, Drivdahl RH, et al: Overexpression of interleukin-15 induces skeletal muscle hypertrophy in vitro: implications for muscle wasting disorders. Exp Cell Res 2002;280: 55–63. Neary NM, Small CJ, Wren AM, et al: Ghrelin increases energy intake in cancer patients with impaired appetite: acute, randomised, placebocontrolled trial. J Clin Endocrinol Metab 2004;89:2832–2836. Onuma E, Tsunenari T, Saito H, et al: Parthyroid hormone-related protein (PTHrP) as a causative factor of cancer-associated wasting: possible involvement of PTHrP in the repression of locomoter activity in rats bearing human tumor xenografts. Int J Cancer 2005;116: 471–478.

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in advanced pancreatic cancer: correlation of clinical data with pharmacokinetic and pharmacodynamic endpoints. Ann Oncol 2000;11:1165–1170. 77. van Halteren HK, Bongalrts GPA, Verhagen CAM: Recombinant human erythropoietin attenuates

weight loss in a murine cancer cachexia model. J Cancer Res Clin Oncol 2004;130:211–216. 78. Busquets S, Figueras MT, Fuster G, et al: Anticachectic effect of formoterol: a drug for potential treatment of muscle wasting. Cancer Res 2004; 64:6725–6731.

39

Nausea and Vomiting John D. Hainsworth

S U M M ARY

Incidence • Most common chemotherapyassociated toxicities • More frequent and severe with repetitive doses of chemotherapy • Significant impact on quality of life and can influence patient compliance with treatment

O F

K EY

P OI NT S

• Mechanism of delayed nausea and vomiting (more than 24 hours after chemotherapy) unknown

Evaluation of the Patient • Risk factors, including type of chemotherapy (drugs, doses, schedule), age, sex, and prior alcohol use, should be assessed before treatment.

Etiology of Complication

Grading of Complication

• Acute nausea and vomiting often mediated by activation of serotonin type 3 receptors in the gastrointestinal tract

• Episodes of vomiting should be recorded (number, duration, time to onset); severity of nausea can be graded using visual analog scale.

INTRODUCTION Nausea and vomiting are common side effects associated with systemic chemotherapy, and are among the adverse effects most feared by patients.1,2 Although these complications of treatment are usually self-limiting and seldom life-threatening, the deleterious effects on nutritional status and quality of life can be substantial. Many of the recently introduced targeted agents have less potential to produce nausea and vomiting. However, intensive combination chemotherapy continues to be the cornerstone of treatment for many types of cancer, ensuring that antiemetic therapy will continue to be an integral aspect of supportive care. Antiemetic therapy has improved dramatically during the last 20 years. With optimum treatment, most patients receiving highly emetogenic chemotherapy do not experience any nausea or vomiting during the 24 hours after treatment.3–11 However, delayed symptoms are more common and are often underestimated by treating physicians and nurses.12 Accurate assessment of delayed nausea and emesis is essential in providing maximal intervention with recently available agents. The identification of potent new antiemetics has been made possible by an improved understanding of the physiology of the emetic reflex. Critical assessment of the optimal use of new agents for patients receiving chemotherapy has been facilitated by the development of reproducible methods of assessing nausea and vomiting, and by the conduct of carefully designed, randomized clinical trials.

PHYSIOLOGY OF THE VOMITING REFLEX The pioneering work of Borison and Wang13 more than 40 years ago provided the basis for understanding the vomiting reflex. In studies

Treatment • Optimal treatment provides complete control of acute nausea and vomiting in most patients receiving highly emetogenic chemotherapy regimens; only 15% of patients have severe nausea and vomiting. • Treatment of delayed nausea and vomiting has recently improved with the introduction of aprepitant and palonosetron; however, as many as one-third of patients still experience this complication.

using ablative techniques and electrical stimulation with microelectrodes (primarily in decerebrate cats), these investigators proposed the existence of two distinct sites in the brain stem believed to be critical for the control of emesis. The first of the sites, the so-called vomiting center, was thought to be located in the lateral reticular formation of the medulla. Electrical stimulation of this site triggered the vomiting reflex, whereas ablation prevented the vomiting induced by a variety of stimuli. The vomiting center was thought to be located adjacent to the other structures involved in the coordination of vomiting, including the respiratory, vasomotor, and salivary centers, and cranial nerves VIII and X. More recent studies have suggested that the “vomiting center” is actually not anatomically discrete but that the initiation of the vomiting reflex is controlled by a complex system of networks located in the nucleus tractus solitarius.14,15 The networks in this area control complex patterns of motor activity such as the vomiting reflex and are more accurately described as “central pattern generators.” The second important center identified by Borison and Wang was the chemoreceptor trigger zone (CTZ), located in the area postrema at the ventral aspect of the fourth ventricle. This center, located outside the blood-brain barrier, is exposed to various noxious agents borne in the blood or cerebrospinal fluid. Although electrical stimulation of the CTZ does not produce vomiting, intimate connections to the vomiting center permit stimulation of this center after exposure to blood-borne toxins. Ablation of the CTZ abolishes vomiting induced by these agents. Although these concepts have been retained and are integral to the current understanding of the vomiting reflex, several other important components have also been recognized. Input from the gastrointestinal tract, predominantly through afferent vagal fibers, is critical in initiating the vomiting reflex after ingestion of noxious

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Cerebral cortex

• Anticipatory emesis

• Motion sickness • Inner ear disorders Chemoreceptor trigger zone

Vestibular center

Vomiting center • Drugs opiates anesthetic agents cardiac glycosides cancer chemotherapy • Metabolic abnormalities uremia ketoacidosis hypoxia

Figure 39-1 • Schematic diagram of the various pathways for initiation of the vomiting reflex. Clinical syndromes mediated by each mechanism are illustrated.

• Ingested toxins • Cancer chemotherapy • Radiation therapy

Peripheral receptors (vagal, splanchnic)

substances.16 Incoming vagal afferents connect with the vomiting center directly; an intact CTZ is not essential when vomiting is initiated by this mechanism. It is now known that in addition to ingested substances, some blood-borne substances, including chemotherapeutic agents, can trigger the vomiting reflex through activation of the vagal afferent mechanism. Two additional components of this complex system involve the vestibular apparatus and the higher brain stem and cortical structures. The vestibular system is involved primarily in initiating the vomiting reflex in motion sickness. Input from higher cortical centers seems to be critical in a variety of conditions, including anticipatory emesis seen for patients who have previously experienced chemotherapy-induced emesis. The various components of the vomiting reflex are illustrated diagrammatically in Figure 39-1, along with the clinical situations in which they are operative. Given the complexity of this system, it is not surprising that different pharmacologic approaches are necessary to control vomiting of different etiologies. Improved understanding of the neurochemistry of the emetic reflex has been important in developing antiemetics with new mechanisms of action. The initial focus of such investigation was the area postrema, where receptors for a large number of neuroactive agents have been identified.17–19 Many of these neurotransmitters (e.g., dopamine, histamine, acetylcholine, norepinephrine, substance P) are in themselves emetogenic agents. The development of pharmacologic agents that block specific sets of receptors (e.g., dopamine, neurokinin-1) has resulted in the identification of valuable antiemetics, and it is likely that continued efforts in this area will be productive of additional valuable agents in the future. In addition to neurotransmitters located in the CTZ, type 3 serotonin (5-hydroxytryptamine 3 or 5-HT3) receptors are present in large quantities on vagal and splanchnic afferents within the gastrointestinal tract.20 These peripheral receptors are pivotal in the initiation of the acute nausea and vomiting caused by cisplatin and other strongly emetogenic chemotherapeutic agents; inhibition of this pathway by specific 5-HT3 receptor antagonists results in highly effective antiemetic therapy.21

CLINICAL FEATURES OF CHEMOTHERAPYINDUCED EMESIS Clinical Syndromes Chemotherapy-induced nausea and vomiting can be subdivided into three distinct clinical syndromes, each having specific therapeutic implications. These syndromes and their clinical correlates are defined here; treatment approaches are considered later in the chapter. Because nausea and vomiting are common symptoms among patients with cancer, etiologies other than chemotherapy should also be considered. Among the diverse causes of nausea and vomiting among patients with cancer are intestinal obstruction, liver metastases, central nervous system involvement, and other medications (particularly narcotic analgesics). These etiologies should be considered especially when the time course or duration of nausea and vomiting is unusual for the known chemotherapy-induced syndromes.

Acute Nausea and Vomiting Acute nausea and vomiting after the administration of chemotherapy occur within 24 hours after the chemotherapy dose. The nausea and vomiting during this phase are the most severe, hence the emphasis on therapeutic intervention during this phase. With most chemotherapeutic agents, acute nausea and vomiting begin 1 to 2 hours after intravenous administration. This delay in onset argues against a direct effect at the CTZ, which would be expected to produce emesis within minutes of intravenous drug administration. A peripherally mediated vomiting reflex, probably serotonin release from small intestinal mucosa, offers a better explanation of the delayed onset of emesis.22 The onset of nausea and vomiting after the intravenous administration of cyclophosphamide is delayed even longer than with other agents, typically occurring 9 to 18 hours after administration of the drug.23 The mechanism of cyclophosphamideinduced nausea and vomiting is unclear; the difference in the time of onset suggests that the mechanism might differ from that of other agents.

Nausea and Vomiting • CHAPTER 39

Delayed Nausea and Vomiting

Chemotherapeutic Agents

Delayed nausea and vomiting occur 24 or more hours after chemotherapy administration. Although the severity is decreased in comparison with acute nausea and vomiting, the course can be more protracted, resulting in significant difficulties with hydration, nutrition, and performance status. Delayed emesis is most severe and frequent after administration of high-dose cisplatin; most patients treated with this drug experience some degree of delayed emesis, with onset most frequently 24 to 72 hours after chemotherapy.24 In some patients onset can occur as late as 4 to 5 days after treatment, persisting for several days. Patients who have poor control of acute nausea and vomiting are more likely to experience delayed nausea and vomiting as well; however, delayed emesis can occur among patients who have complete emetic control during the first 24 hours after administration of chemotherapy. The pathophysiology of delayed emesis remains unclear, but it seems likely that this syndrome is mediated centrally by different neurotransmitters. 5-HT3 receptor antagonists, which are highly effective in the prevention of acute emesis, have less activity in the treatment of delayed emesis. Conversely, the neurokinin-1 (NK-1) receptor antagonists, which block the action of substance P, have consistently shown activity against delayed emesis. Peripheral factors, including residual metabolites of chemotherapeutic agents or gastrointestinal mucosal damage, might also play a role.

The commonly used chemotherapeutic drugs are separated into five groups according to emetic potential in Table 39-2. Drugs in category 5 produce emesis in greater than 90% of patients, whereas drugs in category 1 produce emesis in fewer than 10%. The drugs that cause emesis most frequently also cause the most severe emesis. Emesis is most severe during the first 8 hours after onset, but with strongly emetogenic drugs patients are often ill throughout the 24-hour period after administration. In general, the potential for acute nausea and vomiting increases with the dose of chemotherapy. Schedule of administration is also important with certain agents: Large intravenous bolus doses, or doses administered intravenously over a short period of time, are more likely to cause emesis than are smaller divided doses or continuous infusion. The use of chemotherapeutic agents in combination increases the emetogenic potential of a treatment regimen. On the basis of the information in Table 39-2, Hesketh and colleagues26 have proposed a model for predicting the emetogenic potential of a combination regimen (Table 39-3). As new combination regimens are introduced into clinical practice, application of this algorithm can result in optimum, cost-effective antiemetic therapy.

Anticipatory Nausea and Vomiting

Several patient characteristics are important predictors of the development and severity of acute chemotherapy-induced nausea and vomiting. These are age, gender, history of alcohol intake, and history of previous chemotherapy.

Anticipatory nausea and vomiting often occur among patients who have experienced poor control of emesis during previous courses of chemotherapy.25 The onset can occur before or during chemotherapy administration. Because this is a conditioned response, certain associations with chemotherapy administration, such as the hospital environment or the oncologist’s office, might trigger the onset of emesis.

Prognostic Factors Multiple clinical factors that are important in determining the incidence and severity of chemotherapy-induced nausea and vomiting have been identified. These factors include the type of chemotherapy administered, certain patient characteristics, and the antiemetic regimen employed (Table 39-1).

Table 39-1 Determinants of ChemotherapyInduced Nausea and Vomiting

Patient Characteristics

AGE. Data are conflicting regarding the effect of patient age on the severity of chemotherapy-induced nausea and vomiting. However, increasing evidence indicates that chemotherapy-induced emesis occurs more frequently in younger patients.21,22 Fortunately, the best current antiemetic agents are effective and well tolerated by patients of all ages. GENDER. Data from large prospective studies indicate that females have more severe and frequent chemotherapy-induced nausea and vomiting than males, even after controlling for chemotherapy regimen. In one study all patients received cisplatin-containing regimens and were treated with ondansetron; more women receiving high-dose cisplatin with either 5-fluorouracil or etoposide for lung cancer or head and neck carcinomas had poor control of emesis than did men receiving these same regimens (49% vs. 29%, respectively).27

CHEMOTHERAPY

HISTORY OF ALCOHOL INTAKE. Patients with a history of

Emetic potential of drug(s) used

chronic alcohol intake (four to five mixed drinks per day) have more effective control of chemotherapy-induced nausea and vomiting when optimal antiemetics are used.28,29 In a prospective study of 52 patients receiving high-dose cisplatin along with combination antiemetic therapy, 93% of those with a high alcohol intake experienced no emesis, as opposed to 61% of patients without this history.28 It is important to emphasize, however, that the administration of highly emetogenic chemotherapy to these patients, in the absence of appropriate antiemetic therapy, still results in a high incidence of severe acute nausea and vomiting. The mechanism of the alcohol effect is unclear; it is possible, however, that various receptor sites are less sensitive among patients with a history of alcohol intake and that blockade of these receptors is relatively easy with appropriate antiemetics.

Dose Schedule of administration Route of administration

PATIENT CHARACTERISTICS Age Gender Alcohol use Emesis control during prior chemotherapy

ANTIEMETICS Dose Schedule

PREVIOUS CHEMOTHERAPY. Patients who have experienced

Combination regimens

poor control of emesis during previous chemotherapy are more likely to have unsatisfactory results with subsequent antiemetics.27 The development of an anticipatory component to the nausea and

Route of administration

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Table 39-2 Acute Nausea and Vomiting without Prophylactic Treatment with Commonly Used Chemotherapeutic Agents Level 5

Frequency of Emesis (%) >90

Agent

Level

Frequency of Emesis (%)

Carmustine >250 mg/m2

2 (con’t)

10–30 (con’t)

Cisplatin ≥50 mg/m2 2

90

Mechlorethamine Streptozocin

Mitomycin Paclitaxel

Carboplatin Carmustine ≤250 mg/m

Panitumomab

Cisplatin 750 mg/m2 ≤1500 mg/m2

Sunitinib

Cytarabine >1 g/m2

Topotecan

2

2

Temozolomide

Doxorubicin >60 mg/m2

Tositumomab/iodine-131 tositumomab

Epirubicin >90 mg/m2

3

30–60

1

Alemtuzumab Asparaginase

Methotrexate >1000 mg/m2

Bevacizumab

Procarbazine (oral)

Bleomycin Bortezomib

Arsenic trioxide 2

Chlorambucil (PO)

Cyclophosphamide (PO)

Cladribine

Doxorubicin 20–60 mg/m2

Denileukin diftitox

Epirubicin ≤90 mg/ m2

Erlotinib

Hexamethylmelamine (PO)

Fludarabine

Idarubicin

Gemtuzumab ozogamicin

Ifosfamide

Hydroxyurea

Irinotecan

Imitanib

Lomustine

Melphalan (low dose, oral dosing)

Methotrexate 250–1000 mg/m2

Methotrexate ≤50 mg/m2

Mitoxantrone

Pentostatin

Oxaliplatin >75 mg/m2 10–30

50 mg/m2

Cyclophosphamide ≤750 mg/m

2

Y ibritumomab tiuxetan

Methotrexate >50 mg/m2 11 g/dL, 12 g/dL

Hemoglobin ≤11 g/dL?

Follow hemoglobin, reevaluate if the hemoglobin decreases.

Symptoms such as fatigue? yes

Treat any reversible causes identified, reevaluate if anemia persists.

Initiate ESP therapy • epoetin alfa 40,000 U/wk or • darbepoetin alfa 200 U/2wk (or every 3 week ESP regimen)

Response adequate? yes

Continue ESP therapy titrated to maintain hemoglobin level of approximately 12 g/dL

no

yes

no

Iron parameters: • TSAT 1 L/day) tend to originate more proximally in the GI tract. Because of the specialized composition of gastric juice, bile, and succus pancreaticus, these more proximal fistulae not only tend to lead to dehydration but also are more prone to cause electrolyte and acid-base imbalances. Lowoutput fistulae generally originate more distally in the alimentary canal, resulting in low-volume outputs that are generally isotonic. For these reasons, the classification of fistulae according to their daily output volume can help to predict the likelihood and kinds of complications to be expected. Resuscitation of patients with GI fistulae follows the principles of general fluid and electrolyte assessment and management. A urinary catheter and, if necessary, central venous pressure monitoring are quite helpful. Specific fluid, electrolyte, and acid-base disturbances, organ dysfunction, and nutritional status should be identified with the initial bloodwork and treated appropriately. Subsequent diagnostic and therapeutic maneuvers must await completion of the resuscitation phase, so these issues must be dealt with promptly, over the initial 12 to 48 hours after presentation.

Patients who exhibit signs of sepsis must be evaluated aggressively to identify the source. Intra-abdominal and perifistular abscesses are common and must be identified quickly. Thorough physical examination (including digital examination of the rectum, vagina, stomas, and wounds) is absolutely necessary. CT scanning of the abdomen and pelvis using intravenous and enteric contrast and contrast in the rectum, fistula tract, and drainage tubes is often the most enlightening radiologic study (Fig. 53-4). Any undrained foci of infection must be addressed aggressively and drained either percutaneously or operatively. Once the acute electrolyte imbalances have been corrected and the fistula output has stabilized, intravenous fluid and nutritional infusions may be combined to simplify fluid management. Definitive treatment of fistulae ultimately requires the reestablishment of normal skin integrity; macerated skin and large open wounds complicate and delay spontaneous or operative closure. Techniques such as sump drainage, stoma bag application, and barrier protection of the skin using special adhesives or pastes should be used liberally. Malnutrition either pre-exists or will develop in nearly all patients with GI fistulae unless specific, aggressive nutrition support is initiated early in the treatment course. Either enteral or parenteral nutrition support can generally be started as soon as the initial resuscitative and stabilization measures have been accomplished. Formal nitrogen balance studies and indirect calorimetric measurement of energy expenditure are the gold standards for adjusting protein and calorie intake to meet needs. Once requirements have been determined, the route of administration that is chosen depends on overall patient status and the nature of the fistula. In general, early initiation of parenteral nutrition is helpful to ensure adequate support without delay. Subsequent aggressive attempts should be made to meet some or all of the patient’s nutritional requirements enterally. Fistulae can be managed appropriately only when their anatomic features are well defined. Reversible causes for fistulae failing to close spontaneously must be identified. Careful examination of the fistula with appropriate biopsies is necessary to rule out epithelialization or the presence of cancer. Generally, the entire GI tract should be investigated with radiographic contrast studies to rule out distal obstruction, to determine the origin of the fistula accurately, and to identify all involved organs and poorly drained associated abscesses. A fistulogram, obtained in the presence of an experienced surgeon, can provide valuable anatomic information and can help in the formulation of surgical plans. In patients with cancer, a thorough search for recurrent and metastatic disease is also important. In patients who are stable and in whom no factors preventing spontaneous fistula closure are present, a trial period of nonsurgical management is generally indicated. The availability of parenteral and enteral nutrition support programs should be coordinated with home care services to allow for spontaneous closure. In some patients, adequate stabilization cannot be achieved. Ongoing fistula output or sepsis in these patients precludes nutritional repletion, adequate skin care, and protection of end-organ function. In this setting, early operation is mandatory. The operative goals for treating intestinal fistulae are to effect excision of the fistula tract and associated diseased tissue, restore continuity of the GI tract, and prevent recurrent fistulae. Use of vascularized omental, mesenteric, bowel, or muscular flaps to fill inflamed cavities and protect anastomoses is often helpful and should be planned for in conjunction with appropriate consultants preoperatively. The definitive operation is a good opportunity to simplify subsequent management of the patient through the insertion of enteric tubes for feeding and GI drainage. Bypass of fistulae can be a useful palliative technique when definitive resection is technically impossible or is thought to be associated with prohibitive morbidity.

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Figure 53-4 • Arterioenterocutaneous fistula. Patient with a multiply recurrent high-grade liposarcoma of the spermatic cord treated with radical surgical resection and radiation therapy. After the most recent surgery, the patient developed an enterocutaneous fistula that was managed conservatively. He later presented with brisk bleeding from the fistula and per rectum. A, CT scan of the pelvis demonstrating recurrent tumor and tract (arrows) of enterocutaneous fistula. B, Arteriogram with contrast extravasation from the left external iliac artery and communication of the bowel (arrow).

A

B

REFERENCES 1. Turnbull ADM: Abdominal and upper gastrointestinal emergencies. In Turnbull ADM (ed): Surgical Emergencies in the Cancer Patient. Chicago, Year Book, 1987, pp 152–194. 2. Schnoll-Sussman F, Kurtz RC: Gastrointestinal emergencies in the critically ill cancer patient. Semin Oncol 2000;27:270–283. 3. Miner TJ, Brennan MF, Jacques DP: A prospective, symptom related, outcomes analysis of 1022 palliative procedures for advanced cancer. Ann Surg Oncol 2004;240:719–727. 4. Leschka S, Alkadhi H, Wildermuth S, et al: Multidetector computed tomography of acute abdomen. Eur Radiol 2005;15:2435–2447. 5. McCahill LE, Krouse R, Chu D, et al: Indications and use of palliative surgery: results of Society of Surgical Oncology survey. Ann Surg Oncol 2002;9: 104–112. 6. Krouse RS, Nelson RA, Farrell BR, et al: Surgical palliation at a cancer center: incidence and outcomes. Arch Surg 2001;136:773–778. 7. Carraro PG, Segala M, Orlotti C, et al: Outcome of large-bowel perforation in patients with colorectal cancer. Dis Colon Rectum 1998;41:1421–1426. 8. Koch P, del Valle F, Berdel WE, et al: Primary gastrointestinal non-Hodgkin’s lymphoma: I.

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Anatomic and histologic distribution, clinical features, and survival data of 371 patients registered in the German Multicenter Study GIT NHL 01/92. J Clin Oncol 2001;19:3861–3873. List AF, Greer JP, Cousar JC, et al: Non-Hodgkin’s lymphoma of the gastrointestinal tract: an analysis of clinical and pathologic features affecting outcome. J Clin Oncol 1988;6:1125–1133. Yanchar NL, Bass J: Poor outcome of gastrointestinal perforations associated with childhood abdominal non-Hodgkin’s lymphoma. J Pediatr Surg 1999; 34:1169–1174. Hiramoto JS, Terdiman JP, Norton JA: Evidencebased analysis: postoperative gastric bleeding: etiology and prevention. Surg Oncol 2003;12: 9–19. Dutcher JP, Schiffer CA, Aisner J, et al: Incidence of thrombocytopenia and serious hemorrhage among patients with solid tumors. Cancer 1984;53: 557–562. McCarthy DM: Prevention and treatment of gastrointestinal symptoms and complications due to NSAIDs. Best Pract Res Clin Gastroenterol 2001;15:755–773. Elting LS, Rubenstein EB, Martin CG, et al: Incidence, cost and outcomes of bleeding and

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chemotherapy dose modification among solid tumor patients with chemotherapy-induced thrombocytopenia. J Clin Oncol 2001;19:1137–1146. Koutras AK, Makatsoris T, Paliogianni F, et al: Oxaliplatin-induced acute-onset thrombocytopenia, hemorrhage, and hemolysis. Oncology 2004;67:179– 182. Kemeny N, Daly J, Oderman P et al: Hepatic artery pump infusion: toxicity and results in patients with metastatic colorectal carcinoma. J Clin Oncol 1984; 2:595–600. Chang AE, Schneider P, Sugarbaker PH, et al: A prospective randomized trial of regional versus systemic continuous 5-fluorodeoxyuridine chemotherapy in the treatment of colorectal liver metastases. Ann Surg 1987;206:685–693. Barnett KT, Malafa MP: Complications of hepatic artery infusion: a review of 4580 reported cases. Int J Gastrointest Cancer 2001;30:147–160. Talamonti MS, Dawes LG, Joehl RJ, Nahrwold DL: Gastrointestinal lymphoma: a case for primary surgical resection. Arch Surg 1990;125:972–977. Hurwitz H, Fehrenbacher L, Novotny W, et al: Bevacizumap plus irinotecan, fluorouracil and leucovorin for metastatic colorectal cancer. N Engl J Med 2004;350:2335–2342.

Acute Abdomen, Bowel Obstruction, and Fistula • CHAPTER 53 21. Kabbinavar F, Hurwitz HI, Fehrenbacher L, et al: Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer. J Clin Oncol 2003;21:60–65. 22. Heinzerling JH, Huerta, S: Bowel perforation from Bevacizumab for the treatment of metastatic colon cancer: incidence, etiology, and management. Curr Surg 2006;63:334–337. 23. Dagher R, Cohen M, Williams G, et al: Approval summary: imatinib mesylate in the treatment of metastatic and/or unresectable malignant gastrointestinal stromal tumors. Clin Cancer Res 2002;8: 3034–3038. 24. Schwartzentruber D, Lotze MT, Rosenberg SA: Colonic perforation: an unusual complication of therapy with high-dose interleukin-2. Cancer 1988;62:2350–2353. 25. Smith F, Goff SL, Klapper JA: Risk of bowel perforation in patients receiving interleukin-2 after therapy with anti-CTLA 4 monoclonal antibody. J Immunother 2007;30:130–135. 26. Jones GT, Abramson N: Gastrointestinal necrosis in acute leukemia: a complication of induction therapy. Cancer Inv 1983;1:315–320. 27. Seewaldt V, Cain JM, Greer BE, et al: Correspondence: Bowel complications with taxol therapy. J Clin Oncol 1993;11:1198. 28. Seewaldt VL, Cain JM, Goff BA, et al: A retrospective review of paclitaxel-associated gastrointestinal necrosis in patients with epithelial ovarian cancer. Gyn Oncol 1997;67:137–140. 29. Glenn J, Funkhouser WK, Schneider PS: Acute illnesses necessitating urgent abdominal surgery in neutropenic cancer patients: description of 14 cases and review of the literature. Surgery 1989;105:778– 789. 30. Mower WJ, Hawkins JA, Nelson EW: Neutropenic enterocolitis in adults with acute leukemia. Arch Surg 1986;121:571–574. 31. Katz JA, Wagner ML, Gresik MV, et al: Typhlitis: an 18 year experience and postmortem review. Cancer 1990;65:1041–1047. 32. Cunningham SC, Fakhry K, Bass BL, et al: Neutropenic entercolitis in adults: case series and review of the literature. 2005;50:215–220. 33. Horowitz NS, Cohn DE, Herzog TJ, et al: The significance of pneumatosis intestinalis or bowel perforation in patients with gynecologic malignancies. Gynecol Oncol 2002;86:79–84. 34. Kurbegov AC, Sondheimer JM: Pneumatosis intestinalis in non-neonatal pediatric patients. Pediatrics 2001;108:402–406. 35. Heng Y, Schuffler MD, Haggitt RC, et al: Pneumatosis intestinalis: a review. Am J Gastroenterol 1995; 90:1747–1758. 36. Safdar A, Armstrong D: Infectious morbidity in critically ill patients with cancer. Crit Care Clin 2001;17:531–570, vii–viii. 37. Angel CA, Rao BN, Wrenn E, et al: Acute appendicitis in children with leukemia and other

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malignancies: still a diagnostic dilemma. J Pediatr Surg 1992;27:476–479. Rao PM, Rhea JT, Novelline RA, et al: Effect of computed tomography of the appendix on treatment of patients and use of hospital resources. N Engl J Med 1998;338:141–146. Miller SD, Andrassy RJ: Complications in pediatric surgical oncology. J Am Coll Surg 2003;197:832– 837. Runzi M, Layer P: Drug-associated pancreatitis: facts and fiction. Pancreas 1996;13:100–109. North JH Jr, Weber TK, Rodriguez-Bigas MA, et al: The management of infectious and noninfectious anorectal complications in patients with leukemia. J Am Coll Surg 1996;183:322–328. Grewal H, Guillem JG, Quan SHQ, et al: Anorectal disease in neutropenic leukemic patients. Dis Colon Rectum 1994;37:1095–1099. Krouse RS, McCahill LE, Easson AM: When the sun can set on an unoperated bowel obstruction: management of malignant bowel obstruction. J Am Coll Surg 2002;195:117–128. Skibber JM, Matter GJ, Pizzo PA, Lotze MT: Right lower quadrant pain in young patients with leukemia: a surgical perspective. Ann Surg 1987;206:711–716. Balthazar EJ: George W. Holmes lecture: CT of small bowel obstruction. AJR Am J Roentgenol 1994;162:255–261. Tang E, Davis J, Silberman H: Bowel obstruction in cancer patients. Arch Surg 1995;130:832–837. Turnbull AD, Guerra J, Starnes HF: Results of surgery for obstructing carcinomatosis of gastrointestinal, pancreatic, or biliary origin. J Clin Oncol 1989;7:381–386. Ripamonti C, Twycross R, Baines M, et al: Clinicalpractice recommendations for the management of bowel obstruction in patients with end-stage cancer. Support Care Cancer 2001;9:223–233. Idelevich E, Kashtan H, Mavor E, et al: Small bowel obstruction caused by secondary tumors. Surg Oncol 2006;15:29–32. Tilney HS, Lovegrove RE, Purkayastha S, et al: Comparison of colonic stenting and open surgery for malignant large bowel obstruction. Surg Endosc 2007;21:225–233. Blair SL, Chu DZ, Schwarz RE: Outcome of palliative operations for malignant bowel obstruction in patients with peritoneal carcinomatosis from nongynecological cancer. Ann Surg Oncol 2001;8:632–637. Baines M, Oliver DJ, Carter RI: Medical management of intestinal obstruction in patients with advanced malignant disease: a clinical and pathological study. Lancet 1985;2:990–993. Yagi T, Karasuno T, Hasegawa T, et al: Acute abdomen without cutaneous signs of varicella zoster virus infection as a late complication of allogeneic bone marrow transplantation: importance of empiric therapy with acyclovir. Bone Marrow Transplant 2000;25:1003–1005.

54. Horak DA, Forman SJ: Critical care of the hematopoietic stem cell patient. Crit Care Clin 2001;17: 671–695. 55. Iwasaki T. Recent advances in the treatment of graft-versus-host disease. Clin Med Res 2004;2:243– 252. 56. Takatsuka H, Iwasaki T, Okamoto T, et al: Intestinal graft-versus-host disease: mechanisms and management. Drugs 2003;63:1–15. 57. Kaur S, Cooper G, Fakult S, Lazarus HM: Incidence and outcome of overt gastrointestinal bleeding in patients undergoing bone marrow transplantation. Dig Dis Sci 1996;41:598–603. 58. Maile CW, Frick MP, Crass JR, et al: The plain abdominal radiograph in acute gastrointestinal graftvs-host disease. Am J Roentgenol 1985;145:289– 292. 59. Day DL, Ramsay NKC, Letourneau JG: Pneumatosis intestinalis after bone marrow transplantation. Am J Roentgenol 1988;151:85–87. 60. Chirletti P, Caronna R, Arcese W, et al: Gastrointestinal emergencies in patients with acute intestinal graft-versus-host disease. Leuk Lymph 1998;29:129–137. 61. StPeter SD, Abbas MA, Kelly KA: The spectrum of pneumatosis intestinalis. Arch Surg 2003;138:68– 75. 62. Vogelsang GB, Dalal J: Hepatic venoocclusive disease in blood and bone marrow transplantation in children: incidence, risk factors, and outcome. J Pediatr Hematol Oncol 2002;24:706–709. 63. Falconi M, Pederzoli P: The relevance of gastrointestinal fistulae in clinical practice: a review. Gut 2001;49S:iv2–10. 64. Lloyd DA, Gabe SM, Windsor AC: Nutrition and management of enterocutaneous fistula. Br J Surg 2006;93:1045–1055. 65. Tarazi R, Steiger E: Enterocutaneous fistulas. In Kinney JM, John M, Hill GL, Owen OE (eds): Nutrition and Metabolism in Patient Care. Philadelphia, WB Saunders, 1988, pp 243–257. 66. Jahnson S, Westerborn O, Gerdin B: Prognosis of surgically treated radiation-induced damage to the intestine. Eur J Surg Oncol 1992;18:487–493. 67. Coia LR, Myerson RJ, Tepper JE: Late effects of radiation therapy on the gastrointestinal tract. Int J Radiat Oncol Biol Phys 1995;31:1213–1236. 68. Bentzen SM: Preventing or reducing late side effects of radiation therapy: radiobiology meets molecular pathology. Nat Rev Cancer 2006;6:702–713. 69. Spalding AC, Lawrence TS: New and emerging radiosensitizers and radioprotectors. Cancer Invest 2006;24:444–456. 70. Intensity Modulated Radiation Therapy Collaborative Working Group: Intensity-modulated radiotherapy: current status and issues of interest. Int J Radiat Oncol Biol Phys 2001;51:880–914. 71. Zmora O, Tulchinsky H, Gur E, et al: Gracilis muscle transposition for fistulas between the rectum and urethra or vagina. Dis Colon Rectum 2006;49: 1316–1321.

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54

Superior Vena Cava Syndrome David H. Johnson, Janessa Laskin, Anthony Cmelak, Steven Meranze, and John Robert Roberts S U M M ARY

O F

K EY

P OI NT S

Etiology

Clinical Features

• Superior vena cava (SVC) syndrome is usually due to neoplastic process— predominantly primary lung carcinoma, with a disproportionate number of patients having small cell histology; non-Hodgkin’s lymphoma and metastatic tumors are the next most common. • SVC syndrome can be iatrogenic— sometimes seen as a complication of central venous line or cardiac surgery.

• Usual symptoms are head “fullness,” dyspnea, cough, and chest pain, typically with insidious onset. • More severe symptoms are infrequent, and life-threatening, neurologic symptoms are rare. • Diagnosis is based on clinical findings.

Anatomy and Physiology • Junction of the brachiocephalic veins forms the thin-walled, low-pressure SVC, which is subjected to obstruction from a variety of mediastinal components. • External compression often precedes direct tumor invasion or thrombus formation. • The SVC has an extensive collateral network.

Evaluation • Chest radiograph typically shows mediastinal widening; a mass is often seen in the region of the SVC. • Small-dose cavagrams can be safely accomplished to define exact location and routes of collateral flow. • Computed tomography (CT) scanning identifies the mass and collateral flow and is the most helpful study to guide treatment. • Treatment of an identified mass before histologic diagnosis is rarely justified unless prior diagnosis is established. • Methods used to define histology are sputum cytology, bronchoscopy, lymph node biopsy, thoracentesis, percutane-

INTRODUCTION Obstruction of the superior vena cava (SVC) may occur as an acute or subacute process producing a syndrome with characteristic features including facial edema and plethora, dilation of chest wall and neck veins, mild to moderate respiratory difficulty, and, less commonly, conjunctival edema, central nervous system complaints such as headache, or, more rarely, visual disturbances and signs of altered states of consciousness.1–4 The first recorded description of SVC obstruction (SVCO) occurred in 1757 when William Hunter described the entity in a patient with syphilitic aortic aneurysm.5 For nearly two centuries thereafter, nonmalignant processes such as aortic aneurysms, syphilitic aortitis, or chronic mediastinitis due to tuberculosis were the predominant etiologic factors.1,3,6,7 However, these diseases are now quite rare, and cancer has become the leading cause of SVCO primarily because of the rapid increase in the incidence of bronchogenic carcinoma after World War II.1,3,6,8–10 Although SVCO was once considered a medical emergency, it is now well established that patients with SVCO rarely experience immediate, life-threatening

ous biopsy, mediastinoscopy, and thoracotomy; previously reported high risks associated with these procedures are not borne out in current data.

Treatment • Radiation therapy with or without chemotherapy is the preferred treatment in most malignant causes of SVC obstruction, particularly small cell lung cancer (SCLC) and non-Hodgkin’s lymphoma. • Radiation therapy fractionation schedule depends on tumor histology, stage, prognosis, patient’s general condition, and whether obstruction is acute or subacute. • Surgery is usually reserved for selective patients with benign causes of obstruction and consists of a bypass procedure. • Percutaneously placed, self-expanding intravascular wire stents provide an option or adjunct to other procedures in the palliative treatment of patients (usually with malignant disease).

complications.6,11–13 Consequently, in cases in which a diagnosis is not known, it is appropriate to proceed with a biopsy to establish the underlying cause, because optimal management is dependent on etiology.14

ANATOMY AND PATHOPHYSIOLOGY The SVC is formed by the junction of the brachiocephalic veins, which in turn are formed by the joining of the internal jugular and subclavian veins. Thus, the SVC represents the major drainage system of venous blood from the head, neck, arms, and upper thorax.15 The right and left brachiocephalic veins join at about the level of the sternal angle to form the SVC. The SVC descends on the right side of the ascending aorta and empties into the right atrium, with its distal 2 cm lying within the pericardial sac (Fig. 54-1). Because of its mediastinal location surrounded by several rigid structures including the sternum, trachea, pulmonary artery, right main-stem bronchus, and numerous lymph nodes, the SVC is particularly vulnerable to obstruction. Despite being a relatively large 803

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Right internal jugular

Left internal jugular

Left subclavian

Right subclavian Right brachiocephalic SVC

Left brachiocephalic

Azygous

Hemiazygous vein

vessel, its thin vascular walls and low intravascular pressure contribute to the ease with which the SVC can be obstructed.15 SVCO can be caused by external compression due to tumor or by lymph nodes enlarged by inflammation or metastases (Fig. 54-2). SVCO also can be caused by direct tumor invasion or by a thrombus. Secondary thrombus is reported to occur in up to 50% of cases2 and may contribute to the lack of response to appropriate therapeutic maneuvers. The azygous vein represents an important collateral system of the SVC and is formed by the junction of the right subcostal and right ascending lumbar veins. Additional routes of collateral flow include the mammary, vertebral, lateral thoracic, paraspinous, and esophageal vessels. The azygous vein ascends through the posterior and superior mediastinum, arches over the hilum of the right lung, and ends in the SVC. Fortunately, extensive anastomoses are formed between the SVC, azygous, and vertebral systems, providing multiple routes of collateral blood flow. Therefore, an obstruction of the SVC above the orifice of the azygous vein is better tolerated than is blockage below this level, because blood can be diverted through chest-wall veins into the thoracic and iliac veins and enter the heart by way of the inferior vena cava and azygous systems.9,15,16 Blood from the head and neck also can return to the heart via the vertebral plexus. If the SVC is obstructed between the azygous vein and the heart, the only route of blood return is via the inferior vena cava.

ETIOLOGY Figure 54-1 • Normal anatomy and drainage pattern of the superior vena cava.

Since the middle part of the 20th century, cancer has been the principal cause of SVCO, with bronchogenic carcinoma accounting for up to 85% of cases (Table 54-1).1–3,8,11,17–21 The two most frequent lung cancer histologic types associated with SVCO are small cell and

Intercostal veins Superior vena cava

Trachea Esophagus

Right mainstem bronchus Azygous vein

Enlarged nodes or tumor blocking superior vena cava with azygous vein patent Enlarged nodes or tumor blocking superior vena cava distal to junction with azygous vein Heart

Diaphragm

Figure 54-2 • Lateral view of the thorax with superior vena cava obstruction.

Superior Vena Cava Syndrome • CHAPTER 54

Table 54-1 Causes of Superior Vena Cava Syndrome Parish et al

Yellin et al

Lochridge et al

Davenport et al

Bell et al

Armstrong et al

Scarantino et al

Little et al

Total patients

86

63

66

35

159

125

60

42

Lung cancer

45

30

52

26

129

99

36

35

Non-small cell

33

26

44

6

64

57

23

28

Small cell

12

4

8

20

65

42

13

7

Lymphoma

8

13

8

1

3

18

8

3

Metastases

12

4

4

4

4

8

4

3

2

4

—

1

—

—

—

1

Benign

19

11

2

—

2

—

—

—

Biopsy not done

—

—

—

3

21

—

12

—

Thymoma/Thyroid

Data compiled from references 1–3, 11, 17–19, and 21.

squamous cell carcinoma.1,15,22–24 Although small cell lung cancer (SCLC) accounts for just 15% to 20% of newly diagnosed lung cancers, it is the underlying cause of up to 65% of all cases of SVCO.6,9,22,25,26 The tendency of SCLC to occur centrally within the lung, as well as its high incidence of mediastinal lymph node metastases, most likely accounts for this consistent observation. Although lung cancer is the leading cause of SVCO, the incidence of this syndrome in patients with lung cancer ranges from 3% to 30%, with most series tending toward the lower figure.4,12,27 Non-Hodgkin’s lymphoma is the second most common cause of SVCO.3,6,28,29 Perez-Soler and colleagues29 identified 36 cases among 915 lymphoma patients treated at the M.D. Anderson Cancer Center (University of Texas, Houston). SVCO was most commonly observed with diffuse large cell and lymphoblastic lymphomas.29 The frequency of mediastinal presentations with the latter histologic types may account for this association, because up to 65% of patients with lymphoblastic lymphomas are first seen with a mediastinal mass. The incidence of SVCO in these categories of non-Hodgkin’s lymphoma is reported to be 7% and 20%, respectively.29 Metastatic cancers account for approximately 5% to 10% of SVCOs (see Table 54-1).1,3,8,11,18,21,30–32 The most common primary tumor sites are, in approximate order of frequency: breast cancer, germ cell malignancies, and gastrointestinal cancers. Less common primary sites include sarcomas (including primary sarcomas of the great vessels),33–35 transitional cell carcinoma, prostate cancer, and melanomas.32 However, virtually any cancer capable of metastasizing to the mediastinum can result in SVCO. Nonmalignant causes of SVCO account for up to 5% of cases. An increasingly common benign cause of SVCO is central venous catheter-induced thrombosis, which may occur with cardiac pacemakers, LaVeen shunts, hyperalimentation lines, and Swan-Ganz catheters, as well as those used for chemotherapy administration.8,36–43 SVCO seems to be more common when the tip of the catheter is placed in the left subclavian vein in the upper part of the vena cava.40 The incidence of catheter-related thrombosis may be reduced with the administration of very-low-dose warfarin (1 mg/day) before insertion of the catheter and continuation afterward.44,45 Additional rare benign causes of SVCO include chronic mediastinitis secondary to histoplasmosis, retrosternal goiters, Nocardia infection, and congestive heart failure.3,46–50 In children, SVCO is most frequently related to iatrogenic causes secondary to cardiovascular surgery for congenital heart disease or ventriculoatrial shunts for hydrocephalus.51–53 The most common malignant causes of SVCO in children are non-Hodgkin’s lymphoma, acute lymphoblastic leukemia, Hodgkin’s disease, neuroblastomas, and yolk sac tumors.51,53

CLINICAL FEATURES Although the duration of symptoms may range from a few days to several weeks, a majority of patients have symptoms of 4 weeks’ duration or less.1 The physical findings accompanying SVCO are diagnostic (Fig. 54-3). Patients frequently complain of a sense of head “fullness,” mild dyspnea, cough, chest pain, and occasionally dysphagia (Table 54-2).1,3,13,21,25,53 Less frequently arm edema, stridor, upper body cyanosis, and neurologic symptoms (e.g., headaches or lethargy) may occur. All symptoms may be aggravated by positional changes, particularly those associated with lowering of the head, for example bending to put on shoes. The prospect of catastrophic neurologic events has led to the characterization of SVCO as an “oncologic emergency.”4,9,53 However, experimental studies in dogs as well as several recent reviews have conclusively demonstrated that life-threatening neurologic symptoms such as seizures, syncope, or coma rarely occur.6,8,13,14,16

RADIOGRAPHIC FINDINGS AND DIAGNOSTIC STUDIES Imaging Studies A standard chest radiograph is the first radiographic procedure performed when SVCO is suspected, with the most common abnormality being mediastinal widening. Typically, a mass is found in the superior mediastinum, right hilum or perihilar region, or right upper lobe3,4; however, a normal chest radiograph is not inconsistent with the diagnosis of SVCO.3 A contrast-enhanced chest computed tomography (CT) scan provides visualization of extravascular and intravascular tumor, as well as thrombus formation within the SVC, and also demonstrates collateral flow.54–61 A CT diagnosis depends on diminished or absent contrast opacification of central venous structures such as the innominate vein or the SVC inferior to the obstruction, and opacification of collateral venous routes,59 especially anterior subcutaneous collaterals (Fig. 54-4).54 Because dilution of the contrast medium by unopacified blood or the displacement of blood by laminar flow may simulate an intraluminal filling defect, both criteria must be present for the diagnosis of SVCO to be made.59 The anatomy defined by CT scan may help to guide a fine-needle aspiration biopsy or another diagnostic procedure if a histologic diagnosis has not been previously established. The current-generation helical CT scans also have been used to diagnose SVCO with results that correlate well with regular contrast CT scans.58 In addition, helical scans can potentially reveal more information regarding the site and extent of disease

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Figure 54-4 • Chest computed tomography venogram scan of a patient with superior vena cava obstruction. Note the abrupt blockage of contrast dye indicative of the superior vena cava obstruction.

Figure 54-3 • A patient with characteristic venous dilation and facial edema.

and the collateral pathways involved, as well as define soft-tissue abnormalities. Contrast venacavograms may still play an occasional role in determining management strategy, particularly when surgical bypass or stenting is being considered.62 Current techniques using low-osmolarity contrast involve the positioning of a small catheter in the desired vascular location under fluoroscopic guidance with injection of only small amounts of contrast dye necessary to define the pattern of venous flow and degree of SVCO.62 Collateral circulation is usually identified readily, and complications are uncommon. Cavography also is possible by using nuclear medicine techniques.63–66

In the majority of cases of SVCO, a contrast CT scan will be the most useful radiographic study; noncontrast studies are of limited value, because the vessels are difficult to distinguish. However, occasionally other imaging studies may also provide helpful data.56,57 For example, transesophageal echocardiography can be used to distinguish thrombus formation from extrinsic compression of the SVC.67 Single-photon emission CT has been used to identify obstruction of the SVC by an intravascular metastasis from an adenocarcinoma.68

DIAGNOSTIC APPROACH In the absence of a known cause of SVCO, every effort should be made to obtain a histologic diagnosis before the initiation of any therapy (Box 54-1) for two reasons.6,8,13,14 First, a definitive diagnosis is necessary to plan therapy, and second, even a brief course of radiation therapy before establishing a diagnosis can make histologic diagnosis difficult or even impossible.14,69,70 The least invasive diagnostic technique should be performed initially, followed by more invasive procedures as necessary (Box 54-2). Procedures commonly used to establish a tissue diagnosis include

Table 54-2 Signs and Symptoms of Superior Vena Cava Obstruction Parish et al

Yellin et al

Lochridge et al

Maddox et al

Bell et al

Armstrong et al

Scarantino et al

Little et al

Total patients

86

63

66

56

159

125

60

42

Suffusion

69

54

55

49

62

56

55

35

Dyspnea

54

19

55

—

112

69

47

22

Cough

47

13

46

—

11

29

11

26

Pain

17

4

—

—

20

19

—

—

Dysphagia

10

4

1

—

—

16

6

3

6

—

2

—

6

—

—

—

Arm edema

3

20

—

19

13

49

—

—

Stridor

1

—

22

—

1

—

—

—

Neurologic

2

—

23

—

0

0

—

—

Hemoptysis

—

5

—

—

—

—

—

5

Syncope

Data compiled from references 1, 3, 11, 17–19, 21, and 31.

Superior Vena Cava Syndrome • CHAPTER 54 Box 54-1.

HISTOLOGIC CONFIRMATION OF UNDERLYING CAUSE OF SUPERIOR VENA CAVA OBSTRUCTION

In most cases the distinction between carcinoma and lymphoma is readily accomplished with routine hematoxylin and eosin stains. Special studies sometimes required to distinguish these entities include the following: 1. Immunoperoxidase stains a. Common leukocyte antigen; positive in lymphoma, negative in carcinomas b. Epithelial membrane antigen or keratin; positive in carcinomas, negative in lymphomas c. Surface immunoglobulins; positive in B-cell lymphomas, negative in carcinomas 2. Electron microscopy a. Desmosomes and intracellular junctions typical of carcinomas b. Microvilli typical of adenocarcinomas c. Dense-core granule (neurosecretory) present in neuroendocrine tumors (e.g., small cell lung cancer) d. Transformed lymphocytes, identified from a paucity of organelles, abundant free ribosomes, prominent nucleoli, and occasional presence of nuclear blebs Immunoperoxidase studies can usually be obtained relatively quickly and are most helpful in distinguishing a lymphoma from a carcinoma. However, expensive pathology studies are avoided unless necessary to help guide therapeutic decisions.

sputum cytology, bronchoscopy, lymph node biopsy, thoracotomy, and mediastinoscopy, although the diagnosis is sometimes obtained through other means such as thoracentesis and percutaneous lung biopsy with or without ultrasound guidance.6,17,71–73 The complication rate of invasive procedures in the face of SVCO is fairly modest. Schraufnagel and associates13 reviewed the outcome of 93 invasive procedures in 62 patients with diagnostic problems, and none of the procedures, including bronchoscopies and mediastinoscopies, was associated with a fatal outcome. Yellin and coworkers21 performed 27 invasive diagnostic procedures in 63 patients with SVCO. No mortality or major bleeding episodes were observed, and diagnostic material was obtained in 89% of patients. Ahmann6 reported that complications of bronchoscopy and lymph node biopsies are virtually nonexistent and that contrast studies, such as nuclear medicine venography, are remarkably safe in the presence of SVCO. Of the various invasive procedures used to obtain tissue in patients with SVCO, mediastinoscopy seems to be the most risky. However, even this procedure has a relatively low complication rate. Mineo and colleagues73 reviewed the outcome of 80 patients who underwent diagnostic mediastinoscopy for SVCO by a single surgeon over a 23-year period. Five patients had

Box 54-2.

APPROACH TO PATIENTS WITH SUPERIOR VENA CAVA OBSTRUCTION

Diagnosis is established by physical examination and clinical presentation (see text). • Respiratory status should be assessed promptly. Only patients in extremis should be treated urgently with radiation without a histologic diagnosis. Emergency radiation therapy is necessary in fewer than 5% of all SVCO cases. Stent insertion can also be considered as first-line therapy for patients with malignant cases of SVCO. • Diagnostic evaluation should proceed with least invasive procedures performed initially followed by more invasive procedures as needed to obtain histologic diagnosis: • Chest radiograph • Sputum cytology • Thoracentesis with cytologic evaluation of fluid • Node biopsy if palpable node is present, avoid fine-needle aspiration if lymphoma suspected • Fiberoptic bronchoscopy • Mediastinoscopy • Thoracostomy • Evaluation may vary depending on the age and sex of the patient. • In older adults (i.e., ≥50), the most common cause of SVCO is lung cancer; lymphoma or metastatic cancer are less common and benign processes are uncommon. • In young adults (12.0

8.5

11.0

(P < 0.001)

(P = 0.61)

(P = 0.39)

(30 Gy/10 fx) Patchell62 (1989–1997)

(50.4 Gy/28 fx) 50

Kondziolka (1985–1988)

WBRT

14

71

6

—

7.5

WBRT + RS

13

62

36

—

11.0

(P = 0.0005)

—

(P = 0.22)

WBRT

167

69

[71% 1-year LC]

—

5.7

WBRT + RS

164

68

(36 Gy/12 fx) Andrews49 (1996–2001)

(37.5 Gy/15 fx) Aoyama90 (1999–2003)

[82% 1-year LC]

—

6.5

(P = 0.013)

—

(P = 0.136)

RS

67

43

[73% at 1 year]

[27% at 1 year]

8.0

WBRT + RS

65

37

[89% 1-year LC]

[34% at 1 year]

7.5

(P = 0.002)

(P = 0.53)

(P = 0.42)

(30 Gy/10 fx)

FFP, freedom from progression; fx, fractions; LC, local control; NS, not significant; RS, radiosurgery; WBRT, whole-brain radiotherapy.

metastasis. The observation arm had a significantly increased risk of local failure (46% for observation versus 10% for WBRT), distant brain failure (37% versus 14%), and any brain failure (70% versus 18%); shorter time to local failure (median: 27 weeks versus more than 52 weeks [6.2 months versus more than 12 months]; P < 0.001; hazard ratio: 6.03); and shorter time to any brain failure (median: 26 weeks versus more than 70 weeks [6.0 months versus more than 16.1 months]; P < 0.001; hazard ratio: 4.94). Patients who were randomized to observation were more likely to die neurologic deaths (44% versus 14%; P = 0.003) but, interestingly, had similar duration of functional independence (median: 35 weeks [8.0 months] for observation versus 37 weeks [8.5 months] for WBRT; P = 0.61) and similar survival time (median: 43 weeks versus 48 weeks [9.9 months versus 11.0 months]; P = 0.39).62 Topics that were not addressed in the report included the use or success of salvage therapy and acute and late toxicity of WBRT and salvage therapies.

SURGERY FOR MULTIPLE METASTASES. Surgical resection may also be used successfully to manage selected patients with more than one brain metastasis. Bindal and colleagues reported a median survival time of 14 months among 26 patients with multiple brain metastases who underwent resection of all of their brain lesions in a single operation, identical to the survival time of matched patients who had had resection of a single metastasis.63 The complication rate was 9% per craniotomy, the 30-day mortality rate was 4%, and only 6% of symptomatic patients worsened, while 83% improved and 11% remained stable.63 A different group describing results of surgery and WBRT had noted significantly poorer survival among 18 patients with multiple brain metastases compared with 28 patients with a

single metastasis, but apparently, only one patient with multiple metastases had undergone gross total resection of all (two) metastases, and this patient survived 46 months.64 In a recent series, Paek and colleagues reported a median survival time of 8 months after surgery for approximately 103 patients with a newly diagnosed single metastasis versus 11 months after surgery for 46 patients with newly diagnosed multiple brain metastases (only 9 of whom had multiple brain metastases resected). Most patients received postoperative WBRT.65

TOXICITY OF SURGERY. The morbidity and mortality associated with surgical resection of brain metastases have decreased over the years as techniques have improved. Lang and colleagues estimated the 30-day mortality rate to be about 4% to 5% after surgery for brain metastasis (essentially identical to the 30-day mortality rate in patients who were managed with WBRT alone).56 The most common types of postoperative morbidity include wound infection, hemorrhage, meningitis, pneumonia, deep venous thrombosis, and pulmonary embolism, which occur in about 10% to 15% of patients on average.56,66 Most patients are symptomatic preoperatively; one surgical series reported that 0% to 13% of patients worsened neurologically, 65% to 84% improved, and 11% to 22% remained stable after resection of single or multiple brain metastasis.63 Paek and colleagues recently reported outcomes in 208 patients who underwent resection of one (N = 191) or multiple (N = 17) newly diagnosed (N = 149) or recurrent (N = 59) brain metastases at a single institution using modern neurosurgical techniques.65 The 30-day mortality rate was 1.9%, with two deaths from hemorrhage in the resection cavity, one from pulmonary embolism secondary to deep venous thrombosis, and one from bowel perforation with sepsis. The median hospital

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lower doses are generally prescribed for larger target volumes, and radiosurgery targets tend to be limited to about 2.5 to 3 cm in diameter.

RETROSPECTIVE RESULTS OF RADIOSURGERY. Table 56-4 summarizes results of selected retrospective series of radiosurgery for single or multiple newly diagnosed or recurrent brain metastases treated with or without adjuvant WBRT.69–83 For median target volumes ranging from 0.9 to 7.5 mL and median prescribed doses ranging from 15.0 to 27.0 Gy in a single fraction, the crude local control rates were 85% to 96% and the 1-year actuarial local control probabilities were 82% to 94% by lesion, with a 0% to 17% risk of symptomatic radiation necrosis (average risk, 4%). In most of the series, the median survival times ranged from 7 to 11 months. Figure 56-4 • Postcontrast T1-weighted magnetic resonance imaging of a brain metastasis shown on the day of radiosurgery with superimposed 50% and 25% isodose contours (left) and follow-up imaging 11 months later, showing near complete response (right). A dose of 17.5 Gy was prescribed at the 50% isodose contour.

RADIOSURGERY DOSE-RESPONSE RELATIONSHIPS. An RTOG dose escalation trial in patients with recurrent primary or metastatic brain tumors concluded that the maximum tolerated doses of single fraction radiosurgery (without WBRT) were 24 Gy for tumors 2 cm or less in maximum diameter, 18 Gy for tumors 2.1 to 3.0 cm, and 15 Gy for tumors 3.1 to 4.0 cm.84 A dose-response analysis specifically in newly diagnosed or recurrent brain metastases 2 cm in diameter or smaller found crude local control rates of 91% using a radiosurgery dose less than 20 Gy (N = 46), 99% using a dose of 20 Gy (N = 158), and 96% using a dose higher than 20 Gy (N = 24) when radiosurgery was combined with planned WBRT. Because the risk of complications was 1.9% for radiosurgery doses of 20 Gy or less and 5.9% for doses above 20 Gy, the authors concluded that 20 Gy was the optimal radiosurgery dose for metastases 2 cm or less in diameter. Of note, the crude local control rates were 97% for radiosurgery with WBRT versus 87% for radiosurgery without planned WBRT (P = 0.0001). On the basis of a data set of 518 newly diagnosed or recurrent brain metastases treated with radiosurgery with or without WBRT at

stay was 3 days after surgery, and KPS improved postoperatively in 33%, remained stable in 61%, and decreased in 6% of patients.65

Radiosurgery Radiosurgery implies the delivery of carefully targeted, very focal radiation to one or more intracranial targets, usually using a specially adapted linear accelerator67 or a gamma knife.68 A stereotactic frame may be applied prior to the procedure under local anesthesia to help allow very precise targeting. Multiple beams or arcs provide for very steep falloff of dose outside of the target or targets, minimizing dose to surrounding normal brain tissue, but a thin shell of tissue around the target receives a potentially damaging dose of radiation (Fig. 56-4). Because the risk of radiation injury increases with increasing volume,

Table 56-4 Results of Radiosurgery with or Without Whole-Brain Radiotherapy for Newly Diagnosed or Recurrent Brain Metastases No. of Metastases/ No. of Patients

Mean or Median Dose

Median Target Volume

Auchter69

122/122

17.0 Gy

2.7 mL

86*/85†

Flickinger70

116/116

17.5 Gy

—

85*

71

237/237

21.5 Gy

7.5 mL

95*

9

First Author

Local Control by Lesion (%)

Median Survival Time (Months)

Necrosis (%)

12.9

0

11

4

Single metastases

Simonova

2.5

Single or multiple metastases Deinsberger72

161/110

18.3 Gy

3.1 mL

89*

12.4

2

Flickinger73

229/157

16.0 Gy

3.0 mL

89*

10

1 5

Fukuoka

>215/130

5.5 mL

≥96*

8

Gerosa75

1307/804

20.6 Gy

4.8 mL

94†

13.5

Goodman76

682/258

18.5 Gy

1.7 mL

82†

9.1

Joseph77

189/120

26.6 Gy

5.3 mL

94*

7.4

Kihlstrom78

235/160

27.0 Gy

4.5 mL

94*

7

Moriarty79

643/353

15.0 Gy

2.5 mL

88†

10.5

3–6

Petrovich80

1305/458

18 Gy

0.9 mL

87†

9

4.7

5.5

2

74

>25 Gy

Pirzkall81

311/236

20 Gy

—

92*

82

411/193

20 Gy

—

82 by patient

Young83

669/250

—

91*

Sansur

*Crude. † 1-year actuarial.

—

7.5 7

— — 17 5

2 50%) tumor shrinkage (a complete or partial objective response) after endocrine treatment occurs in one-third of unselected patients but is more likely among those with either steroid-receptorpositive tumors, a long disease-free interval from diagnosis to relapse, or bone or soft tissue metastases rather than visceral disease. Selection of specific endocrine treatment for patients is based on menopausal status. Premenopausal patients are now usually treated with a combination of tamoxifen and ovarian ablation, the latter achieved by the use of LHRH agonists, surgical bilateral oophorectomy or in some centres ovarian irradiation. For postmenopausal patients the choice of agents is large with aromatase inhibitors, tamoxifen, fulvestrant, and progestogens the most frequently used. Although in general the median duration of response to endocrine therapy is around 15 months, prolonged responses to first-line hormone treatments lasting several years are not uncommon in patients with bone metastases. Numerous recent developments in cytotoxic and biological treatments are of relevance to the patient with metastatic bone disease. Patients with disease progressing after endocrine therapy, and those with rapidly progressive life-threatening disease or those who are known to have estrogen receptor- and progestogen receptor-negative tumors, should be considered for cytotoxic chemotherapy. This is usually combined with trastuzumab in patients with tumors overexpressing the HER2neu growth factor receptor. Objectively responding patients usually gain relief of symptoms (including bone pain) and might become able to resume their previous activities. Responses among women with bone metastases are nearly always only partial, with a median duration of response of 9 to 12 months. The precise choice of drugs and schedule of administration to obtain the best results is not yet certain and vary from one patient or clinical problem to another. The anthracyclines (doxorubicin and epirubicin) and the taxanes (docetaxel and paclitaxel) are particularly active but at full doses are sometimes too toxic for elderly or frail patients. Chemotherapy can be especially hazardous for patients with extensive bone disease because of both poor bone marrow tolerance after replacement of functioning marrow by tumor and the effects of previous irradiation. In view of this, regimens with relatively little myelotoxicity are usually preferable. The use of hematopoietic growth factors may be required to permit chemotherapy to be administered safely.

cytotoxic agent in endocrine-resistant prostate cancer and is able to improve median survival by about 3 months. Cautious use of this agent in appropriate patients is now recommended.78

Prostate Cancer

In the last two decades the bisphosphonates have become established as a valuable additional approach to the range of current treatments. All bisphosphonates are pyrophosphate analogs, characterized by a P-C-P containing central structure rather than the P-O-P of pyrophosphate, and a variable Rc chain that determines the relative potency, side effects, and the precise mechanism of action.81 The P-C-P backbone renders bisphosphonates resistant to phosphatase activity and promotes their binding to the mineralized bone matrix. The structures of the commonly used bisphosphonates are illustrated in Figure 57-14. After administration, bisphosphonates bind avidly to exposed bone mineral around resorbing osteoclasts, leading to very high local concentrations of bisphosphonate in the resorption lacunae (up to 1000 mM). During bone resorption, bisphosphonates are internalized by the osteoclast, where they cause disruption of several biochemical processes involved in osteoclast function, ultimately leading to apoptotic cell death. These include destruction of the cytoskeleton, disruption of the sealing zone at the bone surface, and loss of the ruffled border across which the hydrolytic enzymes and protons necessary for bone dissolution are normally secreted. The molecular mechanism of action of the bisphosphonates are now established, with nitrogen-containing bisphosphonates having been shown to inhibit enzymes of the mevalonate pathway, which are responsible for events that lead to the post-translational modification of a number of proteins including the small guanosine triphosphatases such as Ras and Rho.81 Non-nitrogen-containing bisphosphonates, such as

In prostate cancer, bone is the dominant site for metastatic disease and in many patients is the only symptomatic problem. The appearances on plain radiography are predominantly osteoblastic and because of this, radiologic response is notoriously difficult to evaluate. Nevertheless, at least 80% of prostate tumors show some degree of hormone responsiveness. Worldwide, the luteinizing hormone-releasing hormone (LHRH) agonists are the most commonly used form of endocrine therapy, although surgical castration remains a first-line treatment in some parts of the world. Stilbestrol is no longer appropriate first-line therapy because of its feminizing effects and cardiovascular risks. The role of combined endocrine therapy causing total androgen blockade (LHRH + antiandrogen) and the timing of endocrine interventions have, and continue to be, areas of intense clinical trial investigation. Patients with advanced prostate cancer tend to be elderly and often of poor performance status. Because of this and the presence of widespread bone involvement, their tolerance of toxic chemotherapy regimens is often poor, and this has significantly limited the use of this treatment modality. Until recently there was little evidence that cytotoxic drugs prolonged survival for patients with advanced prostate cancer. Mitoxantrone and prednisone has been shown in a randomized trial to provide better palliation of symptoms and improved quality of life compared with that achieved with prednisone alone.77 More recently, docetaxel has been shown to be an active

Other Tumors Skeletal morbidity is a major problem in multiple myeloma, and either widespread lytic metastases or diffuse osteopenia can occur. Around 50% of patients respond to chemotherapy, with a reduction in paraprotein levels and subjective improvement. Alkylating agents, anthracyclines, vinca alkaloids, and the corticosteroids are the most frequently prescribed first-line agents. Despite the subjective improvement that is seen, bone healing is rare, with lytic lesions persisting despite control of the disease for months or years. Survival in multiple myeloma has been shown to be improved by the selective use of high-dose chemotherapy with bone marrow or peripheral blood stem cell support, and this is part of standard first-line treatment in fit patients under the age of 65.79 Newer agents including thalidomide and related analogs, bortezomib, and arsenic trioxide provide many more options that have transformed the clinical course of multiple myeloma in recent years into that of a chronic disease. Bone involvement in curable malignancies is uncommon. In patients with germ cell tumors, bone involvement is an adverse prognostic feature, but despite this, cure with chemotherapy is usual. Bone involvement at diagnosis in lymphoma is relatively uncommon. When localized it does not significantly affect the prognosis in Hodgkin’s disease but does carry an adverse prognosis in non-Hodgkin’s lymphoma. Curative therapy is still possible, however, and there is no evidence that bone represents a “sanctuary site.” Chemotherapy is of only limited and temporary benefit in relatively chemotherapy-resistant solid tumors such as non–small cell lung cancer or melanoma. Patients with skeletal metastases from these tumors derive most benefit from local palliative radiotherapy and bisphosphonates. Alternative (and more effective) systemic treatment approaches are urgently needed for many of these maligancies. Biologic agents, especially the angiogenesis inhibitors sorafinib and sunitinib, are showing great promise in renal cell cancer, and small molecules and antibodies are being developed at a rapid pace across a range of tumors.80

Bisphosphonates

859

860

Part II: Problems Common to Cancer and Its Therapy OH O

P

OH O

OH

P

O

OH

Pyrophosphate OH O

P

OH C

Bisphosphonates to Prevent Skeletal Morbidity and Relief of Bone Pain

OH P

O

OH CH3 OH Etidronate (Ethane-1-hydroxy-1, 1-diphosphonate)

O

OH

Cl

OH

P

C

P

OH

Cl

OH

O

Clodronate (Dichloromethane diphosphonate)

O

OH

OH

OH

P

C

P

OH

CH2

OH

metastases. Biochemical data indicate that bone resorption is of importance not only in classic “lytic” diseases such as myeloma and breast cancer but also in prostate cancer, with values of resorption markers in the latter at least as high as those seen in breast cancer and other solid tumors.10,53 As a result, the osteoclast is a key therapeutic target for skeletal metastases irrespective of the tissue of origin.

O

CH2 NH3 Pamidronate (3-amino-1-hydroxypropylidene-1, 1-diphosphonate)

Figure 57-14 • Structural formulas of commonly used bisphosphonates in relation to pyrophosphate.

clodronate, have been found to induce osteoclast apoptosis through the generation of cytotoxic adenosine triphosphate analogs.82 Recent studies also suggest that bisphosphonates could have direct apoptotic effects on tumor cells, and that this effect may be enhanced by combination with other anticancer agents.83 After intravenous administration of a bisphosphonate, approximately 25% to 40% of the injected dose is excreted by the kidney, and the remainder is taken up by bone.84 All bisphosphonates suffer from poor bioavailability when given by mouth. They must be taken on an empty stomach, because they bind to calcium in the diet and can cause gastrointestinal toxicities such as nausea, vomiting, indigestion, and diarrhea.85 Irrespective of the mechanism(s) of action, bisphosphonates have been used successfully in the treatment of conditions characterized by increased osteoclast-mediated bone resorption, such as Paget’s disease of bone or osteoporosis. In oncology they have become the standard treatment for tumor-induced hypercalcemia and a valuable, new form of medical therapy for bone metastases.86

Rationale for the Wider Use of Bisphosphonates As indicated previously, it is now generally accepted that osteoclast activation is the key step in the establishment and growth of bone

Although radiotherapy is the treatment of choice for localized bone pain, many patients have widespread, poorly localized bone pain, whereas others experience recurrence of bone pain in previously irradiated sites. The bisphosphonates provide an additional treatment approach for the relief of bone pain across a range of tumor types, and the effects seems to be independent of the nature of the underlying tumor or radiographic appearance of the metastases, with sclerotic lesions responding similarly to lytic metastases.87 Additionally, based on the results of large randomized controlled trials conducted in the late 1990s, the bisphosphonates became the standard of care for the treatment and prevention of skeletal complications associated with bone metastases in patients with breast cancer and multiple myeloma. More recently, they have demonstrated benefits in patients with bone metastases secondary to other cancers including prostate cancer,88 lung cancer,88 and other solid tumors (Table 57-4).89,90

BREAST CANCER. The greatest experience with bisphosphonates is in the management of bone metastases from breast cancer, and the value of the agents is now undisputed.91 Oral Bisphosphonates. The absorption of bisphosphonates from the gut is poor, variable, and dramatically inhibited by food intake. To make matters worse, the absorbed fraction of oral bisphosphonates decreases even further when the absolute ingested amount is lower; thus, the more potent bisphosphonates are even less well absorbed than etidronate and clodronate. Nevertheless, both oral clodronate and ibandronate have been shown in randomized trials to have some clinical efficacy (see Table 57-4).92,93 Paterson and coworkers randomized 173 patients with bone metastases from breast cancer to receive either clodronate capsules, 1600 mg daily, or placebo capsules of identical appearance in addition to appropriate anticancer treatment(s).92 In the patients who received clodronate, there was a significant reduction in skeletal morbidity (219 vs. 305 events per 100 patient years). Most of the benefit was accounted for by a reduction in hypercalcemic episodes and the incidence of vertebral fractures, with no significant effect on nonvertebral fractures, radiotherapy requirements, changes in antitumor therapy, or survival. Oral ibandronate is the newest and most potent oral agent and is available in Europe and many other countries outside North America. A film-coated formulation of ibandronate has been developed that has been shown to produce a dose-dependent reduction, at doses that are generally well tolerated, in both urinary calcium and collagen cross-link excretion.93 Phase III placebo-controlled trials of the oral formulation have been completed. The endpoint of a significant reduction in the proportion of patients experiencing a SRE was not met. However the skeletal morbidity rate was significantly less with ibandronate, and the investigators concluded that the activity of oral ibandronate is similar to other bisphosphonates.94 This new oral agent has obvious attractions to both patients and health care providers, but the place of ibandronate cannot be clearly defined until comparative data with other bisphosphonates are available. Intravenous Bisphosphonates. In the first randomized study of intravenous pamidronate, Conte and associates95 randomized 295 patients with breast cancer and bone metastases to chemotherapy

Bone Metastases • CHAPTER 57

Table 57-4 Effects of Bisphosphonates on Skeletal Morbidity: Results of Randomized Trials BREAST CANCER Agent and Route

N

Results

Investigator

Clodronate 1600 mg PO vs. placebo

173

Reduced SMR

Paterson92

305 vs. 219 events/100 woman years (P < 0.001) Pamidronate 45 mg IV vs. control

295

Increased time to bone progression

Conte95

168 vs. 249 days (P = 0.02) Pamidronate 90 mg IV vs. placebo

382

Reduced proportion experiencing SRE

Hortobagyi97

65% vs. 46% (P < 0.001) Delay in first SRE 7.0 vs. 13.1 months (P = 0.0005) Pamidronate 60 mg IV vs. control

401

Pamidronate 90 mg IV vs. placebo

374

Median time to skeletal progression

Hultborn96

9 vs. 14 months (P < 0.01) Reduced proportion experiencing SRE

Theriault98

67% vs. 56% (P = 0.027) Delay in first SRE 6.9 vs. 10.4 months (P = 0.049) Ibandronate 2/6 mg IV vs. placebo

467

Zoledronic acid 4 mg IV vs. placebo

227

Reduced SMR with 6-mg dose, 2 mg ineffective

Body104

SMR 2.18 vs. 1.61 (P = 0.03) Reduced proportion experiencing SRE

Kohno101

50% vs. 30% (P = 0.003) Reduced SMR by 43% (P = 0.016)

MULTIPLE MYELOMA Agent and Route

N

Results

Investigator

Clodronate 1600 mg PO vs. placebo

350

Improved 2-year progression-free survival

Lahtinen105

24% vs. 12% (P < 0.05) Pamidronate 90 mg IV vs. placebo

392

Clodronate 1600 mg PO vs. placebo

614

Reduced proportion experiencing SRE

Berenson107

24% vs. 41% (P < 0.001) Less skeletal morbidity and pain on progression

McCloskey106

MULTIPLE MYELOMA AND BREAST CANCER Agent and Route Zoledronic acid 4/8 mg IV vs. pamidronate 90 mg IV

N 1648

Results

Investigator

Zoledronic acid (4 mg) showed clinical activity equivalent to that of pamidronate (90 mg)

Rosen102,103

In breast cancer patients, 43% had a SRE with 4 mg zoledronic acid, compared with 45% with pamidronate. In myeloma, 47% of patients had a SRE with zoledronic acid and 49% with pamidronate

PROSTATE CANCER Agent and Route

N

Results

Investigator

Clodronate (4 × 520) mg oral vs. placebo

311

Reduction in number of SREs vs. placebo not significant (49% vs. 41%, P = NS)

Dearnaley110

Pamidronate 90 mg IV vs. placebo

378

Number of SREs equal in pamidronate and placebo arms, P = 1.0

Small112

Zoledronic acid 4/8 mg vs. placebo

643

Proportion of patients experiencing at least one SRE during the study was 25% lower in the zoledronate arm than in the placebo arm (P = 0.021)

Saad88,113

OTHER TUMOR TYPES Agent and Route

N

Results (Bisphosphonate vs. Placebo/Control)

Investigator

Zoledronic acid 4/8 mg vs. placebo

773

Significant delay to time of first skeletal event in Zoledronic acid arm compared with placebo (P = 0.023)

Rosen89

Significant reduction in proportion of patients having an event (47% vs. 38%, P = 0.039) SRE, skeletal related event; SMR, skeletal morbidity rate.

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with or without intravenous pamidronate, 45 mg every 3 weeks (a dose intensity of pamidronate that is now considered suboptimal). Blinded, extramural review of serial radiographs was performed and identified a 48% increase in the median time to progression in bone in favor of the patient group who received pamidronate (249 vs. 168 days, P = 0.02). The other major endpoint of this trial was bone pain. A marked improvement in pain was seen more often in the pamidronate group (44% vs. 30%, P = 0 .025), indicating that intravenous pamidronate adds to the symptom relief achieved by chemotherapy alone. Similar results were reported in a Scandinavian trial, in which 401 patients receiving chemotherapy for advanced breast cancer were randomly allocated to receive either an intravenous pamidronate infusion (60 mg every 4 weeks) or a placebo infusion of the same dose intensity of pamidronate that was given in the Conte study.96 Subsequently, the results of two double-blind, placebo-controlled trials of 90 mg pamidronate infusions every 3 to 4 weeks in addition to cytotoxic or endocrine treatments for patients with breast cancer and lytic bone metastases established bisphosphonate treatment as the standard of care in breast cancer.97,98 These two studies were of similar design, with the exception of the systemic anticancer treatment at study entry. As in all subsequent bisphosphonate trials the primary endpoint of these studies was the influence of pamidronate on the number of patients experiencing SREs as well as the time to first SRE and the rate of SREs as determined by either a simple annual rate or more complex multiple event analysis techniques. SREs were defined as • Occurrence of pathologic long bone and vertebral fractures • Development of spinal cord compression • Need for radiation for pain relief or to treat or prevent pathologic fractures or spinal cord compression • Requirement for surgery to bone • Episodes of hypercalcemia of malignancy In the chemotherapy study,97 382 patients received either pamidronate 90 mg or placebo every month in combination with systemic chemotherapy. The median time to the SRE was significantly longer in the pamidronate group than in the placebo group (13.1 vs. 7.0 months, P = 0.005), and the proportion of patients in whom any SRE occurred was significantly lower (43% vs. 56%, P = 0.008). Benefits were maintained for at least 2 years. Pain, analgesic use, and Eastern Cooperative Oncology Group (ECOG) performance status were monitored throughout the study period. Because there was inevitably a tendency for the underlying cancer to progress during the study period, there was an overall deterioration in mean performance status, pain, and analgesic consumption. The deterioration, however, was significantly less in the pamidronate group for all of these endpoints. Quality of life was also better maintained in the pamidronate group. Survival was similar in other treatment groups. In the endocrine study, 374 patients were randomized to receive hormone therapy with pamidronate 90 mg or placebo every month.97 As in the chemotherapy study, pamidronate reduced the number and rate of SREs. The time to first SRE (excluding hypercalcemia of malignancy) was 6 months for the placebo group and 10 months for those receiving pamidronate (P < 0.049). The benefits of pamidronate were slower to appear than in the chemotherapy study, but again the effect was maintained for at least 2 years. The effects on pain and analgesic consumption were even more clearly evident in this study. Again, there was no difference in survival by treatment group. Zoledronic acid is the most potent bisphosphonate available. A phase I study of 30 patients with hypercalcemia indicated that dose levels as low as 0.02 mg/kg (1–2 mg total dose) were effective in achieving normocalcemia.99 Following a dose-finding, phase II study of zoledronic acid at doses of 0.5 g, 2 g, and 4 mg zoledronic acid given on a 4-weekly schedule, 4 mg zoledronic acid was selected for phase III evaluation.100 A plaeebo-controlled trial of zoledronic acid was performed in Japan.101 In this study, Kohno and coworkers randomly assigned women with bone metastases from breast cancer

to treatment with zoledronic acid (n = 114) or placebo (n = 114) every 4 weeks. After 1 year, the percentage of patients with at least one SRE (excluding hypercalcemia of malignancy) was significantly reduced by 20% by zoledronic acid (29.8% vs. 49.6% for placebo; P = 0.003). Zoledronic acid also significantly delayed the time-tofirst SRE (P = 0.007) and reduced the risk of SREs by 41% in multiple event analysis (P = 0.019) compared with placebo. Elsewhere in the world zoledronic acid was compared to pamidronate in a randomized, double-blind, phase III trial.102 The trial was designed as a noninferiority trial, in which the primary efficacy variable was the proportion of patients experiencing at least one SRE. A group of 1130 patients with advanced breast cancer and at least one metastatic bone lesion were randomized to receive either 4 mg zoledronic acid or 8 mg zoledronic acid via a short intravenous infusion, or 90 mg pamidronate via a 2-hour infusion. Treatments were administered every 3 to 4 weeks. Initially, zoledronic acid was administered as a 5-minute infusion in 50 mL of 0.9% saline or 5% dextrose. This was amended to a 15-minute infusion in 100 mL of saline or dextrose because of concerns over renal toxicity. Similarly, the 8mg dose of zoledronic acid was reduced to 4 mg because of continuing concerns over renal safety. After 25 months of follow-up, the reduction in the overall proportion of patients with a SRE and the skeletal morbidity rate were similar in patients receiving zoledronic acid and pamidronate. However, zoledronic acid 4 mg reduced the risk of developing skeletal complications (including hypercalcemia of malignancy) as determined by multiple event analysis by an additional 20% compared with pamidronate in the overall population (P = 0.025).103 Thus, the risk of skeletal complications with zoledronic acid is approximately one-half the rate experienced in the prebisphosphonate era (Fig. 57-15). All markers of bone resorption or formation decreased from baseline to the end of the study, but at all time points the urinary marker of bone resorption NTX was significantly less in the zoledronic acid 4-mg group than in the pamidronate group. Median overall survival was similar at approximately 2 years in the study groups. The most common adverse events were bone pain, nausea, fever, and fatigue, and as with the other adverse effects, they occurred generally with a similar frequency in each group. The incidence of renal dysfunction among the patients receiving 4 mg zoledronic acid (given on the 15-minute schedule) was indistinguishable from that for the pamidronate patients. Ibandronate is another highly potent amino-bisphosphonate that is licensed in Europe for the treatment of hypercalcemia of malignancy, the treatment of metastatic bone disease, and the prevention

64% risk of skeletal complication with no bisphosphonate Approx 33% risk reduction with pamidronate Further 20% risk reduction with zoledronic acid

64%

43%

34%

Figure 57-15 • Incremental improvement in the risk of skeletal-related events with use of pamidronate and more recently zoledronic acid in the treatment of bone metastases from breast cancer.

Bone Metastases • CHAPTER 57

and treatment of osteoporosis. A phase III placebo-controlled trial of monthly infusions in breast cancer has shown a significant reduction in skeletal-related morbidity with ibandronate 6 mg.104 Additionally, improvements in pain and quality of life were clearly demonstrated at this dose. However, as with the oral formulation, the clinical value of intravenous ibandronate is unclear until comparative trials with established bisphosphonates are completed.

MULTIPLE MYELOMA. Multiple myeloma is typically characterized by a marked increase in osteoclast activity and proliferation. This excessive resorption of bone can be detected histomorphometrically at an early phase in the development of the disease, and this itself could, through the release of interleukin-6 by the osteoclasts, play a contributory role to the growth of myeloma cells in bone. Bisphosphonates could thus be of great benefit in these patients. Oral Bisphosphonates. In a randomized, placebo-controlled trial of 350 patients with newly diagnosed myeloma, it was demonstrated that 2.4 g of clodronate daily for 2 years results in a significant reduction in the proportion of patients developing progression of osteolytic bone lesions (24% vs. 12%). There was only a mild, albeit significant, effect on the incidence of bone pain, however, and no effect on the occurrence of fractures or overall survival.105 Another randomized, placebo-controlled trial of 614 patients evaluated the efficacy of 1600 mg daily of clodronate given from the time of diagnosis. Treatment with clodronate was associated with a 50% decrease in the proportion of patients with severe hypercalcemia (5.1% vs. 10.1%, P = 0.06) and a reduction in reported nonvertebral fractures (6.8% vs. 13.2%, P = 0.04). Additionally, a 30% reduction in the number of vertebral fractures (80 vs. 146, P = 0.012) was observed in a subset of patients with serial spine radiograms available for review.106

Intravenous Bisphosphonates. In the last 10 years, intravenous bisphosphonates have become routine clinical management for most patients with multiple myeloma. This followed the very clear results demonstrated in a 21-month placebo-controlled trial of pamidronate 90 mg conducted in 392 patients.107 The proportion of patients developing SRE(s) was significantly lower in the group receiving pamidronate than the group receiving placebo (24% vs. 41%, P < 0.001). Quality-of-life scores, performance status, pain scores, the incidence of pathologic fractures, and the need for radiotherapy were all favorably influenced by pamidronate therapy. Zoledronic acid has also been evaluated in multiple myeloma; 450 patients with advanced myeloma were included in a randomized trial comparing zoledronic acid with pamidronate.102 No significant differences between the two agents were identified. Of the patients treated with zoledronic acid 4 mg, 50% experienced one or more SREs, compared with 54% in the group receiving pamidronate. The risk of an SRE was 7% lower with zoledronic acid, but this difference was not statistically significant.103 Intravenous ibandronate has been investigated in a randomized trial; however, the 2-mg dose chosen was unfortunately inactive and not statistically different from placebo.108 It is now known that a dose of ibandronate 6 mg is required to reduce skeletal morbidity from metastatic bone disease.104 PROSTATE CANCER. Bisphosphonates have been shown to reduce biochemical markers of bone resorption in patients with osteoblastic bone lesions that are associated with advanced prostate cancer. Additionally, several phase II studies have assessed bone pain and analgesic use with some benefit in these acute endpoints.86 However, these trials were statistically underpowered to detect significant effects on skeletal complications, and the results were not sufficiently convincing to lead to either regulatory approval for or widespread use of bisphosphonates for metastatic bone disease in prostate cancer. Furthermore, until recently, randomized, placebocontrolled trials of bisphosphonates had failed to demonstrate a sig-

nificant reduction in skeletal complications from bone metastases in patients with advanced prostate cancer.

Oral Bisphosphonates. In a study of 57 patients with hormonerefractory prostate cancer and bone pain at study entry, Smith109 concluded that etidronate had no significant effects on pain levels or analgesic usage over and above placebo. The Medical Research Council in the United Kingdom performed a phase III trial of oral clodronate (Loron, 1040 mg twice daily) in 311 men with metastatic bone disease from prostate cancer.110 A slight reduction in the proportion of patients receiving clodronate who experienced a SRE, an improvement in time to progression and increased median survival were observed, but none of these differences was statistically significant. Intravenous Bisphosphonates. A more recent clinical trial involving 208 patients investigated both pain and analgesic usage. In this study, intravenous clodronate was added to a background treatment of mitoxantrone and prednisolone. The study also included objectively measurable skeletal complications as clinical endpoints. No significant differences between clodronate and placebo were seen.111 Pamidronate has also been studied in 236 patients with prostate cancer and bone metastases treated with intravenous pamidronate (90 mg) or placebo every 3 weeks for 9 months. This trial assessed bone pain as the primary endpoint and included an assessment of SREs as a secondary endpoint. Patients in this trial had very advanced disease (median baseline PSA = 97.8 ng/mL in the pamidronate group), very high levels of bone resorption, and substantial bone pain at study entry. Pamidronate did not reduce the incidence of SREs and had only a slight effect on bone pain.112 Despite the failure of all other bisphosphonates, zoledronic acid was investigated in patients with advanced prostate cancer to determine whether the increased potency of this compound would translate into improved clinical benefit. In this study, 643 patients with hormone-refractory prostate cancer and documented bone metastases were randomized to receive either placebo or zoledronic acid at a dose of 4 mg or 8 mg administered every 3 weeks.88 In the 8-mg arm, the dose was reduced by a protocol amendment to 4 mg because of concerns over renal safety, and conclusions on the efficacy of this cohort are difficult to make. Zoledronic acid was significantly more effective than placebo across all primary and secondary endpoints. The zoledronic acid 4 mg treatment group experienced significantly fewer SRE(s) (33% vs. 44% with placebo; P = 0.021). Furthermore, there were consistent reductions in the proportion of patients with each type of skeletal complication, including nonvertebral fractures. Zoledronic acid also prolonged the time to first skeletal complication by more than 4 months (P = 0.011). Zoledronic acid 4 mg remained superior to placebo when fractures were excluded, indicating that the beneficial effect was not simply as a result of the prevention of osteoporotic fractures. Using the Andersen-Gill multiple-event analysis, it was calculated that zoledronic acid 4 mg reduced the overall risk of skeletal complications by 36%.113 Zoledronic acid reduced bone pain at all time points. Despite the favorable effects on skeletal morbidity, however, there were no significant effects on disease-related endpoints such as time to progression and survival. Treatment was generally well tolerated, and in particular the risk of renal function deterioration in patients treated with zoledronic acid 4 mg via a 15minute intravenous infusion was found to be similar to that of placebo-treated patients. The only adverse events that occurred at increased frequency with zoledronic acid were fatigue, anemia, myalgia, and pyrexia. OTHER TUMORS. Until recently there had only been anecdotal reports of the use of bisphosphonates in other tumors associated with bone metastases. The pathophysiology of bone metastases is broadly similar in all tumor types, however, and bisphosphonates could thus be expected to be of value in preventing skeletal morbidity, especially

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if metastatic bone disease was a patient’s dominant site of disease. As part of the development program for zoledronic acid, a phase III randomized, placebo-controlled trial was performed in the management of bone metastases from solid tumors other than breast or prostate cancer.89 The study found that 4 mg of zoledronic acid significantly reduced the proportion of patients with at least one SRE (39% vs. 48%, P = 0.039) and significantly prolonged the time to the first SRE compared with placebo (314 days vs. 168 days, respectively, P = 0.021). This is a particularly important result in a population of patients with a very short median survival time (6 months). Overall, zoledronic acid reduced the risk for SRE(s) by about 30% (hazard ratio 0.693 vs. placebo, P = 0.003). Of particular note was a 58% reduction in the risk of an SRE in the subgroup of patients with renal cell cancer accompanied by an apparent increase in time to progression and overall survival, albeit in an unplanned subset analysis.90

Optimum Use of Bisphosphonates in Metastatic Bone Disease Criteria need to be determined regarding when in the course of metastatic bone disease bisphosphonates should be started and stopped.114,115 Because of the logistics and cost of delivering monthly intravenous infusions for all patients with metastatic bone disease, certain empiric recommendations on who should receive treatment are needed.116 These should take into account the underlying disease type and extent, the life expectancy of the patient, the probability of the patient experiencing a SRE, and the ease with which the patient can attend for treatment (or be treated by a domiciliary service). Consensus guidance recommendations indicate that all patients with multiple myeloma116 and radiologically confirmed bone metastases from breast cancer117 should receive bisphosphonates from the time of diagnosis and continued indefinitely. The development of an SRE is not necessarily a sign of treatment failure or signal to stop treatment; evidence is now available to confirm that bisphosphonates delay second and subsequent complications, not just the first event. However, a recent report suggested that switching to zoledronic acid may be an appropriate option in breast cancer patients in whom pamidronate or clodronate therapy proves unsatisfactory. Clemons and colleagues conducted a phase II clinical trial involving 31 patients with breast cancer who had experienced progressive bone metastasis or SREs while on pamidronate or clodronate therapy. Subjects were switched to monthly administration of zoledronic acid 4 mg. After 8 weeks, 13 of 31 subjects (42%) reported a reduction in pain (P < 0.001). The switch to zoledronic acid was also associated with significant reductions in urinary markers of bone turnover (P = 0.008).118 Bisphosphonate treatment—specifically zoledronic acid—is also appropriate for patients with endocrine-resistant metastatic bone disease from prostate cancer. Patients with other tumors and symptomatic metastasis to bone should be considered for bisphosphonate treatment if bone is the dominant site of metastasis, especially if the prognosis is reasonable (longer than 6 months). Patients with renal cell cancer particularly seem to benefit from treatment. Despite the obvious clinical benefits of bisphosphonates, it is clear that only a proportion of events is prevented, and some patients do not experience a skeletal event despite the presence of metastatic bone disease. It is currently impossible to predict whether an individual patient needs or will benefit from a bisphosphonate. Overall, bisphosphonates reduce the frequency of skeletal events by 25% to 40%; however, bisphosphonates are a relatively costly additional intervention in cancer care that is now potentially applicable to a very large proportion of patients with advanced malignancy. The cost effectiveness of routine long-term treatment has been questioned, and prioritization of bisphosphonate use is essential.119,120 A report on the use of the bone resorption marker NTX suggests that biochemical monitoring could be useful to identify patients at high risk of skeletal complications. In this study of 121 patients with bone metastases, monthly measurements of urinary NTX were made during treatment with a range of bisphosphonates.121 All SREs, hos-

pital admissions for control of bone pain, and deaths during the period of observation were recorded. NTX was strongly correlated with the number of SREs and/or deaths (P < 0.001). Patients with NTX values above 100 nmol/mmol creatinine were many times more likely to experience an SRE or death than those with NTX below this level (P < 0.01). Thus, a more cost-effective use of bisphosphonates, particularly in patients with additional extensive visceral metastases or solid tumors associated with a short life expectancy, might be to reserve them until patients have raised NTX levels, and to adjust the dose and schedule to maintain a normal rate of bone resorption. Randomized trials to assess this approach are planned. Uncertainty remains about the appropriate duration and schedule of treatment. Bone marker–directed therapy is under evaluation in randomized clinical trials, and the value of maintenance therapy after 1 to 2 years of treatment may become clear from the Optimize trial ongoing in the United States. Long-term, full-dose monthly intravenous treatment is associated with the development of osteonecrosis of the jaw (a hitherto rarely encountered problem characterized by painful bone destruction, secondary infection, and delayed healing in the mandible and or maxilla).122 The risk of osteonecrosis of the jaw is related to duration of treatment at around 1% per year on therapy.123 Good dental hygiene and pretreatment restorative treatment are recommended to reduce the risk of this unpleasant complication of treatment.124

New Targeted Therapies in the Treatment of Metastatic Bone Disease As our understanding of the signaling mechanisms between bone cells themselves and bone cells and tumor cells increases, several targeted agents have entered clinical development. These include inhibition of RANKL, cathepsin K (an osteoclast-derived enzyme that is essential for the resorption of bone), PTHrP, and Src (a key molecule in osteoclastogenesis). Of all these, inhibition of RANKL seems the most promising. Denosumab is a fully human monoclonal antibody that binds and neutralizes RANKL with high affinity and specificity, thereby inhibiting osteoclast function and bone resorption. Denosumab could potentially be used to treat bone loss caused by bone metastases, multiple myeloma, or osteoporosis. Following a single subcutaneous dose, denosumab caused rapid and sustained suppression of bone turnover in postmenopausal women with low bone mass,125 as well as in multiple myeloma and breast cancer patients.126 A subsequent dose-finding phase II study has defined a dose and schedule for phase III development of 120 mg four times weekly. Currently a broad program in metastatic disease, treatment-induced bone loss, and bone metastasis prevention studies are underway.

Protecting the Skeleton Prevention of Bone Metastases Bone is the most frequent site of distant relapse, accounting for around 40% of all first recurrences.1 In addition to the well-recognized release of bone cell-activating factors from the tumor, it is now appreciated that release of bone-derived growth factors and cytokines from resorbing bone can both attract cancer cells to the bone surface and facilitate their growth and proliferation.127 Inhibition of bone resorption could therefore have an effect on the development and progression of metastatic bone disease and is an adjuvant therapeutic strategy of potential importance. Encouraging animal studies with a variety of animal tumor models and a range of bisphosphonates have shown inhibition of bone metastasis development and a reduction in tumor burden within bone.128 More recently, several clinical trials have been reported using the relatively low-potency oral bisphosphonate, clodronate. In the largest study, 1079 women with primary operable breast cancer were randomized to receive either clodronate 1600 mg daily or placebo for 2

Bone Metastases • CHAPTER 57

years in addition to standard adjuvant systemic treatment. With a median follow-up time of 5 years, a modest reduction in the frequency of bone metastases in the patients treated with clodronate (63 [12%] vs. 80 [15%] patients) was seen.129 There was no significant effect on non-bone recurrence (112 [21%] vs. 128 [24%] patients), but patients randomized to the clodronate arm had a higher probability of survival (82% vs. 77%, P = 0.047). In a second study, Diel and colleagues130 studied 302 patients with breast cancer selected on the basis of immunocytochemical detection of tumor cells in the bone marrow, a known risk factor for the subsequent development of distant metastases. Patients received appropriate adjuvant chemotherapy and endocrine treatment. The incidence of osseous metastases was significantly lower in the clodronate group, and there was also a large, somewhat unexpected, reduction in the incidence of visceral metastases in the clodronate group. Survival was significantly extended. These results have subsequently been updated and show similar results, although the striking effect on extraskeletal visceral relapse is no longer statistically significant.131 A third study produced conflicting results; the overall 5-year survival was significantly lower in the clodronate group (70% vs. 83%, P = 0.009). although there were some prognostic imbalances favoring the control group that may explain this unexpected result.132 A definite adjuvant role for bisphosphonates will require the results from much larger randomized studies. The National Surgical Adjuvant Breast Project has completed accrual (n = 3300) to a placebo-controlled trial of oral clodronate in stage I to III breast cancer in an attempt to resolve the value or otherwise of adjuvant clodronate. Similarly a large trial of adjuvant zoledronic acid (n = 3360) in stage II/III breast cancer has completed accrual. First efficacy results from these studies are not expected before 2008. It is hoped that the added potency of zoledronic acid might have beneficial effects, not only through the inhibition of bone resorption, but also through direct effects on tumor cells in the bone marrow. There is increasing evidence, from a range of cell line and animal model experiments, that zoledronic acid can inhibit tumor cell adhesion and invasion.133 Additionally, zoledronic acid promotes apoptosis both directly and, more importantly, a schedule-dependent synergy with chemotherapy.134 These effects are mediated through the mevalonate pathway, using the same molecular pathway that aminobisphosphonates exploit to inhibit osteoclast function. Finally, there are experimental data from animal models indicating that zoledronic acid can suppress angiogenesis.135

Effects of Cancer Treatments on Skeletal Health There are now increasing numbers of long-term survivors from cancer who have received combination chemotherapy, radiotherapy, and hormonal cancer treatment. Many of these individuals are at increased risk of osteoporosis, largely because of the endocrine changes induced by treatment. There might also be clinically relevant, direct effects of cytotoxic drugs on bone. Cancer treatment-induced bone loss is a particularly important long-term problem for women with breast cancer and men receiving ADT.136 Peak bone mass minus the bone loss associated with age and estrogen deficiency are the main determinants of osteoporotic fracture risk. However, other factors, including genetics, lifestyle, concomitant medication, and nutrition, influence the risk for bone loss.

Bone Loss in Breast Cancer Cancer treatment-induced bone loss is an increasingly recognized complication in women receiving long-term estrogen-reducing therapies. Estrogen is known to be critical in the maintenance of normal bone mass in women. After menopause a reduction in bone mineral density (BMD) occurs, with the loss most pronounced during the first 3 years, when the rate can be as high as 5% annually, before reducing to a rate of about 0.5% annually thereafter. In the adjuvant setting, all third-generation aromatase inhibitors have demonstrated

increased loss of BMD, which may lead to osteoporosis and skeletal complications.136 In fact, spinal function is negatively affected by bone loss regardless of detectable fractures. Compared with naturally occurring bone loss, aromatase inhibitor-associated bone loss may result in greatly increased BMD loss at 1 year, with rates averaging 5% to 6% in the immediate postmenopausal period.136 Several clinical trials have demonstrated increased fracture rates in postmenopausal women with breast cancer receiving aromatase inhibitors.137 However, a significant proportion of this excess rate of fractures may be due to the absence of the bone-protective effects of tamoxifen. Tamoxifen is known to have a modest estrogenic effect on bone, at least in postmenopausal women, and placebo-controlled trials, for instance in the breast cancer prevention setting, have shown a modest reduction in fracture incidence compared with placebo. Furthermore, in a study of breast cancer patients who received either letrozole or placebo as extended adjuvant therapy, although more patients in the letrozole group had fractures after 5 years, the difference did not reach significance.138 A few studies have evaluated women with breast cancer and a treatment-induced premature menopause. Saarto and colleagues139 studied the effect of clodronate 1600 mg in 148 premenopausal women receiving adjuvant chemotherapy for breast cancer. They observed that rapid bone loss occurred in the women who became amenorrheic after chemotherapy (6% and 2% losses at 2 years in the spine and hip, respectively). Among those receiving clodronate, however, the bone effects of chemotherapy-induced premature menopause were attenuated (2% loss and 1% gain at 2 years in the spine and hip, respectively). In a comparison of risedronate with placebo in a postmenopausal group of patients receiving tamoxifen, Delmas and associates140 observed an approximate 2.5% increase in BMD at the lumbar spine and femoral neck in the risedronate group compared with the group receiving placebo. Intravenous bisphosphonate administration is used widely in oncology in the treatment of metastatic disease and could be an attractive option for preventing bone loss in a cancer population. Among patients with breast cancer, 6-monthly zoledronic acid has also been shown to reverse the bone loss induced by a combination of the LHRH analog goserelin plus further estrogen suppression with anastrozole. In this study, the mean loss of bone in the lumbar spine over 3 years in the absence of a bisphosphonate was 17%. However, the bone loss was abrogated by administration of 6-monthly zoledronic acid.141 In older postmenopausal women the strategy of immediate 6monthly zoledronic acid alongside aromatase inhibitor therapy has been compared with initiation of bone protection treatment if osteoporosis or significant bone loss occurs on treatment. The difference in lumbar spine BMD at 1 year was approximately 5%.142

Bone Loss in Prostate Cancer Many men with prostate cancer are at risk of developing osteoporosis, largely because of the ADT they receive for their cancer. ADT may be achieved either by bilateral orchiectomy or, increasingly, by use of a gonadotropin-releasing hormone agonist. These treatments are being introduced earlier and earlier in the course of the disease with the result that men may experience many years of androgen suppression. ADT results in substantially reduced serum concentrations of testosterone (to less than 5% of normal level) and estrogen (to less than 20% of normal level). It might be expected that androgen deprivation would lead to increased bone loss and increased fracture rate. As yet there seem to have been no large prospective studies of the relationship between fracture rate and ADT, but retrospective studies indicate a significantly increased fracture rate with an estimated 10-year probability of fracture at around 40%.143 As the potential scale of the problem is being realized, attention is not only focusing on measuring bone density in such patients to assess those at risk, but also on therapeutic options such as bisphosphonates to prevent or treat

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therapy-related bone loss in prostate cancer. Pamidronate given on a 3-monthly schedule has been shown to prevent loss in BMD in patients with locally advanced prostate cancer.144 A multicenter prospective study has evaluated the potent bisphosphonate, zoledronate, in locally advanced or recurrent prostate cancer.145 At 1 year, in the zoledronic acid arm, BMD increased by 5.3% at the lumbar spine and 1.1% in the total hip. In the placebo arm the BMD decreased by 2% in the lumbar spine and 2.8% in the total hip.

COMPLICATIONS OF BONE METASTASES Bone metastases cause considerable morbidity: pain, impaired mobility, hypercalcemia, pathologic fracture, spinal cord or nerve root compression, and bone marrow infiltration. In two large randomized trials that included patients with breast cancer and multiple myeloma receiving chemotherapy, the mean skeletal morbidity rates (number of skeletal events per year) in the absence of bisphosphonates were 3.5 and 2.0, respectively, indicating that a skeletal event occurs in metastatic bone disease from breast cancer on the average of every 3 to 4 months and in multiple myeloma every 6 months.97,107 Despite the clinical importance of metastatic bone disease and the huge expenditure on medical care for skeletal complications, however, there has until recently been relatively little thought given as to how best to coordinate clinical management and deliver optimum care for patients with bone metastases. Extensive infiltration of the bone marrow by metastatic disease causes leukoerythroblastic anemia and pancytopenia predisposing to infection and hemorrhage. Radiotherapy, often needed for the treatment of bone metastases, can exacerbate this problem, which in turn can compromise the ability to give chemotherapy effectively. Animal experiments have shown that cytotoxic drugs can interfere with osteoblastic function and new bone formation, but the clinical significance of these findings is unknown. Other iatrogenic factors might also aggravate morbidity from bone metastases including corticosteroids and endocrine ablation.

Bone Pain Bone pain is the most common type of pain from cancer and a significant problem in both hospital and community practice. Pain is usually the presenting symptom and is caused by a variety of factors, including periosteal stretching, compression or infiltration of nerve roots, reflex muscle spasm, and the local effects of cytokines. Features of bone pain are that it is often poorly localized, has a deep, boring quality that aches or burns, and is accompanied by episodes of stabbing discomfort. It is often worse at night, being little helped by sleep and not necessarily relieved by lying down. There is often disturbance of the highly innervated periosteum—possibly giving this pain its neurogenic-like qualities and adding to its intractability. Spinal instability is the cause of back pain in 10% of patients with cancer.146 This instability can cause excruciating pain that is mechanical in origin. The patient is comfortable only when lying still, and any movement reproduces severe pain. Consequently, the patient might not be able to sit, stand, or walk. Because the pain is mechanical in origin, radiation therapy or systemic treatment cannot help; the only solution is stabilization of the spine. Stabilization requires major surgery, with risks of significant morbidity and mortality, but with careful selection of patients, excellent results can be obtained.

Hypercalcemia of Malignancy Hypercalcemia (see also Chapter 48) is another emergency associated with metastatic bone disease. Its clinical features include nausea, vomiting, dehydration, and confusion. Although malignant hypercalcemia is usually associated with demonstrable bone metastases, this is not always the case. Hypercalcemia causes a number of signs and symptoms, which vary considerably from patient to patient. These are often nonspecific, affecting many systems in the body, and can

be mistaken for symptoms of the underlying cancer or associated treatment if there is not an astute awareness of the possibility of hypercalcemia. If untreated, a progressive rise in serum calcium leads to deterioration in renal function and level of consciousmess. Death ultimately ensues as a result of cardiac arrhythmias and renal failure. It is now clear that various mechanisms are involved in the pathogenesis of malignant hypercalcemia. These include increased bone resorption (osteolysis) and systemic release of humoral hypercalcemic factors. Bone metastases are common but not invariably present. In some tumors, such as squamous cell cancers, humoral mechanisms are dominant, increasing both renal tubular calcium reabsorption and phosphate excretion. In others—multiple myeloma and lymphoma, for example—osteolysis predominates, whereas in breast cancer both osteolysis and humoral mechanisms seem to be important. Doubt about the etiology of hypercalcemia in patients with cancer is unusual, but nonmalignant causes must be considered, particularly in the absence of metastases. In the community, hyperparathyroidism is the most common cause of hypercalcemia and may be encountered also in patients with cancer. Measurement of PTH using a modern, specific radioimmunoassay is worthwhile if there is any doubt about the diagnosis; levels of PTH tend to be low or undetectable in malignancy and inappropriately high in hyperparathyroidism. Intravenous bisphosphonates, in conjunction with rehydration, are now established as the treatment of choice for hypercalcemia. Approximately 70% to 90% of patients will achieve normocalcemia, resulting in relief of symptoms and improved quality of life. Zoledronic acid is the most effective bisphosphonate for the acute treatment of this metabolic emergency.147

Pathologic Fractures Metastatic destruction of bone reduces its load-bearing capabilities, resulting initially in trabecular disruption and microfractures and, subsequently, in total loss of bony integrity. Rib fractures and vertebral collapse are the most common occurrences, resulting in loss of height, kyphoscoliosis, and a degree of restrictive lung disease. The most severe disability, however, is caused by fracture of a long bone or epidural extension of tumor into the spine. The incidence of pathologic fracture in patients with bone metastases is somewhat uncertain and is dependent on whether rib and vertebral fractures are included and the method of evaluation. In recent series in which regular skeletal surveys were performed the annual risk of a pathologic fracture (in the absence of bisphosphonates) was between 20% in prostate cancer85 and 40% in breast cancer.97 Not all of these fractures are symptomatic, and undoubtedly some are due to treatment-induced osteoporosis rather than metastatic infiltration, but nevertheless structural damage to bone is clearly extremely common in metastatic bone disease. Paterson and colleagues,92 who systematically reviewed serial radiographs in patients participating in a clinical trial of oral clodronate, showed that a woman with bone metastases from breast cancer can expect to experience an average of 1.3 vertebral and 0.4 nonvertebral fractures a year. The probability of developing a pathologic fracture increases with the duration of metastatic involvement and is therefore, somewhat paradoxically, more common in those patients with bone-only disease who have a relatively good prognosis. A retrospective analysis of 859 patients with bone metastases from breast cancer showed that those with bone-only disease had almost a fourfold increase in the incidence of subsequent pathologic long-bone fractures compared with patients who also had concomitant liver metastases. This finding was attributed to the different survival outcomes between the two groups; the median survival from diagnosis of bone metastases for patients with bone-only disease was 2.2 years, compared with 5.5 months for those with concomitant liver disease.148 The study also showed that scintigraphic evidence of metastases in the femora and humeri at the

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time of diagnosis significantly predicted an increased risk of future fractures. Because the development of a fracture is so devastating to a patient with cancer, increased emphasis is now being placed on attempts to predict metastatic sites at risk of fracture and the use of prophylactic surgery and long-term administration of bisphosphonates to reduce fracture risk. Assessment of patients with symptomatic bone metastases by a specialist orthopedic and/or spinal surgeon should be a much more frequent component of multidisciplinary management than has been the case until now. Fractures are common through lytic metastases and weightbearing bones, the proximal femora being the most commonly affected sites. Damage to both trabecular and cortical bone are structurally important, but it is the relevance of cortical destruction that is most clearly appreciated. Several radiologic features that could predict imminent fracture have been identified. Risk factors that have been taken into account include pain, the anatomic site of a lesion, its radiologic characteristics, and its size. Although intensity of pain, which is difficult to quantify, is not clearly associated with fracture risk, pain that is exacerbated by movement seems to be an important factor in predicting impending fracture. Presumably, such functional pain indicates diminution in the mechanical strength of a bone and in one series was followed invariably by fracture. As far as radiologic appearances are concerned, there is a general consensus that lytic lesions carry a much higher risk of fracture than either mixed or osteosclerotic lesions. Accordingly, a particularly high fracture rate is found in association with metastases from lung cancer. Given the poor prognosis of this tumor, however, such fractures rarely lead to prolonged disability. By contrast, in breast cancer, which follows a much more protracted course, pathologic fracture is a major cause of prolonged disability. Radiologic assessment also yields information about the size of a lesion and the extent to which the bone is destroyed. When less than two thirds of the diameter of a long bone is affected, pathologic fracture is relatively unusual but above this limit the fracture rate increases markedly, with an incidence of approximately 80% for such lesions. A practical scoring system incorporating anatomic, radiographic, and symptom-related factors has been described to give valuable guidance in the selection of patients for prophylactic fixation.149 Prophylactic internal fixation is usually the treatment of choice for such lesions, followed by radiotherapy to inhibit further tumor growth and avoid further bone destruction. It is easier to stabilize a bone while it is still intact, and the rehabilitation and convalescence are shorter and easier. Providing the lesion is irradiated, there is no evidence to suggest that surgery increases the risk of disseminating tumor cells either locally or into the circulation. Indeed, there is some experimental evidence that pathologic fractures are associated with an increased incidence of pulmonary metastases and that prophylactic stabilization decreases this incidence. If a given patient is not fit for surgery, radiotherapy and avoidance of weight-bearing activity are indicated. Before surgery, a radionuclide bone scan and radiographs of the entire length of the affected bone should be obtained. These measures ensure that any other metastases that might subsequently develop into a pathologic fracture are also stabilized and included in the radiotherapy field. A pathologic fracture at the edge of a plate or of an intramedullary nail, particularly when fixed with methylmethacrylate, is more difficult to treat than if there were no implant in the bone. Pathologic fractures are not necessarily a manifestation of terminal disease, and primary internal stabilization followed by radiotherapy are usually the treatments of choice, and certainly the only modalities likely to both restore mobility and relieve pain. Untreated pathlogic fractures rarely heal, and although radiotherapy might achieve local tumor control, bony union remains unlikely. Radiotherapy inhibits chondrogenesis (a prerequisite for fracture healing), and with large

areas of bone destruction there could be insufficient matrix remaining for adequate repair. The type of internal stabilization chosen depends on the site of the lesion, and the range of stabilization devices and custom-made prostheses increases year on year. When feasible, closed intramedullary nailing is preferred; however, at the end of the long bones, intramedullary nailing alone is inadequate, and alternative techniques are necessary. It is essential that the internal stabilization provide sufficient strength to allow unsupported use of the limb and for the legs to bear weight. Fulfilling this demand could require supplementation with methylmethacrylate (which is inserted into the tumor cavity with the implant fixed across the methylmethacrylate while still soft) and bridging normal bone above and below the lesion. Pathologic femoral neck fractures do not unite despite internal fixation, and this situation requires replacement arthroplasty. Careful preoperative assessment of the pelvis and femur is necessary, and for this, CT scanning or MRI is often helpful. If there is no metastatic involvement of the acetabulum, a hemiarthroplasty could be all that is required. If the acetabulum is involved, however, total-replacement arthroplasty is indicated, and sometimes pelvic reconstruction is necessary. Many patients have metastases in the distal femur together with proximal involvement, and for these patients, a long-stemmed femoral prosthesis is recommended. For humeral fractures, internal fixation is also useful, providing more rapid and greater pain relief compared with conservative treatment. Although patients with a very short life expectancy can be managed adequately with conservative treatment, those patients expected to survive longer than 3 months are best managed by internal fixation to ensure pain relief and restoration of function. Replacement arthroplasty could be necessary if the proximal humerus is involved, but most pathologic fractures of the humerus can be treated by intramedullary nailing. Occasionally, patients present with an isolated metastasis in the distal skeleton. If on careful evaluation there is no other evidence of dissemination of the tumor, resection of the lesion should be considered. Local resection and prosthetic replacement are usually possible, but occasionally, amputation is indicated.

Spinal Instability Spinal instability can cause excruciating pain that is mechanical in origin and not relieved by radiotherapy or systemic treatment. As with pathologic fractures of long bones, stabilization is required for pain relief and involves major surgery, which is associated with significant morbidity and mortality. There are several methods for spinal stabilization, but in general, the posterior approach is technically easier and allows stabilization of a larger area of the spine. With careful selection of patients, excellent results can be obtained. An associated neurologic deficit is not a contraindication to these procedures. Percutaneous vertebroplasty and kyphoplasty, a new approach to treating spinal pain and instability, involves injecting an acrylic polymer into a diseased vertebral body. The technique was developed initially for the treatment of painful vertebral hemangiomas, and considerable experience with it has been obtained in the treatment of osteoporotic compression fractures. Its use has now been extended to the treatment of malignant spinal disease.150,151 The technique provides effective pain relief, which is achieved more rapidly than with radiotherapy, and it confers the added benefit of providing structural support to the spinal column, thus reducing the risk of vertebral collapse and instability. Although generally a safe procedure, vertebroplasty can be complicated by leakage of the polymer, which predisposes to spinal cord or nerve root compression. The risk of this is less with kyphoplasty.151 The technique seems to have the potential for wider use, particularly among patients with limited vertebral disease and those for whom major surgical spinal stabilization procedures are unsuitable.

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Compression of the Spinal Cord or Cauda Equina Compression of the spinal cord or cauda equina in patients with metastatic disease of the spine is a medical emergency necessitating prompt diagnosis and treatment (see also Chapter 55). Its causes include pressure from an enlarging extradural mass, spinal angulation after vertebral collapse, vertebral dislocation after pathologic fracture, or, rarely, pressure from intradural metastases. The most common primary tumors producing this complication, in decreasing order of frequency, are carcinoma of the breast, lung cancer, prostatic cancer, lymphoma, and renal carcinoma. Back pain is the most common initial symptom of spinal cord compression; two types of pain can occur—local spinal or radicular. Radicular pain varies with the location of the tumor, being common in the cervical (79%) and lumbosacral (90%) regions and less common with thoracic lesions (55%). Both local spinal and radicular pain are experienced close to the site of the lesion identified at myelography. Motor weakness, sensory loss, and autonomic dysfunction are all common at presentation of spinal cord or cauda equina compression. The development of back pain in a patient with cancer, coincident with an abnormality on a plain spinal radiogram, should serve as a warning for the possible development of spinal cord compression. Compression of the spinal cord or cauda equina can occur in association with spinal stability or in isolation. When there is a greater than 50% vertebral collapse, compression of the spinal cord becomes more likely. The keys to successful rehabilitation are early diagnosis, high-dose corticosteroids, rapid assessment, and urgent referral for either decompression and spinal stabilization or radiotherapy. Neurologic recovery is unlikely if the spinal compression is not relieved within 24 to 48 hours. In a retrospective analysis of 70 patients with spinal cord compression secondary to breast cancer, the most frequent symptom was motor weakness (96%), followed by pain (94%), sensory disturbance (79%), and sphincter disturbance (61%).152 Of these 70 patients, 91% had at least one symptom for more than 1 week. The ability to walk was maintained by 96% of those ambulant before therapy. In those unable to walk, 45% regained ambulation, with radiotherapy and surgery equally effective. Median survival was 4 months. The most important predictor of survival was the ability to walk after treatment. These findings stress the importance of prompt presentation, diagnosis, and treatment and suggest that earlier diagnosis and intervention should improve outcome. The choice between surgical decompression and radiotherapy depends on a variety of clinical features. Surgical decompression is indicated for patients with recent onset of symptoms and with progressive paraplegia and urinary retention of less than 30 hours’ duration. The site of compression should be localized to no more than two or three vertebral segments, and the patient should have a life expectancy of at least several weeks. For patients in whom the para-

plegia has been established for several days or urinary retention has been present for more than 30 hours, surgical decompression rarely results in the recovery of bladder or motor function. Radiotherapy is indicated for those who are either unfit for surgery or do not meet the criteria for surgical decompression. Several studies in the past suggested that surgical decompression had no advantage over radiotherapy.153 It should be appreciated, however, that these studies compared dorsal laminectomy—an outdated and now inappropriate procedure—with irradiation. However, surgical decompression should be followed by spinal stabilization. More recently a randomized trial was performed that compared modern surgical techniques with radiotherapy. Patchell and associates randomized 101 patients to surgery plus radiotherapy or radiotherapy alone. Significantly more patients in the surgery group (42/50, 84%) than in the radiotherapy group (29/51, 57%) were able to walk after treatment (odds ratio 6.2 [95% confidence interval 2.0–19.8] P = 0.001). Patients treated with surgery also retained the ability to walk significantly longer than did those with radiotherapy alone (median 122 days vs. 13 days, P = 0.003). Thirty-two patients entered the study unable to walk; significantly more patients in the surgery group regained the ability to walk than patients in the radiation group (10/16 [62%] vs. 3/16 [19%], P = 0.01). The need for corticosteroids and opioid analgesics was also significantly reduced in the surgical group.154 Therefore, the choice of management should be decided on an individual basis, and there are undoubtedly patients who will benefit greatly from appropriate and prompt surgical management.

SUMMARY The management of bone metastases requires an experienced multidisciplinary team to ensure timely diagnosis and the appropriate integration of local and systemic treatments. The effects of tumor cells on bone cell function (especially on osteoclast activity) underpin the rationale for the use of bisphosphonate treatment to reduce skeletal morbidity. These bone-specific treatments are now an accepted part of routine clinical management. Additionally, the disruption of bone remodeling results in release of collagen fragments, which seems to have value in predicting skeletal events, prognosis, and monitoring of response. Further developments in our understanding of the pathophysiology of bone metastases can be expected to provide new therapeutic strategies. Already, improved knowledge of the signaling molecules involved in regulating osteoclast function—notably OPG and RANK ligand—has led to the development of highly active targeted therapies for bone diseases, including cancer and other novel therapeutic approaches in clinical development. Over the next 5 years several of these compounds can be expected to gain regulatory approval, and ultimately combinations of bone-targeted therapies may be recommended to further reduce the clinical burden of metastatic bone disease.

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52. Terpos E, Szydlo R, Apperley JF, et al: Soluble receptor activator of nuclear factor kappaB ligandosteoprotegerin ratio predicts survival in multiple myeloma: proposal for a novel prognostic index. Blood 2003;102:1064–1069. 53. Coleman RE, Major P, Lipton A, et al: Predictive value of bone resorption and formation markers in cancer patients with bone metastases receiving the bisphosphonate zoledronic acid. J Clin Oncol 2005;23:4925–4935. 54. Cleeland C: The measurement of pain from metastatic bone disease: capturing the patient’s experience. Clin Cancer Res 2006;12(Suppl): 6236s–6242s. 55. Brown JE., Coleman RE: Assessment of the effects of breast cancer on bone and the response to therapy. Breast 2002;11:375–385. 56. Fogelman I, Cook G, Israel O, van der Waal H: Positron emission tomography and bone metastases. Semin Nucl Med 2005;35:135–142. 57. Duffy MJ: Serum tumour markers in breast cancer: are they of clinical value. Clin Chem 2006;52: 345–351. 58. Robertson JFR, Jaeger W, Syzmendera JJ, et al: The objective measurement of remission and progression in metastatic breast cancer by use of serum tumor markers. Eur J Cancer 1999;35:47–53. 59. Vicini FA, Vargas C, Abner A, et al: Limitations in the use of serum prostate specific antigen levels to monitor patients after treatment for prostate cancer. J Urol 2005;173:1456–1462. 60. Smith MR, Kabbinvar F, Saad F, et al: Natural history of rising serum prostate-specific antigen in men with castrate non metastatic prostate cancer. J Clin Oncol 2005;23:2918–2925. 61. Villalon AH, Tattersall MH, Fox RM, Woods RL: Hypercalcaemia after tamoxifen for breast cancer: a sign of tumor response. Br Med J 1979;20:1329– 1330. 62. Walls J, Assiri A, Howell A, et al: Measurement of urinary collagen cross-links indicate response to therapy in patients with breast cancer and bone metastases. Br J Cancer 1999;80:1265–1270. 63. Vinholes J, Coleman R, Lacombe D, et al: Assessment of bone response to systemic therapy in an EORTC trial: preliminary experience with the use of collagen cross-link excretion. European Organization for Research and Treatment of Cancer. Br J Cancer 1999;80:221–228. 64. Costa L, Demers LM, Gouveia-Oliveira A, et al: Prospective evaluation of the peptide-bound collagen type I cross-links N-telopeptide and Ctelopeptide in predicting bone metastases status. J Clin Oncol 2002;20:850–856. 65. Vinholes JJ, Purohit OP, Abbey ME, et al: Relationships between biochemical and symptomatic response in a double-blind trial of pamidronate for metastatic bone disease. Ann Oncol 1997;8:1243–1250. 66. Jagdev SP, Purohit OP, Heatley S, et al: Comparison of the effects of intravenous pamidronate and oral clodronate on symptoms and bone resorption in patients with metastatic bone disease. Ann Oncol 2001;12:1433–1438. 67. Agarawal JP, Swangsilpa T, van der Linden Y, et al: The role of external beam radiotherapy in the management of bone metastases. Clin Oncol 2006;18:747–760. 68. Blitzer PH: Reanalysis of the RTOG study of the palliation of symptomatic osseous metastasis. Cancer 1985;55:1468–1472. 69. Van der Linden Y, Steenland E, van Howelingen HC, et al: Patients with a favourable prognosis are equally palliated with single and multiple fractionation radiotherapy; results on survival in the Dutch Bone Metastasis Study. Radiother Oncol 2006;78:245–253. 70. Wu JS, Wong R, Johnston M, et al: Meta-analysis of dose fractionation trials for the palliation of

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tumors: a randomized, phase III, double-blind, placebo-controlled trial. Cancer 2004;100:2613– 2621. Lipton A, Colombo-Berra A, Bukowski RM, et al: Skeletal complications in patients with bone metastases from renal cell carcinoma and therapeutic benefits of zoledronic acid. Clin Cancer Res 2004; 10:6397S–6403S. Pavlakis N, Schmidt R, Stockler M: Bisphosphonates for breast cancer. Cochrane Database Syst Rev 2005;3:CD003474. Paterson AH, Powles TJ, Kanis JA, et al: Doubleblind controlled trial of oral clodronate in patients with bone metastases from breast cancer. J Clin Oncol 1993;11:59–65. Coleman RE, Purohit OP, Black C, et al: Doubleblind, randomised, placebo-controlled study of oral ibandronate in patients with metastatic bone disease. Ann Oncol 1999;10:311–316. Body JJ, Diel IJ, Lichinitzer M, et al: Oral ibandronate reduces the risk of skeletal complications in breast cancer patients with metastatic bone disease: results from two randomised, placebo-controlled phase III studies. Br J Cancer 2004;90:1133–1137. Conte PF, Mauriac L, Calabresi F, et al: Delay in progression of bone metastases treated with intravenous pamidronate: results from a multicentre randomised controlled trial. J Clin Oncol 1996;14:2552–2559. Hultborn R, Ryden S, Gunderson S, et al: Efficacy of pamidronate on skeletal complications from breast cancer metastases. A randomised prospective double blind placebo controlled trial. Acta Oncol 1996;35(Suppl 5):73–74. Hortobagyi GN, Theriault RL, Porter L, et al: Efficacy of pamidronate in reducing skeletal complications in patients with breast cancer and lytic bone metastases. N Engl J Med 1996;335: 1785–1791. Theriault RL, Lipton A, Hortobagyi GN, et al: Pamidronate reduces skeletal morbidity in women with advanced breast cancer and lytic bone lesions: a randomised, placebo-controlled trial. J Clin Oncol 1999;17:846–854. Body JJ, Lortholary A, Romieu G, et al: A dosefinding study of zoledronate in hypercalcaemic cancer patients. J Bone Miner Res 1999;14:1557– 1661. Berenson JR, Rosen LS, Howell A, et al: Zoledronic acid reduces skeletal-related events in patients with osteolytic metastases. Cancer 2001;91:1191–1200. Kohno N, Aoqi K, Minami H, et al: Zoledronic acid significantly reduces skeletal complications compared with placebo in Japanese women with bone metastases from breast cancer: a randomized, placebo-controlled trial. J Clin Oncol 2005;20: 3299–3301. Rosen LS, Gordon D, Kaminski M, et al: Zoledronic acid versus pamidronate in the treatment of skeletal metastases in patients with breast cancer or osteolytic lesions of multiple myeloma: a phase III, double-blind, comparative trial. Cancer J 2001;7:377–387. Rosen LS, Gordon D, Kaminski M, et al: Longterm efficacy and safety of zoledronic acid compared with pamidronate disodium in treatment of skeletal complications in patients with advanced multiple myeloma or breast cancer: a randomized, double-blind, multicenter, comparative trial. Cancer 2003;98:1735–1744. Body JJ, Diel IJ, Lichinitser MR, et al: MF 4265 Study Group. Intravenous ibandronate reduces the incidence of skeletal complications in patients with breast cancer and bone metastases. Ann Oncol 2003;14:1399–1405. Lahtinen R, Laakso M, Palva I, et al, for the Finnish Leukaemia Group: Randomised, placebo-

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controlled multicentre trial of clodronate in multiple myeloma. Lancet 1992;340:1049–1052. McCloskey EV, Maclennan ICM, Drayson M, et al: A randomised trial of the effect of clodronate on skeletal morbidity in multiple myeloma. Br J Haematol 1998;100:317–325. Berenson JR, Lichtenstein A, Porter L, et al: Efficacy of pamidronate in reducing skeletal events in patients with advanced multiple myeloma. N Engl J Med 1996;334:488–493. Menssen HD, Sakalova A, Fontana A, et al: Effects of long-term intravenous ibandronate therapy on skeletal-related events, survival, and bone resorption markers in patients with advanced multiple myeloma. J Clin Oncol 2002;20:2353– 2359. Smith JA Jr: Palliation of painful bone metastases from prostate cancer using sodium etidronate: results of a randomized, prospective, double-blind, placebo-controlled study. J Urol 1989;141:85–87. Dearnaley DP, Sydes MR, Mason MD, et al: A double-blind placebo-controlled randomised trial of oral sodium clodronate for metastatic prostate cancer (MRC PR05). J Natl Cancer Inst 2005;95: 1300–1311. Ernst DS, Tannock IF, Winquist EW: Randomised, double-blind controlled trial of mitoxantrone/prednisone and clodronate versus mitoxantrone/prednisone and placebo in patients with hormone refractory prostate cancer and pain. J Clin Oncol 2003;21:3335–3342. Small EG, Smith MR, Seaman JJ, et al: Combined analysis of two multicenter, randomized, placebocontrolled studies of pamidronate disodium for the palliation of bone pain in men with metastatic prostate cancer. J Clin Oncol: 2003;21:4277– 4284. Saad F, Gleason DM, Murray R, et al: Long-term efficacy of zoledronic acid for the prevention of skeletal complications in patients with advanced prostate cancer and bone metastasis. J Natl Cancer Inst 96:879–882, 2004. Plunkett TA, Rubens RD: Bisphosphonate therapy for patients with breast carcinoma: who to treat and when to stop. Cancer 2003;97(Suppl 3): 854–858. Body J-J: Effectiveness and cost of bisphosphonate therapy in tumor bone disease. Cancer 2003; 97(Suppl 3):859–865. Hillner BE, Ingle JN, Berenson JR, et al: American Society of Clinical Oncology 2003 update on the role of bisphosphonates and bone health issues in breast cancer. J Clin Oncol 2003;21:4042–4057. Kyle RA, Yee GC, Somerfield MR, et al: American Society of Clinical Oncology 2007 clinical practice guideline update on the role of biophosphonates in multiple myeloma. J Clin Oncol 2007;25:2464– 2472. Clemons MJ, Dranitsaris G, Ooi WS, et al: Phase II trial evaluating the palliative benefit of secondline zoledronic acid in breast cancer patients with either a skeletal-related event or progressive bone metastases despite first-line bisphosphonate therapy. J Clin Oncol 2006;20:4895–4900. Hillner BE: Pharmacoeconomic issues in bisphosphonate treatment of metastatic bone disease. Semin Oncol 2001;28 (Suppl 11):64–68. Delea T, Langer C, McKiernan J, et al: The cost of treatment of skeletal-related events in patients with bone metastases from lung cancer. Oncology 2004;67:390–396. Brown JE, Thomson CS, Ellis SP, et al: Bone resorption predicts for skeletal complications in metastatic bone disease. Br J Cancer 2003;89: 2031–2037. Ruggiero S, Gralow J, Marx RE, et al: Practical guidelines for the prevention, diagnosis, and treatment of osteonecrosis of the jaw in patients with cancer. J Oncol Pract 2006;2:7–14.

Bone Metastases • CHAPTER 57 123. Hoff AO, Toth BB, Altundag V, et al: Osteonecrosis of the jaw in patients receiving intravenous bisphosphonate therapy (abstract 8528). J Clin Oncol 2006;24(Suppl):475s. 124. Migliorati CA, Casiglia J, Epstein J, et al: Managing the care of patients with bisphosphonate-associated osteonecrosis. An American Academy of Oral Medicine position paper. J Am Dent Assoc 2005;136:1658–1668. 125. Bekker PJ, Holloway DL, Rasmussen AS, et al: A single-dose placebo-controlled study of AMG 162, a fully human monoclonal antibody to RANKL, in postmenopausal women. J Bone Miner Res 2004;19:1059–1066. 126. Body JJ, Facon T, Coleman RE, et al: A study of the biological receptor activator of nuclear factorkappa B ligand inhibitor, Denosumab, in patients with multiple myeloma or bone metastases from breast cancer. Clin Cancer Res 2006;12:1221– 1228. 127. Hiraga T, Williams PJ, Ueda A, et al: Zoledronic acid inhibits visceral metastases in the 4T1/luc mouse breast cancer model. Clin Cancer Res 2004;10:4559–4567. 128. Ottewell PD, Coleman RE, Holen I: From genetic abnormality to metastases: murine models of breast cancer and their use in the development of anticancer therapies. Breast Cancer Res Treat 2006;96:101–113. 129. Powles TJ, Paterson AE, McCloskey E, et al: Reduction in bone relapse and improved survival with oral clodronate for adjuvant treatment of operable breast cancer. Breast Cancer Res Treat 2006;8:R13, E-pub Mar 15. 130. Diel IJ, Solomayer EF, Costa SD, et al: Reduction in new metastases in breast cancer with adjuvant clodronate treatment. N Engl J Med 1998;339; 357–363. 131. Jaschke A, Bastert G, Solomayer EF, et al: Adjuvant clodronate treatment improves the overall survival of primary breast cancer patients with micrometastases to bone marrow—a longtime follow-up (abstract 529). ASCO Annual Meeting Proceedings 2004;22(14S):9S. 132. Saarto T, Vehmanen L, Virkkunen P, Blomqvist C: Ten-year follow-up of a randomized controlled trial of adjuvant clodronate treatment in node-

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positive breast cancer patients. Acta Oncol 2004; 43:650–656. Woodward JK, Neville-Webbe HL, Coleman RE, Holen I: Combined effects of zoledronic acid and doxorubicin on breast cancer cell invasion in vitro. Anticancer Drugs 2005;16:845–854. Neville-Webbe HL, Rostami-Hodjegan A, Evans CA, et al: Sequence- and schedule-dependent enhancement of zoledronic acid induced apoptosis by doxorubicin in breast and prostate cancer cells. Int J Cancer 2005;113:364–371. Wood J, Schnell C, Green J: Novel anti-angiogenic effects of the bisphosphonates compound zoledronic acid. J Pharmacol Exp Ther 2002;302: 1055–1061. Lester J, Dodwell D, McCloskey E, Coleman R: The causes and treatment of bone loss associated with carcinoma of the breast. Cancer Treatment Rev 2005;31:115–142. McCloskey E: Effects of third-generation aromatase inhibitors on bone. Eur J Cancer 2006;42:1044– 1051. Perez EA, Josse RG, Pritchard KI, et al: Effect of letrozole versus placebo on bone mineral density in women with primary breast cancer completing 5 or more years of adjuvant tamoxifen: a companion study to NCIC CTG MA.17. J Clin Oncol 2006:24;3629–3635. Saarto S, Blomqvist C, Valimaki M, et al: Chemical castration induced by adjuvant cyclophosphamide, methotrexate, and fluorouracil chemotherapy causes rapid bone loss which is reduced by clodronate: a randomised study in premenopausal patients. J Clin Oncol 1997;15:1341–1347. Delmas PD, Balena R, Confravreux E, et al: Bisphosphonate risedronate prevents bone loss in women with artificial menopause due to chemotherapy of breast cancer: a double-blind, placebo-controlled study. J Clin Oncol 1997;15:955–962. Gnant MF, Mineritsch B, Luschin-Ebengreuth G, et al: Zoledronic acid prevents cancer treatment induced bone loss in premenopausal women receiving adjuvant endocrine therapy for hormoneresponsive breast cancer: a report from the Austrian Breast and Colotrectal Cancer Study Group. J Clin Oncol 2007;25:820–828.

142. Brufsky A, Harker WG, Beck JT, et al: Zoledronic acid inhibits adjuvant letrozole-induced bone loss in postmenopausal women with early breast cancer. J Clin Oncol 2007;25:829–836. 143. Allain TJ: Prostate cancer, osteoporosis and fracture risk. Gerentology 2006;52:107–110. 144. Smith MR, McGovern FJ, Zietman AL, et al: Pamidronate to prevent bone loss during androgen-deprivation therapy for prostate cancer. N Engl J Med. 2001;345:948–955. 145. Smith MR, Eastham J, Gleason DM, et al: Randomized controlled trial of zoledronic acid to prevent bone loss in men receiving androgen deprivation therapy for nonmetastatic prostate cancer. J Urol 2003;169:2008–2012. 146. Harrington KD: Orthopaedic surgical management of skeletal complications of malignancy. Cancer 1997;80(Suppl):1614–1627. 147. Major PP, Lortholary A, Hon J, et al: Zoledronic acid is superior to pamidronate in the treatment of hypercalcemia of malignancy—a pooled analysis of two randomized, controlled clinical trials. J Clin Oncol 2001;19:558–567. 148. Plunkett TA, Smith P, Rubens RD: Risk of complications from bone metastases in breast cancer: implications for management. Eur J Cancer 2000;36:476–482. 149. Mirels H: Metastatic disease in long bones. A proposed scoring system for diagnosisng impending pathological fracture. Clin Orthop Rel Res 1989;249:256–264. 150. Liberman I, Reinhardt MK: Vertebroplasty and kyphoplasty for osteolytic vertebral collapse. Clin Orthop 2003;415(Suppl):S176–S186. 151. Jensen ME, Kallmes DF: Percutaneous vertebroplasty in the treatment of malignant spine disease. Cancer J 2002;8:194–206. 152. Hill ME, Richards MA, Gregory WM, et al: Spinal cord compression in breast cancer: a review of 70 cases. Br J Cancer 1993;68:969–973. 153. Findlay GFG: Adverse effects of the management of malignant spinal cord compression. J Neurol Neurosurg Psychiatry 1984;47:761–768. 154. Patchell RA, Tibbs PA, Regine WF, et al: Direct decompressive surgical resection in the treatment of spinal cord compression caused by metastatic cancer: a randomised trial. Lancet 2005;366:643–648.

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Lung Metastases Valerie W. Rusch

S U M M ARY

Incidence • Lungs are the second most common site of metastases. • Lungs are the sole site of metastasis in 80% of patients with sarcoma and in 2% to 10% of patients with carcinoma.

Etiology • Hematogenous spread • Lymphangitic spread in carcinomas can occur early or late in the natural progression of all cancers. • It is not well understood why lung metastases take several years to develop.

Evaluation • Few lung metastases are symptomatic; only 15% to 20% of patients complain of cough or pain. All patients with isolated pulmonary metastasis from extrathoracic malignancy should be evaluated for the possibility of resection. • Initial imaging studies should consist of a plain chest radiograph followed by computed tomographic (CT) examination to predict resectability. Newer imaging modalities, such as 18Ffluorodeoxyglucose positron emission tomography (FDG-PET) and magnetic resonance imaging (MRI), have not

• • • •

O F

K EY

P OI NT S

been shown to be as accurate or cost effective as CT. CT is unable to distinguish reliably between malignant and benign lesions. CT differs from the final pathology report in 42% of cases. CT underestimates the number of malignant lesions in 25% of cases. The accuracy of radiologic imaging is only 37%, underestimating the number of lesions by 39% and overestimating them by 25%, for patients undergoing bilateral exploration.

•

Prognostic Factors • Number of metastases • Disease-free interval (36 months). • Histology/organ site of primary tumor

Surgical Treatment • First described case of pulmonary metastasectomy was by Weinlechner in 1882. • Alexander and Haight described the first series of patients; 12 patients remained disease-free for 1 to 12 years. • General guidelines that should be met before undertaking a resection include the following:

INTRODUCTION The first described case of pulmonary metastasectomy was reported in 1882 by a German surgeon named Weinlechner, who removed two incidental pulmonary nodules during a chest wall resection for sarcoma.1 In 1939 Barry and Churchill2 reported the first long-term survivor from pulmonary metastasectomy, a patient with metastatic renal cell cancer. Their patient survived 23 years after surgery. Subsequently, Alexander and Haight described the first series of patients undergoing pulmonary metastasectomy and its correlation to survival.3 Twelve patients in their study remained free of disease for 1 to 12 years. Most important, from this early study came the first generally accepted criteria for pulmonary metastasectomy:

• •

•

• Control of the primary tumor, or ability to resect the primary tumor • Ability to resect metastatic disease completely • Ability of the patient to withstand the extent of pulmonary resection required to remove all gross tumor • Absence of extrathoracic metastasis • Absence of better alternative treatment The location of metastases determines the extent and type of resection: • Peripheral metastasis—parenchymal sparing • Central metastasis—lobectomy or pneumonectomy • Solitary endobronchial metastasis— lobectomy, sleeve lobectomy, or pneumonectomy Ensure that all grossly palpable tumors are resected with clear margins. More radical resection (lobectomy, pneumonectomy) does not increase survival. Bilateral metastases and recurrence of pulmonary metastases are not contraindications to resection and should not deter resection in lesion(s) that can be removed completely.

• The primary tumor should be completely removed. • There should be no evidence of extrapulmonary disease. • The patient should be able to tolerate the planned operation from the standpoint of overall medical condition. Subsequently these criteria were modified by other authors to reflect our improved understanding of the management of pulmonary metastases. Current additional criteria include the following:4 • Control of the primary tumor, or ability to resect the primary tumor completely simultaneous with resection of metastasis • Ability to resect metastatic disease completely • Ability of the patient to tolerate the extent of pulmonary resection required to remove all gross tumor

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Table 58-1 Prognostic Factors after Pulmonary Metastasectomy Disease-free interval Mediastinal lymph node metastases Number of pulmonary nodules Tumor doubling time Size of pulmonary nodules Bilateral disease

The role of surgery in the treatment of pulmonary metastases will continue to evolve as better systemic therapies become available. Currently, only a minority of patients with metastatic disease from any source are candidates for pulmonary metastasectomy; however, improved imaging studies and the widespread use of computed tomography (CT) might detect more patients who have small-volume pulmonary metastases and are therefore candidates for metastasectomy. A current perspective of the evaluation and treatment of patients with isolated lung metastases is presented in this chapter.

DIAGNOSIS

Surgical margins Histologic subtype

• Absence of extrathoracic metastasis • Absence of better alternative treatment Although these criteria are widely used, there are continued attempts to refine the selection of patients for pulmonary metastasectomy. Several prognostic factors that are not universal across all tumor histologies have been reported to affect outcome after pulmonary metastasectomy (Table 58-1). The largest series of pulmonary metastectomies reported to date is from the International Registry of Lung Metastasis, which analyzed 5206 cases.5 The overall 5-year survival after pulmonary metastasectomy without stratifying for tumor type was 36%. Factors associated with better prognosis included a long disease-free interval, complete resection, and a small number of lung nodules. A staging system based on these prognostic factors was proposed (Fig. 58-1). 100 Group Patients Deaths I=no risk factors 819 349 II=1 risk factor 1,720 903 III=2 risk factors 1,553 972 IV=unresectable 581 421

80

Logrank chi2=328.2 (3df)

60

40

Few patients with pulmonary metastasis are symptomatic. It is estimated that only 15% to 20% of patients present with cough or nonspecific chest pain and even fewer still with hemoptysis. Traditionally, chest radiography has been the most commonly used and cost-effective modality for screening patients for metastasis from extrathoracic malignancy. The most frequent radiographic appearance of a pulmonary metastasis is a peripherally located, wellcircumscribed nodule (Fig. 58-2).6,7 Several less common radiographic characteristics have also been described. Cavitating lesions are associated with a differential diagnosis that includes benign, infectious, and malignant causes (Fig. 58-3). When malignant, cavitary lesions are usually squamous cell carcinomas. The frequency of cavitation in metastatic nodules is approximately 4%; however, squamous cell malignancy is responsible for 69% of these lesions. Spontaneous pneumothorax also can occur with metastatic lung lesions and is thought to be caused by cavitation and erosion into a bronchiole wall. Spontaneous pneumothorax is most frequently seen in patients with sarcoma. It is said that a spontaneous pneumothorax in a patient with history of a sarcoma should prompt an evaluation for possible occult metastatic lung lesions. Calcification of pulmonary nodules is usually related to a benign process such as a hamartoma; however, metastatic lesions of many types (especially osteogenic sarcoma) are known to produce calcification (Fig. 58-4).6,7 Calcification in metastatic lesions are thought to be produced by several processes in different tumor types, including bone formation in osteogenic sarcoma, mucinous calcification of adenocarcinomas, or dystrophic calcification of lesions such as synovial sarcoma or giant cell tumors of the bone.6 Hemorrhage around lung nodules is also seen more frequently in benign lung lesions (e.g., fungal or mycobacterial infections) and is visualized as a halo around the lung nodule. This can also be seen in metastatic lesions and should raise the suspicion for metastasis in patients with a history of malignancy.

20

Computed Tomography 0 0

60

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180

65 85 60 5

20 30 18 1

Months Patients at risk: I II III IV

198 296 189 31

Figure 58-1 • Survival of the four prognostic groups based on the analysis of 5206 patients entered into the International Registry of Lung Metastases. Group I includes patients who had completely resectable disease with a single metastasis and a disease-free interval after resection of the primary tumor (DFI) of 36 months or more; group II, patients who had completely resectable disease with multiple metastases or a DFI of less than 36 months; group III patients who had completely resectable disease with multiple metastases and a DFI of less than 36 months; and group IV patients with unresectable disease. (From Pastorino U, Buyse M, Friedel G, et al: Long-term results of lung metastasectomy: prognostic analyses based on 5206 cases. J Thorac Cardiovasc Surg 1997;113:37.)

The use of CT as an adjunct to plain film radiography for the diagnosis of pulmonary metastases has increased dramatically during the last decade. This has been facilitated by the development of highspeed helical scanners. It is clear that CT is able to visualize more lesions than chest radiography.5,8–17 Chang and coworkers11 reported that when compared with conventional radiography, CT was able to visualize nearly twice as many nodules.18 CT is not able to distinguish reliably between malignant and benign lesions, however. McCormack and colleagues8 retrospectively studied 144 patients who had both a chest x-ray and a CT to identify metastatic lesions. The CT results differed from the final pathology reports in 42% of cases, with CT scan underestimating the number of malignant nodules in 25% of patients. Pastorino and associates5 reported results of imaging on 2988 patients undergoing pulmonary metastasectomy. The overall accuracy of radiologic assessment of the number of metastatic nodules was 61%, underestimating metastasis in 25% of patients. Interestingly, in those patients (1134) who had bilateral exploration, the accuracy of imaging was only 37%, underestimating the number of lesions in 39%, overestimating in 25%. The accuracy and sensitivity of CT also depends on the size of the lesions (Table 58-2): the larger

Lung Metastases • CHAPTER 58

A

B

Figure 58-2 • Two patients with metastatic sarcoma demonstrating typical findings of well-circumscribed peripheral lesions. A, Plain chest radiograph. B, Computed tomographic image.

the lesion, the greater the sensitivity and accuracy. Munden and colleagues14 reported on the clinical significance of pulmonary lesions less than 1 cm in diameter, finding malignant pulmonary lesions in 81% of patients with a history of malignancy. Multiple authors have described the ability of CT to detect a greater number of pulmonary nodules, while acknowledging a decreasing specificity of identifying malignant nodules with this diagnostic tool.11,15 Therefore, not all small pulmonary nodules in patients with a history of cancer can be assumed to represent metastatic disease. Currently, there are no established guidelines for the routine screening for pulmonary metastases. Many institutions still use periodic chest radiography as the only imaging modality to rule out pulmonary metastasis. Other authors

suggest that the use of CT for routine screening is indicated in groups of patients whose primary tumors have an unusually high propensity to spread to the lungs.13,16 As the quality and speed of CT scanning progresses, it will probably become the sole screening tool for identifying pulmonary metastases.11,15

Magnetic Resonance Imaging Magnetic resonance imaging (MRI) provides the benefits of reduced radiation exposure (of particular interest for cases involving younger patients) and the ability to detect lesions at lung-mediastinal interfaces. MRI has not gained wide acceptance as a screening tool, however, mainly because of its increased time constraints and cost. Further technical considerations that are unfavorable include motionrelated artifacts and an inability to detect calcified lesions. Kersjes and colleagues19 performed a study comparing MRI and helical CT in the detection of pulmonary metastasis and showed MRI to have an overall accuracy of 84%. For lesions smaller than 5 mm, however, the sensitivity of MRI was only 36%. The routine use of MRI is currently not advocated as a screening tool for patients with pulmonary metastasis.

Nuclear Imaging

Figure 58-3 • Chest CT scan demonstrating cavitary lesion in a patient with metastatic colorectal cancer.

Imaging with 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) is being used more frequently to assist in the staging of primary tumors. Most often, this is used at the time of diagnosis to rule out distant metastasis but is occasionally used to assess response to therapy. Currently FDG-PET is not used as a screening tool to identify pulmonary metastasis, but multiple authors support the eventual use of this modality as a screening tool.20,21 Dose and associates21 studied 50 patients with breast cancer who had FDG-PET evaluation to determine presence of metastatic disease. FDG-PET had a sensitivity of 78.6% in identifying pulmonary metastasis as compared with conventional chest radiography, which had a sensitivity of 41.6%.21 Other authors report that FDG-PET may not be superior to conventional imaging techniques in identifying pulmonary metastasis from bone and soft-tissue sarcomas.22,23 Lucas and coworkers22 studied 62 patients with soft-tissue sarcoma who had FDG-PET during initial evaluation. The sensitivity of FDG-PET in detecting lung metastasis was 86.7% as compared with 100% for CT, leading the authors to conclude that CT is a superior imaging tool

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A

B

Figure 58-4 • A and B, Chest CT scans of patient with metastatic osteosarcoma demonstrating the presence of calcifications in metastatic lesions.

Table 58-2 Detection of Pulmonary Metastasis by Computed Tomography Year

Author

1998

Waters et al18,*

No. of Nodules

Sensitivity for Small Lesions

Sensitivity for Larger Lesions

144

44% (≤5 mm)

91% (>5 mm)

90

69% (≤6 mm)

95% (>6 mm)

188

48% (≤6 mm)

87% (>6 mm)

9

1999

Diederich et al

2002

Margaritora et al10,†

*Canine model † High-resolution CT group

Table 58-3 Surgical Approaches to Resection of Pulmonary Metastases Surgical Approach

Advantages

Disadvantages

Unilateral Disease Posterolateral thoracotomy

Superior exposure and palpation of lung

Painful, large incision

Videothoracoscopy (VATS)

Less painful

Inability to palpate, poor ability to detect deep lesions

Clamshell thoracotomy

Excellent bilateral exposure

Painful; sacrifice of both internal mammary arteries

Simultaneous bilateral thoracotomies

One hospital stay/procedure

Painful

Staged (sequential) bilateral thoracotomy

Allows technically complex procedures

Two hospitalizations and procedures

Median sternotomy

Bilateral exposure, less painful

Poor exposure to posterior lung fields

Bilateral Disease

for identifying these lesions. Further study is warranted to determine the true benefit, if any, of using FDG-PET as a routine screening tool for identifying patients with pulmonary metastasis. Because PET is currently unable to detect sub-centimeter lung lesions reliably, it is likely that CT will remain the most sensitive imaging modality.

SURGICAL APPROACHES TO LUNG METASTASIS The goal of pulmonary metastasectomy is to achieve complete resection of all visible and palpable tumors in the lung. The surgical approach is dictated by the extent and location of disease and by the

patient’s performance status. There are several approaches available depending on the size, number, and location of the lesions. The advantages and disadvantages of these approaches are outlined in Table 58-3. The standard approach to the patient with disease localized to one hemithorax is a unilateral posterolateral thoracotomy. The thorax is usually entered through the fifth intercostal space. Several variations of this include muscle-sparing incisions, axillary incisions, or an anterior thoracotomy. All of these approaches allow full visual inspection and manual palpation of the entire lung. During the past decade, the use of video-assisted thoracoscopy (VATS) has been described for pulmonary metastasectomy. This

Lung Metastases • CHAPTER 58

approach is controversial, however.24–28 McCormack and colleagues25 performed a prospective study evaluating the role of VATS to treat pulmonary metastasis. Eighteen patients had preoperative CT followed by VATS resection of all visible and CT-detected lesions. All patients then immediately underwent thoracotomy with resection of any additional lung nodules. Additional malignant lesions were found in 56% of patients after attempted VATS resection of the nodules found on preoperative imaging. The authors concluded that this high failure rate of CT and VATS warranted closure of the study before the intended 50 patients could be enrolled. Recently, other authors have advocated the use of VATS for patients with a solitary pulmonary metastasis. Mutsaerts and associates24,28 described their experience with 20 patients undergoing either VATS or thoracotomy for resection for single pulmonary metastasis. The 5-year survival and recurrence rates seemed to be similar to those seen with thoracotomy.28 Other authors have described localization methods using radiotracer injection to help identify small or deeply located lesions during VATS resections.26 Currently, the practice in our institution is to use VATS primarily for the diagnosis of metastatic disease, or as a therapeutic procedure in highly selected patients for whom thoracotomy may be a higher risk procedure. Thoracotomy or other open procedures remain our preferred approach to pulmonary metastasectomy because of the prognostic importance of complete resection and the potential for missing small metastases by VATS. However, a VATS approach may be appropriate for patients with one or two metastases clearly defined on high-quality imaging studies. The approach to bilateral disease is more variable, but the principles remain the same. Median sternotomy, “clamshell” thoracotomy (bilateral anterior thoracotomy with transverse sternotomy), sequential bilateral thoracotomies, and simultaneous bilateral posterolateral thoracotomies are used as standard surgical approaches to the resection of bilateral metastases.29–31 In general, resection of bilateral metastases is preferably done as a single operation. Sequential thoracotomies are performed only when the anatomic location of a lesion requires a complex or extensive operation, or when the patient’s comorbidities dictate a more conservative approach to management. In contrast to primary lung cancers, pulmonary metastases require only a local excision with a surrounding rim (1–2 cm) of benign lung tissue. This is accomplished most frequently by wedge resection performed by precision electrocautery or with a stapling instrument (Fig. 58-5). Segmentectomy, lobectomy, or pneumonectomy are used less commonly. It is important to note that survival after metas-

Detection of lung lesions on CXR in patient with Hx of malignancy

Noncontrast helical CT of chest

Unilateral lesion

Multiple ipsilateral lesions

Bilateral single or multiple lesions

FNA or VATS for diagnosis

Exploratory thoracotomy with removal of all grossly palpable lesions±lymph node sampling

Simultaneous bilateral thoracotomy or Clamshell thoracotomy or Sequential thoracotomy

±

Figure 58-6 • Management algorithm for patients with isolated pulmonary metastasis from extrathoracic malignancy. CXR, chest radiography; FNA, fine-needle aspiration; Hx, history; VATS, video-assisted thoracoscopy.

tasectomy is not increased by a more radical resection such as lobectomy or pneumonectomy. These procedures might be required technically to remove the lesion, however, and should be applied to do so if needed. The most important principle of these techniques is a clear margin of resection. The risk of resection of pulmonary metastasis using standard wedge resection is very low, with mortality generally 1% or less. Complications include bleeding, infection in the wound, or pneumonia and prolonged air leak. More extensive resections such as lobectomy or pneumonectomy carry only slightly higher morbidity rates, and these operations are very well tolerated by most patients. The preoperative evaluation for these patients is similar to those undergoing lung resection for any other cause (Fig. 58-6). Pulmonary function testing should be obtained for all patients to assure that the volume of lung resection will not compromise overall respiratory function. Thorough evaluation of underlying cardiovascular disease should also be undertaken, with preoperative stress testing as clinically indicated.

PULMONARY METASTASECTOMY FOR SPECIFIC TUMOR TYPES Colorectal Cancer

A

B

C

Figure 58-5 • A–C, Method of wedge resection by using a stapling device. This technique is most suitable for peripherally located metastases adjacent to the fissures or edges of the lung. (From Rusch VW: Surgical techniques for pulmonary metastasectomy. Sem Thorac Cardiovasc Surg 2002;14:4.)

It is estimated that 10% to 25% of patients with primary colorectal tumors will have detectable metastases at the time of diagnosis.32 Despite advances in adjuvant therapy and surgery, 50% of all patients with colorectal cancer will develop some form of metastasis during their lifetimes.33 Approximately 15% of patients having curative resection of their primary colorectal tumor will develop distant metastasis, including metastasis to the lung.34 Pulmonary metastasis can occur even with favorable primary tumor characteristics. Okumura and coworkers35 reported that 26% of pulmonary metastectomies were performed in patients with Duke’s A or B primary colorectal cancer. Since Blalock36 reported the first pulmonary metastasectomy for colorectal cancer in 1944, several authors have reported their experience regarding overall survival and prognostic factors. The 5- and 10-year survival rates range from 30% to 40% and 27% to

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Table 58-4 Survival of Patients Undergoing Pulmonary Metastasectomy for Colorectal Cancer* Year

Author

Number

5-Year Survival (%)

1992

McAfee et al37

139

30.5

N/A

1996

Okumura et al35

159

40.5

27.7

1998

McCormack and Ginsberg38

287

40.0

32.0

39

10-Year Survival (%)

2001

Zink et al

110

32.6

N/A

2002

Saito et al40

165

39.6

37.2

*Studies represent analysis of 100 patients or more.

37%, respectively (Table 58-4).37–40 It is clear from the literature that pulmonary metastasectomy for colorectal cancer is associated with long-term survival, especially when a complete resection is performed. McCormack and associates41 reviewed 144 patients who underwent pulmonary metastasectomy for colorectal cancer and showed that survival for patients who underwent complete resection was approximately 40% at 5 years, whereas incomplete resection was associated with a poor prognosis. As for other primary tumors, studies evaluating the disease-free interval, the number of lesions (multiple vs. single), and the presence of lymph node involvement have shown these to be significant prognostic factors after pulmonary metastasectomy for colorectal cancer.35,37–46 Colorectal cancer is also unique in the fact that the serum tumor marker carcinoembryonic antigen (CEA), used as a marker for follow-up, has been shown to be a prognostic indicator for patients with pulmonary metastases.37–46 There is some disagreement as to whether single, ipsilateral metastases are associated with a better prognosis than either multiple ipsilateral or bilateral metastases. The presence of bilateral metastases was previously thought to be a contraindication to metastasectomy. Recently, though, several authors have reported that the survival for patients with bilateral lesions is not significantly reduced as compared with patients who have multiple ipsilateral lesions.38,43 Although a solitary metastasis might be associated with a better prognosis than multiple metastases (either unilateral or bilateral), the main criteria used to select patients with multiple lesions is whether removal of these lesions is technically feasible and removal of the volume of lung parenchyma does not seem to compromise the patient’s lung function to a significant extent. CEA levels are elevated in 40% to 70% of patients before pulmonary metastasectomy. Most authors report elevated CEA levels to be an adverse prognostic factor.39,40,44,45,47 The reason for this is unclear, although it has been postulated that the presence of CEA may promote adhesion or attachment of tumors cells or could be due to undetected extrathoracic metastasis.48 Although an elevated CEA is not used currently to exclude patients from resection, it could be useful to consider this in the context of other known prognostic factors. For instance, a patient who rapidly develops pulmonary metastases after resection of the primary tumor, and who has multiple lung nodules and an elevated CEA level, might be treated initially with chemotherapy instead of going directly to pulmonary metastasectomy. Hilar or mediastinal lymph node metastases occur in 1% to 28% of patients with colorectal pulmonary metastasis.35,40–43,45 Saito and colleagues40 reported that 20 of 138 (14.5%) patients who underwent lymph node sampling had positive nodes. The 5-year survival was 48.5% for the patients without hilar or mediastinal lymph node metastasis, vs. 6.2% at 4 years for the patients with lymph node metastasis. More recently, Welter and associates49 reported that 28 of 169 patients undergoing resection of colorectal pulmonary metastases had hilar or mediastinal nodal metastases. Nodal metastases had a highly significant adverse impact on survival. Okumura and coworkers35 performed systematic lymph node dissection on 100 patients

with colorectal pulmonary metastasis. Fifteen of these patients had positive lymph nodes with a 5-year survival of 6.7% as compared with a 50% survival for those with negative lymph nodes, indicating that complete lymph node dissection does not increase survival. The routine use of mediastinal lymph node dissection in patients with pulmonary metastases seems to be the exception rather than the norm. The presence of malignant lymph nodes could indicate a group of patients otherwise thought to be disease-free who might benefit from additional adjuvant therapy. We favor doing mediastinal lymph node sampling or dissection in these patients. Although these procedures might not have a therapeutic effect, they certainly provide important prognostic information. Previously, the presence of both liver and lung metastases from colorectal cancer was thought to be a contraindication to metastasectomy. Synchronous or metachronous lung and liver metastasis occurs in approximately 5% of patients with colorectal cancer. Headrick and colleagues50 reported 5- and 10-year survivals of 30% and 16%, respectively, for 58 patients who underwent resection of both liver and lung metastases from colorectal cancer. Decreasing morbidity and mortality rates for liver resection now make resection of both lung and liver metastases a viable option in carefully selected patients.

Bone and Soft-Tissue Sarcoma Bone and soft-tissue sarcomas compose a histologically diverse group of tumors accounting for 1% of all adult malignancies, with approximately 6600 cases occurring in the United States annually.51,52 Metastasis will occur in 25% to 70% of patients with localized disease, and 10% will present with metastasis upon diagnosis.52 Isolated pulmonary metastases occur in up to 20% of sarcoma patients during the course of their disease, with the lung being the site of failure after treatment in up to 90% of cases.53,54 Factors associated with an increased risk of pulmonary metastasis include high tumor grade, primary tumor size greater than 5 cm, lower extremity site, and histologic subtype.53 The lack of effective systemic therapy for most soft-tissue sarcomas makes surgical resection the best treatment for pulmonary metastases. Several studies have shown that the complete resection of pulmonary metastases is associated with long-term survival.5,51,53,55 Billingsley and coworkers51 reported that the 3-year actuarial survival was 46% in patients who had complete resection as compared with 17% (P < 0.001) for those with incomplete resection of pulmonary metastasis. The cumulative 5-year survival for patients with bone and soft-tissue sarcomas after pulmonary metastasectomy ranges from 31% to 40% (Table 58-5).5,51,55–57 The histologic subtype of sarcoma influences both the development of pulmonary metastasis and overall survival. High-grade, undifferentiated, and alveolar soft-part sarcomas produce pulmonary metastasis in approximately 60% of patients.51 Tumors that are high grade and histologic variants, such as liposarcoma, malignant fibrous histiocytoma, and malignant peripheral nerve tumor, have all been reported to be unfavorable prognostic factors.51,53,58 Longer diseasefree interval, fewer lesions, and unilaterality have also been reported to be favorable prognostic factors.

Lung Metastases • CHAPTER 58

Table 58-5 Survival of Patients Undergoing Pulmonary Metastasectomy for Bone and Soft-Tissue Sarcomas* Number

5-Year Survival (%)

Choong et al55

214

40

1996

van Geel et al56

255

38

1997

Pastorino et al5

1917

31

1999

Billingsley et al51

138

37

Year

Author

1995

*Studies represent analysis of 100 patients or more.

The incidence of hilar or mediastinal lymph node metastasis in patients with lung metastasis from sarcoma is 2%.5 This low propensity for lymph node involvement is also a feature of primary sarcomas. Therefore, it seems that routine mediastinal lymph node sampling or dissection is unlikely to offer significant prognostic information or therapeutic benefit. The rate of recurrence after pulmonary metastasectomy for sarcoma ranges from 45% to 83%; however, the role of repeat resection of pulmonary metastases has been studied by few authors.53,57–60 Weiser and associates61 reported experience from Memorial SloanKettering Cancer Center in 86 patients who underwent repeat resection of pulmonary metastases for soft-tissue sarcoma. The 5-year survival after undergoing at least two operations for pulmonary metastasectomy was 36%. Patients who had a complete repeat resection had a median survival of 51 months, as compared with 6 months in those who could not be completely repeat resected. Poor prognostic indicators for repeat resection included three or more nodules, lesions greater than 2 cm in size, and high-grade primary tumors. This study strongly suggested a benefit for repeat or multiple procedures for clearance of pulmonary disease in carefully selected patients. Pulmonary metastases from osteosarcoma occur most frequently in pediatric patients and respond better to chemotherapy than do soft-tissue sarcomas. Pulmonary metastasectomy within the context of a multimodality therapy program is a well-accepted approach to treatment and is associated with a 5-year survival of approximately 30%. Response to chemotherapy, the disease-free interval from resection of the primary tumor, and the number of metastases and complete resection are reported to influence overall survival.62–64

Melanoma Patients with metastatic melanoma have an especially poor prognosis. The most common sites of metastasis from melanoma are the lungs, distant subcutaneous tissue, and distant lymph nodes, with isolated lung metastasis occurring in 1.9% to 11% of patients.65,66

Table 58-6 Survival of Patients Undergoing Pulmonary Metastasectomy for Malignant Melanoma Number

5-Year Survival (%)

Thayer et al70

18

11.1

1990

Karp et al68

22

4.5

1991

Gorenstein et al66

54

25.0

1995

Tafra et al67

106

27.0

1998

Ollila et al72

45

15.6

2002

Dalrymple-Hay et al20

121

22.1

Year

Author

1985

Tafra and coworkers67 reported their experience with 106 patients with metastatic melanoma who underwent pulmonary metastasectomy. Sixty-five of these patients underwent a complete resection. Although the benefit of a complete (vs. an incomplete) resection on survival could not be demonstrated in a univariate analysis, a multivariate analysis found that surgical resection (vs. no operation) was associated with a significantly better survival (P = 0.0001). Other authors have reported that complete resection is associated with prolonged survival.68,69 Overall, the 5-year survival rates after resection of pulmonary metastasis from malignant melanoma vary from 4.5% to 27% (Table 58-6).20,66–68,70–72 This wide range of values probably reflects the relatively small numbers of patients included in some studies. Prognostic factors that have been reported include operation, number of nodules, prior immune therapy, histologic type, disease-free interval, and tumor-doubling time.20,65–72 Harpole and colleagues69 reported on a large series of patients who had pulmonary metastases from melanoma. Of the total of 945 patients, 112 (11.8%) underwent pulmonary metastasectomy. Histologic type (nodular and acral lentiginous lesions), high Clark level, and thicker primary tumors were significantly associated with pulmonary metastasis. The overall 5-year survival in this group was 4%. Patients who had a complete resection had a significantly higher median survival rate as compared with those who had only partial resection, although all patients who underwent operations survived longer than those who had no operation. An analysis of the subset of patients who had a solitary metastasis found that patients who underwent resection had a significantly better median survival than patients managed nonsurgically. Important prognostic factors after surgery included complete resection, a long disease-free interval from treatment of the primary tumor to the diagnosis of the pulmonary metastasis, treatment with chemotherapy, and the total number of nodules. These data suggest that appropriately selected patients with metastatic melanoma confined to the lungs can benefit from pulmonary metastasectomy.

Renal Cell Carcinoma Barry and Churchill2 performed the first pulmonary metastasectomy from renal adenocarcinoma in 1938. Approximately 30% of patients with renal cell carcinoma will present with metastasis, and approximately 30% to 50% of patients with initially localized tumors will develop distant metastases.73 One-half of patients who have a radical nephroureterectomy will develop pulmonary metastases later and only 16% of these patients will have disease confined to the lung.74,75 The 5-year survival of patients with unresected metastasis is approximately 2.7%.76 The 5-year survival after resection of isolated pulmonary metastasis is reported to range from 36% to 44% (Table 58-7).75–79 Several prognostic factors for survival have been identified, including complete resection, the disease-free interval between primary tumor treatment and metastasis, the number of metastases, and the presence or absence of lymph node metastases.75,76,78 Pfannschmidt and associates76 reported one of the largest series of pulmonary metastasectomy for renal cell cancer; they found that complete resection was possible in 78% of patients and that these patients had a 5-year survival of 41.5% vs. 22.1% for those with incomplete resection. This survival rate of 22.1% in incompletely resected patients was better than that for patients who had no resection at all. Hilar and mediastinal lymph node metastases are seen in 22% to 30% of patients with metastatic renal cell cancer.75,76 Fourquier and colleagues75 and Pfannschnidt and associates76 performed systematic mediastinal lymph node dissection on 50 and 191 patients, respectively. Both studies found that the presence of lymph node involvement was associated with a poorer survival. Although it is unknown whether lymph node dissection is therapeutic, it offers important prognostic information and should probably be performed in these patients.

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Table 58-7 Survival of Patients Undergoing Pulmonary Metastasectomy for Renal Cancer* Year

Author

1994

Cerfolio et al79 75

1997

Fourquier et al

1999

Friedel et al77 76

Table 58-8

Survival of Patients Undergoing Pulmonary Metastasectomy for Head and Neck Cancer

Number

5-Year Survival (%)

Year

Author

96

36

1992

Finley et al83,*

18

29

50

44

1996

Wedman et al84

21

59

77

39

1997

Nibu et al81

32

32

83

50

2002

Pfannschmidt et al

149

42

2002

Piltz et al78

105

40

2006

Marulli et al101

59

53

*Studies represent analysis of 50 patients or more.

Synchronous metastases are traditionally thought to be a relative contraindication to resection. The true incidence of synchronous versus metachronous metastases is in renal cell cancer is unclear, although in reported series of resected patients, synchronous lesions are less frequent and are thought to indicate a worse prognosis.75,76 On the other hand, Fourquier and colleagues75 examined survival in completely resected patients with synchronous lung metastases. Although the overall survival rate was lower in patients with synchronous lesions (48% vs. 20%), this was not statistically significant and again appeared to offer a survival benefit relative to no resection at all. The recent development of more effective systemic therapy for renal cell cancers through the use of antiangiogenesis agents may alter the need or indications for pulmonary metastasectomy. This is an evolving area of cancer management, and the impact of these drugs on pulmonary metastasectomy warrants study.

Head and Neck Cancer Approximately 60,000 new cases of head and neck cancer occur each year in the United States.80 The potential for metastatic spread is dependent on the stage of the primary tumor, with the rate of lung metastasis ranging from 4.3% to 25.1%.81 Head and neck tumors and especially squamous cell cancers have a predilection for metastasizing to the lung, which is often the only site of metastasis.82 The diagnosis of lung nodules in these patients becomes even more challenging when one realizes that 10% to 40% of lung nodules in these patients are actually second primary lung tumors.83 There are few effective systemic therapy options for the treatment of lung metastasis from head and neck tumors, leaving surgical resection as the most viable option. The estimated 5-year survival after pulmonary metastasectomy in these patients ranges from 29% to 59% (Table 58-8).80,81,83,84 The wide range of survival rates may be related to the heterogeneous histologic groups reported in most series. Squamous cell tumors of head and neck origin have a worse prognosis than their glandular counterparts such as thyroid, adenoid cystic, and mucoepidermoid tumors.80,81,84 Liu and coworkers80 reported on 83 patients undergoing pulmonary metastasectomy from head and neck tumors. In their series the 5-year overall survival for squamous cell tumors was 34% as compared with 64% for tumors of glandular origin (P = 0.14). Bilateral metastasis and recurrence of metastasis are not contraindications to resection and have not been shown to be adverse prognostic factors at this time.

Germ Cell Tumors Germ cell tumors compose only 1% of cancers but are the most common neoplasm in men aged 15 to 35 years. The vast majority of these tumors arise in the testis, with an annual incidence of 5 cases per 100,000.85 The survival of patients with germ cell tumors has increased dramatically during the past 30 years because of cisplatinbased chemotherapy.85 Monitoring of treatment and recurrence has

1999

Number

80

Liu et al

5-Year Survival (%)

*Included only patients with squamous cell carcinoma metastasis.

been made possible by the use of sensitive tumor markers, including α-fetoprotein and human chorionic gonadotropin. Pulmonary metastasis at the time of presentation in these patients is common, approaching 50% in patients with retroperitoneal disease.86 Residual masses after chemotherapy are present in approximately 50% of patients and could contain viable malignancy, mature teratoma, or only fibrosis and/or necrosis.86 Surgical resection of these lesions is crucial to identifying which of the preceding components is present, to predicting outcome, and to determining if any further therapy is warranted. The estimated 5-year survival rate after pulmonary metastasectomy ranges from 59% to 77% (Table 58-9).85–87 The most significant prognostic factor is the presence of viable tumor cells in the resected specimen. Liu and coworkers85 reviewed the experience at Memorial Sloan-Kettering Cancer Center over a 28-year period of 157 patients undergoing pulmonary metastasectomy for germ cell tumors. After resection, viable tumor was found in 70 patients (44.5%), necrosis in 47 patients (29.9%), and mature teratoma in 40 patients (25.4%). Survival was significantly poorer in those patients with viable tumor cells (43% over 10 years) as compared with those patients with necrosis/fibrosis (86% 10-year survival) or mature teratoma (84% 10-year survival).85 The presence of mature teratoma in a specimen did not significantly worsen prognosis. The inability to make an accurate determination of the presence or absence of viable tumor cells in all residual lesions after treatment for germ cell tumors mandates that all of these lesions be resected to determine overall prognosis and potential for further therapy. More recently, Kesler and colleagues88 reported the results of resection in 134 patients with either lung or mediastinal metastases. The overall survival at 5 years was 42.3%. Older patient age, lung rather than mediastinal metastases, and the number of metastases (four or more) adversely influence survival.

Breast Cancer Breast cancer is the most prevalent cancer among women in the United States, with approximately 100,000 cases occurring annually.89 Approximately 15% to 25% of patients with metastatic disease will have their disease confined to the thorax. The data regarding

Table 58-9

Survival of Patients Undergoing Pulmonary Metastasectomy for Germ Cell Tumors Number

5-Year Survival (%)

Cagini et al86

141

77

1994

Anyanwu et al87

104

59

1998

Liu et al85

157

68

134

42.3

Year

Author

1998

2005

88

Kesler et al

Lung Metastases • CHAPTER 58

pulmonary metastasectomy are controversial, with most studies analyzing the outcome of encompassing small groups of patients treated over several decades. The largest series reported to date is by Friedel and colleagues90 from the International Registry of Lung Metastases. They reported on 467 patients undergoing pulmonary metastasectomy for breast cancer. Complete resection of all metastasis was possible in 84% of patients. The 5-year overall survival was 38% in patients with complete resection, as compared with 18% of patients with incomplete resection (P = 0.0009). A long disease-free interval and fewer lesions were associated with a longer survival in this group.90 McDonald and associates91 reported on 60 patients undergoing pulmonary metastasectomy for breast cancer and failed to show a survival benefit for surgical management. Because breast cancer also frequently progresses to extrathoracic disease and is sensitive to current systemic therapies, pulmonary metastasectomy is rarely an appropriate treatment option. Indeed breast cancer is an example of the evolution of the role of pulmonary metastasectomy. Before the advent of effective hormonal and chemotherapy, pulmonary metastasectomy was commonly performed for breast cancer

with metastases confined to the lungs. Surgery is now infrequently considered for treatment.

Nonsurgical Approaches to Lung Metastasis Two therapeutic options are emerging as potentially effective alternatives to resection in selected patients with lung metastases: stereotactic body radiation therapy (SBRT) and radiofrequency ablation (RFA). SBRT is a method for delivering focused radiation to a tumor while excluding tissues not grossly involved with the tumor. It is usually delivered as high-dose hypofractionated treatment over just a few days and is therefore an attractive option for patients who have advanced disease or who may also require chemotherapy within a short interval of local therapy. Several small series now report excellent local tumor control rates with low toxicity (Table 58-10). Small, solitary peripheral tumors have been considered the ideal target for SBRT, although some series include patients with larger tumors, multiple lesions, or tumors that include the midline and the hilum.92 The length of follow-up after SBRT is relatively short in most series,

Table 58-10 Published Treatment Concepts and Results of Stereotactic Radiotherapy of Targets in Thorax

Author

No. of No. of Patients Targets

Average No. of Targets (per patient)

Median Lesion Volume (range)

Central Tumor Dose (Gy)/ No. of Fractions

Crude Isodose Local Treated Control (%) (%)

Median Follow-up Time in Months (range)

Acute Toxicity (Grade 3–5) (%)

NA

NA

Blomgren et al, 1998102

13

17

1.3

48 mL (3–198)

21–66/1–3

66

94

Uematsu et al, 1993103

45

66

1.5

2.5 cm (0.8–4.8)

38–94/5–15

80

97

11 (3–31)

11

0

Wulf et al, 2001104

26

27

1

57 mL (5–277)

45/3

65

85

8 (2–33)

22

8 (Grade 5)

Nakagawa et al, 2000105

15

22

1.5

Chest wall 15–45/1* 40 mL (5– 18–25/1* 126), Central lung 4.5 mL (0.8–13)

NA

95

10 (1–82)

100 (Grade 1)

0

Uematsu et al, 2001106

50

50

1

3.2 cm (0.8–5)

38–75/5–10

94

36 (22–66)

—‡

0

Nagata et al, 2002107

40

43

1.1

12.6 mL (0.5–39)

40–48/4

100

94

18 (3–29)

95 (Grade 1)

0

Hara et al, 2002108

19

23

1.2

4 mL (1–16)

23–36/1

100

79

13 (3–24)

5 (Grade 2)

5 (Grade 3)

Onimaru et al, 2003109

45

57

1.3

2.6 cm (0.6–6)

48–60/8

100

88

18 (2–44)

2.2 (Grade 2)

2.2 (Grade 5)

Hof et al, 2003110

10

10

1

12 mL (5–19)

19–26/1

100

80

15 (8–30)

70 (Grade 1)

0

Lee et al, 2003111

28

34

1.2

41.4 mL (4.4–230)

30–40/3–4

100

91

18 (7–35)

100 (Grade 1)

0

Timmerman et al, 2003112

37

37

1

22.5 mL (1.5–157)

24–60/3

100

84

15.2 (2–30)

95 (Grade 1–2)

5 (Grade 3)

Wulf et al, 2004113

61

71

1.2

17 mL (1–277)

33–56/1–3

66–80

92

7

0

Okunieff et al, 200692

49

125

2.6

4.7 mL (0.1–125)

50/10

100

94

41 (Grade 1– 2)

2 (Grade 3)

80†

8.2 (3.5–25)

Acute Toxicity (Grade 1–2) (%)

9–11 (2–61) 18.7 (4–61)

NA, not available. *Only tumor peripheral dose reported. All but one patient also received 20–40 Gy fractionated radiation. † Prescribed doses were 30–60 Gy. The isodose line used varied and for this table it was assumed to be 80% based on the examples published. ‡ “Most” patients had Grade 1. No patients experienced toxicity greater than Grade 1. From Okunieff P, Petersen AL, Philip A, et al: Stereotactic body radiation therapy (SBRT) for lung metastases. Acta Oncol 2006;45:808–817.

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and there are no randomized trials comparing SBRT to surgical pulmonary metastasectomy. The optimal total radiation dose and dose per fraction, and the need for respiratory gating during treatment, are not yet fully defined.93 RFA is a thermal energy system delivered via percutaneous needle placed under imaging guidance into a tumor. RFA causes tumor destruction by coagulation necrosis. There is even less experience with RFA than with SBRT for the treatment of pulmonary metastases. Currently most institutions limit the use of RFA to peripheral tumors less than 5 cm in size. However, local recurrence rates of up to 50% are reported, and serious complications including hemorrhage, pneumothorax, severe pleuritic pain, and effusions occur with RFA. Longterm follow-up data in large numbers of patients are lacking.94–97 Both SBRT and RFA are potentially promising alternatives to surgical pulmonary metastasectomy in selected patients. However, well-designed prospective clinical trials are needed to define the indications for these nonsurgical approaches along with the associated risks and long-term outcome. Certainly, either of these modalities may be appropriate options for patients who are not surgical candidates.

Other Investigational Approaches to Lung Metastases Two additional approaches have been investigated for the treatment of unresectable pulmonary metastases: isolated lung perfusion (ILP),

and transpulmonary chemoembolization.98,99 ILP is an extension of the technique of isolated organ perfusion originally developed for limb perfusion of patients with malignant melanoma or sarcoma. First developed by Johnston and coworkers100 in 1983, ILP requires surgical exploration via thoracotomy or median sternotomy, isolation and cannulation of the pulmonary artery and veins, and chemotherapy perfusion of the isolated lung. However, clinical trials in humans have shown that ILP can be associated with significant pulmonary toxicity. The technical complexity and potential morbidity of this approach to treatment have prevented widespread acceptance of ILP. It remains an investigational treatment modality that should only be used within the context of clinical trials.98 Transpulmonary chemoembolization is a method of delivering chemotherapy to the lung without surgery. Selective percutaneous image-guided catheterization of segmental pulmonary arteries is performed with a balloon catheter that occludes blood inflow. Chemotherapy is injected into the segmental pulmonary artery, which is then occluded by a second injection of microspheres. In a small trial involving 23 patients, Vogl and colleagues99 performed transpulmonary embolization of 26 lung metastases. Tumor regression was observed in 8 patients and tumor stabilization in 6 patients. Treatment was well tolerated with only minor complications. Though clearly an investigational approach to treatment, transpulmonary chemoembolization seems to warrant further study for patients with unresectable pulmonary metastases.

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Lung Metastases • CHAPTER 58

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Part II: Problems Common to Cancer and Its Therapy 98. Hendriks JMH, Van Putte BP, Grootenboers M, et al: Isolated lung perfusion for pulmonary metastases. Thorac Surg Clin 2006;16:185–198. 99. Vogl TJ, Wetter A, Lindemayr S, Zangos S: Treatment of unresectable lung metastases with transpulmonary chemoembolization: preliminary experience. Radiology 2005;234:917–922. 100. Johnston MR, Minchin R, Shull JH, et al: Isolated lung perfusion with adriamycin. A preclinical study. Cancer 1983;52:404–409. 101. Marulli G, Sartori F, Bassi PF, et al: Long-term results of surgical management of pulmonary metastases from renal cell carcinoma. Thorac Cardiovasc Surg 2006;54:544–547. 102. Blomgren H, Lax I, Göranson H, et al: Radiosurgery for tumors in the body. Clinical experience using a new method. J Radiosurg 1998;1:63–74. 103. Uematsu M, Shioda A, Tahara K, et al: Focal, high dose, and fractionated modified stereotactic radiation therapy for lung carcinoma patients: a preliminary experience. Cancer 1998;82:1062– 1070.

104. Wulf J, Hadinger U, Oppitz U, et al: Stereotactic radiotherapy of targets in the lung and liver. Strahlenther Onkol 2001;177:645– 655. 105. Nakagawa K, Aoki Y, Tago M, et al: Megavoltage CT-assisted stereotactic radiosurgery for thoracic tumors: original research in the treatment of thoracic neoplasms. Int J Radiat Oncol Biol Phys 2000;48:449–457. 106. Uematsu M, Shioda A, Suda A, et al: Computed tomography-guided frameless stereotactic radiotherapy for stage I non-small cell lung cancer: a 5-year experience. Int J Radiat Oncol Biol Phys 2001;51:666–670. 107. Nagata Y, Negoro Y, Aoki T, et al: Clinical outcomes of 3D conformal hypofractionated single high-dose radiotherapy for one or two lung tumors using stereotactic body frame. Int J Radiat Oncol Biol Phys 2002;52:1041–1046. 108. Hara R, Itami J, Kondo T, et al: Stereotactic single high dose irradiation of lung tumors under respiratory gating. Radiother Oncol 2002;63:159– 163.

109. Onimaru R, Shirato H, Shimizu S, et al: Tolerance of organs at risk in small-volume, hypofractionated, image-guided radiotherapy for primary and metastatic lung cancers. Int J Radiat Oncol Biol Phys 2003;56:126–135. 110. Hof H, Herfarth KK, Münter M, et al: Stereotactic single-dose radiotherapy of stage I non-small cell lung cancer (NSCLC). Int J Radiat Oncol Biol Phys 2003;56:335–341. 111. Lee SW, Choi EK, Park HJ, et al: Stereotactic body frame based fractionated radiosurgery on consecutive days for primary or metastatic tumors in the lung. Lung Cancer 2003;40:309–315. 112. Timmerman R, Papiez L, McGarry R, et al: Extracranial stereotactic radioablation: results of a phase I study in medically inoperable stage I nonsmall cell lung cancer. Chest 2003;124:1946– 1955. 113. Wulf J, Haedinger U, Oppitz U, et al: Stereotactic radiotherapy for primary lung cancer and pulmonary metastases: a noninvasive treatment approach in medically inoperable patients. Int J Radiat Oncol Biol Phys 2004;60:186–196.

59

Liver Metastases Nancy Kemeny, Margaret Kemeny, and Laura Dawson

S U M M ARY

Incidence • Liver metastases are most frequently seen in patients with colorectal cancer (CRC; nearly 15% of patients presenting and an additional 60% developing subsequent spread); they are less common in patients with breast cancer (4% of initial failures), lung cancer (15%), and melanoma (24%).

Etiology • The liver has a rich blood supply from both the hepatic artery and the portal vein; metastases can reach the liver from any organ, but the direct passage of blood from the gastrointestinal tract to the liver via the portal circulation plays a critical role in explaining the high rate of liver metastases from these sites.

Detection • Contrast computed tomography (CT) and magnetic resonance imaging (MRI) can detect approximately two-thirds of liver metastases. • CT angiography, CT portography, and intraoperative ultrasound seem to have increased sensitivity as compared with standard techniques. • Positron emission tomography (PET) is more useful to detect extrahepatic disease. • Of laboratory tests, carcinoembryonic antigen (CEA) can be useful for patients with metastatic CRC to the liver.

Treatment Hepatic Resection • There is general agreement that surgical resection is the treatment of choice for patients with one to three metastases from CRC, producing a 5-year survival of about 30%.

O F

K EY

P OI NT S

• Significant advances in surgical technique include the ability to perform metastasectomies (rather than formal lobectomies) and total vascular exclusion. • The liver is the chief site of relapse after hepatic resection (50% of all patients). • Prognostic variables that influence survival after hepatic resection include the presence of extrahepatic disease, the stage of the primary colon cancer, the time interval between primary and the development of hepatic metastases, and the number of metastases and positive margins. • The use of adjuvant hepatic artery infusion (HAI) after liver resection has produced positive results in two American trials. There is a clear decrease in hepatic recurrence with adjuvant HAI.

Systemic Chemotherapy • The objective response rate with intravenous chemotherapy is improving, with response rates of 40% to 50% among patients with breast cancer, gastric cancer, and now even colon cancer.

Hepatic Artery Infusion • Eight randomized trials demonstrate a higher response rate for HAI than for systemic infusion (42% to 64% vs. 0 to 38%, respectively). Survival advantage is difficult to interpret: two large U.S. studies allowed a crossover from systemic therapy to HAI after tumor failure. New CALGB study without crossover showed a survival advantage. The chief toxicity of HAI is biliary enzyme elevations in 40% and biliary sclerosis in 5% to 35% of patients.

Hepatic Artery Embolization • Embolization could play a role in highly vascular tumors (neuroendocrine tumors and hepatocellular carcinoma, HCC). • Embolization agents include Gelfoam, lipiodol, and degradable starch microspheres. • Embolization is rarely useful for patients with metastatic CRC. • Chemoembolization involves the local entrapment of drug in the embolization agent in an attempt to provide a prolonged exposure of the tumor to drug locally, with less systemic exposure.

Ablative Techniques • Cryosurgery involves destruction of tissue using a freezing probe. • Limitations include the difficulty of controlling freezing and the typical requirement for laparotomy. • Radiofrequency ablation involves destruction by frictional heat and can be used percutaneously but is rarely good for lesions larger than 3 cm.

Absolute Ethanol Injection • Ethanol is injected into the tumor under ultrasound guidance and could be of use for small HCCs.

Radiation • Whole-liver external-beam irradiation therapy alone is limited by the occurrence of radiation hepatitis to about 30 Gy in 15 fractions. • Parts of the liver can be treated to far higher doses using yttrium-90 (90Y) microspheres, interstitial brachytherapy, and external-beam irradiation therapy guided by three-dimensional treatment planning.

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INTRODUCTION

tion of the liver and adjacent abdominal architecture. A second setting with a narrow window and a lower width (100–150 Hounsfield units) is then used to evaluate the liver, because this setting increases contrast differences between the normal liver parenchyma and abnormalities.

Not so long ago an oncology textbook would not have devoted an entire chapter to liver metastases. Oncologists were so pessimistic about the appearance of such metastases that “no treatment” was often the recommendation. Enormous changes have occurred, so that now, diagnosing liver metastases early can lead to effective treatment and even cure for a growing percentage of patients. The liver is the primary site of metastases for many malignant neoplasms. Gastrointestinal malignancies are especially prone to spread to the liver because of its portal venous drainage. Extraabdominal tumors such as bronchogenic carcinoma, breast cancer, and malignant melanoma often spread hematogenously to the liver. For gastrointestinal tumors, differences are seen in the natural history of the hepatic metastases. In some circumstances, hepatic metastases are a sign of disseminated disease. When gastric and pancreatic cancers metastasize to the liver, the mean survival is short, and widespread metastases often exist, so that radical measures such as hepatic resection or hepatic artery infusion (HAI) are rarely appropriate. In contrast, for colorectal cancer (CRC) the liver might be the sole site of metastatic disease, and a sizeable number of these patients might have isolated liver metastasis. In this setting, progress has been significant in the areas of hepatic resection, regional chemotherapy, and radiation therapy as discussed in this chapter. For nongastrointestinal tumors, metastases to the liver are less common as the initial site of relapse. Although breast, lung, and melanoma are the main extragastrointestinal cancers to metastasize to the liver, initial isolated metastases in the liver occur in 4%, 15%, and 24% of these patients, respectively. Treatment of these types of metastases varies according to the sensitivity of the tumor type to chemotherapy. Those that are more sensitive to antineoplastic agents might benefit from systemic therapy or aggressive regional approaches (breast), whereas those that have limited response to chemotherapy (such as melanoma) may be approached regionally in the setting of a clinical trial. Special care must be exercised in choosing patients for regional therapy who do not have CRC, because diseases such as breast cancer and melanoma are rarely confined to the liver. The last 20 years have witnessed new and more accurate methods of detecting and quantifying liver metastases, more advanced surgical techniques facilitating hepatic resection, and biologic advances that have increased the spectrum of available regional therapies. This chapter discusses advances in detection and treatment of liver metastases.

The rapidity with which multidetector CT scanners can acquire images makes possible imaging of the entire liver during the peak of the bolus of intravenous contrast, supporting CT angiography and portography.3 This noninvasive imaging can provide images of hepatic vasculature of superb quality, avoiding catheter angiography. This technique is useful to detect vascular anomalies that may help in planning of hepatic resection or hepatic arterial chemotherapy. Most often, imaging is acquired during an arterial phase and a portal venous phase.

DETECTION

Magnetic Resonance Imaging

The need for early and accurate detection of liver metastases has become more critical as the guidelines for resectability of liver metastases have become more liberal. Furthermore, ablative therapies and conformal radiation therapy are more likely to control unresectable liver metastases detected at an earlier stage, with a lower burden of disease. The most common imaging modalities for liver metastases imaging and their usefulness are described in the sections that follow.

Magnetic resonance imaging (MRI) exploits differences in magnetic properties of atomic nuclei to produce images. When placed in a magnet, protons have a nuclear spin that aligns with the magnetic field and give off radiowaves (MR signal) on returning to equilibrium. As the chemical environment changes, there is a change in the frequency of the signal, and the image. Differences in the time to return to equilibrium (relaxation times) are exploited in MRI, using T1- and T2-weighted pulse sequences. Most commonly, axial imaging is used for liver metastases imaging; however, coronal or sagittal views can define better a tumor’s proximity to adjacent vessels. The most common pulse sequences used for liver metastases imaging are a T1or T2-weighted spin echo. The T1 images generally show metastases as low-intensity lesions, whereas in T2-weighted images, metastases are of high signal intensity (Fig. 59-1). T2-weighted sequences are generally superior for detection and characterization of liver masses. Benign cysts and hemangiomas usually appear homogeneous and bright, whereas metastatic lesions are less bright and more heterogeneous.2,4 Although MRI inherently provides for soft-tissue contrast, contrast agents, such as gadolinium (Gd) chelates, can improve diagnostic accuracy. Similar to CT, MR angiography can be used as a

Imaging Techniques Computed Tomography Computed tomography (CT) has been used for more than 20 years to image hepatic metastases. Modern CT scanners are capable of rapid scanning and high-resolution three-dimensional images of the liver that can be acquired during one breath hold.1 Projection and reformatted views can be useful when assessing the proximity of liver metastases to the vessels. CT images are routinely evaluated by two window levels to maximize detection of lesions. A soft-tissue window (width of 300–500 Hounsfield units) is used for the initial examina-

Noncontrast Computed Tomography Contrast CT is sometimes not possible because of contrast allergic reactions or renal impairment. Although the sensitivity and specificity of noncontrast CT is far reduced as compared to contrast CT, it may help in identifying hypervascular metastases (especially carcinoid tumors, islet cell tumors, and renal cell carcinomas) or visualizing calcifications or hemorrhage. Noncontrast CT often fails to distinguish hypovascular tumors from the liver parenchyma. Nonenhanced blood vessels may also appear as low-attenuation masses and be confused with metastases.2

Contrast Computed Tomography Intravenous contrast is infused over approximately 2 min, with repeat imaging acquired during the infusion to capture the different enhancing phases of tumors. The two most useful phases for hepatic metastases are the arterial phase (25-sec delay) and portal venous phase (60-sec delay). During portal venous imaging, the liver parenchyma enhances and hypovascular metastases, such as those from CRC, appear as filling defects.2 Most metastases are seen best in the portal venous phase, but some are best seen in delayed venous and occasionally arterial phases. Hypervascular liver metastases may be missed with CT, because very vascular metastases may enhance to the same degree as normal liver parenchyma. Delayed contrast CT, referring to scanning 4 to 6 hr after contrast injection, is most useful as an adjunct to increase the sensitivity and accuracy of contrast CT, because malignant lesions usually do not retain contrast and appear as hypodense areas within the enhanced normal liver parenchyma.2

Computed Tomography Angiography and Portography

Liver Metastases • CHAPTER 59

A

B Figure 59-1 • A, T2-weighted spin-echo image (left); T1-weighted spin echo image (right). In T2-weighted images the metastases are areas of high signal intensity. In T1-wieghted images metastases have a low intensity. B, Dynamic gadolinium-enhanced MRI demonstrating peripheral nodular enhancement in a hepatic hemangioma. Enhancement increases as the study advances over time from upper left to lower left and then to upper and lower right.

noninvasive method to evaluate hepatic vasculature. Novel MR contrast agents have the potential for improving detection of liver metastases.5 Advantages of MRI include the lack of radiation exposure to the patient and the low frequency of reactions to contrast agents. MRI is particularly useful for patients with contraindications to iodinated contrast, and MRI can detect hypervascular lesions that are not well visualized on CT. Limitations of MRI include inherent warping and the time required for imaging, which may lead to artifacts in patients who cannot hold their breath. Parallel imaging allows for high-resolution imaging of the entire liver within one breath hold, with the potential for more sophisticated sequences (such as diffusion-weighted imaging and MR spectroscopy) to improve liver metastases detection in the future.

most useful in detecting superficial liver metastases in small patients. Contrast-enhanced US, using intravascular microbubble contrast agents, has shown similar accuracy for liver metastases detection compared to CT and MR.6 An advantage of contrast-enhanced US is the potential for characterization of liver lesions based on morphologic evaluation as well as temporal vascular enhancement pattern.7 During the portal venous phase, benign lesions typically enhance more than the liver, whereas malignant lesions enhance less.8 Expertise in contrast-enhanced US is not yet widespread. Intraoperative US can also be useful at detecting small, deep hepatic metastases not palpable. In a study of 84 patients undergoing colon resection, intraoperative US detected 14 hepatic metastases that were missed on surgical palpation.9 In a similar study, intraoperative US detected seven nonpalpable lesions in 70 patients.10

Ultrasonography Ultrasonography (US) relies on sound waves to generate an image. US is most commonly used for screening for metastases because of its wide availability and lack of the need for radiation exposure. It is

Positron Emission Tomography Positron emission tomography (PET), in which a radioactively labeled tracer is administered to the patient and the scanner collects

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the emitted positron radioactivity to generate an image, allows imaging of cellular processes (such as cellular proliferation (18Flabeled thymidine), hypoxia (18F-labeled Miso), and blood flow ([15O]water) to be visualized. The majority of clinical experience has been with fluorodeoxyglucose ([18F]FDG), which images cellular glucose metabolism. A limitation of PET scans of the liver is that they are acquired over many breathing cycles, leading to blurring due to respiratory motion and lower spatial resolution as compared with CT. This has driven the development of dual-modality scanners capable of CT and PET scanning. Scanners capable of respiratory sorting are also being developed. FDG PET scans have been done to stage patients before resection of liver metastases11–13 and to evaluate response to treatment.14 In one study, 6 of 34 liver metastases treated with chemoembolization demonstrated persistent FDG PET activity, which led to further treatment.15 FDG PET has also been found to be useful in diagnosing peritoneal recurrences in patients with elevated serum carcinoembryonic antigen (CEA), with a sensitivity and accuracy for peritoneal disease detection of 88% and 78%, respectively (as compared with 38% and 44% for CT).16 In patients with colorectal liver metastases staged planned for resection, Ruers and colleagues found that 10 of 51 patients (20%) had a change in treatment plan due to unresectable metastases or extrahepatic metastases detected on FDG PET.12 Other studies have also demonstrated a change in treatment resulting from FDG PET scans obtained before planned liver resection in 20% to 58% of patients.13,17–19 In contrast, 15 out of 52 pathologically proven liver metastases were missed with FDG PET in another study, emphasizing that FDG PET cannot be the sole method used to evaluate liver metastases. Five-year survival of a series of 100 patients with liver metastases, imaged with FDG PET and treated with hepatic resection, was 58% (46% to 72%), demonstrating excellent outcomes in well-selected patients.20 A continuing Canadian randomized trial of PET versus no additional imaging in resectable liver metastases, funded by the Ontario Clinical Oncology Group (OCOG), has the potential to more clearly define which patients with liver metastases planned for resection are most likely to have a change in treatment and benefit from PET scans obtained before resection (personal communication, Steven Gallinger, Toronto, March 2007).

detection were 88% and 96% and for extrahepatic disease detection were 92% and 85%, respectively, as compared to 83% and 84% for hepatic disease and 61% and 91% for extrahepatic disease in CT. The management was changed based on FDG PET scans in 32% of cases on average. Overall, most studies have confirmed increased sensitivity of FDG PET as compared with MRI and CT, with increased sensitivity on a per-patient analysis as compared with a per-lesion analysis.5 FDG PET is less sensitive for detection of small liver metastases (less than 1–2 cm) than state-of-the-art MRI or CT.5,29,30 At present, the standard of care for imaging of hepatic metastases is helical multiphasic intravenous contrast CT scan, with MRI or contrast-enhanced US alternatives for patients with contraindications to CT contrast. For patients whose treatment plan includes resection, extra imaging should be considered to help best select patients with liver-confined metastases.

Differential Diagnosis for Liver Metastases The majority of liver metastases enhance on the portal venous phase of imaging. Arterial and portal venous phase imaging, using CT, MRI, or US, can help in distinguishing malignant from benign lesions and hepatic metastases from primary cancers. However, several benign lesions cannot always be reliably distinguished from metastases. Hemangiomas may be confused with highly vascular tumors and cysts. Dynamic Gd-enhanced MRI can help distinguish hemangiomas from tumors. Hemangiomas can also be mistaken for metastases in noncontrast CT scans and on portal phase contrast CT (see Fig. 59-1). Although portal phase contrast CT is sensitive in detecting small hepatic lesions, it has a high false-positive rate because benign lesions such as hemangiomas, cysts, adenomas, or flow artifacts may be confused with metastases. Technetium-99 (99mTc) red blood cell scintigraphy is the most specific noninvasive test to diagnose hepatic hemangioma (Fig. 59-2).31 Table 59-1 lists some lesions that may be confused with liver metastases and suggests the tests to differentiate among those entities. A rare entity that can on occasion be confused with metastases is focal fatty infiltration of the liver (Fig. 59-3). This may be seen in patients receiving hyperalimentation. MRI is helpful for distinguishing these lesions.

Biochemical Laboratory Tests Comparison of Modalities Many studies have compared imaging modalities for the detection of liver metastases. A German study21 prospectively compared dynamic CT with MRI and US in 75 patients with known gastrointestinal tumors before exploratory laparotomy. Ninety-five liver metastases were detected in 32 patients.22 Of the 95 lesions, 68% were detected by CT, 63% by MRI, and 53% by US. Another prospective study involving 69 patients came to the same conclusion, and also demonstrated the superiority of MRI T2-weighted images over T1.23 Other studies have shown improved detection of liver tumors with MRI,24,25 and differences in the shape and size of hepatic metastases depending on which CT or MR sequence is used for imaging.26 Overall, CT scans and MRI seem to have similar sensitivity and specificity for detection of liver metastases using state-of-the-art techniques at experienced centers. Noncontrast external US has a lower rate of accuracy for liver metastases detection as compared with contrast CT or MRI. A unique study of US before liver transplantation allowed the accuracy to be determined pathologically.27 Only 36 of 80 tumors in 34 patients were detected, indicating a sensitivity of 45%. The majority of lesions not detected were smaller than 1 cm. Contrast-enhanced US has demonstrated similar accuracy for liver metastases detection compared to CT and MRI.6 FDG PET can detect disease not seen on CT or MRI in patients with liver metastases.12,13,28 In a comprehensive review of the literature,11 the sensitivity and specificity of FDG PET for hepatic disease

Unlike imaging techniques, the laboratory tests that are available for liver function assessment are not very sensitive. The basic liver function tests include the alkaline phosphatase, bilirubin, albumin, prothrombin time, lactate dehydrogenase (LDH), and serum transaminases. For patients who have metastatic colon cancer, CEA is also extremely useful (see Fig. 59-3). Several studies have looked at the usefulness of these tests to detect liver metastases, especially metastases from CRC.32–34 CEA remains the most sensitive test for CRC, but even this test can be normal in the presence of liver metastases, especially with minimal hepatic disease. In a prospective study at the City of Hope Hospital (Durante, CA, USA) in patients with metastatic liver disease deemed resectable by CT, the average alkaline phosphatase and LDH levels were within normal limits, whereas CEA was elevated in 73% of the patients.35 Serum LDH is useful as a prognostic indicator, with high serum LDH denoting a poorer survival.36

HEPATIC RESECTION Because of recent advances in techniques for liver surgery, hepatic resection for metastatic disease has become increasingly safe and used more frequently in treatment over the last two decades. Hepatic resection of metastases was first attempted just before World War II. Experience gained from trauma centers during the war led to the emergence of techniques applicable to resection of metastatic hepatic lesions. In recent years many new devices have helped in the technical aspects of the surgery and in the overall safety of the procedures.

Liver Metastases • CHAPTER 59

A

B

Figure 59-2 • Hepatic hemangiomas. A, Transaxial single-photon-emission computed tomography (SPECT) image obtained 2 hours after injection of 99mTc-labeled red blood cells. Normal blood activity is seen in the liver, and increased blood pooling activity is seen in the aorta and spleen (which is normal). There is a large area of increased blood pooling activity in the posterior section of the right lobe of the liver that corresponds to an area of reduced attenuation on the CT scan. B, The CT scan is obtained from the same plane as the SPECT scan. An increase in blood pooling activity with an increase in intensity from earlier images is specific for hemangioma.

In the last few decades hepatic resections have gone from formal lobectomies to resections along nonanatomic lines. Metastasectomies, or removal of the tumor plus a rim of normal hepatic tissue, can be done rather than a formal lobectomy. With the greater familiarity of the hepatic anatomic segments of Couinaud (Fig. 59-4),37 current resections are more frequently being performed along these lines especially with the use of intraoperative US. These segment divisions cannot be seen by the naked eye on the surface of the liver but can be mapped out with the help of intraoperative US. In the modern operating room the use of intraoperative US is a necessary skill for

Table 59-1 Benign Liver Lesions That May Be Confused with Malignancy Technique

Benign Lesions

Malignancy

MRI (T2-weighted)

Vessels

Small metastatic lesions

Hemangiomas

Highly vascular tumor (islet cell, renal, carcinoid)

CT

CTP

Cysts

Cystic tumors

Cysts

Highly vascular tumors (islet cell, renal, carcinoid)

Fatty infiltration

Metastases

Fatty infiltration

Hepatocellular cancer

Nonenhanced vessels

Metastases

Adenoma

Abscess

Hemangioma

Metastases

Cysts Adenoma Flow artifacts Angiography

Focal nodular hyperplasia Hemangioma

Metastases

CT, computed tomography; CTP, CT portography; MRI, magnetic resonance imaging.

the operating surgeon. This technique allows for both a reduction in the amount of normal liver removed and resection of disease from both lobes. Several new devices have been introduced to help with the resection itself. Probably the most commonly used device is the cavitron ultrasonic aspirator, which allows dissection of the liver parenchyma without entering the bile ducts. A new technique for dividing the parenchyma is the multiprobe bipolar radiofrequency device, which was introduced in 2004. This device uses radiofrequency waves to coagulate vessels and bile ducts in the liver parenchyma before dividing the tissue.38 Other devices are available, but in the end it depends on the operating surgeons and their own techniques, which vary from center to center. No clear advantage has been established for one product over the other. The operative technique of total vascular exclusion for hepatic resections involving tumors near to the vena cava was introduced in the 1980s by surgeons from the liver transplant community. With this technique the vena cava is cross-clamped in two areas (suprahepatic and subhepatic), and the porta hepatis is also occluded.39,40 In Bismuth and associates’41 report on 54 patients using total vascular exclusion, the average duration of cross-clamping was 46 min, and the average operating time was 6 hr. The transfusion requirements were low, and no intraoperative deaths occurred. The application of vascular occlusion seemed to be useful for tumors close to the vena cava or for large central tumors. Again, it is a technique to be used in special circumstances by surgeons who are familiar with the procedure. The use of the autotransfuser, a technique pioneered in trauma patients, had been avoided initially for patients with malignancies because of fear that tumor cells might be disseminated into the bloodstream with this instrument. Prospective studies on patients undergoing hepatic resection for tumors have not shown this to be true.42,43 Animal studies also support the concept that metastatic cells are organ specific and would not disseminate if introduced into the bloodstream. Because of these studies, many centers are using the autotransfusers during hepatic resection and reducing the need for multiple blood transfusions. Another technical advance for hepatic surgery has been the use of fibrin glue, which can aid in sealing the large, raw surfaces of the liver left after major resections.44

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Figure 59-3 • Fatty infiltration. This lesion was thought to represent metastatic disease; surgery revealed it was only fatty replacement.

The question of drainage after hepatic resection has been addressed prospectively in a study that showed no difference in complications in the drained group versus the undrained group. When the data are analyzed more carefully, however, it becomes clear that patients with lobectomies or greater did benefit from drains, whereas those patients with smaller resections did not need drains placed.45,46 However, this study was done before the advent of fibrin glue, which may reduce the need for drains. In general, a major surgery such as a liver resection has been reserved for a situation in which the operation can be curative. Experience has shown that the resection of colorectal metastases to the liver can be curative in at least one-quarter of patients with certain requirements. The curability by resection of liver metastases from other primary cancers is not quite as clear. The resection of metastases from other gastrointestinal malignancies, such as stomach and pancreas, has been disappointing because of the aggressive nature of these tumors by the time they become metastatic.47,48 The performance of liver resections for metastatic disease from a breast cancer is controversial. Liver metastases from gastrointestinal

II VII

VIII III

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V

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VI

Figure 59-4 • Couinaud’s eight hepatic segments. (From Iwalsuki S, Sheahan DG, Starzl TE: The changing face of hepatic resection. Curr Probl Surg 1989;25:281.)

tumors can be considered regional spread, but for the metastases to go from the breast to the liver requires release of the tumor cells into the systemic circulation. Thus, the concept that the liver could be the only site of spread is harder to prove, and as a result, curative intent is more difficult to achieve. Most of the literature about hepatic resections for breast cancer metastases has come from France. In a recent publication one hospital retrospectively reviewed their experience in 108 patients from the time of 1984 to 2004. Twenty-three (21%) of the patients were found to be unresectable at laparotomy because of intra-abdominal metastases or hepatic lesions that were technically not resectable. Of the 85 patients who had a liver resection, 38% had a solitary metastasis, 32% had two or three lesions, and 31% had more than three lesions. The metastases were small with an average size of 2.8 cm. The median follow-up of the patients was 38 months, with a median survival of 32 months. Only 18 patients were alive after 5 years. A multivariate analysis of factors showed only response to chemotherapy, the absence of extrahepatic metastases, and a clear margin at resection correlated with survival.49 Another Parisian team reported their experience from 1988 to 1997 with 49 liver resections for metastatic breast cancer. They reported no mortality and a morbidity of 11.5%. The 1-, 2-, and 3-year survival rates were 86%, 79%, and 65%, respectively. Recurrence in the remaining liver was seen in 49% of the patients at 3 years after resection. Recurrent disease anywhere in the body was seen in 63.8% of patients at 3 years after the resection. The only factor that correlated with survival was the disease-free interval between diagnosis of the primary breast cancer and the appearance of the liver metastases, with a 3-year survival of 45% if the liver metastases appear in less than 4 years after the primary breast cancer versus a 3-year survival of 82% if the breast cancer was more than 4 years from the appearance of the liver metastases.50 Another study from Paris reported on a smaller number of resections of solitary hepatic metastases from breast cancer.51 Of the 32 patients with isolated liver metastases, 27 were found to have actual metastatic disease, whereas 5 had benign disease. Six of the 27 had diffuse disease that was not amenable to resection, whereas of the 21 who underwent hepatic resection, the average survival time was 26 months. For both of these studies there is no way of judging the usefulness of hepatic resection versus chemotherapy for these patients. It is interesting to note that the more recent study has a superior

Liver Metastases • CHAPTER 59

survival time. This could signify several factors, including better drugs and improved selection. That the disease-free survival (DFS) is still quite low, however, emphasizes that even with strict patient selection, the chance of cure with a liver resection is quite limited, but real. Thus patients who have metastatic disease to the liver due to breast cancer need to be screened carefully before they are offered resection as a therapeutic option. Issues such as other sites of metastatic disease, response to chemotherapy, and disease-free interval between breast cancer and liver metastases all must be considered before hepatic resection. Gastrointestinal neuroendocrine tumors frequently metastasize to the liver but their growth is often slow, and patient survival can be prolonged even without surgical intervention. Both the rarity of these tumors and their prolonged course make them poor candidates for prospective trials. Thus, the role of hepatic resection for metastatic neuroendocrine tumors can only be evaluated in a retrospective manner. At the Mayo Clinic, over a 20-year period from 1970 to 1990, only 37 patients with hepatic metastases from a neuroendocrine tumor had a resection (17 curative resections and 20 palliative resections).52 Eleven of the 17 patients with curative resections were alive 1 to 92 months after surgery (median 19 months) without evidence of disease. Of the 20 patients who underwent palliative resection, 1 died in the postoperative period, 8 died of disease 9 to 76 months after resection, and 19 patients had some relief of symptoms. The authors concluded that resection is reasonable in cases in which the bulk of tumor can be removed and the patients are symptomatic. A second report from the Mayo Clinic reviewed their hepatic resections for neuroendocrine tumors from 1984 to 1992, with a total of 74 cases.53 Because this is a report from the same institution and there is some overlap of the time period, clearly many more resections were done in the later years (1990–1992) than in the two decades before. The study included patients whose primary tumors were either completely resected or potentially completely resectable. Patients with carcinoid syndrome were injected with subcutaneous somatostatin preoperatively. Most patients54 had nonanatomic resections, although 36 had a lobectomy or greater. The mortality rate was 2.7%, and the morbidity was 24.3%. Overall survival at 4 years was 73%, with a mean follow-up of 2.2 years. All of the 12 patients who died had tumor progression. There was no significant survival difference among those patients who had curative resections (that is, removal of all gross disease) versus those who had palliative resections. Symptom relief was seen in 90% of patients. The authors concluded that resection should precede hepatic arterial occlusion and systemic chemotherapy, because it provides an excellent response rate and good survival. Other studies with considerably fewer patients also support this conclusion.55–57 A review of the literature in 2002 reported on 227 patients with hepatic surgery for metastatic malignancies. The operative mortality for the 212 patients with carcinoid tumors was 2.3% and morbidity was 71%. The overall 5-year survival was 71%, and when looking at only the metastatic islet cell tumors, the survival was even higher at 82%. Symptomatic relief from carcinoid syndrome was 86% with a duration of 4 to 120 months. Some discussion of ablative techniques was offered, but only with very preliminary results. The analysis of these sorts of data suggest that for selected patients liver resection is safe, appropriate, and can result in long-term survival and relief from endocrinopathy symptoms. Guidelines for resection include the preoperative assessment that the primary and metastatic disease is completely resectable (Box 59-1).59 The other noncolorectal tumors metastatic to the liver that have been resected enough to have reported studies are the sarcomas. The largest retrospective review comes from M.D. Anderson Cancer Center with 66 patients having liver surgery for metastatic sarcoma. The mean size of the lesions was 3.9 cm and mean number of lesions was 3. The majority of patients had a gastrointestinal stromal tumor (GIST) (36 patients), 18 had a leiomyosarcoma, and the rest had various other sarcomas. Thirty-five patients had resection, 13 patients

Box 59-1.

SURGICAL GUIDELINES FOR HEPATIC RESECTION

The existence of extrahepatic intra-abdominal metastases should be excluded before attempting a hepatic resection. Because the periportal lymph nodes are the most common site for intra-abdominal extrahepatic metastases, biopsy samples from them should be taken and sent for frozen section. If extrahepatic disease is present the hepatic resection should not be carried out. The extent of a hepatic resection can span from one small nodule to a trisegmentectomy by which 75% of the liver is removed. The assessment of patients for trisegmentectomy is very difficult, because no tests are currently available to delineate accurately which patients can survive with a 75% loss of liver mass. In general, however, if a patient has cirrhosis, an extensive resection is discouraged. Debilitated (poor performance status) patients are not good candidates for major hepatic resection. Age alone should not preclude a patient’s eligibility for hepatic resection.

had radiofrequency ablation (RFA), and 18 patients had the combination of resection and RFA. The operative mortality and morbidity were 4.5% and 15.2% respectively. The recurrence rates were high with 85% of patients, with RFA recurring, 89% with the combination of resection and RFA recurring, and 57% of patients with resection only recurring (statistically better for this group). The 5-year DFS and overall survival were 16% and 27%, respectively. The article discussed the issue of the GIST tumors and the targeted therapy with Gleevec (imatinib mesylate). The authors felt that resection was still recommended for the GIST tumors, because the rate of complete response with imatinib mesylate was only around 5%, and the bulk of the patients progressed after 2 years on treatment. The conclusions of the report were that although recurrence is high, hepatic resection in selected patients is indicated and will lead to longer survival.60 The group at Memorial Sloan-Kettering (MSKCC) reviewed their experience with hepatic resections for sarcomas in the period from 1982 to 2000. There were 331 patients with liver metastases from a variety of primary sarcomas, 56 of whom had a hepatic resection. Thirty-four of the 56 patients had a GIST. Ten of the 56 patients have actually survived for 5 years, but only 2 are disease free. The disease-specific survival rate for 3 and 5 years was 50% and 30%, respectively. The patients with GIST had the same survival as those with other types of sarcoma. The time interval (less than or greater than 2 years) between the appearance of the primary sarcoma and that of the liver metastases had a significant influence on survival and was the only independent prognostic variable in a multivariate analysis.61 An analysis of hepatic resection for leiomyosarcomas from Germany reported a 40-month median survival in patients who had all tumor removed with negative margins. The conclusions from this study, similar to the other reports, were that hepatic resection for leiomyosarcomas could be done safely and in selected cases could prolong survival. Most of the other reports in the literature about resection of hepatic metastases from sarcomas are anecdotal, with far fewer patients in each study.62,63 The use of hepatic resection for metastases from melanoma is moderately rare as seen in a report from two major melanoma centers, where 1750 patients with hepatic metastases from melanoma were identified and only 34 of them had a surgical exploration with the intent to resect the tumor from the liver. Of the 34 patients explored, 24 went on to hepatic resection and 18 of them actually had a resection with curative intent. The median DFS and overall survival in the 24 patients with resection were 12 months and 28 months, respectively. When this was compared with the 6-month overall median survival in the 899 patients with hepatic metastases treated

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nonoperatively, it was significantly better. The two major contributors to improved survival seemed to be longer disease-free interval between the primary melanoma and the development of hepatic metastases, and limited hepatic disease that could be completely removed. For the select patients who would fit these criteria—and obviously there are not many—they seemed to benefit from hepatic resection.64 Unlike these other primary tumors, the data on the resection of colorectal metastases to the liver has been advancing exponentially over the last 30 years. In the United States, there are more than 50,000 patients each year with liver metastases from CRC. The rate of resection of these metastases has been increasing because of the expanding patient eligibility criteria. In the last decade there have been several retrospective reviews of resection of hepatic metastases from colorectal primaries—some with more than 1000 patients— that have added to the knowledge of which patients will benefit from resection.54,65–69 In the two largest series, the 5-year survival for patients with one to three metastases who had a resection was 30% or greater.65,66 Because of the strong feeling over the last three decades that resection was the optimal treatment for patients with one to three metastases, no randomized study of resection versus any other treatment has been performed. Two early studies compared the survival of matched historical control patients who underwent resection with those who had solitary hepatic lesions but did not undergo surgery.70,71 In both studies, not 1 of the 120 patients without resection survived for more than 3 years, whereas 30% of the resected patients survived for 5 years, underscoring the rationale for resection of solitary lesions. The retrospective series from MSKCC reviewed their experience with 1001 liver resections in patients with colorectal metastases from the years 1985 to 1998.65 Because the study was reported in early 1999, there were many patients who did not have a 5-year follow-up; in fact; the median follow-up of survivors was 32 months, which might not be long enough to tell us which patients would survive to 5 years. The median number of liver tumors was two, with 517 patients having solitary lesions and 330 having two or three lesions (Table 59-2). The operative mortality was 2.8%. The 3-, 4-, and 5-year survival rates were 89%, 57%, and 37%, respectively; however, only 24.6% of patients resected before 1994 are 5-year survivors. The number of tumors removed (one or greater than one), the size of the tumors removed (greater or less than 5 cm), the preoperative CEA level (greater or less than 200 ng/mL), the extent of resection (less than or greater than a lobectomy), the resection margin in the hepatic

specimen (negative or positive), and the presence of extrahepatic disease all were highly significant univariate and multivariate predictors of postsurgical survival. From these data, a clinical risk score was devised using five clinical criteria: the nodal status of the primary CRC, the disease-free interval from the primary CRC to the development of liver metastases, the number of hepatic tumors, the prehepatic resection CEA level, and the size of the hepatic tumors. Each criterion was given 1 point if the inferior condition existed, and then the points were added to give the score. The 5-year actuarial survival for a clinical risk score of zero was 60% as compared with a 14% survival for a score of 5. This gives surgeons a good insight into the prognostic expectations, but the score was not really intended for exclusion of patients from resection. Another large series was a multi-institutional report from France reviewing 1568 patients who underwent resection of liver metastases from CRC (Table 59-3).66,72 Like the previous report, this study also looked at the effect of numerous prognostic indicators on overall survival after liver resection and then combined seven indicators into a prognostic scoring system. In this study, the 5-year survival among patients who had a resection of four or more lesions was 14% as compared with 30% for three or fewer nodules (P = 0.001). Both large studies, together with others, emphasize that the resection of more than four hepatic lesions results in significantly fewer cures, whereas for patients with one to three metastases, agreement exists that resection is worthwhile and can offer at least a 30% 5-year survival.65–70,72–76 The French series found that one of the most significant prognostic variables was the stage of the primary lesion. For patients with CRC and negative lymph nodes (stage II), the 5-year survival after hepatic resection was 35% as compared with 26% for patients with mesenteric lymph node involvement (P < 0.001). Other studies addressing the issue of stage are listed in Table 59-4.73,76–80 In the French study, preoperative serum carcinoembryonic antigen (CEA) was also significant; patients with a CEA value of less than 5 had a 2-year survival of 70%, as compared with a 56% 2-year survival for patients whose CEA was above 30. For patients with tumor nodules smaller than 5 cm, survival was 30% as opposed to 26% for larger nodules (P = 0.002). The age of the patient was not found to be significant in this study or others.81 The time between the primary tumor and the development of liver metastases in this and other studies was significant for prognosis if the time periods were divided to include at least the first year with the synchronous lesions (Table 59-5).76–80

Table 59-2 Five-Year Survival after Hepatic Resection of Colorectal Cancer Metastases Based on Number of Lesions Resected NO. OF METASTASES (% OF 5-YEAR SURVIVAL) Study Group 65

Memorial Sloan Kettering

No. of Patients

1

441

44

2–3

510 France63

1350

Mayo Clinic

187

>3

28

23

30

183 68

>1

30

14

29

9

31

70 23 Italy72

134

Hepatic Registry73

789

Liver Met Survey49

2122

20

78

*60 yr

27

Age

Tumor size 5 cm

26

0.002

Stage of primary Dukes B

35

Dukes C

21

0.001

Disease-free interval 2 years

32

0.002

No. of nodules resected 3

14

0.0001

Resection margin >1 cm

32

12 mo

NS 0.05 0.01

333

42

0.02

Yes

35

52

NS

No

62

52

Yes

85

18

No

113

22

A group from Paris reporting on surgery for patients who had been downstaged by chemotherapy also found that if the tumors’ pathologic response to chemotherapy was less than complete necrosis, the survival after resection was significantly decreased (P = 0.002).84 This group of patients with more than four lesions and progressing disease should probably not be candidates for surgical resection. The use of the PET scan may help increase the survival of patients with liver resections from colorectal primaries because of improved selection of patients. A study of 100 patients who were shown to have disease confined to their liver by PET scan reported a 5-year survival of 58%. PET scan is believed to detect unsuspected tumors in 25% of patients who would have otherwise been considered to have only resectable hepatic metastases. This was not a randomized study, so it is difficult to determine if the PET scan alone was responsible for this improved survival over the usual 30% 5-year survival. However, it may be prudent at this time to perform a PET scan before taking

NS

patients to liver resection, so that those who have incurable disease will be spared major surgery.85 The issue of trying to downstage patients with unresectable liver metastases and then proceed to liver resection is becoming more relevant as newer and more effective chemotherapy agents are being introduced. A report from Paris followed 1104 patients with liver metastases from colorectal primaries considered to be unresectable. Of these patients, 138 (13%) were downstaged sufficiently to go on to surgery. Eighty percent of these patient developed tumor recurrence, 72% of which involved the liver. DFS was 17% at 5 years. These numbers reflect that the overwhelming majority of patients will not be downstaged by chemotherapy sufficiently for resection, and even those that are do not fare particularly well.84 Several studies have addressed the issue of extrahepatic intraabdominal metastases at the time of hepatic resection. In most instances, surgeons do not proceed with liver resection in the presence

Table 59-6 Significance of Hepatic Resection Margin Institution Erlangen80

Mayo69

Pittsburgh68

Memorial

321

Margin of Resection

No. of Patients

5-Year Survival (%)

0–4 mm

67

23

5–9 mm

40

29

>10 mm

65

39

0–1 mm

17

29

1–10 mm

123

30

>10 mm

31

36

None

24

17

0–10 mm

92

25

>10 mm

95

29

None

17

0

1–10 mm

248

43

>10 mm

113

43

65

17

None

P Value NS

NS

0.006

0.00003

Liver Metastases • CHAPTER 59

Table 59-7 Recurrence after Hepatic Resection Author Van Ooijen et al79 77

Codi et al

No. of Patients

No. of Recurrences

No. of Liver Only

No. of Liver and Extrahepatic

No. of Extrahepatic Only

117

69

20 (29%)

14 (21%)

34 (50%) 28 (41%)

93

69

28 (41%)

13 (19%)

Hohenberger et al320

122

80

17 (21%)

55 (69%)

8 (10%)

Hughes et al75

607

424

148 (27%)

154 (27%)

106 (28%)

89

61

25 (41%)

9 (15%)

27 (44%)

465

235

96 (41%)

Rees et al322 321

Fong et al

of extrahepatic metastases. In the series from New York, 88 patients with extrahepatic disease were included.19 More than half of these patients had direct extension into other organs such as the diaphragm. Only 10 patients had positive portal nodal disease. Looking at all 88 patients with extrahepatic disease, the 5-year actuarial survival was 18%. The breakdown between those with discontinuous disease or direct extension was not made. In the Mayo Clinic report on hepatic resections in patients with extrahepatic disease, none of the 22 patients survived for 5 years, and only 1 survived for 3 years.86 In the Hepatic Registry, of the 61 patients with extrahepatic involvement undergoing hepatic resection, none had a 5-year DFS.74 These data support the view that hepatic resection in the presence of noncontiguous extrahepatic intra-abdominal disease, with the exception of a local recurrence, is rarely curative and, in general, is inadvisable. For extrahepatic disease not in the abdomen, the usefulness of resection could be different for disease in the lungs, especially solitary lesions in the lung. A recent study from the Mayo Clinic reviewed their experience with resection of both hepatic and pulmonary metastases from colorectal primaries. There were 58 patients, with no operative mortalities and a 5-year survival rate of 30%. These authors believed that the resection of both lung and liver of selected cases was justified.87 Relapse rates after liver resections are described in Table 59-7. In the larger studies it seems that approximately 40% of patients who have hepatic resections will have recurrent disease in the liver as the first sign of the relapse. Of the 69 patients who had relapse in the Milan study, 28 (41%) had relapse in the liver only, 19 (28%) had only extra-abdominal relapse, 9 (13%) had intra-abdominal extrahepatic relapse, and 13 (19%) had relapse in both the liver and at an extrahepatic site.49 Although extrahepatic failure is of concern, the liver remains the main site of relapse, appearing in more than 60% of patients. Because of the increased use of hepatic resection for metastatic liver disease, the incidence of repeat hepatic resection has also increased. Approximately 10% of patients who have had a hepatic resection can have a repeat resection (Table 59-8). A study from Paris of 116 patients who underwent repeat hepatic resection reported a

16 (7%)

123 (52%)

low operative mortality of 0.9% and a 3-year survival of 33%.54 Of the patients who underwent repeat hepatic resection, 55% had recurrence in their liver after this operation. There were 170 patients in a registry report who had repeat hepatic resections with a 5-year survival of 26%, which was comparable to the 5-year survival for the original hepatic resection.88 A recent study from MSKCC reviewed 126 second liver resections for recurrent colorectal metastases; the 5-year actuarial survival was 34%, with 19 actual 5-year survivors. The operative mortality was 1.6%, and morbidity was 28%.89 A study of recurrence among these patients revealed a liver recurrence in 67% of the patients. Repeat hepatic resection, when feasible, can be done safely, and the outcome is comparable to that for the original hepatic resection.90 Thus, if patients have isolated liver metastases (preferably solitary lesions) after hepatic resection, they should be candidates for repeat resection. The place for ablation of liver metastases whether by radiofrequency (RF) heating or cryotherapy freezing has not yet been delineated adequately. Because extensive reporting on resection has indicated an expected 30% 5-year survival for patients with one to three hepatic metastases from colorectal primaries, the use of ablation in this setting cannot be warranted until a randomized study has shown that the two therapies are equivalent. A retrospective study from M.D. Anderson Cancer Center analyzed a series of 418 patients with liver-only metastases from colorectal primaries, of whom 57 had RF only. The overall recurrence rate and the liver recurrence rate were both significantly higher for the RF group over the resection-only group, with a liver recurrence of four times greater in the RF group. The overall survival and DFS was also significantly better in the resection group. A multivariate analysis continued to show RF as a significant factor in poor prognoses. Interestingly, 31 patients with solitary tumors were treated with RF (we are not told why this happened), and these patients had significantly worse survival than the patients with resection of solitary lesions (P = 0.025). The group of patients who had RF alone when compared with those 70 patients with chemotherapy alone had a significantly improved survival.91 A subsequent report from the same group of investigators compared RF to resection for patients with solitary colorectal metastases. The hepatic recurrence rate was much higher in the RF group than in the

Table 59-8 Survival after Repeat Hepatic Resections for Colorectal Metastases SURVIVAL (%) Author

No. of Patients

Operative Mortality (%)

2-Year

3-Year

Nordlinger et al54

116

0.9

57

33

Petrowsky et al89

126

1.6

51

34

Fernandez-Trigo et al88

170

37

26

21

50

Que et al323

5-Year

895

Part II: Problems Common to Cancer and Its Therapy

resection group, 37% as compared with 5%, respectively. The 5-year recurrence-free survival and overall survival were 40% and 50% as compared with 0% and 0% for resection versus RF, respectively. The conclusions are obvious: resection is the preferred method for treatment of solitary metastases whenever possible.92

1.0

Proportion surviving

0.8

Synchronous Liver Metastases

Adjuvant Therapy after Liver Resection Despite the high curative resection rate of hepatic resections, the recurrence of metastases is around 70%, with 50% recurring in the liver. The use of hepatic artery infusion (HAI) of chemotherapy after liver resection was studied in a small prospective randomized fashion at the City of Hope Medical Center.101,102 The patients with solitary metastases all had resection of their tumors, and half received postoperative continuous HAI of fluorodeoxyuridine (FUDR). For the six patients who had resection only, their median time to failure was 8.7 months, and three of the six metastases recurred in the liver. For the five patients with resection plus pump, none had recurrence in the liver, and their median time to failure was 30.7 months. There was no difference in median survival between the two groups. At MSKCC, 156 patients were randomized after liver resection to either HAI with systemic chemotherapy (HAI + SYS) or systemic chemotherapy alone (SYS). Chemotherapy was administered for 6 months. The endpoint of this study was 2-year survival. HAI therapy used was FUDR and dexamethasone (Dex), and the systemic therapy was 5-fluorouracil (5-FU) and leucovorin (LV) or continuous infusion of 5-FU. Patients were stratified by type of previous chemotherapy or no chemotherapy and the number of liver metastases (one, two to four, more than four). Two-year survival was increased in the group receiving HAI + SYS (86%) versus 72% for SYS (P = 0.03).103A Median survival is presently 68.4 months for the HAI + SYS group and 58.8 months for the group receiving SYS alone. With a median 10-year follow-up, the 10-year survivals are 41% and 27%, respectively (Fig. 59-5).104 Hepatic DFS is clearly better for the HAI + SYS group, with a median survival not reached, as compared with 32.5 months with SYS alone (P = 0.003; Fig. 59-6). Overall, DFS is also increased significantly (31.3 and 17.2 months for the HAI + SYS vs. SYS alone [P = 0.02]; Fig. 59-7). Toxicity was increased in the combined group, with increased diarrhea and increased liver function test abnormalities. A total bilirubin greater than 3 mg/dL occurred in 18% of patients receiving HAI and in 2% of the SYS group.103 The Eastern Cooperative Group (ECOG) and the Southwestern Oncology Group (SWOG) conducted a randomized study of hepatic resection alone versus resection followed by 4 cycles of HAI-FUDR

0.6 Combined therapy 0.4 0.2

Monotherapy

0.0 0

50

100

150

Months after resection

Figure 59-5 • Survival curve. Combined modality treatment: HAI FUDR + systemic 5-FU/LV. Monotherapy: systemic 5-FU/LV.104

Proportion progression free

The question of whether liver metastases can safely be resected at the time of resection of the primary colorectal carcinoma is still uncertain. Small metastases seen at the time of laparotomy for the primary lesion can easily be resected. When patients are evaluated for their primary and are found to have a large burden of metastatic disease, a question may be whether a simultaneous resection of the primary tumor and the metastases should be done, or whether preoperative chemotherapy should be offered. Bolton and Fuhrman reported a 12% operative mortality with simultaneous surgeries that increased to 24% if the surgery included a major liver resection.93 Nordlinger reported a 7% mortality for simultaneous resections compared with 2% for staged resections (P = 0.01).94 Tanaka and coworkers, Martin and colleagues, and Weber and associates, on the other hand, reported that operative mortality and morbidity were comparable whether staged or simultaneous procedures were done.95–97 It is the practice in many institutions to do a simultaneous resection with right colon primaries or when single synchronous metastases are found in the liver, and staged resections for rectal primaries or for patients with multiple liver metastases.98 Some advocate systemic chemotherapy first, followed by resection of liver first, and then resection of the colon99 or simultaneous resection.100

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

SYS alone (median = 32 mo) HAI+SYS (median = NR)

0

1

2

3

4

5

6

7

8

9

10

11

12

From surgery date (yrs)

Figure 59-6 • Hepatic progression-free survival. HAI (FUDR/dex) + SYS (5-FU/LV) versus SYS (5-FU/LV) alone. P < 0.0001.104

Proportion progression free

896

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

SYS alone (median = 17 mo) HAI+SYS (median = 31 mo)

0

1

2

3

4

5

6

7

8

9

10

11

12

From surgery date (yrs)

Figure 59-7 • Progression-free survival. HAI (FUDR/dex) + SYS (5-FU/ LV) versus SYS (5-FU/LV) alone. P < 0.02.104

Liver Metastases • CHAPTER 59

and 12 cycles of systemic infusion of 5-FU. Only patients with three or fewer metastases were enrolled in the study. The study was powered to answer the question of whether HAI would increase DFS. Of the 109 patients randomized, only 75 are actually in the study, because several patients were excluded because of extrahepatic disease, unresectable disease, or no tumor. Four-year liver recurrence-free survival was 67% in the chemotherapy group and 43% in the control group (P = 0.03). Median survival was 63.7 months for the chemotherapy group and 49% for the control group.105 The endpoint was obtained with a 4-year DFS of 46% with HAI + SYS and 25% for the control group (P = 0.035). A German cooperative group enrolled patients from 26 different centers and entered 226 patients into a study of resection followed by adjuvant hepatic arterial therapy with 5-FU and LV via a port as compared with a control group receiving no chemotherapy after resection. Although 113 were entered into the hepatic arterial group, only 87 were treated. Only 64% had chemotherapy data available, and only 30% completed treatment. At an 18-month interim analysis, the relapse rate was 33% in the group receiving adjuvant therapy and 36% in the group treated with resection alone. If one looks only at those who were treated, the median survival was 44.8 months from the treated group versus 39.7 months in the control group.106 A randomized study from Greece used intraoperative randomization to regional mitomycin C, 5-FU/LV with interleukin-2 via a hepatic arterial catheter with the same drugs via a systemic route versus systemic therapy alone. The 2-year survivals in 122 patients were 80% and 71%, and 5-year survivals were 73% and 60%, in the regionalplus-systemic versus systemic-alone groups, respectively (P = 0.004).107 Five-year hepatic-free recurrence was also significantly increased in the regional-plus-systemic group at 82% versus 49% in the systemicalone group (P < 0.001). Table 59-9 summarizes survival data of the randomized studies and the disease-free survival from these studies.

Tono and coworkers108 randomized 19 patients to continuous infusion of 5-FU, 500 mg per day for 4 days via HAI for 6 weeks. Although this is a small study, there was an increase in 3-year DFS, 66.7% for the regional group and 20% for the control group (P = 0.045). The 5-year survivals were 77.8% for the regional group versus 50% for the control group. In a nonrandomized study comparing two groups of patients in Japan,109 the survival was increased with the use of adjuvant regional therapy after hepatectomy for colon cancer. The 5-year survivals were 23% using surgery alone, and 57% when adjuvant chemotherapy with 5-FU, doxorubicin, and mitomycin C were given after surgery. In another nonrandomized study from Japan,110 58 patients who had radical resection of metastatic colorectal carcinoma could select whether they wanted HAI + systemic, or systemic alone after surgery. The 5-year survival was significantly increased for the HAI + systemic group at 59%, as compared with 27% for the systemic-alone group (P < 0.001), as was hepatic recurrence at 7% versus 57% (P < 0.001). In another small study, 4-year survival was 100% as compared with 47% for the HAI and control groups, respectively (P = 0.05).111 Studies are now testing the new systemic chemotherapeutic agents with HAI as adjuvant therapy after liver resection. A phase I study at MSKCC with systemic CPT-11 (irinotecan) and HAI FUDR/Dex had a 2-year survival of 89% and a 5-year survival of 58%.112 Another trial using escalating systemic doses of oxaliplatin (Oxali)/5-FU/LV and HAI therapy,113 with a minimum follow-up time of 26.9 months, had a 2-year survival of 98%. At the Mayo Clinic, FUDR + Dex has been added to systemic Oxali and capecitabine. Presently, the 2-year survival is 86%.114 Ongoing studies to address the usefulness of adjuvant therapy after liver resection include the National Surgical Adjuvant Breast and Bowel Project study of HAI + systemic Oxali + capecitabine versus systemic Oxali + capecitabine alone. A trial at MSKCC is

Table 59-9 Randomized Trials of Adjuvant Therapy and Studies after Liver Resection: HAI versus SYS or Control 2-YEAR SURVIVAL (%) Author

5-YEAR SURVIVAL (%)

No. of Patients

HAI

SYS or Control

HAI

SYS or Control 60

110

58

80

71

73

MSKCC103

Kusonoki et al

156

86

72

57

48

Lorenz et al106

201*

60

62

50

30

75†

70

65

60

35‡

122

92

75

73

60

38

100

60

100

47§

ECOG105 107

Lygidakis et al

111

Asahara et al

DISEASE-FREE SURVIVAL 2-YEAR (%)

5-YEAR (%)

Studies

No. of Patients

HAI

SYS

HAI

SYS

MSKCC104

156

55

40

40

30

0.02

ECOG105

75

60

40

40

20#

0.03

Lorenz et al106 Lygidakis et al Tono et al108

Median 20/12.6#

186 107

P Value

NS

122

66

48

60

35

0.0002

19

75

30

60

20

0.045

*Treated patients, not everyone randomized. † Patients entered in study, not everyone randomized. ‡ Updated figures.107 § 4-year survival. # No treatment in control arm.

897

898

Part II: Problems Common to Cancer and Its Therapy

addressing the question of the safety of bevacizumab (Bev) given with HAI + systemic therapy after liver resection in a randomized study of HAI and systemic therapy with or without bevacizumab. Few studies have evaluated the utility of systemic therapy after liver resection. In the ENG study (EORTC/NCIC CTG/GIVIO), 128 patients were randomized to receive 5-FU and LV chemotherapy for 8 weeks versus no further therapy (control) after liver or lung resection. There were no significant differences in DFS or survival between the two groups. The 4-year survivals were 57% and 47% for the treated and control groups.115 A European intergroup study (FFCD) randomized 173 patients to systemic 5-FU/LV versus no further treatment. The 5-FU and LV were given by the bolus method on a monthly schedule. Two-year DFS was 50.4% for those receiving chemotherapy and 38.1% for the control group (P = 0.058). Negative prognostic factors were synchronous disease, multiple metastases, stage III tumor, preoperative hypertension, postoperative complications, and an elevated preoperative CEA. Two- and five-year survivals were 81% and 51% for the chemotherapy group and 82% and 41% for the control group. A trial combining these two studies to increase power demonstrated a DFS of 27.9 versus 18.8 months (P = 0.058) and overall survival of 62.2 versus 47.3 months (P = 0.095) for the chemotherapy versus surgery-alone groups, respectively. Patient characteristics in this study included one metastasis in 68% of patients, and more than 1 year disease-free interval in 57% of patients. This clearly can affect results, as seen in their own analysis, where DFS was 27 months for one metastasis and 16.8 months for two or more metastases (P = 0.036), and survivals were 64.5 and 40 months for one or more than two metastases, respectively. In the MSKCC adjuvant therapy of HAI + systemic 5-FU/LV after liver resection study, the patients had worse baseline characteristics, with 36% of patients having only one metastasis, whereas 19% had more than four metastases, and only 20% had a disease-free interval longer than 1 year.103 The efficacy of systemic CPT-11 in posthepatic resection was addressed in 29 patients of whom 62% had only one metastasis, 79% had a disease-free interval longer than 1 year, and 62% had a surgical margin larger than 1 cm. Their median DFS was 45 months, and 2-year survival was 85%. Adjuvant therapy following resection has also been attempted in HCC. In patients with HCC, a randomized study compared hepatic arterial injection of epirubicin and oral 1-hexycarbamoyl-5-fluorouracil (HCFU) to no further treatment in 57 patients after liver resection of HCC.116 There was no significant difference between the two groups, and the authors believed that the chemotherapeutic agents chosen might not have been appropriate. Postoperative adjuvant therapy could compromise survival if the treatment is not effective, because it suppresses host immunity. In a small randomized study of intravenous epirubicin with hepatic arterial iodized oil and cisplatin compared to no further treatment after liver resection for HCC, the DFS was shorter in the treated group. There was an increase in extrahepatic metastases in the treated group.117 Factors that inhibit hepatic carcinogenesis might be more useful as adjuvant therapies for HCC. Retinoic acid inhibits chemically induced hepatocarcinogenesis in rats, spontaneous HCC in mice, and the production of α-fetoprotein in human hepatoma cell lines. In a study by the Hepatoma Prevention Study Group, 89 patients were randomized to polyphenolic acid (acyclic retinoid) or placebo for 12 months after liver resection. The polyphenolic group had a significant reduction in recurrent or new hepatomas (P = 0.04). The drug also improved overall survival, but not significantly.118

SYSTEMIC CHEMOTHERAPY Responses of liver metastases to systemic chemotherapy are variable but usually reflect the response of the primary tumor. Most studies of systemic therapy do not differentiate patients who have only liver metastases, making it difficult to draw conclusions about the usefulness of systemic chemotherapy to treat liver metastases. In some

cases, however, the response rates of liver metastases are well documented. In breast cancer, liver metastases represent a poor prognostic indicator, with median survival of 10 months in some series.119 Visceral metastases, including liver metastases, have been reported to have fewer estrogen-positive receptors. Insulin-like growth factors are present in the liver and the lung and could be important to the growth and motility factors for breast cancer and lung cancer.120 In looking at prognostic factors that predict response, patients with liver metastases were the ones who tended to respond the least.121 Carter’s122 review of single agents (5-FU or cyclophosphamide) demonstrated a 20% response in liver metastases vs. 32% and 27% in soft-tissue and osseous metastases, respectively. The combination of halotestin, prednisone, and 5-FU produced objective responses in 26 of 52 patients (50%) with liver metastases from breast carcinoma.84 Although combination chemotherapy has substantially improved the response rates obtained in treating breast cancer, liver metastases still have a lower response rate than soft-tissue or pulmonary disease. In a Southwest Oncology Group study of 262 patients, 41 of 88 patients (47%) with liver metastases responded, as compared with 110 of 154 patients (71%) without liver involvement (P = 0.001).123 There is a suggestion that taxoids might be more effective against liver metastases.124 For metastases from gastric carcinoma, one of the more commonly used regimens (5-FU, doxorubicin, and mitomycin C) produces a mean response of 35%, whereas for those with hepatic metastasis, the response rate was 28%.125 The cumulative response for liver metastases was 34%, while the overall mean response rate was 36% for combination chemotherapy in the treatment of gastric carcinoma.126 For patients with CRC, the liver is the most common site of dissemination, with as many as 70% of patients with metastatic disease developing liver metastases.127 Several new agents are now available, among which are irinotecan (CPT-11) and oxaliplatin (Oxali).128,129 Randomized trials using CPT-11 with 5-FU/LV versus 5-FU/LV alone130,131 produced an increase in response rate and survival. When CPT-11/5-FU/LV (IFL) was compared to Oxali + 5-FU/LV (FOLFOX), the response rate was increased from 35% to 45%, and the survival was increased from 15 to 19.5 months for the IFL and FOLFOX groups, respectively.132 When 5-FU was changed to infusion FOLFIRI (CPT-11/5-FU/LV) it became more effective and produced results that are similar to FOLFOX.133 In the last few years, targeted agents have become available: Bev, a monoclonal antibody to vascular endothelial growth factor and cetuximab (C225), an antibody to epidermal growth factor receptor. The addition of Bev to IFL134 increased response rates and survival (35% to 45%, and 15.6 to 19.5 months, for IFL vs. Bev + IFL, respectively). For almost four decades, the 2-year survival for metastatic colorectal patients treated with 5-FU or 5-FU + LV was 25%; with these new agents, 2-year survival has moved up to 30% to 39%, with a marked improvement in overall survival from 12 to 20 months. When does one start systemic chemotherapy for patients with metastatic disease to the liver from breast, gastric, or colon cancer? For patients with colon or gastric cancer who are asymptomatic and have a small volume of disease (12 cycles) was related to longer hospital stays after liver resection.188 For clearly unresectable disease, patients should enter protocols that are addressing the question of how best to decrease liver metastases to allow resection. In patients with synchronous CRC, there are those who advocate for simultaneous resection of both the primary and the metastatic disease, those who recommend chemotherapy first,189,190 and those who state both surgeries can be done simultaneously. Supporting the concept of controlling micrometastatic disease, the Liver Met Survey group found patients with more than five metastases survived longer if they were given neoadjuvant chemotherapy, with a 5-year survival of 22% versus 12% (P = 0.07) for the preoperatively treated and preoperatively nontreated groups, respectively. In another retrospective review at MSKCC, preoperative chemotherapy did not improve survival. In a review of 230 patients who received HAI + systemic therapy after liver resection,191 the median survivals for patients who did or did not receive preoperative (neoadjuvant) therapy were 63 and 115 months, respectively(P = 0.26). If metastases that disappear on CT remain viable, preoperative chemotherapy may be a disservice. In a review by Benoist and coworkers on 586 treated patients,192 38 patients had disappearance

907

908

Part II: Problems Common to Cancer and Its Therapy

Resectable liver metastases Resect

or

or

Unresectable liver mets

Extrahepatic disease

Regional chemotherapy

Systemic chemotherapy

Systemic chemotherapy

Enter adjuvant clinical trials

HAI + SYS chemotherapy

Tumor growth

Figure 59-12 • Treatment of liver metastases.

of at least one lesion on CT. Pathologic examinations of sites with a complete response showed that in 11 of 15 (80%) there were viable tumor cells. Areas that could not be found were closely followed, and in 23 of 41 sites (47%), tumor reoccurred. As use of preoperative chemotherapy increases, reports are emerging about liver toxicity from preoperative chemotherapy. There is an increased incidence of steatosis, sinusoidal abnormalities, venoocclusive disease, and steatohepatitis. In the Rubbia-Brandt and colleagues193 report on 153 patients undergoing liver resection, 51% of the 87 patients who have received chemotherapy before resection had sinusoidal dilation and 78% who had prior Oxali showed striking sinusoidal alterations, with venoocclusive fibrosis in 48%. Kooby and associates reported on 325 patients with fatty livers undergoing resection at MSKCC and found that those treated with preoperative chemotherapy were more likely to have steatosis (66%), with marked steatosis being an independent predictor of complications following hepatic resection.194 Vauthey and coworkers reported a 20% incidence of steatohepatitis in patients receiving preoperative CPT-11 versus 4.4% in those who received no preoperative chemotherapy, and an 18.9% incidence of sinusoidal dilation in those receiving preoperative Oxali versus 1.9% in those receiving no preoperative chemotherapy. Patients with steatohepatitis had an increased 90-day mortality, 14.7% versus 1.6% (P = 0.001).195 Is neoadjuvant chemotherapy useful for resectable liver metastases? The EORTC study is looking at preoperative and postoperative FOLFOX versus observation. They have randomized 364 patients to (1) six cycles of FOLFOX before and after surgery or (2) surgery alone.196 Presently, in the group receiving preoperative chemotherapy, 7.7% progressed before surgery and 11% were not able to undergo resection. It is too early to look at the DFS and survival. For clearly resectable disease, until we know more from these studies, surgery should be done first, followed by adjuvant therapy. Figure 59-12 provides a treatment algorithm for liver metastases.

HEPATIC ARTERIAL EMBOLIZATION Because both hepatic metastases and primary liver tumors derive their blood supplies from the hepatic artery, hepatic arterial ligation (HAL) and embolization have been used to reduce the tumor’s blood supply.197 In most hepatic tumors, HAL produces only a transient benefit because of the rapid development of collateral vessels. In vascular neuroendocrine tumors, HAL produces objective tumor reduction, whereas in colon metastases (which are typically less vascular), minimal regression occurs.

The development of a collateral blood supply might be minimized by injecting vasoocclusive particles into the hepatic artery (hepatic artery embolization, HAE), which also provides the opportunity for retreatment, because only the microvasculature is occluded.103 In one study of 61 individuals with liver metastases from colorectal carcinoma, patients were randomized to HAE, HAL + microspheres (HAE), or no further treatment. The median survivals were 8.7, 13, and 9.6 months, respectively, suggesting that embolization or ligation alone had no effect on survival for patients with CRC.198 Gerard and colleagues199 randomized 67 patients to HAL alone versus HAL and portal vein infusion of 5-FU. The median survival in both groups was 12 months; among patients whose median extent of liver involvement was 30%, only one patient responded. Both studies suggest that this technique is not useful for CRC. HAE has a definite role in highly vascular tumors, such as neuroendocrine tumors of the liver.200 These tumors usually grow slowly, and a reduction in tumor bulk can result in significant palliation. Ajani and associates201 treated 22 patients who had islet cell carcinoma with HAE using polyvinyl alcohol particles (Ivalon) and gelatin sponge particles (Gelfoam). The median survival in this study was 33 months from initiation of embolization (range, 1 month to 72 months); 12 patients had a partial response associated with subjective improvement and decrease in hormone levels. Other studies using HAE are listed in Table 59-24; all demonstrate an improvement in symptoms and a decrease in hormone levels. Gelfoam (size 1–2 mm), one of the agents used for embolization, does not lead to peripheral vascular occlusion and has an inconsistent duration of occlusion (due to absorption), whereas Ivalon (size 150–500 µm), a smaller particle, allows for more peripheral occlusion and is not absorbable, allowing for a more persistent arterial occlusion.201–203 A collagen particle (Angiostat; size 20–250 µm) and a biodegradable albumin microsphere (Spherex; size 15–40 mm) are currently being tested, especially in combination with chemotherapy. Interference with hepatic blood flow can exacerbate the underlying liver disease and can be dangerous in patients with portal venous thrombosis, which is present more often in patients with primary liver tumors than in those with metastatic disease. In a study by Carr and coworkers,204 four patients with HCC had reversal of tumor-induced portal vein thrombus. The researchers obtained objective responses in 22 of 35 patients (63%) using Spherex (biodegradable) starch microspheres, doxorubicin, and cisplatin. There is clear evidence that arterial embolization causes antitumor effects, but some randomized studies have not shown increased survival.205

Liver Metastases • CHAPTER 59

Table 59-24 Neuroendocrine Tumors Investigators

Agent

No. of Patients

Biochemical or Symptomatic Response (%)

Tumor Response (%)

Hepatic artery ligation Martin et al206 Moertel207

8

76

10

70

6

50

17 —

Melia et al208 Hepatic artery embolization Carrasco et al202

Gelfoam

23

87

Ajani et al201

Ivalon + Gelfoam

22

60

Marlink et al203

Gelfoam

10

100

90

Maton et al385

Lyodura

13

76

—

Complications of Embolization The complications of embolization are nausea, vomiting, fever, pain, and changes in liver function tests (Table 59-25). Problems less commonly encountered include the following: injury of the gallbladder by retrograde flow through the cystic artery, ischemic necrosis of the bowel by embolization of one of the vessels to the intestinal tract, pancreatic infarction and pancreatitis by embolization of one of the pancreatic vessels, and dyspnea by embolization of the lungs.201–203,206–208 In neuroendocrine tumors responding to HAE, rapid cell death can result in tumor lysis syndrome with symptomatic hyperuricemia, leading to uric acid nephropathy and oliguria. Vigorous hydration and prophylactic allopurinol might prevent this problem. In carcinoid tumors, HAE could cause a life-threatening carcinoid crisis from

Table 59-25 Complications and Management of Hepatic Arterial Embolization COMPLICATION Pain Fever Nausea and vomiting ↑WBC ↑LDH ↑SGOT Cholecystitis Hepatic gas formation and abscess Renal insufficiency Ileus

PRE-EMBOLIZATION Hydration Allopurinol Somatostatin (only in neuroendocrine carcinomas)* Analgesics just before procedure

POSTEMBOLIZATION Analgesics Follow WBC, LDH, SGOT, creatinine Treat by appropriate measures nausea, fever, abscess, infection, ileus LDH, lactate dehydrogenase; SGOT, serum glutamic-oxaloacetic transaminase; WBC, white blood cell count. *150–250 mg SC, every 6–8 hrs before procedure.

the rapid release of hormones from tumor cells. Somatostatin analogs may be given, either before the procedure or if a carcinoid crisis should occur.209 To avoid some of the serious complications of HAE, patients with cirrhosis, portal vein occlusion, and biliary tract obstruction are usually excluded.

CHEMOEMBOLIZATION An extension of the work with embolization is chemoembolization. This process involves a local entrapment of drug in the embolization agent and provides a prolonged exposure of the tumor to the drug locally with less systemic drug circulation. A nonrandomized study by Daniels and colleagues210 suggested that the addition of chemotherapy to the embolic agent (angiostat) produced an increase in response rate over the embolic agent alone. In a study by Venook and associates,211 51 patients with unresectable HCC were treated with Gelfoam and a mixture of three drugs—doxorubicin, mitomycin C, and cisplatin—given via a percutaneous hepatic artery catheter. Twelve (24%) had a partial response, and tumor liquefaction was noted in 70% of patients on CT, with a more than 50% reduction in α-fetoprotein in 68% of patients. Using this technique and the same drugs, these investigators also treated liver metastases from neuroendocrine tumors. In 12 patients with a median liver involvement of 60%, 33% had a partial response with a reduction in hormone levels.160 Median survival from treatment initiation was 7 months (range 3 months to 3 years). Lipiodol has been found to remain selectively in the primary and secondary liver cancers when injected into the hepatic artery, allowing visualization of tumors as small as 4 mm.161 Thus, Lipiodol can be used to deliver either chemotherapy or local radiation by combining with an agent such as iodine-131 (131I).212 Chemoembolization is rarely used for CRC, because median survivals are usually not increased; average survival is approximately 9 months.213 Adding systemic therapy to hepatic chemoembolization could improve results. In one trial using regional cisplatin in a polyvinyl alcohol suspension with systemic 5-FU, the partial response rate was 40%, and median survival was 19.3 months.214 Another form of chemoembolization is to enclose the chemotherapeutic agents in a microsphere.215 Degradable starch microspheres injected intra-arterially are trapped in an extracapillary network formed in liver metastases.215,216 Drug dissolved in the microsphere suspension is retained in the blood vessels of the target organ as long as the blood flow is blocked and then gradually releases the chemotherapeutic agents, resulting in a longer duration of tumor exposure to the drug. The most appropriate agents for microspheres would be those that, like mitomycin C, are preferentially toxic to cells under hypoxic conditions. Another useful drug to use with microspheres is doxorubicin. In a rabbit study, the mean tumor drug

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level was significantly higher when this drug was used with the microsphere compared with doxorubicin alone, whereas hepatic uptake of the drug by normal tissue was similar in the two groups.217 Monoclonal antibodies can also be attached to the microspheres.218 Radioembolization attempts have been made using glass microspheres containing 90Y, a β-emitter with tissue penetrance of 2.5 mm. Andrews and coworkers218 reported 5 of 23 responses with yttrium embolization. A study of 131I- labeled lipiodol administered to 20 patients (15 HCC, 5 metastatic) produced an α-fetoprotein drop in 11 of 12 patients and a response in 9.219 The response rates for radioembolization of metastatic tumors are far less than those reported for treatment of primary HCC, perhaps because most metastatic tumors except for neuroendocrine primaries are far less vascular than HCC. Arterial embolization is commonly used for HCC. Responses and increase in time to tumor progression have been documented.205,220–222 Meta-analysis showed an increase in survival with chemoembolization compared with conservative management.223

CRYOSURGERY Cryosurgery is an in situ destruction of tissue using subzero temperatures. The rapid freeze/thaw of tissues results in cellular damage and death. One advantage of cryosurgery is the ability to use local treatment without sacrificing normal tissue. Among the difficulties with this technique are defining the full extent of the tumor and the inability to monitor the amount of freezing, and thus the possibility of overtreatment of surrounding vulnerable normal tissue. Two technical developments have improved the use of cryosurgery: 1. Cryoprobes cooled by liquid nitrogen allow more precise freezing, even within the liver. 2. Intraoperative ultrasound allow precise placement of the cryoprobe and more accurate monitoring of the freezing process. Cryosurgery has been used intraoperatively but also can be used percutaneously. In a series of 32 patients with liver tumors (24 with CRC), 28% remained free of disease for 5 to 60 months.224 In another series using intraoperative cryosurgery on 18 patients who had metastatic CRC with 1 to 12 lesions, Onik and coworkers225 reported that 4 patients had complete remission with a median survival of 28 months, whereas 14 patients were considered inadequately treated and had a 21-month median survival. Weaver and colleagues226 treated 47 patients with cryosurgery with occasional operative resection. The number of metastases ranged from 1 to 12. The 2-year survival was 62%. Morris and Ross,227 reporting on 67 patients, noted that 75% of patients undergoing cryosurgery had an increase in CEA by 6 months later. Occasionally, the surgeon feels that all disease has been destroyed but, as seen by this study’s PET results after cryosurgery, tumor can still be present even though the surgeon feels that no disease has been left behind. The 2-year survival after cryosurgery varies from 72% to 12%. The Boston series reported the highest survival, which might reflect the type of patient being selected for cryosurgery (i.e, lesser extent of disease and a smaller number of metastases).228 Adam and associates229 reported a 2-year survival rate of 50% for patients with colorectal metastases as compared with 67% for patients with HCC. They reported a local recurrence rate of 44% for the colorectal patients. Because local recurrence is high, the use of HAI after cryosurgery could be useful. One small, nonrandomized study doubled survival with the use of HAI after cryosurgery.230 Other trials are now evaluating the use of HAI with or without systemic therapy. In a series of 185 nonrandomized patients, 71 received adjuvant CPT-11 and/or HAI of FUDR after cryosurgery. Two-year survival was 75% for patients receiving postcryosurgery therapy as compared with 35% if no adjuvant therapy was given.231 Cryoablation can also be used after hepatic resection with close margins or to remove central lesions.

Cryoprobes can be used as a handle to assist in segmental resections. An ice ball is produced with 1-cm margins around the tumor; then the probe is used for traction so that a segmental resection can be performed.232 The addition of cryosurgery to conventional surgical procedures was evaluated by Seifert and coworkers118 in a randomized study. Those receiving surgical procedures plus cryosurgery had similar survival but an increase in liver recurrence for the cryo group. Cryosurgery does involve some technical issues, including the following: • Adequate hydration before surgery, because myoglobinuria and tumor lysis can occur. • Attention to bile ducts, because biliary fistula can occur. • Two freeze/thaw cycles are preferred. • The probe should not be pulled or twisted vigorously, because that could cause cracking.233 Complications include hepatic cracking secondary to the thermal stresses that occur during rapid freezing; these are usually associated with hemorrhage, which could require packing. Other complications include biliary fistula requiring percutaneous drainage (which occurred in one patient) and myoglobinuria, resulting in acute tubular necrosis. Published data do not support the use of cryosurgery in patients with resectable disease outside of a clinical trial.234

RADIOFREQUENCY ABLATION AND MICROWAVE COAGULATION Just as tumors can be destroyed by cold, they can also be destroyed by heat. Techniques such as RFA and microwave coagulation (MC) have been used to destroy tumors. RFA involves placing within the tumor a small electrode, which is used to deliver energy to the tissue. The radiofrequency current generates ionic agitation, which is converted into frictional heat and results in breakdown of proteins and cellular membranes. The larger tumors can be destroyed by cryoablation. For RFA, tumors must be less than 4 or 5 cm in size. During the ablation, a hyperechoic area is formed around the tip of the needle, which corresponds to the area treated. It is sometimes difficult to evaluate whether all tumors have been treated. One of the advantages of RFA as opposed to cryoablation is that it can be performed percutaneously, because the probes are 10 mm in length. Solbiati and colleagues235 treated 109 patients with colorectal metastases. He found a local control of 70%. Recurrence was significantly more frequent among patients with lesions larger than 3 cm. New metastases developed in 50% of patients, and survival rates were 67% and 33% at 2 and 3 years, respectively.235 Bilchik and associates236 proposed an algorithm for unresectable hepatic neoplasms, using cryosurgery for larger lesions and radiofrequency for tumors smaller than 3 cm, because local recurrences occurred in 38% of those receiving RFA and only in 17% of patients receiving cryosurgery. Among the most useful situations in which to use RFA is for patients with HCC who also have cirrhosis. Curley and coworkers237 presented a series of 110 patients with cirrhosis who received RFA for HCC, with no recurrences occurring in 50%. Cancer cells could be more sensitive than normal cells to heat due to the decreased vasodilation capacity of the neurovascular bed.238 MC was initially developed for coagulation. When microwaves are applied to living tissue, they act mainly on the watery component. Using a probe to deliver 80-watt output for a 30-sec duration creates a column of coagulated area of 10 mm. In 19 patients with HCC, 28 of 31 nodules underwent complete tumor ablation. Ten of the 19 patients are still free of disease (follow-up, 14–64 months). Advocates of this therapy suggest that MC does not have inhomogeneous distribution within the tumor as seen with percutaneous ethanol injection therapy (PEIT).239 MC is useful only in very small tumors (5 cm) was found to be a significant predictor of survival.244 Other studies have reported size as an important predictor of recurrence.245 At M.D. Anderson Cancer Center 348 patients with liver metastases from CRC were treated for cure: 190 had resection only, 101 had RFA and resection, and 57 had RFA alone. Recurrences were lowest with resection: 52% versus 64% for RFA and resection, and 84% for the RFA alone. Liver-only recurrence after RFA was 44%. Four-year survival was 65% for resection, 36% for resection and RFA, and 22% for RFA alone.91 A multivariate analysis of these patients found the type of procedure (i.e., resection, RFA and resection, or RFA alone) influenced survival, with the RFA-alone group having the lowest survival. Of course, RFA was usually a component of therapy when resection was not possible, especially in cases where the anatomic distribution of tumors made complete resection impossible. Therefore, this is not a true comparison of RFA versus resection. However, 3-year survival for patients with one metastasis treated by resection or RFA was 80% versus 40%, respectively, suggesting RFA cannot replace resection. The combination of RFA and HAI may be useful. In an M.D. Anderson study on 50 patients, 32% remained tumor-free at a 20month median follow-up. Recurrence at the site of RFA was seen in 10% and new liver metastases in 30%.246 Kainuma and coworkers treated nine patients with bilobar disease with RFA and regional chemotherapy with 5-FU, doxorubicin, and cisplatin.247 The local recurrence rate was 55%, and 2-year survival was 39%. Martin and colleagues treated 21 patients with RFA and HAI FUDR. With a median followup time of 24 months, the median survival is 30 months.248 RFA has been used in other tumor types. In a Chinese series on 240 patients with HCC and 44 patients with liver metastases treated with RFA, patients with liver metastases had a higher extrahepatic recurrence (P = 0.019) and shorter DFS (P = 0.007). Patients with multiple liver metastases had a higher local and extrahepatic recurrence249 in neuroendocrine tumors. RFA can relieve symptoms in 95% of patients.250,251 Livraghi and associates reported on 24 breast cancer patients whose liver lesions were treated with RFA, and 10 patients are still alive and free of disease with a median follow-up time of 10 months.252

Toxicity from RFA is clearly outlined in a series on 312 patients that included liver abscesses (7), portal vein thrombosis (3), pleural effusion (5), colon perforation (1), and renal insufficiency (1).253 In conclusion, RFA may have a role during surgical resection when one side of the liver is resected and small disease exists on the other side that cannot be resected. In patients who have undergone a resection and develop a small recurrence, percutaneous or laparoscopic RFA can be used if the lesion can be reached easily and is not close to large vessels. The presence of blood vessels near the tumors causes conduction of thermal energy away from the tumor and spares killing the tumor near the blood vessel.254 Whether these techniques are more useful than chemotherapy, or should be used in combination with chemotherapy, are not known; the EORTC are exploring the use of RFA and chemotherapy versus chemotherapy alone.

PERCUTANEOUS ETHANOL INJECTION PEIT was first performed in 1983 in Japan. Ultrasound guidance is used to place up to 30 mL of absolute ethanol into the lesion.255 In patients with primary hepatoma, this treatment produced 5-year survivals of 43% for small lesions. Suzuki and coworkers256 assigned 42 patients with HCC less than 3 cm to three groups—(1) chemolipiodolization, (2) chemolipiodolization followed by gelatin sponge transcatheter embolization (TAE) or (3) PEIT—and demonstrated a decrease in local recurrence with PEIT. Local recurrences at 1 year were 61%, 29%, and 20% for groups 1, 2, and 3, respectively. Shiina and colleagues257 reported a 10-year survival of 66% using PEIT on single hepatocellular lesions smaller than 2 cm. In pooled data on 11,000 patients with HCC from Japan, 3-year survival for surgical resection, PEIT, or embolization was 58%, 53%, and 20%, respectively. Other researchers, however, report a higher recurrence rate after PEIT as compared with surgical resection. The size of the lesion also affects outcome. In an Italian study on 26 patients with metastatic disease, 13 of 15 patients with lesions smaller than 2 cm had responses, whereas among the 6 patients with lesions larger than 4 cm, no response was seen.257 Yamamoto and associates258 randomized 100 patients to TAE versus TAE + PEIT. The 3-year survival was 20% for the TAE group and 50% for the TAE + PEIT group, respectively (P = 0.05). This technique was also useful for treating small neuroendocrine tumors, possibly because they are highly hypervascular. At present, the technique needs further study to determine where it fits into the therapeutic armamentarium and whether it will increase survival for patients with metastatic liver tumors. Pending further study, it seems that PEIT and cryosurgery could be applicable to patients with small metastatic lesions who cannot undergo surgical resection. Whether these techniques will be more beneficial than regional hepatic arterial therapy is not clear, because they only treat visible disease and do not deal with possible small metastases that are not visible.

ISOLATION PERFUSION To administer high drug concentrations locally, the liver can be isolated by clamping the hepatic arteries, vena cava, and portal vein and then placing a catheter in the hepatic artery to perfuse the liver. A catheter in the retrohepatic vena cava drains the liver, and extracorporeal filters allow removal of chemotherapeutic agents and simplify the technique of isolated perfusion. With a double-balloon inferior vena cava catheter, doses as high as 5,000 mg/m2 of 5-FU and 120 mg/m2 of doxorubicin have been administered. An initial trial using 30 mg/m2 of mitomycin C produced venoocclusive disease in four of the nine patients.259 With the use of melphalan (L-PAM), toxicity was decreased, and with doses of 30 mg/kg, complete responses were seen in 2 of 9 patients.260 Hyperthermic isolation perfusion of tumor necrosis factor and melphalan is being investigated. Melphalan (1.5 mg/kg) and tumor necrosis factor (1 mg) over 60 min of hyperthermic infusion produced a 75% response rate.261

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GENE THERAPY Tumors largely restricted to the liver (primary or metastatic) can potentially be treated by gene therapy. The gene transfer agents can be injected locally or via the hepatic artery. One trial involves the use of an adenovirus vector carrying the wild-type p53 gene. Alteration in p53 function is present in more than half of all malignancies, and reexpression of wild-type p53 can result in apoptosis and in tumor shrinkage in rodents. A phase I study of recombinant adenovirus encoding wild-type p53 administered via the hepatic artery produced no responses in 19 patients.262 Transgene expression in tumor tissue was seen in patients receiving the highest dose levels. Other gene therapies involve the use of prodrug genes to convert innocuous drugs into active chemotherapeutic agents (cytosine deaminase converts 5-fluorocytosine to 5-FU).263 To deliver directed immunotherapy, Rubin and coworkers264 injected HLA-B7 gene on a liposomal vector into liver tumors. No responses were seen in 15 patients in the phase I study, but plasmid DNA was detected in 14 of 15 patients. Another concept being evaluated is to have cells express a gene such as thymidine kinase and then kill the cells by use of a ganciclovir, which is converted by thymidine kinase to an active metabolite.263

RADIATION THERAPY In this section, the evolving role of radiation therapy for liver metastases will be reviewed. The first experience in using radiation therapy for liver metastases was with external-beam whole-liver radiotherapy (with and without systemic or regional chemotherapy), in which the doses that could be delivered safely were not high enough to eradicate metastases. More recently, technologic advances in radiation treatment planning allow high-dose radiation therapy to be delivered conformally around liver metastases safely, with the potential for eradication of disease and cure, in appropriately selected patients. Other types of radiation have also been used to treat liver metastases including brachytherapy and hepatic arterial delivery of 90Y-tagged microspheres. Here, the experience following whole-liver irradiation, conformal radiation therapy, brachytherapy, and hepatic arterial 90Y for treatment of liver metastases will be reviewed.

Whole-Liver Irradiation The approach of using whole-liver irradiation for metastases is limited by the low tolerance of the whole liver to irradiation, with doses

required to be less than 30 Gy over 3 weeks to avoid liver toxicity. Several clinical trials of whole-liver irradiation for liver metastases have established the safe whole-liver doses that can be delivered in a variety of fractionations.265,266 The duration of response and survival rates tend to be short267–269 (Table 59-26). In contrast, low-dose whole-liver irradiation can produce palliation of painful liver metastases in the majority of cases.266,269,270 In an attempt to improve on the poor outcomes following wholeliver irradiation alone, whole-liver irradiation has been combined with systemic or regional chemotherapy. The most widely used drugs in this effort have been the fluoropyrimidines because of their activity against CRC and their radiation-sensitizing properties.271–273 The results of some of these trials are summarized in Table 59-27. In general, the response rates and survival rates following combinedmodality therapy seem to be superior to those obtained following whole-liver irradiation alone. Selection bias may be at least partially responsible for this effect. A recent study demonstrated that wholeliver radiation (20 Gy in 10 fractions) does not improve the efficacy of 5-FU for patients who have diffuse liver metastases.274

Conformal Radiation Therapy Technical advances in liver cancer imaging, radiation planning, methods to account for breathing motion during radiation delivery and image guidance at the time of radiation delivery (Figs. 59-13 and 59-14) have made it possible to deliver high-dose radiation safely to focal liver metastases, while sparing irradiation of the uninvolved liver, using a variety of radiation fractionation schedules.275 In most studies, higher doses of radiation have been associated with more durable local control rates than lower doses, regardless of the fractionation schedule.276–278 Since the late 1980s at the University of Michigan, a series of phase I/II trials for patients with unresectable intrahepatic cancer have investigated dose-escalated conformal radiation therapy delivered concurrently with hepatic arterial chemotherapy (predominantly floxuridine [0.2 mg/kg/day]). In one of the first studies, the objective response rate of 22 patients with unresectable CRC liver metastases, treated with as much as 72.6 Gy at 1.5 Gy twice daily, was 50% (2 complete remission, 9 partial response, 11 stable disease),279 with a median survival of 20 months. Similar results were obtained in subsequent studies,280 including the most recent study in which the prescription dose was individualized based on the volume of liver irradiated and risk of toxicity, allowing higher doses (as much as

Table 59-26 Results of Treatment of Metastatic Cancer to the Liver Treated with Whole-Liver Irradiation Alone No. of Patients

Response (% Total)

Median Survival (mo)

Hepatitis† Toxicity

21–30/7–19

103

55‡

3

0

48% Colorectal

21/7 ( ± misonidazole)

187

80‡/7§

4

0

Reference

Histology*

Dose (Gy/No. of Fractions)

Borgelt et al266

38% Colorectal

RTOG 76-05 Leibel et al269 RTOG 80-03 56% GI

≈20–37.5/8

36

72‡

ND

1

268

Prasad et al

33% Colorectal

≈25/16

27

70‡

4

0

Russell et al265

60% Colorectal

27/15

53

4

0

30/20

69

4

0

33/22

51

4

2

Phillips et al267

RTOG 84-05

GI, gastroimestinal; ND, not determined. *Predominant histology. † Number of patients with ≥ grade 3 radiation hepatitis. ‡ Subjective decrease in pain. § Objective (CT scan).

ND

Liver Metastases • CHAPTER 59

Table 59-27 Results of Treatment of Metastatic Cancer to the Liver Treated with Whole-Liver Irradiation with Chemotherapy Reference

Dose (Gy/Fractions)

Chemo

Ajlouni et al309

21–30/14–20

FUDR

IAH

10

30†

9

0

Byfield et al310

15–30/12‡

FUDR

IAH

28

ND

9

1

13.5–21/5–7

5-FU, Dox

IAH

22

48†

>3

1

Herbsman et al

25–30/15

FUDR

IAH

13

70§

16

0

Lawrence et al313

33/22

FUDR

IAH

19

39†

7

0

13

ND

ND

3

§

0

Friedman et al311 312

No. of Patients

Route

36/24 314

Response (% Total)

Median Survival (mo)

Hepatic* Toxicity

Lokich et al

19.5–30/10–12

5-FU or FUDR

IAH

12

63

ND

McCracken et al206

19.5/13

5-FU, Mito C

IAH

13

(adjuvant)

ND

1

Raju et al315

21/1.5

FUDR or FU

IAH or IV¶

12

83§

14

0

≈22.5–32.3/15

5-FU

IV

27

83§

6

0

Sherman et al

15–30/7–10

5-FU or Pro ± HU ± Cy ± 5-FU

§

4

0

Webber et al318

25/10

FUDR

Rotman et al316 317

319

#

IV

50

90

IAH

25

72§

12

0

†

Wiley et al

25.5/17

5-FU

IAH

19

37

6

0

Volberding et al320

21/7

5-FU, Dox, MTX

IAH

27

33†

7

0

Cy, cyclophosphamide; Dox, doxorubicin; 5-FU, 5-fluorouracil; FUDR-fluorodeoxyuridine; HU, hydroxyurea; IAH, intra-arteria hepatic infusion; IV, intravenous infusion; Mito C, mitomycin C; MTX, methotrexate; ND, not determined; Pro, procarbazine. *Number of patients with ≥ grade 3 radiation hepatitis. † Objective response (CT or radionuclide scan documenting 50% decrease in bidimensional proudct). ‡ Split-course therapy. § Subjective response (e.g., decrease in pain). ¶ FUDR (IAH) in four patients, 5-FU IV in eight patients. # Includes 19 patients who received RT only.

Figure 59-13 • Planning CT showing multiple liver metastases (in blue), treated with multiple radiation beams. Insert is a cutout of a verification CT obtained in the radiation treatment position immediately before therapy, registered to the planning CT scan.

90 Gy at 1.5 Gy twice daily) to be delivered safely to more patients. The median survival of 47 patients with liver metastases (median diameter ∼10 cm) treated on this study was 17.2 months.277 Stereotactic body radiation therapy (SBRT), referring to a limited number of high-dose fractions delivered very conformally to targets, using biologic doses of radiation higher than those used in standard fractionation, has also been used to treat liver metastases.278,281–287 Safety of 1- to 10-fraction SBRT has been described in several retrospective series and more recently confirmed in prospective dose escalation studies (Table 59-28). Blomgren and colleagues from Sweden first reported a response rate of 43% for 14 liver metastases treated with 20 to 45 Gy in one to four fractions,262 with a prolonged time to maximal response (e.g., maximal response at 16 months for a 13cm liver metastases). No liver toxicity was seen in patients with metastases, but hemorrhagic gastritis was seen in one patient. In an update in 1998, the local control rate was 95% with a mean survival of 17.8 months for 21 liver metastases.288 SBRT (20 Gy × 2 or 15 Gy × 3) has also been used safely in patients with recurrent liver metastases following hepatic resection for CRC metastases, with no serious toxicity and local control 13 to 101 months following surgery.289 A prospective study of escalated single-fraction SBRT (14 Gy to 26 Gy) did not find a maximal tolerated dose in 60 liver tumors (56 metastases) with a median tumor size of 10 mL (1–132 mL) and found an actuarial local control rate of 81% at 18 months following SBRT.281,290 SBRT delivered in three fractions (37.5 Gy total) has also been reported to be safe in small liver metastases, with 2-year local control and survival rates of 61% and 41%, respectively.282,291 A North American prospective study confirmed the safety of threefraction SBRT in 18 patients with 25 tumors of maximal diameter 6 cm.278 A Canadian prospective study has shown the feasibility of delivering six-fraction SBRT using an individualized dose allocation approach as first described by the Michigan group, for liver cancers ranging from 3 to 3,000 mL.292

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Target volume Treatment dose: 57Gy, 6 fractions

Figure 59-14 • Conformal radiation dose distribution, with the treatment isodose 57 Gy in six fractions, conforming to the target volume (pink).

More recently, outcomes following SBRT for 174 liver metastases from colorectal, pancreatic, breast, and lung cancer in 69 patients were reported.285 The median dose delivered was 48 Gy (range 30– 55 Gy) at 2 to 6 Gy per fraction. The local control was 76% and 57% at 10 and 20 months, respectively, with an overall medial survival of 14.5 months. No grade 3 toxicity was reported. Based on this experience, 10-fraction SBRT is being studied in a Radiation Therapy Oncology Group study that is now open.

Brachytherapy High-dose radiation can also be delivered to focal liver metastases, with maximal sparing of dose to uninvolved liver, using interstitial brachytherapy, in which radiation is delivered from radioactive sources placed within or near the tumor.293–297 High-dose-rate iridium-192 afterloaded to applicators placed at the time of lapa-

rotomy was used to deliver 20 to 30 Gy in 2 to 13 settings, with an actuarial local control rate of 26% at 26 months and two complete responses seen in lesions subsequently biopsied.296 Iodine-125 seed implants, which deliver low-dose-rate irradiation (less than ∼0.15 Gy/ hr) over several months also have been used to treat liver metastases, with 10 of 11 tumors controlled at 1 year.295 More recently, CTguided placement of iridium-192 for liver metastases has been shown to be feasible in a phase II study294 of 20 patients with liver cancers (19 metastases, 1 cholangiocarcinoma) unsuitable for thermal ablation. The mean tumor diameter was 7.7 cm (5.5–10.8 cm) for peripheral lesions and 3.6 cm (2.2–4.9 cm) for hilar cancers. The dose delivered ranged from 12 to 25 Gy. Two serious complications were observed. One patient had an intrahepatic hemorrhage on removal of the brachytherapy sources. Another patient developed obstructive jaundice 14 days after brachytherapy and subsequent elevated bilirubin and liver failure 9 months later, perhaps associated

Table 59-28 Selected Results of Liver Metastases Treated with Conformal or Stereotactic Radiation Therapy TUMOR TYPE

No. of Patients

Mets

Ben-Josef et al277

47

47+

281

Blomgren et al

23

14

Herfarth et al290

37

60

Reference

Other 9

Wulf et al291

23

23

Schefter et al278

18

25

1 11

286

Mendez Romero et al

25

34

Katz et al285

69

174

NR, not reported.

LOCAL CONTROL

Dose, No. of Fractions

%

50%) should make one more seriously entertain the idea of a carcinomatous pleural effusion, and greater than 85% lymphocytes should make one entertain the diagnosis of lymphoma, sarcoidosis, chylothorax, rheumatoid pleurisy, or yellow nail syndrome.16,37 An increase in pleural fluid eosinophilia (>10% of nucleated cells) might be associated with benign disease (hemo- or pneumothorax), but also can be associated with all types of malignancy.38 The presence of mesothelial cells is not helpful in terms of diagnosis.37,39 Pleural fluid cytology is the simplest and most definitive method of diagnosing a malignant effusion. The sensitivity depends on the type of malignancy, extent of disease, and experience of the cytopathologist. Fluids should be concentrated first for optimal detection of malignancy.40 In general, the sensitivity is on the order of 62% to 90% and, as the gold standard, pleural fluid cytology is virtually 100% specific in the hands of an expert cytopathologist.41 If an effusion demonstrates carcinoma and breast cancer is a diagnostic possibility, the cytologic specimen can be stained for estrogen and progesterone receptors as a means of both diagnosis and selection of potential treatment. In short, the presence of an abnormal cell population should prompt a further workup for the aforementioned causes, including malignancy. Other tests have been evaluated in numerous studies as a means to refine the diagnosis of pleural effusions. For example, an elevated adenosine deaminase concentration has a high association with tuberculosis.42 Elevated lipids (triglycerides) can be associated with a chylothorax and obstruction of the thoracic duct by any number of means, including malignancies.43 In clinical experience, this and other tests (e.g., creatine kinase, LDH isoenzyme analysis, β2microglobulin, albumin, ferritin, lysozyme, and others) are rarely used, are rarely associated with malignancy, and are therefore discussed elsewhere.23 Other tests including tumor markers (carcinoembryonic antigen [CEA], CA 19-9) have also been evaluated for use in determining the etiology of effusions but have not been found to be sensitive or specific.

Evaluation of a Suspected Malignant Effusion Although most malignant effusions occur among patients with known cancers, they can be the first indication of the presence of malignancy in as many as 30% of patients.44 In some patients an effusion often can be the only site of a potential malignancy after a thorough evaluation. Once a patient has been diagnosed with an exudative effusion, a malignant cause must be high on the list of differential diagnoses. A thorough history and complete physical examination must be performed, with careful attention to any potential causes or risk factors of malignancy. Once this evaluation takes place, the physician must gather some definitive evidence to institute appropriate evaluation. Frequently, such evidence involves consultation between the patient’s primary physician and either a

pulmonologist or an oncologist. Many clues about the etiology of the effusion are obtained with the performance of further evaluations, such as chest radiographs, CT scans, or mammograms. Alternatively, cytologic evaluation of an exudative effusion can reveal the presence and type of malignancy directly. It is important to keep in mind the most common malignancies that cause effusions. Not surprisingly, a majority of these are caused by lung cancer. Breast cancers and lymphomas also cause a significant number of these, with approximately one-third of all malignant pleural effusions being caused by other types of malignancies (see Table 60-2).45 Appropriate evaluations should be performed as indicated (e.g., careful breast examination and mammogram in women with effusions; particular attention for lung cancer among patients with a history of smoking). It should be noted that there is a recently established entity, primary effusion lymphoma. This is found in patients positive for the human immunodeficiency virus; it is associated with human herpesvirus 8 infection and has a pathogenesis similar to Kaposi’s sarcoma.46 This syndrome also can include pericardial effusions and ascites. Patients with mesothelioma often have a history of asbestos exposure and show evidence of both effusion and pleural thickening on CT scan. These patients can be considered for CT-guided biopsy of the appropriate areas, which is often effective in making a diagnosis.47 A small percentage will be diagnosed with carcinoma of unknown primary, whose management, usually via chemotherapy, is also discussed in Chapter 98. Even after an extensive evaluation, results can be nondiagnostic, and the patient must undergo further evaluation to determine the etiology of the effusion because no other cause has been identified although malignancy is still suspected. These further evaluations will now be discussed.

Closed Pleural Biopsy These refer to blind, percutaneous biopsies of the parietal pleura using a special needle, such as a Cope’s needle. Unfortunately, among patients with a cytology-negative malignant effusion, the yield of this procedure is only about 7%.48 Novel approaches to this procedure, such as the use of brushings or a Tru-cut needle, might ultimately prove to be better diagnostically, but the procedure is currently rarely used in such situations because of its poor yield.49,50

Thoracoscopy Video-assisted thoracoscopic surgery (VATS) is the most commonly used procedure for thoracoscopy in the United States and is usually performed by thoracic surgeons. This procedure, using several ports and trochars, requires general anesthesia with single-lung ventilation and many single-use disposable instruments.51 Although VATS is well tolerated, it does have some risks and carries with it a significant expense, due to general anesthesia and the requirement for expensive instruments. On occasion, it requires conversion to an open procedure if there are significant adhesions or if there are undue risks noted with the insertion of a thoracoscope.45 Single-lung ventilation is also required and might be difficult for patients with significant lung disease. With these techniques, fewer than 10% of malignant pleural effusions go undiagnosed, and thus thoracoscopy of some sort has become part of the evaluation algorithm when necessary if other methods fail.52 Interestingly, a recent technique uses a semi-rigid pleuroscope, which is similar to bronchoscopes currently in use by pulmonologists but easier to use. These can be used to drain and pleurodese effusions and might ultimately decrease the need for VATS in diagnosing potentially malignant effusions if comparison studies demonstrate its effectiveness.53

Treatment For most malignancies, the existence of a malignant effusion places the patient into a noncurable, advanced staging category, but one

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that is often treatable nonetheless. In light of this a palliative approach is often the mainstay of therapy, with several important exceptions that will be addressed in the discussion that follows. For patients with relatively small effusions that do not cause a high degree of dyspnea or impairment of functional status, consideration of systemic therapy of the malignancy, usually chemotherapy, is indicated. If, on the other hand, dyspnea is a primary concern, immediate management of the effusion is necessary. Unfortunately, for many patients with advanced disease, chemotherapy might not be able to provide rapid enough resolution of dyspnea or other symptoms, and thus one must use mechanical means of reducing the effusion. First and foremost, the clinician should consider the individual patient’s situation, with particular attention to overall prognosis, prior therapies (if any), age, and performance status. The physician must also weigh the likelihood of progression of other systemic disease during the time required for resolution of an effusion. For patients with large effusions who are in the terminal stage of their disease, supplemental oxygen with hospice referral might be the only appropriate intervention.

Systemic Therapy in Specific Malignancies Many advanced solid tumors do not respond well to chemotherapy, so primary management of the effusion is often required. There are notable exceptions. Small cell lung cancer is unlikely to be curable, but effusions from this cause respond well to chemotherapy.54,55 Testicular and other germ cell neoplasms are not only responsive but also very curable, even at an advanced state, and thus early systemic treatment should be used if at all possible.56 Even patients with advanced germ cell tumors have good responses and should be considered for aggressive systemic therapy. Breast cancer might respond well to hormonal therapy or chemotherapy, particularly among patients who have not received prior systemic therapy. Mesothelioma is a unique tumor that arises directly from pleural or peritoneal surfaces. Effusions are often associated with the tumor and are not included in the staging system. Patients amenable for aggressive local therapy, even with an effusion, should be considered for such therapy. Effusions from hematologic malignancies, such as lymphoma, are often highly responsive to systemic chemotherapy. Treatment of effusions related to hematologic malignancies are generally focused on treatment of the disease as a whole rather than treatment directed at the effusion directly. Because patients with solid-organ tumors and advanced disease have relatively poor responses to chemotherapy overall, early direct management of the effusion among patients with dyspnea should be considered at the outset for patients who are unlikely to achieve rapid responses to chemotherapy.

patient’s symptoms before systemic chemotherapy is given. Similarly, it might also allow for palliation of symptoms for those patients with far advanced disease. Therapeutic thoracenteses are performed similarly to diagnostic procedures. Although a pneumothorax usually occurs when air leaks into the pleural space through the needle, it can also occur if the visceral pleura is lacerated with the needle. The latter complication may be avoided if a plastic catheter is threaded through the needle, directed inferiorly, and the needle is then removed; this procedure minimizes the chances that a pneumothorax will occur via perforation when the visceral and parietal surfaces become opposed after fluid removal. If a pneumothorax occurs the leak usually seals itself quickly, but closure can be hastened by having the patient lie on the affected side, which decreases the pressure gradient between the alveoli and the pleural space.58 The volume of pleural fluid that can be removed safely is unknown. One study by Light and coworkers demonstrated that pleural fluid removal could be considered safe as long as pleural fluid pressure does not decrease below −20 cm H2O.28 Because most clinicians do not measure pleural pressure, current recommendations are that no more than 1.0 to 1.5 L of fluid be removed at any one time, and that amount only if there are no signs of any adverse events such as dyspnea, pleural pain, or cough, which are indications of the rapid restoration of a strong negative pleural pressure. Patients with contralateral mediastinal shift can have larger amounts of fluid removed safely, because these adverse events are unlikely to occur. Rapid reexpansion might bring on the phenomenon of reexpansion pulmonary edema, which is due to the rapid restoration of capillary permeability through unknown mechanisms; this can occur when air or fluid is removed from the pleural space.58 Patients with ipsilateral mediastinal shift are unlikely to obtain significant relief from thoracentesis, because this condition indicates a large amount of either trapped lung or mainstem bronchial occlusion.46 There have been no randomized or comparison trials examining the use of repeated thoracentesis compared with the use of other procedures. It is also difficult to predict the length of time within which an effusion might recur; some patients have rapid recurrence (within days), whereas others might have recurrence over a period of weeks. Most oncologists and pulmonologists often make an initial attempt at a therapeutic thoracentesis to allow immediate symptomatic relief and time for systemic therapy to take effect, as well as to gauge the extent to which a pleural effusion causes dyspnea. For patients whose effusions recur rapidly, more invasive procedures might be required; those whose effusions recur more slowly might be managed solely with repeat thoracentesis.

SYSTEMIC CHEMOTHERAPY AND EFFUSIONS. It should be remembered that the pharmacokinetics of drugs in the presence of effusions are poorly understood, because drug might accumulate in effusions and only slowly redistribute throughout the body. Methotrexate is a classic example of this phenomenon. Methotrexate is slowly released from all of the “third spaces,” and those of large volumes (ascites, pleural effusions, or anasarca) might dramatically prolong the terminal half-life and lead to potential increased toxicity.57 There are no direct guidelines for the use of methotrexate or other chemotherapies when large effusions exist, but the clinician should consider drainage of the effusion before the use of methotrexate, or the use of alternative therapies. Patients with pleural effusions receiving new chemotherapy agents should be monitored closely for treatment-induced toxicity.

Effusion-Targeted Therapy THERAPEUTIC THORACENTESIS. Therapeutic thoracentesis might serve as the main or sole therapy for management of an effusion in many patients. Thoracentesis, unlike other procedures, might permit rapid relief of symptoms without need of hospitalization. If systemic disease is a significant concern, thoracentesis might allow for a window of opportunity in which to gain control over a

RADIATION THERAPY. In general, radiation of the hemithorax, as a means of controlling an effusion, is contraindicated in most patients with malignant pleural effusions. This is because of the high incidence of radiation pneumonitis that is likely when a sufficient dose of radiation is given to large areas potentially involved with malignant effusions.59 Certain situations, such as lymphatic obstruction from focal areas of lymphadenopathy (which may arise in lymphoma or in lung cancer), might benefit from radiation applied to these specific areas. Radiation doses are dependent on the nature of the malignancy. CHEMICAL PLEURODESIS. Pleurodesis is intended to achieve a “symphysis between the parietal and visceral pleura, in order to prevent accumulation of either air (pneumothorax) or fluid (pleural effusion) in the pleural space,”60 and malignant pleural effusions are the largest indications for pleurodesis.60,61 The mechanism by which this symphysis occurs is poorly understood, but in general it depends on pleural irritation to cause a cycle of inflammation, activation of the pleural coagulation cascade, fibrin placement and fibroblast recruitment, and, finally, collagen deposition that ultimately results in the fusion of both pleural surfaces.60,62,63 Nevertheless, the exact

Effusions • CHAPTER 60

mechanisms and factors that influence pleural sclerosis and effect pleurodesis are not well known and require further research. Many agents have been used in the past with various success rates reported; much of this information is based on personal and anecdotal experiences. These agents include, as a minimum list • • • • • • • • • • • • • • • • • • • •

Talc Tetracycline Minocycline Doxycycline Quinacrine Mepacrine Bleomycin Mitomycin Thiotepa Nitrogen mustard 50% glucose and water Interferon-α Interferon-γ Iodopovidone Radioactive colloidal gold Autologous blood Fibrin glue Bacille Calmette-Guérin Silver nitrate Killed Corynebacterium parvum60

The exact mechanism by which these agents effect pleurodesis probably differs slightly from one agent to another, but all lead to a final common pathway that activates the pleural coagulation cascade and the appearance of a fibrin network that yields a symphysis between the two surfaces.61–63 The various agents have different properties and methods of administration.

METHOD OF PLEURODESIS. It should be recalled that patients must demonstrate that symptoms (usually dyspnea) respond to drainage of the pleural fluid, because patients with malignancy often have numerous reasons for dyspnea—pulmonary metastases, anemia, pulmonary embolus, trapped lung, or poor gas exchange— and might be unlikely to benefit from a resolution of the effusion. Thus, it is important for patients to undergo a trial of therapeutic thoracentesis initially rather than proceed directly to chest tube drainage. At the time that pleurodesis is performed, the effusion must be drained. For successful sclerosis, there should be evidence of complete lung reexpansion after initial drainage. Traditionally one gauged the optimal time for sclerosis when there was minimal pleural fluid drainage (30 Gy) is associated with delayed sexual maturation due to gonadotropin deficiency from damage to GnRH secretory neurons.23 Deficiency in other pituitary hormones is less common. Five years after treatment, a study of 251 patients who had been treated for pituitary disease with external radiotherapy described an incidence of thyroid-stimulating hormone (TSH) deficiency of 9% at 20 Gy, increasing to 52% at 42 to 45 Gy.24 A similar trend for incidence of adrenocorticotrophic hormone (ACTH) deficiency relation was seen. Hyperprolactinemia can be seen after high-dose radiotherapy (>40 Gy) and has been described in both sexes and all age groups but is most common in young women.25 Constine and associates26 described a 50% frequency of hyperprolactinemia in 32 patients who were treated with radiation for brain tumor with doses ranging from 39.6 to 70.2 Gy. Other investigators reported rates of 20% after treatment for nasopharyngeal carcinoma. Hyperprolactinemia can cause pubertal delay or arrest in children, galactorrhea and/or amenorrhea in women, and decreased libido and impotence in adult males. Radiation-induced anterior pituitary hormone deficiencies are irreversible and progressive but are treatable with appropriate hormone replacement therapy. Careful surveillance and close follow-up with an endocrinologist are warranted.

Thyroid Irradiation of the thyroid may produce hypothyroidism, Graves disease, silent thyroiditis, benign nodules, and thyroid cancers.27 Hancock and colleagues described their experience with thyroid disease among patients treated with irradiation with or without chemotherapy for Hodgkin’s disease at Stanford University.28 Of 1787 patients, 1677 received irradiation to the thyroid. At 26 years of follow-up, the actuarial risk of thyroid disease was 67%. Hypothyroidism developed in the majority of the patients (47%). The risk of Graves disease was 7 to 20 times higher than that for normal subjects. The risk of thyroid cancer was noted to be 15.6 times the expected risk for normal subjects. The association between thyroid cancer and radiation is discussed in further detail in Chapter 75. These data remind clinicians to follow thyroid function closely in patients who have been treated with upper mantle or cervical irradiation. Similar results were noted in the Childhood Cancer Survivor Study with an evaluable cohort of 1791 (959 males) Hodgkin’s disease survivors. Among patients with Hodgkin’s disease, the risk of hypothyroidism at 20 years from the time of diagnosis in those treated with 45 Gy or more was 50%.29 Total dose of irradiation received has been shown to correlate with the incidence of hypothyroidism in many studies.27–30 There is controversy regarding the effect of age at the time of irradiation, gender, and association with the prior use of lymphangiograms.27,29 Radiation-induced thyroid dysfunction is thought to be caused by damage to small thyroid vessels and to the glandular capsule. Focal and irregular follicular hyperplasia, hyalinization, and fibrosis beneath the vascular endothelium, lymphocytic infiltration, single and multiple adenomas, and thyroid carcinomas are histomorphologic features that are described in such patients.27,28 A rare complication of external neck irradiation is acute radiation thyroiditis.30 It is more commonly associated with therapeutic doses of radioiodine for thyroid diseases. Patients typically present with fever, pain in the anterior cervical region, and transient hyperthyroidism. Hyperthyroidism with a clinical picture that resembles Graves disease may be seen after neck irradiation for Hodgkin’s disease.31 The incidence is uncertain owing to the small number of cases reported. The clinical picture is characterized by diffuse thyroid enlargement, suppressed TSH, high levels of thyroid hormones, and development of thyroid autoantibodies. Ophthalmopathy, with or

without overt hyperthyroidism, may be seen and is thought to be related to autoantibodies, similar to Graves disease.32

Parathyroid Glands There are several studies that link prior head and neck irradiation and hyperparathyroidism.33,34 Cohen and colleagues35 followed a cohort of patients who were treated with radiation to the tonsils before the age of 16 years. Among the 2923 patients, 32 patients were found to have clinical hyperparathyroidism. This is a 2.5-fold to 2.9-fold increase compared with the general population in the same age group. There is a long latency period (>25 years) between exposure and onset of hyperparathyroidism. Clinical presentations vary from asymptomatic increases in serum parathormone levels and hypercalcemia to disabling metabolic bone disease or nephrolithiasis. Individuals with a history of head and neck radiation should be monitored with calcium levels periodically (every 1 to 2 years) and indefinitely.36

ROLE OF SYSTEMIC THERAPY Chemotherapy is not widely recognized to contribute to endocrine dysfunction; however, many data indicate to the contrary. Effects of systemic chemotherapy on ovarian and testicular function are discussed in Chapter 64.

Hypothalamic-Pituitary Axis In children, chemotherapeutic agents alone may disrupt growth hormone (GH) secretion even in the absence of cranial radiation. Roman and colleagues studied growth and GH secretion in 60 children who were in complete remission after treatment with chemotherapy and surgery for solid tumors.37 They observed growth hormone deficiency in 45% of those studied and found that these children were more likely to have received high doses of chemotherapy (actinomycin D), but they could find no correlation with the duration of treatment, length of follow-up, tumor type, sex, or age. Depending on the intensity of chemotherapy, significant height loss can be detected in 40% to 70% of patients at 6-year follow-up.38 Adjuvant chemotherapy can also aggravate growth failure in children with brain tumors receiving craniospinal radiation.39 Rose and colleagues reported hypothalamic dysfunction in patients with non–central nervous system tumors who received chemotherapy but did not receive cranial irradiation or traumatic brain injury.40 Of 31 identified patients, GHD was identified in 15 (48%), central hypothyroidism in 16 (52%), and pubertal abnormalities in 10 (32%) patients. GHD and hypothyroidism were coexistent in eight patients (26%). Overall, 81% (n = 25) had GHD, hypothyroidism, precocious puberty, or gonadotropin deficiency. The syndrome of inappropriate antidiuretic hormone (SIADH) secretion may result from the effects of many chemotherapeutic agents, either by potentiation of antidiuretic hormone (ADH) effect or by increased ADH secretion. The most commonly implicated agents are vinca alkaloids and cyclophosphamide. The vinca alkaloids are reported to stimulate the central release of ADH from the neurohypophyseal system,41 whereas alkylating agents enhance renal tubular sensitivity to ADH.42 Regardless of the mechanism, the result is an increase in water reabsorption by the distal tubules of the kidney, leading to volume expansion and dilutional hyponatremia. Many case reports also implicate platinum agents,43 vinorelbine,44 taxanes,45 and methotrexate.46 Clinically significant hyponatremia may occur with administration of these agents. Management requires fluid restriction and, at times, salt replacement.

Thyroid Clinically evident thyroid dysfunction is rarely associated with the use of standard chemotherapy agents. However, a growing body of

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literature points to the increased prevalence of endocrine dysfunction after bone marrow transplantation, which may be seen following high doses of chemotherapy in absence of any radiation. There are reports of thyroid dysfunction in nearly 50% of allogeneic bone marrow transplant recipients treated with busulphan and cyclophosphamide alone.47 Thyroid dysfunction may present as low T3 syndrome (free T4 normal, TSH normal, and free T3 below normal), chronic thyroiditis, and transient subclinical hyperthyroidism or hypothyroidism. Chemotherapy may potentiate radiation-induced damage to normal tissue. Among 32 patients treated for medulloblastoma in childhood, Paulino found that 18 patients developed hypothyroidism after a median time of 41 months after irradiation. Hypothyroidism was reported in 10 of the 12 patients (83%) who received 23.4 Gy plus chemotherapy (vincristine, N-[2-chloroethyl]-N ′-cyclohexyl-Nnitrosourea [CCNU], cisplatin, or cyclophosphamide) and 6 of 10 (60%) of those who received 36 Gy plus chemotherapy (vincristine, CCNU, prednisone) versus only 2 of the 10 (20%) of those who received 36 GY radiation alone.48 Aminogluthemide, now an infrequently used drug to disrupt adrenal and peripheral steroid hormone synthesis, inhibits cholesterol conversion to pregnenolone. It causes thyroid dysfunction after long-term use due to blockade of iodination of tyrosine.49 Figg and associates reported that 9 of 29 men who were treated with aminogluthemide for metastatic prostate cancer had clinical and biochemical evidence of hypothyroidism.50 Chemotherapeutic agents can also interfere with circulating thyroid hormones, thereby altering their free blood levels. 5-Fluorouracil increases total T3 and T4 levels, but free T4 index and TSH remain normal, indicating increased levels of thyroxine-binding globulin or enhanced binding capacity.51 l-Asparaginase causes transient thyroxine-binding globulin deficiency by diminishing hepatic synthesis and also inhibits TSH secretion from the pituitary, resulting in decreased total T4 as well as free T4 levels.52 Transient hyperthyroidism after l-asparaginase therapy for acute lymphoblastic leukemia has also been observed.53 These thyroid function abnormalities are mild and short-lived and generally do not require specific therapy. In postmenopausal women, tamoxifen therapy is associated with changes in thyroid hormone concentrations, though patients may remain clinically euthyroid.54 Mamby and colleagues undertook a randomized, placebo-controlled trial with tamoxifen 10 mg orally, twice daily, with 14 patients in each group.55 Thyroid function test assessment before and 3 months after initiation of therapy was done. Serum thyroid-binding globulin increased, as did T4 uptake and T4 levels in the tamoxifen-treated group compared to placebo. TSH levels and free thyroxine index remained unchanged; patients were clinically euthyroid and did not require treatment.

or ectopic production of corticotrophin. It has been reported to result in sustained remission for some patients with metastatic adrenal carcinoma with long-term administration.59 Although the mechanism of action is incompletely understood, adrenal necrosis and permanent adrenal insufficiency can result, necessitating lifelong glucocorticoid administration.

Pancreatic exocrine or endocrine insufficiency attributable to chemotherapy is uncommon. Acute pancreatitis has been described as a complication of l-asparaginase therapy and can be fatal.60 Although rare, several cases of diabetes mellitus have been associated with lasparaginase therapy.61 Hyperglycemia is usually transient and responds to intravenous fluids and drug discontinuation. A plausible mechanism may be inhibition of protein synthesis by l-asparaginase leading to interference with insulin production.62 High triglyceride levels have been associated with l-asparaginase use, though it is not clearly associated with incidence of pancreatitis in these patients. Knoderer and colleagues retrospectively described 33 patients (13%) with asparaginase-associated pancreatitis in a cohort of 254 patients over a 5-year period.63 Twelve cases were noted after Escherichia coli asparaginase, and 20 cases were noted after PEG-asparaginase therapy. The incidence of pancreatitis was found to be independent of the individual or cumulative asparaginase dose. The interval to pancreatitis diagnosis was longer for PEG-asparaginase than for E. coli asparaginase (P = 0.02). Patients who received prednisone (P = 0.02) and daunomycin (P = 0.006) were more likely to develop pancreatitis than were those who received dexamethasone (P = 0.04). Use of other chemotherapy agents was not observed to have a significant effect on the incidence of pancreatitis. High-dose cytarabine can rarely result in pancreatitis. Siemers and colleagues described two patients with evidence of pancreatitis among 30 patients treated with cytarabine.64 Prior therapy with l-asparaginase was found in another small series of patients who received cytarabine and developed pancreatitis.65 Streptozotocin is a nitrosurea that is used primarily for the treatment of pancreatic endocrine tumors. In preclinical models, streptozotocin causes beta cell necrosis and insulin-dependent diabetes in many species.66 Mild glucose intolerance has been described in patients receiving this agent; however, specific treatment is rarely required.67 Androgen ablation therapy may also be associated with diabetes. Keating and colleagues showed an increased incidence of diabetes in prostate cancer patients receiving GnRH agonist. A potential mechanism is the increase in body fat mass associated with hypogonadism, which results in insulin resistance.68

Adrenal

ROLE OF BIOLOGIC AGENTS

A method of medically ablating or reducing adrenal function was sought for a number of years as an alternative to surgical resection of the adrenal glands. Surgical adrenalectomy was used primarily for the treatment of advanced breast cancer. Aminoglutethimide and ketoconazole both suppress adrenal function. These drugs appear to have their effect through their ability to inhibit important cytochrome P-450 isozymes, which are necessary for adrenal steroid synthesis.56,57 Aminoglutethimide, at doses of 1000 to 1500 mg/day, and ketoconazole, at doses of 800 to 1200 mg/day, produce adrenal insufficiency in 30% to 40% of patients. Although standard glucocorticoid treatment is generally required in these patients during treatment, mineralocorticoid replacement is usually not required. The antiadrenal effects of ketoconazole and aminoglutethimide are reversible with treatment discontinuation. Full recovery within 1 to 2 weeks is usual. Mitotane (1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane), is an oral chemotherapeutic agent that is used to treat adrenal carcinoma based on its potent antiadrenal effects.58 It is used primarily to treat adrenal hyperfunction associated with adrenal carcinoma

Biologic agents are increasingly important in cancer treatment, and various endocrine complications are being recognized with the use of these agents. Immune therapies may cause thyroid dysfunction. Atkins and colleagues69 were the first to describe an association between therapy with recombinant interleukin-2 and thyroid abnormalities. Interleukin-2 therapy is known to be associated with both hypothyroidism and hyperthyroidism, though the former is more common.70 In Atkins and colleagues’ original report, seven patients (21%) had laboratory evidence of hypothyroidism, a decline in the serum thyroxine concentration and serum free thyroxine index, and an increase in the serum TSH concentration 6 to 11 weeks after treatment.69 All five symptomatic patients had borderline or elevated serum antimicrosomal antibody titers after treatment; two had serum antibodies to thyroglobulin. Five of the seven patients with hypothyroidism (71%) but only 5 of the 27 euthyroid patients (19%) had evidence of tumor regression (P < 0.02). Fifteen patients (47%) became hypothyroid with high serum TSH levels within 60 to 120 days after the

Pancreas

Endocrine Complications • CHAPTER 65

start of treatment. The proposed mechanism is autoimmune with development of antithyroid antibodies. Proposed mechanisms are that either the interleukin-2 treatment itself triggers autoreactive Bcell clones or cellular and/or cytokine-mediated thyroid destruction leads to activation of autoreactive B-cell clones. Hypothyroidism is a recognized complication of tyrosine kinase inhibitors. Sunitinib maleate is an oral tyrosine kinase inhibitor that was recently approved for the treatment of gastrointestinal stromal tumors and renal cell carcinoma. Desai and colleagues reported hypothyroidism in patients undergoing sunitinib therapy.71 One potential mechanism may be sunitinib-induced destructive thyroiditis through follicular cell apoptosis. Sunitinib is also a RET/PTC tyrosine kinase inhibitor that is involved in pathogenesis of papillary thyroid cancer and perhaps affects normal thyroid function as well. Imatinib also interacts with thyroid hormone replacement and results in increased TSH levels in hypothyroid individuals who are on levothyroxine therapy. However, it does not appear to have a direct effect on the thyroid but alters the levels of thyroxine binding protein.72 Berman and colleagues reported hypophosphatemia and associated changes in bone mineral metabolism in patients taking imatinib for either chronic myelogenous leukemia or gastrointestinal stromal tumors.73 Patients were found to have high levels of urinary phosphate and markedly decreased serum levels of osteocalcin and N-telopeptide, indicative of reduced bone turnover. Imatinib inhibits platelet-derived growth factor receptor, which in a rat model has been demonstrated to have a critical role in skeleton development. Interferon-beta (IFN-β) and interferon-alpha (IFN-α) may both increase ACTH, prolactin, growth hormone, and cortisol levels in patients.74 An assessment of IFN-α-induced endocrine stimulation in patients with myeloproliferative disorders revealed that on day 1 of therapy, a significant stimulation of the hypothalamic-pituitary axis was apparent, an effect that disappeared by the third week of therapy.75 The acute stimulatory effect of IFN-α on cortisol release appears to be mediated by the release of hypothalamic corticotropin-releasing hormone. There are reports of alterations in the levels of sex hormones during IFN therapy, and male sexual dysfunction has been noted.76 Clinicians should keep in mind that there are limited data regarding the effects of many new agents and one must be alert to endocrine dysfunction in patients receiving such drugs.

EVALUATION AND TREATMENT OF COMMON ENDOCRINE DYSFUNCTION A detailed history, including treatment history and physical examination, should be done in any patient who is suspected of having

endocrine dysfunction. Evaluation should be directed by this information and the location and type of tumor. The initial approach to the diagnostic workup is outlined in the following sections. Endocrinology consultation should be sought for more complex and multisystem involvement. Table 65-3 shows a brief outline of evaluation of common endocrine disorders. The workup for gonadotropin deficiency and hormone replacement is discussed in detail in Chapter 64.

Hypothalamic-Pituitary Axis Disorders Growth Hormone Deficiency EVALUATION. The assessment of pituitary GH production is difficult because GH secretion is pulsatile and serum GH levels are often low between the pulses. Therefore, measurement of a single serum GH level is of limited use in diagnosing GHD. Serum insulinlike growth factor (IGF-1) and IGF binding protein-3 (IGFBP-3) concentrations may be measured as a surrogate marker for GH production, and further evaluation is indicated if these results are below the mean for normal children of the same age. An IGF-1 level below 84 ng/mL using the Esoterix assay reportedly is highly indicative of GHD.77 Confirmation can be done by GH secretion provocative tests. According to the consensus guidelines for the diagnosis and treatment of adults with GH deficiency, the insulin hypoglycemia test is the gold-standard GH provocative test.78 According to the Food and Drug Administration, GHD is diagnosed if the maximum stimulated serum GH concentration is less than 5.1 µg/L (polyclonal radioimmunoassay) or less than 2.5 µg/mL (immunochemiluminescent assay).79 Even though reliable, this test requires strict monitoring. The insulin hypoglycemia test is contraindicated in debilitated patients, those with cardiovascular or cerebrovascular disease, and those with a history of seizure, abnormal electroencephalogram (EEG), or history of brain surgery.77–79 In these patients, the combined arginine/GHRH stimulation test may be used. The arginine stimulation test involves intravenous infusion of 0.5 g/kg body weight (to a maximum of 30 g) of arginine given over 30 minutes and measuring serum growth hormone level at 0, 30, 60, 90, and 120 minutes. Though historically the insulin hypoglycemia test was considered the gold standard for GHD, the arginine-GHRH test is much safer, is 95% sensitive and 91% specific (at a cutoff of 4.1 µg/L), and has essentially replaced the former.80 The diagnosis of GHD in childhood is a complex process that requires clinical and growth assessment combined with biochemical tests and radiologic evaluation. GHD may be an isolated finding or a component of multiple pituitary hormone deficiency. In a child

Table 65-3 Diagnostic Evaluation of Common Endocrine Disorders Disease

History

Signs

Screening

Confirmatory Tests

GH deficiency

Fatigue, poor stamina, hypoglycemia

Slow growth velocity, delayed puberty, truncal fat distribution

IGF-1, IGFBP-3, and bone maturation

Insulin hypoglycemia test, arginine, L-dopa, clonidine

Hypothyroidism (primary or secondary)

Fatigue, cold intolerance, weight gain, cognitive dysfunction, mental retardation, constipation, growth failure, dry skin, depression, menstrual disturbances

Slow movement and slow speech, delayed relaxation of tendon reflexes, bradycardia, coarse skin, periorbital edema, macroglossia

Free T4, TSH, and bone maturation

TSH surge

Hyperthyroidism

Hyperactivity, tremors, diarrhea, sweating, weight loss, heat intolerance

Atrial fibrillation, lid lag, proptosis, goiter

Free T4, T3, and TSH

Radioiodine uptake scan

Adrenal insufficiency (primary or secondary)

Dehydration, hyperpigmentation, weakness, fatigue

Electrolyte disturbance, hypotension, nausea, vomiting

Early morning serum cortisol level

Low- or high-dose ACTH stimulation test

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with clinical criteria for GHD, a peak GH concentration less than 10 mg/L has traditionally been used to support the diagnosis after a provocative GH test.81 Supportive evidence is indicated by very short height (more than 2.5 standard deviations below the mean height for normal children of the same age), delayed bone age, poor growth velocity (less than twenty-fifth percentile), and a predicted adult height substantially below the mean parental height.77 IGF-I/IGFBP3 levels and GH provocation tests should be done after hypothyroidism has been excluded as a cause of slow growth. Great care should be taken in using insulin or glucagon provocative tests in a young child, and testing should be monitored by an experienced team.

also be evaluated for coexistent adrenocortical, gonadal, posterior pituitary, and, in children, growth hormone function. As was noted previously, patients with cranial irradiation or traumatic brain injury are at high risk for panhypopituitarism. Primary hyperthyroidism is associated with low TSH and high free T4. Graves disease is associated with uniformly high 24-hour radioiodine uptake, while toxic adenoma is associated with focal high uptake. If free T4 and T3 are high with a normal or high TSH in a clinically hyperthyroid patient, pituitary magnetic resonance imaging should be done to look for a pituitary mass (TSH-secreting adenoma).

TREATMENT. Children with proven GHD should receive GH therapy as soon as possible after diagnosis. The goal of therapy is to maximize height attained before puberty. The usual starting dose of GH is 25 to 50 µg/kg/day, administered subcutaneously in the evening.72,73 Each dose produces a pharmacologic level of GH for approximately 12 hours. Evaluation of the growth response and adjustment of GH dose should occur every 4 to 6 months, and assessment should include measurement of height, weight, and arm span. GH dose is increased as weight gain occurs to maintain a stable dose per kilogram of body weight. Serum IGF-1 measurements are recommended yearly. If IGF-1 increases above the upper limits of normal for age and gender, the GH dose should be decreased. GH therapy in adults is approved by the Food and Drug Administration only if there is evidence of hypothalamic or pituitary disease and a subnormal serum GH response to a provocative test. The goal of the therapy is to improve muscle and cardiac function, restore normal body composition, and improve serum lipids. The usual starting dose for adults between 30 to 60 years of age is 300 µg/day; the dose is increased every 1 to 2 months by 100 to 200 µg/day, guided by clinical response and measurement of serum GH levels.77,78

TREATMENT. Thyroid hormone replacement with levothyroxine

Hyperprolactinemia EVALUATION. The presenting symptoms of hyperprolactinemia include amenorrhea, galactorrhea, impotence, and infertility. The diagnosis of hyperprolactinemia is made by a random serum prolactin level measurement. Dynamic testing of the lactotrophin reserve with thyrotropin-releasing hormone is not useful because it does not differentiate between the different causes of hyperprolactinemia.82 Hypothyroidism must also be ruled out as a cause of clinical findings, and careful review of medications should be done to rule out druginduced hyperprolactinemia.

TREATMENT. Dopamine released from hypothalamic nerve endings acts as a prolactin inhibitory factor, and dopaminergic agonists are useful in the treatment of hyperprolactinemia. Commonly used agents include bromocriptine and cabergoline. The most common side effects of these drugs are nausea, postural hypotension, and mental fogginess. Less common side effects include nasal stuffiness, depression, Raynaud phenomenon, alcohol intolerance, and constipation. The usual starting dose is 0.25 mg of cabergoline twice a week or 1.25 mg of bromocriptine once a day. The doses can be increased on the basis of serum prolactin level and the side effect profile. Thyroid Disorders EVALUATION. Primary hypothyroidism is characterized by a high serum TSH (normal range: 0.5 to 5 mU/L) concentration and a low serum free T4 concentration (normal range- 0.8 to 1.8 ng/dL). Secondary hypothyroidism is characterized by a low serum T4 concentration and a serum TSH concentration that is not appropriately elevated.83 To distinguish between pituitary and hypothalamic causes of hypothyroidism, thyrotropin-releasing hormone stimulation test and/or imaging studies of the sellar and suprasellar region should be done. Patients who are diagnosed with central hypothyroidism should

(T4) is usually sufficient. The goal of therapy is to attain normal TSH (in primary hypothyroidism) and free T4 levels (in secondary hypothyroidism). The average replacement dose of T4 in adults is approximately 1.6 µg/kg body weight per day. Treatment with liothyronine (T3) may be required in patients with no response to levothyroxine therapy or in patients with myxedema coma. Thyroid ablation with surgery, radioiodine, or pharmacologic agents (propylthiouracil, methimazole) should be considered in patients with hyperthyroidism. Beta blocker therapy is used as a clinical adjunct in most patients. The goal of therapy is to keep the TSH and T3 and T4 in the normal range. Surgery is indicated primarily in patients who have large or obstructive goiter.

Syndrome of Inappropriate Antidiuretic Hormone EVALUATION. SIADH is characterized by hyponatremia, a low plasma osmolality, an inappropriately elevated urine osmolality (above 100 mosmol/kg), and a high urinary sodium concentration (usually above 40 mEq/L). Other supportive findings include low BUN and serum uric acid concentration, normal plasma creatinine concentration, normal acid-base and potassium balance, and normal adrenal and thyroid function.84

TREATMENT. Water restriction is the mainstay of therapy in asymptomatic hyponatremia and in chronic SIADH. Severe, symptomatic, or resistant hyponatremia requires the administration of salt tablets or hypertonic saline administration. A loop diuretic (such as furosemide once or twice a day) may be used to enhance the effect of fluid reduction, since it directly interferes with the countercurrent concentrating mechanism by decreasing sodium and chloride reabsorption in the medullary portion of the loop of Henle.85 In patients who remain refractory, demeclocycline (300 to 600 mg twice a day) or lithium may be used.86 These drugs act on the collecting tubule cell to diminish its responsiveness to ADH, thereby increasing water excretion. ADH receptor antagonists are being evaluated that are selective for the V2 (antidiuretic) receptor and may thus reverse the hyponatremia. Conivaptan blocks V2 and V1a receptors and is available in parenteral form.87

Hyperparathyroidism EVALUATION. Primary hyperparathyroidism is characterized by elevated parathormone level and hypercalcemia. Supportive findings include low serum phosphate level, an increase in 24-hour urinary calcium excretion, a decrease in serum calcitriol and 25(OH) cholecalciferol, and an increase in biochemical markers of bone turnover (collagen cross-links, osteocalcin, bone-specific alkaline phosphatase). Patients may present with classic symptoms of the disease (nephrolithiasis or bone disease), or they may have nonspecific symptoms such as fatigue, weakness, mild depression, vague abdominal pain, and constipation.

TREATMENT. Patients should be advised to avoid factors that can aggravate hypercalcemia, including thiazide diuretic and lithium carbonate therapy, volume depletion, prolonged bed rest or inactivity, and a high-calcium diet (>1000 mg/day). Daily vitamin D intake

Endocrine Complications • CHAPTER 65

of 400 to 600 IU daily should be maintained, as vitamin D deficiency stimulates parathormone secretion and bone resorption and therefore is deleterious in patients with primary hyperparathyroidism. Surgical removal of the parathyroid glands remains the mainstay of therapy in most patients. Medical therapy involves estrogen plus progestin, bisphosphonates, or raloxifene. These drugs inhibit bone resorption and increase bone density and possibly lower serum calcium concentrations in patients with hyperparathyroidism. Calcimimetics and vitamin D analogs act by suppressing parathyroid hormone release and counteract the effects of hyperparathyroidism at the level of the parathormone receptor. Calcimimetics are currently being studied for primary and secondary hyperparathyroidism.88

Adrenal Disorders EVALUATION. There is controversy about the optimal biochemical approach to diagnosis of corticotropin deficiency. In moderate to severe corticotropin deficiency, the early morning serum cortisol levels are consistently less than 250 nM. A corticotropin stimulation test may be used to confirm the diagnosis. Both low-dose (1 µg) and high-dose (250 µg) corticotropin stimulation tests are useful to distinguish primary from pituitary (secondary) causes of adrenal insufficiency. Serum cortisol levels are measured at 0, 30, and 60 minutes after intravenous administration of corticotropin. If corticotropin and adrenal secretion are normal, the serum cortisol levels should increase to 20 mg/dL or higher. In patients with severe corticotropin deficiency, the serum cortisol response will be lower or absent as a result of adrenal atrophy. Primary adrenal insufficiency is characterized by low response to both low-dose and high-dose corticotropin stimulation tests. Acute adrenal crisis is an oncologic emergency. Electrolyte disturbances such as hyponatremia, hyperkalemia, azotemia, hypercalcemia, and hypoglycemia are common.

TREATMENT. Adrenal insufficiency requires glucocorticoid supplementation and at times mineralocorticoid supplementation. Pituitary or isolated ACTH deficiency is not characterized by mineralocorticoid deficiency. Patients with symptomatic adrenal insufficiency should be treated with hydrocortisone or cortisone in the early morning and afternoon. The usual initial oral dose is 25 mg of hydrocortisone (15 mg in the morning and 10 mg in the afternoon). This may be decreased over time, with the goal of using the minimal effective dose to prevent weight gain and osteoporosis.89 Patients with primary adrenal insufficiency require mineralocorticoid replacement with fludrocortisone (0.05 to 2 mg orally each day).

Box 65-1.

TREATMENT OF ACUTE ADRENAL CRISIS

1. Check airway, breathing and circulation, and baseline vital signs. Establish intravenous access with a large-gauge needle. 2. Assess serum electrolytes, glucose, and random plasma cortisol and ACTH levels. 3. Replace intravascular volume with isotonic saline (at least 2 to 3 liters), and maintain hydration. 4. Intravenous corticosteroid administration preferably with dexamethasone (4 mg intravenous every 8 hrs) intravenously. Hydrocortisone 100 mg IV every 6 hours may be used but can interfere with ACTH stimulation test. 5. Treat underlying causes of the adrenal crisis (e.g., infection). 6. Perform a short ACTH stimulation test to confirm the diagnosis of adrenal insufficiency if the patient does not have known adrenal insufficiency. 7. Begin mineralocorticoid replacement with fludrocortisone (0.1 mg by mouth daily) after volume replacement.

Box 65-2.

YEARLY SURVEILLANCE FOR ENDOCRINE DISORDERS IN CHILDHOOD CANCER SURVIVORS

1. Detailed history and physical examination, including accurate height and weight measurements (arm span measurement if unable to assess height) 2. Determination of bone age (radiograph of left hand and wrist) in children who are growing too fast or too slowly 3. Ascertainment of Tanner stage of pubertal development and interpretation of whether the pubertal status and rate of progression are appropriate for chronologic age 4. Measurement of IGF-I and IGFBP-3 levels in children who are growing too slowly (to assess for GHD) 5. Measurement of serum LH, FSH, and sex hormone levels (testosterone or estradiol) in children with delayed or interrupted progression of puberty 6. Measurement of free T4 and TSH levels

During periods of stress, patients with adrenal insufficiency require higher-than-usual doses of hydrocortisone, and in severe illness, they might require intravenous high-dose hydrocortisone therapy due to acute adrenal crisis. Box 65-1 outlines a treatment algorithm for acute adrenal crisis, which should be treated as an oncologic emergency.

SURVEILLANCE OF CHILDHOOD CANCER SURVIVORS With improved therapy for most childhood cancers, there is an increasing population of childhood cancer survivors.90 Such individuals are at risk for long-term endocrine complications related to the tumor and/or the treatment received. The risk of a particular endocrinopathy depends on the tumor location and the dose and duration of radiotherapy and chemotherapy received. Box 65-2 depicts a summary of recommended yearly surveillance in childhood cancer survivors for endocrine complications.91 Close follow-up should be performed every 4 to 6 months if the initial tests are normal but the child remains symptomatic. Children typically exhibit catch-up growth and weight gain after completion of therapy. Some children transiently develop breast buds corresponding to this period of newly improved nutrition. These children should be examined every 3 to 6 months to evaluate for precocious puberty. Assessment of Tanner stage of pubertal development is useful to assess for precocious or delayed puberty. Further testing should be guided by the physical findings. Surveillance for adrenocortical deficiency is indicated primarily for high-risk patients such as those who received cranial irradiation in excess of 40 Gy. There is controversy regarding how long the surveillance should be carried out. The surveillance is guided largely by the pattern of growth and development. If normal growth and pubertal development are attained, the surveillance can be stopped.

CONCLUSION Endocrine disorders are common in patients with cancer and are related primarily to cancer therapy. Careful clinical examination and yearly surveillance should be done in cancer survivors. A high degree of clinical suspicion is necessary in patients who are on newer therapies with which there is limited experience. Most endocrine disorders are readily treatable, and an accurate diagnosis should be pursued aggressively.

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Second Malignant Neoplasms John P. Plastaras, Daniel M. Green, and Giulio J. D’Angio

S U M M ARY

Epidemiology • Pediatric patients who survive their primary cancer are at increased risk of developing a new malignancy. • The magnitude of this risk is modulated by several factors, including the patient’s genetic susceptibility, the type of surgical procedure used for removal of the tumor, the use of radiation therapy as part of the treatment plan, the chemotherapeutic agents that are employed, the severity of immune suppression that is present at the completion of all treatment, and environmental exposures. • The potential risk of a second malignant process following treatment of adults should not lead to therapeutic compromises when treatment, often aggressive, is of known benefit.

General Recommendations • All former cancer patients should remain under a physician’s care indefinitely.

O F

K EY

P OI NT S

• All former cancer patients should undergo frequent physical examinations (preferably by a physician or other specifically trained health care worker who is familiar with the problem of therapy-induced malignancy). • Physicians should counsel cancer survivors regarding the potential for the adverse effects of tobacco use, including the potential to interact with the adverse effects of their prior therapy, such as irradiation of the oropharynx, esophagus, and/or lungs.

•

•

•

Specific Recommendations • Plain radiographs should be obtained for patients who have been irradiated whenever local pain occurs in a previously irradiated bone. • Patients who have received irradiation to volumes that include the breast,

INTRODUCTION Second malignant neoplasms (SMNs) develop in patients as a result of genetic and iatrogenic factors and their interplay. The therapies that are employed are different for children and adults because of the differences in the primary cancers that are encountered. Primary cancers in adults are usually of epithelial origin, unlike the embryonal and sarcomatous neoplasms encountered at earlier ages. In addition, some of the cancers that are encountered in adults are the result of therapies they received for nonmalignant conditions that were treated when they were children. This discussion therefore has been divided into two parts according to the age group being considered.

CHILDREN AND ADOLESCENTS SMNs are a recognized complication of successful treatment of children and adolescents for cancer. The frequency of these had been reported to be 1.9% at 10 years after diagnosis, 5.0% to 12.0% at 25 years after diagnosis, 3.3% to 4.9% at 25 years after diagnosis among 3-year survivors, 3.2% to 12.0% at 20 years after diagnosis among 5-year survivors, and 4.2% to 7.8% at 25 years after diagnosis among 5-year survivors.1–8 These series differed with respect to the time period during which the patients were treated,

•

uterine cervix, or intestine should undergo routine evaluation with available screening tests, such as mammography, Pap smear, and stool examination for the presence of occult blood. Annual mammography should be initiated no later than 10 years after breast irradiation. Careful physical examination of irradiated patients will facilitate the early identification of thyroid nodules and skin cancer. All patients who have been treated with an alkylating agent, procarbazine, or a topoisomerase II inhibitor, should have a complete blood count every 6 to 12 months for a minimum of 12 years after diagnosis. Presence of macrocytosis and/or cytopenia should prompt evaluation of the bone marrow.

the completeness of follow-up, and the treatment exposures that the patients experienced. SMNs are a major threat to the long-term survivors of childhood cancer, in whom they are looming ever larger as a cause of death. Moller and colleagues reported results of a survey in the Nordic countries, where the proportions of deaths from second tumors increased from 3% to 22% between 5 and 10 years and 20+ years.9 In fact, Lawless and colleagues found SMNs to be more often the cause of death than was relapse of the primary tumor.10 In their single-institution study, they found that SMNs were the leading cause of death (39%) in 15+-year survivors; greater than the rates for primary cancer (21%) and cardiac (16%) deaths. Increasingly, the data suggest that although specific exposures (whether to a particular chemotherapeutic agent or to ionizing radiation) might be linked to the occurrence of a new malignancy, the most important factor in the pathogenesis of many SMNs could well be the patient’s genetic susceptibility. We will review the genetic and treatment factors that have been associated with the occurrence of SMNs and discuss the followup and evaluation of the successfully treated pediatric cancer patient.

Genetic Factors The importance of genetic predisposition to the occurrence of a SMN has been demonstrated most clearly in patients with hereditary

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Percentage

retinoblastoma. Among 1604 1-year survivors of retinoblastoma in Boston and New York City between 1914 and 1984, 961 had the hereditary form of the disease.11 The cumulative percentage of those who developed a SMN was 51.0% (±6.2%) 50 years after retinoblastoma diagnosis, compared with 5.0% (±3.0%) among those with nonhereditary retinoblastoma (Fig. 66-1A). Among patients with hereditary retinoblastoma, the cumulative percentage that developed SMNs was 58% among those whose treatment included radiation therapy (RT), compared with 26.5% among those whose treatment did not include RT (Fig. 66-1B). Kleinerman and colleagues12 updated this series to focus on secondary sarcomas and found that there was a significantly increased risk of sarcomas within the RT field as well as an increased risk outside of the RT field, with a 13% cumulative risk of sarcoma 50 years after RT. Leiomyosarcomas were frequently diagnosed over 30 years later, indicating that older carriers remain at risk. Fletcher and colleagues13 described the long-term risks of SMNs in retinoblastoma survivors born before 1950, a group that was not routinely treated with high-dose RT. The cumulative incidence of SMNs from age 25 to 84 was 69% in the hereditary cases and 48% in the sporadic cases, but the majority were epithelial cancers and not sarcomas. These studies indicate that retinoblastoma carriers have both an inherent risk of other SMNs and a RT-

60 55 50 45 40 35 30 25 20 15 10 5 0

Hereditary retinoblastoma Nonhereditary retinoblastoma

Treatment Factors Surgery Surgical procedures can increase the risk of subsequent malignancy. Adenocarcinoma of the colon has been reported in several patients after ureterosigmoidostomy. The incidence of adenocarcinoma in these patients was approximately 9.9 per 1000, compared with an incidence rate of 9.9 per 100,000 in the general population.22 The majority of reported patients have undergone this procedure for treatment of exstrophy of the bladder. The median interval between ureterosigmoidostomy and the diagnosis of colon carcinoma was 22 years.23

51.0% (± 6.2%)

5.0% (±3.0%)

0

5

10

15

20

25

30

35

40

45

50

Time after retinoblastoma diagnosis (yrs) 60 55 50 45 40 35 30 25 20 15 10 5 0

Radiotherapy No radiotherapy

58.3% (± 8.9%)

26.5% (± 10.7%)

0

B

dependent risk of secondary sarcomas, which affects how these patients should be followed into adulthood. Li-Fraumeni syndrome consists of sarcoma diagnosed in the proband before age 45 years, with additional cancers (frequently softtissue sarcoma or breast cancer) diagnosed in other children and young adults within the family.14 The genetic defect in some families with the Li-Fraumeni syndrome was demonstrated to be a mutation within the p53 gene.15–18 Because the pattern of first and second malignant tumors in some patients with SMNs resembles the distribution that is observed within some families with Li-Fraumeni syndrome, a series of patients with SMNs was evaluated for the occurrence of mutations at this locus. Mutations were identified in 5.1% of 59 patients who were examined.19,20 Pediatric patients with cancer who have neurofibromatosis have a relative risk of 8.1 of developing a SMN compared with pediatric patients with cancer who do not have neurofibromatosis.21 Future research could demonstrate that neurofibromatosis type 1 patients who develop SMNs have coexistent germline p53 mutations.

Radiation Therapy

A

Percentage

1024

5

10

15

20

25

30

35

40

45

50

Time after retinoblastoma diagnosis (yrs)

Figure 66-1 • A, The cumulative incidence of second malignant tumors is increased in children with familial retinoblastoma. B, The cumulative risk of second malignant tumors is increased in children with familial retinoblastoma who are treated with radiation therapy. (Data from Wong GL, Boice JD Jr, Abramson DH, et al: Cancer incidence after retinoblastoma: radiation dose and sarcoma risk. JAMA 1997;1262:278.)

The risk of various SMNs has been linked to the use of both RT and chemotherapy.8,24,25 As treatment of childhood malignancies has intensified over the past several decades, the cure rate has increased, but so has the risk of SMNs.8 In the cohort of 13,136 patients from the Childhood Cancer Survivor Study, 59% of the nonbreast, nonskin, nonthyroid SMNs occurred within RT fields.26 A European case-control study of 4581 survivors by Guerin and colleagues demonstrated a radiation dose-response for the excess risk of SMNs that best fit a linear model of 0.13 per Gy.24 This study also noted that the relative risk of SMN was increased when chemotherapy and RT were delivered concomitantly. Patients who receive neck irradiation for malignant diseases are at risk for the subsequent occurrence of thyroid malignancies. These have been reported after treatment of patients with medulloblastoma, acute lymphoblastic leukemia, and Hodgkin’s disease (HD).27–29 The incidence of thyroid cancer in survivors of HD was 0.8% (1 in 119) among children who were treated at Stanford University.30 Malignant thyroid tumors occurred at lower RT doses than did benign lesions.31 Ron and colleagues reported that a linear-exponential model fit the dose-response data for thyroid cancer after treatment for childhood cancer better than a linear model did.32 This finding is consistent with the data of Upton, who demonstrated that the dose-response relationship for some radiation-induced experimental tumors was quadratic rather than linear.33 These data led Gray to hypothesize that the shape of the dose-response curve was the sum of two radiation-induced processes: mutation induction and cell death.34 Radiation-associated SMNs in other sites, however, have been fit to linear models (see later discussion). Central nervous system tumors, including meningiomas and gliomas, have been reported with increasing frequency after direct or incidental irradiation of the brain, which is not surprising when the occurrence of brain tumors in children treated with low doses of RT for tinea capitis is recalled. Neglia and colleagues35 reported that the excess radiation-associated risk for secondary gliomas was 6.8 and

Second Malignant Neoplasms • CHAPTER 66

1200 1000 800 600 400 200 90

Relative risk

80 70 60 50 40 30 20 10 0 0

0

10

20

30

40

50

60

Dose in Gy

Figure 66-2 • The risk of subsequent glioma (closed squares, purple line) and meningioma (open squares, blue line) increases with radiation dose. (Data from Neglia JP, Robison LL, Stovall M, et al: New primary neoplasms of the central nervous system in survivors of childhood cancer: a report from the Childhood Cancer Survivor Study. J Natl Cancer Inst 2006;98:1528.)

6 5

Percentage

that for meningiomas was 9.9. There was a linear dose response, with a steeper slope for meningiomas (1.1) than for gliomas (0.3; Fig. 66-2). The excess risk for gliomas was greatest in children who were irradiated before age 5. Other researchers have confirmed the increased risk of central nervous system tumors in children whose treatment for acute lymphoblastic leukemia included cranial irradiation.36 In children with neurofibromatosis type 1, there was a threefold relative risk of second nervous system tumors after RT for optic pathway gliomas compared to unirradiated patients.37 Genetic loci associated with the occurrence of Wilms’ tumor have been identified on chromosomes 11, 17, and 19.38–40 Some patients have germline mutations in WT1, the only Wilms’ tumor-associated gene that has been sequenced.41,42 Li and associates reported that the frequency of SMNs in a cohort of successfully treated Wilms’ tumor patients was 6% 20 years after diagnosis.43 SMNs were diagnosed only in irradiated patients. Breslow and colleagues reviewed the occurrence of SMNs among patients entered on the National Wilms Tumor Studies.44 The cumulative risk of SMN was 1.6% 15 years after diagnosis (Fig. 66-3). The relative risk of developing SMN was increased in patients who had received RT, the relative risk increasing with increasing radiation dose. Administration of doxorubicin increased the relative risk at each level of radiation exposure.44 Other researchers have reported a cumulative frequency of SMN after treatment for Wilms’ tumor at 3.9% at 20 years, with a relative risk of 11.0.45 Patients with bilateral Wilms’ tumor were not at increased risk for SMNs, according to the National Wilms Tumor Study Group analysis.44 Sarcomas of bone have been reported both in patients with hereditary retinoblastoma and in those who survive other types of childhood cancer. The cumulative risk of SMN in bone was estimated to be 2.8% among 9170 patients who were evaluated but was 14.1% among those with retinoblastoma and 22.1% among those who had

4 3 2 1 0 0

5

10

15

20

25

Time since Wilms’ diagnosis (yrs)

Figure 66-3 • Children who have been successfully treated for Wilms’ tumor have a significant risk of developing a second malignant tumor. (Data from Breslow NE, Takashima JR, Whitton JA, et al: Second malignant neoplasms following treatment for Wilms’ tumor: a report from the National Wilms Tumor Study Group. J Clin Oncol 1995;13:1851.)

been treated for Ewing sarcoma (ES) at 20 years after diagnosis (Fig. 66-4).46 The relative risk was 2.7 among patients whose treatment had included RT; the relative risk increased with increasing RT dose and more intensive use of alkylating agents.46 Other researchers reported a relative risk for osteosarcoma of 88–1515 in patients who had been treated for retinoblastoma; the relative risk rises to 800 after treatment for ES.3,47 Hawkins and colleagues calculated the cumulative frequency of bone cancer in previously irradiated childhood cancer survivors as 0.5% among those who were not treated for retinoblastoma and as 7.2% among those who were treated for heritable retinoblastoma.48 A dramatically high risk of sarcoma of bone after treatment of ES was observed in a multi-institutional study.49 The cumulative frequency of a SMN in ES patients who were treated successfully was 9.2% at 20 years after diagnosis, and that of a secondary sarcoma was 6.5%. No secondary sarcomas developed in patients who had received less than 48 Gy.49 A case-control study of bone sarcoma as a SMN found no difference in relative risk between patients treated with orthovoltage and megavoltage RT.46 A decreased risk might have been expected because of the lower absorbed doses in bone after high-energy RT. Successfully treated patients are at risk of developing carcinomas (e.g., of the skin) within prior RT treatment volumes given at a very early age.50 Irradiation of breast tissue increases the risk of breast cancer, as was demonstrated in women who were exposed to diagnostic radiograph for pulmonary tuberculosis and survivors of the atomic bomb detonations in Hiroshima and Nagasaki.51,52 The reported relative risks of breast cancer in girls who were treated for HD have varied widely. The largest study with the longest follow-up reported a relative risk of 11.5.53 In this large, population-based study of 383 HD survivors, all 16 women who developed breast cancer had supradiaphragmatic irradiation. The cumulative risk of breast cancer at 25 years was 9.9% for all HD patients and 12.2% for those who were treated with supradiaphragmatic RT. Total body irradiation, a component of most preparative regimens for allogeneic bone marrow transplantation for malignant diseases, is associated with a cumulative risk for the occurrence of a second solid neoplasm of 6.1% at 10 years after treatment.54

Hormone Therapy Growth hormone (GH) treatment is necessary for the management of some children who received cranial irradiation as part of their

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Percentage

1026

16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

Entire cohort Retinoblastoma

14.1%±4.3%

5.5%±2.1%

2

5

10

15

20

25

Figure 66-4 • The cumulative incidence of bone sarcoma as a second malignant tumor is increased in children who were treated for retinoblastoma compared with those who were treated for other forms of childhood cancer. (Data from Tucker MA, D’Angio GJ, Boice JD Jr, et al: Bone sarcomas linked to radiography and chemotherapy in children. N Engl J Med 1987:317:588.)

30

Years from diagnosis of the first malignancy Entire cohort Retinoblastoma

9170 5524 319 214

288 979 296 122 58 23 Number of patients at risk

therapy for acute lymphoblastic leukemia or brain tumors. The question of whether use of GH increases the risk of second cancers was addressed in the Childhood Cancer Survivor Study cohort.55 Using a time-dependent Cox multivariate model, the authors found that GH increased the risk of SMNs by 2.1, which was less than previous estimates. The majority of SMNs were intracranial, especially meningiomas. Although the use of radiation was taken into account in their model, the radiation dose was not, so it is possible that the need for GH was a surrogate for radiation dose. The possible risk identified by these two studies must be weighed in the context of the substantial benefits that accrue to these patients as the result of GH therapy, including improved linear growth and bone mineral accretion.

Chemotherapy The significance of prior treatment with chemotherapy in the pathogenesis of SMNs was first evaluated in detail in cohorts of adults who had been treated successfully for HD. The risk factors for the occurrence of SMNs in pediatric patients after treatment for HD have been evaluated less thoroughly. Bhatia and coworkers reported that the cumulative risk of developing any SMN after treatment for HD in childhood was 7% at 15 years after diagnosis.56 The risk of developing non-Hodgkin’s lymphoma (NHL) was 1.1%, and the risk of developing any type of leukemia was 2.8% at 15 years after diagnosis. The investigators found that the relative risk of leukemia was proportional to the alkylating agent dose score, that is, the cumulative dose of those drugs. The actuarial risk of developing acute myelogenous leukemia 10 years after diagnosis was 11% among pediatric patients treated at Stanford University with low-dose (2500 cGy) RT and MOPP (nitrogen mustard [M], vincristine [O], procarbazine [P], and prednisone [P]) chemotherapy, which has an alkylating agent dose score of 2.57 The risk was 1.1% 15 years after diagnosis among pediatric patients treated with involved or extended field RT and various chemotherapy regimens that did not contain nitrogen mustard (vincristine, prednisone, doxorubicin, with or without procarbazine; cyclophosphamide, vincristine, prednisone, procarbazine, or methotrexate).58

59

Other investigators have studied the risk of developing SMNs after various chemotherapeutic agent exposures. Tucker and colleagues reported that prior treatment with an alkylating agent increased the risk of developing bone cancer or leukemia (Fig. 66-5) as SMN.46 De Vathaire and coworkers demonstrated that dactinomycin increased the risk of a bone or soft tissue SMN (relative risk: 8.7).59 Garwicz and associates reported that treatment with classical alkylating agents (nitrogen mustard, cyclophosphamide, lomustine), nonclassical alkylating agents (procarbazine), vinca alkaloids (vinblastine, vincristine), or prednisone each increased the relative risk of SMN.21 Only procarbazine increased the relative risk of SMN when it was included in a two-factor multivariate model.21 Klein and colleagues reported an increased relative risk for SMN with higher doses of several agents, including cyclophosphamide (RR 6.3 for doses >8000 mg/m2), cisplatinum (relative risk: 2.8 for doses >435 mg/ m2), and 6-mercaptopurine (relative risk: 4.5 for doses >5000 mg/ m2).60 Neglia and associates reported that treatment with an anthracycline (relative risk: 1.51 for doses of 101–300 mg/m2; relative risk: 1.44 for doses >300 mg/m2) or epipodophyllotoxin (relative risk: 2.78 for doses >4001 mg/m2) did not demonstrate an increased relative risk with increasing alkylating agent dose score or cisplatinum exposure.6 The epipodophyllotoxins have been identified as important leukemogens. Pui and coworkers reported the risk of secondary acute myelogenous leukemia (AML) as 4.7% at 6 years after diagnosis among patients treated for acute lymphoblastic leukemia.61 The risk was substantially higher (19.1%) among patients with T-cell leukemia. These investigators subsequently demonstrated that the risk of secondary AML in this population was related to the administration of epipodophyllotoxins, with the cumulative frequency of AML being 12.3% among those who were treated twice weekly and 12.4% among those who were treated weekly, compared with a frequency of 1.6% among those who were treated with the drug less frequently or not at all (Fig. 66-6).62 The risk of secondary leukemias after the treatment of solid pediatric tumors has been linked to treatment with radiation, epipodophyllotoxins, and vinca alkaloids.25 The onset of secondary leukemia

Second Malignant Neoplasms • CHAPTER 66

Entire cohort Hodgkin’s disease

4.2%±1.9%

4

Percentage

3

Figure 66-5 • The cumulative incidence of leukemia as a second malignant tumor is increased in children who were treated for Hodgkin’s disease compared with those who were treated for other forms of childhood cancer. (Data from Tucker MA, Meadows AT, Boice JD Jr, et al: Leukemia after therapy with alkylating agents for childhood cancer. J Natl Cancer Inst 1987;78:459.)

2

1

0.8%±0.2%

0 2

5

10

15

20

Years from diagnosis of the first malignancy Entire cohort Hodgkin’s disease

is generally considered an early event, but Haddy and coworkers noted peaks in the first 10 years and again after 20 years.25 The risk of secondary AML depends on the cumulative dose of drug administered and the schedule of administration; the frequency was reported to be 0% among germ cell tumor patients who were treated with less than 2000 mg/m2, 5.9% among childhood acute lymphoblastic leukemia patients who were treated with 1800 to 9900 mg/m2, 11.3% among germ cell tumor patients who received more than 2000 mg/ m2, and 18.4% among pediatric NHL patients who received 4200 to 5600 mg/m2.63–65 Hawkins and associates reported an increased risk of secondary leukemia with increasing cumulative dose of epipodophyllotoxin.66

9170 1036

5524 659

2288 979 212 47 Number of patients at risk

296 12

The carcinogenic potential of the anthracycline doxorubicin was suggested by the results of a previous case-control study of risk factors for leukemia as a SMN. Increasing doxorubicin dose was found to be associated with an increasing relative risk of leukemia as a SMN after adjustment for the cumulative dose of alkylators given concomitantly.67 More recently, prior treatment with anthracyclines or epipodophyllotoxins has been shown to increase the risk of therapyrelated acute promyelocytic leukemia and of secondary leukemia or myelodysplasia.68,69 Patients who received 1.2 to 6.0 g/m2 of epipodophyllotoxin had a relative risk of developing leukemia of 3.9, whereas those who received more than 170 mg/m2 of anthracycline had a relative risk of developing leukemia of 3.0.68 This finding is of

14 12.4% (6.1%−24.4%) 12

Figure 66-6 • The cumulative incidence of acute myelogenous leukemia after treatment for acute lymphoblastic leukemia is increased among children whose therapy includes weekly epipodophyllotoxin. (Data from Pui C-H, Behm FG, Raimondi SC, et al: Secondary acute myeloid leukemia. N Engl J Med 1989;321:136.)

Cumulative risk (%)

10 8 6 4 1.6% (0.4%−6.1%)

2 0 0

1

2

3

4

5

6

25 32

8 8

Years since diagnosis of ALL Weekly 84 Biweekly 148

76 130

73 55 41 121 98 60 Number of patients at risk

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interest, as doxorubicin is now known to have topoisomerase II as one of its targets—the same target as that of the epipodophyllotoxins. A case-control study of risk factors for bone sarcoma as a SMN did not identify any effect of doxorubicin therapy on the risk of developing such SMNs.46 An analysis of risk factors for any SMN in a large cohort of pediatric patients with cancer demonstrated that treatment with doxorubicin was the only factor that was identified (other than treatment with carmustine) that increased the risk of a SMN. (This finding apparently extended the earlier suggestion that doxorubicin was leukemogenic.2) The evidence in adults will be reviewed later in the chapter. The leukemogenicity of topoisomerase II inhibitors could be related to the high degree of specificity of these agents for specific DNA targets, including the myeloid-lymphoid leukemia gene and the acute myeloid leukemia 1 (AML1) gene.70–72 Although most studies of carcinogenicity of chemotherapeutic agents have focused on the development of leukemia after treatment, it is clear that solid tumor induction is also possible after exposure to one or more chemotherapeutic agents. The best example of this is the occurrence of solid SMNs in genetically predisposed retinoblastoma patients who were treated only with cyclophosphamide after enucleation.73 As our ability to identify genetically predisposed patients improves, our understanding of the apparent anomaly of solid tumor induction after systemic exposure to a carcinogenic agent will increase.

Immune Suppression Immune suppression is a component of allogeneic bone marrow transplantation. To prevent graft-versus-host disease, antithymocyte globulin can be administered to the recipient, or the bone marrow can be manipulated to remove T cells. These manipulations and bone marrow transplantation from an unrelated bone marrow donor increase the risk of Epstein-Barr virus-associated B-cell lymphoproliferative disorder. The cumulative incidence rates at 10 years after bone marrow transplantation were 11.3% among those who were treated with antithymocyte globulin, 11.4% among those who received T-cell-depleted bone marrow, and 2.3% among those who received bone marrow from unrelated donors.74

survivors are only slightly less likely to use tobacco.76,77 Physicians should counsel childhood cancer survivors about the potential for the adverse effects of tobacco use to interact with the adverse effects of their prior therapy, such as irradiation of the oropharynx, esophagus, and/or lung.

ADULTS Genetic Factors Travis provides an excellent, comprehensive overview and update of research concerning SMNs focusing on solid tumors in the adult age group.78 She stresses that several factors besides chemotherapy, RT, and their interplays need to be considered. For example, the complex potential interactions of alkylating agents, RT, and tobacco smoking are developed extensively in her discussion of lung cancers as SMNs, especially in HD patients. She points out the additional variable in HD patients (i.e., impaired immunologic responses), so that data derived from long-term survivors of HD cannot be extrapolated with confidence to patients with other SMNs. Studies of second cancers can help point to common genetic mechanisms. For example, a study of secondary pancreatic cancers showed an increased propensity of pancreas cancer to occur not only after tobacco-associated cancers, but also after breast cancer (male and female) and ovarian cancer, which are associated with BRCA2 mutations.79 The potential importance of immunodeficiency in the appearance of SMNs was the hypothesis explored by Hemminki and coworkers in an imaginative epidemiologic investigation.80 They noted that skin cancers and NHLs were the most frequent malignant lesions to appear in immune-deficient patients (e.g., in renal transplant patients who were given needed post-transplant immune-suppressing agents). Using the Swedish nationwide database, they found 4301 secondary skin cancers and 1672 NHLs in a period of about 40 years among 10.2 million survivors of a primary cancer. The researchers found increased risks—up to 12-fold depending on patient sex and primary tumor site—for these two neoplastic entities. These results suggest that immunodeficiency could play a role in the appearance of SMNs.

Recommendations Survivors of childhood cancer are at increased risk of developing a new malignancy. The magnitude of this risk is modulated by several factors, including the patient’s genetic susceptibility, the type of surgical procedure that is employed for removal of the tumor, the use of RT as part of the treatment plan, the chemotherapeutic agents that are employed, the severity of immune suppression present at the completion of all treatment, and environmental exposures.75 All former patients should remain under a physician’s care indefinitely and should undergo frequent physical examinations, preferably by a physician or other health care worker who is familiar with the problem of therapy-induced malignancy. Patients who have been irradiated should have plain radiographs obtained whenever local pain occurs in a previously irradiated bone. Patients who have received irradiation to volumes that include the breast, uterine cervix, or intestine should undergo routine evaluation with available screening tests, such as mammography, Pap smear, and stool examination for the presence of occult blood. Annual mammography should be initiated no later than 10 years after breast irradiation. Careful physical examination of irradiated patients will facilitate the early identification of thyroid nodules and skin cancer. All patients who have been treated with an alkylating agent, procarbazine, or a topoisomerase II inhibitor should have a complete blood count every 6 to 12 months for a minimum of 12 years after diagnosis. The presence of macrocytosis and/or cytopenia should prompt evaluation of the bone marrow. The adverse health consequences of tobacco use, including carcinogenesis, are well documented. Unfortunately, childhood cancer

Observed no. of cases 2 6

4

1

2

2

3

2

7.6

5 Relative risk

1028

4 3 2

ANLL CLL

1 0 1–4

5–9

10–14

15–19

Time since first radiation treatment (yrs)

Figure 66-7 • Schematic illustration of risk factors for second primary cancers. ANLL, acute nonlymphoblastic leukemia; CLL, chronic lymphoblastic leukemia. (Data from Storm HH: Second primary cancer after treatment for cervical cancer. Later effects after radiotherapy. Cancer 1998;61:679.)

Second Malignant Neoplasms • CHAPTER 66

Several sites of primary tumors in both males and females were encompassed in their study, making therapy-induced immunosuppression unlikely. This conclusion points to possible underlying genetic factors as contributory influences (Box 66-1). The importance of the Li-Fraumeni syndrome as a risk factor for cancer in young adults has been discussed previously.75 Cancer families provide evidence supporting laboratory studies of the genetic bases of adult cancers. Cancer predisposition genes for breast cancer (BRCA1, BRCA2), colon cancer (MSH1, MSH2, APC, DCC), malignant melanoma (CDK2), and renal cell carcinoma (RCC) have all been identified.

Box 66-1.

EVALUATION FOR PATIENTS AT RISK FOR SECOND MALIGNANT NEOPLASMS

Risk Factors • Treatment with radiation therapy, an alkylating agent, and/or a topoisomerase II inhibitor • History of hereditary retinoblastoma (unilateral with a positive family history or bilateral) • Carrier of ataxia-telangiectasia • Postsurgical chronic lymphedema • Ureterosigmoidostomy • Estrogen treatment

History • • • • • • • • • • • • • • • •

Pain in any previously irradiated area Bruising Gum bleeding Pallor Easy fatigability Breast lump Cough Chest pain Hemoptysis Blood in stool Constipation Tenesmus Hematuria Increased urinary frequency/incontinence Difficulty voiding Intermenstrual bleeding

Physical Examination • • • • • • •

Pallor Petechiae Chronic skin ulceration Presence of lump (mobility, tenderness, consistency) Asymmetric breath sounds Nodule in prostate Abnormal uterine cervix

Evaluation • Plain radiographs of any painful area or mass in a previously irradiated area • Stool examination for occult blood (any patient who received any abdominal irradiation) • Pap smear • Urine cytology (in any patient with hematuria and a history of bladder irradiation and/or treatment with a cyclophosphamide or ifosfamide) • Mammogram • Additional tests as indicated by the history and physical examination

At the clinical level, carriers of the ataxia-telangiectasia gene (ATM) are more likely to develop cancer; Swift and colleagues estimate a 3 to 4 excess relative risk of cancer in male and female carriers.81 Female breast cancer was the most frequent cancer reported (excess risk of about 5) and was more likely to occur in those exposed to ionizing radiation. Little and coworkers compared the relative risks of developing post-therapeutic irradiation SMNs to those among Japanese survivors of the atomic bomb blasts.82 They found the relative risks of developing leukemia or lung, bone, or ovarian cancer to be higher among the Japanese survivors than among treated patients. Neither chemotherapy nor underlying genetic factors seemed to play a role in their results. The cytogenetics of SMNs have been the subject of intensive investigation. Le Beau and associates described characteristic abnormalities of chromosome 5(del (5q)) and 7(del (7q)) in patients with treatment-associated acute myeloid leukemia.83 (The terms acute myeloid leukemia [AML] and acute nonlymphoblastic leukemia will be used interchangeably in this discussion. In most cases, the term that is used will be the one employed by the authorities being cited.) Detourmignies and colleagues found t(15;17) in treatment-associated acute promyelocytic leukemia (t-APL) and the other forms of acute myeloid leukemia (t-AML), the same translocation as is found in those diseases de novo.84 In addition, t(8;21), t(9;11) and inv16 were found in t-APL and other t-AMLs. They tend to arise in patients with solid tumors, have short latent periods, and are associated with prior therapy with drugs that inhibit topoisomerase II. Cytogenetic evaluations of solid SMNs have demonstrated chromosome 22 deletions in meningiomas and deletions in chromosomes 10 and 17 in malignant astrocytomas.85,86 These cytogenetic findings could have important implications regarding early identification and early treatment of SMNs.

Treatment Factors Surgery In adults, perhaps the most common SMN ascribed to surgery is angiosarcoma in a lymphedematous structure (Stewart-Treves syndrome), a complication that has been described most often after radical mastectomy.87 The tumors tend to arise in the edematous arm rather than in irradiated areas.88 Marchal and coworkers conducted a survey of breast angiosarcomas that develop in women who were treated with breast-conserving techniques.89 They found 9 cases among almost 20,000 women but found no conclusive evidence linking therapy as a causative factor.

Radiation Therapy Lindsay and colleagues have reviewed the factors involved in radiation carcinogenesis.90 Breast, thyroid, bone, soft tissues, and organs and tissues that are prone to develop SMNs after RT given in childhood are also vulnerable to radiation oncogenesis when treatment is given during the adult years (Fig. 66-7). The dose-response relationship has classically been described as bell-shaped: an initial increase with low and moderate radiation doses to a peak followed by a rapid decrease at higher doses due to killing of the vulnerable cells. The observed rate for second cancers after radiation of Hodgkin’s disease has been higher than predicted, and Sachs and Brenner proposed a model using normal cell repopulation to account for high cancer rates after higher radiation doses.91 Precise RT dose-response relationships remain murky, in part owing to limited data about the true RT doses at observed SMN sites.

LYMPHOMA. Survivors of Hodgkin’s disease are at increased risk for a variety of cancers, especially lung, colorectal, and breast cancer, all of which were more common in those who were treated with chemotherapy and radiation.92 In fact, a leading cause of death among long-term survivors of HD is second cancer. Treatment of HD has served as a model for therapeutic radiation-induced carcinogenesis,

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especially the risk for breast cancer.91,93,94 Wendland and colleagues studied secondary breast cancers in 8036 females with HD from Surveillance, Epidemiology and End Results (SEER) registries. Second breast cancers were seen in 2.3%, and the standardized incidence ratio was 1.9 for women treated with RT.94 Interestingly, the breast cancer-free survival curves crossed for irradiated and nonirradiated patients. There was a paucity of early breast cancers in the radiated group, but an increased incidence over time. This was modeled best with a nested proportional hazard model, possibly owing to a therapeutic effect on pre-existing tumors combined with induction of latent tumors. Travis and colleagues93 studied 3817 female 1-year survivors of HD and noted an age- and dose-dependent cumulative risk of breast cancer development that was lower in patients who were treated with alkylating agents. In their model, women who were treated at age 25 would have an estimated cumulative absolute risk of breast cancer of 1.4% by age 35, 11% by age 45, and 29% by age 55. Projections from these studies should be taken with caution, however, owing to decreases in the dose and volume radiated in the modern era. A SEER study of 77,876 patients with NHL showed that irradiated patients had a similar risk of SMNs compared with unirradiated patients (relative risk: 1.04).95 The SMN types differed, with more sarcomas, breast cancers, and mesotheliomas occurring in irradiated patients. Younger patients had an increased relative risk for SMNs, which was more pronounced in irradiated patients.

BONE AND SOFT TISSUE. The largest follow-up study of second cancers in patients with soft-tissue sarcoma primaries (N = 6,671) comes from the Swedish Family-Cancer Database, which identified 650 second cancers (9.7%).96 The median time to second cancer was 7 years, and the standardized incidence ratio was 1.42 (95% CI: 1.31 to 1.53). The most common second cancer was another soft-tissue sarcoma. Although 10 of 39 were at the same primary site, the majority occurred at different anatomic sites. A weakness of this database is the lack of treatment information, so Ji and colleagues did a reverse analysis, which showed an increased rate of second soft-tissue sarcomas after any primary tumor as well (1.92, 95% CI: 1.78 to 2.07).96 This suggests that factors other than treatment, such as genetic predisposition and environmental risk, also play an important role. Lagrange and coworkers reported 80 radiation-associated sarcoma cases collected by a consortium of French cancer centers.97 The median dose of RT was 50 Gy (range: 9 to 110 Gy) delivered to adults (median age: 44 years) for a variety of primary diagnoses. Of the histologically proven secondary sarcomas, 70% were bone and 30% were soft-tissue sarcomas, osteosarcomas and malignant fibrous histiocytomas predominating among them. Unlike the earlier study by Tountas and colleagues,98 these authors could not demonstrate a correlation with increasing dose. The outcomes were poor despite aggressive treatment based largely on surgical maneuvers. HEAD AND NECK. In the head and neck, second primary tumors are commonly observed as a result of premalignant field changes due to environmental risk factors, such as tobacco. A multicentric, casecontrol study of laryngeal and hypopharyngeal cancers showed an average second cancer rate of 2.1% per year. A higher risk was associated with tobacco, alcohol, and butter consumption, whereas citrus fruit consumption was protective.99 An attempt has been made to prevent second cancers with antioxidant therapy in a randomized, placebo-controlled trial.100 Unfortunately, α-tocopherol did not lower the overall recurrence/second cancer rate and actually increased the rate in the first 3.5 years. In an analysis of 326 consecutive patients with nasopharyngeal carcinoma who were treated with definitive RT, 5.2% developed second primary cancers at a rate of 1% per year.101 Only about one third of these were in-field, and there was not an association with the total prescribed dose. The only second tumors that occurred after 5

years were within the radiation field, supporting the concept of a lag time for radiation-induced solid cancers.

GYNECOLOGIC. Storm reviewed the frequency of SMNs in a cohort of 24,970 Danish women with invasive cervical cancer and 19,470 who had carcinoma in situ followed for 30 or more years.102 Taken together, there was an increased relative risk of 1.9 in irradiated patients who survived 30 or more years, representing an excess of 64 cases per 10,000 women annually (see Fig. 66-7). WernerWasik and coworkers analyzed the frequency and patterns of SMNs in women with cervical cancer and came to a different conclusion.103 They found 11 SMNs among 10 of the 125 women with FIGO stage I and II cervical carcinoma who received RT in a recent 10-year period (1980 to 1990). All of the SMNs were outside the fields of irradiation, and none of the women had received chemotherapy. The researchers concluded that the increased relative risk of a SMN might be genetically based, as the administered treatments did not appear to be factors. The Werner-Wasik report again emphasizes the need to consider the multiple possible contributing factors to oncogenesis (see Fig. 66-7). Sturgeon and associates found an excess of SMNs among women with vulvar or vaginal cancers.104 Most of the SMNs were smokingrelated (lung, upper airways) or in patients infected with the human papillomavirus, which is known to be associated with cancers of the genital tract. A related observation by Hemminki and Dong is of interest; they found an increase in anal cancers in both women with cervical cancer and their husbands, implicating human papillomavirus in the etiology of anal cancer.105 Hall and colleagues found an increased risk of ovarian cancer as a SMN in certain sets of women.106 These were women younger than 50 years of age with melanoma or cancer of the colon, breast, cervix, uterine corpus, or ovary. The relative risks ranged from about 5 to almost 20. Bergfeldt and coworkers could not substantiate a reputed increase in breast cancers among women with ovarian cancer.107 Their casecontrol study from a pool of 5060 Swedish women led them to conclude that increased surveillance (mammography) of surviving patients with ovarian cancer was not warranted. Travis and associates likewise attributed secondary breast cancers to factors other than therapy, although they attribute soft-tissue, bladder, and rectal malignancies to RT and leukemia to chemotherapy.108 Buiatti and colleagues reported a population-based study of second primary cancers.109 They considered only the 463 metachronous SMNs that developed among the 19,252 adults with primary cancers of the colon, rectum, lung, stomach, and female breast who constituted the study population. Significantly higher risks of developing another cancer were found in patients under 65 years of age. Associations of three types were found: 1. Between primary rectal and secondary kidney cancers 2. Between colon and later ovarian malignancies 3. Between female breast and subsequent rectal cancers, although cancer in the opposite breast constituted the highest risk in this group No correlations were made between the treatments that were used for the primary tumor and the secondary cancer. This report, together with that of Werner-Wasik and associates,103 highlights the fact that SMNs should not all be assumed to have an iatrogenic basis.

GENITOURINARY TRACT. The incidence of second neoplasms resulting from RT for prostate cancer is controversial. Movsas and associates found no increase in the risk of SMN among 543 of their patients when compared with a matched set of 18,135 men derived from the Connecticut Tumor Registry.110 Most of the SMNs developed outside the RT fields and were associated with lifestyles that were predisposing to cancer. Johnstone and colleagues also found no definite increase.111 Groups using the SEER database have found

Second Malignant Neoplasms • CHAPTER 66

conflicting results.112–115 In comparison to patients who were treated only with surgery, there was a small but significantly increased risk of in-field tumors, especially bladder and rectal cancers, following RT.112,113 However Moon and colleagues also found an increased risk of second cancers outside the field and in men who were treated with transurethral resection of prostate alone.115 These results suggest the researchers did not adjust for all relevant factors. In fact, when Cox modeling was used and adjusted for attained age, no significant excess risk from RT was found.114,116 SMNs occurring in men with testicular cancers have been studied by several investigators. Wanderas and associates found an increased risk of second germ cell cancer, usually of the same histology, among 2201 patients, the risk being highest among men younger than 30 years of age at first diagnosis.117 More SMNs than expected were found by Ruther and colleagues in their multicenter collection of men with pure seminomas.118 These included both nontesticular and testicular SMNs. Other researchers, who have studied larger numbers of patients, have documented increased relative risks for gonadal and nongonadal carcinomas of 1.2 to 2.3. Higher risks were recorded for leukemia (relative risk: 2.4) and soft-tissue sarcomas (relative risk: 3.0).119 The SMNs tended to develop in irradiated fields. Travis and coworkers showed that men who were treated for testicular cancer had a persistent increased risk of second cancers (relative risk: 1.9) in a large cohort study of 40,576 survivors.120 SMNs of the lung, colon, bladder, pancreas, and stomach accounted for almost 60% of the excess cancers. The risks of cancer 40 years after treatment were 36% for seminomas and 31% for nonseminomas compared to 23% for the general population to age 75. Richiardi and associates also found an elevated risk of second cancers with a standardized incidence ratio of 1.65 compared to population controls in a cohort of 29,511 patients with testicular cancer.121 Interestingly, they found a markedly increased risk of myeloid leukemia, especially in nonseminomas that were diagnosed after 1990 (standardized incidence ratio: 38). The elevated risk in these patients could be due not only to genetic and environmental factors, but also to outdated RT techniques in which extended fields were used even for early-stage disease.122

BREAST CANCER. After a diagnosis of breast cancer, the risk of second cancers is increased in general in comparison to population controls. A study of 335,191 women with either invasive or noninvasive breast cancer revealed a second primary rate of 12% for women who were diagnosed before age 50 and 17% for women who were diagnosed over age 50.123 Noteworthy is the fact that the standardized incidence ratios for second cancers actually decreased with age, suggesting that genetic mechanisms play a role. The patterns of second cancers, namely, second breast, bone, colorectal, sarcoma, leukemia, lung, ovarian and thyroid cancer, have a shared pattern of risks with BRCA1, BRCA2, p53, and PTEN mutations.123 In a study of 491 female carriers of either BRCA1 or BRCA2 mutations, the risk of contralateral breast cancer was 29.5% at 10 years, with BRCA1 carrying a higher hazard than BRCA2.124 Studies of second malignancies after male breast cancer have similarly shown increased rates compared to population controls, especially breast, gastrointestinal, and prostate cancers, but unlike female breast cancer, the risks increased over time.125,126

In women who had been treated uniformly with wide local excision, axillary dissection, and postoperative irradiation, the cumulative 10-year SMN rate was 16% in the 1253 women who were reviewed, about half of whom had second breast malignancies.127 A 20-year analysis of 1801 similarly treated women showed a contralateral breast cancer rate of 15.4%, the majority (83%) of which were the invasive type.128 The contralateral breast does receive some radiation dose during breast RT. It is in the range that is known to be carcinogenic, especially in young women.129 A case-control study of 41,109 women with breast cancer showed an attributable contralateral breast cancer risk of only 2.7% from prior irradiation.130 Some of the more important variables that were considered in the analyses are shown in Table 66-1. An increase in relative risk was observed in irradiated women surviving 10 or more years, but only in those who had been treated when under 45 years of age (relative risk: 1.85). The risk among this sample increased with increasing RT dose, the analyzed range extending from 1.99 Gy to 4 Gy, and the relative risks from 1.54 to 2.35, respectively (P = 0.003). The use of adjuvant RT appears to increase the risk of both contralateral breast and other cancers (skin, lung, colon, and esophagus) in some,131–134 but not all studies.135 Additional evidence for an increased risk of esophageal cancer after breast cancer has been found in population-based studies.136,137 This may be attributable to the radiation field that was used to include the internal mammary nodes. Although the absolute risk from RT of breast cancer is small, the doses to the contralateral breast and mediastinum can and should be reduced by using modern RT techniques.

NERVOUS SYSTEM. Jones conducted an extensive retrospective analysis of brain tumors occurring in patients after RT for pituitary lesions, a unique group that receives relatively high doses of RT for nonmalignant lesions.138 He concluded that the risk of RT oncogenesis in adults is low after small-field RT in the doses usually used for the control of pituitary disorders. When both irradiated and unirradiated adults with pituitary tumors who developed regional fibrosarcomas, gliomas, and meningiomas were compared, the role of therapeutic RT in the genesis of such SMNs became questionable. Erfurth and associates not only find no clear evidence implicating RT, but also again raise the possibility that underlying genetic factors are responsible.139 LEUKEMIA. The risk of secondary leukemia is greatest within the first several years after radiation, whereas the risk for solid tumors generally becomes evident thereafter. Although relatively rare, an excess absolute risk of AML was detected in patients who have been treated for HD.140 The excess risk was greatest in the first 10 years after treatment and subsequently remained elevated. In cervical cancer patients, Storm described a significantly increased relative risk of acute nonlymphoblastic leukemia (relative risk: 3.5), manifest in the early postirradiation years but not of chronic lymphoblastic leukemia (CLL).102 The relative risk decreased by the 10th year after diagnosis, and the incidence approximated that of the general population by the 15th year after diagnosis. This was unlike the experience with solid tumors, in which relative risks continued to increase with time.

Table 66-1 Relative Risk of Contralateral Breast Cancer after Radiation Therapy Age at Treatment (10-Year Survivors)

No. Exposed

Total No.

Relative Risk (95% Confidence Interval)

45 yrs old

55

298

1.08 (0.74–1.57)

Data from Boice JD, Harvey EB, Blettren M, et al: Cancer in the contralateral breast after radiotherapy for breast cancer. N Engl J Med 1992;326:781.

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Intensity-Modulated Radiotherapy and Proton Radiotherapy Technological advancements in RT over the past several decades have led to safer, more tolerable treatment. Intensity-modulated radiotherapy (IMRT) has been popularized in recent years owing to the ability to shape the irradiated volume in three dimensions. This permits both a limitation of doses to adjacent normal structures and an increase in the dose to critical diseased sites that need a boost, allowing for dose escalation opportunities. A disadvantage of IMRT is higher integral body doses, resulting both from the spreading of a low dose to a greater volume and from longer treatment times with the attendant increase in incidental irradiation due to machine head leakage. Higher integral doses have led to concerns about a possible increased risk of fatal SMNs; indeed, these have been calculated to be 1.7% for conventional treatment compared to 2.1% for IMRT.141 These worries were echoed by Hall, who also pointed out a potentially increased risk of SMNs from proton RT due to neutron contamination of the proton beam.142 Proton RT has the advantage of no exit dose, but the most common method of proton field generation, passive scattering, is associated with a higher level of neutron contamination. Active scanning proton RT has less neutron contamination than passive scattering and is predicted to deliver a lower integral dose than photon IMRT. Hall’s neutron contamination calculations and SMN risk for passive scattering proton RT were widely criticized as being based on outdated equipment.143 The point still stands, however, that active scanning proton RT may provide the best conformality with the lowest risk of SMNs, despite a very high price tag.144

Radionuclide Therapy Unlike external beam RT that is aimed to a particular site, radionuclide therapy is delivered intravenously and any localization is a result of specific affinities. Although the use of radioimmunotherapy for lymphoma with radiolabeled anti-CD20 antibodies is on the rise, the most experience with radionuclide therapy has been with I-131 for thyroid cancer. In a pooled European cohort of patient with primary thyroid cancers, patients who were treated with I-131 were found to have an increased incidence of bone, soft-tissue, colorectal, and salivary gland cancers.145 A multinational record linkage study of 39,002 patients with primary thyroid cancer demonstrated an increased risk of second cancers both after and before treatment for the thyroid cancers.146 A similar observation was seen in both the SEER database and a cohort from Leiden University Medical Center, indicating that common genetic and environmental risk factors are probably more important than I-131 causation.147,148

Chemotherapy LEUKEMIA–LYMPHOMA. Most of the SMNs reported after chemotherapy have been AML or NHL. The alkylating agents were the first to be implicated in the etiology of these SMNs and remain the most frequently implicated agents.149,150 Subsequently, the leukemogenicity of the nitrosoureas was recognized.151 Greene and colleagues estimated the increased relative risk of AML in patients with brain tumors who had been given carmustine to be about 25.152 More recently, other agents have been associated with AML. These include the epipodophyllotoxins, the platinum compounds, and their combinations.25,153 AML that developed in women with ovarian cancer who had been given alkylating agents was one of the first iatrogenic chemotherapyrelated SMNs to be reported in convincing numbers.150 The relative risk reported by Reimer and associates was more than 170 for women who received alkylating agents compared with those who did not. That estimate was updated by Greene and colleagues, who found a relative risk of 110 and the excess risk of acute nonlymphoblastic leukemia to be 5.8 cases per 1000 women per year.154 Melphalan and chlorambucil were the two agents that were most strongly implicated

(relative risks of 122 and 159, respectively), the risk of leukemia being dose-related. Kaldor and coworkers published an international case-control study in 1990 of women with ovarian cancer and reported increased risks of leukemia in those who had received cyclophosphamide, chlorambucil, melphalan, thiotepa, or dihydroxybusulfan (treosulfan) as single agents.155 The relative risks, which increased with larger administered doses, ranged from 2.2 for low-dose cyclophosphamide to 23.0 for chlorambucil and melphalan and 33.0 for treosulfan in high doses as defined by the researchers. The risk estimates were relative to those of women who received only RT or surgery, no increased leukemia risk being identified in patients who had RT alone. The risk of leukemia was also increased in patients who were treated with the combination of doxorubicin and cisplatin. Kaldor and coworkers concluded that at least one of the two drugs was leukemogenic.155 The controversies that are engendered by large-scale studies of this kind are reflected in the editorial that accompanied the article and the brisk correspondence that followed.156,157 Travis and associates underscore these issues by their findings regarding the 4402 10-year survivors among the 32,251 women with ovarian cancer on whom they collected data.108 They found 1296 SMNs rather than the expected 1014 in such a cohort, the cumulative risk at 20 years of follow-up being 18.2% versus the 11.5% expected in the general population. Although leukemias appeared to be related to chemotherapy and cancers in infradiaphragmatic sites appeared to be related to RT, there were also increased risks of tumors in other sites—for example, breast cancer and ocular melanoma—that are not obviously related to the treatments that were used. Travis and associates therefore postulated genetic or other factors that predispose to ovarian cancer as being responsible.108 In this way, they echo the surmises of Werner-Wasik and colleagues concerning the genetic bases of SMNs that develop in women with cervical cancer.103 Fisher and coworkers reviewed the extensive experience that had accumulated in women who were treated for breast cancer following the protocols of the National Surgical Adjuvant Breast and Bowel Project (NSABP).158 Using data from the SEER registry for comparison, they reported a relative risk of 24.0 for AML among all patients who were treated with surgery and chemotherapy. The risk was found to be 39.3 among patients under 50 years of age compared with a relative risk of 19.9 among those age 50 or older. The relative risks of AML were 2.6 among all women who were treated with surgery only and 10.3 among those who were treated with surgery and RT. These data confirm the leukemogenicity of melphalan, the alkylating agent that was employed in the chemotherapy trials conducted by the NSABP.155 Curtis and associates reported the relative risks for acute nonlymphoblastic leukemia or myelodysplastic syndrome of 10.0 and 17.4 among unirradiated and irradiated patients with breast cancer, respectively, who were treated with an alkylating agent.159 Greene reviewed the evidence concerning the carcinogenicity of cisplatin in animals and humans and provided a concise survey of the oncogenic potential of other chemotherapeutic agents that are in common use in adults with cancer.153 He pointed out that cisplatin has many of the properties of an oncogene and is carcinogenic in animal systems, where its effects can be reversed by MESNA (2mercaptoethane-sulfonate). The evidence implicating cisplatin as a leukemogene in humans is found in situations in which it has been used together with etoposide or doxorubicin. It is not clear whether cisplatin is a cofactor in leukemogenesis when used with other drugs or whether these other drugs, rather than cisplatin, are responsible. AML, usually of French-American-British M5 morphology, which has a characteristic translocation that involves 11q23, has been detected in patients who were given topoisomerase II inhibitors.160,161 The anthracyclines, epipodophyllotoxins, and dactinomycin are such inhibitors.161 Detourmignies and colleagues implicated inhibitors of topoisomerase II in their report of therapy-associated acute promyelocytic leukemia (t-APL).84 They pointed out that the same translo-

Second Malignant Neoplasms • CHAPTER 66

cation, t(15;17), can be identified in both de novo and treatment-related acute promyelocytic leukemias that develop after therapy with drugs of this class. van Leeuwen provided an extensive analysis of AML and myelodysplasia that developed after cancer treatments of various kinds and at different ages.162 In general, the findings authoritatively confirm the observations of others in that chemotherapy was found to be more leukemogenic than was irradiation, and the latent periods for the appearance of nonlymphatic leukemias after therapy with topoisomerase II inhibitors and with alkylating agents tended to be short (4 involved lymph nodes or stage IV) after surgical resection with no evidence of disease. The outcome was much better in the treated patients compared with historical controls, with a median survival of 37.5 months vs. 12.2 months.140 Based on these results, ECOG designed a double-blinded, randomized, six-arm phase III study of various combinations of a peptide vaccine, GMCSF, and placebos of both (protocol 4697) in resected high-risk stage III and stage IV patients, which recently closed to accrual.

Adjuvant Biochemotherapy With the initial success of biochemotherapy in advanced disease,141 it seemed logical to design a trial for the high-risk adjuvant setting. Intergroup trial S0008 is comparing three cycles of biochemotherapy with 1 year of high-dose interferon alfa-2b administration in veryhigh-risk patients with stage III disease (i.e., ulcerated primary lesion with regional lymph node involvement, grossly involved or clinically palpable nodes, matted nodes, two or more involved nodes, or satellite lesions). The study continues to accrue patients, although the disappointing recently released data on biochemotherapy in patients with stage IV disease casts doubt on the utility of this intensive, toxic approach in stage III disease.142

Neoadjuvant Therapy A newer strategy involves neoadjuvant therapy for regionally advanced melanoma. A dramatic response recently was reported in patients with stage IIIB-C melanoma who were treated with standard highdose interferon alfa-2b: IV infusion daily for 4 weeks prior to curative surgical resection, followed by SC injections three times a week for 11 months.143 Of the 20 enrolled patients, 11 demonstrated an objective clinical response and 3 evidenced a complete pathologic response. Fifty percent of patients were free of recurrent disease at median follow-up of 18.5 months.143 Biochemotherapy in the neoadjuvant setting also has been evaluated in two phase II studies.144,145 Both studies showed promising results that urge further evaluation in randomized phase III setting.

Melanoma • CHAPTER 73

MANAGEMENT OF ADVANCED DISEASE Diagnosis and Evaluation When metastatic disease is suspected, the diagnosis should be pathologically confirmed whenever possible. Often this can be accomplished with a minimally invasive procedure, such as excisional biopsy, fine-needle aspiration, or core biopsy. Routine staining of the pathology slides plus immunohistochemical staining with S100, HMB-45, and anti-MART-1/Melan-A, should confirm the diagnosis of melanoma and differentiate it from other malignancies. In the rare circumstance in which it is necessary to differentiate clear cell sarcoma from melanoma, the presence of the t(12;22)(13;q13) translocation can be used to exclude the diagnosis of melanoma. Patients should be fully staged with appropriate imaging studies before therapy is initiated. All patients should undergo an MRI scan of the brain, and whole-body imaging with either spiral CT scans of the chest, abdomen, and pelvis or combined PET/CT. Patients with bony symptoms should have a bone scan, plain x-rays, or MRI, depending on the clinical assessment.

Role of Surgery in Advanced Melanoma Because highly effective chemotherapy is not available, surgery can be an appropriate treatment for isolated metastases. Surgical excision of isolated metastatic melanoma can provide quick and effective palliation and, in some cases, long-term survival.146,147 Surgical candidates should be selected carefully. Surgery should be used only in cases of accessible lesions and when the risk of perioperative morbidity is acceptable. Isolated visceral metastases, especially to the brain and lung, are amenable to surgical therapy. The same is true for both symptomatic and asymptomatic gastrointestinal metastases, as well as for lesions in the skin, subcutaneous tissues, or distant lymph nodes.

Chemotherapy DTIC is an alkylating agent that is converted to its active metabolite, 5-(3-methyl-1-triazeno) imidazole-4-carboxamide (MTIC), in the liver. As a single agent, it has been studied extensively in metastatic melanoma. In early studies by the Central Oncology Group, DTIC had an overall response rate of 20%, with 5% of patients achieving complete responses.148 Most of the patients responding to this treatment had nodal or cutaneous metastases. Subsequent randomized studies showed objective response rates of 5% to 20%.148,149 With modern antiemetic agents, DTIC is very well tolerated. Although multiple dosage schedules have been studied, 800 to 1000 mg/m2 IV over 1 hour every 3 to 4 weeks is more convenient and at least as effective as any other schedule.149 Unfortunately, DTIC has not been proven to provide a survival benefit compared with supportive care or other treatments. Temozolomide, also an alkylating agent, is a pro-drug of MTIC. In contrast to DTIC, temozolomide is orally available and penetrates the blood–brain barrier. It currently is FDAapproved for the treatment of primary brain tumors, but not for melanoma. In a phase II trial, temozolomide at a dose of 150 to 200 mg/m2 orally on days 1 to 5 in a 28-day cycle had similar activity to DTIC, with a complete response rate of 5% and an overall response rate of 21%, with some responses in the central nervous system (CNS).150 In a head-to-head comparison, the survival rates were similar—6.4 months for DTIC and 7.7 months for temozolomide.151 Both agents are well tolerated. The oral administration route for temozolomide is attractive to patients, but without FDA approval, it is not always adequately reimbursed by insurance. Temozolomide also is used on an extended schedule, 75 mg/m2 orally every day for 6 weeks followed by a 2-week break. This is the preferred dosing when concurrent external beam radiation to the CNS, or elsewhere, is indicated, since temozolomide is radiosensitizing.152,153 Studies currently are in progress to determine whether one dosing regimen is

better than the other. A phase I study showed greater drug exposure with an extended dosing regimen,154 whereas selective CD4+ lymphopenia is more common with the extended regimen.155 Consideration should be given to prophylaxis against Pneumocystis jiroveci (formerly Pneumocystis carinii) pneumonia in these patients, especially those who are receiving concomitant radiation. A long list of chemotherapeutic agents has shown low levels of activity in metastatic melanoma, including the platinum compounds cisplatin156 and carboplatin,157 BCNU,158 vindesine,159 paclitaxel,160 docetaxel,161 and vinorelbine.162 None has been shown to be superior to DTIC as a single agent, in either efficacy or toxicity profile. Twodrug combinations have not yet yielded superior results to singleagent DTIC. A small, randomized trial comparing DTIC with DTIC plus tamoxifen showed almost a doubling of the response rate with the addition of tamoxifen.163 This result, however, was not confirmed in a follow-up study.164 A three-drug combination of cisplatin, vinblastine, and DTIC showed encouraging early results in a phase III trial comparing the combination with single-agent DTIC, but unpublished follow-up data showed no advantage to the combination. Two combination chemotherapy regimens—bleomycin, vincristine, lomustine, and DTIC (BOLD) and a combination of three alkylating agents (DTIC, BCNU, and cisplatin) with tamoxifen, known as the Dartmouth regimen—showed great promise in the treatment of metastatic disease and became quite popular among clinicians during the 1980s and early 1990s. The BOLD regimen demonstrated an objective response rate of greater than 40% in a phase II trial.165 However, in a phase III study, the response rate dropped to the single digits and the regimen fell out of favor.166 In several single-institution studies of the Dartmouth regimen, very high response rates of greater than 50% were consistently reported.167 In one single-institution study, when tamoxifen was removed from the regimen to minimize the risk of deep vein thrombosis, the response rate dropped precipitously.168 In a randomized trial performed by the National Cancer Institute of Canada in which the Dartmouth regimen was compared with the Dartmouth regimen minus tamoxifen, no survival advantage was shown to occur with the addition of tamoxifen.169 However, there were more objective responses in the tamoxifen group, particularly in patients who would now be categorized as stage M1a. In an Intergroup study led by investigators at Memorial Sloan-Kettering Cancer Center, the Dartmouth regimen was compared with single-agent DTIC. In this large randomized study, no survival benefit was seen with the Dartmouth regimen. Of note, again there were more objective responses seen in the combination chemotherapy arm.149 Further, in a randomized trial comparing the Dartmouth regimen with Melacine (Corixa Corp.), a melanoma cell-lysate vaccine, alone, survival rates were equally poor in both arms, but toxicity was significantly less with the vaccine.170

Immunotherapy Since the early 1980s, two biologic therapy agents, interferon-alfa (2a and 2b) and interleukin-2 (IL-2) have been studied extensively in metastatic melanoma. In multiple studies of single-agent interferon alfa in metastatic disease, the objective response rate was approximately 15%. With single-agent interferon alfa, there are very few complete responses, and a survival benefit has never been demonstrated. Most responses are in patients with soft tissue or lymph node involvement (stage M1a).171,172 Higher, more toxic doses appear more active, but low, nontoxic doses (1 to 3 million units/m2 SC) appear inactive in the metastatic setting.172 There is no randomized clinical study showing a survival benefit when interferon-alfa is added to any single agent, such as DTIC164 or IL-2,173 or to any combination therapy regimen, including the BOLD174 and Dartmouth regimens.175 IL-2, originally known as T-cell-derived growth factor, was first reported by Rosenberg and associates176 to have significant response

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rates in metastatic melanoma when given at high doses. While it does activate several types of immune effector cells, including lymphokineactivated killer cells, natural killer cells, B and T lymphocytes, and macrophages, its mechanism of action remains unclear. Its principal anti-melanoma activity is believed to be mediated by the activation of melanoma-specific cytotoxic T cells. High-dose IL-2 has been extensively studied in metastatic melanoma. Two large series have been published using the high-dose schedule of 600,000 to 720,000 units/kg IV every 8 hours for 14 doses originally described by Rosenberg and associates.176 The National Cancer Institute reported an overall response rate of 18% and a complete response rate of 5%, with some of the responses being quite durable.177 Similar results were reported by Atkins and associates.178 The striking finding in both studies was that of a few, very durable responses, some maintained for longer than 120 months. Adoptive immunotherapy with lymphokine-activated killer cells does not appear to improve the response rate seen with high-dose IL-2 alone.179 The response rate with tumor-infiltrating lymphocytes and IL-2 was reported to be 40%, but this has not been confirmed in a phase III trial.180 High-dose IL-2 can cause substantial toxicity. The patient must be hospitalized for administration of the drug and subsequent close monitoring. Major side effects include fluid retention, renal failure, myocardial ischemia, and neurologic changes. Patients with underlying cardiac, renal, or pulmonary disease are not candidates for highdose IL-2 therapy.181 Patients with any risk factors for cardiac disease should have a normal cardiac stress thallium study before receiving high-dose IL-2. Many low-dose, alternative IL-2 regimens have been evaluated, including low-dose bolus administration, continuous infusion, and SC administration. Although these regimens are associated with less toxicity, none has shown a significant objective response rate and none has provided long-term remission. Polyethylene glycol modified-IL-2182 and liposomal IL-2183 have not shown any substantial clinical benefit. Based on the in vitro synergy between IL-2 and interferon alfa-2b in metastatic melanoma, several clinical trials have been conducted combining the two agents. Activity was seen only with doses of IL-2 that required inpatient administration.173 The hope that combined therapy with relatively nontoxic, low-dose, outpatient IL-2 and interferon would yield significant clinical activity in metastatic melanoma has not been realized.

Biochemotherapy In the early 1990s, several investigators began to study intensive regimens that included combination chemotherapy and the biologic agents interferon alfa-2b and IL-2. Single-institution studies showed significant objective response rates. Richards and colleagues184 combined BCNU, cisplatin, and DTIC with high-dose IL-2 and interferon in a sequential fashion and obtained an overall response rate of 55% and a complete response rate of 14%. Legha and colleagues141 combined the cisplatin, vinblastine, and DTIC (CVD) chemotherapy regimen with continuous-infusion IL-2 and SC interferon alfa2b, first sequentially and then in a combined regimen, with all of the drugs given over a 5-day period and the cycle repeated every 3 weeks. The combined regimen consisted of cisplatin, 20 mg/m2 IV on days 1 through 4; vinblastine, 1.5 mg/m2 IV on days 1 through 4; DTIC, 800 mg/m2 IV on day 1 only; IL-2, 9 million units/m2 daily by continuous infusion on days 1 through 4; and interferon alfa-2b, 5 million units/m2 SC on days 1 through 5, and on days 7, 9, 11, and 13; and was repeated every 21 days. The authors reported an overall response rate of 64% and a complete response rate of 21%. The combined regimen seemed as active as the more protracted sequential regimen. Two large single-institution randomized phase III studies have been reported comparing combination chemotherapy with biochemotherapy. The National Cancer Institute Surgery Branch compared

a regimen of cisplatin, DTIC, and tamoxifen with the same regimen followed immediately by high-dose IL-2 and interferon alfa-2b. The overall response rate was higher in the biochemotherapy arm, but median survival was superior in the chemotherapy arm (15.8 vs. 10.7 months).185 At M.D. Anderson Cancer Center, CVD was compared with CVD plus IL-2 and interferon alfa-2b given sequentially. The overall response rate and median survival rate were superior in the biochemotherapy arm (48% vs. 25% and 11.8 months vs. 9.5 months, respectively).186 The encouraging results reported in these studies led to a large randomized Intergroup study led by ECOG (protocol 3695).142 This study compared CVD with the combined CVD, IL-2, and interferon alfa-2b regimen of Legha,141 with several modifications, including reducing the dose of vinblastine by 25%, using prophylactic G-CSF, and limiting the number of chemotherapy cycles to four. This modified Legha biochemotherapy regimen previously was studied in a phase II trial and yielded an objective response rate of 48% and a complete response rate of 20% in 40 patients.187 In ECOG 3695, 416 patients were enrolled between 1998 and 2002 and no previous chemotherapy or IL-2 was permitted. Sixty percent of the patients had received high-dose interferon alfa-2b prior to the development of metastatic disease. The overall response rate was 17.1% in the biochemotherapy arm vs. 11.4% in the chemotherapy arm, and complete response rates were 3% vs. 1.4%, respectively. OS was equally poor in both arms of the study, 8.7 months for biochemotherapy vs. 8.4 months for chemotherapy.142 The results of this large, well-done, randomized trial were very disappointing. It may be that the reduced vinblastine dose and the lack of familiarity by physicians and nurses with the complex biochemotherapy regimen contributed to the low response rates in the cooperative group setting. However, the results show that biochemotherapy, as given in this trial, leads to very few durable responses and should not be considered a standard therapy. It is possible that when this study is published with a more detailed analysis, subsets of patients may be identified who are more likely to benefit from biochemotherapy, such as those who have an excellent performance status, low-volume disease, or no previous treatment with interferon alfa-2b. It seems unlikely that any variation of biochemotherapy currently being evaluated will prove superior to the regimen studied in ECOG 3695. O’Day and colleagues188 explored the use of maintenance biotherapy (IL-2 and GM-CSF) for patients who achieved stable disease or partial remission with biochemotherapy. One hundred thirty-three patients were enrolled and treated on a 1-year program consisting of IL-2, 1 million units/m2 SC Monday through Friday; GM-CSF, 125 µg/m2 SC, 2 weeks on and 2 weeks off; and seven 2-day IV infusions of decrescendo IL-2. The reported 12- and 24-month survival was 57% and 23%, respectively, and 12% of patients were disease-free at a median follow-up of 30 months. Although the results are intriguing, in light of the low response rates to biochemotherapy reported in the ECOG 3695 trial, it seems that only a very small number of patients would benefit from this approach.

New Therapies New treatment options are under development for patients who have advanced locoregional or widely disseminated melanoma. From a clinical and basic research perspective, melanoma occupies the crossroads of molecular biology and immunology. As an externally visible tumor, it offers a unique opportunity to investigate lesions at the earliest stages of carcinogenesis for molecular events or signatures portending progression, invasion, and dissemination. Knowledge of the genetic basis for aggressive melanoma behavior has led to the design of molecularly targeted therapies. In addition, melanoma is among the most immunogenic of all human cancers, and as such has been the prototype for defining cancer-specific antigens and developing anti-cancer immunotherapies.

Melanoma • CHAPTER 73

IMMUNOTHERAPY Cancer vaccines are a form of active immunotherapy, the effects of which depend on target-specific activation of the patient’s immune system. Vaccines directed against melanoma-associated or melanomaspecific proteins (antigens) have proved capable of enhancing antitumor immune responses in patients that can be detected in vitro, and yet have had limited clinical success in the setting of advanced metastatic disease. Most melanoma vaccine trials for advanced melanoma have been nonrandomized phase I/II studies. These have included inoculation with whole melanoma cells or gene-modified cells, heat shock proteins, naked DNA, recombinant viral vectors, recombinant proteins, synthetic peptides, and dendritic cells pulsed with peptides or cell lysates.189 Peptide vaccines have been studied most intensively, due to ease of manufacturing at relatively low cost, low potential for toxicity, and well-developed laboratory techniques for immunomonitoring. The targeted antigens have included commonly expressed cancer-testis antigens (e.g., MAGE, NY-ESO-1) and melanoma differentiation antigens (e.g., tyrosinase, gp100, MART1/Melan-A). Clinical trials have explored the best way to administer peptide vaccines, whether as a single peptide, with multiple peptides binding to HLA class I alone or to both class I and class II (eliciting both cytotoxic and helper T cell responses); with amino acid substitutions that augment their immunogenicity; or in combination with other biologic agents. However, the results of melanoma vaccine trials to date, and cancer vaccine trials overall, have been generally disappointing in the setting of advanced disease, with objective response rates of less than 5%.190 It is possible that melanoma vaccines will be more efficacious in the adjuvant setting, against microscopic disease burdens. The immunosuppressive in vivo milieu of the tumor microenvironment is now thought to play a critical role in determining the outcome of interactions between the immune system and cancer. Recent studies indicate that immune responses are tightly regulated by positive and negative signals, through receptor-ligand interactions on the cell surface. Specific interactions between costimulatory or coinhibitory receptors on resting and activated T lymphocytes (CD28 family of molecules), and their ligands on tumor cells or professional antigen presenting cells (B7 family), trigger biochemical signals leading to cascades of transcription and expression of downstream genes in the cell nucleus. These are among the earliest events regulating the initiation, differentiation, functional maturation and termination of innate and adaptive immune responses. CTLA-4, an inhibitory member of the CD28 family of molecules which binds to B7.1 and B7.2 on antigen presenting cells, has been recently targeted in phase I and II clinical trials via infusions of blocking antibodies to treat patients with advanced stage III/IV melanoma.191,192 Objective response rates (complete + partial responses) of 13%, including durable complete responses, have been observed, but a significant rate of serious autoimmune complications (approximately double the response rate) has limited the use of this agent.193 Current investigations aim to identify molecular markers that might make it possible to select patients most likely to benefit from this therapy and least likely to develop autoimmune responses. However, available clinical data indicate a significant correlation between autoimmunity and tumor regression,192,193 consistent with the mechanism of action of anti-CTLA-4. Blocking antibodies directed against other CD28 and B7 family members are under clinical development, and based on information from preclinical models they may provide a more favorable therapeutic ratio than anti-CTLA-4. These include antibodies blocking PD-1, a co-inhibitory receptor on T cells, and B7-H1, a ligand for PD-1 that is expressed on most melanomas.194 Because preliminary data have revealed PD-1 expression on highly activated melanoma-specific T cells stimulated by cancer vaccines and other means, the combination of PD-1:B7H1 blockade with immunization is envisioned for future clinical trials.

Adoptive immunotherapy, a form of passive immunotherapy involving the transfer of ex vivo-expanded tumor-specific T lymphocytes into immune-replete or depleted patients, has been under study for the past two decades as a treatment for advanced metastatic melanoma.195 Preclinical models have indicated the potential advantages of removing tumor-specific lymphocytes from the immunosuppressive in vivo milieu, and manipulating them in vitro to express a “favorable” phenotype of rapid proliferation and anti-tumor reactivity (cytokine secretion, cytolysis, highly avid T cell receptors) prior to transfer back into the autologous cancer-bearing host. For patients with at least one surgically resectable metastatic lesion, tumor infiltrating lymphocyte (TIL) therapy seems to offer the highest probability of objective clinical response. Adoptive TIL transfer in the context of lymphodepleting chemotherapy and high-dose IL-2 has yielded an objective response rate of 51%, including heavily pretreated patients who have not responded previously to high-dose IL-2 therapy.196 As expected, serious toxicities related to chemotherapy-induced cytopenias and IL-2 administration, as well as some significant autoimmune events, were encountered. For patients without resectable tumors, or whose tumors fail to yield reactive TIL, alternative T cell transfer options have been explored in the clinic. These include melanoma peptide-specific T cell clones and cell lines derived from peripheral blood,197,198 or peripheral blood lymphocytes genetically engineered to express melanoma peptide-specific T cell receptors.199 These approaches have yielded relatively low objective response rates (0 to 15%), possibly due to the lack of T cell help, failure of transferred T cells to traffic to tumor sites, expression of PD-1 or other coinhibitory receptors on T cells, or emergence of antigen-negative tumor variants. Currently, clinical development of adoptive T cell transfer still is restricted to a limited number of medical centers due to the complex and intensive nature of this treatment.

Anti-Angiogenic Agents Melanoma is a highly vascular tumor, and because new blood vessel formation is thought to play an important role in its pathobiology, the clinical use of anti-angiogenic agents is being actively investigated in advanced metastatic disease. Thalidomide, which has anti-angiogenic as well as immunomodulatory properties, is ineffective as a single agent against metastatic melanoma.200 However, recent clinical trials have investigated the activity of thalidomide in combination with extended dosing of temozolomide. In a phase II trial which excluded patients with brain metastases or prior chemotherapy, an objective response rate of 32% was observed among 38 patients with advanced stage IIIC or IV disease.201 A markedly lower response rate of 12% was observed in a phase II study using the same dosing regimen, which included patients with brain metastases and prior chemotherapy.202 Of note, a phase II trial of the same combination regimen in patients with brain metastases, with or without extracranial metastases, was discontinued prematurely because of a high rate of serious or lethal adverse events (31%), particularly thromboembolic events, in the absence of objective tumor regressions.203 Newer anti-angiogenic agents evaluated in early-phase melanoma clinical trials include the thalidomide analog lenalidomide (CC-5013, Revimid)204; the anti-avβ3 integrin antibody MEDI522205; and anti-vascular endothelial growth factor (anti-VEGF, bevacizumab, Avastin).206 Owing to the mechanism of action of this class of antineoplastic agents, it is possible that disease stabilization, rather than objective tumor regression, will prove to be the most appropriate indicator of efficacy.207

Targeted Therapies Recent scientific advances have increased our understanding of the molecular events promoting melanoma carcinogenesis and maintaining the cancer cell phenotype. Most commonly, these involve

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aberrations in the mitogen-activated protein kinase (MAPK) pathway supporting cell proliferation, the phosphatidylinositol 3′ kinase (PI3K) pro-survival pathway, and/or the melanocyte-stimulating hormone (MSH)/microphthalmia-associated transcription factor (MITF) melanocyte lineage-specific survival pathway.35,208 Knowledge of these genetic events has permitted the rational targeting of critical molecules supporting melanoma growth and survival.209 In the largest randomized trial conducted to date in patients with advanced stage III/IV melanoma, Bcl-2 antisense oligonucleotide (oblimersen, Genasense [Genta Incorporated]) was combined with DTIC in an attempt to enhance the chemosensitivity of melanoma cells by blocking pro-survival mechanisms. Studies have shown variable overexpression of Bcl-2, which inhibits the intrinsic apoptosis pathway, in melanomas. Compared to patients treated with DTIC alone, patients receiving the combination regimen showed small but statistically significant increases in objective response rate, durable response, and progression-free survival. Increased benefits were observed in a subset of patients with normal serum LDH, suggesting that such patients should be selected for future trials with oblimersen.210 BRAF is a member of the MAPK signaling pathway, which transduces extracellular signals via cell surface receptor tyrosine kinases to promote cell activation and proliferation. A somatic Val600Glu missense mutation in the kinase domain of BRAF is associated with more than 50% of melanomas, as well as with smaller percentages of some other cancers, and causes constitutive activation of the MAPK pathway contributing to melanoma progression.25 The multikinase inhibitor sorafenib (Nexavar [Onyx Pharmaceuticals]), which targets mutant and wild-type BRAF as well as c-Kit, VEGFR-2, VEGFR-3, and some other proliferation and angiogenesis receptors, recently was shown to be ineffective against advanced melanoma as a single agent.211 In addition, in 2006 the corporate drug sponsor announced failure to demonstrate improvement in PFS, the primary endpoint, in its international randomized phase III trial comparing sorafenib to placebo in combination with carboplatin and paclitaxel in chemotherapy-refractory patients with advanced melanoma.212 An ECOG trial that is of similar design but targets chemotherapy-naïve patients and has OS as its endpoint, is currently in progress. Several clinical trials exploring the efficacy of imatinib (Gleevec) in advanced melanoma have been conducted, despite varied data regarding the expression of c-Kit tyrosine kinase receptor in melanoma.213,214 The results of two phase II trials using high-dose imatinib as a single agent have shown no evidence of clinical efficacy.215,216 However, a recent report has demonstrated the association of genetic abnormalities in c-Kit (mutations, amplifications) with distinct clinical subtypes of melanoma, specifically acral melanoma, mucosal melanoma, and melanomas arising from chronically sun-exposed skin.32 Kit abnormalities were not observed in melanomas arising from skin without chronic sun damage. Of note, this pattern of genetic abnormality is distinct from that observed in melanomas with mutated NRAS/BRAF, underscoring the fact that clear genetic subtypes of melanoma exist. Thus, rational selection of patients for treatment with imatinib as well as other molecularly targeted agents in future clinical trials will depend on the identification of subsets of melanoma patients harboring the relevant genetic alterations.

Ending Treatment Patients with a poor performance status, comorbid conditions, multiple brain metastases, or advanced age are unlikely to benefit from intensive systemic therapy. These patients also may experience more side effects than healthier patients from the currently available therapies. Providing comfort measures only may be a reasonable option in some patients with metastatic melanoma. Likewise, if first-line therapy for metastatic melanoma has been unsuccessful, the patient should undergo a thoughtful assessment of his or her overall status before additional anti-tumor therapy is given. In any case, supportive

care and comfort measures are essential aspects of oncologic practice.

Special Clinical Situations in Stage IV Disease Solitary Metastasis Occasionally, patients have a solitary metastatic lesion. There have been several reports of prolonged survival after surgical resection of solitary brain, lung, and liver metastases, regardless of whether patients also received postoperative adjuvant therapy.146 A number of clinical trials are underway for patients with stage IV melanoma and no evidence of disease, and patient enrollment should be strongly considered. There is no established role for adjuvant therapy in this setting, including interferon alfa-2b, outside of a clinical trial.

Brain Metastasis Patients with brain metastases generally do poorly. Standard therapy is whole-brain irradiation, which offers some palliation. Patients with a solitary brain lesion and no disease elsewhere, or responding or slowly growing systemic disease, should be considered strongly for neurosurgical resection, stereotactic radiosurgery, or gamma-knife therapy.217 For patients with multiple brain metastases and a good performance status, consideration should be given to stereotactic radiosurgery or gamma-knife surgery, after whole-brain radiation. Those patients with measurable brain metastases also should be considered for temozolomide therapy, in addition to local measures, because this is the only systemic treatment that penetrates the blood– brain barrier. In the appropriate setting, these approaches can provide significant local control and palliation.

Unusual Problems Unknown Primary Site Patients can present with stage III or IV disease without a history of previously diagnosed melanoma. All of these patients should have a thorough skin examination, including the anal region. An eye examination is appropriate if the metastatic pattern is consistent with ocular melanoma. Unless there are specific intestinal or gynecologic signs or symptoms to suggest a mucosal primary, invasive tests, such as colonoscopy or upper endoscopy, are not recommended. Often a primary site is not found. Sometimes patients provide a history of an unusual skin lesion arising and disappearing without biopsy or treatment, or a history of a skin lesion that was cauterized or frozen. Patients who appear to have only regional lymph node involvement should undergo a potentially curative regional lymph node dissection, like any other patient with regional metastases. These patients should then be considered for systemic adjuvant therapy. Patients with isolated or disseminated metastatic disease should be treated according to the same paradigms as those with known primaries. Periodic follow-up with a dermatologist still is recommended.

Uveal Melanoma Uveal, or ocular, melanoma is rare. Although it can be morphologically similar to cutaneous melanoma, clinically it behaves differently. Primary ocular melanoma most often is treated with iodine 125 plaque brachytherapy or enucleation.218 Ocular melanoma metastasizes via a hematogenous route, often spreading primarily to the liver. Sometimes the metastatic process moves relatively slowly, with several years passing before recurrence with metastatic disease. It appears to respond less often to chemotherapy and biologic therapy, although this was not confirmed in a review of the SWOG experience.219 Some biologic correlates may explain these differences: human leukocyte antigen expression appears downregulated on ocular melanoma cells,220 the BRAF mutation is not present,221 and distinct chromosomal abnormalities, such as monosomy 3, are present.222 Although initial results using the BOLD plus interferon alfa-2b regimen in metastatic ocular melanoma were promising, these results were not

Melanoma • CHAPTER 73

confirmed in a large trial.223 Some patients with primarily liver metastases appear to benefit from chemoembolization224 or liver perfusion.225 Many clinical trials for metastatic melanoma exclude patients with ocular melanoma.

Mucosal Melanoma Primary mucosal melanoma also is rare. Patients often have advanced disease at the time of initial diagnosis because primary sites in locations such as the gastrointestinal tract or sinuses make early detection difficult. This, too, appears to be a distinct disease, although morphologically similar to cutaneous melanoma.32,35 Interestingly, a retrospective analysis showed a surprisingly good response rate of 44% for biochemotherapy in 18 patients with metastatic anorectal melanoma.226 Currently, treatment options for metastatic disease are the same as those for cutaneous metastatic melanoma.227

TREATMENT COMPLICATIONS Serious postsurgical complications are uncommon. The risks of bleeding and infection after most surgeries for melanoma are small. After sentinel node biopsy, some patients have a transient lymphocele at the site of node excision. The risk of lymphocele can be minimized by tying off the lymphatics during the sentinel node resection. If a lymphocele is large or painful, a simple office aspiration should provide adequate management. Small, asymptomatic lymphoceles can be observed and usually resolve on their own. Lymphedema has been reported in patients after sentinel node biopsy of the axilla and the inguinal area, but the incidence is low.228 Complete lymphadenectomy for regionally metastatic melanoma carries a risk of seroma, sensory loss, and lymphedema. A few patients have a seroma or prolonged drain output. Lymphedema is the most feared common complication of lymph node dissection and can occur after axillary or inguinal lymphadenectomy. The risk of lymphedema in this population in general is low, and most cases are mild to moderate and controllable with diligent care.

FOLLOW-UP AND SURVEILLANCE PLANS Most melanoma recurrences are recognized first by the patient himor herself or on a routine physical examination. A study from the Sydney Melanoma Unit reported that 73% of all first melanoma recurrences were detected by the patients themselves.229 Mooney and colleagues230 reported the results of a surveillance program using physical examination, blood tests, and chest x-rays for 1004 patients with stage I or II cutaneous melanoma. Physical examination detected 72% of recurrences, constitutional symptoms indicated 17% of recurrences, and chest x-ray showed 11% of recurrences. Among the 373 patients followed in a surveillance program at the Yale Melanoma Unit, of the 78 patients who had recurrences, 76% were diagnosed by a complete history and physical examination alone.231 Routine

laboratory or radiologic studies have never been shown to be beneficial for follow-up of patients with early stage melanoma. For patients with stage III or IV disease there is no consensus about utility of, or the optimal frequency of, routine surveillance imaging. Occasionally, false-positive lesion findings on CT scan can lead to anxiety, further diagnostic tests, or even biopsy of lesions that may be unrelated to the melanoma. CT scans are useful for evaluating suspected pulmonary, mediastinal or intra-abdominal metastases, and often are of greatest use in the evaluation of patients with symptoms that raise concern or new findings on physical examination. In patients with known distant metastases, scans are most useful when the presence of additional metastases would alter the treatment plan or when better definition of lesions is required for treatment planning or patient entry into a research protocol. FDG-PET has a sensitivity of 78% to 100% in detecting metastatic melanoma. Falsepositive findings may be seen in patients with inflammatory processes, such as sarcoid, and in those with second primary tumors.57,58,232 PET also can be used to determine the need for further diagnostic procedures, such as radiologically guided needle biopsy of suspicious, accessible lesions. In a patient with symptoms, an abnormal finding on physical examination or laboratory tests, or an abnormal x-ray, the definitive diagnosis of metastatic melanoma can be made only by a biopsy. Excisional or needle biopsy is relatively easy to perform when the suspected metastasis is easily accessible. However, in the right clinical setting, radiologic studies are sufficient for a clinical diagnosis, especially if the metastases involve more than one site and the abnormality was not present on previous studies.

ISSUES FOR THE FUTURE Today, most melanomas are found at an early stage and thus are very treatable. In fact, surgery often is the only necessary treatment. However, some melanomas are at an advanced stage when they are detected, and even some early melanomas eventually will recur. There is no routinely reliable and successful way to treat patients with advanced disease. Today the most important ways to improve melanoma-related survival are prevention and early detection. Although much progress has been made in identifying the molecular defects that are common to malignant melanocytic lesions, few of these discoveries have thus far been translated to effective therapies. It is expected that further research in the molecular genetics and the immunology of melanoma will result in an explosion of information about the events governing melanoma onset and progression. Given the limited pathologic materials available for study, multiinstitutional collaboration will be required to substantially improve our understanding of the pathogenesis of melanoma. Ultimately, these discoveries will provide the framework for the design of targeted therapies for future melanoma patients.

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metastatic malignant melanoma. Clin Res 1977; 25:411. Evans LM, Casper ES, Rosenbluth R: Phase II trial of carboplatin in advanced malignant melanoma. Cancer Treat Rep 1987;71:171–172. Ramirez G, Wilson W, Grage T, et al: Phase II evaluation of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU; NSC-409962) in patients with solid tumors. Cancer Chemother Rep 1972;56:787–790. Retsas S, Newton KA, Westbury G: Vindesine as a single agent in the treatment of advanced malignant melanoma. Cancer Chemother Pharmacol 1979;2:257–260. Legha SS, Ring S, Papadopoulos N, et al: A phase II trial of taxol in metastatic melanoma. Cancer 1990;65:2478–2481. Einzig AI, Schuchter LM, Recio A, et al: Phase II trial of docetaxel (Taxotere) in patients with metastatic melanoma previously untreated with cytotoxic chemotherapy. Med Oncol 1996;13:111– 117. Feun LG, Savaraj N, Hurley J, et al: A clinical trial of intravenous vinorelbine tartrate plus tamoxifen in the treatment of patients with advanced malignant melanoma. Cancer 2000;88:584–588. Cocconi G, Bella M, Calabresi F, et al: Treatment of metastatic malignant melanoma with dacarbazine plus tamoxifen. N Engl J Med 1992; 327:516–523. Falkson CI, Ibrahim J, Kirkwood JM, et al: Phase III trial of dacarbazine versus dacarbazine with interferon alpha-2b versus dacarbazine with tamoxifen versus dacarbazine with interferon alpha-2b and tamoxifen in patients with metastatic malignant melanoma: an Eastern Cooperative Oncology Group study. J Clin Oncol 1998;16: 1743–1751. Seigler HF, Lucas VS Jr, Pickett NJ, et al: DTIC, CCNU, bleomycin and vincristine (BOLD) in metastatic melanoma. Cancer 1980;46:2346–2348. Chemotherapy of disseminated melanoma with bleomycin, vincristine, CCNU, and DTIC (BOLD regimen). The Prudente Foundation Melanoma Study Group. Cancer 1989;63:1676–1680. Del Prete SA, Maurer LH, O’Donnell J, et al: Combination chemotherapy with cisplatin, carmustine, dacarbazine, and tamoxifen in metastatic melanoma. Cancer Treat Rep 1984;68:1403–1405. McClay EF, Mastrangelo MJ, Sprandio JD, et al: The importance of tamoxifen to a cisplatincontaining regimen in the treatment of metastatic melanoma. Cancer 1989;63:1292–1295. Rusthoven JJ, Quirt IC, Iscoe NA, et al: Randomized, double-blind, placebo-controlled trial comparing the response rates of carmustine, dacarbazine, and cisplatin with and without tamoxifen in patients with metastatic melanoma. National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 1996;14:2083–2090. Mitchell MS, Von Eschen KB. Phase III trial of Melacine melanoma vaccine versus combination chemotherapy in the treatment of stage IV melanoma. JCO : ASCO Annual Meeting Proceeding 1997;16:1778. Krown SE, Burk MW, Kirkwood JM, et al: Human leukocyte (alpha) interferon in metastatic malignant melanoma: the American Cancer Society phase II trial. Cancer Treat Rep 1984;68:723– 726. Creagan ET, Ahmann DL, Green SJ, et al: Phase II study of recombinant leukocyte A interferon (rIFN-alpha A) in disseminated malignant melanoma. Cancer 1984;54:2844–2849. Sparano JA, Fisher RI, Sunderland M, et al: Randomized phase III trial of treatment with highdose interleukin-2 either alone or in combination with interferon alfa-2a in patients with advanced melanoma. J Clin Oncol 1993;11:1969–1977.

Melanoma • CHAPTER 73 biochemotherapy (BCT) for patients with 174. Vuoristo MS, Grohn P, Kellokumpu-Lehtinen P, metastatic melanoma. JCO : ASCO Annual et al: Intermittent interferon and Meeting Proceeding 2005:7503. polychemotherapy in metastatic melanoma. J 189. Ribas A, Butterfield LH, Glaspy JA, et al: Current Cancer Res Clin Oncol 1995;121:175–180. developments in cancer vaccines and cellular 175. Feun LG, Savaraj N, Moffat F, et al: Phase II trial immunotherapy. J Clin Oncol 2003;21:2415– of recombinant interferon-alpha with BCNU, 2432. cisplatin, DTIC and tamoxifen in advanced malignant melanoma. Melanoma Res 1995;5:273– 190. Rosenberg SA, Yang JC, Restifo NP: Cancer immunotherapy: moving beyond current vaccines. 276. Nat Med 2004;10:909–915. 176. Rosenberg SA, Lotze MT, Muul LM, et al: 191. Ribas A, Camacho LH, Lopez-Berestein G, et al: Observations on the systemic administration of Antitumor activity in melanoma and anti-self autologous lymphokine-activated killer cells and responses in a phase I trial with the anti-cytotoxic recombinant interleukin-2 to patients with T lymphocyte-associated antigen 4 monoclonal metastatic cancer. N Engl J Med 1985;313:1485– antibody CP-675,206. J Clin Oncol 2005;23: 1492. 8968–8977. 177. Rosenberg SA, Yang JC, White DE, et al: Durab192. Attia P, Phan GQ, Maker AV, et al: ility of complete responses in patients with metaAutoimmunity correlates with tumor regression in static cancer treated with high-dose interleukin-2: patients with metastatic melanoma treated with identification of the antigens mediating response. anti-cytotoxic T-lymphocyte antigen-4. J Clin Ann Surg 1998;228:307–319. Oncol 2005;23:6043–6053. 178. Atkins MB, Lotze MT, Dutcher JP, et al: High193. Beck KE, Blansfield JA, Tran KQ, et al: dose recombinant interleukin 2 therapy for Enterocolitis in patients with cancer after patients with metastatic melanoma: analysis of 270 antibody blockade of cytotoxic T-lymphocytepatients treated between 1985 and 1993. J Clin associated antigen 4. J Clin Oncol 2006;24:2283– Oncol 1999;17:2105–2116. 2289. 179. Rosenberg SA. Immunotherapy of patients with 194. Chen L: Co-inhibitory molecules of the B7-CD28 advanced cancer using IL-2 alone or in family in the control of T-cell immunity. Nat Rev combination with lymphokine activated killer cells. Immunol 2004;4:336–347. In DeVita VT, Hellman S, Rosenberg SA (eds): Important Advances in Oncology. Philadelphia, JB 195. Ho WY, Blattman JN, Dossett ML, et al: Adoptive immunotherapy: engineering T cell Lippincott, 1988, pp 217–257. responses as biologic weapons for tumor mass 180. Rosenberg SA, Yannelli JR, Yang JC, et al: destruction. Cancer Cell 2003;3:431–437. Treatment of patients with metastatic melanoma 196. Dudley ME, Wunderlich JR, Yang JC, et al: with autologous tumor-infiltrating lymphocytes Adoptive cell transfer therapy following nonand interleukin 2. J Natl Cancer Inst 1994;86: myeloablative but lymphodepleting chemotherapy 1159–1166. for the treatment of patients with refractory 181. Schwartzentruber DJ: Interleukin-2: Clinical metastatic melanoma. J Clin Oncol 2005;23:2346– applications, principles of administration and 2357. management of side effects. In Rosenberg SA (ed): 197. Yee C, Thompson JA, Byrd D, et al: Adoptive T Biologic Therapy of Cancer. Philadelphia, cell therapy using antigen-specific CD8+ T cell Lippincott, Williams & Wilkins, 2000, pp 32–50. clones for the treatment of patients with metastatic 182. Yang JC, Topalian SL, Schwartzentruber DJ, et al: melanoma: in vivo persistence, migration, and The use of polyethylene glycol-modified antitumor effect of transferred T cells. Proc Natl interleukin-2 (PEG-IL-2) in the treatment of Acad Sci USA 2002;99:16168–16173. patients with metastatic renal cell carcinoma and 198. Powell DJ Jr, Dudley ME, Hogan KA, et al: melanoma. A phase I study and a randomized Adoptive transfer of vaccine-induced peripheral prospective study comparing IL-2 alone versus IL-2 blood mononuclear cells to patients with metastatic combined with PEG-IL-2. Cancer 1995;76:687– melanoma following lymphodepletion. J Immunol 694. 2006;177:6527–6539. 183. Adler A, Schachter J, Barenholz Y, et al: Allogeneic 199. Morgan RA, Dudley ME, Wunderlich JR, et al: human liposomal melanoma vaccine with or Cancer regression in patients after transfer of without IL-2 in metastatic melanoma patients: genetically engineered lymphocytes. Science clinical and immunobiological effects. Cancer 2006;314:126–129. Biother 1995;10:293–306. 200. Reiriz AB, Richter MF, Fernandes S, et al: Phase II 184. Richards JM, Mehta N, Ramming K, et al: study of thalidomide in patients with metastatic Sequential chemoimmunotherapy in the treatment malignant melanoma. Melanoma Res 2004;14: of metastatic melanoma. J Clin Oncol 1992;10: 527–531. 1338–1343. 201. Hwu WJ, Krown SE, Menell JH, et al: Phase II 185. Rosenberg SA, Yang JC, Schwartzentruber DJ, et study of temozolomide plus thalidomide for the al: Prospective randomized trial of the treatment of treatment of metastatic melanoma. J Clin Oncol patients with metastatic melanoma using 2003;21:3351–3356. chemotherapy with cisplatin, dacarbazine, and 202. Laber DA, Okeke RI, Arce-Lara C, et al: A phase tamoxifen alone or in combination with II study of extended dose temozolomide and interleukin-2 and interferon alfa-2b. J Clin Oncol thalidomide in previously treated patients with 1999;17:968–975. metastatic melanoma. J Cancer Res Clin Oncol 186. Eton O, Legha SS, Bedikian AY, et al: Sequential 2006;132:611–616. biochemotherapy versus chemotherapy for 203. Krown SE, Niedzwiecki D, Hwu WJ, et al: metastatic melanoma: results from a phase III Phase II study of temozolomide and thalidomide randomized trial. J Clin Oncol 2002;20:2045– in patients with metastatic melanoma in the 2052. brain: high rate of thromboembolic events 187. McDermott DF, Mier JW, Lawrence DP, et al: A (CALGB 500102). Cancer 2006;107:1883– phase II pilot trial of concurrent biochemotherapy 1890. with cisplatin, vinblastine, dacarbazine, interleukin 204. Bartlett JB, Michael A, Clarke IA, et al: Phase I 2, and interferon alpha-2B in patients with metastudy to determine the safety, tolerability and static melanoma. Clin Cancer Res 2000;6:2201– immunostimulatory activity of thalidomide 2208. analogue CC-5013 in patients with metastatic 188. O’Day S, Atkins M, Weber J, et al: A phase II malignant melanoma and other advanced cancers. multi-center trial of maintenance biotherapy Br J Cancer 2004;90:955–961. (MBT) after induction concurrent

205. McNeel DG, Eickhoff J, Lee FT, et al: Phase I trial of a monoclonal antibody specific for alphavbeta3 integrin (MEDI-522) in patients with advanced malignancies, including an assessment of effect on tumor perfusion. Clin Cancer Res 2005;11:7851–7860. 206. Carson WE, Biber N, Shah K, et al: A phase II trial of recombinant humanized monoclonal antivascular endothelial growth factor (VEGF)antibody in patients with metastatic melanoma. JCO : ASCO Annual Meeting Proceeding 2003;22:2873. 207. Yang JC, Haworth L, Sherry RM, et al: A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 2003;349:427–434. 208. Garraway LA, Widlund HR, Rubin MA, et al: Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature 2005;436:117–122. 209. Fecher LA, Cummings SD, Keefe MJ, et al: Toward a molecular classification of melanoma. J Clin Oncol 2007;25:1606–1620. 210. Bedikian AY, Millward M, Pehamberger H, et al: Bcl-2 antisense (oblimersen sodium) plus dacarbazine in patients with advanced melanoma: the Oblimersen Melanoma Study Group. J Clin Oncol 2006;24:4738–4745. 211. Eisen T, Ahmad T, Flaherty KT, et al: Sorafenib in advanced melanoma: a Phase II randomised discontinuation trial analysis. Br J Cancer 2006;95:581–586. 212. Bayer Pharmaceuticals Corporation and Onyx Pharmaceuticals. Phase III trial of Nexavar in patients with advanced melanoma does not meet primary endpoint. December 4, 2006. Available from: http://www.onyx-pharm.com/wt/page/pr_ 1165242111. 213. Potti A, Moazzam N, Langness E, et al: Immunohistochemical determination of HER-2/neu, c-Kit (CD117), and vascular endothelial growth factor (VEGF) overexpression in malignant melanoma. J Cancer Res Clin Oncol 2004;130:80–86. 214. Janku F, Novotny J, Julis I, et al: KIT receptor is expressed in more than 50% of early-stage malignant melanoma: a retrospective study of 261 patients. Melanoma Res 2005;15:251–256. 215. Ugurel S, Hildenbrand R, Zimpfer A, et al: Lack of clinical efficacy of imatinib in metastatic melanoma. Br J Cancer 2005;92:1398–1405. 216. Wyman K, Atkins MB, Prieto V, et al: Multicenter Phase II trial of high-dose imatinib mesylate in metastatic melanoma: significant toxicity with no clinical efficacy. Cancer 2006;106: 2005–2011. 217. Gaudy-Marqueste C, Regis JM, Muracciole X, et al: Gamma-Knife radiosurgery in the management of melanoma patients with brain metastases: a series of 106 patients without whole-brain radiotherapy. Int J Radiat Oncol Biol Phys 2006;65:809–816. 218. Collaborative Ocular Melanoma Study Group. The COMS randomized trial of iodine 125 brachytherapy for choroidal melanoma: V. Twelveyear mortality rates and prognostic factors: COMS report No. 28. Arch Ophthalmol 2006;124:1684– 1693. 219. Flaherty LE, Unger JM, Liu PY, et al: Metastatic melanoma from intraocular primary tumors: the Southwest Oncology Group experience in phase II advanced melanoma clinical trials. Am J Clin Oncol 1998;21:568–572. 220. Krishnakumar S, Abhyankar D, Lakshmi SA, et al: HLA class II antigen expression in uveal melanoma: correlation with clinicopathological features. Exp Eye Res 2003;77:175–180. 221. Cohen Y, Goldenberg-Cohen N, Parrella P, et al: Lack of BRAF mutation in primary uveal melanoma. Invest Ophthalmol Vis Sci 2003;44:2876–2878.

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225. Carroll NM, Alexander HR Jr: Isolation perfusion of the liver. Cancer J 2002;8:181–193. 226. Kim KB, Sanguino AM, Hodges C, et al: Biochemotherapy in patients with metastatic anorectal mucosal melanoma. Cancer 2004;100:1478–1483. 227. Tomicic J, Wanebo HJ: Mucosal melanomas. Surg Clin North Am 2003;83:237–252. 228. Wrone DA, Tanabe KK, Cosimi AB, et al: Lymphedema after sentinel lymph node biopsy for cutaneous melanoma: a report of 5 cases. Arch Dermatol 2000;136:511–514. 229. Francken AB, Shaw HM, Accortt NA, et al: Detection of first relapse in cutaneous melanoma patients: implications for the formulation of

evidence-based follow-up guidelines. Ann Surg Oncol 2007;14:1924–1933. 230. Mooney MM, Kulas M, McKinley B, et al: Impact on survival by method of recurrence detection in stage I and II cutaneous melanoma. Ann Surg Oncol 1998;5:54–63. 231. Poo-Hwu WJ, Ariyan S, Lamb L, et al: Follow-up recommendations for patients with American Joint Committee on Cancer Stages I–III malignant melanoma. Cancer 1999;86:2252–2258. 232. Holder WD Jr, White RL Jr, Zuger JH, et al: Effectiveness of positron emission tomography for the detection of melanoma metastases. Ann Surg 1998;227:764–769.

74

Nonmelanoma Skin Cancers: Basal Cell and Squamous Cell Carcinomas Gary S. Wood, Juliet Gunkel, Daniel Stewart, Ellen Gordon, Mamad M. Bagheri, Manish Gharia, and Stephen N. Snow S U M M ARY

Incidence • 1 million new cases occur annually including 80% basal cell carcinomas (BCCs) and 20% squamous cell carcinomas (SCCs). • Incidence is increasing 2% to 3% per year. • SCC incidence is increased 18- to 36fold in organ transplant recipients.

Etiology and Epidemiology • Ultraviolet radiation from sun exposure is a major risk factor and causes mutations in key genes. • Hedgehog signaling pathway mutations are involved in BCC pathogenesis. • p53 mutations are involved in both SCC and BCC pathogenesis, as well as in the development of actinic keratoses, which are the precursors of SCCs.

Pathology and Biology • Several histopathologic subtypes exist. • The more infiltrative or poorly differentiated variants are more clinically aggressive (e.g., morpheaform BCC and spindle cell SCC).

Clinical Findings • BCC and SCC are found mainly on sun-exposed skin. • Classic BCC is a pearly, telangiectatic, variably ulcerated nodule or a pale, sclerotic plaque.

O F

K EY

P OI NT S

• Classic SCC is a flesh-tone or red, variably keratotic, variably ulcerated nodule.

Differential Diagnosis and Staging • Amelanotic melanoma, keratoacanthoma, cutaneous metastasis, cutaneous lymphoma, cutaneous lymphoid hyperplasia, adnexal tumor, Merkel cell carcinoma, and sebaceous gland carcinoma are included in the differential diagnosis. • BCCs that are large, deep, or infiltrative may be locally aggressive and recurrent but metastasize only rarely (40

1

≤40

1.89

1.12–3.19

0.026

1.11–2.16

0.012

0.024

Method of detection X-ray finding only

1

Clinical symptoms

1.55

Histologic type Well

1

Intermediate

1.85

1.18–2.90

Poor

1.61

0.93–2.79

Architecture Clinging/micropapillary

1

Cribriform

2.39

1.41–4.03

Solid/comedo

2.25

1.21–4.18

0.002

Margins Free

1

Not free

1.84

1.32–2.56

0.0005

1.33–2.49

0.0002

Treatment LE + RT

1

LE

1.82

LE, local excision; RT, radiotherapy. From Bijker N, Meijnen P, Peterse JL, et al: Breast-conserving treatment with or without radiotherapy in ductal carcinoma-in-situ: ten-year results of European Organisation for Research and Teatment of Cancer randomized phase III trial 10853— a study by the EORTC Breast Cancer Cooperative Group and EORTC Radiotherapy Group. J Clin Oncol 2006;24:3381–3387.

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Part III: Specific Malignancies

DCIS at the genetic level is the goal of many ongoing studies. However, little is known about this biology.

Treatment Most cases of DCIS are diagnosed by mammography. Approximately 72% of cases are detected by calcification alone, 12% by calcification and a soft tissue density, and 10% by a soft tissue abnormality. The surgeon, when offering the patient a breast-conserving approach, must be confident that the remaining breast is free of suspicious mammographic abnormalities. Other clusters of microcalcification within the breast take on greater significance after a diagnosis of DCIS is established, and biopsy of these may be appropriate. Coordination between the pathologist and the breast-imager is very important in documenting the relationship between calcium observed on film and that observed in the specimen. When a patient has extensive areas of suspicious calcification, the entire area must be cleared surgically to ensure the success of breast conservation. When this is not possible, simple mastectomy with reconstruction, if the patient desires, is appropriate local treatment. Recent experiences with combined breast excision and plastic restoration (oncoplastic tecniques) showed the possiblity of complete removal of relatively large areas of microcalcifications with acceptable cosmetic results.168 Guidelines for DCIS therapy have been influenced significantly by a number of retrospective reports on follow-up of patients treated with excision only or with excision plus irradiation. More importantly, these retrospective study results have been used to design large, prospective phase III trials. The need for radiation after wide excision first was tested prospectively by the NSABP group in a randomized trial.169 This study randomized 818 women with DCIS to receive lumpectomy, with or without radiation. Patients were stratified by method of detection (i.e., mammogram only or palpable mass) as well as by age. Negative tumor margins were required, and were defined as no DCIS seen at the inked margin. After a median of 12 years of follow-up, the local failure rate in the index breast was 17% in women randomized to receive radiation, compared with 39.7% in the observed group. However, this significant difference in local control did not translate to any difference in survival for the women in this trial.170 A similar large phase III trial comparing radiation with observation after wide local excision for women with DCIS was undertaken by the EORTC group (see Biology of DCIS). This study randomized 1010 women to lumpectomy and radiation versus lumpectomy only, showing a rate of local control similar to that of the NSABP B-17 study. With a median follow-up of 10 years, the local control rate was 74% in the excision-only group, compared with 85% in women randomized to radiation. The risk of DCIS and invasive LR was reduced by 48% (P = 0.0011) and 42% (P = 0.0065), respectively. Both groups had similar low risks of metastases and death. At multivariate analysis, factors significantly associated with an increased LR risk are shown in Table 95-11, and the effect of the radiation was seen across all risk factors.167 A randomized trial of 1046 women, all undergoing mammographic screening in Sweden, comparing whole breast radiation to observation, showed similar results as well.171 With a median followup time of 5.2 years, the cumulative incidence of local recurrences was 7% in the RT group compared with 22% in the observed group. As with both the NSABP and the EORTC trials, no differences were seen in the SweDCIS study in terms of disease-specific survival. Several breast surgeons published individual series of patients with DCIS treated with wide excision only. Patient selection clearly plays a role in this kind of reporting. Nonetheless, local control rates in these retrospective studies are quite good, and the results generated another series of cooperative group trials. Silverstein’s series from Van Nuys, using an index of the same name, related outcome to the following factors: size, pathology grade, patient age, and surgical margin width.172,173 Several U.S. trials testing these hypotheses recently were closed. This included the Eastern Cooperative Oncology Group

(ECOG) 5194 observation-only registry trial, which showed that carefully selected patients with low- or intermediate-grade DCIS treated with lumpectomy without irradiation have an acceptably low risk of ipsilateral events at 5 years.174 The Radiation Therapy Oncology Group (RTOG) recently closed the accrual of a phase III trial that randomized selected low-risk patients with DCIS to wide excision, with or without radiation. Final sample size trial was less than initially planned due to low accrual rate (600 women enrolled compared to the target of 1800). In a Danish nationwide prospective study of in situ carcinoma of the breast, a total of 275 women treated with excision alone were registered from 1982 to 1989. Within a median follow-up of 120 months, a crude recurrence rate of 28% was found, of which 53% recurred as invasive carcinomas and 47% as in situ cancers.175 Thus, this issue of identifying a “good risk” group of women with DCIS who can be observed without the addition of radiation remains controversial. In general, a woman’s risk of local failure in the index breast after wide excision appears to be reduced by a factor of approximately one half by the addition of whole-breast radiation. Because survival remains excellent, whatever local treatment is selected, involving the patient in a decision, based on her assessed risk and the relative benefits and risks of radiation, appears appropriate for women in lowerrisk groups. These groups are defined by older age, tumor grade, size, and margin width, and likely by other factors yet to be described. Several trials comparing lumpectomy plus radiation versus mastectomy are summarized in Table 95-12. The NSABP B-24 study tested the role of tamoxifen, in addition to lumpectomy and radiation therapy, in a prospective trial of 1804 women. With a median follow-up of 74 months, with the addition of tamoxifen to 50 Gy radiation, the risk of ipsilateral invasive recurrence was further reduced by approximately 50%. However, the absolute risk reduction was only 3%, and no significant reduction in noninvasive recurrence was seen. Predictably, the addition of tamoxifen decreased the risk of a contralateral breast event by approximately 1.5%.176 A 2 × 2 factorial design randomized trial in the United Kingdom, Australia, and New Zealand randomized 1701 patients to receive, after lumpectomy, either tamoxifen alone, tamoxifen plus radiation therapy, radiation therapy alone, or nothing. At a median of 52.6 months, ipsilateral invasive disease was not reduced by tamoxifen, but recurrence of overall ductal carcinoma in situ was decreased (hazard ratio 0.68 [0.49–0.96]. Radiotherapy reduced the incidence of ipsilateral invasive disease (0.45 [0.24–0.85]) and ipsilateral ductal carcinoma in situ (0.36 [0.19–0.66]).177 The NASBP and the International Breast Cancer Intervention Study (IBIS) groups are independently evaluating the aromatase inhibitors in preventing local recurrences after lumpectomy and RT for DCIS. In these trials the patients are randomized to take anastrazole or tamoxifen for 5 years. Male breast cancer accounts for only approximately 1% of all cases of breast cancer. Men with DCIS are estimated to be only 7% of that already small group, so aside from case reports, little information exists on the management of this unusual entity.178 Total mastectomy or wide excision with free margin may be considered a reasonable treatment.

Sentinel Lymph Node Biopsy in Ductal Carcinoma in Situ Investigators are now questioning whether the SLNB technique, well established for the staging of invasive breast cancer, may have a role in the staging of DCIS. Several studies have documented a high incidence of lymph node micrometastases detected by SLN biopsy in patients with high-risk DCIS and DCISM (DCIS with microinvasion). Although the biologic significance of breast cancer micrometastases remains unclear at this time, these findings suggest that SLNB should be considered in patients with high-risk DCIS and DCISM. One reason for the high rate of detection of positive SLNs in DCIS is that DCIS of the breast is defined as stage 0 disease, without invasion, but its diagnosis is subject to sampling errors. A positive SLN

Cancer of the Breast • CHAPTER 95

Table 95-12 Randomized Trials Comparing Lumpectomy Plus Radiation to Mastectomy SURVIVAL (%) Trial

No. of Patients

Maximum Tumor Size (cm)

Follow-up (yr)

Mastectomy

Lumpectomy

NSABP

62

1217

4

12

60

Institut Gustave-Roussy

179

2

14.5

65

73

Milan

701

2

16

71

72

EORTC

874

5

8

73

71

Danish

618

5

6

82

79

NCI

237

5

10

75

77

SENTINEL LYMPH NODE MAPPING Radioisotope Europe: 99m Tc colloidal albumin (unfiltered) [ideal particle size 10–200 nm] 0.1–0.6 mCi/4 mL normal saline injected subdermally or into the breast. United States: 99 mTc sulfur colloid 0.3–1.0 mCi in 4 mL normal saline is injected subdermally or into the breast 1 to 4 hours before surgery; a handheld gamma probe is used. Between 4 and 5 mL isosulfan blue dye is injected subdermally beneath the areola or into the breast parenchyma on the axillary side or at the tumor site. Breast is massaged for 5 minutes before the low axilla is explored. EORTC, European Organization for Research on the Treatment of Cancer; NCI, National Cancer Institute; NSABP, National Surgical Adjuvant Breast and Bowel Project.

may raise the question of whether invasion was missed at the breast pathology examination, and thereby reduce the sampling error. Another point in favor of SLNB in DCIS is the fact that the diagnosis of pure DCIS often is made subsequent to surgical excision of the lesion. Some patients with lesions considered to be at high risk for invasion based on clinical, pathological, or mammographic criteria may benefit from a SLNB if final pathology shows invasion. Some criteria for predicting invasion can help decide whether to perform a SLNB. On mammography, DCIS is generally evident as calcifications, whereas a mass often is seen in the setting of an invasive carcinoma. In patients with DCIS, the presence of extensive calcification or an associated mass/lesion at mammography suggests a greater likelihood of an invasive component. High-grade DCIS with comedo necrosis on core biopsy frequently shows invasion at the final pathology of the surgical excision. Consideration should be given to SLNB in patients who are at high risk for invasion or who are undergoing a mastectomy. This would include patients with pathology that is either suspicious or diagnostic for microinvasion and cases that involve the presence of lymphovascular invasion, or the presence of a palpable or mammographic mass. In women who undergo mastectomy, a SLNB can be justified due to its associated lower morbidity, additional pathology attention to the lower number of lymph nodes submitted, and the potential avoidance of a formal ALND if the SLNB procedure shows no nodal involvement should a focus of invasion be identified in the breast specimen.179

MANAGEMENT OF EARLY-STAGE BREAST CANCER The management of early invasive breast cancer is multidisciplinary and must involve specialists in breast imaging, pathology, surgical oncology, radiation oncology, medical oncology, and reconstructive surgery. More than half of all women in the United States (including African-American women) who are diagnosed with breast cancer present with early-stage breast cancer,3 often 2 cm or less, and most are potential candidates for breast conservation (lumpectomy and radiation) assuming adequate surgical margins and cosmetic outcome.180 Preoperative systemic therapy (PST) is a reasonable option to consider for women with operable breast cancer who are not candidates for breast conservation due to large tumor size and/or small breast size at presentation. However, the absolute increase in

the number of women ultimately treated with breast conservation after PST is less than 10%. Available data show no clear survival benefit for preoperative systemic therapy.181 Not all breast cancers have systemic involvement at presentation,182 and maximization of local control with radiation therapy also may reduce the subsequent risk of distant relapse and improve survival.183 Decisions about adjuvant systemic therapy first must take into account the accurate determination of the expression of any predictive markers that identify potential candidates for therapy centered around anti-estrogens (if ER- and/or PR-positive disease),184 trastuzumab (if HER2-positive disease),75 or chemotherapy (if triple negative disease). Estimates of risk based on more traditional prognostic markers (e.g., tumor size and degree of lymph node involvement) are then added to help estimate the absolute improvement offered by the specific systemic therapies in question. Some measures of gene expression profiling appear to offer predictive utility to help with therapy selection.103

Breast-Conservation Therapy with Surgery and Radiation Fortunately for most patients, the appropriateness of breast conservation is one of the most studied treatment decisions in modern medicine. Pioneering investigators in the 1920s and 1930s began to treat groups of women with breast-conserving partial mastectomy, followed by irradiation to the intact breast, challenging the need for total mastectomy. Results from these early studies were promising. Many single institutions initiated programs, including excision plus radiation therapy, for their patients with breast cancer, first in European and Canadian centers and later in the United States. In the early 1970s, several European reports created worldwide interest in nonmastectomy treatment based on individual series that suggested local recurrence rates of 5% to 10% and similar survival. These single-institution studies caused considerable controversy between breast surgeons who believed in the mastectomy as a local treatment and those that embraced breast conservation. This, in turn, led to six randomized prospective trials using megavoltage radiation techniques comparing lumpectomy plus radiation with mastectomy, which were carried out with cooperative groups in both Europe and the United States. A meta-analysis of these trials noted that survival at the 10-year mark in the conserved group was similar to the mastectomy group with a survival trend among lymph node-positive

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Part III: Specific Malignancies

patients.185 Both the NSABP-B 06 Trial180 and the Milan Cancer Institute Trial186 have reported 20-year results showing equivalent outcomes. The NSABP B-06 Trial was unique, in that it randomized women to three treatment arms—the mastectomy arm, the breast conservation arm with radiation, and a breast conservation arm without radiation. The in-breast local failure rate without the additon of radiation approached 40% in that trial. Although initial analyses did not demonstrate a relationship between local in-breast failure and distant metastases, a recent report from the NSABP, focusing on node-positive women receiving breast conservation therapy including radiation, treated on a series of five trials, now shows a significant hazard ratio for mortality associated with local in-breast failure.187 Thus, maximization of the chance of local control in this group of women is now associated with survival as well as in-breast failure. The BCT data were so convincing that in 1990, the NCI held a consensus development conference on the treatment of early breast cancer and declared that breast-sparing therapy not only was equivalent to mastectomy but actually was the “preferable” treatment, because it preserved the breast, with all of the attendant psychological and body image advantages associated with a lesser surgical procedure.

Resection of the Primary Lesion Resection of the primary cancer for therapeutic purposes has different definitions accordingly to different authors. Lumpectomy, wide excision, tumorectomy, segmentectomy, and quadrantectomy are different terms to indicate excision of the cancer with “healthy “ surrounding parenchyma. The European segmentectomy and quadrantectomy were characterized by a larger amount of breast parenchyma removal compared with surgical procedures in the United States, at least in the past.188 The concept of large excisions has been progressively replaced by the concept of free margins. An excision that achieves negative margins for invasive cancer usually is considered sufficient. Numerous reports demonstrate that the margin status appears to influence the risk of recurrence, but still controversial is a definition of the free margin distance, defined as the safe distance of the cancer from the margins. Some authors have correlated the microscopic margin distance to the risk of local recurrences, showing that the distance is proportional to the risk of local recurrence.189 The concept of free margins has been extended to the intraductal component when associated with the invasive cancer. Although the risk of local recurrence decreases in direct proportion to the extent of resection, as expected, the cosmetic result also declines with more aggressive resection. When margins are positive or close (many authors define close margins as < 2 mm), the ideal approach is reexcision of the previous biopsy site or reexcision of the involved/close margin, when specified. For reexcision, sharp dissection is preferred. Electrocautery should be avoided until the specimen is removed to avoid cautery artifacts that might obscure margin information. Patients must be thoroughly informed about the indications for reexcision and the possibility that the histologic features of the specimen will dictate a change in therapy. For example, a reexcision specimen sometimes contains extensive or multifocal intraductal cancer. Some of these patients have intraductal cancer that is not marked by calcification extending into unknown areas of the breast. If all apparent intraductal cancer cannot be removed surgically, the risk of local failure can become unacceptably high and dictates planning for mastectomy and reconstruction. Once the tumor has been removed, sutures or numbered tags are placed on the specimen so that it can be oriented, and the entire specimen has its surface inked in some fashion to allow evaluation of the histologic margins. Different colored inks may be used to represent different surfaces. The specimen is then cut and histologic sections prepared. Extra attention is paid to areas near the surgical margin wherever tumor is seen to encroach grossly. A thorough histopathologic evaluation can be done in a reproducible fashion, and

a template for reporting surgical pathology in patients who undergo lumpectomy can be used to ensure that no important details are neglected. As mentioned before, surgical excision is a balance between complete removal af the cancer with surrounding foci and an accetable cosmetic result. When this goal cannot be achieved, mastectomy and reconstruction may be a better option unless the patient strongly desires conservation of the breast, accepting a possible unsatisfactory cosmetic outcome. Small titanium surgical clips may be placed in the tumorectomy wall to guide the radiation oncologist for precise placement of a radiation boost when indicated. Reapproximation of breast tissue may be used when it does not distort the breast shape. Closure of the parenchyma of the lumpectomy cavity may prevent seroma formation. Although most women today can have breast-conserving surgery, there are some contraindications, which are based on the premise that all invasive or preinvasive cancer should be removed surgically and that radiation therapy is an essential component of breast conservation, thereby reducing the risk of local failures. The contraindications are as follows: • Scleroderma, cutaneous lupus erythematosus, and other active collagen disease of these tissues. • Diffuse calcifications indicating an extensive intraductal component: biopsy of two separate areas of microcalcifications should be performed to confirm the diagnosis of multicentricity. • Multicentric invasive cancers or DCIS may preclude breast conservation. Biopsy of two separate lesions should be performed to confirm the multicentricity pathologically. • Large residual disease after induction chemotherapy may make it difficult to achieve acceptable cosmesis with breast conservation. This is a highly personal decision that the patient must make in conjunction with her physician. Patients with previous chest wall radiation require evaluation by a radiation oncologist to determine the feasibility of breast conservation.

Axillary Staging The second aspect of breast-conservation therapy involves staging the axilla (Fig. 95-30). Although many studies have suggested that a panel of prognostic indicators and molecular markers of the primary tumor might eventually be a better predictor of outcome than the number of involved lymph nodes, no combination of such parameters has been validated. When suspicious axillary lymph nodes are present, axillary dissection should take place. Originally, axillary lymph nodes, including level III nodes medial to the medial border of the pectoralis minor muscle, were carefully removed. These dissections were associated with a higher incidence of lymphedema of the arm than limiting dissection to levels I and II. Level I and II dissection is now the standard for axillary clearance. This type of axillary dissection is associated with the lowest risk of regional failure. When bulky lymph nodes at level I and II are present, or suspicious level III nodes are detected, the removal of the level III nodes, bordered medially by Halsted’s ligament, laterally by the medial edge of the pectoralis minor muscle, superiorly by the anterior aspect of the axillary vein, and posteriorly/inferiorly by the anterior chest wall, should be performed to avoid possible axillary recurrences, which are difficult to treat with chemotherapy and radiation and sometimes are associated with axillary vein thrombosis and invasion of the axillary plexus. During the axillary dissection, care is taken to preserve the lateral and medial pectoral nerves, the long thoracic nerve (to avoid a “winged scapula” appearance), and the thoracodorsal nerve. When possible, preserving the upper branches of the inner costobrachial sensory nerve can avoid paresthesia and numbness in the inner posterior arm. Careful sparing of the brachial lymphatics that lie anterior

Cancer of the Breast • CHAPTER 95 Pectoralis major muscle fold

Previous surgery or site of lumpectomy

Pectoralis major muscle Pectoralis minor muscle

Incision Serratus fascia

Latissimus dorsi muscle fold

Arm extended Axillary vein

Pectoralis major muscle under fascia

A Pectoralis major and minor muscle

Latissimus dorsi muscle

Axillary contents dissected out

B

Level III nodes excised

Intercostobrachial nerve

First rib

Thoracodorsal nerve Long thoracic nerve bundle

Long thoracic nerve

Thoracodorsal nerve bundle

C

D

Drain

Figure 95-30 • A, The incision for axillary node dissection is placed at the inferior aspect of the axillary hairline, and extends from the lateral border of the pectoralis major muscle to the anterior border of the latissimus dorsi muscle. Nylon traction sutures are placed in the dermis. Flaps are raised superiorly and inferiorly. B, The pectoralis major and minor muscles are retracted to facilitate dissection of the lymph node-bearing fatty tissue from beneath the muscles and away from the axillary vein and chest wall. C, Excision of level III lymph nodes (highest level near the entrance of the axillary vein into the chest) is easily accomplished, even through the small axillary incision, with the arm rotated upward. D, The completed dissection, showing the isolated nerves to the serratus muscles, the latissimus dorsi muscle, the pectoral muscles, and the sensory nerve to the inner aspect of the upper arm.

to the axillary vein and brachial plexus minimizes the risk of lymphedema. Lymphedema is associated with body habitus, and people with obesity are at higher risk, despite optimal surgical technique.

Sentinel Lymph Node Biopsy in Invasive Disease SLN biopsy has largely replaced axillary dissection for patients with clinically negative axillae and is becoming the standard surgery for staging breast cancer.The concept of the sentinel lymph node (SLN)

originated with a 1977 description of mapping of the first draining lymph node in penile carcinoma. Detection of a sentinel “blue” node was described in melanoma in 1992 and in breast cancer the year after using blue dye and radioisotope localization. Published reports validated the sentinel lymph nodes by concurrent axillary dissection, and the use of isotope and blue dye simultaneously appear to shorten the learning curve and result in slightly better identification of sentinel lymph nodes. However, some surgeons prefer using the blue dye alone.

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Axillary lymph node biopsy (ALND) and SLN biopsy in breast cancer are done for staging/prognosis, local control of disease, and the possibility of a survival benefit from removing involved nodes. The premise of SLNB is equivalent to ALND in all three aspects, with the added advantage of sparing node-negative patients the morbidity of a larger operation from which they could not benefit. The debate over SLN biopsy is whether current data, largely observational, are sufficient to adopt SLN biopsy as standard of care in the absence of level I evidence from the two ongoing U.S. trials (ACOSOG Z0011, a randomized trial of axillary node dissection in women with clinical T1 or T2 N0 M0 breast cancer who have a positive SLN; and NSABP B-32: a randomized phase III clinical trial designed to compare SLN resection to conventional axillary dissection in clinically nodenegative breast cancer patients) and six European clinical trials (a Milan Trial,190 the ALMANAC trial,191 the EORTC 10981–22023 AMAROS trial, the German clinical interdisciplinary sentinel study-KiSS protocol, the French randomised SLN study (Fransenod) trial, and the International Breast Cancer Study Group randomized 23–01 trial. For lymph node staging, SLNB is best regarded as a diagnostic test for the detection of nodal metastases and, as such, should not require validation by randomized trials. More than 60 observational studies (in over 6000 patients) of SLN biopsy validated by a “backup” ALND have established a 95% success rate for SLN identification and a 95% sensitivity for detecting nodal metastases (i.e., SLNB is “falsely negative” in 5% of node-positive patients, or 2% to 3% of all cases). While this observation suggests a loss of prognostic information for those few false-negative patients, it must be balanced against studies showing that following ALND, 9% to 20% of patients initially deemed node-negative by conventional pathology (a single H&E-stained section) are found on further sectioning and/or immunohistochemical (IHC) staining to have nodal metastases, and that these missed nodal metastases are prognostically significant.192 Such exhaustive pathologic analysis is not logistically feasible for an entire ALND specimen. One study alone required the examination of 1600 slides to identify each additional node-positive patient, but it is feasible for the two or three nodes obtained at SLN biopsy. Enhanced pathologic analysis of SLN finds nodal metastases in an additional 10% of cases and is associated with a lower false-negative rate compared with SLN examination by the conventional singlesection method. Viewed in this way, SLN biopsy with enhanced pathology generates fewer false-negative results than ALND with conventional pathology, and so it may turn out to have similar accuracy rates when compared to ALND. If SLN biopsy is falsely negative in a small proportion of nodepositive cases, this would seem to pose an increased risk for the development of axillary local recurrence (LR) among patients staged by SLNB alone. In fact, data from several observational studies193–199 and one randomized trial190 demonstrate that LR following a negative

SLN biopsy is a rare event, occurring in 0.2% of cases, results which compare favorably to those of axillary sampling and of ALND (Table 95-13).200 Of particular interest is the observation from the single randomized trial199 of a 9% false-negative rate in the control arm (SLN biopsy plus ALND) but no axillary LR in either study arm, including the 167 patients staged by SLN biopsy alone. Possible explanations for this consistently low rate of axillary LR across all 10 studies includes selection for early-stage disease, a local effect of systemic therapy, and most importantly the use of breast radiotherapy, which inevitably treats the axillary nodes closest to the breast. While all reports of LR following SLN biopsy are limited by relatively short follow-up, a significant future increase in LR is unlikely, as evidenced by an earlier study of mastectomy without ALND (NSABP B-04201) in which 75% of axillary LR appeared within the first 2 years. In preventing axillary LR, SLN biopsy is at least equivalent both to ALND and to axillary sampling. There is considerable controversy about the staging of patients whose SLNs appear negative by standard H&E examination, but who have isolated tumor cells found by immunohistochemistry or reverse transcriptase polymerase chain reaction evidence of a few cancer cells. The latest TNM staging manual suggests classifying these patients as node-negative, with a subscript to indicate these additional findings. Of note, some European centers consider these patients node-positive. Also controversial is the surgical management of patients with positive SLN biopsy: is completion of the axillary dissection necessary? Some of the clinical trials just described will, we hope, answer this question. A nomogram to predict the likehood of additional positive nodes after positive SLNs has been developed.202 Initial contraindications for SLN biopsy, including clinically suspicious axillary lymph nodes, tumor arising from the axillary tail of the breast, multicentricity, large cancers, large biopsy cavity, previous axillary sampling, previous breast and/or axilla radiation therapy, and pregnancy, have been progressively cleared by evidence specific to these patient subsets.203 Still controversial is the use for SLN biopsy after PST, but data from the NSABP B-27 study shows that SLN biopsy appears to be a reliable procedure following preoperative chemotherapy.204

Irradiation of the Intact Breast No subsets of women have been identified who do as well following conservation surgery without radiation therapy as with it, although such subsets of patients have been aggressively sought.205 Current recommendations are to irradiate the breast after lumpectomy to achieve the lowest possible recurrence rate. The only possible exception is elderly women with comorbid conditions, as discussed later in this chapter. When radiation therapy is elected, the whole breast is treated through a pair of tangentially directed fields to a dose of 4500 to 5000 cGy over 5 to 6 weeks. Traditionally, the patient is

Table 95-13 Axillary Local Recurrence after Axillary Sampling, Axillary Lymph Node Dissection, and Sentinel Lymph Node Biopsy Axillary sampling

No. of Patients

Median Months Follow-up (range)

Axillary Local Recurrence No. (%)

443

91 (49–132)

18/437 (4.1)

5825

64 (27–180)

52/5825 (0.9)

4981

27 (14–46)

11/4981 (0.2)

2 series (1995–2000) ALND 9 series (1986–2004) SLN biopsy 10 series (2000–2004) ALND, axillary lymph node dissection; SLN, sentinel lymph node. Data from Naik AM, Fey J, Gemignani M, et al: The risk of axillary relapse after sentinel lymph node biopsy for breast cancer is comparable with that of axillary lymph node dissection: a follow-up study of 4008 procedures. Ann Surg 2004;240:462–468.

Cancer of the Breast • CHAPTER 95

specifically irradiating the dissected axilla. Microscopic extracapsular nodal extension appears to have little effect on the local control rate in the axilla after surgery alone, and can safely be ignored. A clinically negative axilla that is undissected is well controlled by axillary irradiation of 4500 to 5000 cGy. In patients with positive axillary nodes found on axillary dissection, most investigators use a field that irradiates the supraclavicular nodes and the apex of the axilla (level III nodes). Some institutions prefer to use this field only in patients with four or more positive nodes, whereas other institutions apply it to all axillary-positive patients, and the recent meta-analysis from the Oxford Overview would support the later practice.183 The Early Breast Cancer Trialists Collaborative Group (EBCTCG; www.ctsu. ox.ac.uk/~ebctcg) compared radiation therapy to none in two patient groups: those with breast conservation therapy (approximately 8,300 women) and those treated with mastectomy (approximately 10,000 women). For all node-positive women, the addition of radiation therapy not only reduced the risk of a local failure, but also reduced mortality at 15 years follow up, by about 5%. Figure 95-32 demonstrates the positive effect of breast or breast and nodal radiation for women on breast cancer study trials.

Elderly Patients

Figure 95-31 • Left breast irradiation using prone breast technique can spare lung, left ventricle, and coronary arteries.

treated supine, with fields designed to skim along the lung/chest wall interface, and irradiate the smallest volume of underlying lung. In left-sided primary lesions, the fields should result in the lowest possible exposure of the heart. Alternatively, the patient may be treated in the decubitus or prone position (Fig. 95-31).206 Following the completion of whole breast treatment, an additional series of radiation treatments (or “boost”) usually is directed to the lumpectomy cavity only. A striking exception to the use of the boost has been the series of NSABP trials, starting with the B-06 trial; no boost was used until the opening of the B-39 Trial. A randomized prospective EORTC trial evaluated the need for a boost in patients undergoing lumpectomy.207 In this trial, 5318 women were randomly assigned to a boost of 1600 cGy or no boost, after completing 5000 cGy whole-breast radiation. The results demonstrated that the boost reduced the local failure rate by a factor of 2; looking at age, this translated to a reduction from 19% to 9.5% for those women under age 40, but an absolute difference in those over 60 years of only 1.9%. Most institutions currently recommend a boost for all patients, although based on the EORTC study, considering omitting the boost for women with negative margins over the age of 60 is reasonable. This set of data also confirms the favorable prognosis in the elderly patient. The boost is accomplished quickly and easily on a linear accelerator equipped with electron-beam capability, and requires five to eight additional treatments. Localizing the target within the breast for boost purposes can be accomplished by CT, by ultrasound, or by clips left in the tumor bed. Morbidity associated with a boost is limited to temporary skin tanning. With either a complete axillary dissection or a successful SLNB and negative results, the axilla is considered treated by the surgeon. Much clinical experience indicates that the axilla is the site of first breast cancer failure in fewer than 1% of patients who have had an axillary dissection. Axillary recurrences occur in approximately 2% of patients with negative nodes or one to three positive nodes and in patients with axillary dissection who received irradiation to the breast only after lumpectomy. Widespread extracapsular extension and gross tumor left behind in the axilla are the only indications for

A North American intergroup trial focused on women 70 years of age or older with stage I ER-positive disease, all of whom underwent lumpectomy followed by 5 years of tamoxifen and were randomized to receive radiation therapy or not. Two thirds did not undergo an axillary dissection, and half were 75 years of age or older. With a median follow-up of 0.82 years,there was a statistical difference in local failure rates: 1% of the radiated women versus 69% of the tamoxifen-only women had a recurrence in the ipsilateral breast. However, no differences were found in the rates of mastectomy, distant recurrence, and overall survival.208

Partial Breast Irradiation Despite the excellent results of breast-conserving surgery with wholebreast radiation, a study from the American Colleges of Radiology and Surgeons noted only 42.6% of women in the United States. with stage I and II breast cancer were treated in this manner.209 The study also demonstrated that 11% of women with breastconservation surgery never received radiation. Thus, considering ways of making this treatment more available to women who may not live near a center equipped with radiation facilities was addressed in the early 1990s, especially as data from Milan showed that almost all local failures in women treated with “standard” BCT were located in the original quadrant of diagnosis.210 The concept of “partial accelerated breast irradition” (PBI) was thus proposed and tested. The goals were two-fold: to increase the proportion of women who had access to both the surgery and the radiation, by dramatically shortening the time course of the radiation; and to limit the radiation to the lumpectomy cavity with a small margin of normal tissue. In the United States, two centers, the Ochsner Clinic and William Beaumont Hospital, independently initialized pilot PBI studies using catheter-based brachytherapy and low-dose rate sources. After reports of initial success, the RTOG Cooperative Group opened a phase II trial, allowing either high- or low-dose-rate sources and the catheter technique. The dose used was 3400 cGy, given over 5 days using twice-per-day treatments for the 33 high-dose patients. Results in the 99 patients in the trial, with a median follow-up time of 6.1 years, showed an in-breast failure of 3% for the high-dose-rate group, and 6% for the 33 women in the low-dose-rate group.211 Catheter-based PBI is technically demanding. Although most radiation oncologists are not trained to use it, in 2002 the FDA approved a new catheter for deliver of brachytherapy, the MammoSite Radiation Therapy System (Cytyc Surgical Products)

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Part III: Specific Malignancies

6097 women with BCS and node-negative disease 60

60 5-year gain 16.1% (SE 1.0)

15-year gain 5.1% (SE 1.9) Logrank 2p = 0.006 50

40 29.2

30

BCS 22.9

20

Breast cancer mortality (%)

Isolated local recurrence (%)

50

40 31.2% BCS

30

26.1% BCS + RT

20.3

20 17.4

BCS + RT

10

8.9

10

10.0

8.0

6.7

0

0 0

5

10

15

0

5

Time (years)

10

15

Time (years)

1214 women with BCS and node-positive disease 60

60 5-year gain 30.1% (SE 2.8)

15-year gain 7.1% (SE 3.6) Logrank 2p = 0.01

46.5

50

50

55.0% BCS

45.2

47.9% BCS + RT

41.1

40

30

20 BCS + RT

10

11.0

Breast cancer mortality (%)

BCS

Isolated local recurrence (%)

1912

40 36.5

30

24.3

20 20.9

10

13.1

0

0 0

5

10

15

Time (years)

0

5

10

15

Time (years)

Figure 95-32 • Effect of radiation therapy (RT) after breast cancer study on local recurrence and on breast cancer mortality—15-year probabilities. Data from 10 trials. Vertical lines indicate 1 SE above or below the 5-, 10-, and 15-year percentages. (From Lancet 366:2092.)

Fig. 95-33. This device was easy to insert into the lumpectomy cavity, and rapidly appeared as a treatment in clinical practices. In order to address whether these PBI therapies are equivalent to standard whole-breast external beam radiation, the NSAPB and the RTOG together opened a phase III trial addressing this issue, in 2005. Women are randomized between whole-breast radiation or PBI using the RTOG schedule of twice per day treatments to a total dose of 3400 cGy. The PBI may be given by catheter brachytherapy, the MammoSite device, or an external beam PBI technique.212 With more than half of the planned 4200 patients accrued by 2007, the

PBI technique using the external beam, noninvasive system has proved to dominate the PBI arm. Other researchers have approached the technical issues with PBI by developing intraoperative radiation techniques. The group in Milan is working on a randomized trial using a portable linear accelerator that can be brought into the operating room, and the group at Memorial Sloan-Kettering Cancer Center has devised a special appplicator that uses high-dose-rate afterloading therapy. Both deliver a dose of about 2000 cGy directly to the lumpectomy cavity in the operating room before the surgeon closes the wound.213

Cancer of the Breast • CHAPTER 95

LR as first event (%)

15 Patients Events 847 67 851 66

MRM BCT

10

5 Failure analysis EORTC 10801 and DBCG-82TM 0 0

Figure 95-33 • Elliptical balloons for delivery of brachytherapy to the lumpectomy site (MammoSite; Cytyc Surgical Products).

With increasingly sophisticated treatment planning and delivery systems, late complications of lumpectomy and radiation frequently seen 20 years ago are no longer common. Approximately 5% to 20% of patients have measurable arm edema with either lumpectomy or mastectomy, which suggests that the axillary procedure is the key factor in itself. Rib fractures are seen in approximately 2% to 5% of patients treated with radiation; most of these are asymptomatic and detected when a bone scan or chest x-ray is done for other reasons. Therefore, rib fracture should always be included in the differential diagnosis of a previously irradiated patient with breast cancer who has chest wall or rib tenderness and whose bone scan shows an area of tracer uptake in the ribs of the treated chest wall. No specific therapy is indicated, because most of these fractures heal spontaneously. Approximately 1% or fewer of patients treated with radiation have symptomatic radiation pneumonitis. This complication is more common in patients who have received chemotherapy and radiation that included a supraclavicular field. In almost all patients, pneumonitis resolves either spontaneously or with a short course of corticosteroid therapy, and no long-term sequelae occur. Sometimes, radiation causes scarring in the small rim of lung treated in the tangential fields that can appear as a density in the lung field underlying the treated breast on routine chest x-ray and is confirmed by CT scan showing lung changes confined to the area of high-dose irradiation just underneath the anterior chest wall. Usually, no intervention is needed. Rarely, breast irradiation leads to late cardiac damage.214 Much of this information comes from treatment of the postmastectomy chest wall, especially when radiation was directed specifically at the internal mammary nodes. Recent studies suggest that few patients have sufficient cardiac volume within the radiation port to place them at risk for later damage and that these patients can be recognized in advance so that such complications may be avoided with sophisticated treatment planning. A supraclavicular portal sometimes results in brachial plexus injury, and the risk appears increased if large daily dose fractions are used. In rare cases, years later, radiation results in a soft tissue sarcoma within the radiation portal. The incidence of this extremely serious complication is approximately 0.1%. Recent studies have confirmed an increase in the risk of developing an ipsilateral lung cancer in women smokers with breast cancer who opted for lumpectomy with radiation.215

Local Recurrences In most centers, local in-breast failure is managed with mastectomy. Although there are reports of successful treatment either by excision alone, especially for late recurrences, or by excision plus additional

10

644 657

At risk

265 279

Figure 95-34 • Actuarial time to locoregional recurrence (as a first event) by original treatment group (MRM versus BCT) for the 1677 patients in trials EORTC 10801 and DBCG-82TM. Patients at risk at 5 and 10 years are indicated. BCT, breast-conserving therapy; MRM, modified radical mastectomy. (From van Tienhoven G, Voogd A, Peterse JL, et al: Prognosis after treatment for loco-regional recurrence after mastectomy or breast conservation in two randomised trials (EORTC 10801 and DBCG-82TM). EORTC Breast Cancer Cooperative Group and the Danish Breast Cancer Cooperative Group. Eur J Cancer 1999;35:32–38.)

local radiation, these techniques are not considered part of standard care. An outstanding study of local recurrences came from pooling two randomized studies from the EORTC 10801 and the Danish study group DBCG-82TM prospective randomized trials comparing breast conservation surgery and radiation with mastectomy. Both therapies were associated with a similar risk of local failure and overall survival following salvage treatment, which in most cases consisted primarily of mastectomy in the conserved group and radiation in the mastectomy group.216 Figures 95-34 and 95-35 illustrate the data for both BCT and mastectomy patients. There is increased interest in MRI for surveillance after breast conservation, but data are lacking

100 80 Survival (%)

Complications of Treatment

5 Time (years)

MRM BCT

Patients Events 66 33 67 32

60 40 20

Failure analysis EORTC 10801 and DBCG-82TM

0 0

At risk

2

4 6 Time (years) 44 40

8

10 15 12

Figure 95-35 • Actuarial overall survival from salvage treatment by original group (MRM or BCT). Patients at risk at 4 and 8 years are indicated. BCT, breast-conserving therapy; MRM, modified radical mastectomy. (From van Tienhoven G, Voogd A, Peterse JL, et al: Prognosis after treatment for loco-regional recurrence after mastectomy or breast conservation in two randomised trials (EORTC 10801 and DBCG-82TM). EORTC Breast Cancer Cooperative Group and the Danish Breast Cancer Cooperative Group. Eur J Cancer 1999;35:32–38.)

1913

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on improved outcomes when compared with clinical examination and routine imaging with mammography and ultrasound, as appropriate.217

FACTORS THAT AFFECT OUTCOME Patient Age Younger patients (e.g., under 35 years of age) have a higher rate of lumpectomy failure (Table 95-14) as well as a higher rate of chest wall failure after mastectomy than do older patients. It is possible that age serves as a surrogate for higher risk tumors as younger women are more likely to have extensive intraductal carcinoma (EIC), high nuclear grade, lymphatic space invasion, and tumor necrosis.218 It is likely that the recurrence patterns seen in these young women are related to a combination of these adverse histologic features.

Tumor Size When lumpectomy and the use of radiation are examined as a function of tumor size, there is a dramatic fall-off in the application of breast-sparing therapy for T2 tumors in comparison with smaller T1 tumors. The important factor in the decision should not be tumor size, however, but cosmetic results. Women whose primary tumor size makes it difficult to perform a cosmetically acceptable excision can have their tumor reduced with preoperative chemotherapy and may then become candidates for breast-conserving therapy. In the NSABP B-18 preoperative trial, 67.8% of women in the preoperative arm vs. 59.8% in the postoperative arm underwent breast-conservation therapy, with no difference in survival outcome.219,220

Histology Schnitt and associates in Boston noted that a new histologic pattern within a breast tumor was a strong predictor for subsequent in-breast failure after radiation. They observed a pattern containing intraductal carcinoma present both within and adjacent to the invasive breast cancer lesion, defined as an extensive intraductal component (EIC). DCIS had to be a prominent feature of the invasive cancer, in at least

25% of the tumor area, and no specific amount of DCIS adjacent to the tumor was required. Tumors that were predominantly DCIS with small areas of minimal invasion were classified as EIC-positive and are 4 times as likely to develop local recurrence. This suggested that there might be a greater tumor burden inadvertently left behind at the site of the primary excision in patients with EIC-positive disease. Follow-up studies demonstrated that this negative prognostic effect of the EIC could be overcome with the surgical removal of a larger volume of tissue in this patient subset. In other words, with wider margins, the effect of the EIC was removed.189

MASTECTOMY For many decades, Halsted’s radical mastectomy was the treatment of choice for all stages of breast cancer. This operation, which consists on the removal of a large portion of the breast skin, all the breast parenchyma, and the major and minor pectoral muscles en bloc with the axillary lymph nodes, is now limited to locally advanced tumours (T3, T4b) when preoperative chemotherapy is not an option. A less aggressive modified radical mastectomy, with sparing of the pectoralis major muscle, was described by Patey in 1948 and has proved to be equally effective in the local control of most tumors. SLN biopsy has made axillary dissection for staging unnecessary in women with no suspicious palpable lymphadenopathy and negative findings. Total or simple mastectomy with removal of skin, including nippleareolar complex and all breast tissue without the axillary lymph nodes, has supplanted modified radical mastectomy for this group of node negative patients who require mastectomy. Reconstruction of the breast is a valid option after mastectomy and can be done immediately or after the procedure (i.e., delayed). With attempts to improve overall cosmesis after mastectomy and breast reconstruction, more “conservative” mastectomy procedures have evolved. Skin-sparing mastectomy, in which removal of the skin is limited to the nipple–areola complex, sometimes with a small portion of the surrounding skin, has come to be accepted as an oncologically safe approach that minimizes deformity and improves cosmesis. The risk of local recurrences after skin-sparing mastectomy and breast reconstruction was compared to that of conventional

Table 95-14 Age as a Factor in Local Breast Cancer Recurrence* Author

Definition of “Young Age” (yr)

No. Young/No. Recurrences (%)

No. Older/No. Recurrences (%)

Boyages et al.†

≤34

61/15 (25)

722/76 (11)

Delouche et al.‡

≤40

71/14 (20)

339/26 (8) 383/44 (11)

Forquet et al.

≤32

35/12 (34)

Haffty et al.||

≤50

135/24 (18)

248/26 (10)

Kurtz¶

≤39

210/41 (20)

1172/106 (9)

§

Ryoo et al.**

≤40

51/8 (16)

346/18 (5)

Solin et al.††

≤35

88/12 (14)

808/42 (5)

651/126 (19)

4014/338 (8)

Total

*Any locoregional failure. † Boyages J, Recht A, Connolly I, et al: Factors associated with local recurrences as a first site of failure following the conservation treatment of early breast cancer. Recent Results Cancer Res 1989;115:92. ‡ Delouche G, Bachelot F, Premont M, Kurtz JM: Conservation treatment of early breast cancer: long term results and complications. Int J Radiat Oncol Biol Phys 1987;13:29. § Fourquet A, Campana F, Zafrani, et al: Prognostic factors of breast recurrences in the conservative management of early breast cancer: a 25-year follow-up. Int J Radiat Oncol Biol Phys 1989;17:719. || Haffty BG, Fischer D, Rose M, et al: Prognostic factors for local recurrence in the conservatively treated breast cancer patient: a cautious interpretaion of the data. J Clin Oncol 1991;9:997. ¶ Kurtz JM, Jacquemier J, Amalric R, et al: Why are local recurrences after breast-conserving therapy more frequent in younger patients? J Clin Oncol 1990;8:591. **Ryoo MC, Kagan AR, Wollin M, et al: Prognostic factor for recurrence and cosmesis in 393 patients after radiation therapy for early mammary carcinoma. Radiology 1989;172:555. †† Solin LJ, Fowble B, Schultz DJ, Goodman RL: Age as a prognostic factor for patients treated with definitive irradiation for early stage breast cancer. Int J Radiat Oncol Biol Phys 1989;16:373.

Cancer of the Breast • CHAPTER 95

mastectomy in some retrospective series showing similar rates of local control, although selection bias may have partially influenced the results.221 A few retrospective studies with relatively long follow-up reported good results after treatment with nipple-sparing mastectomy, in which only the breast parenchyma is removed without skin or the nipple and areola in selected early-stage breast cancer and high-risk women pursuing mastectomy.222–224 The ductal tissue below the nipple represents the major oncologic concern in the preservation of the nipple during mastectomy, and complete removal of this tissue may affect the vascularization of the nipple, increasing the probability of necrosis. Occult nipple involvement ranged from 0% to 50% in several retrospective studies of nipple-sparing mastectomy, in which the pathology blocks of the nipple were reanalyzed, but the percentage has been lower in more recent publications. Careful pathologic examination of the retro-areolar ducts must be performed in nipple-sparing mastectomy. Removal of these structures should be considered the most conservative approach. In modified radical or total mastectomy, when surgery is performed through incisions other than circumareolar incisions, the incision is placed so that the scar will not be visible when the patient wears a bathing suit or low-cut dress. When elliptical or transverse incisions are used for a total mastectomy, it is ideal to excise any previous biopsy scar as well as the nipple-areolar complex. Skin flaps are carefully developed with a combination of scalpel and electrocautery dissection. The flaps are sufficiently thin to remove all apparent breast tissue, but it is not necessary to remove the subcutaneous tissue of the flaps. This tissue carries the blood vessels to the skin and is of cosmetic importance. Especially in young women, the cleavage plane between breast tissue and subcutaneous tissue may not be evident, and complete removal of the breast tissue is the priority. Depending on the patient’s body habitus, the flaps may contain from 1 to 8 mm of subcutaneous fat. In a standard modified radical or total mastectomy, the inferior flap extends inferiorly below the inframammary crease for approximately 2 cm onto the interior fascia of the rectus muscle. If immediate reconstruction is to be performed, the dissection usually ends at the inframammary crease, unless additional breast tissue is present inferiorly to the crease. The superior flap is then dissected similarly, just to beneath the clavicle. The medial extension of the skin flaps reaches the lateral edge of the sternum, and the lateral extension reaches the anterior edge of the latissimus dorsi. In bilateral mastectomy, attention should be paid to not crossing the sternum, creating a tunnel between the two medial flaps, because this may affect the cosmesis of the breast reconstruction. Once the flaps have been developed, the breast is dissected from the chest wall by dissecting the pectoralis major fascia off the muscle superiorly and medially, progressing inferiorly. If total mastectomy is performed and the axilla is not to be dissected, as the breast is dissected from the lateral edge of the pectoralis major muscle, the pectoralis muscle is seen beneath it. The surgeon should spare the medial pectoral nerves, which wrap aound the lateral border of the pectoralis minor muscle and insert into the posterior aspect of the pectoralis major muscle. Division of these nerves leads to atrophy in the central portion of the pectoralis major muscle. If total mastectomy is performed, the dissection may progresses into the axilla above any apparent breast tissue, and sometimes may include some lower axillary lymph nodes, which are present in the axillary tail of Spence. During total or simple mastectomy, no attempt is made to remove the axillary lymph nodes. The breast is then swept inferiorly, with care taken to spare the thoracodorsal and long thoracic nerves in their lower extent as the breast tissue is removed. When the specimen is removed, a suture is used to identify the axillary tail and the retro-areolar ducts in case of nipple-sparing mastectomy so that the pathology department can orient it properly for assessing marginal clearance. If the axilla is to be dissected, either in continuity or as a separate axillary dissection, the axilla is best entered from within the fascia of the pectoralis major muscle posteriorly. Coming down two finger-

breadths from the uppermost extent of the pectoralis major fascia posteriorly, a transverse incision is made. This incision goes through the pectoralis major fascia and 1 mm beneath it, through the clavipectoral fascia. Fat from the axilla then pops through this division of the clavipectoral fascia. The inferior border of the axillary vein is two fingerbreadths below the highest extent of the pectoralis major fascia. This is important because the lymphatics of the arm pass anterior to the axillary vein. Staying below the axillary vein minimizes the risk of subsequent lymphedema of the arm. The landmark for the axillary anatomy is the thoracodorsal vein, which is one fingerbreadth out from the chest, 2 cm lateral to the chest wall and passing dorsally into the dorsal inferior aspect of the axillary vein. When it is identified, the thoracodorsal nerve is seen to emerge from behind the axillary vein, just medial to the thoracodorsal vessels. It joins these vessels, continuing onto the anterior surface of the thoracodorsal vein. Between 1 and 2 cm below the axillary vein, the highest branch of the intercostobrachial nerve is seen coming from the chest wall and going to the arm. It is important and usually possible to spare and clear this nerve branch to avoid the dysesthesias associated with its division and total numbness of the inner posterior arm. These technical steps minimize the sensory complications and lymphedema associated with axillary dissection. When the thoracodorsal nerve has been identified, sweeping the axillary fat downward off of the chest wall moves the level II axillary nodes out from behind the pectoralis minor muscle and clears the long thoracic nerve safely. The long thoracic nerve is always found in the same anteriorposterior plane as the thoracodorsal nerve, but is immediately applied to the chest wall. At this point, the three major midaxillary nerves have been identified. If the medial pectoral nerves were not previously identified by sweeping the fatty tissue off of the lateral border of the pectoralis minor muscle, the medial pectoral nerves will not be seen coming around the lateral border of the pectoralis minor and entering the posterior aspect of the pectoralis major. The axillary contents can now be cleared inferiorly, with all of the important nerves and vessels in view. When axillary dissection is complete, the remainder of the breast flap division allows the specimen to be handed off. If the mastectomy was performed through a circumareolar incision, the specimen may be too large to be delivered and a tennis racket incision 1 to 2 cm lateral to the circumareolar incision will allow the specimen to be retracted. When suspicious palpable lymph nodes are present at level III, removal of these nodes is advised. If possible, the pectoralis minor muscle is preserved. The lateral borders of the pectoralis major and minor muscles are dissected, thereby preserving the medial pectoral nerve. In this way it is possible in nearly every case to dissect the apex without division or removal of the pectoralis minor. Both pectoral muscles are retracted medially to maintain the exposure of the level III region. After incising the surrounding fascia anteriorly to the axillary vein at its junction with the chest wall, just lateral to Halsted’s ligament, careful traction in an inferiolateral direction makes the dissection of the specimen from the surrounding structures easily possible. Small branches from the axillary vein and lymphatic vessels usually can be cauterized. For larger blood vessels, clamping and cutting is mandatory. The content of level III can be removed in continuity with the specimen of levels I and II. For the purpose of the pathohistologic examination, each level should be marked separately with a metallic tag. If division of the tendon, with or without removal of the pectoralis minor muscle, is indicated or unavoidable, dissection ends as soon as the border of the pectoralis major is visualized. The pectoralis minor remains in continuity with the breast and the axillary tissue. When hemostasis is complete, the plastic surgeon can reconstruct the breast according to the patient’s body habitus. For a very smallbreasted woman, an expander can be placed beneath the pectoralis major and partially expanded to allow the skin anterior to the muscle to achieve normal tension. For a fuller breast, the latissimus dorsi can

1915

1916

Part III: Specific Malignancies

be freed, swung around anteriorly, and joined to the lateral border of the pectoralis major muscle with a prosthesis lying deep to the muscle. This muscle cover provides the reconstructed breast with a very natural feel. For a larger-breasted woman, optimal reconstruction may involve various forms of pedicled or free (myo)cutaneous flaps.

ADJUVANT POSTMASTECTOMY IRRADIATION At one point, irradiation of the chest wall after mastectomy was almost universally applied in the treatment of breast cancer, largely because of the more advanced nature of breast cancer in the early decades of the 20th century, with an attendant high rate of local chest wall failure. As surgical techniques improved and clinicians began to take a more rigorous scientific look at breast cancer therapy, the need for postoperative chest wall irradiation began to be questioned. Possibly the first randomized prospective clinical trial testing one form of cancer therapy against another was the Manchester, England, trial of immediate versus delayed chest wall irradiation in postmastectomy patients, begun in 1948. Since that time, more than 30 randomized trials of postmastectomy irradiation have been performed. Although chest wall radiation dramatically reduced the risk of subsequent chest wall recurrence and decreased the chance of dying of breast cancer, the overall survival rate in irradiated women remained unchanged, without the addition of systemic therapy. As systemic therapy improved, postmastectomy radiation was prescribed only in very locally advanced cases (Box 95-1). However, two recent randomized trials of postmastectomy radiation versus none, with all women in the study receiving systemic CMF chemotherapy, did show a survival advantage for the addition of radiation in premenopausal women with 1 to 3 positive nodes, the trial target population.225,226 With this new outcomes information, the added value of postmastectomy radiation, in addition to systemic therapy, became a controversial topic, with questions such as “Which patients are at highest risk for local failure?” and “What anatomic areas should be targeted for treatment?” To address these issues, a guideline panel from the American Society of Clinical Oncology (ASCO) recommended postmastectomy radiation for patients with four or more involved nodes, and suggested the treatment for those with T3 tumors as well. However, it concluded that there was insufficent evidence to make a formal recommendation in the subsets of one to three involved nodes, women with reconstruction, and the use of internal mammary node radiation as part of the target volume.227 The 2000 EBCTCG overview analyzed almost 10,000 women on trials of postmastectomy radiation (Fig. 95-36) and showed significant improvement of both local control and survival at 15 years in all groups, except nodenegative women treated with mastectomy.228

PREDICTIVE AND PROGNOSTIC FACTORS FOR INVASIVE DISEASE Although it is possible to describe the average risk of recurrence and potential benefit offered by systemic therapy for a population of patients using clinical nomograms like Adjuvant! Online,229,230 this exercise is difficult in an individual patient. A pure prognostic factor like TNM predicts patient outcome in the absence of therapy while a pure predictive factor predicts the likelihood of response to a specific therapy. HER2 and ER are examples of moderate prognostic factors with a strong predictive utility in predicting response to specific therapies that can help to individualize treatment recommendations for women with early stage breast cancer75,184 at high risk for recurrence. Unfortunately, even commonly used markers such as ER/PR and HER2 suffer from lack of assay standardization and reproducibility, and many assays have not been properly validated (Table 95-15).231 Other commonly used prognostic markers include tumor grade, lymphatic or vascular invasion, and specific histologies like mucinous and tubular cancers. Bone marrow micrometastases appears to be of prognostic significance, but assays lack standardization, while circulating tumor cells do not have sufficient sensitivity at present for use in the adjuvant setting.232 Women whose tumors overexpress or amplify the HER2 receptor may be less likely to respond to endocrine manipulations with tamoxifen or aromatase inhibitors. These women may consider the addition of adjuvant chemotherapy, even if their tumors do not exceed 1 cm

Table 95-15 Criteria for Evaluating Clinically Useful Tumor Markers IDENTIFICATION OF A POTENTIAL MARKER What is the distribution of the marker in normal and abnormal tissues? What is the prevalence of the marker in the patient population of interest? What is the source of specimens examined? Institutional or cooperative group tissue or serum banks? Does the marker appear to predict outcome (prognostic factor) or response to therapy (predictive factor)?

DEVELOPMENT AND VALIDATION OF A CLINICAL ASSAY Has the assay target that best correlates with the intended marker objective been identified (e.g., gene amplification, protein expression)? Has the optimal specimen source for the assay been identified (e.g., paraffin block, fresh tumor tissue, peripheral blood, urine)? Have the conditions of the assay been optimized and standardized? Has its reproducibility in other labs been tested? Has a standardized and cross-validated scoring system been developed?

Box 95-1.

INDICATIONS FOR POSTMASTECTOMY RADIATION

Tumor >5 cm T4 tumor Involvement of 4 or more axillary lymph nodes Gross extracapsular nodal disease Residual disease after mastectomy

Have the sensitivity and specificity of the assay been validated against a gold standard?

VALIDATION OF THE CLINICAL USEFULNESS OF THE MARKER Has this marker been validated in a patient population other than the one used to develop the predictive and prognostic model? Does the presence of the marker discriminate between patient subsets according to the outcome of interest?

Additional Considerations

Is the prognostic or predictive information provided by the marker independent of other established markers?

Involvement of 1 to 3 axillary lymph nodes Gross multifocality Extension into the nipple or skin

Has a prospective randomized trial using the proposed marker been performed using the information provided by the assay to stratify patients according to risk or to select planned therapy?

Cancer of the Breast • CHAPTER 95 1428 women with mastectomy with AC and node-negative disease 60 5-year gain 4.0% (SE 1.1) 15-year loss 3.6% (SE 2.6) Logrank 2p = 0.01 (excluding data beyond year 15: logrank 2p = 0.18) 50 50 Breast cancer mortality (%)

Isolated local recurrence (%)

60

40

30

20

10

6.3

8.0

2.3

3.1

Mastectomy + AC + RT

5

10

15

0 0

40 31.3% Mastectomy + AC + RT

30 22.3

20 12.5

10

Mastectomy + AC

27.7% Mastectomy + AC

20.8

11.3

0 0

5

Time (years)

10

15

Time (years)

8505 women with mastectomy with AC and node-positive disease 60 5-year gain 17.1% (SE 0.9) 15-year gain 5.4% (SE 1.3) Logrank 2p = 0.0002 50.9 50 50

60.1% Mastectomy + AC 54.7% Mastectomy + AC + RT

40 27.6

30

29.2% Mastectomy + AC

22.8

20

10 5.8

7.5

7.8% Mastectomy + AC + RT

0

Breast cancer mortality (%)

Isolated local recurrence (%)

60

46.7

40 34.0

30

32.1

20

10

0 0

5

10

15

Time (years)

0

5

10

15

Time (years)

Figure 95-36 • Effect of radiation therapy (RT) after mastectomy and axillary clearance AC on local recurrence and on breast cancer mortality—15-year probabilities. Data from 25 trials. Vertical lines indicate 1 SE above or below the 5-,10-, and 15-year percentages. (From Lancet 366:2092.)

and carry ER or PR, although the adjuvant role of trastuzumab has not been tested in these small tumors. HER2 overexpression also identifies patients in the adjuvant78,79,233 and metastatic234 settings who might benefit from trastuzumab therapy. Many other individual prognostic or predictive factors are under investigation. Levels of urokinase-type plasminogen activator and its inhibitor PAI-1 had strong prognostic value in women with node-negative breast cancer who did not receive adjuvant systemic therapy and had more than 10 years of follow-up.235 In addition to individual factors, powerful emerging technologies, such as proteomics or gene arrays, hold the potential to identify patterns of expression of genes or proteins at baseline or in response to therapy that may have prognostic or predictive value, and to assist in treatment decision-making for individual women.94,100,101,236 Initial studies with array-based expression profiling showed the ability of the technology to classify breast cancer according to five gene clusters: luminal subtype A, luminal subtype B, HER2-positive, basal, and normal breast-like subtypes (see Fig. 95-2) and that BRCA-

1 genotype predisposes to the basal tumor subtype.97 The ER-positive group was characterized by high expression of many genes expressed by breast luminal cells, and the ER-negative group showed gene expression characteristic of basal epithelial cells. However, a third group showed genes related to HER2 overexpression, suggesting that this molecular characteristic may have equal or greater weight than ER expression in subclassifying breast cancers. Finally, a small group of breast cancers cluster with normal breast epithelium and are referred to as normal breast-like. These distinct subtypes of breast tumors, described as basal-like, ERBB2 (HER2), luminal A, luminal B, and normal breast-like, show distinctive molecular signatures and appear to represent diverse biologic entities associated with distinct clinical outcome, and the comparison of several independently developed gene signatures appears to show similar prognostic information suggesting the existence of a common set of biologic phenotypes.104 These patterns of gene expression appear to provide more specific information than identification of a single gene with a specific effect.

1917

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Part III: Specific Malignancies

By late 2007, three gene expression profiles were available for commercial testing in the United States: the Oncotype DX (Genomic Health, Inc.) assay for women with ER-positive lymph node-negative disease based on a 21-gene profile developed by Paik and coworkers102,103; the MammaPrint (Agendia) assay based on a 70-gene prognostic signature developed by van’t Veer and colleagues100,101,237; and the Breast Cancer Profiling (BCP) assay (AviaraDx) based on the two-gene ratio signature developed by Ma and associates.238,239 Retrospective studies showed that the Oncotype DX assay has prognostic utility and identifies which patients who are lymph node-negative and ER-positive will do well or not (low vs. high recurrence score [RS], respectively). Of greater importance, this assay also has predictive utility in determining in a similar group of patients who will benefit from the addition of a non-anthracycline regimen to tamoxifen and who will not (high vs. low RS, respectively). The Oncotype DX also has been shown to predict the likelihood of response to preoperative systemic chemotherapy.240 However, several questions remain about the utility of these various assays,241 and ongoing trials will prospectively confirm and test, respectively, the clinical utility of the Oncotype DX (TAILORx trial, see http://www.cancer.gov/clinicaltrials/digestpage/TAILORx) and MammaPrint (MINDACT trial, see http://www.breastinternationalgroup.org/TransBIG/Mindact. aspx) as a sole determinant of therapy selection. Gene expression profiles with predictive utility such as the Oncotype DX103 can help individualize the therapy benefit in a very specific subset of patients with ER-positive, lymph node-negative disease. Preliminary data presented at the San Antonio Breast Cancer Symposium in the fall of 2007 (see www.sabcs.org) suggest that the Oncotype DX assay also may help identify among postmenopausal women with ER-positive, lymph node-positive disease those who would benefit from the addition of anthracycline-based chemotherapy to tamoxifen.

ADJUVANT SYSTEMIC THERAPY While adjuvant systemic therapy significantly reduces the odds of recurrence and death, mammography screening and earlier diagnosis are responsible for at least half of the breast mortality reduction observed between 1990 and 2003,242 suggesting that the ability of tumors to metastasize may be acquired over time and would counter the prevailing so-called systemic theory.182 Large databases also indicate that the 5-year survival rate in women with small endocrine-responsive tumors is not likely to be affected by their disease, and chemotherapy offers minimal potential benefit,243 although data from the NSABP suggest improvements in both recurrence-free and overall survival in women with ER-positive and ER-negative tumors no more than 1 cm in size.244 These uncertainties are due to the modest treatment benefits obtained with available systemic therapies on average and the considerable heterogeneity observed in breast cancer even when considering tumors with similar profile using standard pathologic parameters (e.g., tumor size, nodal status, and ER/PR expression). While systematic reviews and computerized nomograms have been quite useful to demonstrate the average benefit for specific patient subgroups, especially when the absolute benefit is otherwise small, these efforts fail to recognize this individual variability. It is now understood that the small to modest therapeutic effects noted in individual clinical studies are of great value if applied to the large population of women with breast cancer. Since 1985, the EBCTCG (see www.ctsu.ox.ac.uk/~ebctcg) has performed, at 5-year intervals, an ongoing combined analysis, or meta-analysis, of all available randomized trials to detect whether a specific treatment modality used for patients with operable breast cancer had an effect on overall survival and to determine the magnitude of this effect.228 General conclusions can be reasonably drawn about the effectiveness of various adjuvant systemic therapies. A significant survival advantage after polychemotherapy was unequivocally shown in all adequately studied age categories. However, the magnitude of benefit appears to be less in older women. Likewise, chemotherapy has been

effective in patients with node-negative or node-positive disease. Polychemotherapy is superior to monochemotherapy, and chemotherapy administered for 12 months or longer has not been associated with greater benefit than shorter duration of treatment (e.g., 6 months). Increasing evidence indicates additional benefit of combining chemotherapy and tamoxifen in receptor-positive patients, and anthracycline-containing regimens appear to have greater effects on recurrence and survival than standard CMF regimens. The Oxford Overview also shows that adjuvant tamoxifen improves survival, irrespective of age or menopausal status. The hormone-receptor status of the primary tumor is the strongest predictor of the magnitude of the treatment benefit of tamoxifen. In contrast to chemotherapy, more prolonged administration of tamoxifen (i.e., 5 years) provides greater benefit than a single year of administration. Some studies show no additional benefit when tamoxifen is continued beyond 5 years, which is now the standard in most centers, but this question has not been fully resolved. Ovarian function suppression reduces mortality rate in women younger than 50 years of age when compared with no therapy and is similar to the benefit offered by CMF-based chemotherapy, but its additive role after chemotherapy, in addition to tamoxifen, and its use with aromatase inhibitors are now the subject of large international clinical trials started in 2003 and led by the International Breast Cancer Study Group (IBCSG). However, it is now accepted that breast cancer is a heterogeneous disease. Decisions about whether to consider adjuvant systemic therapy should first take into account the tumor phenotype according to predictive markers of response that help select therapy (e.g., ER, PR, and HER2) and provide estimates of relative risk reduction, followed then by traditional prognostic markers of risk (e.g., tumor size, node involvement, and grade) to help estimate the actual absolute risk reduction benefit, and finally the assessment of existing comorbidities that may affect toxicity risks. Approximately 70% of newly diagnosed breast cancers express ER and/or PR, and one fifth overexpress HER2 (half of them [10% of the total] are ER/PR negative), while the remaining 15% to 20% express none of them (the so-called triple negative phenotype). Published data from 2000 systematic reviews by the EBCTCG suggest a reduction in mortality in patients with ER-positive disease through the next 15 years of 38% (age < 50 years) and 20% (age 50–69 years) with chemotherapy, and an additional reduction of 31% of the residual risk with tamoxifen, thereby approaching a final mortality reduction that ranges from 57% to 45%, respectively.228 However, it is important for clinicians to recall that the overview results provide information about predicted outcomes for populations, but do not provide insight into the distribution of benefit among individual patients. Breast cancer patients traditionally have overestimated the absolute value of systemic therapy, and a common misinterpretation is that the treatment benefit is shared among patients, with most having some benefit, and it is important to consider the estimated individual risk, comorbidity, and personal patient preferences when discussing the potential benefits of adjuvant systemic therapy. Quantitative tools like Adjuvant! Online (www.adjuvantonline. com) were developed to help patients and health care providers estimate the potential actual benefit from adjuvant systemic therapy,229,230 although estimates of benefit from adjuvant trastuzumab have not yet been included in this model. Clinical practice guidelines often are used, such as those from the National Comprehensive Cancer Network in the United States (available at www.nccn.org/professionals/physician_gls/default.asp).154 Another good example comes from the St. Gallen International Expert Consensus Panel Meeting (Tables 95-16 and 95-17).245 The fourth cycle of the Oxford Overview 2000 published in 2005 included approximately 150,000 patients in 194 unconfounded randomized clinical trials of adjuvant chemotherapy or endocrine therapy started by 1995.228 These were trials of CMF, FAC or FEC chemotherapy, and tamoxifen or ovarian suppression as endocrine

Cancer of the Breast • CHAPTER 95

Table 95-16 St. Gallen International Experts Consensus Meeting 2007 Definition of Risks for Patients with Operable Breast Cancer LOW

INTERMEDIATE

HIGH

NEGATIVE

NEGATIVE

POSITIVE (1–3 LNS)

POSITIVE (1–3 LNS)

Node Status

Plus All Factors

Plus Any Factor

Plus All Factors

Plus All Factors

Path T size

≤2 cm

>2 cm

Grade

1

3-Feb

Vascular invasion

No

Yes

ER/PR

Positive

Negative/negative

Positive

Negative/negative

HER2

Negative

Positive

Negative

Positive

Age (yr)

≥35

1 cm. † Only if other concomitant risk factors (grade > 1, hormone receptors lacking). ‡ Only if size >20 mm and PgR negative. Adapted from Piccart-Gebhart MJ: Adjuvant trastuzumab therapy for HER2-overexpressing breast cancer: what we know and what we still need to learn. Eur J Cancer 2006;42:1715–1719.

and lower toxicity,269 but they have not been approved in the adjuvant setting. Tamoxifen is associated with an increase in bone mineral density in the axial skeleton and with stabilization in the appendicular skeleton in postmenopausal women. While it leads to bone mineral loss in the lumbar spine and hip in premenopausal women, the NSABP P-01 study showed a reduction (relative risk, 0.81; 95% CI, 0.63 to 1.05) in fractures of the hip, radius, and spine in all age groups, especially among those 50 years of age and older.270 Data from the 2005 EBCTCG showed that 5 years of adjuvant tamoxifen for ERpositive disease reduced the annual breast cancer death rate by 31% regardless of age or chemotherapy use. Duration of tamoxifen (5 years versus 2 years) is important, and the annual breast cancer mortality rates are similar during years 0 to 4 and 5 to 14, with a cumulative reduction in mortality twice as large at 15 years as after 5 years since diagnosis.228 Duration of adjuvant tamoxifen is limited to 5 years,271,272 but two large, ongoing international trials (Adjuvant Tamoxifen Long vs. Short [ATLAS] and Adjuvant Tamoxifen Treatment, Offer More? [aTTom]) may help to answer this question. Host factors such

as polymorphisms of the P450 CYP2D6 gene may identify patients who are poor metabolizers (*4/*4 genotype variant) and have reduced levels of the active tamoxifen metabolite endoxifen,273 but the clinical utility of this information is not fully settled, other than perhaps to identify patients who should avoid use of potent CYP2D6 inhibitors, including some of the selective serotonin reuptake inhibitors commonly used as antidepressants.

Ovarian Function Suppression Adjuvant endocrine therapy arguably is the most effective targeted therapy in women with early-stage, ER-positive breast cancer, regardless of age or nodal status. Although breast cancer is primarily a disease of older women, up to 25% of all patients newly diagnosed with invasive disease are younger than age 50 (half with ER-positive disease).274 It is unfortunate, therefore, that the survival benefit of tamoxifen was not fully recognized till the mid-1990s.275 The ovary is the primary site of estrogen production in premenopausal women. In 1896, Sir George Beatson first reported the benefits of oophorec-

Cancer of the Breast • CHAPTER 95

Table 95-21 Adjuvant Trastuzumab Trials: Efficacy Results B31 + N9831

HERA

FINNISH TRIAL BCIRG006

Observation H ¥ 1 yr (N = 1693) (N = 1694)

Control (N = 1679)

H ¥ 1 yr (N = 1672)

Patients with events

220

127

261

133

147

77

98

27

12

Distant events

154

85

193

96

113

52

67

26

8

Events for OS

37

29

92

62

36

20

28

14

6

HR for DFS

0.54

AC-T

AC-TH

Control (N = 115)

TCH

H ¥ 9 wk (N = 116)

Events* for DFS

0.48

0.61 (0.47–0.79)

(95% CI)

(0.43–0.57)

(0.39–0.59)

0.49 (0.37–0.65)

P-value

50

70

Pawlicki et al.

CT ± S + RT

72

NA

NA

NA

Loprinzi et al.

S + CT + RT + CT

9

100

>25

55

Keiling et al.

CT + S + CT

41

100

NR

63

Jacquillat et al.

CT + RT + CT + H

66

100

NR

66

Alberto et al.

CT + S + CT + RT

22

95

26

10

Ferriere et al.

CT + RT ± S + CT

75

93

NR

54

Pourny et al.

CT + S ± RT + CT

33

82

70

60

Chevallier et al.

CT + RT ± CT ± S

178

83

37

32

Rouesse et al.

CT + RT + CT + H

91

41

36

40

Israel et al.

CT + S + CT

25

96

NR

62

Krutchik et al.

CT + RT + CT

32

NA

24

NA

Brun et al.

CT + RT + S + CT

26

NA

31

NA

Thoms et al.

CT + S + CT + RT

61

NA

61

35

Swain et al.

CT + RT + S + CT + H

45

NA

36

NR

Fields et al.

CT + S + RT + CT

37

NA

49

44

Maloisel et al.

CT + S + CT + RT + H

43

NA

46

75

Koh et al.

CT + RT + CT

40

NA

39

37

CT + S + CT + RT

23

NA

38

30

CT + S + CT + RT

43

NA

31

40

CT, chemotherapy; H, hormone therapy; NA, not available; NR, not reached; RT, radiation therapy; S, surgery. Adapted from Hortobagyi G, Singletary S, Strom E: Treatment of locally advanced and inflammatory breast cancer. In Harris J, Lippman M, Morrow M, Osborne CK (eds): Diseases of the Breast, 2nd ed. Philadelphia, Lippincott Williams & Wilkins, 2000, p 651.

sarily signal great risk of systemic disease. It may represent actual recurrence at or near the site of the original primary lesion, or it may be a new primary lesion, especially when located in a different quadrant of the breast. The incidence of breast recurrence in patients treated with adequate local lumpectomy and breast radiation is 10% to 20% at 10 years, and the preferred treatment for patients who have a failure in the conserved breast is salvage mastectomy, with or without reconstruction. Other approaches, such as tumorectomy, result in lower rates of subsequent local control. In the surgical management of these women, it is important to consider the decreased blood supply and decreased skin elasticity from the previous radiation. Skin closure should be accomplished at salvage mastectomy, keeping the possibility of delayed healing in mind. For some of the same reasons, reconstruction with a tissue expander often results in increased incidence of necrosis, infection, and capsular contracture, and reconstruction with autologous tissue often is preferred. Systemic staging is recommended at the time of diagnosis of an in-breast recurrence. The decision to use additional multidrug chemotherapy or hormone therapy is based on the nature of the breast recurrence as well as on whether the patient previously received adjuvant chemotherapy. Prospective studies to evaluate the optimal locoregional and systemic management of these patients are difficult to conduct, and these patients benefit from management by an experienced multidisciplinary breast team. In the absence of definitive data, adjuvant systemic treatment often is considered. Selection of

regimens frequently accounts for prior adjuvant regimens and pathogy findings (e.g., ER/PR and HER2 expression).

Special Problems Patients who undergo unsuccessful radiation therapy, chemotherapy, or a combination of the two in an attempt to control locoregional disease present a special challenge for the surgeon. Surgery should be attempted in these patients, because failure to control local disease results in a considerable decrease in quality of life. These patients often have painful, ulcerating, bleeding, and chronically infected local tumors. Surgery should be aggressive, frequently including the ribs and intercostal muscles. As emphasized, the extent of locoregional recurrence is a major influence on the design of a treatment plan. Therapy decisions also are significantly affected by the recognition that such disease is a marker for distant disease. For these reasons, a multimodality approach appears to have the best chance of providing real patient benefit.

MANAGEMENT OF METASTATIC DISEASE The primary goal of therapy in patients with metastatic breast cancer is palliation of symptoms and prolongation of high-quality life, because most patients with metastatic (advanced) breast cancer ultimately die of their disease. At the same time, there has been an

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improvement in the survival of of these patients with metastatic disease over the last few decades as a result of more effective therapies and diagnosis at earlier phases of metastatic disease (stage migration).376 Chemotherapy trials with taxanes treating a mixed population of patients showed a median survival, on average, of approximately 2 years.377 However, some patients survive long-term, and a very small number of patients with “oligometastatic” disease may even benefit from multimodality therapy that includes surgical resection of an isolated visceral metastasis with curative intent.378 Also, specific therapies like trastuzumab have changed the natural history of HER2positive metastatic breast cancer.234 Approximately 75% of metastases occur within the first 5 years after the diagnosis of early-stage disease, especially among patients with hormone receptor-negative disease. Unfortunately, a smaller risk of recurrence persists, and metastases have been documented as late as 20 to 30 years after the initial diagnosis. Although most patients with metastatic disease are expected to progress at some point, certain clinical and tumor characteristics are useful in predicting prognosis. Patients with a long interval since initial diagnosis, excellent performance status, hormone receptor-positive disease that primarily involves bone or soft tissue, and only a few sites of visceral involvement are likely to have a better long-term prognosis. Available locoregional systemic and supportive care treatments can result in significant regression of disease, relief of symptoms, and, in some cases, prolongation of survival. Although the goal of treatment of metastatic breast cancer seldom is cure, palliation with improved quality of life can be achieved in many patients. Preliminary evidence indicates a role for combined multimodality therapy in patients with small-volume (preferably isolated) metastatic disease.379,380 If confirmed, this also could have significant implications and force re-examination of the current recommendations for no surveillance in the absence of specific symptoms. Also, more effective surveillance tools would be required. Previous exposure to adjuvant therapy predicts a lower response to first-line chemotherapy in patients with metastatic breast cancer. However, retrospective data suggest that patients who have a recurrence long after completing adjuvant therapy may respond to similar regimens.381 IBCSG data also suggest that quality-of-life scores may correlate with outcome in metastatic breast cancer,382 and improvement in symptoms such as pain and shortness of breath may correlate with greater response to therapy.383

Evaluation of Suspected Metastases Many patients present with nonspecific symptoms, such as new pain, weight loss, or dyspnea. Whenever possible, tissue acquisition for diagnostic confirmation and reassessement of receptor status (ER, PR, and HER2) should be considered. Clinicians should be wary of solitary lesions seen on bone scintigraphy or CT scan because they may not represent metastatic breast cancer. In the appropriate clinical setting, imaging studies without tissue confirmation may be acceptable evidence of metastatic disease, such as multiple areas of osseous lytic or blastic metastases or multiple sites with visceral involvement. Baseline imaging studies, including bone scintigraphy, CT, and plain x-rays, will provide a baseline for the evaluation of response to the planned treatment modality. Prompt initiation of supportive measures and specific anticancer therapy in patients with significant symptoms or life-threatening complications (e.g., spinal cord compression, destructive bone lesions in weight-bearing areas, hypercalcemia, and symptomatic pleural or pericardial effusions and ascites) can offer significant palliation of symptoms. Skeletal scintigraphy (bone scan) remains the primary modality for screening for bone metastasis, but often needs to be complemented by other modalities (plain x-rays, CT, and MRI) as it primarily focus on bone metabolism.384 Other metabolic studies like FDG-PET may have a role specially when integrated with conventional CT imaging for anatomic information (albeit frequently

without iodide contrast),385 but FDG-PET is inferior to bone scan for the assessment of blastic lesions,386 and cost remains a barrier for many centers. Recent reports suggest an apparent increase in the prevalence of central nervous system (CNS) involvement, especially in patients with HER2-positive disease.387 This is in great part due to improved control of systemic disease and lack of penetration of antibody therapy in sanctuary sites. This has also been observed in otherwise unselected patients who respond to conventional chemotherapy drugs and appear to be at risk for CNS relapse.388

Endocrine Therapy Effective therapies with minimal toxicity, such as endocrine therapy, are highly desirable and should be considered a primary option over cytotoxic chemotherapy in patients with hormone receptor-positive disease. There is some evidence patients whose tumors co-express HER2 have less responsive disease to aromatase inibitor therapy alone and may benefit from the addition of trastuzumab,389 but single-agent oral therapy with an anti-estrogen remains a viable option. Although the clinical factors discussed earlier are important in the selection of systemic therapy, routine measurement of ER and PR expression to identify patients who may respond to endocrine therapy may be the single most important initial test. Patients with ER/PR-positive and bone/soft tissue only or asymptomatic visceral disease should be considered for initial palliation with endocrine therapy. Examples of palliative endocrine regimens are listed in Table 95-25.

Selective Estrogen-Receptor Modulators Tamoxifen, 20 mg daily, still is the most commonly used SERM in many countries. Toremifene is now available for use in this patient

Table 95-25 Examples of Commonly Used Endocrine Regimens in the Metastatic Setting* INITIAL ENDOCRINE THERAPY OPTIONS No prior endocrine therapy or over one year since endocrine therapy Premenopausal → Anti-estrogen or OFS with endocrine therapy like postmenopausal women Postmenopausal → Aromatase inhibitor or anti-estrogen Prior endocrine therapy Second-line endocrine therapy (see below)

SUBSEQUENT ENDOCRINE THERAPY OPTIONS Premenopausal patients Add OFS and treat like postmenopausal women Postmenopausal patients Nonsteroidal (anastrozole, letrozole) or steroidal (exemestane) aromatase inhibitor Note: men of any age should not receive an aromatase inhibitor without concomitant use of an LH-RH agonist Fulvestrant Tamoxifen or Toremifene Megestrol acetate Fluoxymesterone Ethinyl estradiol *Consider chemotherapy if no clinical benefit (response or stable disease) after three endocrine regimens or symptomatic visceral disease. Adapted from NCCN Clinical Practice Guidelines in Oncology: Breast Cancer. V.2.2008. www.nccn.org/professionals/physician_gls/PDF/breast.pdf.

Cancer of the Breast • CHAPTER 95

population, but has a similar profile and is cross-resistant with tamoxifen. PET with either FDG or the estrogen analog 16α-18fluoroestradiol-17β before and after treatment with tamoxifen can assess the functional status of the ER in vivo (metabolic flare) and may predict response to tamoxifen in patients with ER-positive disease.390 Acquired resistance to tamoxifen may be explained by a variety of mechanisms, such as mutations in the ER, changes in tamoxifen metabolism, levels of intracellular tamoxifen, and differential expression of steroidreceptor transcriptional coactivators and corepressors.391

Aromatase Inhibitors In postmenopausal women with no or distant previous exposure to antiestrogen agents, the aromatase inhibitors show similar or modestly superior efficacy compared with tamoxifen.297 These drugs are ineffective in premenopausal women and should be avoided as single agents in this setting. Aromatase inhibitors are more active and less toxic than megestrol acetate as second-line therapy; they also are more active and less toxic than the first-generation drug aminoglutethimide. Letrozole appears to be a more potent suppressor of total-body aromatization and plasma estrogen levels than anastrozole in patients with breast cancer,392 but direct comparison as second-line therapy in metastatic breast cancer show no convincing clinical advantage of one aromatase inhibitor over another.393 Circulating levels of estrone and estradiol decrease similarly in both responders and nonresponders after therapy with an aromatase inhibitor, suggesting that intratumoral aromatase activity could play a more significant role in differentiating these two groups of patients.394 There are few data regarding the optimal sequence of therapy (i.e., a SERM followed by an aromatase inhibitor, or vice versa), and most patients will be treated with both at some point. Aromatase inhibitors should be avoided as a single agent in men with metastatic disease, as their chronic administration may lead to a significant increase in levels of follicular stimulating hormone and testosterone without any change in levels of estradiol, but appear to be effective when combined with an LH-RH agonist as chemical castration.395

Table 95-26 Examples of Commonly Used Endocrine Regimens in Metastatic Breast Cancer PREFERRED SINGLE AGENTS Doxorubicin Epirubicin Pegylated liposomal doxorubicin Paclitaxel Docetaxel Capecitabine Vinorelbine Gemcitabine Albumin-bound paclitaxel Ixabepilone

PREFERRED AGENTS WITH BEVACIZUMAB Paclitaxel

PREFERRED AGENTS WITH TRASTUZUMAB Paclitaxel ± carboplatin Docetaxel Vinorelbine

PREFERRED COMBINATIONS CAF/FAC (cyclophosphamide/doxorubicin/fluorouracil) FEC (fluorouracil/epirubicin/cyclophosphamide) AC (doxorubicin/cyclophosphamide) EC (epirubicin/cyclophosphamide) AT (doxorubicin/docetaxel; doxorubicin/paclitaxel) CMF (cyclophosphamide/methotrexate/fluorouracil) Docetaxel/capecitabine

Ovarian Ablation

GT (gemcitabine/paclitaxel)

The prefered endocrine therapy in premenopausal women with endocrine-responsive disease and recent exposure to tamoxifen is ovarian function suppression with surgical or medical techniques (LHRH agonist).396 Radiation ablation is less reliable and technically more challenging, and the results are not as immediate.397 Available data show both an overall survival advantage and a progression-free survival advantage with the addition of tamoxifen to an LHRH agonist,398,399 but there are limited data on ovarian function suppression and an aromatase inhibitor.

OTHER ACTIVE AGENTS Cisplatin Carboplatin Etoposide (oral) Vinblastine Fluorouracil continuous infusion Adapted from NCCN Clinical Practice Guidelines in Oncology: Breast Cancer. V.2.2008; www.nccn.org/professionals/physician_gls/PDF/breast.pdf.

Other Antiestrogens The nonsteroidal pure antiestrogen fulvestrant downregulates ER without the agonistic activity of tamoxifen. It appears to have inhibitory effects in breast and uterus with neutral effect in bones and lipids,400 and clinical effects appear similar to aromatase inhibitors.401 It is currently only approved in postmenopausal women as a monthly, deep IM injection. Data on the clinical benefit from a loading disease are conflicting.

Chemotherapy Patients with symptomatic visceral disease, ER- and PR-negative disease, or disease that is resistant to endocrine therapy should receive chemotherapy. Given the palliative goal and the toxicities of cytotoxic therapy, the challenge for the oncologist is in deciding when to initiate chemotherapy. Many appropriate chemotherapy regimens are available, and little evidence recommends com-

bination therapy over sequential single-agent chemotherapy (Table 95-26). There often is a fine line between premature use of chemotherapy in the asymptomatic patient without disease-related complications versus delaying therapy until deterioration of performance status significantly decreases the likelihood of response. There is considerable interest in identifying biologic parameters that may predict the success of specific chemotherapy regimens such as the identification of patients with HER2-positive diseases who could be offered trastuzumab. Patients with disease that does not express hormone receptors are more likely to respond to chemotherapy.402 The organ distribution of metastases and the patient’s symptoms, history of exposure to chemotherapy, and general medical condition are helpful considerations when determining the time of initiation of chemotherapy. Age alone should never be a contraindication.

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Single-Agent Chemotherapy Commonly used single-agent chemotherapy drugs in patients with advanced disease include anthracyclines, taxanes, vinorelbine, and capecitabine. If no data suggest a markedly strong benefit of one class of drugs over another, decisions should be made based on patient convenience and toxicity profile. Other active agents include gemcitabine, platinum compounds, etoposide, vinblastine, and continuous-infusion 5-fluorouracil. The activity of taxanes in patients with anthracycline-resistant disease is well documented,403 and may extend to patients previously treated with another taxane. Taxanes appear to have a better toxicity profile compared with either doxorubicin404 or a CMF. Capecitabine is active in patients who showed disease progression after previous taxane regimens.405 The epothilone B drug ixabepilone also has clinical activity in patients with disease resistant to paclitaxel, doxorubicin, and capecitabine.406,407

Combination Chemotherapy Commonly used combination chemotherapy regimens include FAC/ CAF (5-fluorouracil, doxorubicin, and cyclophosphamide), FEC (with epirubicin), AC, epirubicin and cyclophosphamide, AT (doxorubicin plus docetaxel or paclitaxel), and CMF. A survival advantage was seen in randomized trials of first-line therapy with paclitaxel or docetaxel against combination regimens. However, such survival advantage correlates with whether or not patients who showed disease progression while receiving the nontaxane regimen were subsequently crossed over to a taxane drug. Similar findings were seen in a study of docetaxel with or without capecitabine in patients with metastatic breast cancer who were previously treated with an anthracycline.408 In this study, only a small fraction of patients who were initially treated with docetaxel alone subsequently crossed over to capecitabine at progression. Combinations between anthracyclines and taxanes have been tested, but paclitaxel interferes with doxorubicin elimination and lowers the threshold for cardiac toxicity. Dexrazoxane appears to be cardioprotective in patients treated with FAC. Gemcitabine plus paclitaxel significantly improves progression-free survival when compared to paclitaxel alone,409 and a similar study design led to FDA approval in 2007 of ixabepilone in combination with capecitabine.410 High-dose chemotherapy with autologous or allogeneic stem cell support remains investigational. Available data do not support its use as a standard approach.411

HER2-Targeted Therapy Anti-HER2 therapy with the recombinant monoclonal antibody trastuzumab is another recent example of targeted therapy. Its potential benefits are restricted to women whose tumors overexpress HER2, and evidence does not support its use against HER2-negative disease. Although data do not show a survival advantage with combined cytotoxic therapy in metastatic disease whenever there is sufficient cross-over to the investigational agent, this benefit was seen when trastuzumab was added to anthracycline and cyclophosphamide or paclitaxel.234 Excessive cardiac toxicity limits the ability to combine anthracyclines with trastuzumab.412 Many consider trastuzumab the mainstay of therapy in HER2positive disease. However, the sequential approach of trastuzumab followed by chemotherapy (or the combination) versus upfront combined therapy has not been tested. Combination of an aromatase inhibitor with trastuzumab appears to offer improved survival.389 Single-agent therapy with trastuzumab is an active option413 and can be given once every 3 weeks.414 The dual tyrosine kinase inhibitor lapatinib recently was approved by the FDA in combination with capecitabine for the treatment of patients who previously were treated with trastuzumab,415 and also prolongs time to tumor progression, with superior results when combined with paclitaxel versus paclitaxel

alone.416 This drug also is active in HER2-positive patients who have not been treated with trastuzumab.417

Therapies Targeting Angiogenesis New blood vessel formation plays a key role in breast cancer growth locally and at distant sites, and angiogenesis appears to be essential for tumor development, invasion, and metastasis.418 Evidence exists that angiogenesis precedes transformation of mammary hyperplasia to malignancy. Hypoxia is a strong angiogenesis signal, and expression of the hypoxia-inducible factor HIF-1α increases from normal breast tissue through usual ductal hyperplasia, DCIS, and invasive disease.419 Expression of HIF-1α is increased in poorly differentiated tumors and is associated with proliferation and expression of vascular endothelial growth factor (VEGF). Microvessel density (MVD) is associated with higher-grade DCIS and greater risk of developing metastatic disease.418 Angiogenesis inhibition has been observed with drugs such as tamoxifen and commonly used chemotherapy drugs in preclinical models, and these observations led to increased interest in the socalled “metronomic” schedules of chemotherapy administration. Agents targeting various angiogenesis receptors and ligands have been developed. The small-molecule tyrosine kinase inhibitor sunitinib modulates VEGF receptor (VEGFR)-1, VEGFR-2, platelet-derived growth factor receptor (PDGFR), c-kit, and Flt-3. It is approved by the FDA for the treatment of advanced renal cell cancer and has single-agent activity in refractory metastatic breast cancer.420 The humanized monoclonal antibody bevacizumab, which targets the VEGF-A ligand, is approved in the United States for patients with metastatic colon and lung cancer, and has a small risk of complications including bleeding, healing impairment, hypertension, and proteinuria. Bevacizumab has single-agent activity in patients with refractory advanced breast cancer421 and when combined with the chemotherapy drug capecitabine.422 Bevacizumab was shown to increase progression-free survival significantly when added to paclitaxel in a randomized first-line trial for women with metastatic disease (ECOG 2100),423 and it is now the subject of a prospective randomized trial testing a sequential anthracycline/paclitaxel regimen with or without bevacizumab for patients diagnosed with high-risk, earlystage HER2-negative breast cancer (ECOG 5103).

Bisphosphonates Bone is the most common site of metastatic disease and ultimately is affected in most patients.424 Monthly injections of bisphosphonates for up to 2 years can reduce the risk of skeletal events in patients who have lytic bone metastases and are receiving systemic therapy. Zoledronate is an effective alternative to pamidronate because of its shorter infusion time.425 However, these more potent third-generation bisphosphonates are associated with an increased risk of osteonecrosis of the jaw.426

New Approaches The marked increase in understanding of the cellular and molecular biology of breast cancer led to a striking increase in novel therapies for breast cancer and a significant shift away from the high-dosechemotherapy hypothesis that “more is better,” to a more rational, targeted approach that first was attempted with endocrine therapy and now also is being used with anti-HER2 therapy. Other examples in patients with advanced disease include therapies targeting the epidermal growth factor family of receptors, VEGF receptors, and other angiogenesis targets. The redundancy of many of these pathways suggests the potential need for a combination of drugs targeting multiple steps or more promiscuous drugs that interact with multiple targets.427 These studies include combinations of existing hormonal and cytotoxic drugs, and the design of these studies is challenging. Patients must have access to high-quality, well-conducted studies to

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ensure that useful data are generated, are properly interpreted, and lead to improved cancer care. An important challenge in the treatment of patients with breast cancer is how best to identify those who are most likely to benefit from specific interventions while avoiding unnecessary toxic exposure in those who are unlikely to benefit. Circulating tumor cells, at baseline and after just 4 weeks of therapy, can identify patients early on who are not responding so that a potentially toxic therapy can be discontinued.108,155 Ongoing trials are now examining whether therapeutic decisions made based on these findings will result in improved clinical outcome.

UNUSUAL PROBLEMS ENCOUNTERED IN BREAST CANCER Male Breast Cancer Male breast cancer is rare, accounting for 0.2% of male cancers and fewer than 1% of new breast cancers.428 Mutations in the BRCA2 gene predispose men to breast cancer and may account for up to 40% of all cases. Most present as infiltrating ductal carcinoma with unilateral, firm, painless masses. Nipple discharge should be taken seriously and is an indication for FNA or core or excisional biopsy. Mammography and ultrasound may be of help and may contribute to differentiating a breast cancer from a gynecomastia. A negative finding on FNA or core biopsy requires an excision procedure, and cytologic findings that show gynecomastia require close follow-up. The tumor phenotype appears similar to that observed in women. Treatment is modeled after female breast cancer, both locoregional and systemic therapies. There is evidence that SLNB can be safely applied in male breast cancer.429 Because most patients have endocrine-responsive disease, orchiectomy or chemical castration with LH-RH agonists often is used in patients with advanced disease. Tamoxifen is the most common endocrine therapy used, although androgens, antiandrogens, corticosteroids, estrogens, and progestins have also been used. Aromatase inhibitors are of potential interest in men with metastatic disease. However, approximately 80% of circulating estrogens are derived from the aromatization of precursor androgens with the remaining estrogens come from direct testicular secretion. This explains the apparent lower response rate observed with the first-generation aromatase inhibitor aminoglutethimide compared with tamoxifen and with anastrozole.430 These observations suggest that orchiectomy and tamoxifen remain the endocrine therapies of choice in men with breast cancer, although a recent report also suggests a role for aromatase inhibitors if given with an LH-RH agonist.395

Breast Cancer and Pregnancy Breast Cancer during Pregnancy Carcinoma of the breast, although rare in pregnant women, occurs in about 1 to 3 patients per 10,000 deliveries and is the most common malignancy associated with pregnancy. It is expected to become more common with the observed increase in the average age at first fullterm pregnancy. The advanced level of disease and the poor prognosis associated with gestational breast cancer appear to be due primarily to delay in diagnosis. For this reason, a thorough examination of the breast should take place during the first obstetric visit, before the breast becomes engorged or hypertrophic, and the obstetrician must pursue early signs of breast cancer actively via a thorough breast examination. Diagnosis and staging are far more difficult in pregnant women because of physiologic changes in the mother and radiation risk to the fetus. Mammograms are not routinely performed—little information can be gained because of pregancy-related increased breast density. Ultrasonography and MRI can be used,

although safety of the gadolinium for the fetus has not yet completely been determined. Routine bone scintigraphy is contraindicated in pregnant women. Biopsy of a mass during pregnancy is difficult and must be undertaken with extreme care to avoid infection and milk fistulas in the lactating breast. FNA and stereotactic NCB are the initial procedures used in evaluating a breast mass, with lactation often suppressed preoperatively with bromocriptine. Biopsy can almost always be performed under local anesthesia; however, no evidence shows that general anesthesia poses significant risk to either the mother or the fetus if proper precautions are taken. There are some data in favor of the safety of SLNB in pregnant women.431 Modified radical mastectomy is the treatment of choice for breast cancer during pregnancy. Depending on the extent of disease and the predicted delivery date, breast conservation may be considered. Tumor excision and axillary dissection are performed during pregnancy, followed by breast irradiation after delivery. Survival is not improved by therapeutic abortion, and pregnancy-associated breast cancer should be treated using the same decision tree that would be appropriate for a patient who is not pregnant. While therapeutic abortion early in pregnancy greatly simplifies the treatment of early-stage breast cancer, it does not improve treatment outcome, and there are no reports show that breast cancer is harmful to the fetus, because breast cancer cells cannot traverse the placenta, as in some cases of melanoma, lymphosarcoma, or leukemia. In most cases, chemotherapeutic agents are not recommended for use in pregnant women because of concerns about teratogenesis. The effect on the fetus also is related to drug dosage, gestational age, and the individual patient’s tolerance. However, cytotoxic chemotherapy has been administered, mostly after the first trimester, without identifiable damage to the fetus. Data from the M.D. Anderson Cancer Center suggest that women can be safely treated with FAC chemotherapy during the second and third trimesters without sugnificant short-term complications, though little is know about long-term effects on fertility and cardiac function on these children.432 Administration of paclitaxel also appears feasible, as the drug is a substrate for placental p-glycoprotein and little is transferred to the fetal circulation.433

Pregnancy after Breast Cancer Many women now emerge from treatment for breast cancer with fertility intact, and some will become pregnant. The IBCSG identified in their clinical trial database 94 patients who became pregnant after a diagnosis of breast cancer, including 8 women who had a relapse during pregnancy. The investigators compared the outcome of these patients with the outcome of 188 matched, controlled patients from the same database, and found no adverse effect from pregnancy on survival.434 Thus far, there is no evidence that pregnancy after breast cancer treatment increases the risk of a worse outcome. However, women who remain premenopausal after chemotherapy are at risk for premature menopause.339

Axillary Metastases with Occult Breast Cancer A woman with clinically suspicious axillary lymph nodes despite negative breast examination and mammogram requires careful evaluation for breast cancer. FNA often allows the diagnosis of adenocarcinoma versus other tumor types. Breast is the most common primary source when dealing with adenocarcinoma, although gastrointestinal (GI) sources or the thyroid often may be involved. It is not unreasonable, therefore, to order a CT scan of the chest and abdomen if further breast imaging with ultrasound and MRI is unrevealing. The definition of “occult breast carcinoma” is a breast cancer presenting with metastatic axillary nodes without evidence of the primary tumor at the physical, mammographic, or ultrasound examination. Occult breast carcinoma presenting as axillary metastases is

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rare, accounting for less than 0.40% of primary operable breast cancer. Occult breast cancer should be differentiated from a contralateral dissemination to the contralateral axillary lymph nodes, accounting for 4%, based on physical examination only. The Memorial Sloan-Kettering experience with 69 patients with occult primary showed that MRI was able to detect a breast carcinoma in 63% of the patients, and no carcinoma was found in the mastectomy specimen when the MRI was negative.435 For a long time, ipsilateral mastectomy with complete axillary dissection has been the therapy of choice for patients presenting with axillary lymph node metastasis with occult primary.436 Some reports are showing that conservation of the breast is possible. The Milan group reported, at 43 months’ follow-up, a disease-free survival of 84% and two breast recurrences (supraclavicular and multiple metastasis).437 The M.D. Anderson Cancer Center reported local recurrence in 4 of their 32 patients (13%).438 These data are in favor of breast conservation in an occult primary. The routine use of MRI in this rare presentation of breast cancer allows detection of the primary tumor in a large number of patients. The 5- and 10-year survival rates are essentially the same as those for T1N1 and T2N1 tumors, and even patients in whom a primary breast cancer is not identified are treated in the same way as patients with N1 breast cancer.

Paget’s Disease of the Breast Paget’s disease of the breast is a cutaneous manifestation of underlying breast malignancy. This eczematoid lesion (often weeping, red, and crusting in late presentation) occurs in approximately 1% to 4% of patients with breast cancer, and as many as 50% of patients with Paget’s disease may have an underlying cancer. Paget’s disease is a carcinoma of glandular origin, and Paget’s cells are spread through the epidermis as a result of motility induced by a chemotactic factor released by epidermal cells. The greatest prevalence of Paget’s disease of the breast occurs in the sixth decade of life. The infrequency of early diagnosis is associated with a delay in recognition of early symptoms. Although an early sign of Paget’s disease is severe itching accompanied by erosive nipple changes, the median delay between first symptom and diagnosis is approximately 6 months, and often longer. Paget’s disease associated with a palpable underlying tumor can be treated with breast conservation with removal of the nipple-areolar complex. This procedure usually leaves an acceptable breast mound for subsequent reconstruction of the nipple-areolar complex. SLN biopsy is an appropriate method for evaluating axillary status. This disease shows the importance of physical examination and physician-patient education. Early-stage Paget’s disease of the breast is almost universally associated with prolonged disease-free survival. Several conditions mimic Paget’s disease of the breast secondary to underlying breast cancer, including erosive adenomatosis of the nipple and pemphigus vulgaris of the nipple. Although mastectomy has been the traditional surgical approach to this entity, breast conservation, with removal of the nipple-areolar complex, followed by whole-breast radiation, appears equally effective for Paget’s disease. Of note in this collaborative study, the group had did not have mammographic or physical evidence of an underlying breast mass.

Cystosarcoma Phyllodes and Sarcomas Sarcomas of the breast are rare, representing fewer than 1% of malignant breast tumors. Included in this group are benign and malignant forms of cystosarcoma phyllodes (CSP), accounting for approximately 0.5%; carcinosarcoma; and sarcoma. CSP tumors have a characteristic leaf-like architecture, with clefts lined by epithelial cells, and about 25% of the time are associated with fibroadenomas. Some authors refer to benign CSP as giant fibroadenomata, reserving the

term CSP for the malignant lesion. The average age at presentation is the mid-40s. Distinguishing benign from malignant lesions has been emphasized in describing CSP. Several histologic characteristics are considered indicators of malignant CSP, including increased cellularity, subepithelial stromal overgrowth, stromal anaplasia, tumor size, contour, degree of cellular atypia, and mitotic activity. Older series suggested a low risk of death when tumors had fewer than three mitotic figures per high-power field and that stromal overgrowth was associated with a risk of metastases. Barrio and colleagues439 at Memorial Sloan-Kettering Cancer Center reviewed the pathologic features and clinical course of 293 women who had CSP. Of these patients, 206 (70%) had benign CSP and 87 (30%) had malignant CSP. Breast-conserving surgery was the local therapy in 242 breasts; mastectomy was chosen in the remaining 48 breasts. With a median follow-up of 7.9 years, 35 patients recurred, for an actuarial 10-year rate of 14.4%. In this series, the classification into “benign” versus “malignant” types was not related to the risk of local failure. Using univariate analysis, the risk of a local failure was increased in women with positive margins, fibroproliferation, and necrosis.439 CSP tumors usually are treated with wide local excision to include a sufficient margin of normal breast tissue from the tumor bed. Even benign tumors have a high incidence of local recurrence if they are simply shelled out of the breast tissue, and meticulous effort is required to obtain an adequate tissue margin. Recurrences of benign CSP may be re-excised, again with wide margins, but some cases are better managed by mastectomy and reconstruction. In a SEER series of 821 women diagnosed with malignant CSP, 52% were treated with mastectomy and the remaining 48% with lumpectomy. In this nonrandomized series, the patients treated with lumpectomy were younger, had smaller tumors, and had an improved cause-specific survival, but the role of radiation is not adequately described.440 Aside from malignant CSP, sarcomas of the breast include a wide range of histologic types, such as carcinosarcoma, osteosarcoma, liposarcoma, angiosarcoma, malignant histiocytoma, leiomyosarcoma, stromal sarcoma, and mixed types. Carcinosarcoma is different, in that it is composed of a combination of malignant epithelial cells, as would be found in breast adenocarcinoma, in addition to malignant stromal cells characteristic of sarcoma. These tumors may behave somewhat differently from pure sarcomas and can spread to axillary lymph nodes. Treatment of carcinosarcoma usually includes mastectomy with adequate tissue margins, including muscle and skin, if necessary, and decisions about chest wall irradiation are influenced by tumor size, location, and margins. North and colleagues at the Roswell Park Cancer Institute reported 25 patients treated for breast sarcoma (10 with angiosarcomas) between 1964 and 1995.441 These patients had a median age of 55 years, 10 had a mastectomy, and their 10-year overall survival rate was 36%. In general, the type of surgery is determined by breast size, and, while wide local excision is adequate primary surgical treatment for most lesions, attention should be given to obtaining wide margins, including deep muscle, if needed. Tumors larger than 5 cm and high-grade tumors may require mastectomy, possibly with chest wall resection, and the surgeon should approach these tumors according to the established guidelines for sarcoma surgery. Lymph node dissection is not usually recommended due to the hematogenous nature of its dissemination. Imaging studies are recommended to rule out systemic dissemination (e.g., lung) at presentation. Adjuvant radiation therapy should be considered on a case by case basis, although data are limited, and little is known on the role of adjuvant chemotherapy. Therefore, while the rarity of breast sarcomas limits the rigorous evaluation of therapeutic options, it seems appropriate to approach sarcomas of the breast using therapeutic methods derived from the soft tissue sarcoma literature.

Cancer of the Breast • CHAPTER 95

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patients with advanced breast cancer: phase III trial results. J Clin Oncol 2002;20:2812–2823. Moinpour C, Wu J, Donaldson G, et al: Gemcitabine plus paclitaxel (GT) versus paclitaxel (T) as first-line treatment for anthracycline pretreated metastatic breast cancer (MBC): Quality of life (QoL) and pain palliation results from the global phase III study [abstract 621]. ASCO Meeting Abstracts 2004;22. Vahdat LT, Thomas E, Li R, et al: Phase III trial of ixabepilone plus capecitabine compared to capecitabine alone in patients with metastatic breast cancer (MBC) previously treated or resistant to an anthracycline and resistant to taxanes [abstract 1006]. ASCO Meeting Abstracts 2007:25. Hortobagyi GN: What is the role of high-dose chemotherapy in the era of targeted therapies? J Clin Oncol 2004;22:2263–2266. Seidman A, Hudis C, Pierri MK, et al: Cardiac dysfunction in the trastuzumab clinical trials experience. J Clin Oncol 2002;20:1215–1221. Vogel CL, Cobleigh MA, Tripathy D, et al: Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 2002;20:719–726. Leyland-Jones B, Arnold A, Gelmon K, et al: Pharmacologic insights into the future of trastuzumab. Ann Oncol 2001;12 Suppl 1:S43– S47. Geyer CE, Forster J, Lindquist D, et al: Lapatinib plus capecitabine for HER2-positive advanced breast cancer. N Engl J Med 2006;355:2733– 2743. Leo AD, Gomez H, Aziz Z, et al: Lapatinib (L) with paclitaxel compared to paclitaxel as first-line treatment for patients with metastatic breast cancer: a phase III randomized, double-blind study of 580 patients [abstract 1011]. J Clin Oncol, 2007 ASCO Annual Meeting Proceedings 2007;25(18S):1011. Gomez HL, Chavez MA, Doval DC, et al: A phase II, randomized trial using the small molecule tyrosine kinase inhibitor lapatinib as a first-line treatment in patients with FISH positive advanced or metastatic breast cancer [abstract 3046]. J Clin Oncol, 2005 ASCO Annual Meeting Proceedings 2006;23:abstract 3046. Schneider BP, Miller KD: Angiogenesis of breast cancer. J Clin Oncol 2005;23:1782–1790. Bos R, Zhong H, Hanrahan CF, et al: Levels of hypoxia-inducible factor-1 alpha during breast carcinogenesis. J Natl Cancer Inst 2001;93:309– 314. Miller KD, Burstein HJ, Elias AD, et al: Phase II study of SU11248, a multitargeted tyrosine kinase inhibitor (TKI) in patients (pts) with previously treated metastatic breast cancer (MBC) [abstract 1066]. Breast Cancer Res Treat 2005;94(suppl 1): S61. Cobleigh MA, Langmuir VK, Sledge GW, et al: A phase I/II dose-escalation trial of bevacizumab in previously treated metastatic breast cancer. Semin Oncol 2003;30:117–124. Miller KD, Chap LI, Holmes FA, et al: Randomized phase III trial of capecitabine compared with bevacizumab plus capecitabine in patients with previously treated metastatic breast cancer. J Clin Oncol 2005;23:792–799. Miller KD, Wang M, Gralow J, et al: A randomized phase III trial of paclitaxel versus paclitaxel plus bevacizumab as first-line therapy for locally recurrent or metastatic breast cancer: a trial coordinated by the Eastern Cooperative Oncology Group (E2100). Breast Cancer Res Treat 2005;94: A3. Solomayer EF, Diel IJ, Meyberg GC, et al: Metastatic breast cancer: clinical course, prognosis

Cancer of the Breast • CHAPTER 95

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and therapy related to the first site of metastasis. Breast Cancer Res Treat 2000;59:271–278. Berenson JR, Rosen LS, Howell A, et al: Zoledronic acid reduces skeletal-related events in patients with osteolytic metastases. Cancer 2001;91:1191–1200. Khosla S, Burr D, Cauley J, et al: Bisphosphonateassociated osteonecrosis of the jaw: report of a task force of the American Society for Bone and Mineral Research. J Bone Miner Res 2007;22: 1479–1491. Arteaga CL: Molecular therapeutics: is one promiscuous drug against multiple targets better than combinations of molecule-specific drugs? Clin Cancer Res 2003;9:1231–1232. Giordano SH, Buzdar AU, Hortobagyi GN: Breast cancer in men. Ann Intern Med 2002;137:678–687. Port ER, Fey JV, Cody HS 3rd, et al: Sentinel lymph node biopsy in patients with male breast carcinoma. Cancer 2001;91:319–323. Giordano SH, Valero V, Buzdar AU, et al: Efficacy of anastrozole in male breast cancer. Am J Clin Oncol 2002;25:235–237.

431. Mondi MM, Cuenca RE, Ollila DW, et al: Sentinel lymph node biopsy during pregnancy: initial clinical experience. Ann Surg Oncol 2007;14:218–221. 432. Hahn KM, Johnson PH, Gordon N, et al: Treatment of pregnant breast cancer patients and outcomes of children exposed to chemotherapy in utero. Cancer 2006;107:1219–1226. 433. Nekhayeva IA, Nanovskaya TN, Hankins GD, et al: Role of human placental efflux transporter P-glycoprotein in the transfer of buprenorphine, levo-alpha-acetylmethadol, and paclitaxel. Am J Perinatol 2006;23:423–430. 434. Gelber S, Coates AS, Goldhirsch A, et al: Effect of pregnancy on overall survival after the diagnosis of early-stage breast cancer. J Clin Oncol 2001;19: 1671–1675. 435. Buchanan CL, Morris EA, Dorn PL, et al: Utility of breast magnetic resonance imaging in patients with occult primary breast cancer. Ann Surg Oncol 2005;12:1045–1053. 436. Khandelwal AK, Garguilo GA: Therapeutic options for occult breast cancer: a survey of the

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American Society of Breast Surgeons and review of the literature. Am J Surg 2005;190:609–613. Galimberti V, Bassani G, Monti S, et al: Clinical experience with axillary presentation breast cancer. Breast Cancer Res Treat 2004;88:43–47. Vlastos G, Jean ME, Mirza AN, et al: Feasibility of breast preservation in the treatment of occult primary carcinoma presenting with axillary metastases. Ann Surg Oncol 2001;8:425–431. Barrio AV, Clark BD, Goldberg JI, et al: Clinicopathologic features and long-term outcomes of 293 phyllodes tumors of the breast. Ann Surg Oncol 2007;14:2961–2970. Macdonald O, Lee C, Tward C, et al: Malignant phyllodes tumor fo the female breast: determinants of cause-specific survival from the Surveillance, Epidemiology and End Results: (SEER) Program. Int J Radiat Oncol Biol Phys 2006;66:S121. North JH Jr, McPhee M, Arredondo M, et al: Sarcoma of the breast: implications of the extent of local therapy. Am Surg 1998;64:1059– 1061.

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Sarcomas of Bone Mark C. Gebhardt, Dempsey Springfield, and James R. Neff*

S U M M ARY

Incidence and Epidemiology • More than 2400 new cases of bone sarcoma are diagnosed annually in the United States. • No specific etiologic agents are identified in the majority of cases. • Secondary neoplasms are related to known oncogenic factors (e.g., ionizing radiation, alkylating chemotherapy agents, combinations of both). • Hereditary cancer syndromes (tumor suppressor genes) are responsible for some cases.

Diagnosis and Radiographic Staging • Plain radiographs are recommended. • Magnetic resonance imaging (MRI) scan of primary tumor is the best radiographic study to obtain. • Chest x-ray is indicated; chest computed tomography (CT) is indicated for suspected malignant lesions. • Whole-body technetium-99m (99mTc) bone scan is indicated. • Positron emission tomography (PET) scanning is controversial and has yet to be generally accepted.

O F

K EY

P OI NT S

• Needle or open biopsy is necessary for a tissue-specific diagnosis and to determine histologic grade. • In the pathology review, immunohistochemistry and cytogenetics are important. • Electron microscopic tissue occasionally is required.

and extracompartmental), grade (high and low), and metastasis (“skip” lesions, nodal, bone, and lung are all lumped together).

Primary Therapy

• Metastasis at presentation is a worse prognostic finding. • Histologic grade is the next most significant prognostic indicator. • Size is less significant, but lesions larger than 10 cm in diameter have a poor prognosis. • Tumor response to neoadjuvant chemotherapy • Surgical margins of resection (minimum of a “wide” margin)

• A wide surgical margin is recommended. • Limb-sparing procedures are appropriate for 70% to 90% of patients. • Adjuvant irradiation is not routinely used for bone sarcomas. • Local recurrence rates for limb-sparing procedures approach 5% or less. • Reconstruction methods can be tailored to patients’ needs. • New and improved biocompatible implant materials and improved designs are available.

Staging System

Future Trends

• The American Joint Committee on Cancer now monitors location, grade (high and low), depth, and size (8 cm); designates “skip” lesions (T3); and separates metastasis to bone from other sites (Mla, Mlb). • The Musculoskeletal Tumor Society monitors location (intracompartmental

• The search continues for new drugs, drug schedules, potentiating agents, and improved dose intensity. • Identification of risk factors (e.g., cytogenetic, molecular genetic, and signal transduction abnormalities) will improve to identify new methods of potential treatment.

Prognostic Factors

INTRODUCTION Approximately 2400 new malignant tumors of bone (excluding multiple myeloma) are diagnosed each year in the United States. The femur is the most common site, but primary sarcoma can occur in any bone. Osteosarcoma, Ewing’s sarcoma, and chondrosarcoma account for approximately 90% of all primary sarcomas of bone. The management of osteosarcoma and Ewing’s sarcoma includes chemotherapy and surgery, while chondrosarcoma is treated by surgery *Deceased. The authors dedicate this revised and updated chapter to the memory of James Russell Neff (1940–2005) who was the original author. Dr. Neff was a leading specialist in treating cancer of the bone and soft tissues who was known internationally for performing innovative procedures to help people lead more normal lives following cancer.

alone.1 The management of these patients, from initial evaluation and biopsy through surgical therapy and long-term follow-up, is labor intensive and technically demanding. Patients with a bone sarcoma should be treated in a center that has expertise in the management of these tumors.

Surgical Staging System Currently, the staging system adopted by the Musculoskeletal Tumor Society (MSTS) in 1980 and modified in 1986 is accepted by most musculoskeletal oncologists.2–4 Malignant tumors are divided into only two histologic grades: low-grade malignant (G1) and high-grade malignant (G2). Low-grade malignant lesions (G1), comprising Broder’s I and II lesions, have a low probability of metastasis (25%). The majority of these tumors can be managed by relatively conservative surgical procedures and do not require chemotherapy.

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Part III: Specific Malignancies

High-grade lesions (G2), Broder’s III and IV tumors, have a significantly higher incidence of metastases, requiring more radical surgical procedures and possibly neoadjuvant and/or adjuvant chemotherapy. Table 96-1 is a representative grouping of both low- and high-grade malignant tumors of bone and soft-tissue origin. Bone sarcomas that are totally intraosseous are intracompartmental (T1 or A). Those that penetrate the cortex are considered extracompartmental (T2 or B; Table 96-2). Patients without evidence of metastatic disease after radiographic staging are designated M0. In general, metastatic disease that is evident in the lung, in the lymph nodes, or as an intramedullary “skip” lesion indicates a poor prognosis and is designated M1. Surgical procedures are defined by the relationship of the circumferential surgical plane of dissection and the pseudocapsule. Surgical margins are defined as intralesional, marginal, wide, and radical. Examples of intralesional margins include curettage of a presumed benign tumor and cytoreductive debulking procedures. Marginal margins, achieved when the plane of dissection passes through the reactive zone of the pseudocapsule, are suitable for management of the majority of benign tumors. Such margins are accomplished when the surgeon “shells out” a neoplasm, cleaving the tissue between the reactive zone and the zone of compression. This technique leaves behind viable tumor satellites at the periphery of the lesion; thus,

Table 96-2 Surgical Sites (T) Intracompartmental (T1)

Extracompartmental (T2)

Intraosseous

Soft-tissue extension

Intra-articular

Soft-tissue extension

Superficial to deep fascia

Deep fascial extension

Parosseous

Intraosseous or extrafascial

Intrafascial compartments

Extrafascial planes or spaces

Ray of hand or foot

Midfoot and hindfoot

Anterolateral leg

Popliteal “space”

Posterior leg

Groin-femoral triangle

Middle thigh

Intrapelvic (retroperitoneal)

Posterior thigh

Midhand

Buttocks

Antecubital fossa

Dorsal forearm

Axilla

Volar forearm

Periclavicular

Anterior arm

Paraspinal

Posterior arm

Head and neck

Periscapular Adapted from Enneking WF, Spanier SS, Goodman MA: A system for the surgical staging of musculoskeletal sarcoma. Clin Orthop 1980;153:106.

Table 96-1 Surgical Grade (G) Low (G1)

High (G2)

Parosteal osteosarcoma

Classic osteosarcoma

Periosteal osteosarcoma

High-grade surface

Low-grade central osteosarcoma

Paget’s sarcoma of bone Radiation sarcoma

Intraosseous osteosarcoma Secondary chondrosarcoma

Primary chondrosarcoma Dedifferentiated chondrosarcoma Mesenchymal chondrosarcoma

Clear cell chondrosarcoma Fibrosarcoma, Kaposi’s sarcoma

Fibrosarcoma

Atypical malignant fibrous histiocytoma

Malignant fibrous histiocytoma (MFH) MFH of bone Undifferentiated primary sarcoma Giant cell sarcoma, bone

Hemangioendothelioma

Angiosarcoma

Hemangiopericytoma

Hemangiopericytoma

Myxoid liposarcoma

Pleomorphic liposarcoma Neurofibrosarcoma (schwannoma)

Clear cell sarcoma

Rhabdomyosarcoma

Epithelioid sarcoma

Synovial sarcoma

Chordoma

Ewing’s sarcoma of bone

Adamantinoma

PNET (primitive neuroepithelial tumor)

Alveolar cell sarcoma Other and undifferentiated

marginal margins are not sufficient for local control of malignant or benign “aggressive” lesions. A wide margin is obtained when the plane of dissection passes through absolutely normal nonreactive tissue that is well removed from the pseudocapsule. Wide margins are sufficient for virtually all bone sarcomas. Radical margins of a bone sarcoma are achieved when the entire bone is removed. This usually requires an amputation. A radical margin is rarely necessary.5 The American Joint Committee on Cancer (6th edition) has adapted the TNM staging system to bone. The topography (T) of the primary tumor now includes size based on relevant published reviews, in which the greatest dimension (8 cm for Ewing’s tumor, 9 cm for conventional osteosarcoma) has replaced the compartment concept. Also, T3 has now been assigned to patients who develop “skip” metastases (Table 96-3). The problem of defining histopathologic grade (G) has been addressed and now essentially consists of low- and high-grade lesions (Table 96-4). G1 and G2 have been combined into low-grade and G3 and G4 into high-grade histopathology. Currently, all Ewing’s tumors are classified as G4 or high-grade. This grouping is now identical to the G1 and G2 of the MSTS staging system. The stage groupings are shown in Table 96-5. Here, the committee has appro-

Askin’s tumor Alveolar cell sarcoma

Table 96-3 Definition of TNM Primary Tumor (T) TX

Primary tumor cannot be assessed

T0

No evidence of primary tumor

T1

Tumor ≤8 cm in greatest dimension

T2

Tumor >8 cm in greatest dimension

T3

Discontinuous tumors in the primary bone site

Adapted from Greene FL, Page DL, Fleming ID, et al (eds): AJCC Cancer Staging Manual, 6th ed. New York, Springer Verlag, 2002.

Sarcomas of Bone • CHAPTER 96

Table 96-4 Histologic Grade (G)

Table 96-6 Radiographic Techniques Available

GX

Grade cannot be assessed

G1

Well differentiated–low grade

1. Plain films: loss of trabeculation, matrix identification, calcification, etc.

G2

Moderately differentiated–low grade

G3

Poorly differentiated–high grade

G4*

Undifferentiated–high grade

*Ewing’s sarcoma is classified as G4. Adapted from Greene FL, Page DL, Fleming ID, et al (eds): AJCC Cancer Staging Manual, 6th ed. New York, Springer Verlag, 2002.

priately addressed the difference in prognosis of patients who have sustained metastases to lung (Mla) and to other sites, including bone (Mlb).

Radiographic Staging

2. Polytomes: evaluate margination, matrix identification, calcification, etc. 3. Bone scan: three phases to evaluate vascularity, static skeletal survey 4. Rapid whole-body STIR MRI: excellent survey study with cooperative patients and in institutions using these techniques 5. CT: margination, matrix identification, calcification, cortical disruption, axial localization of lesion 6. MRI: excellent soft-tissue contrast, sometimes nearly diagnostic; T1 best for anatomy, contrast enhancement, magnetic resonance angiography capability, best overall single study when properly monitored by a physician 7. PET: adds metabolic parameter used to monitor effectiveness of neoadjuvant chemotherapy 8. Other: 201T1 scan, gallium scan, PET scan

Conventional bone radiography remains the single most useful initial study for bone tumor evaluation. The study should include anteroposterior (AP) and lateral projections of the lesion. Malignant neoplasms usually result in ill-defined or “poorly marginated” radiographic margins with little or no reactive bone, loss of medullary trabeculation, and endosteal cortical erosion, suggesting an active and destructive process at the tumor/host bone interface. The pathologic process biologically overwhelms the normal time-dependent reactive processes of bone formation. Therefore, the radiographic presence or absence of a reactive rim of bone is often useful in predicting the biologic aggressiveness of the pathologic process (Table 96-6). Neoplastic bone formation is often seen in osteosarcoma, and calcification is often seen in chondrosarcoma. The use of technetium-99m (99mTc) bone scintigraphy remains the standard for surveying the skeleton for multiple osseous lesions. The test can be administered as a single delayed static study or can be displayed in multiple timed phases to evaluate the vascularity of the lesion. It is important to obtain a whole-body bone scan. Computed tomography (CT) is superior to magnetic resonance imaging (MRI) only to evaluate a small lesion in the cortex, subtle bone formation, or calcification; otherwise, MRI is the study of choice. CT remains the standard for evaluation of the chest for occult metastases. The cross-sectional display usually provides sufficient resolution (80

Yes

t(21;22)(q12;q12)

EWS-ERG fusion

t(2;22)(q33;q12)

EWS-FEV fusion

25

No

>90

Yes

Tumor Type

Ewing’s sarcoma/peripheral primitive neuroectodermal tumor

Fibrosarcoma, infantile Gastrointestinal stromal tumor

Inflammatory myofibroblastiv tumor

2p23 rearrangement

KIT mutation

Leiomyosarcoma

Deletion of 1p

ALK fusion genes

Frequency (%)

5–10

Diagnostic Utility?

Yes

50

Yes

>50

No

>75

Yes

>75

Yes

90

No

Malignant peripheral nerve sheath tumor

Complex

>90

No

Myxofibrosarcoma (myxoid malignant fibrous histiocytoma)

Ring form of chromosome 12

?

?

Neuroblastoma Good prognosis

Hyperdiploid, no 1p deletion

90

Yes

Poor prognosis

1p deletion

90

Yes

Rhabdoid tumor

Double minute chromosomes

N-myc amplification

>25

Yes

Deletion of 22q

INII inactivation

>90

Yes

t(2;13)(q35;q14)

PAX3-FKHR fusion

>75

T(1;13)(p26;q14), double minutes

PAX7-FKHR fusion

Rhabdomyosarcoma Alveolar

Embryonal

10–20

Yes Yes

>75

Yes

Loss of heterozygosity at 11p15

>75

Yes

Trisomies 2q, 8, and 20

Synovial sarcoma Monophasic

t(X;18)(p11;q11)

SYT-SSXI or SYT-

>90

Yes

Biphasic

t(X;18)(p11;q11)

SSX2 fusion

>90

Yes

STY-SSXI fusion

Sarcomas of Soft Tissue • CHAPTER 97

syndrome develop, to varying degrees, skin hyperpigmentation, urticaria pigmentosa, and cutaneous mast cell disease in addition to one or more GISTs.75 Activating KIT mutations have been shown to lead to ligand-independent activation of the KIT receptor tyrosine kinase pathway, which results in dysregulated cell growth, and are thought to be the first step in the pathogenesis of GISTs.72 Interestingly, the identification of the important role of KIT in the pathogenesis of GISTs has led to treatment with imatinib mesylate.76 A small molecule drug specifically inhibits the KIT pathway (see sections on Prognostic Factors as Therapeutic Targets and Gastrointestinal Stromal Tumors).

Genetics of Sporadic Soft-Tissue Sarcomas Sarcomas tend to fall into two major subsets. In one group, the tumors are cytogenetically simple and are characterized by near diploid karyotypes with few chromosomal rearrangements, resulting in the formation of fusion proteins. These translocations are highly diagnostic of specific histologic subtypes of STSs. Also included within the cytogenetically simple STSs would be GIST tumors, which are characterized by a specific activating mutation of the c-Kit gene but few other genetic abnormalities.

Chromosomal Rearrangements A large number of sarcomas have been found to have consistent chromosomal abnormalities (see Table 97-3).77 These chromosomal rearrangements are important diagnostically, may be important prognostically (see the section on Potential Molecular Prognostic Factors), have shed light on the pathogenesis of sarcomas, and may provide targets for pharmacologic therapy (see the section on treatment of GISTs). Benign soft-tissue neoplasms also harbor chromosomal rearrangements. Chromosomal translocations are the most common cytogenetic abnormality in soft-tissue neoplasms and are likely responsible for the initiation of tumorigenesis in most cases.77 Deletions and trisomies have also been reported and are thought to represent secondary changes involved in tumor progression. Deletions tend to represent loss of tumor suppressor genes, whereas trisomies indicate the presence of an oncogene. Although we know a lot about the primary tumorigenic events in many sarcomas, defining the secondary changes has been much more problematic and is an area of intense study. Cloning and molecular analysis of the various genetic aberrations that characterize different sarcomas have revealed the different pathogenetic mechanisms that underlie these tumors. Translocations typically create chimeric transcription factors or growth factors that result in deregulation of transcription or growth control. A typical example of a chimeric transcription factor is the PAX3-FKHR fusion protein, which has been shown to activate a complex myogenic transcriptional program when the protein is expressed in a fibroblast cell line.78 Infantile fibrosarcoma is characterized by a translocation involving chromosomes 12 and 15 that encodes a chimeric ETV6-NTRK3 constitutively activated growth factor receptor.79 Other oncogenic proteins appear to act by a mechanism that remodels chromatin structure, which is known to have a profound influence on gene expression (e.g., INI1 mutations in rhabdoid tumors).69 Specific chromosomal rearrangements are very useful in the diagnosis of STSs. Beyond the obvious benefit of providing further objective proof of a diagnosis in morphologically typical cases, the detection of chromosomal aberrations may facilitate the diagnosis of lesions that are difficult to characterize by standard histopathologic, ultrastructural, and immunohistochemical techniques.80,81 For example, the presence of the translocation t(X;18)(p11;q11) has been used to confirm the diagnosis of synovial sarcoma in poorly differentiated cases that were diagnostically very challenging.82–84 Similarly, the finding of the characteristic translocation t(11;22)(q24;q12) in a small round blue cell tumor supports the diagnosis of Ewing’s sarcoma/primitive neuroectodermal tumor (PNET).85

Translocations can be identified by cytogenetic analysis, fluorescence in situ hybridization, or reverse transcriptase polymerase chain reaction. A detailed description of these techniques is beyond the scope of this chapter, but each technique has its advantages and disadvantages. Cytogenetic analysis requires fresh (living) tissue, since the cells need to be cultured before karyotypic analysis. This technique is becoming more widely available than before, owing to the availability of overnight transport of biologic specimens and for-profit core facilities. Fluorescence in situ hybridization is a technologically sophisticated technique that does not require fresh or frozen tissue. Fluorescence in situ hybridization is easier to perform on cytogenetic cultures or frozen tissue, however, so these are still preferable to paraffinembedded material. Reverse transcriptase polymerase chain reaction is an extremely sensitive technique that can be performed on fresh, frozen, or paraffin-embedded tissues. The major drawback of reverse transcriptase polymerase chain reaction is the relatively high falsepositive rate, which results from its sensitiveness. Meticulous care is required to prevent problems from contamination. Although reverse transcriptase polymerase chain reaction can be performed on paraffin-embedded tissue, it is preferable to perform the analysis on fresh or frozen tissue. Since all of these techniques either require or are easier to perform on fresh or frozen tissue, it is advisable to freeze and store a portion of any suspected sarcoma or poorly differentiated neoplasm for potential molecular analysis. Many sarcomas are characterized by several different translocations, some of which are mutually exclusive (see Table 97-3). For instance, alveolar rhabdomyosarcoma is characterized by a translocation involving chromosomes 2 and 13, which results in fusion of the PAX3 and FKHR genes, or by a translocation involving chromosomes 1 and 13, which results in fusion of the PAX7 and FKHR genes.86–89 Sarcomas within a subtype may also have differences in the specific exons that are involved in each of these different translocations. It has been proposed that this heterogeneity may result in differences in prognosis (see section on Potential Molecular Prognostic Factors). For example, Ewing’s sarcoma/PNET and synovial sarcoma possess genetic variations that have been suggested to have prognostic significance.90–93 Future research might establish whether cytogenetic and molecular factors can be used as a basis for therapeutic decisions and the prediction and evaluation of response to treatment. The identification of genetic alterations with high specificity for different sarcomas will also identify specific therapeutic targets. This has already resulted in the successful treatment of two different sarcomas: GISTs and dermatofibrosarcoma protuberans (DFSP).76,94 About 90% of GISTs harbor activating mutations in the KIT oncogene, which result in ligand-independent activation of the KIT receptor tyrosine kinase pathway.74 Imatinib mesylate, a small molecule drug that is administered orally and inhibits the KIT pathway, has been shown to be very efficacious in the treatment of GIST (see the section on investigational new drugs in the Chemotherapy section). DFSP is characterized by translocations involving the COL1A1 and PDGF-β genes, which result in activation of the platelet-derived growth factor-β PDGF-β pathway. Imatinib mesylate is also active against the PDGF-β pathway and has been shown to be effective in the treatment of a small number of DFSPs. It is noteworthy to mention two common benign soft-tissue tumors—leiomyomas and lipomas—that have a high frequency of chromosomal rearrangements of chromosome 12q that involves the high-mobility protein group gene HMGIC.95 These translocations are not seen in the corresponding leiomyosarcomas or liposarcomas, indicating that in these tumors, the benign form is not a precursor of the malignant counterpart.

Sarcomas with Complex Karyotypes A second major subset of STSs is characterized by aneuploidy and the lack of specific fusion genes. This group of sarcomas includes

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Part III: Specific Malignancies

Table 97-4 Sarcomas with Complex Karyotypes Type of Sarcoma

Resembles

Fibrosarcoma (other than congenital)

Fibrous tissue

Leiomysarcoma

Smooth muscle

Malignant fibrous histiocytoma

Poorly differentiated

Osteosarcoma

Bone

Chondrosarcoma (types other than extraskeletal myxoid)

Cartilage

Liposarcoma (types other than myxoid)

Fat

Embryonal rhabdomyosarcoma

Skeletal muscle

Malignant peripheral nerve sheath tumor*

Nerve sheath

Angiosarcoma

Blood vessels

*Some have NF1 mutations.

such tumors as leiomyosarcomas, MPNST, and fibrosarcomas (see Tables 97-3 and 97-4). These tumors tend to occur in the older age group and appear to have a relatively high frequency of mutations in the p53 and RB signaling pathways.96,97 These tumors are characterized by chromosomal gains and losses that presumably target tumor suppressor genes (in the event of losses) and oncogenes (in the event of gains). Currently, the best-characterized targets of amplifications

are the cell cycle regulatory genes CDK4 and HDM2, but others remain to be identified and remain a current challenge in the field. Thus, it appears that alterations disrupting chromosomal mechanics and DNA repair can lead to sarcomagenesis.

PATHOLOGY STSs have been described at virtually all anatomic sites. The anatomic sites and site-specific histologic subtypes of 4207 sarcomas treated at a single referral institution are outlined in Figure 97-1. Approximately half of all STSs occur in the extremities (lower: 34%; upper: 14%), where the most common histopathologic subtypes are welldifferentiated, myxoid, and round cell liposarcoma (28%) and malignant fibrous histiocytoma (pleomorphic undifferentiated sarcoma) (24%). Retroperitoneal sarcomas make up 15% of all STSs, welldifferentiated or dedifferentiated liposarcoma being the predominant histologic subtype (42%). The visceral sarcomas make up an additional 14%, while the head and neck sarcomas make up approximately 4%.

Classification In broad terms, sarcomas can be classified into neoplasms that arise in bone and those that arise from the soft tissues. However, even this seemingly simple distinction is fraught with difficulty, as is illustrated by Ewing’s sarcomas. These tumors often arise in close association with bone and are often classified as bone sarcomas. However, it is now clear that these tumors can also arise in soft tissues, and it is

N=460 (14%)

Upper extremity 32%

N=141 (4%)

Head and neck

8% 12%

15%

14%

5% 12%

37%

6%

18% 16%

9%

N=491 (14%)

16%

Visceral

3%

59% Retroperitoneal/ N=489 Intra-abdominal (15%) 28% 17% 42%

3%

8% 3%

6% 6%

N=1,098 (34%)

Lower extremity

26% 19%

13% 8%

28% Other ERMS Synovial Fibrosarcoma

MFH Leiomyosarcoma MPNT Liposarcoma

24% 7%

Figure 97-1 • Anatomic distribution and site-specific histiotypes of 4207 adult patients with soft-tissue sarcomas seen at the University of Texas M.D. Anderson Cancer Center, 1996–2003.

Sarcomas of Soft Tissue • CHAPTER 97

becoming increasingly clear that these tumors arise from primitive cells of mesenchymal origin. So are they bone sarcomas or soft-tissue sarcomas, and does it really matter in terms of classification? Sarcomas of the soft tissues can be further grouped into those that arise from viscera (gastrointestinal, genitourinary, and gynecologic organs) and those that arise from nonvisceral soft tissues (muscle, tendon, adipose, pleura, and connective tissue). An alternative way to index STSs is by their differentiation. Tumors can be grouped broadly into adipocytic tumors, fibroblastic/ myofibroblastic tumors, so-called fibrohistiocytic tumors, smooth muscle tumors, pericytic (perivascular) tumors, PNETs, skeletal muscle tumors, vascular tumors, osseous tumors, and tumors of uncertain differentiation (Table 97-5).98 Classification is based on clinical, histologic, ultrastructural, immunohistochemical, and genetic features. Electron microscopic evidence of cellular substructures, neurofibrils, microfilaments, actin-myosin complexes, dense bodies, and so on often helps to clarify the tissue of origin.99 However, the widespread availability of commercial antibodies for immunohistochemical analysis has diminished the need for electron microscopic examination in many cases. Immunohistochemical staining for proteins that are characteristic of smooth muscle (smooth muscle actin and desmin), skeletal muscle (muscle-specific actin, desmin, and myogenin), blood vessels (factor VIII, CD34, and CD31), and epithelial tissue (epithelial membrane antigen and cytokeratins) often facilitates reliable classification.100,101 The tissue of origin classification scheme is the most commonly used scheme and is the basis for the recent World Health Organization classification system for sarcomas.98,102 The World Health Organization classification system is reproducible for most sarcomas. As the degree of histologic differentiation declines, however, the determination of the tissue of origin becomes increasingly difficult. In particular, despite advanced immunohistochemical techniques, electron microscopy, and molecular analysis, determining the tissue of origin for some soft-tissue tumors is difficult, occasionally arbitrary, and sometimes impossible. This leads to significant disparities in diagnoses among pathologists. Discrepancies between the original histologic diagnosis and the subsequent diagnosis by an expert reviewer have been noted in as many as 25% of cases.103,104 Review of tissue specimens by an expert at a regional sarcoma center is therefore imperative, because the degree of expertise in correctly diagnosing rare and unusual sarcomas is directly related to the number of sarcomas that a pathologist has seen. It is important to classify STSs as precisely as possible because of major differences in their clinical behavior and in their susceptibility to different therapies. For example, a few STSs, including epithelioid sarcoma, clear cell sarcoma, angiosarcoma, and rhabdomyosarcoma, have a greater risk of regional lymph node metastasis.105,106 In one single-institution study, the overall rate of nodal metastasis at the time of sarcoma presentation was only 2.7%; however, the rate was much higher for angiosarcoma (13.5%), embryonal rhabdomyosarcoma (13.6%), and epithelioid sarcoma (16.7%).105 Patterns of distant metastases also differ for subtypes of sarcoma. For example, myxoid liposarcoma tends to metastasize to soft-tissue sites, including the retroperitoneum,107 and patients with myxoid liposarcoma often present with metastatic disease. Therefore, if a myxoid liposarcoma is identified in the abdomen, the thighs should be examined for an occult primary tumor. The vast majority of so-called primary myxoid liposarcomas of the abdomen and retroperitoneum are actually metastatic myxoid liposarcomas or misdiagnosed dedifferentiated liposarcomas, which often mimic myxoid liposarcoma.108 Patterns of local spread also differ dramatically among subtypes of sarcoma. For example, DFSP has a propensity to infiltrate subcutaneous adipose tissue in a manner that is very difficult to detect; therefore, wide surgical excision of DFSP is essential. When planning a surgery for DFSP, the surgeon should regard the grossly observable lesion as the tip of the iceberg. Angiosarcoma also spreads very diffusely and is difficult to define grossly.

Table 97-5

Histologic Classification of Soft-Tissue Sarcoma

ADIPOCYTIC SARCOMAS Atypical lipomatous tumor/well-differentiated liposarcoma Dedifferentiated liposarcoma Myxoid liposarcoma Pleomorphic liposarcoma

FIBROBLASTIC AND MYOFIBROBLASTIC SARCOMAS Malignant solitary fibrous tumor Inflammatory myofibroblastic tumor Myxoinflammatory fibroblastic sarcoma Infantile fibrosarcoma Adult fibrosarcoma Myxofibrosarcoma (myxoid malignant fibrous histiocytoma) Low-grade fibromyxoid sarcoma Sclerosing epithelioid fibrosasrcoma

SO-CALLED FIBROHISTIOCYTIC SARCOMAS Pleomorphic malignant fibrous histiocytoma/undifferentiated highgrade pleomorphic sarcoma Giant cell malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma with giant cells Inflammatory malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma with prominent inflammation

SMOOTH MUSCLE SARCOMAS Leiomyosarcoma

SKELETAL MUSCLE SARCOMAS Embryonal rhabdomyosarcoma Alveolar rhabdomyosarcoma Pleomorphic rhabdomyosarcoma

VASCULAR SARCOMAS Epithelioid hemangioendothelioma Angiosarcoma

OSSEOUS SARCOMAS Extraskeletal osteosarcoma

SARCOMAS OF UNCERTAIN DIFFERENTIATION Synovial sarcoma Epithelioid sarcoma Alveolar soft part sarcoma Clear cell sarcoma of soft tissue Extraskeletal myxoid chondrosarcoma Desmoplastic small round cell tumor Extarenal rhabdoid tumor Intimal sarcoma

Histologic Grading Histologic classification alone does not always provide enough information to predict the clinical behavior of STSs. For many sarcomas, histologic grading provides additional information that can aid in predicting biologic behavior and planning treatment. The spectrum of grades varies among specific histologic subtypes (Fig. 97-2). For

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Histologic type I

II

III

Fibrosarcoma Infantile fibrosarcoma Dermatofibrosarcoma protuberans Malignant fibrous histiocytoma Liposarcoma Well-differentiated liposarcoma Myxoid liposarcoma Round cell liposarcoma Pleomorphic liposarcoma Leiomyosarcoma Rhabdomyosarcoma Angiosarcoma Malignant hemangiopericytoma Synovial sarcoma Malignant mesothelioma Malignant schwonnoma Neuroblastoma Ganglioneuroblastoma Extraskeletal chondrosarcoma Myxoid chondrosarcoma Mesenchymal chondrosarcoma Extraskeletal osteosarcoma Malignant granular cell tumor Alveolar soft part sarcoma Epithelioid sarcoma Clear cell sarcoma Extraskeletal Ewing’s sarcoma

example, leiomyosarcomas exhibit wide variations in grade and should always be graded, while Ewing’s sarcomas/PNETs are always high-grade and therefore do not require grading. In careful comparative multivariate analyses, histologic grade has been the most important prognostic factor in assessing the risk for distant metastasis and tumor-related mortality.109–111 Several grading systems have been proposed, but there is no consensus regarding the specific morphologic criteria that should be employed in the grading of STSs. The two most important criteria appear to be the mitotic index and the extent of tumor necrosis. Two of the most commonly used grading systems are the U.S. National Cancer Institute (NCI) system developed by Costa and colleagues112 and the FNCLCC system (Federation Nationale des Centres de Lutte Contre le Cancer) developed by the French Federation of Cancer Centers Sarcoma Group.113 The NCI system is based on the tumor’s histologic subtype and amount of tumor necrosis, but cellularity, nuclear pleomorphism, and mitotic index are considered for certain subtypes. The FNCLCC system employs a score generated by evaluation of three parameters: tumor differentiation, mitotic rate, and amount of tumor necrosis. The prognostic values of these two grading systems were retrospectively compared in a population of 410 adult patients with nonmetastatic STS.114 Significant discrepancies were observed in one third of cases. An increased number of grade III tumors, a reduced number of grade II tumors, and better correlation with overall and metastasis-free survival were observed in favor of the FNCLCC system.114 Thus, in the absence of other comparative data, the FNCLCC system may be the best presently available grading system.

CLINICAL PRESENTATION AND DIAGNOSIS The majority of patients present with a painless mass, although pain is noted at presentation in up to one third of cases.115 Delay in diagnosis of sarcomas is common, the most common incorrect diagnosis for extremity and trunk lesions being lipoma or hematoma.

Figure 97-2 • The spectrum of grades observed among histologic subtypes of soft-tissue sarcoma. (Reprinted with permission from Enzinger FM, Weiss SW: Malignant tumors of uncertain type. In Enzinger FM, Weiss SW [eds]: Soft Tissue Tumors, 3rd ed. St. Louis, Mosby, 1995, p 1067.)

Physical examination should include an assessment of the size and mobility of the mass. Its relationship to the fascia (superficial versus deep) and nearby neurovascular and bony structures should be noted. A site-specific neurovascular examination and assessment of regional lymph nodes should also be performed.

Biopsy Biopsy of the primary tumor is essential for most patients presenting with soft-tissue masses. In general, any soft-tissue mass in an adult that is asymptomatic or enlarging, is larger than 5 cm, or persists beyond 4 to 6 weeks should be biopsied. The preferred biopsy approach is generally the least invasive technique required to allow a definitive histologic diagnosis and assessment of grade. In most centers, core-needle biopsy provides satisfactory tissue for diagnosis116–118 and has been demonstrated to result in substantial cost savings compared to open biopsy.118 Direct palpation can be used to guide needle biopsy of most superficial lesions, but less accessible sarcomas often require an image-guided biopsy to safely sample the most heterogeneous component of the mass. Needle tract tumor recurrences after closed biopsy are rare but have been reported,119 leading some surgeons to advocate tattooing the biopsy site for subsequent excision or for inclusion in radiotherapy treatment volumes (Fig. 97-3). In some centers, fine-needle aspiration may be an acceptable biopsy technique for primary soft-tissue masses, provided that an experienced sarcoma cytopathologist is available.120–122 Owing to the frequent difficulty in accurately diagnosing these lesions even when adequate tissue is available, however, the major utility of fineneedle aspiration in most centers is in the diagnosis of suspected recurrent sarcoma. Incisional or excisional biopsy is rarely required but may be performed when a definitive diagnosis cannot be achieved by less invasive means. Several technical points merit comment. Relatively small, superficial masses that can easily be removed should be biopsied by complete excision with microscopic assessment of surgical margins.

Sarcomas of Soft Tissue • CHAPTER 97

Imaging

Figure 97-3 • Axial CT image of a recurrent well-differentiated low-grade liposarcoma of the retroperitoneum. Note the tumor nodule (arrow) representing a tumor implant within muscle from a previous needle biopsy that was obtained through the posterior abdominal wall. It is beneficial to consider the location of biopsy tracts so that subsequent surgery or radiotherapy includes the area of the biopsy (see text for details).

Incisional and excisional biopsies should be performed with the incision oriented longitudinally (for extremity lesions) to facilitate subsequent wide local excision. The incision should be centered over the mass at its most superficial point. Care should be taken not to raise tissue flaps. Meticulous hemostasis should be ensured to prevent dissemination of tumor cells into adjacent tissue planes by hematoma. All excisional biopsy specimens should be sent fresh, sterile, and anatomically oriented for pathologic analysis. At definitive resection of a previously biopsied sarcoma, the previous surgical biopsy scar should be excised en bloc with the tumor.

Optimal imaging of the primary tumor is dependent on the anatomic site. For soft-tissue masses of the extremities, magnetic resonance imaging (MRI) has been regarded as the imaging modality of choice (Fig. 97-4). This is because MRI enhances the contrast between tumor and muscle and between tumor and adjacent blood vessels and provides multiplanar definition of the lesion.123,124 Despite the fact that a study by the Radiation Diagnostic Oncology Group that compared MRI and computed tomography (CT) in patients with malignant bone (N = 183) and soft-tissue (N = 133) tumors showed no specific advantage of MRI over CT from a diagnostic standpoint,125 the majority of musculoskeletal radiologists and almost all oncologists prefer MRI for soft-tissue tumors. For pelvic lesions, the multiplanar capability of MRI may provide superior single-modality imaging (Fig. 97-5). The multiplanar capability is also helpful for visualization of disease in noncoplanar ways when conformal radiotherapy technique is being employed and is an especially helpful adjunct in using image fusion techniques or to visualize peritumoral edema that may harbor sarcoma cells. In the retroperitoneum and abdomen, CT usually provides satisfactory anatomic definition of the lesion (Fig. 97-6). Occasionally, MRI with gradient sequence imaging can better delineate the relationship of the tumor to midline vascular structures, particularly the inferior vena cava and aorta. More invasive studies such as angiography or cavography are almost never required for the evaluation of STSs. The utility of [18F]fluorodeoxyglucose positron emission tomography (FDG PET) in the evaluation and treatment of STS has been a subject of recent studies and has been reviewed in detail elsewhere. The technique utilizes radiolabeled glucose analogs,19 which are taken up at increased rates by malignant tumors. Pilot studies of PET in STS suggest that by evaluating tumor metabolic activity, PET scans may allow for noninvasive assessment of tumor grade.126 Recent preliminary studies have demonstrated that PET may be helpful in

Figure 97-4 • A 47-year-old male with malignant fibrous histiocytoma of the left thigh. Axial contrast-enhanced T1-weighted (left) and flow sensitive gradient (right) images reveal a large mass in the vastus intermedius muscle. A plane is identified between the mass and the profunda femoris and superficial femoral vessels (arrows).

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Part III: Specific Malignancies

A

B Figure 97-5 • A, A 74-year-old female with retroperitoneal malignant fibrous histiocytoma extending into the upper pelvis. Axial contrastenhanced T1-weighted images reveal a large heterogeneous mass with foci of necrosis (large arrows). Note the relationship to the common iliac vessels (small arrows). B, The same patient as in (A). Coronal T1-weighted (left) and source MR images (right) from contrast-enhanced 3D MR angiogram reveal a large abdominal mass (large arrows) closely abutting the right renal capsule. Note the relationship to the aorta. The right renal artery is visualized (small arrow) as is the portal vein (arrowhead).

the assessment of locally recurrent STS127 and in the evaluation of response to therapy.128,129 The role and cost-effectiveness of PET in the staging of STS remain incompletely defined; therefore, further studies will be required to fully define the role for FDG PET in the diagnosis, evaluation, and treatment of STS.

STAGING The relative rarity of STSs, the anatomic heterogeneity of these lesions, and the presence of more than 30 recognized histologic subtypes of variable grade have made it difficult to establish a functional system that can accurately stage all forms of this disease. The recently revised staging system (sixth edition) of the American Joint Committee on Cancer (AJCC) and the International Union Against Cancer (UICC) is the most widely employed staging system for STSs.130 This staging system is a revision of the original AJCC system, which was first published in 1977; it incorporates histologic grade into the conventional TNM system (Table 97-6). The 2002 sixth edition

classification has T and N categories that are identical to those of the 1997 fifth edition TNM, but some modifications have been made to the stage groupings. For the detailed background to these changes, the reader is referred to a more complete discussion. All STS subtypes are included except dermatofibrosarcoma protuberans, a condition that is considered to have only borderline malignant potential. Four distinct histologic grades are recognized, ranging from well-differentiated to undifferentiated. Histologic grade and tumor size are the primary determinants of clinical stage (see Table 97-6). Tumor size is further substaged as “a” (a superficial tumor that arises outside the investing fascia) or “b” (a deep tumor that arises beneath the fascia or invades the fascia). The system is designed to optimally stage extremity tumors but is also applicable to torso, head and neck, and retroperitoneal lesions; it should not be used for sarcomas of the gastrointestinal tract. This staging system has been validated by analysis of 1146 patients presenting with primary extremity STS at the Memorial Sloan-Kettering Cancer Center. Stage-specific survival plots are outlined in Figure 97-7.

Sarcomas of Soft Tissue • CHAPTER 97

Table 97-6

AJCC/UICC Staging System for Soft-Tissue Sarcoma ≤5 cm

T1 T1a

Superficial to muscular fascia

T1b

Deep to muscular fascia >5 cm

T2 T2a

Superficial to muscular fascia

T2b

Deep to muscular fascia

N1

Regional nodal involvement

G1

Well-differentiated

G2

Moderately differentiated

G3

Poorly differentiated

G4

Figure 97-6 • Contrast-enhanced CT scan of the abdomen demonstrating a retroperitoneal malignant fibrous histiocytoma. Note the large mass (large arrow) between the aorta and inferior vena cava with abutment and displacement of celiac axis and hepatic artery (small arrows). The portal vein (arrowheads) is well visualized, and low attenuation foci in the liver, which are unopacified hepatic veins, are incidentally visualized.

A major limitation of the present staging system is that it does not take into account the anatomic site of STSs. Anatomic site, however, is an important determinant of outcome. Patients with retroperitoneal and visceral sarcomas have a worse overall prognosis than do patients with extremity tumors. Although site is not incorporated as a specific component of any present staging system, outcome data should be reported on a site-specific basis.

PROGNOSTIC FACTORS Conventional Clinicopathologic Factors A thorough understanding of the clinicopathologic factors that are known to affect outcome is essential in formulating a treatment plan for the patient with STS. Over the past decade, many multivariate analyses of prognostic factors for patients with localized sarcoma have been reported. With few exceptions,109,110,131,132 most studies have analyzed fewer than 300 patients (range: 82 to 297 patients). At least three detailed analyses of prognostic factors in STS merit comment.109,110,131 The initial study of prognostic factors in extremity

Undifferentiated

Stage IA

G1, 2

T1a, b

N0

M0

Stage IB

G2, 2

T2a, b

N0

M0

Stage IIA

G3, 4

T1a, b

N0

M0

Stage IIB

G3, 4

T2a

N0

M0

Stage III

G3, 4

T2b

N0

M0

Stage IV

Any G

Any T

N1

M0

Any G

Any T

Any N

M1

Modified from Greene FL et al (eds.): UICC TNM Classification of Malignant Tumors, 6th ed. [LOC.], Springer Verlag, 2002.

sarcoma from Memorial Sloan-Kettering Cancer Center evaluated clinicopathologic prognostic factors in a series of 423 patients with localized extremity STS seen from 1968 to 1978.128 This analysis, among the first to discriminate between specific clinical endpoints, clearly established the clinical profile of what is now accepted as the high-risk patient with extremity STS: the patient with a large (>5cm), high-grade, deep lesion. The adverse prognostic significance of a high tumor grade, deep tumor location, and tumor size greater than 5 cm was also noted in the recent report of the French Federation of Cancer Centers study of 546 patients with sarcomas of the extremities, head and neck, trunk wall, retroperitoneum, and pelvis.110 A follow-up report from Memorial Sloan-Kettering evaluated clinicopathologic prognostic factors that had been documented

1.0 UICC stage I (N=168) UICC stage II (N=500) UICC stage III (N=478)

Figure 97-7 • Overall survival by AJCC stage in a population of 1146 patients with primary extremity sarcoma treated at the Memorial SloanKettering Cancer Center.

Proportion surviving

0.8

0.6

0.4

0.2

0 0

12

24

36

48

60

72

84

Time (mo)

96

108

120

132

144

156

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Part III: Specific Malignancies

Table 97-7 Multivariate Analysis of Prognostic Factors in Patients with Extremity Soft-Tissue Sarcoma Relative Risk

Endpoint

Adverse Prognostic Factor

Local recurrence

Age > 50 years

1.6

Local recurrence at presentation

2.0

Microscopically positive margin

1.8

Fibrosarcoma

2.5

Malignant peripheral nerve tumor

1.8

Distant recurrence

Disease-specific survival

Size 5.0–10.0 cm

1.9

Size > 10.0 cm

1.5

High-grade

4.3

Deep location

2.5

Local recurrence

1.5

Leiomyosarcoma

1.7

Other nonliposarcoma histology

1.6

Size > 10.0 cm

2.1

Deep location

2.8

Local recurrence at presentation

1.5

Leiomyosarcoma

1.9

Malignant peripheral nerve tumor

1.9

Microscopically positive margin

1.7

Lower-extremity site

1.6

Adverse prognostic factors identified are independent by Cox regression analysis. Modified from Pisters PWT, Leung DHY, Woodruff JM, et al: Analysis of prognostic factors in 1041 patients with localized soft tissue sarcomas of the extremities. J Clin Oncol 1996;14:1679.

prospectively in a population of 1041 patients with extremity STS.109 The endpoints for the multivariate analyses were local recurrence, distant recurrence (metastasis), and disease-specific survival. Results of the regression analyses for each of these endpoints are summarized in Table 97-7. These results, using prospectively acquired data, confirm the initial observations made at that institution using an independent data set.131 In addition, the previously unappreciated prognostic significance of specific histologic subtypes and the increased risk for adverse outcome associated with a microscopically positive surgical margin or locally recurrent disease were noted. Unlike for other solid tumors, the adverse prognostic factors for local recurrence of a STS are different from those that predict distant metastasis and tumor-related mortality (see Table 97-7).109 In other words, patients with a constellation of adverse prognostic factors for local recurrence are not necessarily at increased risk for distant metastasis or tumor-related death. Therefore, staging systems that are designed to stratify patients according to risk of distant metastasis and tumor-related mortality using these prognostic factors (such as the AJCC/UICC system) will not stratify patients according to risk of local recurrence. The results of these multivariate analyses should be incorporated in the design of new staging systems and clinical trials for STS, and the identification of individual patients who are at high risk for distant recurrence and death. It should be emphasized that the prognostic factors that have been identified have been derived primarily from studies of patients with localized extremity sarcomas. Despite the fact that extremity sarcomas make up the majority of sarcomas, these results may not be optimally generalized to the greater population of STS patients. Separate reviews of prognostic factors for sarcomas of the retroperitoneum,133,134 head

and neck,135–138 gastrointestinal tract,139,140 colon and rectum,141 uterus,142 synovial sarcomas,143,144 malignant fibrous histiocytoma,145–147 and Ewing’s sarcoma148–151 have been reported.

Potential Molecular Prognostic Factors Attention has recently been focused on the evaluation of molecular pathologic prognostic factors. Specific molecular parameters that have been evaluated for prognostic significance have included p53,152 mdm2,152 Ki-67,152 altered expression of the retinoblastoma gene product (pRb)58,153 in high-grade sarcomas, and the presence of the SYT-SSX fusion transcripts in synovial sarcoma92 or EWS-FL11 fusion transcripts in Ewing’s sarcoma.90,91 A preliminary report evaluating the prognostic role of pRb expression in 44 primary and 12 metastatic high-grade human sarcomas by immunohistochemical methods and Western blotting demonstrated that alterations in pRb are more commonly associated with highgrade tumors, metastatic lesions, and decreased survival.58 However, a subsequent report from the same group in an expanded population of 174 adult patients with STS revealed that pRb alterations were frequently observed in both low- and high-grade lesions and that altered pRb expression did not correlate with known predictors of survival and was not an independent predictor of long-term outcome.153 These studies and the now well-documented phenomenon of late (>5 years post-treatment) recurrence of STS154 underscore the importance of long-term follow-up and relatively large sample size in these types of analyses. p53 is a tumor suppressor gene located on chromosome 17. Somatic p53 mutations have been reported in 4% to 65% of patients with STS.155–159 Detection of p53 has also been correlated with reduced overall survival in immunohistochemical studies of paraffinembedded STSs. Data on the underlying prognostic significance of p53 status are conflicting; some investigators report no independent adverse prognostic significance by regression analysis,152,160 and others report a highly significant correlation between p53/mdm2 status and outcome.161 Ki-67, an antigen that is expressed throughout the majority of the cell cycle, is utilized as a measure of dividing cells.162 Preliminary reports of series of heterogeneous sarcomas in adults suggested that proliferative index as measured by Ki-67 nuclear staining correlated with histologic grade but was not of independent prognostic significance when histologic grade was taken into account.160,163 However, additional studies in larger numbers of patients have demonstrated that Ki-67 status is an independent prognostic factor.152,164,165 An initial immunohistochemical analysis of a cohort of 65 STSs and a subsequent analysis of 132 STSs from the French Federation of Cancer Centers Sarcoma Group demonstrated the adverse prognostic significance of increased Ki-67 activity.164,165 Heslin and colleagues evaluated the potential prognostic significance of pRb, p53, mdm2, and Ki-67 by immunohistochemical techniques in a population of 121 patients with primary, high-grade extremity sarcomas and compared these factors to conventional clinicopathologic prognostic factors (median follow-up: 64 months).152 Clinicopathologic and molecular factors that were found to be statistically significant adverse prognostic factors in both univariate and multivariate analyses for the separate endpoints of distant metastasis and tumor-related mortality included tumor size greater than 5 cm, microscopically positive surgical margin, and a Ki-67 score greater than 20 (>20% nuclear staining). Overexpression of p53 or mdm2 or deletion of pRb did not correlate with an increased risk of distant metastasis or tumor-related mortality. Synovial sarcoma is characterized by a specific chromosomal translocation, t(X;18)(p11;q11), which is seen in more than 90% of these tumors.82,84 This had led to studies evaluating the potential prognostic significance of SYT-SSX fusion transcripts, which arise from this translocation.92 The t(X;18)(p11;q11) translocation fuses the SYT gene from chromosome 18 to either of two homologous genes at

Sarcomas of Soft Tissue • CHAPTER 97

evident in a recent study of GIST cases not treated with imatinib.74,172 Of interest, c-Kit was highly phosphorylated in all cases, even in those few that lacked demonstrable sequence mutations.74 Mutations were found in specific regions of the c-Kit gene, and specific mutations appear to be associated with differing prognosis (see following discussion). The subset of patients with exon 11 mutations resulting in single amino acid substitutions (i.e., missense codon mutations) fared much better than did patients with deletion/insertion mutations of exon 11 (5-year recurrence-free survival rate of 89% ± 11% versus 37% ± 10%, respectively). A potential explanation for this finding is that exon 11 missense mutations are detected in lower-grade, favorable outcome GISTs.172 Against this explanation is the fact that the vast majority of metastatic GISTs have exon 11 mutations. While it is conceivable that the type of mutation is a surrogate for the behavior of a GIST, however, it is also plausible that the type of mutation represents the initial pathogenetic mechanism, making it a true prognostic marker and target. Finally, in vitro studies suggest that GISTs with regulatory region KIT mutations are more likely to respond to imatinib than are GISTs with enzymatic region mutations.173

Xp11, SSX1, or SSX2. The fusion transcripts SYT-SSX1 and SYTSSX2 are believed to function as aberrant transcriptional regulators. The prognostic significance of these alternative forms of the SYT-SSX fusion gene and the relationship of these fusion transcripts and synovial sarcoma tumor morphology (monophasic versus biphasic subtype) were examined in 45 patients with synovial sarcoma.92 There was a significant correlation (P = 0.003) between histologic subtype and fusion transcript type; all 12 biphasic synovial sarcomas had an SYTSSX1 fusion transcript, whereas 17 (52%) of 33 monophasic tumors were positive for SYT-SSX1. Moreover, the presence of the SYTSSX1 transcript was an independent adverse prognostic factor for metastasis-free survival. Thus, SYT-SSX fusion transcripts may be used as a diagnostic marker for synovial sarcoma, and transcript subtype should be confirmed as an independent prognostic factor. Ewing’s sarcomas are characterized by a translocation involving chromosomes 22 and 11: t(11;22)(q24;q12).85 Recent studies have evaluated the prognostic significance of transcripts produced by the fusion of the EWS and FL11 genes from chromosomes 22 and 11. The most common EWS-FL11 fusion, designated as type 1, is found in 65% of Ewing’s sarcomas.166,167 Two groups have demonstrated that type 1 EWS-FL11 fusion transcripts are associated with a more favorable prognosis.90,91 De Alava and colleagues91 have demonstrated that the prognostic significance of type 1 EWS-FL11 fusion transcripts is independent of tumor site, stage, and size. However, these observations were made with a short follow-up (median: 26 months, range: 1 to 140 months); therefore, additional studies and longer follow-up are needed to further substantiate these interesting observations. The biologic basis for these observations is unknown. Additional studies that examine the relationship between EWS-FL11 transcript subtype and proliferative rate, apoptosis, and response to treatment are warranted. With the increasing use of cDNA expression profiling, it is possible that expression profile-based stratification may be possible within specific histologies, in a manner analogous to current use of this technique to stratify breast cancer.168 Additionally, these profiles have recently been applied to determining metastatic potential of primary tumors, and this approach could clearly be applicable to STSs, in which the presence or absence of metastases remains the most important prognostic factor.169 Most recently, phosphoprotein profiling has been employed to stratify patients with stage III rhabdomyosarcoma and predict outcome.170 This approach also holds future promise for both prognostic and therapeutic approaches (see later discussion). Although specific cellular and molecular parameters have been identified as having independent prognostic significance, there is currently no consensus on how specific molecular prognostic factors should be utilized in clinical practice. Until more data are available, molecular prognostic factors that have proven to be of prognostic significance (e.g., Ki-67) should be considered for inclusion as stratification criteria in clinical trials.

We have just now begun to see the implications and success of targeting genetic alterations in sarcomas that drive oncogenesis. GIST tumors were previously thought to be gastrointestinal leiomyosarcomas that were particularly resistant to cytotoxic chemotherapy. It was subsequently demonstrated that these tumors were derived from interstitial cells of Cajal and were frequently characterized by point mutations in the c-Kit receptor tyrosine kinase and were clearly distinct from leiomyosarcomas.174,175 Subsequently, the tumors were treated with imatinib mesylate that targets the c-Kit kinase with dramatic results.176 The c-Kit mutations that were seen were located in exon 11 (juxtamembrane domain, seen in 71% of tumors), exon 9 (the extracellular region, 13%), exon 13 (first lobe of the splitkinase domain, 4%), and exon 17 (phosphotransferase domain, 4%). The subset of patients with exon 11 mutations resulting in point single amino acid substitutions (i.e., missense codon mutations) fared much better than did patients with deletion/insertion mutations of exon 11 (5-year recurrence free survival rate of 89% ± 11% versus 37% ± 10%, respectively).172 In addition, tumors that lack c-Kit mutations appear to have mutations in PDGF-α receptor, and these mutations appear to be mutually exclusive of c-Kit mutations.177 Finally, the type of mutation that is identified appears not only to predict response to imatinib mesylate, but also to suggest whether other kinase inhibitors such as sunitinib may have beneficial effects.178 While it is unlikely that most sarcomas will be driven by mutations in a kinase that is amenable to target inhibition, it is likely that as more information is acquired regarding activation of specific signaling pathways in STSs, most will have treatment options that are not currently available.

Prognostic Factors as Therapeutic Targets

Predicting Individual Prognosis

The prognosis of GISTs is poor when they are treated by surgery alone,171 and these tumors rarely respond to conventional systemic chemotherapy. The most exciting discovery in GIST research in recent years is targeted molecular therapy, which will be discussed later in the chapter. The proto-oncogene c-Kit is the cellular homolog of the oncogene v-Kit (a feline sarcoma virus). c-Kit encodes a transmembrane tyrosine kinase receptor, KIT (CD117), that is structurally similar to platelet-derived growth factor and provides selective targets of key aberrations in the molecular signaling implicated in the pathogenesis of GISTs and other tumors (e.g., DFSP). An interesting additional feature of c-Kit expression in GIST is that different types and locations of mutations in c-Kit appear to be independently significant for predicting disease-free survival irrespective of treatment with kinase receptor inhibitors.172 CD117 expression was uniformly

Kattan and colleagues have observed that information that is appropriate for researchers is not as helpful for patients with cancer who must plan in a different way for the future.179 Patients’ main preoccupation is to obtain a predicted probability of individual (i.e., personal) survival unencumbered by specific knowledge of prognostic factors, relative risk, or the risk group in which the person may belong. Kattan and colleagues have constructed and validated a nomogram to predict the probability of 12-year sarcoma-specific death based on a prospective series of patients (Fig. 97-8).179 This tool is useful for individual patient counseling, follow-up scheduling, and clinical trial eligibility assessment and is further facilitated by being also available for personal handheld computer devices. This sarcoma-specific computer application is available at www. nomograms.org.

Molecular Therapeutic Targets in Sarcomas

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0

10

20

30

40

50

60

70

80

90

100

Points 5–10 Size (cm) 10 Deep

Depth Superficial Lower extremity

Thoracic/trunk

Head/neck

Site Upper extremity

Visceral Retro/Intra-abdominal Lipo

Leiomyo Synovial

Histology Fibro Age (years)

MFH

16 20 0

20

30

40

40

60

50

60

70

Other

MPNT

90

80

80 100 120 140 160 180 200 220 240 260 280 300 320

Total points 0.04 0.06 0.08 0.1 0.15 0.2

0.3

0.4 0.5 0.6 0.7 0.8 0.88

Figure 97-8 • Postoperative nomogram for 12-year sarcoma-specific death risk. Fibro, fibrosarcoma; GR, grade; Leiomyo, leiomyosarcoma; Lipo, liposarcoma; MFH, malignant fibrous histiocytoma; MPNT, malignant peripheral nerve sheath tumor; SSD, sarcoma-specific death. (Reproduced with permission from Kattan M: Statistical prediction models, artificial neural networks, and the sophism “I am a patient, not a statistic.” J Clin Oncol 2002;20:885.)

12-yr low gr. SSD 0.04 0.06 0.08 0.1 0.15 0.2

0.3 0.4 0.5 0.6 0.7 0.8 0.88 0.95 0.99

12-yr high gr. SSD Instructions for Physician: Locate the patient's tumor size on the Size axis. Draw a line straight upwards to the Points axis to determine how many points towards sarcoma-specific death the patient receives for his tumor size. Repeat this process for the other axes, each time drawing straight upward to the Points axis. Sum the points achieved for each predictor and locate this sum on the Total points axis. Draw a line straight down to either the Low Grade or High Grade axis to find the patient's probability of dying from sarcoma within 12 years assuming he or she does not die of another cause first. Instruction to Patient: “If we had 100 patients exactly like you, we would expect betweenandto die of sarcoma within 12 years if they did not die of another cause first, and death from sarcoma after 12 years is still possible.”

TREATMENT OF LOCALIZED PRIMARY SOFT-TISSUE SARCOMA Surgery Limb-Sparing Surgery versus Amputation Surgical resection remains the cornerstone of therapy for localized STS, and the prototypical situations concern the management of lesions arising in the extremity, the most common anatomic site. Over the past 20 years, there has been a marked decline in the rate of amputation as the primary therapy for extremity STS. With the widespread application of multimodality treatment strategies, fewer than 10% of patients currently undergo amputation.180,181 The current use of limb-sparing multimodality treatment approaches for patients with extremity sarcoma is largely based on a randomized prospective study from the U.S. NCI in which patients with extremity sarcomas that were amenable to limb-sparing surgery were randomized to receive amputation or limb-sparing surgery with postoperative radiotherapy.182,183 Both arms of this trial included postoperative chemotherapy with doxorubicin, cyclophosphamide, and methotrexate. With more than 9 years of follow-up evaluation, 5 (19%) of 27 patients randomly assigned to receive limb-sparing surgery and postoperative radiation with chemotherapy had local recurrences, as compared to 1 (6%) of 17 patients in the amputation

plus chemotherapy arm (P = 0.22; Fig. 97-9).183 The disease-free survival rate was 63% for limb-sparing surgery versus 71% for amputation (P = 0.52; Fig. 97-10), and the overall survival rate was 70% for limb-sparing surgery versus 71% for amputation (P = 0.97). This study established that for patients for whom limb-sparing surgery is an option, a multimodality approach employing limb-sparing surgery combined with postoperative radiotherapy yields disease-related survival rates comparable to those for amputation while simultaneously preserving a functional extremity. Currently, at least 90% of patients with localized extremity sarcomas can undergo limb-sparing procedures.180,184 Most surgeons consider definite major vascular, bony, or nerve involvement to be relative indications for amputation. Complex en bloc bone, vascular, and nerve resections with interposition grafting can be undertaken, but the associated morbidity is high. Therefore, for a few patients with critical involvement of major bony or neurovascular structures, amputation remains the only surgical option but offers the prospect of prompt rehabilitation with excellent local control and survival.183

Completeness of Resection Satisfactory local resection involves resection of the primary tumor with a margin of normal tissue around the lesion. The width of the margin should differ depending on whether or not adjuvant radiotherapy is used. It is clear that dissection along the tumor pseudo-

Sarcomas of Soft Tissue • CHAPTER 97

100 90

Remission (%)

80 70 60 50 40 30 20 Total 17 27

10

Fail 1 5

Amputation Limb-sparing surgery

0 0

1

2

3

4

5

6

7

8

9

10

Years

Figure 97-9 • Local recurrence rates in patients with high-grade extremity sarcomas randomized to receive amputation or limb-sparing surgery. All patients were treated with adjuvant chemotherapy using doxorubicin, cyclophosphamide, and methotrexate. Median follow-up: >9 years (P = 0.22). (Reprinted with permission from Yang JC, Rosenberg SA: Surgery for adult patients with soft tissue sarcomas. Semin Oncol 1989;16:289.)

capsule (enucleation) is associated with local recurrence rates ranging between 33% and 63%.185–187 Wide local excision with a margin of normal tissue around the lesion is associated with local recurrence rates in the range of 10% to 31%, as was noted in the control arms (surgery alone) of the randomized trials evaluating postoperative radiotherapy.188,189 In contrast to malignant melanoma, a disease for which there are randomized data to address adequate margin size, no comparable data are available to define what constitutes a satisfactory gross resection margin for a sarcoma. In general, every effort should be made to achieve a wide margin (2 cm is a frequently cited arbitrary choice) around the tumor mass, except in the immediate vicinity of

100

Disease-free survival (%)

90 80 70 60 50 40 30 20 Total 17 27

10 0 0

1

2

3

4

Fail 5 10

5

Amputation Limb-sparing surgery

6

7

8

9

10

Years

Figure 97-10 • Disease-free survival rates for patients with high-grade extremity sarcomas randomized to receive amputation or limb-sparing surgery. All patients were treated with adjuvant chemotherapy using doxorubicin, cyclophosphamide, and methotrexate. Median follow-up: >9 years (P = 0.52). (Reprinted with permission from Yang JC, Rosenberg SA: Surgery for adult patients with soft tissue sarcomas. Semin Oncol 1989;16:289.)

functionally important neurovascular structures, where, in the absence of frank neoplastic involvement, dissection is performed in the immediate perineural or perivascular tissue planes. The 2-cm choice is unnecessary if radiotherapy is also used, since substantial modification of the surgical approach with much closer margins of resection (e.g., 1 to 2 mm) is made possible, and even large lesions can be managed conservatively in that setting. Technical details of the surgical approach to extremity sarcomas are beyond the scope of this chapter but are comprehensively reviewed in a surgical atlas.190 At the same time, it is also important to bear in mind that involved (i.e., positive) resection margins remain an adverse finding even when adjuvant radiotherapy is used, notwithstanding the amelioration of risk that radiation treatment provides. Data from Memorial SloanKettering Hospital, Princess Margaret Hospital, and Massachusetts General Hospital suggest an additional absolute reduction of local control of approximately 10% to 15% for patients with positive margins compared to those with microscopically negative surgical margins.190–193 In considering the existing outcome data, it is important to bear in mind that these data consider the rubric “positive margins” in a uniform way, although in reality, this is unlikely to be the case. In fact, positive resection margins have different causes. One is oncologically inadequate surgery in which positive resection margins might have been avoidable in another surgeon’s hands. When this is the case, microscopically positive surgical margins can be considered a technical failure. Alternatively, positive resection margins may arise in anatomically adverse presentations in which locally advanced disease challenges the goals of conservative resection from the outset. In another study from the Princess Margaret Hospital, Gerrand and colleagues evaluated the type of microscopically positive margin as a prognostic factor and defined four groups in this setting.194 Patients with low-grade liposarcomas and microscopically positive surgical margins (group 1) have a low risk of local failure (4.2%), as do those in whom a positive margin is anticipated before surgery to preserve critical structures and radiotherapy is given to sterilize the minimal residual disease (group 2). However, two categories of positive margins are associated with a higher risk of local recurrence: (1) patients who present after prereferral unplanned excision and who have a positive margin on subsequent reexcision (group 3) and (2) patients with unanticipated positive margins occurring during primary sarcoma resection (group 4; Fig. 97-11). For group 3, an “unplanned excision” is defined as an excisional biopsy or resection that is carried out without adequate preoperative staging or consideration of the need to remove normal tissue around tumor, an adverse feature reported by the same authors previously.195 These data appear to support the premise that, provided that adjuvant radiotherapy is administered, a very small amount of residual disease resulting from a “planned” positive margin at the site of a critical anatomic structure (group 2, local recurrence rate of 3.6% and 95% confidence interval 0 to 10.4) is not associated with the same deleterious risk that occurs with a positive margin that follows major contamination due to “shell out” intralesional surgery (group 3, local recurrence rate of 31.6%, 95% confidence interval: 10.7 to 52.5) or inadvertent contamination of the wound (group 4, local recurrence rate of 37.5%, 95% confidence interval: 13.8 to 61.2).194 These data seem particularly relevant to anatomic sites where achievement of adequate resection margins is a perennial problem, such as the head and neck, as evidenced by recent results from a prospective series where the outcome approaches that of extremity and body wall sarcomas.196 We would caution, however, that such results are probably not attainable without a defined management protocol and joint multidisciplinary assessment before treatment is undertaken, since there exist issues that merit discussion at the individual case level (e.g., the complex relationship between tissues to be resected and reconstructed and the radiotherapy volumes and doses, all of which can influence each other in the decision algorithm).

2023

Part III: Specific Malignancies Local recurrence-free rate 1.0 0.9 Cumulative survival (%)

2024

0.8 0.7 0.6 0.5 0.4 0.3 0.2 Group 2 Group 3 Group 4

0.1 0.0 0

1

2

3

4

5

Time (yrs)

Figure 97-11 • Kaplan-Meier estimate for local recurrence-free rate for three different groups of positive margin categories (see text for details). Group 1 (low-grade liposarcoma with positive resection margins) is not shown. Tick marks represent censored cases. (Reproduced with permission from Gerrand CH, Wunder JS, Kandel RA, et al: Classification of positive margins after resection of soft-tissue sarcoma of the limb predicts the risk of local recurrence. J Bone Joint Surg Br 2001;83:1149.)

Lymph Node Dissection Given the low (2% to 3%) prevalence of lymph node metastasis in adults with sarcomas,102,103 there is no role for routine regional lymph node dissection. Patients with angiosarcoma, embryonal rhabdomyosarcoma, and epithelioid histiotypes have an increased incidence of lymph node metastasis and should be carefully examined for adenopathy. Therapeutic lymph node dissection (curative) results in a 34% actuarial survival rate;105 therefore, the rare patients with regional nodal involvement who have no evidence of extranodal disease should undergo therapeutic lymphadenectomy. Patients with adverse features at the time of dissection (i.e., extracapsular extension beyond the lymph nodes into perinodal fat or positive or doubtful margins on the neurovascular bundle) or in whom treatment into the next grossly uninvolved lymph node echelon is not feasible with surgery should also be considered for additional adjuvant nodal irradiation. The principles underlying this approach have recently been outlined.197

Surgery Alone Although the majority of patients with extremity STS should be treated with preoperative or postoperative radiotherapy, recent reports

suggest that concomitant radiotherapy might not be required for selected patients with completely resected, small, primary STSs (Table 97-8).198–201 Rydholm and colleagues have reported their experience with 70 patients with subcutaneous or intramuscular extremity sarcomas treated with wide surgical resection and microscopic assessment of surgical margins.200 Negative histologic margins were obtained for 32 of 40 subcutaneous and 24 of 30 intramuscular tumors. The 56 patients with microscopically negative margins received no postoperative radiotherapy, yet only 4 (7%) developed local recurrence. A study from Brigham and Women’s Hospital reported similar results for a selected group of 74 patients with primary extremity STS treated by surgery without radiotherapy.201 The 10-year actuarial local control rate was 93% plus or minus 4%. The absolute gross margin was a significant predictor of local recurrence; patients with a close gross margin of less than 1 cm had a 10-year local control rate of 87% ± 6% compared to 100% for patients with a closest gross margin of 1 cm or greater (P = 0.04). The generally favorable local control rates with surgery alone that these and other198,202 investigators reported in these series of highly selected patients are comparable to local recurrence rates observed for more heterogeneous patient populations treated with conventional multimodality therapy incorporating preoperative or postoperative radiotherapy (Table 97-9).66,188,203–209 These data support the hypothesis that selected patients with small, primary STSs can be treated with surgical resection alone without preoperative or postoperative radiotherapy. It is difficult to define the precise selection criteria that should be used to identify patients with primary sarcoma who can safely undergo treatment by surgery without radiotherapy. Most investigators have limited this approach to patients with carefully selected T1 tumors that can be resected with clear margins (see Table 97-8). In contrast, Karakousis and colleagues did not consider absolute tumor size but instead utilized surgical resection alone for all patients in whom a minimum intracompartmental margin of 2 cm could be maintained circumferentially, irrespective of tumor size.198 Karakousis and colleagues recently updated the results for high-grade STS of the policy of limiting the use of postoperative radiation treatment for tumors resected with positive or “narrow” (less than 2 cm) resection margins.210 This approach has yielded useful data because the consistent application of this treatment approach resulted in a local recurrence rate of 19% with “wide” margin surgery alone compared to 24% after “narrow” margin surgery and adjuvant radiotherapy.210 Although the results provide some clarity about the 2 cm or greater margin benchmark, it would be useful to also have similar data from other groups for a variety of margin widths to draw conclusions about when it is safe to withhold radiotherapy. Moreover, the authors acknowledge the potential to treat a greater proportion of cases with radiotherapy and lower the 19% local recurrence rate in some of those “favorable cases” that are currently treated with surgery alone by widening the indication for adjuvant radiotherapy in a proportion of these patients.210 This view would certainly be consistent with

Table 97-8 Results of Surgery Alone for Selected Patients with Soft-Tissue Sarcoma First Author

Institution

No. of Patients

Geer199

MSKCC

Rydholm200

Lund, Sweden

Baldini201

BWH

74

Karakousis198

RPCI

116

Fabrizio563

Mayo

34

Selection Criteria

174

T1 size, primary tumor

56

G/M margin negative

Adjuvant Radiation (No.)

Local Recurrence (%)

Distant Recurrence (%)

117

10

5

0

7

NR

T1 size, G/M margin negative

0

7

12

2 cm G margin

0

10

NR

Not stated

0

15

12

BWH, Brigham and Women’s Hospital; G/M, gross/microscopic; Mayo, Mayo Clinic; MSKCC, Memorial Sloan-Kettering Cancer Center; NR, not reported; RPCI, Roswell Park Cancer Institute.

Sarcomas of Soft Tissue • CHAPTER 97

Table 97-9 Local Control with Surgery and Radiotherapy for Localized Soft-Tissue Sarcoma Radiotherapy Approach

First Author

Radiation Dose (GY)

Study Design

No. of Patients

Local Failure (%)

Preoperative EBRT

Suit203

50–56

Retrospective

89

17

Barkley204

50

Retrospective

110

10

Brant205

50.4

Retrospective

58

9

O’Sullivan211

50

RCT

94

7

Brachytherapy

Pisters193

42–45

RCT

119

9

(high-grade)

45

23

(low-grade)

Postoperative EBRT

Lindberg207

60–75

Retrospective

300

22

Karakousis208

45–60

Retrospective

53

14

Suit203

60–68

Retrospective

131

12

45 + 18

RCT

Yang

188

O’Sullivan211

RCT

Subset

91

0

(high-grade)

50

5

(low-grade)

96

7

EBRT, external beam radiotherapy; RCT, randomized controlled trial. Randomized controlled trials and selected nonrandomized retrospective series.

contemporary observations such as the local recurrence rate of 7% in patients who are undergoing combined modality treatment such as those in the recent Canadian randomized trial (see section on Preoperative or Postoperative Radiotherapy).211 Factors other than anatomic location, tumor size, and the feasibility of achieving an R0 resection (macroscopically and microscopically complete) should be considered in selecting patients for treatment by surgery alone. For example, the issue of whether the patient has had a prior “unplanned” excision (referred to earlier) is important. At the Princess Margaret Hospital, a significantly higher rate of local recurrence was apparent in patients who were treated after unplanned excision on the outside than in patients who received their treatment at their institution (22% versus 7%, P = 0.03).195 It is important to remember that unplanned excision is very common in the community setting, where small soft-tissue lesions are often excised without image guidance under the presumption that they are benign. Therefore, while it is reasonable to attempt a reexcision if it is considered feasible, patients who have undergone unplanned excision should also be strongly considered for adjuvant radiation.

Preoperative or Postoperative Radiotherapy Conservative (limb-sparing) surgery and radiotherapy have been combined to optimize local control for patients with localized STS. Radiotherapy can be administered preoperatively,203–205,212,213 postoperatively,207,214,215 or by interstitial techniques (brachytherapy).66,206,216–220

Local Control Data from two randomized controlled trials (RCTs)71,188 have confirmed earlier retrospective reports suggesting that surgery combined with radiotherapy results in superior local control compared to surgery alone.204,207,209 Yang and colleagues from the NCI recently reported on a RCT of postoperative external beam radiotherapy (EBRT).188 In this trial, 141 patients with localized extremity STSs amenable to limb-sparing resection were randomly assigned to receive postoperative EBRT or no radiotherapy. All patients with high-grade lesions received postoperative chemotherapy. In the subset of 91 patients with high-grade lesions, no local recurrences have been noted in the 44 patients who received postoperative radiotherapy (with chemotherapy) versus 9 local recurrences (19%) in the 47 patients who received postoperative chemotherapy alone (P = 0.0003). In the 50 patients with low-grade sarcomas, 1 (4%) of 26 patients who

received adjuvant radiotherapy has had a local recurrence versus 8 (33%) of 24 patients treated by surgical resection alone (P = 0.016). However, no improvement in survival was noted with adjuvant radiotherapy in the entire cohort of patients or in any subgroup. The second RCT of postoperative radiotherapy was conducted at Memorial Sloan-Kettering Cancer Center, where investigators studied adjuvant brachytherapy for patients with extremity and superficial trunk STSs.193 One hundred sixty-four patients with extremity or superficial trunk STSs were randomly assigned to receive adjuvant brachytherapy (42 to 45 Gy with an iridium-192 implant) or no postoperative radiotherapy after complete resection of their sarcomas. Randomization took place in the operating room after gross total resection, thereby limiting the potential bias that might influence the extent of surgical resection in a comparative trial. Sixty-eight of 119 patients with high-grade tumors also received chemotherapy. With a median follow-up of 76 months, 5-year actuarial local control rates were significantly better in the group treated with adjuvant brachytherapy (82%) than in those who received surgery alone (69%). Subset analysis demonstrated that the local control advantage of brachytherapy was confined to patients with high-grade lesions, for whom the 5-year local control rate was 89% (versus 66% in the surgery-only group; Fig. 97-12). Patients with low-grade STSs did not appear to experience the same local control benefit with adjuvant brachytherapy.193,220 As was noted in the NCI RCT,177 the improvement in local control did not translate into any detectable survival difference between the brachytherapy and no-brachytherapy arms of the trial. Local failure rates with combined-modality regimens incorporating surgery and radiotherapy are generally less than 15% (see Table 97-9). Despite theoretical advantages that may favor preoperative radiation, brachytherapy, or postoperative radiation, there does not appear to be a major difference in local control rates among these radiation techniques, although at present, data comparing the approaches are sparse.

Relationship Between Local Control and Survival Whether local control affects overall survival for patients with STS remains unclear and highly controversial.221–225 Only an adequately powered prospective randomized trial can assess the precise nature of any relationship between local control and overall survival. Three RCTs have evaluated local control and survival in the context of defining treatment approaches for STS. In a randomized trial of

2025

Part III: Specific Malignancies

1.0 Proportion free of local recurrence

0.9

Figure 97-12 • Local recurrencefree survival in patients with high-grade sarcoma treated in the Memorial SloanKettering Cancer Center randomized trial of postoperative brachytherapy versus surgery alone. A statistically significant difference was noted in local recurrence-free survival with brachytherapy (P = 0.0025). (Reprinted with permission from Pisters PW, Harrison LB, Leung DH, et al: Long-term results of a prospective randomized trial of adjuvant brachytherapy in soft tissue sarcoma. J Clin Oncol 1996;14:859.)

0.8 0.7 0.6 0.5 0.4 0.3

P=0.0025

0.2

This mark ( ) indicates last follow-up Brachytherapy (56 pts. 51 censored) No brachytherapy (63 pts. 44 censored)

0.1 0.0 0

12

24

36

48

60

72

84

96

108

120

132

Months after surgery

amputation versus conservative surgery plus radiation from the NCI, local recurrence rates were 19% in the limb-sparing arm versus 6% in the amputation arm (P = 0.022).182,183 Despite this, overall survival rates were equivalent at 70% for limb-sparing surgery and 71% for amputation (P = 0.97). In the randomized trials of postoperative radiotherapy,188,193 the improvement in local control that was noted in patients who were treated with surgery plus radiotherapy did not translate into any detectable survival advantage. Thus, none of the currently available data from prospective RCTs support the hypothesis that better local control enhances survival in patients with sarcoma. Methodologically, the available trials are problematic for this issue because the outcome of interest (i.e., a difference in survival consequent on a differential in local control) would require a prohibitively large sample size. Thus, it is most improbable for these trials to be capable of demonstrating an effect with their modest sample sizes that were intended for evaluation of different outcomes. Indeed, it is most unlikely if even a meta-analysis of the trials could demonstrate this. Furthermore, data from nonrandomized studies support the concept that there is little, if any, relationship between local control and survival. In a recent series from Sweden, the outcome of patients who were treated with an inadequate excision was compared with that of patients who had an adequate operation.224 Local recurrence was 3.5 times more common after inadequate excision, but there was no difference in the incidence or timing of distant metastases. The power of the RCTs that have been reported so far to detect a difference in survival is relatively small, and a large number of patients may be required to demonstrate that prevention of local recurrence affects survival.222 Stotter and colleagues have argued that local recurrence is a time-dependent variable and should be considered as such in multivariate studies.221 Analysis in this fashion of the data from a nonrandomized study demonstrates a statistically significant relationship between local control and survival. Other retrospective analyses have yielded similar conclusions.226,227 For a more detailed description of the methodologic problems associated with time-dependent variables and the use of surrogate endpoints that emerge after the initial sarcoma treatment, the reader is referred elsewhere.228 In this context, it is clearly important to distinguish between the well-defined adverse prognostic impact of subsequent local recurrence on survival109,225,229 and the unproven positive effect of improved local control (i.e., prevention of local recurrence with improved local therapy) on survival. The former phenomenon might be a manifestation of more aggressive tumor biology; that is, biologically more aggressive lesions might recur locally and metastasize more frequently.

Treatment Sequencing: Preoperative versus Postoperative Treatment Of further interest, an improvement in overall survival (a crude rate of 85% versus 72% in favor of postoperative radiotherapy, P = 0.0481) has emerged and is only partially explained by increased deaths in the postoperative arm unrelated to sarcoma (Fig. 97-13).211 This observation is of obvious oncologic interest. Longer follow-up is clearly required, and a 5-year analysis is currently under way. In summary, the final results of the SR2 trial are expected to provide insight into the comparative efficacy, functional outcome, economic costs, and complication rates of these two options for EBRT and potentially on survival outcome if the preliminary results are sustained.

Overall survival 100 90 Event free (%)

2026

80 60 40 Log-rank P=0.0481

20 Preoperative RT Postoperative RT

0 0.0

1.0

2.0

3.0

Time (yrs) Patients at risk Preoperative RT Postoperative RT

92 94

87 90

81 74

51 48

Figure 97-13 • Actuarial probability of overall survival for preoperative versus postoperative radiotherapy in extremity soft-tissue sarcoma. (Reproduced with permission from O’Sullivan B, Davis AM, Turcotte R, et al: Preoperative versus postoperative radiotherapy in soft-tissue sarcoma of the limbs: a randomised trial. Lancet 2002;359:2235.)

Sarcomas of Soft Tissue • CHAPTER 97

Until the mature data from the Canadian Sarcoma Group RCT are available, it appears reasonable to treat patients with postoperative EBRT, since local control rates are comparable to preoperative techniques but major wound complication rates are significantly lower. On the other hand, the maturing data on late tissue effects for these respective approaches in the Canadian trial are salutary, and the emerging potential influence on survival, pending 5-year analysis, is awaited with interest. Also, in anatomic sites where wound complications are rarely seen (e.g., the upper extremity), the rationale for wound complication avoidance as a reason to favor the use of postoperative radiotherapy is not as sound. In particular, the obvious advantage to preoperative radiotherapy in the proximal arm and shoulder is apparent where avoidance of large volume and higher dose irradiation that may treat the lung or brachial plexus can be achieved. These principles are also reasonable in the head and neck based on the recent Princess Margaret Hospital data.196 On the other hand, with brachytherapy, the patient’s entire local treatment (surgery plus radiation) can be completed in 10 to 14 days. This has significant cost advantages230 and also has significant implications in terms of overall patient convenience. In the absence of comparative data addressing the efficacy of these techniques in achieving local control, these additional considerations assume increased importance. Where the necessary expertise is available for brachytherapy, this technique provides an excellent, cost-effective alternative for patients with high-grade lesions. Brachytherapy should not be used for patients with low-grade sarcomas.220 Recently, an RCT of EBRT for patients with localized extremity STS was reported by the National Cancer Institute of Canada Clinical Trials Group/Canadian Sarcoma Group in which 190 patients with extremity STS were randomized to preoperative versus postoperative radiation.211 The radiotherapy parameters for this protocol required a field margin of 5 cm around the gross tumor volume for the initial phase of treatment (i.e., treatment to 50 Gy in 25 fractions), and this generally included any peritumoral edema that was seen on MRI, irrespective of the grade or size of the tumor. Subsequently, a reduced-volume field was treated to a total combined dose of 66 Gy in all postoperative cases and in those preoperative patients in whom the resection margins were involved. The results of this trial are complex because the primary endpoint that powered the trial, and hence its sample size, was the cumulative incidence of acute wound complications 120 days after protocol surgery in both arms of the study. Nevertheless, the local control rates after 3.3 years of median follow-up are identical in both arms of the study (7%).

Wound Complication Rates and Post-Treatment Function The results of the Canadian RCT may provide insight into the comparative efficacy, functional outcome, economic costs, and complication rates of preoperative and postoperative treatment sequencing for EBRT. In the absence of a clear local control advantage to any specific radiation technique, clinicians have considered other factors in formulating standards of care. Such factors have included wound complication rates, financial costs, patient convenience, health-related quality of life and physical function, radiotherapy toxicity, and perhaps even overall survival. It is clear that while field size and radiation dose may be minimized with preoperative radiotherapy,231 major wound complications after preoperative radiotherapy and surgery have been reported to be in the 20% to 35% range.232,233 In the Canadian Sarcoma Group RCT, wound complications were defined as secondary wound surgery, hospital admission for wound care, deep packing, or prolonged dressings within 120 days after tumor resection. By these criteria, preoperative radiation had a significantly higher rate (35% versus 17%, P = 0.01) of wound complications than did postoperative EBRT. Of note, the risk was confined to the lower extremity.211 Taken in isolation, the wound complication fact alone can be expected to cause some groups to continue to favor postoperative

radiotherapy, though this could change, as data have now emerged from the Canadian RCT that late tissue outcomes strongly favor the preoperative approach and a putative survival advantage needs to await mature follow-up.234 Thus, the radiotherapy toxicity rates after 2 years differed between the arms of the study. The rates of grade 2 or greater fibrosis and edema were significantly higher in the postoperative arm compared to preoperative radiotherapy and were independently associated with the larger irradiation volumes and doses used in postoperative radiotherapy.234 Short-term functional outcome in the SR2 trial has also been reported and continues to be collected prospectively.235 Two validated instruments—the Toronto Extremity Salvage Score (TESS) and the Short Form-36 quality of life instrument (SF-36)—were applied, as was the observer-based Musculoskeletal Tumor Society Rating Scale (MSTS).235 Patients who were treated with postoperative radiotherapy had better function with higher MSTS, TESS, and SF-36 bodily pain scores at 6 weeks after surgery than did those who were treated with preoperative radiation, but there were no differences at later time points up to 1 year. Thus, the timing of radiotherapy has minimal impact on the function of STS patients in the first year after surgery, but thereafter, significant factors likely come into play. These include the apparently deteriorating late tissue sequelae caused by larger doses and volumes. Of interest, patients who experience wound complications appear to continue to suffer some impaired function. Further follow-up will be required to assess the ongoing evolution of these competing risks.

Conformal Radiotherapy and Intensity-Modulated Radiotherapy STSs present in virtually any anatomic site, and the capacity for unusual presentation is almost limitless. This can result in circumstances in which conventionally delivered radiotherapy is impossible owing to the magnitude of the volume to be treated, uncertainty in defining the target for radiotherapy, or, more usually, because of the proximity of normal tissues to the intended target volume. While some presentations are extremely problematic (e.g., uncertain targets due to organ mobility or imprecise anatomic issues resulting from poor definition of tumor location related to imaging limitations or inadequate surgical and/or pathologic description), others can be addressed by novel methods of radiotherapy delivery. Leading the advances in this field is intensity-modulated radiotherapy, an advanced form of three-dimensional conformal radiotherapy in which radiation beams are not only shaped at their perimeters, but also include variable intensity across the profiles of the beams. This permits the creation of exquisite conformation of dose to targets of irregular shape while generating high-dose gradients between tumor and normal tissues. A full discussion of the potential uses of intensity-modulated radiotherapy in STS is beyond the scope of this chapter but is discussed in detail elsewhere.236 It may be administered preoperatively, postoperatively, or as a sole modality with specific indications. Some applications include its use in lesions adjacent to the spine or critical anatomic structures of the head and neck and in the retroperitoneum to permit liver avoidance (as well as to permit spinal cord, kidney, and intestinal dose limitation), especially in lesions involving the right upper abdominal quadrant. Avoidance of late toxicity to anatomic structures such as weight-bearing bone that are at risk for fracture after treatment of extremity sarcomas seems also to be feasible.237 Although these approaches are promising, their precise contribution and role need to be evaluated.238

Conventional Radiotherapy Without Surgery Radiotherapy alone has been employed as primary therapy for patients with locally advanced, inoperable STS and patients who present with stage IV disease. Efforts to use radiation as the primary treatment have demonstrated that high doses (more than 65 Gy) are required to achieve local control rates between 30% and 60%239,240 and that

2027

2028

Part III: Specific Malignancies

there appears to be an inverse relationship between tumor size and local control rates. In a series of 35 patients treated with high-dose (>65 Gy) primary radiotherapy for tumor sizes less than 5 cm, 5 to 10 cm, and larger than 10 cm, the local control rates were 88%, 53%, and 33%, respectively.241 In general, local control rates with radiation alone are inferior to those after surgery; therefore, primary radiation should be reserved for patients who are medically unfit for surgery, have technically unresectable tumors, or refuse surgery as initial therapy. However, some caution is necessary in interpreting such results in a scientifically valid manner. Encumbered with such adverse selection factors, the outcome of radiotherapy would never be comparable to that of surgery. Moreover, surgery has the added advantage over radiotherapy alone because its use for locally advanced cases is ordinarily also combined with adjuvant radiotherapy.

Neutron Radiotherapy Neutron radiotherapy has been emphasized by certain groups because of the lower oxygen enhancement ratio compared to x-rays and the consequent attractive possibility of overcoming the biologic phenomenon that hypoxic cells generally limit the curability of malignancy with x-rays. Additional differences from x-rays are the reduced repair of sublethal and potentially lethal damage. These neutron effects are less vulnerable to the differential radiosensitivities associated with different phases of the cell cycle. It should be apparent that some of these repair phenomena also negatively affect the tolerance of normal tissues. Neutrons have been employed in a number of pilot studies, primarily in patients with locally advanced disease, with 60% to 70% local control rates.242–244 In a recent large series, 220 patients with locally advanced sarcomas were treated with neutron radiotherapy. Ninety-four patients with gross residual disease after resection were treated with neutron therapy alone; among these patients, 27% had major morbidity, 26% had 5-year survival, and 56% had local control. One hundred four patients with microscopically positive margins of resection received a neutron boost dose; they had 7% morbidity, 65% 5-year survival, and 78% local control rates. These results suggest that for patients with gross residual disease, neutron beam radiation may provide improved local control compared to conventional external beam treatment, but these data should continue to be interpreted with caution when one considers the late tissue sequelae that appear to result from neutron beam radiation use. Comparative studies are needed to define the precise role of neutron beam therapy in the treatment of STS. Apart from unresectable disease, it seems unclear where the benefit of neutron therapy might accrue when one considers the exceptionally favorable results of conventional x-ray treatment combined with surgery and the more adverse normal tissue tolerance to neutron therapy.

Adjuvant Chemotherapy In the past 30 years, improvements in surgical and radiotherapy techniques have led to impressive rates of local control, particularly

in extremity STS, with concomitant sparing of normal tissues and preservation of limb and/or organ function. Regrettably, in the same period, much less progress has been made in finding ways to eradicate the micrometastases that are the ultimate cause of death in many individuals who present with apparently localized STS. The discovery that doxorubicin had significant antitumor activity against adult STS prompted the initiation of multiple RCTs during the period 1973 to 1990 to evaluate the benefit of doxorubicin alone or in combination with other agents after the completion of local treatment. However, most of these trials were too small to detect moderate treatment effects reliably. The statistical technique of meta-analysis may overcome the problem of inadequate power of small RCTs, and meta-analyses based on individual patient data (IPDMA) can minimize other potential biases (e.g., exclusion of unpublished trials, variable followup, postrandomization exclusions, and differing definition of endpoints) that are inherent in analyses that are limited to published results. In 1997, the Sarcoma Meta-Analysis Collaboration (SMAC) published an IPDMA of outcomes for 1568 STS patients included in 14 RCTs that completed accrual by December 1992.245 As is outlined in Table 97-10, significant improvements were found in local and distant relapse-free intervals and recurrence-free survival for all patients, and these improvements did translate into a significant overall survival benefit in the prospectively defined subgroup—almost 60% of the patients—with extremity sarcomas but not in all patients. When interpreting the SMAC results, one should compare them with the IPDMAs that have provided conclusive evidence of the benefits of adjuvant chemotherapy and hormone therapy in early breast cancer.246,247 In contrast with the approximately 1500 patients who were included in the SMAC IPDMA, the Early Breast Cancer Collaborative Trials Group identified 47 trials that recruited 18,000 patients for comparisons of adjuvant chemotherapy versus no adjuvant chemotherapy. The power of large numbers, even in the setting of meta-analysis, is self-evident. Compounding the problem of small numbers of STS cases, STSs as a group show marked heterogeneity in pathology and site of origin; this is much less evident in breast cancer. For example, it has been suggested that in the SMAC metaanalysis, chemotherapy benefits for extremity (57% of total) and high-grade STSs were obscured by inclusion of sarcomas at other locations (head and neck, trunk, and uterus) and those of low (5%) or unknown grade (28%). Another point of contrast with breast cancer is the relative paucity of drugs that are active against STS. At the time of the SMAC meta-analysis, only two agents (doxorubicin and ifosfamide) have reproducibly produced overall response rates exceeding 20% in patients with advanced disease. This provides limited opportunity to exploit the potential advantages of combination chemotherapy. Of the 14 trials that were included in the SMAC meta-analysis, 6 used doxorubicin alone, and the remaining 8 were trials of combination chemotherapy. Only one unpublished STS trial (29 patients) examined the combination of doxorubicin with ifosfamide.

Table 97-10 Adjuvant Doxorubicin-Based Chemotherapy for Localized Soft-Tissue Sarcoma: Sarcoma Meta-Analysis Collaboration Results Survival Outcome

No. of Trials

No. of Patients

Hazard Ratio (95% CI)

Overall, All patients

14

1544

0.89 (0.76–1.03)

P Value 0.12

10-Year Survival Benefit (%) 4

Overall, Extremity only

12

886

0.80 (NA)

0.029

7

Recurrence-free

14

1366

0.75 (0.64–0.87)

0.0001

10

Local recurrence-free

13

1315

0.73 (0.56–0.94)

0.016

6

Metastasis-free

13

1315

0.70 (0.57–0.85)

0.0003

10

Adapted from Tierney JF: Adjuvant chemotherapy for localized respectable soft-tissue sarcoma of adults: meta-analysis of individual data. Lancet 1997;350:1647.

Sarcomas of Soft Tissue • CHAPTER 97

Table 97-11 Post-1992 Randomized Controlled Trials of Adjuvant Chemotherapy versus Observation in Soft-Tissue Sarcoma SURVIVAL Study

Chemotherapy Regimen

Italian Cooperative*249

Epirubicin + Ifosfamide + G-GSF (×5)

53

Control

51

Australian Cooperative†250

No. of Patients

Ifosfamide + Doxorubicin + Dacarbazine (IFADIC) + G-CSF ×6

31

Control

28

Disease Sites

Follow-up

RFS

OS

Limb (grade III > 5 cm)

59 mos (median)

48 mos

75 mos

16 mos

46 mos

P = 0.04

P = 0.03

77%

NR

57%

NR

P = 0.1

P = 0.4

Limb 47, Trunk 12 (grade II/III)

41 mos (mean)

G-CSF, granulocyte colony stimulating factor; mos, months; NR, not reported at defined follow-up time; OS, overall survival; RFS, recurrence-free survival. Outcome data provided in original papers. *Median recurrence-free plus overall survival. † (1) comparison % recurrence-free plus overall survival after mean observation period 41 (8–84) mos. (2) overall survival plotted but actuarial results not reported at defined follow-up time(s).

Since the SMAC meta-analysis, full reports have been published on two additional RCTs (Table 97-11) examining high-dose anthracycline and ifosfamide-based regimens supported by granulocyte colony-stimulating factor (G-CSF). In the Italian Cooperative Group study,248 accrual was terminated, based on an early stopping rule, after half the planned number of patients had been recruited. The median recurrence-free survival and overall survival (see Table 97-11) were significantly better for the chemotherapy group, but a high cumulative incidence of late distant relapses was noted (2 year: 28% versus 45%, P = 0.08; 4 year: 44% versus 45%, P = 0.94 for chemotherapy versus control groups, respectively), although 4-year overall survival remained better for the chemotherapy group (69% versus 50%, P = 0.04). At the last published analysis, after a median follow-up of 89.6 months (range: 56 to 119), the intention-to-treat analysis still reveals a difference in overall survival (P = 0.07).249 The 5-year overall survival estimates, a reasonable endpoint for the survival analysis of adjuvant treatment in STSs, were 66.0% and 46.1% for the treatment and the control groups, respectively (P = 0.04).249 The authors of the second study, a prospective randomized feasibility trial, concluded that a regimen of six cycles of ifosfamide, doxorubicin, and dacarbazine given concurrently with postoperative hyperfractionated radiotherapy (during cycles 3 and 4 when doxorubicin was omitted from the regimen) was manageable and tolerable.250 It did not translate into significant benefits in recurrence-free survival (P = 0.1 versus control arm), time to local failure (P = 0.09), or overall survival (P = 0.4), but the small number of patients (31 versus 28) precludes any meaningful conclusions regarding benefit. A third RCT, from another Italian center, has been published in abstract form.251 Only 19 of 41 patients in the chemotherapy arm received an intensive epirubicin/ifosfamide combination (the remainder received single-agent epirubicin), and the study was underpowered for efficacy endpoints. So can any definite recommendations be made regarding the use of adjuvant chemotherapy in adult STS? In an editorial252 that accompanied the report of the Italian Cooperative Group Trial,248 Bramwell concluded that a specific standard of care was not yet clear and that the situation would take some time to change. A Canadian practice guideline253 on this topic has suggested: “It is reasonable to consider anthracycline-based adjuvant chemotherapy in patients who have had removal of a sarcoma with features predicting a high likelihood of relapse (deep location, size >5 cm, high histological grade).” With the current state of knowledge, we believe that this is a reasonable approach. One of the high-dose anthracycline and ifosfamide regimens used in recent RCTs seems a logical choice for adjuvant

treatment but might not be suitable for the substantial minority of patients who are older than 70 years. (This group has been excluded from RCTs evaluating these regimens.) Ultimately, the decision of whether to administer adjuvant chemotherapy and the regimen that is chosen will depend on a number of considerations, including risk of relapse, physician preferences (which may also depend on patient age and comorbid conditions), referral practices, and available resources. While there are very strong opinions on whether or not chemotherapy has added sufficiently to local therapy for routine use in high-risk patients, all agree on two facts: (1) High-risk patients die far too often of metastatic disease, so effective systemic treatment is needed; and (2) there is no current regimen that is anywhere near good enough.

Neoadjuvant Chemotherapy Given that the role of postoperative adjuvant chemotherapy for patients with adult STS remains controversial, it is hardly surprising that the advantages of chemotherapy given before surgery (neoadjuvant therapy) are even less clear, particularly as there have been no adequately powered RCTs addressing this issue. Nonetheless, neoadjuvant chemotherapy has theoretical benefits that include the following: • Destruction of the primary tumor may reduce the risk of contamination at surgery and permit closer margins with less tissue loss and functional disability but improved local control. • Extensive delays in initiating chemotherapy resulting from complex surgery and/or radiotherapy are avoided; for rapidly growing tumors, this earlier elimination of micrometastases may improve survival, although this has not been proven. • Treating with an intact tumor allows the medical oncologist to assess the effects of preoperative therapy and thus judge whether the chosen chemotherapeutic regimen has activity against the specific tumor in the specific patient being treated. The importance of this opportunity to determine whether an empirically chosen regimen will or will not be beneficial cannot be underestimated, especially in dealing with marginally effective chemotherapy. Numerous neoadjuvant treatment approaches and preoperative drug regimens have been explored. Intra-arterial (IA) administration of drugs such as doxorubicin or cisplatin has been evaluated, in some cases in conjunction with radiotherapy, isolated limb perfusion, and/ or hyperthermia. The IA route delivers drugs more directly to the tumor but is more complex, expensive, and prone to complications

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than is the intravenous (IV) route. In the one small RCT on route of administration, there were no differences in rates of local control or overall failure between neoadjuvant chemotherapy given IA or IV.254,255 In a series of nonrandomized studies reflecting the evolution of neoadjuvant treatment at their center over a 20-year period, the University of California, Los Angeles (UCLA) group treated a total of 498 patients with neoadjuvant chemotherapy.256 The combination of chemotherapy and 28 Gy of radiotherapy before surgery provided the best local control with the lowest complication rate. On the basis of results of the RCT described earlier, IA doxorubicin was replaced by IV doxorubicin, and cisplatin and ifosfamide were added to the most recent protocol.256 In the whole group of 498 patients, the overall local recurrence rates were 11% at 5 years and 15% at 10 years, and corresponding overall survival rates were 71% and 66%. The local recurrence rate was lower and overall survival rate higher for patients who had no residual tumor (38%) or greater than 95% necrosis (14%) after neoadjuvant chemotherapy, compared with those who had less than 95% necrosis. In a multivariate analysis, pathologic necrosis was an independent predictor of local recurrence and overall survival. The percentage of patients with 95% or greater necrosis increased to 48% with the addition of ifosfamide, compared to 13% for patients in all other protocols combined. Pisters and colleagues have reviewed the long-term results of neoadjuvant chemotherapy given at the University of Texas M.D. Anderson Cancer Center for stage IIIB extremity sarcomas between 1986 and 1990.257 All patients received doxorubicin-based regimens; at that time, ifosfamide was rarely used (3 patients). In 75 patients, the overall clinical objective response rate (complete response plus partial response) was 27%. At a median follow-up of 85 months, the 5-year actuarial local recurrence-free survival, overall recurrence-free survival, and overall survival rates were 83%, 52%, and 59%, respectively. In contrast with the UCLA group’s results256 for pathologic response, there were no differences in any outcomes between responding and nonresponding patients, as defined at that time. In the M.D. Anderson experience, complete pathologic response was an infrequent event, occurring in only 8% of patients treated between 1984 and 1992, although patients receiving preoperative radiation were excluded.258 Five of the six patients with pathologic complete response were long-term survivors after a median follow-up of 76 months, supporting the findings from UCLA. In a separate analysis at M.D. Anderson, 65 patients (42 extremity sarcomas and 23 retroperitoneal sarcomas) were treated at the same center between 1991 and 1996 with doxorubicin- or ifosfamide-based neoadjuvant chemotherapy; 34% achieved a radiographic partial response and 9% a minor response.259 Patients having partial response had higher rates of negative-margin resections, local recurrence-free survival, and overall survival than did nonresponders. Postoperative morbidity was also evaluated in a larger cohort of 105 patients (71 extremity and 34 retroperitoneal STS) treated at M.D. Anderson during the same period, of whom 50 received ifosfamide as well as doxorubicin.260 The authors found no evidence that preoperative chemotherapy increased surgical complications (e.g., wound infections and other wound problems), length of hospital stay, rate of readmission, or rate of reoperation. A number of other institutions have recently reported, mostly in abstract form, their experience with neoadjuvant chemotherapy using doxorubicin- and ifosfamide-based regimens that in many cases included cisplatin.261–264 In most of these studies, radiotherapy followed chemotherapy and was given preoperatively or postoperatively. The European Organization for the Research and Treatment of Cancer (EORTC) has performed the only RCT assessing preoperative chemotherapy for STS (three cycles of doxorubicin and ifosfamide plus G-CSF) versus no preoperative chemotherapy (control).265 A total of 150 patients with high-risk STS (≥8 cm any grade, or grade II/III tumors 8 cm) localized, high-grade extremity STSs.272 This treatment protocol involved interdigitating courses of chemotherapy and radiotherapy: three courses of doxorubicin, ifosfamide, mesna, and dacarbazine and two 22-Gy courses of radiation (11 fractions each) for a total preoperative radiation dose that is lower than is usually used (44 Gy). This was followed by surgical resection with microscopic assessment of surgical margins. An additional 16-Gy (8 fractions) boost dose was delivered for microscopically positive surgical margins. The strategy therefore usefully addresses the dual problems of local control and metastatic risk. The outcomes of 48 patients who were treated with this regimen between June 1989 and March 1999 have been compared to those of a matched series of historic controls (treated between January 1988 and March 1997).272 The 5-year actuarial local control, distant metastasis-free survival, and overall survival rates for the sequential chemoradiation group are 92%, 75%, and 87%, respectively. For the matched historic controls, these rates are 86%, 47%, and 58%,

Hyperthermic Isolated Limb Perfusion and Whole-Body Hyperthermia with Chemotherapy Hyperthermic isolated limb perfusion and whole-body hyperthermia are two investigational techniques that continue to receive considerable attention, particularly in Europe. Hyperthermic isolated limb perfusion (with tumor necrosis factor-α [TNF-α], interferon-α [IFN-α], and melphalan) has been used as an neoadjuvant therapy to render tumors resectable and as a primary therapy to avoid amputation for nonresectable extremity STS.275–277 Overall response rates in three series, which recruited 55, 35, and 41 patients, respectively, ranged from 72% to 91%, and limb salvage surgery was possible in 84% to 91% of cases. Small numbers of patients experienced subsequent local recurrence, and some of these patients needed later amputations. Not surprisingly, many patients ultimately developed and died of distant metastases. Hyperthermic isolated limb perfusion has also been advocated to facilitate palliative limb salvage in patients with regional and/or distant metastases from unresectable stage IVA or IVB STS.278 Various techniques of regional or whole-body hyperthermia have been combined with a variety of chemotherapy regimens.279–284 A group from Munich has evaluated preoperative chemotherapy (four cycles of doxorubicin, ifosfamide, and etoposide) combined with

1.0

Figure 97-14 • Actuarial probability of overall survival for MAIDchemoradiotherapy and surgery versus control patients. The significant difference between the groups with respect to cause of death was distant metastasis (see text for details). (Reproduced with permission from Delaney TF, Spiro IJ, Suit HD, et al: Neoadjuvant chemotherapy and radiotherapy for large extremity soft-tissue sarcomas. Int J Radiat Oncol Biol Phys 2003;56: 1117.)

Probability of survival

0.8

0.6

0.4

P=0.0003

0.2

Control (N=48) MAID-ON (N=48)

0.0 0

20

40

60

80

100

120

Follow-up time (months) 48 48

38 45

30 37

26 16

17 6

8 5

Control MAID-ON

2031

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regional hyperthermia (RHT) followed by surgery and adjuvant treatment (same chemotherapy ± radiation). Median follow-up times were 58 months for the RHT-91 protocol (59 patients) and 30 months for the RHT-95 protocol.283 All patients had grade II/III tumors 5 cm or larger with extracompartmental extension. Clinical response rates were 42% and 33% for the two protocols, with respective local progression-free survival rates of 58% and 57%. Corresponding overall survival rates were 42% and 48%, respectively. These results are now being evaluated in a phase III RCT (EORTC 62961/ESHO RHT-95). The assumption has been made that preoperative chemotherapy with doxorubicin, ifosfamide, and etoposide is effective for patients with high-risk localized disease in this setting, and patients are being randomized to receive preoperative etoposide, ifosfamide, and doxorubicin (EIA, four cycles) plus RHT (two fractions) versus EIA chemotherapy alone.

TREATMENT OF SARCOMA PATIENTS AT SPECIALTY CENTERS Recent data on other tumor types have demonstrated improved outcomes for patients who required complex treatment and are treated at specialty centers.285 The most comprehensive data addressing this issue in STS come from Sweden, where Gustafson and colleagues analyzed the quality of treatment in a population-based series of 375 patients with primary STSs arising in the extremities (N = 329) or the trunk (N = 46).286 Comparisons were made between patients who were referred to a specialty soft-tissue tumor center before surgery (N = 195), those who were referred after surgery (N = 102), and those who were not referred for treatment of the primary tumor (N = 78). The total number of operations for the primary tumor was 1.4 times higher in the patients who were not referred and 1.7 times higher in the patients who were referred after surgery than in patients who were referred before surgery. Of greatest significance, however, was the finding that the local recurrence rate was 2.4 times higher in the patients who were not referred and 1.3 times higher in the patients who were referred after surgery than in patients who were referred to a specialty soft-tissue tumor center before any manipulation of their tumor. These findings support the principle of centralizing treatment of these rare tumors, which frequently require complex multimodality therapy.

TREATMENT OF LOCALLY RECURRENT SOFT-TISSUE SARCOMA Incidence of Local Recurrence Despite optimal multimodality therapy, at least 20% to 30% of STS patients will develop recurrent disease, with a median disease-free interval of 18 months.109,287 Not surprisingly, local recurrence rates are a function of the primary tumor site and are highest for retroperitoneal and head and neck sarcomas. This is due in part to the fact that adequate surgical margins are technically more difficult to attain in these locations. Indeed, while acknowledging our earlier discussion relating the type of positive margin,194 by multivariate analysis, an unqualified positive surgical margin is an adverse prognostic factor associated with local recurrence operation for recurrent disease.109,110 In addition, employment of conventional standard-dose postoperative radiotherapy (60 to 65 Gy) is often limited in the retroperitoneum and the head and neck by the relative radiosensitivity of surrounding structures. These factors result in local recurrence rates of 38% for high-grade retroperitoneal sarcomas133 and 48% for high-grade head and neck sarcomas,288 compared to 5% to 25% for extremity lesions (see Table 97-9). It should also be acknowledged, however, that the rarity of nonextremity lesions probably results in a more varied approach to management, which may influence the ultimate outcome and may also have influenced the ability to design

and execute clinical trials in the past. It would seem that properly applied principles of local management can achieve similar results even in sites with traditionally poor results, such as the head and neck, where disease access is limited by proximity to critical local anatomy.196

Surgery and Radiotherapy Locally recurrent STS generally presents as a nodular mass or series of nodules arising in the surgical scar or radiation port. Patients with retroperitoneal recurrences usually present with nonspecific symptoms, often after the recurrence has reached a substantial size. Treatment approaches for patients with locally recurrent STS need to be individualized on the basis of local anatomic constraints and the limitations on present treatment options imposed by prior therapies. In general all such patients should be evaluated for reresection of their local recurrence. The results of such “salvage surgery” are good, two thirds of patients surviving long-term.289,290 If no prior radiotherapy was employed, adjuvant radiation should be utilized after surgery for locally recurrent disease. Occasionally, subtherapeutic or low-dose radiation was previously employed, and such patients might be candidates for additional adjuvant radiation by external beam or brachytherapy approaches. Patients who have had a full course of prior radiation should be managed on an individual basis. In a recent series of 40 patients with recurrent extremity sarcoma, limb salvage was possible by combining limb-sparing re-resection with adjuvant brachytherapy.291 A median dose of 45 Gy was possible with this technique, despite the fact that most patients had received prior external beam radiation. The 5-year actuarial local control rate was 68%, with satisfactory limb preservation. However, brachytherapy should be used with caution in patients who have locally recurrent low-grade sarcomas, since it appears to be ineffective against low-grade sarcomas.193,220 Catton and colleagues from the Princess Margaret Hospital also employed conservative surgery and reirradiation (external beam or brachytherapy) for treatment of local recurrences arising in a previous radiation field in a subset of 10 extremity sarcoma patients.292 With a relatively short median follow-up of 24 months for the entire cohort, local control in the patients treated with further surgery and reirradiation was 100%.292 Similarly, Nori and colleagues at Memorial Sloan-Kettering Cancer Center and Pearlstone and colleagues at the M.D. Anderson Cancer Center reported a local control rate of 82.5% (33 of 40) and 65% (17 of 26), respectively, when using conservative surgery and reirradiation with brachytherapy.291,293 However, despite these encouraging findings, amputation or protocol-based hyperthermic isolated limb perfusion might be the only options for local control in some patients who were previously treated with radiation and have recurrent extremity sarcoma.

TREATMENT OF METASTATIC SOFT-TISSUE SARCOMA The most common site of metastasis from STS of the extremity is the lung. Indeed, the lungs are the only site of recurrence in approximately 20% of all patients with primary extremity and trunk STSs.287,284 Primary visceral and gastrointestinal sarcomas also commonly metastasize to the liver. Extrapulmonary metastases are uncommon forms of first metastasis and usually occur as a late manifestation of widely disseminated disease.287 An obvious exception is in myxoid liposarcoma, in which unpredictable and aberrant recurrences to any area containing fat (the pelvis, retroperitoneum, mediastinum, paraspinal and subcutaneous soft tissue, and bone marrow) are a hallmark of disease behavior.104,295 Evidence strongly suggests that apparently isolated soft-tissue masses that manifest in this disease are metastases, sharing the same molecular lineage with the original primary tumor.296 For sarcomas in general, the median

Sarcomas of Soft Tissue • CHAPTER 97

survival from the time of development of metastatic disease is 8 to 12 months. An obvious exception is alveolar soft part sarcoma, in which metastatic disease may persist for more than 10 years. Optimal treatment of patients with metastatic STS requires an understanding of the natural history of the disease and individualized selection of treatment options based on specific patient factors, disease factors, and limitations imposed by prior treatment.

Surgical Resection The current surgical approach for pulmonary metastases from STS is based on an extrapolation of the observations of Martini, Marcove, and colleagues in a series of patients with osteosarcoma treated at Memorial Sloan-Kettering Cancer Center in the 1960s. It had been observed that in a series of 184 patients undergoing amputation for osteosarcoma, 75% developed metastatic disease to the lungs within 18 months of amputation; and there were no 5-year survivors among this group.297 In the absence of any effective systemic therapy for this disease, efforts were made to resect such metastatic lesions in later patients. Martini and colleagues reported successful complete resection in 22 of 28 patients, with a substantial 5-year overall disease-free survival rate of 32%.298 Multiple investigators have since reported their experience with pulmonary metastasectomy for metastatic STS in adults.299–310 Threeyear overall disease-free survival rates after thoracotomy for pulmonary metastasectomy have ranged from 23% to 54%, as outlined in the selected series summarized in Table 97-12.299–303,310,311 With the exception of a study that evaluated the development and treatment of pulmonary metastases in patients with extremity sarcomas using a prospective sarcoma database (3-year survival rate of 23% after complete resection),303 most studies have been retrospective analyses of the results of pulmonary resection in populations of carefully selected patients with metastatic sarcoma from heterogeneous primary sites. This could account for some of the variability in the reported survival rates. Many investigators believe that repeated thoracotomies to render patients free of disease from pulmonary STS metastases are justified in the absence of effective systemic therapy. Several series of reoperative pulmonary metastasectomy have been published.312,313 In an NCI series, 72% of 43 patients could be rendered free of disease at the second thoracotomy, with a median survival duration from the time of the second thoracotomy of 25 months.312 In a report from the M.D. Anderson Cancer Center of a series of 34 patients undergoing reoperation for a second pulmonary metastasis after successful initial metastasectomy, factors predicting long-term survival included the presence of a solitary metastasis and the ability to perform a

complete resection.313 This study also illustrated the significant survival duration many of these patients enjoy: The median survival in the 19 patients who had unifocal recurrent metastatic disease was 65 months compared to 14 months in the 15 patients with complete resection of two or more sites of recurrent disease. It remains difficult to predict which patients will benefit from pulmonary resection. A number of different clinical criteria have been evaluated by univariate analysis, including the disease-free interval,299,301,311,314 number of metastatic nodules,311,314–317 and tumor doubling time.311,317,318 Multivariate analyses from both the NCI and Roswell Park Cancer Institute confirm that a short disease-free interval (as a surrogate endpoint of adverse behavior) and incomplete pulmonary resection are adverse prognostic factors for survival of patients with pulmonary metastases.301,319 A multivariate analysis from the M.D. Anderson Cancer Center suggested that, in addition, the presence of more than three metastatic pulmonary nodules on preoperative chest CT is an adverse prognostic sign.301 The ability to completely resect all pulmonary disease is perhaps the most important prognostic factor affecting survival after pulmonary metastasectomy; patients with residual pulmonary disease have a median survival of 9 months versus 27 months (P < 0.0001) for patients who are rendered completely free of disease at thoracotomy.301,303 In a series of 65 patients with metastatic pulmonary lesions from extremity sarcoma from the Memorial Sloan-Kettering Cancer Center, the median survival after complete resection was 19 months versus 10 months for patients who had incomplete resections and 8 months for patients who did not undergo surgery (P = 0.005).303 The 3-year overall survival rate after complete resection was 23% compared to 2% in those who were treated nonsurgically (P < 0.001). The clinical criteria of disease-free interval, tumor doubling time, and number of nodules can serve as general prognostic indicators in patients who are being considered for pulmonary metastasectomy, but no single criterion should be used to exclude patients from surgery. The ability to achieve complete resection and the number of pulmonary nodules that are present appear to best define the prognosis for patients postoperatively. Although carefully selected patients may benefit from surgical resection of pulmonary metastases, this treatment approach is feasible in only a small fraction of patients who develop pulmonary metastases. This is best illustrated by data from Memorial Sloan-Kettering, where a population of 716 patients with primary extremity sarcoma were followed for the subsequent development and treatment of pulmonary metastases (Fig. 97-15). Of the initial cohort, 148 patients (21%) developed pulmonary metastases. Isolated pulmonary metastases occurred in 135 (91%) of these 148 patients. Of the 135 patients with pulmonary-only metastases, 78

Table 97-12 Survival Following Complete Resection of Pulmonary Metastases from Soft-Tissue Sarcoma in Adults NO. OF PATIENTS First Author/ Institution

Total

Pulmonary Metastases

Surgical Treatment

Creagan/Mayo299

112

112

112

64 (57%)

18

29

Putnam/NCI300

487

93

68

51 (75%)

23

32

Jablons/NCI301

74

57

57

49 (86%)

27

35

Casson/MDACC302

68

68

68

58 (85%)

25

42

Verazin/Roswell319

78

78

78

61 (78%)

21

21.5, (5 yr)

716

135

78

65 (83%)

19

23

255

255

255

255 (100%)

NR

54

Gadd/MSKCC303 310

Van Geel/EORTC

Complete Resection (%)

Median Survival (Mo)

3-Year Survival (%)

EORTC, European Organization for Research and Treatment of Cancer; Mayo, Mayo Clinic; MDACC, University of Texas M.D. Anderson Cancer Center; MSKCC, Memorial SloanKettering Cancer Center; NCI, U.S. National Cancer Institute; Roswell, Roswell Park Cancer Institute.

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Chemotherapy Admitted 716

First-Line Chemotherapy SINGLE AGENTS. The single-agent activity of doxorubicin

Pulmonary metastases 148 (20%)

Pulmonary metastases only: 135 (19%)

Operation 78 (58%)

Complete resection 65 (83%)

3-year survival 15/65 (23%)

3-year survival 15/135 (11%)

Figure 97-15 • Risk for and subsequent management of pulmonary metastases in 716 patients with primary or locally recurrent extremity softtissue sarcoma. (Reprinted with permission from Brennan MF: The surgeon as a leader in cancer care: lessons learned from the study of soft tissue sarcoma. J Am Coll Surg 1996;182:520.)

(58%) were considered to have operable disease, and 65 (83%) of those who were taken to thoracotomy were able to undergo complete resection of all their pulmonary metastatic disease. This group represents only 44% of patients with pulmonary metastases. Median survival from the time of complete resection was 19 months, and the 3-year overall survival rate was 23%. All patients who did not undergo thoracotomy died within 3 years. For the entire cohort of 135 patients who developed pulmonary-only metastases, the 3-year overall survival rate was only 11% (see Fig. 97-15). The rather disappointing overall results of treatment for pulmonary metastases underscore the importance of careful patient selection for resection of pulmonary metastases. The following criteria are generally agreed upon: (1) the primary tumor is controlled or is controllable; (2) there is no extrathoracic disease; (3) the patient is a medical candidate for thoracotomy and pulmonary resection; and (4) complete resection of all disease appears possible.320 With careful patient selection, the morbidity of thoracotomy can be limited to the subset of patients who are most likely to benefit from this aggressive treatment approach. Wedge excision with negative margins is the procedure of choice for patients who undergo surgery for isolated pulmonary metastases. Formal segmentectomy, lobectomy, and mediastinal lymph node dissection are not necessary and do not contribute to improved local control. Occasionally, lobectomy or even pneumonectomy is required because of the proximity of the metastatic lesion to a pulmonary artery, vein, or major bronchus. Multiple ipsilateral lesions do not represent a contraindication, nor, in fact, do bilateral pulmonary metastases. Bilateral lesions can be approached by staged thoracotomies, median sternotomy, or simultaneous bilateral anterior thoracotomies, depending on the surgeon’s preference and the number and location of the pulmonary metastases. In such cases, the preferred approach for most thoracic surgeons is simultaneous bilateral wedge resections via a sternotomy or “clamshell” anterior thoracotomy. Isolated unilateral lesions may be amenable to thoracoscopic resection.

against metastatic STS is well established, with response rates usually reported as being in the range of 20% to 30%,321–323 but response rates as low as 10%324 and as high as 41%325 have also been noted. The dose-response curve for doxorubicin in sarcomas was demonstrated to be steeper than that for any other tumor in the first RCT to address a dose-response question; the response rate increased from 18% at 45 mg/m2 to 37% at 75 mg/m2 (see later discussion).326 Epirubicin, which was developed as an active but minimally less cardiotoxic analog of doxorubicin, produced an objective response rate slightly lower than that of doxorubicin (18% versus 25%, P = 0.33) in an EORTC RCT of 167 patients receiving equimolar doses (75 mg/m2) of the drugs.327 There is some evidence of a doseresponse relationship with epirubicin as with doxorubicin; a doseescalation study showed response rates of 17%, 44%, and 100% for 140 mg/m2, 160 mg/m2, and 180 mg/m2 of epirubicin, respectively.328 Only three patients were entered at the maximum tolerated dose of 180 mg/m2, and 160 mg/m2 was recommended for routine clinical use. However, the EORTC group, in a three-arm RCT of 334 patients, was unable to demonstrate any benefit from either of two schedules of epirubicin (150 mg/m2) compared with doxorubicin (75 mg/m2); all regimens produced response rates of 14% to 15%.329 Furthermore, there was considerably more myelosuppression in the two epirubicin arms, with two toxic deaths. Nevertheless, particularly in Europe, epirubicin has commonly been substituted for doxorubicin in high-dose regimens. A number of studies of liposomal anthracyclines have suggested that these agents have lower rates of cardiotoxicity but variable activity.330–334 An EORTC phase II RCT 324 demonstrated low activity for both doxorubicin and liposomal doxorubicin (Doxil)—9% versus 10%—but different spectrums of toxicity, with less myelosuppression but with palmar-plantar erythrodysthesia (grade 3: 20%) as the dose-limiting toxicity in the Doxil arm. Lack of response rather than cardiotoxicity is the main limiting factor for anthracycline use in palliative chemotherapy of STS. Nonetheless, strategies to reduce anthracycline toxicity are of particular importance in the adjuvant setting.335 Many American investigators routinely use continuous-infusion regimens with doxorubicin, which have been shown to be equally effective but less cardiotoxic. 336–338 Dexrazoxane is undoubtedly cardioprotective when given with doxorubicin.339 Concerns about the possibility of tumor protection are theoretical and have not been noted in ongoing studies. After the reports of several randomized studies documenting activity ranging from 24% to 67%,340–342 Bramwell and colleagues performed an RCT comparing ifosfamide (5 g/m2 by 24-hour infusion) with cyclophosphamide (1.5 g/m2).342 Respective response rates were 18% and 8%, but the difference was not statistically significant, owing to the inadequate power of the study (P = 0.13). Nonetheless, the authors correctly concluded that ifosfamide was the more active drug, and all sarcoma medical oncologists agree with that assessment. Indirect data from several RCTs in which ifosfamide and/or cyclophosphamide were added to doxorubicin have provided additional evidence that ifosfamide is a more active analog than is cyclophosphamide.321 Questions about the optimal scheduling of ifosfamide (multiple daily bolus doses versus continuous infusion) have never been satisfactorily resolved and are confounded by dose differences in many studies. Two consecutive phase II studies by investigators at M.D. Anderson evaluated ifosfamide (14 g/m2) given as a 72-hour continuous infusion or a 2-hour infusion for 3 consecutive days. Respective response rates were 19% and 42%.343 In an EORTC RCT comparing 5 g/m2 ifosfamide over 24 hours with 3 g/m2 ifosfamide over 4 hours on days 1 to 3, response rates were 3% and 17.5%, respectively. However, a subsequent EORTC study showed no dif-

Sarcomas of Soft Tissue • CHAPTER 97

ference in response rates for 9 g/m2 ifosfamide by continuous infusion or intermittent bolus injection344 (see the section on high-dose ifosfamide).

COMBINATION CHEMOTHERAPY. In the 1970s and early 1980s, before the widespread availability of ifosfamide, most combination chemotherapy regimens were based on doxorubicin and dacarbazine. The addition of cyclophosphamide and vincristine, which are active against childhood sarcomas, created a regimen called CyVADIC, for which the Southwest Oncology Group reported response rates as high as 59% in patients with metastatic disease.345 Later investigators were unable to reproduce such high response rates with the same regimen, however, and summary data on variants of the CyVADIC regimen revealed an overall response rate of 35% in 2092 patients.346 CyVADIC was the control for the three-arm EORTC study discussed later.333 Despite the authors’ conclusions that singe-agent doxorubicin should remain the standard, CyVADIC had the highest response rate (not statistically significant), with significantly lower toxicity than either of the other arms of the trial. Most regimens now used for first-line chemotherapy are based on the combination of doxorubicin and ifosfamide. A recent systematic search of the literature347 found 3 phase III RCTs and 16 phase II trials (excluding phase I studies and those recruiting fewer than 25 patients) in adult STS that used combination regimens including an anthracycline and ifosfamide. Although the response rate varied widely, from 25% to 56%, in the phase II studies, they were at the lower end of this range in the three RCTs.348–350 In the Eastern Cooperative Oncology Group (ECOG) study332 of 178 patients, the response rate was significantly higher for doxorubicin/ifosfamide than for doxorubicin alone (34% versus 20%, P = 0.03), although median survivals were similar. In an EORTC study of 471 patients, however, there were no significant differences in response rate (28% versus 23%) or median survival for doxorubicin/ifosfamide versus doxorubicin alone.349 In an intergroup trial of 340 patients, the ifosfamidecontaining regimen MAID was shown to produce a significantly higher response rate (32% versus 17%, P < 0.002) than doxorubicin/dacarbazine, but with no overall survival benefit.350 Bramwell and colleagues performed a meta-analysis of eight RCTs that compared doxorubicin-based combinations with single-agent doxorubicin.322 In these eight studies with a total of 2281 patients, ten combination regimens were evaluated; five included ifosfamide (two) or dacarbazine (three), and the remaining five used other drugs with low known single-agent activity. There were no significant benefits in terms of response rate (odds ratio: 0.79, P = 0.10) or overall survival (odds ratio: 0.84, P = 0.13) for combination chemotherapy. Inclusion of a small RCT (106 patients)351 that compared epirubicin (180 mg/m2) with epirubicin (180 mg/m2) plus cisplatin (120 mg/ m2) and reported respective response rates of 29% and 54% (P = 0.025) did not significantly alter the meta-analysis results. On the basis of single-agent doxorubicin’s lower overall toxicity than that of combination regimens, Bramwell and colleagues suggested that for chemotherapy given with palliative intent, doxorubicin alone was a reasonable first-line option,322 a conclusion that was also reached in a commentary by Santoro.352 This leaves open the possibility of further second-line chemotherapy with ifosfamide in fit patients progressing on or relapsing after a response to doxorubicin therapy. Whether patients with advanced sarcomas should be treated with chemotherapy at all is also a topic of debate in Europe.353 Interestingly, the European investigators who debate the issue place such patients on clinical trials, and the majority, if not all, of European experts who are members of the Connective Tissue Oncology Society treat patients both on trials and as standard practice. One reason for the lack of convincing evidence that standard-dose combination chemotherapy improves outcomes compared with single-agent doxorubicin for metastatic STS might be that the doses of the drugs in combination regimens are often reduced below optimum levels to limit toxicity. In the first RCT of different doses

of doxorubicin, a steep dose-response relationship was demonstrated for doxorubicin in sarcomas, with an increase in response rate from 18% at 45 mg/m2 to 37% at 75 mg/m2.326 A similar dose-response relationship has been suggested for ifosfamide, the response rate increasing from 8 to 22% with an increase from 6 to 10 g/m2 in consecutive series.354 Myelosuppression, particularly neutropenia, limits the doses of these drugs that can be safely delivered in combination. Use of hematopoietic growth factors or autologous stem cell transplantation permits substantial dose escalation of these agents. In a systematic review, Verma and Bramwell identified seven phase I trials, five phase II trials, and two RCTs exploring doseescalated regimens of doxorubicin and ifosfamide, with and without other agents for metastatic STS.347 Most regimens included the growth factors G-CSF or GM-CSF, but in two trials, autologous stem cell transplantation was used in addition. In the phase I studies, the maximum tolerated doses were in the range of a twofold increase of the doxorubicin or epirubicin dose (over a standard dose of 75 mg/m2) and a 2.4- to 2.8-fold increase of the ifosfamide dose (over a standard dose of 5 g/m2). Significant toxicities included anemia, thrombocytopenia, nephrotoxicity, and neurotoxicity; severe neutropenia and febrile neutropenia were also seen at the higher doses. In a recently reported study,355 De Pas and colleagues reported no nephrotoxicity or neurotoxicity with ifosfamide infused over 12 days at 15 g/m2 (given with doxorubicin, 75 mg/m2), although myelosuppression was dose limiting. Although not the primary objective, response rates were reported for all these phase I studies and were in the range of 28% to 58%. Additionally, for the five phase II studies of dose-escalated doxorubicin and ifosfamide (only studies with more than 20 evaluable patients were included), response rates were 31% to 65%. In a phase II study that was not included in the review, a response rate of 40% in 70 patients was documented.356 Patel and colleagues were the first to maximize the doses of each drug used in the combination.338 In a series of small studies utilizing growth factors to support patients through the intense expected myelosuppression, they delivered doxorubicin at doses of 75 to 90 mg/m2 together with ifosfamide at 10 g/m2 divided as 2- to 3-hour infusions daily for 4 to 5 days. Of note, they identified that the regimen was suitable only for patients younger than 65 years old with two kidneys, normal renal function, and good performance status. Dose reductions were not employed for myelosuppressive toxicity unless it was accompanied by extreme morbidity. Patel reported a 62% response rate overall with a 57% response rate, a 10-month progression-free survival, and a 20-month survival in those with metastatic disease. The EORTC is attempting to confirm the results of the 75/10 regimen in a randomized study versus single-agent doxorubicin. Patel and colleagues attributed their good results to the doseintensive chemotherapy. In contrast, Worden and colleagues randomized patients to doxorubicin at 60 mg/m2 plus either 6 or 12 g/m2 of ifosfamide and showed no advantage for the higher dose.357 Careful review of that study reveals that the careful selection criteria that Patel and colleagues used were not used in the latter study. Several older patients were entered, and in fact, the disadvantage of the higher dose regimen might have been due to unacceptable toxicity. In contrast to these promising results in phase I/II studies, response data in the cooperative group RCTs have been disappointing. In an EORTC trial involving 314 patients with metastatic STS, standarddose doxorubicin (50 mg/m2) plus ifosfamide (5 g/m2) was compared with higher-dose doxorubicin (75 mg/m2) with the same dose of ifosfamide plus GM-CSF.358 Respective response rates were 21% and 23%, with similar median overall survivals (56 versus 55 weeks); but median progression-free survival was significantly longer in the highdose arm (19 versus 29 weeks, P = 0.03). In the other RCT that was included in Verma and Bramwell’s review, as yet reported only in abstract form,359 162 patients were randomized to receive

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standard-dose MAID or MAID with doses escalated 25% plus GCSF support. Respective response rates were 37% versus 43% (P not significant), but no survival data were reported. There were five toxicity-related deaths, however, all in the dose-escalated MAID arm. In a third RCT, 180 mg/m2 epirubicin and 150 mg/m2 epirubicin, each combined with cisplatin but with no growth factor support, were compared in 151 patients.351 There was a higher response rate (51% versus 28%, P = 0.004) and a marginal effect on overall survival (P = 0.06) with the higher-dose regimen. As all three RCTs were quite limited tests of dose escalation, more RCTs are needed before it can be concluded that such regimens are a more effective option than conventional-dose chemotherapy. Verma and Bramwell discuss possible reasons for the lack of a clear benefit of dose-escalated therapy in the RCTs;347 these include tumor heterogeneity, difficulties in eradicating large tumor burdens, and the appropriateness of the drugs, doses, and schedules used. It is interesting that even within the same group, results may differ between phase II and phase III trials. When the EORTC performed a pilot study of doxorubicin 75 mg/m2 + ifosfamide 5 g/m2 with GM-CSF support, the response rate was 45%.360 When the group repeated the study as a phase III study, the response rate was 23%.358 Why is there a discrepancy, and which value is closer to the truth? Phase III studies are supposed to be more representative of the overall population than are studies of highly selected patients from referral centers. Is it not possible, on the other hand, that the pilot study was conducted by a group that was more expert at treating sarcomas? Is it not possible that negative selection bias goes into the phase III trials, that is, investigators treat their younger, potentially curable patients with combination therapy off study and enter only those who are less likely to benefit into the randomized phase III studies? We know that referral to a specialty center improves outcomes of early-stage patients; why not those with metastatic disease? Furthermore, all of the randomized studies that have been reported occurred before there was effective treatment of GISTs. Many GIST patients were entered in front-line chemotherapy studies as having metastatic leiomyosarcoma without attention to its primary site. Inclusion of such patients, especially when they are rarely accounted for in the publications, dilutes the data and makes much of it uninterpretable.

Second-Line Chemotherapy HIGH-DOSE IFOSFAMIDE. Although ifosfamide is often used as a first-line agent, it is clearly active as second-line therapy in patients who are progressing or relapsing after doxorubicin-based regimens. Early studies of ifosfamide suggested that there was a dose-response relationship,354 and several groups have documented responses to high-dose ifosfamide in patients who had not responded to lower doses of the drug.337,361–363 Nevertheless, dose-escalation studies of ifosfamide have produced conflicting results. Doses of 12 g/m2 without and 14 to 18 g/m2 with growth factor support seem achievable and have produced response rates of 33% to 45%, but nephrotoxicity and neurotoxicity are considerable.343,359,363 Frustaci and colleagues found high-dose ifosfamide to be well tolerated when infused at 1 g/m2/day over 21 days.364 In 36 patients, they were able to administer up to three cycles with a median duration of 15 days, producing a response rate of 24%. Myelosuppression was dose limiting, but there was no significant nephrotoxicity or neurotoxicity. Pharmacokinetic data reported by Cerny and colleagues demonstrated that ifosfamide doses greater than 14 to 16 g/m2 given over 5 days resulted in a relative decrease of the active metabolite iphosphoramide mustard, suggesting dose-dependent saturation or inhibition of ifosfamide metabolism.365 Despite encouraging phase II trial results, the advantages of increasing the dose of ifosfamide are far from clear on the basis of recent EORTC studies. The response rate rates for 9 g/m2 (3 g/m2 over 4 hours, days 1 to 3) and 5 g/m2 (24-hour continuous IV) were 3% and 17.5%, respectively, in an RCT of 101 patients.366 Escalation

to 12 g/m2 as a 3-day continuous IV infusion produced a response rate of 16% in a phase II study of 124 patients.367 In the most recent EORTC RCT of first-line chemotherapy, the response rates were 11%, 6.5%, and 9.4% for 75 mg/m2 doxorubicin, 3 g/m2 ifosfamide over 4 hours on days 1 to 3, and 9 g/m2 ifosfamide 24-hour continuous infusion, respectively. In addition to these disappointingly low and similar response rates, there were no differences in progression-free survival between the three arms.344

OTHER MARKETED DRUGS ALONE OR IN COMBINATION. Collected response rate data for many drugs that were studied in phase II trials for metastatic STS have been published in a number of reviews.321,368,369 Dacarbazine has been used most extensively, either as a first-line agent in combination with doxorubicin and ifosfamide (MAID) or as a second-line salvage treatment. Demetri and colleagues found an overall 16% response rate for dacarbazine in 109 patients from collected phase II studies.321 Although dacarbazine is commonly given in divided doses over 3 to 5 days, Buesa and colleagues showed that doses of 1.2 g/m2 over 20 minutes are feasible and active, as well as more convenient, now that effective antiemetics are available.370 The current availability of portable infusion pumps means that prolonged infusions are also feasible. Although Rosen and colleagues reported a response rate of 27% lasting 2 to 18 months or longer, Reichardt and colleagues were not able to confirm this high rate of activity when they gave 12- to 14-day infusions of 200 to 225 mg/m2/day dacarbazine, observing only disease stabilization in 8 of 25 heavily pretreated patients.371 There is conflicting evidence on the activity of cisplatin and carboplatin against metastatic STS, although most reviews have reported response rates of less than 15%.321,368,369 Low response rates are also seen for etoposide given as a single agent.321,342,372 Promising early data on docetaxel373 could not be reproduced in later studies,352,374–377 and paclitaxel similarly has little activity.378–380 Objective response rates were also very low (3% to 5%) in three phase II studies of gemcitabine,381–383 although the M.D. Anderson group described a response rate of 18% in 39 patients if GISTs were excluded378 (they represented the majority of patients in some of the other studies). The M.D. Anderson study also confirmed the importance of timed delivery of gemcitabine, since it cannot be activated faster than 10 mg/m2/min and the remainder of a more rapidly administered dose is simply excreted unchanged in the urine. Hensley and colleagues utilized timed infusion of gemcitabine at 900 mg/m2 over 90 minutes on days 1 and 8 followed by docetaxel 100 mg/m2 on day 8 with growth factor support and a 25% dose reduction for patients with prior pelvic radiation in a series of patients with leiomyosarcoma, almost exclusively of uterine origin, and observed a 53% response rate.384 Leu and colleagues confirmed the activity of the regimen at the lower dose in patients with a variety of other sarcomas.385 Since the response rate among patients with leiomyosarcoma in the M.D. Anderson study of gemcitabine was 4 out of 10, it was unclear whether the activity of the gemcitabine-docetaxel regimen was due to the timed infusion of gemcitabine, the large number of patients with uterine leiomyosarcoma, or the addition of docetaxel. To answer this question, SARC (the Sarcoma Alliance for Research through Collaboration) carried out a RCT comparing equimyelosuppressive doses of timed-infusion gemcitabine versus the combination of gemcitabine and docetaxel. The study employed a Bayesian adaptive randomization design with continuous feedback of data into the randomization model so that more patients were entered in the arm with greater efficacy.386 Success was defined as a RECIST response at any time or freedom from progressive disease at 24 weeks, based on the assessment of the investigators that prolonged freedom from progression was more beneficial to patients than response. Results reported by Maki and colleagues at ASCO 2006 showed a statistically significant superiority of the combination regimen in terms of success rate, progression-free survival, and overall survival.387

Sarcomas of Soft Tissue • CHAPTER 97

Despite poor levels of activity as single agents, some of the preceding drugs have been incorporated into nonanthracycline-based salvage regimens. On the basis of encouraging data in pediatric sarcomas, etoposide has been combined with ifosfamide, although with variable results.377,388–391 All but one such study produced response rate in the range of 38% to 46%; because ifosfamide given alone has produced up to 67% in phase II studies, however, these results are difficult to interpret. A combination of paclitaxel with gemcitabine and cisplatin was evaluated, with preliminary reports of encouraging synergistic activity.392 Temozolomide,393–395 raltitrexed,396 irinotecan,397 sargramostim,398 topotecan,399,400 and vinorelbine401 seem to have minimal activity in STS, despite their proven value in other tumor types.

INVESTIGATIONAL NEW DRUGS. Of drugs that are currently in phase II development, trabectidin (ET743, ecteinascidin 743), a DNA guanine-specific minor groove-binding agent, seems to have clear-cut activity against sarcomas. Hints of activity in bone and STSs were observed in phase I trials402,403 and appeared to be confirmed in phase II trials of this agent. Garcia-Carbonero and colleagues reported a response rate of 17.1% in 35 chemonaive sarcoma patients and 8% in 34 patients who had received prior chemotherapy.405 George and colleagues reported a lower progression rate (5%) but a substantial proportion (19%) of patients with minor responses or stable disease.406 Two European trials described response rates of 11% to 12% in previously treated sarcoma patients.407,408 Occasional severe toxicities, sometimes lethal, seemed to be related to elevated baseline liver function tests; and with careful attention to alkaline phosphatase levels, toxicity other than myelosuppression and fatigue has not been a major problem. In the attempt to avoid the inconvenience of a continuous-infusion schedule, weekly administration has been evaluated. In a randomized study reported at ASCO 2004, the continuous infusion was more toxic, but it was significantly more effective in terms of time to progression and survival.409 This study indicates that the drug is clearly active and that either the continuous-infusion schedule is superior or the dose that was chosen for the weekly schedule was too low. Another interesting observation from the various studies of trabectidin is that it has high activity against a single subtype of sarcomas, myxoid liposarcoma, made by Grosso and colleagues at the Istituto Nazionale Tumori in Milan.410 They noted a response rate of 43% by RECIST and an even higher response rate when changes in tumor density were considered—a so-called tissue response. We and others have verified their observation (unpublished observation), and it is clear that the responsiveness of myxoid liposarcoma to trabectidin is very high. It is a subtype that is rather responsive to doxorubicin plus ifosfamide as well, but the second-line (and sometimes fifth-line) activity of trabectidin is truly remarkable. Myxoid liposarcoma is characterized by a specific translocation putting either FUS or less commonly EWS with CHOP. It is possible that trabectidin specifically blocks transcription of the fusion DNA. It is also evident that trabectidin is more active against tumors with altered DNA repair mechanism. Tumors with low expression of BRCA1 and high expression of ERCC1 have substantially longer progression-free survival than do those with high expression of BRCA1 and low expression of ERCC1.411 Whether the altered DNA repair is a specific feature of myxoid liposarcoma or other translocation-related sarcomas or whether the altered DNA repair is an independent predictor of response is to be determined. Another class of drugs with activity against sarcomas are the mTOR inhibitors, the best studied of which is an investigational agent AP23573 from a small company, ARIAD. Data reported at ASCO by Chawla and colleagues showed an objective benefit rate, defined as RECIST response or stable disease at 16 weeks, of 28% in 193 patients.412 At the same time, anecdotal responses to rapamycin were reported,413 but a phase II study of an alternative mTOR inhibitor414 showed no activity when the endpoint was only RECIST response. Note that if only RECIST response were counted for

AP23573, it would have been declared inactive with a response rate of 2.6%.412 The observation that patients with growing tumor may benefit from therapy when the growth stops is not new, but its importance is becoming increasingly recognized. Initially described in a phase II study of doxorubicin as improvement415 that included both responses that were inadequate to qualify by standard criteria and stabilization of previously progressive disease, improvement has traditionally been accepted as a “second-class” response at best. Trabectidin is the first modern drug for which the clinical observation seemed to take on added meaning. The EORTC has defined rates of time to progression at 3 and 6 months for active and inactive regimens.416 Clinicians who treat osteosarcoma have known that standard responses might not exist despite a complete histologic response. The experience with GIST (see later discussion) has indicated that many patients who are clearly benefiting from therapy and have objectively measurable responses do not qualify as responders by RECIST or World Health Organization criteria. Therefore, the activity of gemcitabine-docetaxel that led to improved progression-free and overall survival, the activity of trabectidin, and the extent of the activity of imatinib in GIST were discovered by using modified response criteria. It make sense to look at new drugs such as the mTOR inhibitors using similar expanded criteria for drug activity and to pursue their study. The identification of a specific molecular target (the tyrosine kinase receptor KIT) in a rare type of STS (GIST) and successful treatment with a drug (imatinib) that inhibits that target provide a model for future drug development in STS. Although it is unlikely that the pathogenesis of most STS will prove to be driven by a single genetic mutation, better molecular differentiation of STSs into categories with similar molecular characteristics could facilitate future studies of highly targeted drugs. At an NCI-sponsored “State of the Science” meeting on STS, Demetri pointed out that the ideal target would meet four conditions: (1) a single validated molecule that is critical to STS pathogenesis in humans, (2) expressed and active, (3) a target for which there are no alternative pathways to bypass the blockade, and (4) necessary and sufficient for sarcoma survival.238 Other potential targets are discussed elsewhere in this chapter. Complex sarcomas with diverse karyotypes and/or drug-resistance mutations are likely to require drugs used in combination to block multiple targets. Recent reviews have described signal transduction pathways in sarcoma as therapeutic targets,417 the potential use of antiangiogenesis agents,418 and new approaches to immunotherapy.419 Early reports of antiangiogenesis therapies have shown limited benefit in STS,420,421 but many agents remain to be evaluated. Although the majority of patients with metastatic STS will not have access to phase II studies of investigational agents, where these are available, trial entry should be encouraged. Conventional wisdom suggests that use of an investigational agent as a first- or second-line therapy for metastatic disease should be considered, since investigational agents that are given as third- or fourth-line treatments could be doomed to failure because of acquired drug resistance. This concept has now been proven incorrect. Whenever there is an effective agent or regimen (gemcitabine-docetaxel, imatinib in GIST, ifosfamide in synovial sarcoma, trabectidin in myxoid liposarcomas), activity is obvious in multiply-treated patients.

UNIQUE ROUTES OF DELIVERY. Some experimental studies have evaluated intraperitoneal delivery of cytotoxic agents, usually doxorubicin and cisplatin, sometimes with hyperthermia, in sarcomas that are confined to the peritoneal cavity after resection of all visible abdominal disease.368,422,423 Evaluation of any benefit is a major challenge in these studies, however, and this technique may be less suitable for sarcomas than for epithelial cancers. Another novel approach is isolated lung perfusion with doxorubicin after resection of pulmonary metastases, in general a more common situation in STSs. To date, studies of isolated pulmonary perfusion have focused on feasibility rather than outcomes.424

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SPECIAL SITES AND SUBTYPES OF SARCOMA Retroperitoneal Sarcomas Retroperitoneal sarcomas are relatively uncommon, accounting for approximately 15% of all sarcomas (see Fig. 97-1). The most common histologic subtypes are well-differentiated and dedifferentiated liposarcoma and leiomyosarcoma, found in 42% and 26% of cases, respectively (see Fig. 97-1). Nearly 80% of patients present with an abdominal mass, and 50% of patients report pain at the time of presentation.189 Patients commonly describe nonspecific gastrointestinal symptoms. Other commonly noted symptoms include neurologic symptoms (primarily sensory) in 27% of patients and weight loss in 7%.189,425 These tumors often grow to substantial size before a patient’s nonspecific complaints are evaluated or an abdominal mass is noted on physical examination. CT and MRI are the primary methods that are used to image retroperitoneal tumors (see Figs. 97-3, 97-5, and 97-6).426–428 These modalities allow assessment of the consistency of the mass (fat, cystic or solid components, associated necrosis), the precise anatomic location of the mass, and the extent of any regional disease, and they confirm function of the contralateral kidney. CT of the abdomen and pelvis usually provide images that are satisfactory for treatment planning. Occasionally, MRI with gradient sequence imaging can be helpful in defining long-segment vascular anatomy for surgical planning. For patients with an abnormal chest radiograph, chest CT should be performed to exclude the possibility of metastatic disease. The differential diagnosis for a retroperitoneal mass is relatively limited. Physical examination should include a testicular examination in men to evaluate the possibility of a primary testicular neoplasm. Laboratory tests should include the common serum markers for germ cell tumors, beta-human chorionic gonadotropin, and α-fetoprotein. If physical examination is suggestive of testicular malignancy or biochemical markers are elevated, testicular ultrasonography should be performed. This may obviate laparotomy for patients with metastatic testicular tumors and allow identification of primary retroperitoneal germ cell tumors. In general, preoperative biopsy is not necessary when radiographic appearance makes the diagnosis of well-differentiated liposarcoma (atypical lipomatous tumor) and surgical resection is planned for a resectable primary retroperitoneal mass. If there is a question of diagnosis (lymphoma) or if preoperative chemotherapy or radiation is considered, a core-needle biopsy is essential. Surgical resection with negative margins remains the standard primary treatment for patients with localized retroperitoneal sarcoma. Unfortunately, the data are extremely difficult to interpret because most series lump all patients, the majority or whom may have atypical lipomatous tumors (also called well-differentiated liposarcoma)

with patients with true high-grade sarcomas. Patients should be thoroughly evaluated with multidisciplinary input before there is any attempt at surgery. If ifosfamide might be required at some point in the patient’s management, for example, and if adequate surgery would remove a kidney, strong consideration should be given to preoperative chemotherapy. Similarly, if radiation will be required, it is preferably given preoperatively (see later discussion). If primary surgery is chosen for the patient, all patients should have preoperative bowel preparation and assessment of bilateral renal function by CT, because en bloc multiorgan resection might be required to achieve negative margins. Resectability rates in recent series combining patients with primary and recurrent lesions have ranged from 25% to 96% (Table 97-13).189,425,429–434 Resectability rates at different institutions are difficult to compare and interpret because these rates are a function of the referral pattern, the criteria that are used to determine which patients will undergo surgical exploration, and the surgeons’ skill and experience.433,435,436 For patients with primary lesions, grossly complete resection is possible in up to 78% of cases.133,433 The most common reasons for unresectability are the presence of major vascular involvement (aorta or vena cava), peritoneal implants, or distant metastases.189 Resection of adjacent retroperitoneal or intraabdominal organs, frequently the kidney, colon, or pancreas, is required in 50% to 80% of cases to permit complete resection.189,431,437 Partial resections or debulking procedures have been performed, but there is no evidence that partial resection improves survival (Fig. 9716).189,437 Deliberate partial resection should be reserved for relief of bowel obstruction or palliation of other critical manifestations of advanced disease. Results from published series demonstrate 5-year overall survival rates in the range of 54% to 64% for patients with completely resected retroperitoneal sarcoma.133,189,431,433,434 Overall survival rates for patients with incompletely resected disease range from 10% to 36%. Adequate margins are often difficult to obtain in retroperitoneal sarcoma surgery because of the proximity of critical organs, vascular structures, and the spine. Consequently, recurrent disease remains a significant problem, recurrence developing alone or with systemic relapse in 46% to 59% of patients with completely resected tumors.134,189,431,433,438 A number of recent studies have evaluated prognostic factors for retroperitoneal sarcomas by univariate and multivariate analysis.133,134,425,431,438 For patients who presented without metastatic disease, complete surgical resection and histologic grade were the primary determinants of survival in several multivariate analyses.133,425,431,434,438 Some investigators have also found by multivariate analysis that large tumor size (>10 cm) and fixation to adjacent retroperitoneal structures other than neurovascular bundles or bone were significant adverse factors for survival.431 Patients who undergo a grossly complete resection have a 60% 5-year overall survival rate and a median survival of 64 months (see Fig. 97-16). These results

Table 97-13 Resectability Rates for Retroperitoneal Sarcomas in Selected Series Accrual Period (Yrs)

Total No. of Patients

No. Completely Resected

Restability Rate (%)

NCI

19

50

37

74

Roswell Park

24

68

27

40

19

116

63

54

5

114

67

59

First Author

Institution

Glenn429 Karakousis430 431

Dalton

Mayo Clinic

Jaques189

MSKCC

Alvarenga425

Royal Marsden

20

110

28

25

Karakousis433

Roswell Park

17

87

83

95

University of Florida

25

63

49

78

434

Kilkenny

MSKCC, Memorial Sloan-Kettering Cancer Center; NCI, U.S. National Cancer Institute.

Sarcomas of Soft Tissue • CHAPTER 97

1.0 CR versus PR (P70

197 (17.8)

9289 (16.9)

Female

537 (48.4)

26,970 (49.1)

Male

572 (51.6)

27, 969 (50.9)

962 (86.8)

43, 788 (79.7)

Sex

Ethnicity White Hispanic

78 (7.0)

6267 (11.4)

Black

48 (4.3)

3909 (7.1)

Other

21 (1.9)

975 (1.8)

Smoking history Smokers

608 (54.8)

N/A

Nonsmokers/unknown

501 (45.2)

N/A

N/A, not available. *Total number of patients referred with diagnosis of malignancy recorded from 1/1/87 to 6/30/95: 54,939.

chosen for study. For example, most recent investigators have excluded the well-characterized group of patients with metastatic squamous carcinomas to cervical lymph nodes.10–12 The specific histologic diagnoses identified in a series of 1109 consecutive patients with CUP are outlined in Table 98-2 and are contrasted with another reported series.13 The low frequency of squamous carcinoma reflects the direct referral of patients with squamous cell carcinoma involving high or midcervical lymph nodes to head and neck oncologists for management.

BIOLOGIC CHARACTERISTICS The biology of CUP has been partially characterized through the evaluation of patient subsets using as primary endpoints responsiveness to therapy and survival. When all patients are considered, CUP is a highly aggressive neoplasm with an overall median survival time of 3 to 4 months in older series.14 More recent studies have documented median survival times of 9 to 12 months.15–18 In our series of 1109 consecutive patients, the median survival period was 11 months. The survival curve for these patients is presented in Figure 98-1. The survival times for the four most commonly encountered pathologic subtypes are presented in Figure 98-2. The median survival times were as follows:

HISTOLOGIC PRESENTATIONS

• For patients with squamous carcinoma (exclusive of patients with mid-high cervical adenopathy), 24 months • For patients with adenocarcinoma, 9 months • For patients with carcinoma, 12 months • For patients with neuroendocrine carcinoma, 33 months.

The frequency with which specific histologic diagnoses are established in CUP depends to some extent on the patient population

The state of differentiation or mucin production did not appear to have a significant influence on the poor survival of patients with

Carcinoma of Unknown Primary • CHAPTER 98

Table 98-2 Histologic Types Identified in 1109 Consecutive Patients with Carcinoma of Unknown Primary HAINSWORTH ET AL13

M.D. ANDERSON CANCER CENTER Histologic Type Adenocarcinoma

No. of Patients*

Percentage of Total

No. of Patients†

Percentage of Total

646

58.3

—

31.8

Well differentiated

14

Moderately differentiated Poorly differentiated

45

—

220

70

46

—

Mucinous No descriptor/other

321

Carcinoma

—

317

Poorly differentiated

28.6

44.1

161

97

21

—

Undifferentiated Large cell

9

—

Small cell

14

—

No descriptor/other

112

—

Squamous

68

6.1

5

2.3

Neuroendocrine

48

4.3

25

11.4

Adenosquamous

7

0.6

0

0

23

2.1

23

10.4

Pathology not available for review/other *Total number of patients, 1109. + Total number of patients, 220.

adenocarcinoma (Fig. 98-3). Using univariate and multivariate analyses, various groups have assessed the influence of other clinicalpathologic features of CUP on survival (Table 98-3). Culine and associates19 also developed and validated a prognostic model to predict the length of survival in patients with CUP. Univariate and multivariate prognostic factor analyses were conducted in a population of 150 unselected patients and led to the construction of two successive classification schemes. When studying the clinical variables only, poor performance status and presence of liver metastases were retained in the multivariate analysis. The first classification scheme

consisted of three groups of patients with median survival times of 10.8, 6.0, and 2.4 months, according to the number of adverse prognostic factors. When serum lactate dehydrogenase (LDH) was introduced in a further step, liver metastases were no longer of significance. The second classification scheme, therefore, included performance status and elevated serum LDH. Good- and poor-risk patients were identified, with median survival times of 11.7 months and 3.9 months and 1-year survival rates of 45% and 11%, respectively. Validation of the second classification was obtained using an external data set; and the median survival times of patients assigned

1.00 1.00

0.75

0.50

P=0.0125

0.25

Survival probability

Survival probability

Uknown primaries, N=1,109, median=11 Primaries found, N=413, median=12

0.75

Adenocarcinoma, N=653, median=9 Carcinoma, N=317, median=12 Neuroendocrine carcinoma, N=48, median=33 Squamous carcinoma, N=68, median=24

1 vs. 2: P=0.0111 2 vs. 3: P=0.0008 2 vs. 4: P=0.0018 2 vs. 3: P=0.4836

0.50

0.25

0.00 10 20 30 40

50 60 70 80 90

Months UPT: Primaries found vs. unknown primaries

Figure 98-1 • Survival curves for 1109 patients with carcinoma of unknown primary (CUP) versus 413 patients referred with CUP in whom the primary cancer site was found.

0.00 10 20 30 40

50 60 70 80 90

Months UPC by histology

Figure 98-2 • Survival curves of the major histologic subtypes of carcinoma of unknown primary.

2059

Part III: Specific Malignancies 1.00

Survival probability

Poorly differentiated adenocarcinoma, N=220, median=9 All other adenocarcinoma, N=433, median=10

0.75

0.50 P=0.3923

0.25

0.00 10

20

30

40

50

60

70

80

90

Months UPC: Adenocarcinoma–influence of differentiation

A 1.00

Poorly differentiated carcinoma, N=161, median=12 All other carcinoma, N=156, median=11

Figure 98-3 • A, Influence of cellular differentiation on the survival of patients with unknown primary adenocarcinoma. B, Influence of cellular differentiation on the survival of patients with carcinoma of unknown primary.

0.75 Survival probability

0.50 P=0.5267

0.25

0.00 10

B

20

30

40

50

60

70

80

90

Months UPC: Carcinoma–influence of differentiation

to the good-risk group and poor-risk group were 12 months and 7 months with 1-year survival rates of 53% and 23%, respectively. This simple prognostic model using performance status and serum LDH allows assignment of patients into two subgroups with divergent outcomes. Additional prospective trials will be designed using this prognostic model. To assess the impact of disease extent on survival, the number of organ sites involved in the metastatic process was assessed at presentation in our series to provide a crude quantitation of tumor burden. For this analysis, involvement of an organ, even when there were multiple individual metastases within the site, was counted as involvement of one organ site. Using this definition, 133 patients (37.5%) had a single involved site and 122 patients (34.5%) had two sites involved. The remaining 99 (28.0%) patients had three or more sites involved in the metastatic process. The survival curves for patients with one, two, and three or more organ sites involved are displayed in Figure 98-4. The median survival time for patients with one site of involvement was 10 months, with two sites of involvement 8.0 months, and with three or more sites of involvement 6.0 months. These survival curves are statistically different by the Cox-Mantel logrank test (P = 0.028). Other studies have documented similar results.18 The question of whether the biology of CUP is fundamentally different from known primary carcinoma with systemic metastases remains controversial.20 Nystrom and associates21 have argued that

1.00 1 site only, N=446, median=14 2 sites, N=319, median=11 3 or more sites, N=344, median=8

Survival probability

2060

0.75 1 vs. 3: P80

75

75

0

0

0

0

75

75

>80

>80

75

>80

75

>80

75

0

30

0

M5

80

>80

75

>80

75

>80

75

0

75

0

M6

30

30

75

>80

75

75

30

75

0

>80

75

0

M7

30

30

30

0

30

75

30

50 chromosomes TEL-AML1 t(12;21) MYC t(8;14), t(2;8), t(8;2) E2A-PBX t(1;19) MLL rearrangements [e.g., t(4;11), t(11;19), t(9;11)] BCR-ABL t(9;22) Others Hypodiploidy30,000/µL), late achievement of CR (>3 to 4 weeks), and Ph/BCR-ABL-positive status (see Table 103-11) have a survival rate of 25% or less, whereas standard risk patients without any of those features have a 5-year survival rate greater than 50%.134 Philadelphia chromosome/BCR-ABL-positive ALL had until recently been associated with the worst prognosis in children, as well as in adults. Outcome has now improved dramatically (see later discussion). Adults with the subtype pro-B ALL or the t(4;11) translocation have a poor prognosis, as do infant ALL patients.147 With intensive regimens, including HDAC and mitoxantrone as consolidation therapy, the results for adults seem to be improving.55 Pro-B-ALL patients benefit from allogeneic SCT in CR1, with survival rates of 60% in transplanted patients.148 The adverse impact of the translocation t(4;11) seems to have changed with new treatment modalities (see earlier). In mature B-cell ALL, CR remission rates were low (40%) a decade ago, and remission duration was short (11 months).149 A change was brought about by innovative childhood B-cell ALL studies that significantly improved outcome, with CR rates of 80% to 94% and an LFS rate of 63% (weighted mean).149 The drugs that were responsible for the improvement were high doses of fractionated cyclophosphamide, HDM (0.5 to 8 g/m2), and HDAC in conjunction with the conventional drugs for remission induction in ALL, given in short cycles at frequent intervals over a period of 6 months. The application of these childhood B-cell ALL protocols in original or modified form also brought a substantial improvement in adult patients with B-cell ALL. CR rate reached 75% (62% to 83%) and the LFS rate of 55% (20% to 71%).149–151 Adverse outcome prognostic factors were late CR (more than two cycles of chemotherapy), high WBC (>30 × 109/L), and age older than 50 years. B-cell ALL has a higher incidence of CNS involvement at diagnosis and of CNS relapse. Therefore, effective measures against CNS disease, such as HDM and HDAC, as well as intrathecal therapy, are important components of treatment regimens. Maintenance treatment has been omitted. Because relapses occur almost exclusively within the first year in childhood, as well as in adult B-cell ALL studies, patients thereafter can be considered to be cured.

Further significant improvement was achieved by the addition of antibody therapy with anti-CD20 (rituximab) because more than 80% of the patients are CD20-positive. With these regimens, survival rates above 70% to 90% can be achieved in adults with mature BALL or Burkitt’s lymphoma.152.153 The translocations t(8;14), t(8;2), and t(8;22) or c-myc aberrations are present in most cases of mature B-cell ALL and in Burkitt’s lymphoma. They may add information in cases with uncertain diagnosis. They have lost their poor prognostic significance, however, because of the improved treatment for patients with B-cell ALL or Burkitt’s lymphoma.

Treatment Response and Minimal Residual Disease Beside age, the most relevant prognostic factor in ALL is still the achievement of CR. Further prognostic factors related to treatment response are delayed time to CR or response to prednisone therapy. A more accurate approach to assess individual response is evaluation of MRD,62,154 since this is an independent prognostic factor that reflects primary drug resistance as well as individual completion of therapy and unknown host factors. The methods for detecting the burden of MRD have been described earlier and are summarized in Table 103-14.

Identification of High-Risk Patients as Candidates for Stem Cell Transplant or Experimental Therapy After the start of consolidation, a high burden of MRD (>10−4) at any time point is associated with a high relapse risk of 66% to 88%,53 and the predictive value increases at later time points (months 6 to 9).155 In the GMALL studies, patients with a high burden of MRD (>10−4) after induction and first consolidation were identified as high risk and were considered candidates for SCT in CR1.156

Identification of Low-Risk Patients in Whom Treatment Intensity Reduction May Be Justified This aim is more difficult to reach. An early and rapid decrease of MRD during induction is associated with a relapse risk rate of only 8%.53 However, this course is observed in only 10% of the patients. In the GMALL studies, patients with negative MRD status after induction, which is repeatedly confirmed during first year and measured with two sensitive markers, are considered as MRD low risk. Assessing molecular CR, thereby evaluating different induction therapies and detecting molecular relapses, are two important new items for follow-up analysis in adult ALL. Molecular response provides a more individual impression of the response and is particularly important, since nowadays, 85% to 90% of adult ALL patients achieve a cytologic CR. Even in phase II studies, molecular relapse is already an inclusion criteria. This makes sense, since patients with an increase of MRD burden above 10−4 after achievement of a molec-

Table 103-14 Methods for Detection of Minimal Residual Disease in Acute Lymphocytic Leukemia Patients Method

Target

Flow-Cytometry

Leukemia-specific immunophenotype

Sensitivity

Application B-Lineage ALL (%)

T-Lineage ALL (%)

∼35

>90

−6

10−4 −4

PCR

Fusion transcripts

10 –10

∼30

∼10

PCR

Ig rearrangements (IgH, IgK)

10−4–10−6

>90

∼20

PCR

TCR rearrangements (TCR-β, -δ, -γ)

10−4–10−6

∼50

>90

IgH, immunoglobulin gene; PCR, polymerase chain reaction; TCR, T-cell receptor gene. Data from Campana D, Pui C-H: Detection of minimal residual disease in acute leukemia: Methodological advances and clinical significance. Blood 1995;85:1416–1434; Beishuizen A, van Wering E, Breit TM, et al: Molecular biology of acute lymphoblastic leukemia: Implications for detection of minimal residual disease. In Hiddemann W, et al (eds): Acute Leukemias: V. Experimental Approaches and Management of Refractory Disease. Berlin, Springer-Verlag, 1996, p 460.

Acute Lymphocytic Leukemia in Adults • CHAPTER 103

ular CR are at high risk of relapse (>80%), and therapeutic action should be taken.157

Table 103-15

Risk Stratification According to Minimal Residual Disease The approaches to integrate MRD analysis in prospective risk stratification of adults can be different in terms of (1) in the time points sampled, (2) the selection of patients for MRD risk stratification, (3) combination of MRD-based and conventional risk factors, and (4) the MRD-based treatment decisions. It is hardly possible to identify adult low-risk patients in whom reduction of the intensity of therapy would be justified. In the GMALL study, these patients are defined according to very strict criteria (see previous discussion) in order to omit maintenance therapy. However 20% to 30% of these patients relapsed. The major aim in MRD-based studies is therefore to identify patients who have a high risk of relapse for treatment intensification with SCT. It remains to be demonstrated that this is an effective strategy, since patients with high MRD before SCT have an increased risk of relapse and might benefit from additional conventional therapy, or even experimental therapy, to reduce tumor load. On the other hand, it has to be questioned whether patients who are candidates for SCT on the basis of conventional risk factors, including Ph+ ALL, should receive allogeneic SCT if they are MRD-negative. The best strategy remains unknown. Evaluation of the burden of MRD has not been without problems. The technical procedure is time-consuming, expensive and requires highly specialized staff. The predictive value depends on the technical quality, such as sensitivity (10−4), the number of targets (at least two for immunoglobulin or T-cell receptor rearrangements), and the frequency of evaluations (3-monthly). At least in multicenter studies, these prerequisites often cannot be fulfilled. Sensitivity of the evaluation of MRD, with the exception of BCR-ABL-based analysis, is also insufficient to evaluate the efficacy of consolidation cycles because, in most patients, MRD is below the detection limit.

Drug Resistance MDR-1 function has been associated with a poorer prognosis.137,158 In vitro sensitivity testing was able to identify patients who had resistance to conventional cytostatic drugs, which was associated with an inferior prognosis. More recently, it was demonstrated that in vitro resistance is associated with distinct gene expression profiles.159 In vitro resistance testing is also increasingly used for effectiveness testing of new cytostatic drugs. In the future, a prediction of response to induction regimens might be possible in order to adapt therapy to individual susceptibility.

NEW THERAPEUTIC APPROACHES IN ADULT ALL TREATMENT WITH MONOCLONAL ANTIBODIES ALL blast cells express a variety of specific antigens, such as CD20, CD19, CD22, CD33, and CD52, which may serve as targets for treatment with monoclonal antibodies (Table 103-15). Monoclonal antibody therapy is an attractive approach, since it is targeted, subtype specific, and, in comparison to chemotherapy, has different mechanisms of action and side effects. One prerequisite for antibody therapy might be the presence of the target antigen on at least 20% to 30% of the blast cells. Application might be most promising in combination with MRD (reviewed by Gökbuget and Hoelzer160).

Anti-CD20 Most experience exists so far with rituximab, which is a chimeric monoclonal antibody to CD20 that is expressed on normal and malignant B-lymphocytes. It exerted significant antitumor activity, and its use has led to an improvement of results in B-cell NHL. however, CD20, defined as expression on more than 20% of the blast

Expression of Surface Antigens on Acute Lymphocytic Leukemia Blast Cells

Subgroup

Antigen

Expression on More Than 20% of Lymphatic Blast Cells*

B-lineage

CD19

95% precursor 94% mature

CD20

41% precursor

cyCD22

17%

86% mature T-lineage

CD25 CD7

Both

99%

CD3

33%

CD52

66%–78%†

CD33

16%

*Data from the GMALL central immunophenotyping, E. Thiel, S. Schwartz, Berlin. † Data from Faderl S, Kantarjian HM, O’Brien S, et al: A broad exploratory trial of Campath-1H in the treatment of acute leukemias. Blood 2000;96:1397a.

cells, is also present on one third of B-precursor ALL blasts, particularly in elderly patients (40% to 50%), and the majority of mature B-ALL blast cells (80% to 90%). The anti-CD20 antibody has been successfully integrated in therapy of mature B-ALL and Burkitt’s lymphoma. It is now also being explored in several pilot studies for CD20-positive B-precursor ALL. In a GMALL protocol for elderly patients, Rituximab is administered prior to chemotherapy cycles starting during induction for a total of eight treatments. Also the combination of hyper-CVAD regimen with Rituximab in B-precursor ALL was feasible and a favorable outcome with CD20-positive ALL was reported (reviewed by Gökbuget and Hoelzer161).

Anti-CD52 The CD52 antigen is expressed by most lymphatic cells and to a higher degree in T-lymphoblasts compared with B-lymphoblasts. CD52-antibodies were first used for ex vivo T-cell depletion of allogeneic bone marrow grafts to prevent GvH disease without further GvH disease prophylaxis. The humanized antibody Campath-1H has antitumor activity in CLL, T-PLL, and other T-NHL. Several studies with anti-CD52 therapy in adults with ALL are ongoing, either during relapse or at the time of MRD. The CALGB has integrated anti-CD52 therapy as consolidation in their frontline therapy and demonstrated its feasibility in a dose-finding study. Efficacy data are not available.162 Additional monoclonal antibodies (B43[anti-CD19]-Genistein, B43[anti-CD19]-PAP, anti-B4-bR [anti-CD19]) have been investigated in phase I to II pilot trials in ALL. Antibodies that were developed for other diseases, such as anti-CD22 in lymphoma and anti-CD33 in AML, could be applicable in ALL because these antigens are expressed in 17% and 16% of adult ALL cases, respectively. Antibody treatment could be administered as single agents or in combination with chemotherapy, for purging, and as post-transplant therapy and might be particularly effective in low-burden disease (MRD-positive patients).

Imatinib in Ph/BCR-ABL-Positive Acute Lymphocytic Leukemia In Ph/BCR-ABL-positive leukemia, the BCR-ABL fusion gene is causally involved in leukemogenesis and is considered to be essential for

2207

2208

Part III: Specific Malignancies

leukemic transformation. With a selective inhibitor of the Abl tyrosine kinase (STI571, imatinib), cellular proliferation of BCR-ABL-positive chronic myeloid leukemia and ALL cells can be inhibited.

Clinical Experience with Imatinib in Advanced Ph-Positive Acute Lymphocytic Leukemia In a multicenter phase II study, 56 patients with relapsed or refractory Ph+ ALL received imatinib at an initial daily dose of 400 mg orally, which was later increased to 600 mg; 60% of Ph+ ALL patients achieved a hematologic response. A complete hematologic remission with normalization of peripheral blood counts (absolute neutrophil count >1.5/nL, platelet count >100/nL) was noted in 19% of patients. Rapid blast cell clearance occurred within 1 week of treatment in the majority of patients. It is noteworthy that the peripheral blood response did not necessarily correspond with a bone marrow response. Median estimated time to progression for ALL patients was 2.2 months.163 Despite the rapid development of relapse that occurs within weeks in many patients, some of these patients went on to SCT.164 Response to imatinib therapy can be closely monitored by quantitative PCR. Hematologic toxicity (grades III and IV) was frequent but was rarely associated with serious infectious or hemorrhagic complications. Nonhematologic toxicity attributed to imatinib consisted primarily of mild-to-moderate gastrointestinal discomfort, peripheral and facial edema, and muscle cramps and was readily manageable. No patient discontinued therapy because of nonhematologic adverse events. There were no imatinib-related deaths. Therefore, imatinib was well tolerated even in heavily pretreated patients.

Imatinib in the Treatment of Ph-Positive Acute Lymphocytic Leukemia In younger patients, imatinib was first administered between chemotherapy cycles. However, with this approach, no molecular remissions were achieved. Therefore, studies with parallel application of chemotherapy and imatinib were started, leading to CR rates above 91% to 96% and molecular CR rates of 38% to 50%.52,165–167 All studies reported an improved OS rate of 55% to 65% compared to 15% in studies before the imatinib era. No trial described increased toxicity compared to chemotherapy alone or negative effects on subsequent SCT. In older patients with de novo Ph+ ALL, treatment results have been previously extremely poor with particularly high induction mortality. Therefore, induction chemotherapy was replaced by singledrug therapy with imatinib. The remission rate was 92% in an Italian trial.168 The German study group (GMALL) conducted a randomized trial comparing dose-reduced chemotherapy and imatinib monotherapy. After induction, all patients received chemotherapy combined with imatinib. The remission rate for the imatinib arm was 93% compared to 54% with chemotherapy.169 The survival was superior to that in previous trials without imatinib, but in both arms, the relapse rate was high, and there was no difference in outcome. At present, a combination of chemotherapy and imatinib is the standard of the treatment of younger patients with Ph+ ALL. Patients are still referred for SCT in CR1 if possible. In elderly patients, imatinib monotherapy for induction seems reasonable, at least if a rapid response is observed. However, it remains unknown whether the high relapse rate can be decreased by a combination of imatinib with mild chemotherapy during induction or by intensification of consolidation. Furthermore, transition to the use of treatment with other tyrosine kinase inhibitors in case of molecular relapse or detection of mutations of BCR/AB2 (see later discussion) is important.

Imatinib and Allogeneic Stem Cell Transplantation in Ph-Positive Acute Lymphocytic Leukemia It is known that MRD after SCT in Ph+ ALL is associated with a relapse probability exceeding 90%. Starting imatinib in the setting

of MRD could decrease this high relapse rate. In a prospective study that was conducted by the GMALL, 27 Ph+ ALL patients received imatinib on detection of MRD after SCT. MRD became undetectable in 52% of patients after a median of 1.5 months. Each of these patients remained in remission at least for the duration of imatinib treatment. Failure to achieve MRD negativity shortly after starting imatinib predicted relapse, which occurred in 92% of these patients after a median of 3 months. LFS rate was 91% after 1 year in the molecular responders compared to 8% in the nonresponders.170 It remains unknown whether imatinib should be started in all patients after SCT or only in case of MRD detection. In any case, continued MRD-positivity after 2 to 3 months of imatinib identifies patients who will ultimately experience relapse and in whom additional or alternative antileukemic treatment should be initiated.

Mechanisms of Resistance to Imatinib MRD detection often leads to early detection of molecular resistance or molecular relapse. Additional treatment can then be initiated before overt relapse occurs. Nowadays, an additional search for mutations of the tyrosine kinase domain of BCR/ABL is required, since these mutations can confer resistance to imatinib and partly to the second-generation tyrosine kinase inhibitors dasatinib and nilotinib.171,172 Both drugs have increased efficacy in comparison to imatinib and are active in the majority of mutations, with the exception of the T315I mutation. The remission rate that is achieved with these drugs in patients who fail imatinib is approximately 30%. These second-generation drugs are currently being evaluated in patients who have relapsed, but trials for de novo Ph+ ALL are starting.

New Cytotoxic Drugs In the past 10 years, a variety of new drugs has been developed for use in ALL (reviewed by Pui and Jeha,83 Thomas,84 and Gökbuget and Hoelzer85). Nelarabine is a purine analog that acts specifically on T-lymphoblasts, with a remission rate between 30% and 40% in relapsed T-ALL.132,173 Treatment was generally well tolerated, although neurotoxicity occurred in some patients. At present, application of nelarabine in frontline therapy is being evaluated. BCX1777 (forodesine) is an inhibitor of the enzyme purine nucleoside phosphorylase and thereby acts similarly to purine analogs.146 Clofarabine is a purine analog without subgroup specific effects. Other new drugs are liposomal preparations that might improve feasibility of treatment, for example, liposomal vincristine, daunorubicin, or liposomal cytarabine for intrathecal application.

FUTURE RISK STRATIFICATION AND TREATMENT CONCEPTS FOR ADULT ACUTE LYMPHOCYTIC LEUKEMIA The treatment of adult ALL has already become more sophisticated and complicated and will be even more so in the future. Treatment strategies depend on factors that are unrelated to the disease, such as the availability of a stem cell donor, patient-related factors, disease markers, treatment response, and availability of targeted drugs. Prognostic factors and patient characteristics therefore no longer serve only as the basis for identification of candidates for SCT in CR1 but also define individualized treatment approaches, which are discussed in the following examples: • Subgroup-adjusted and targeted treatment: Targeted drugs such as tyrosine kinase inhibitors, subgroup-specific purine analogs such as nelarabine, or monoclonal antibodies such as CD20 in mature B-ALL are used to increase subgroup-specific activity of treatment. These approaches have already led to significant improvement of outcome and will be refined in the future. • Age-adapted treatment: In ways that are similar to subgroup-adapted treatment, therapies have to be defined for patients at both ends of the age spectrum: elderly and adolescent patients. In elderly

Acute Lymphocytic Leukemia in Adults • CHAPTER 103

•

•

•

•

patients, the major focus is on effective targeted therapy with as much quality of life as possible, whereas in adolescents, the major aim is to deliver time- and dose-intensive chemotherapy based on pediatric protocols. Individualized treatment: New methods offer the option to adopt intensity and duration of therapy to individual response as measured by the presence of MRD or to add or omit specific drugs according to results of an evaluation for drug resistance. New integrated risk classification: A variety of molecular markers that have been newly detected by microarray analysis have been proposed as prognostic factors.174 They might possibly be integrated in a conventional risk model that aims to identify patients for SCT in CR1 and might rather stimulate analysis of underlying mechanisms, drug targets, or invention of treatment adaptations. Risk-adapted indications for SCT: Indications for SCT have to be defined carefully, taking into account not only long-term results but also acute and long-term toxicities into account. At present, the majority of study groups stick to risk-adapted indications for SCT. Evaluation of new cytostatic drugs: Many of these drugs fit in subtype-adjusted, targeted therapies. Taking the number of targets and drugs into account, evidence-based priorities for clinical eval-

uation in relapsed ALL and for integration in frontline therapy have to be set. Risk- and subtype-adjusted treatment strategies have led to considerable improvement in outcome in patients with mature B-ALL, T-ALL, and Ph+ ALL but less improvement in adult patients with B-precursor ALL. Future strategies will integrate a variety of additional factors, thereby resulting in a more complex, flexible, and patient-specific treatment approach.85 Besides these sophisticated approaches, a better adherence to protocols, support of patients to improve their compliance, and documentation of compliance would be warranted in adult ALL. Treatment should be done at experienced centers, and closer cooperation between internal medicine and pediatric physicians, including cooperative studies, would be desirable. The design of prospective trials will be challenging, since they will focus on even smaller subgroups of ALL and phase I studies with new drugs. These trials will be possible only in larger, international study groups that are able to recruit sufficient patient numbers. To enable any intergroup comparison, international efforts similar to that utilized to study childhood ALL are required to define uniform criteria for diagnostic classification, definition of subgroups, and even prognostic factors.

REFERENCES 1. Ries LAG, Harkins D, Krapcho M, et al: SEER Cancer Statistics Review, 1975–2003. Bethesda, MD, National Cancer Institute. 2. European Group for the Immunological Characterization of Leukemia, Bene MC, Castoldi G, et al: Proposals for the immunological classification of acute leukemias. Leukemia 1995;9:1783–1786. 3. Faderl S, Kantarjian HM, Talpaz M, Estrov Z: Clinical significance of cytogenetic abnormalities in adult acute lymphoblastic leukemia. Blood 1998; 91:3995–4019. 4. Charrin C: Cytogenetic abnormalities in adult acute lymphoblastic leukemia: Correlations with hematologic findings and outcome: A collaborative study of the Groupe Francais de Cytogénétique Hématologique. Blood 1996;87:3135–3142. 5. Moorman AV, Harrison CJ, Buck GA, et al: Karyotype is an independent prognostic factor in adult acute lymphoblastic leukemia (ALL): Analysis of cytogenetic data from patients treated on the Medical Research Council (MRC) UKALLXII/ Eastern Cooperative Oncology Group (ECOG) 2993 trial. Blood 2007;109:3189–3197. 6. Secker-Walker LM, Prentice HG, Durrant J, et al: Cytogenetics adds independent prognostic information in adults with acute lymphoblastic leukaemia on MRC trial UKALL XA. Br J Haematol 1997;96:601–610. 7. Wetzler M, Dodge RK, Mrozek K, et al: Prospective karyotype analysis in adult acute lymphoblastic leukemia: The Cancer and Leukemia Group B experience. Blood 1999;93: 3983–3993. 8. Maurer J, Jannsen JWG, Thiel E, et al: Detection of chimeric BCR-ABL genes in acute lymphoblastic leukemia by the polymerase chain reaction. Lancet 1991;337:1055–1058. 9. Westbrook CA, Hooberman AL, Spino C, et al: Clinical significance of the BCR-ABL fusion gene in adult acute lymphoblastic leukemia: A Cancer and Leukemia Group B study (8762). Blood 1992;80:2983–2990. 10. Gleissner B, Gökbuget N, Bartram CR, et al: Leading prognostic relevance of the BCR-ABL translocation in adult acute B-lineage lymphoblastic leukemia: A prospective study of the German Multicenter Trial Group and confirmed

11. 12. 13. 14.

15.

16.

17.

18.

19. 20.

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Part III: Specific Malignancies 129. Sallan SE: Myths and lessons from the adult/ pediatric interface in acute lymphoblastic leukemia. Hematology Am Soc Hematol Educ Program 2006;2006:128–132. 130. Gökbuget N, Arnold R, Böhme A, et al: Risk adapted treatment of adolescents with acute lymphoblastic leukemia (ALL) according to the German Multicenter Study Group (GMALL) Studies 06/99 and 07/03 yields significantly different outcome for subgroups. Haematologica 2007. 131. Huguet F, Raffoux E, Thomas X, et al: Towards a pediatric approach in adults with acute lymphoblastic leukemia (ALL): The GRAALL2003 Study [abstract]. Blood 2006;108:147. 132. Deangelo DJ, Yu D, Johnson JL, et al: Nelarabine induces complete remissions in adults with relapsed or refractory T-lineage acute lymphoblastic leukemia or lymphoblastic lymphoma: Cancer and leukemia group B study 19801. Blood 2007; 109:5136–5142. 133. Storring JM, Brandwein J, Gupta V, et al: Treatment of adult acute lymphoblastic leukemia (ALL) with a modified DFCI pediatric regimen: The Princess Margaret experience [abstract]. Blood 2006;108:1875. 134. Gökbuget N, Arnold R, Buechner T, et al: Intensification of induction and consolidation improves only subgroups of adult ALL: Analysis of 1200 patients in GMALL study 05/93 [abstract]. Blood 2001;98:802a. 135. Arnold R, Beelen D, Bunjes D, et al: Phenotype predicts outcome after allogeneic stem cell transplantation in adult high risk ALL patients [abstract]. Blood 2003;102:1719. 136. Grabher C, von BH, Look AT: Notch 1 activation in the molecular pathogenesis of T-cell acute lymphoblastic leukaemia. Nat Rev Cancer 2006;6:347–359. 137. Vitale A, Guarini A, Ariola C, et al: Adult T-cell acute lymphoblastic leukemia: Biologic profile at presentation and correlation with response to induction treatment in patients enrolled in the GIMEMA LAL 0496 protocol. Blood 2006;107: 473–479. 138. Asnafi V, Buzyn A, Thomas X, et al: Impact of TCR status and genotype on outcome in adult Tcell acute lymphoblastic leukemia: A LALA-94 study. Blood 2005;105:3072–3078. 139. Baldus CD, Burmeister T, Martus P, et al: High expression of the ETS transcription factor ERG predicts adverse outcome in acute T-lymphoblastic leukemia in adults. J Clin Oncol 2006;24:4714– 4720. 140. Baldus CD, Martus P, Burmeister T, et al: Low ERG and BAALC expression identifies a new subgroup of adult acute T-lymphoblastic leukemia with a highly favorable outcome. J Clin Oncol 2007;25:3739–3745. 141. Burmeister T, Gokbuget N, Reinhardt R, et al: NUP214-ABL1 in adult T-ALL: The GMALL study group experience. Blood 2006;108:3556– 3559. 142. Schorin MA, Blattner S, Gelber RD, et al: Treatment of childhood acute lymphoblastic leukemia: results of Dana-Farber Cancer Institute/ Children’s Hospital acute lymphoblastic leukemia consortium protocol 85–01. J Clin Oncol 1994;12:740–747. 143. Feickert HJ, Bettoni C, Schrappe M, et al: Eventfree survival of children with T-cell acute lymphoblstic leukemia after introduction of high dose methotrexate in multicenter trial ALL-BFM 86. Proc ASCO 1993;12:317. 144. Arico M, Basso G, Mandelli F, et al: Good steroid response in vivo predicts a favourable outcome in children with T-cell acute lymphoblastic leukemia. Cancer 1995;75:1684–1693.

145. Rohatiner AZS, Bassan R, Battista R, et al: Highdose cytosine arabinoside in the initial treatment of adults with acute lymphoblastic leukemia. Br J Cancer 1992;62:454–458. 146. Furman R, Gore L, Ravandi F, Hoelzer D: Forodesine IV (Bcx-1777) is clinically active in relapsed/refractory T-cell leukemia: Results of a phase II study (interim report) [abstract]. Blood 2006;108:1851. 147. Pui C-H, Carroll AJ, Raimondi SC, et al: Childhood acute lymphoblastic leukemia with the t(4;11)(q21;q23): An update. Blood 1994;83:2284–2285. 148. Arnold R, Bunjes D, Ehninger G, et al: Allogeneic stem cell transplantation from HLA-identical sibling donor in high risk ALL patients is less effective than transplantation from unrelated donors [abstract 279]. Blood 2002;100:77a. 149. Hoelzer D, Ludwig W-D, Thiel E, et al: Improved outcome in adult B-cell acute lymphoblastic leukemia. Blood 1996;87:495–508. 150. Thomas DA, Cortes J, O’Brien S, et al: HyperCVAD program in Burkitt’s-type adult acute lymphoblastic leukemia. J Clin Oncol 1999;17: 2461–2470. 151. Hoelzer D, Arnold R, Diedrich H, et al: Successful treatment of Burkitt’s NHL and other high-grade NHL according to a protocol for mature B-ALL [abstract 595]. Blood 2002;100:159a. 152. Hoelzer D, Baur K-H, Giagounidis A, et al: Short intensive chemotherapy with rituximab seems successful in Burkitt NHL, mature B-ALL and other high-grade B-NHL [abstract]. Blood 2003;102:#236. 153. Thomas DA, Faderl S, O’Brien S, et al: Chemoimmunotherapy with hyper-CVAD plus rituximab for the treatment of adult Burkitt and Burkitt-type lymphoma or acute lymphoblastic leukemia. Cancer 2006;106:1569–1580. 154. Hoelzer D, Gökbuget N: New approaches in acute lymphoblastic leukemia in adults: Where do we go? Semin Oncol 2000;27:540–559. 155. Mortuza FY, Moreira I, Papaioannou M, et al: Immunoglobulin heavy chain gene rearrangement in adult acute lymphoblastic leukemia reveals preference of JH-proximal variable gene segments. Blood 2002;97:2716–2726. 156. Gökbuget N, Raff R, Brugge-Mann M, et al: Risk/ MRD adapted GMALL trials in adult ALL. Ann Hematol 2004;83(suppl 1):S129–S131. 157. Raff T, Gökbuget N, Luschen S, et al: Molecular relapse in adult standard risk ALL patients detected by prospective MRD-monitoring during and after maintenance treatment: Data from the GMALL 06/99 and 07/03 trials. Blood 2007;109:910– 915. 158. Mancini M: An integrated molecular-cytogenetic classification is highly predictive of outcome in adult acute lymphoblastic leukemia (ALL): Analysis of 395 cases enrolled in the GIMEMA 0496 Trial [abstract]. Blood 2001;98:3492a. 159. Holleman A, Cheok MH, den Boer ML, et al: Gene-expression patterns in drug-resistant acute lymphoblastic leukemia cells and response to treatment. N Engl J Med 2004;351:533–542. 160. Gökbuget N, Hoelzer D: Treatment with monoclonal antibodies in acute lymphoblastic leukemia: Current knowledge and future prospects. Ann Hematol 2003;83:201–205. 161. Gökbuget N, Hoelzer D: Rituximab in the treatment of adult ALL. Ann Hematol 2006;85:117–119. 162. Stock W, Yu D, Sanford B, et al: Incorporation of alemtuzumab into front-line therapy of adult acute lymphoblastic leukemia (ALL) is feasible: A phase I/II study from the Cancer and Leukemia Group B (CALGB 10102) [abstract]. Blood 2005;106:145.

163. Ottmann OG, Druker BJ, Sawyers CL, et al: A phase II study of imatinib mesylate (Glivec) in Patients with relapsed or refractory philadelphia chromosome-positive acute lymphoid leukemias. Blood 2002;100:1965–1971. 164. Wassmann B, Pfeifer H, Scheuring U, et al: Therapy with imatinib mesylate (Glivec) preceding allogeneic stem cell transplantation (SCT) in relapsed or refractory Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL). Leukemia 2002;16:2358–2365. 165. Thomas DA, Kantarjian H, Cortes J,et al: Outcome with the hyper-CVAD and imatinib mesylate regimen as frontline therapy for adult Philadelphia (Ph) positive acute lymphocytic leukemia (ALL) [abstract]. Blood 2006;108:284. 166. Yanada M, Takeuchi J, Sugiura I, et al: High complete remission rate and promising outcome by combination of imatinib and chemotherapy for newly diagnosed BCR-ABL-positive acute lymphoblastic leukemia: A phase II study by the Japan Adult Leukemia Study Group. J Clin Oncol 2006;24:460–466. 167. de LA, Rousselot P, Huguet-Rigal F, et al: Imatinib combined with induction or consolidation chemotherapy in patients with de novo Philadelphia chromosome-positive acute lymphoblastic leukemia: Results of the GRAAPH2003 study. Blood 2007;109:1408–1413. 168. Vignetti M, Fazi P, Cimino G, et al: Imatinib plus steroids induces complete remissions and prolonged survival in elderly Philadelphia chromosomepositive acute lymphoblastic leukemia patients without additional chemotherapy: Results of the GIMEMA LAL0201-B protocol. Blood 2007;109:3676–3678. 169. Ottmann OG, Wassmann B, Pfeifer H, et al: Imatinib compared with chemotherapy as frontline treatment of elderly patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL). Cancer 2007;109:2068–2076. 170. Wassmann B, Pfeifer H, Stadler M, et al: Early molecular response to posttransplantation imatinib determines outcome in MRD+ Philadelphiapositive acute lymphoblastic leukemia (Ph+ ALL). Blood 2005;106:458–463. 171. Kantarjian H, Giles F, Wunderle L, et al: Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive ALL. N Engl J Med 2006;354:2542–2551. 172. Talpaz M, Shah NP, Kantarjian H, et al: Dasatinib in imatinib-resistant Philadelphia chromosomepositive leukemias. N Engl J Med 2006;354:2531– 2541. 173. Gökbuget N, Arnold R, Atta J, et al: Compound GW506U78 has high single-drug activity and good feasibility in heavily pretreated relapsed Tlymphoblastic leukemia (T-ALL) and Tlymphoblastic lymphoma (T-LBL) and offers the option for cure with stem cell transplantation [abstract]. Blood 2005;106:#150. 174. Armstrong SA, Look AT: Molecular genetics of acute lymphoblastic leukemia. J Clin Oncol 2005;23:6306–6315. 175. Ludwig WD, Raghavachar A, Thiel E: Immunophenotypic classification of acute lymphoblastic leukemia. Bailliere’s Clin Haematol 1994;7:235. 176. Hoelzer D, Thiel E, Ludwig WD, et al: Follow-up of the first two successive German multicentre trials for adult ALL (01/81 and 02/84). Leukemia 1993;7(suppl 2):130–134. 177. Durrant IJ: Results of Medical Research Council trial UKALL IX in acute lymphoblastic leukaemia in adults: Report from the Medical Research Council Working Party on Adult Leukaemia. Br J Haematol 1993;85:84–92. 178. Larson RA, Dodge RK, Burns CP, et al: A fivedrug remission induction regimen with intensive consolidation for adults with acute lymphoblastic

Acute Lymphocytic Leukemia in Adults • CHAPTER 103 leukemia: Cancer and Leukemia Group B study 8811. Blood 1995;85:2025–2037. 179. Mandelli F, Annino L, Rotoli B: The GIMEMA ALL 0183 trial: Analysis of 10-year follow-up. Br J Haematol 1996;92:665–672. 180. Wernli M, Abt A, Bargetzi M, et al: A new therapeutic strategy in adult acute lymphoblastic leukemia: Intensive induction/consolidation, early transplant, maintenance-type therapy in relapse only [abstract]. Proc Am Soc Clin Oncol 1997;16:6a. 181. Ribera JM, Ortega J, Oriol A, et al: Treatment of high-risk acute lymphoblastic leukemia.

Preliminary results of the protocol PETHEMA ALL-93. In Hiddemann et al (eds): Acute Leukemias: VII. Experimental Approaches and Novel Therapies. Berlin, Springer-Verlag, 1998, pp 755–765. 182. Linker C, Damon L, Ries C, Navarro W: Intensified and shortened cyclical chemotherapy for adult acute lymphoblastic leukemia. J Clin Oncol 2002;20:2464–2471. 183. Annino L, Vegna ML, Camera A, et al: Treatment of adult acute lymphoblastic leukemia (ALL): Long-term follow- up of the GIMEMA ALL 0288 randomized study. Blood 2002;99:863–871.

184. Rowe JM, Buck G, Burnett AK, et al: Induction therapy for adults with acute lymphoblastic leukemia (ALL): Results of nearly 1,400 patients from the International ALL Trial (MRC UKALL XII / ECOG E2993) [abstract]. Blood 2003;102:785a. 185. Cornelissen JJ, Carston M, Kollman C, et al: Unrelated marrow transplantation for adult patients with poor-risk acute lymphoblastic leukemia: Strong graftversus-leukemia effect and risk factors determining outcome. Blood 2001;97:1572– 1577.

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104

Acute Myeloid Leukemia in Adults Frederick R. Appelbaum

S U M M ARY

Epidemiology and Etiology

O F

K EY

P OI NT S

• AML is a clonal disease arising in a primitive hematopoietic progenitor cell. • Leukemogenesis is a multistep process requiring some mutations that block differentiation and others that promote proliferation.

• The 2002 World Health Organization (WHO) classification recognizes four categories of AML: (1) AML with recurrent genetic abnormalities; (2) AML with multilineage dysplasia; (3) AML and myelodysplastic syndrome (MDS), therapy-related; (4) AML not otherwise categorized. • Cytogenetics is the most powerful single indicator of outcome, and cases can be defined as favorable, intermediate, or unfavorable according to cytogenetic subtype. Translocation t(15;17) is diagnostic of acute promyelocytic leukemia (APL), the M3 subtype of AML in the FrenchAmerican-British (FAB) classification (see Table 104-3), which requires a very specific form of therapy.

Diagnosis and Classification

Treatment

• The incidence of acute myeloid leukemia (AML) in adults is 3 cases per 100,000 population per year in the United States. • Incidence increases with age; median age at diagnosis is 60 years. • Known causes of AML include exposure to benzene or ionizing radiation and previous chemotherapy; AML also is associated with a few uncommon inherited syndromes.

Tumor Biology

• Diagnosis requires greater than 20% blasts of myeloid origin in marrow or peripheral blood.

• Younger patients with non-M3 AML • Induction—Anthracycline plus cytarabine induces complete remission in approximately 70% of patients.

• Postinduction therapy—In patients with good-risk disease, treatment with repetitive doses of cytarabine is associated with greater than 50% cure rates. In patients with intermediaterisk disease, treatment consists of either continued chemotherapy or hematopoietic cell transplantation (HCT). Patients with poor-risk disease should receive transplantation in first remission if possible. • Older patients with non-M3 AML • Induction—Anthracyclines plus cytarabine will induce complete remission in 50% of patients. • Postinduction therapy—Continued chemotherapy may cure 15% of patients. • Patients with APL • All-trans-retinoic acid (ATRA) should be added to the induction regimen and used as maintenance, with an expectation of cure in two thirds of patients.

INTRODUCTION

EPIDEMIOLOGY AND ETIOLOGY

AML is the result of a genetic event or series of events occurring in an early hematopoietic precursor that both blocks differentiation and allows uncontrolled proliferation. The abnormally proliferating leukemic cells accumulate in the marrow space, eventually replacing normal marrow progenitors, with consequent diminished production of red cells, white cells, and platelets. This, in turn, leads to the common clinical manifestations of AML—namely, anemia, infection, and bleeding. As the disease progresses, leukemic blasts pour into the bloodstream, leading to the “weisses Blut” described by Virchow in 1845.1 Eventually, the leukemic cells accumulate in the spleen, lung, brain, and other vital organs. If left untreated, AML is rapidly fatal, with most patients dying within a few months of diagnosis. With appropriate treatment, however, a substantial proportion of patients can be cured. Remarkable growth in our understanding of AML has occurred over the past decade. One of the major lessons arising from this new knowledge is the complexity of the leukemic process—a lesson that can be daunting but one that also provides multiple new targets for prevention, detection, and treatment.

Incidence Approximately 35,000 Americans were diagnosed with leukemia in 2005.2 Of these, AML developed in 32%, chronic lymphocytic leukemia (CLL) in 26%, chronic myeloid leukemia (CML) in 15%, and acute lymphocytic leukemia (ALL) in 11%; the remaining 16% had unclassified types. The male-to-female ratio is approximately 1.3 : 1. The incidence of AML is constant during the first 30 years of life but then begins to increase almost exponentially (Fig. 104-1). The overall incidence of leukemia in the United States has remained stable over the last 30 years.3

Geographic Clustering Although leukemic clusters within a given geographic area occasionally have been described, no compelling studies suggest that these represent more than chance events.

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Part III: Specific Malignancies 18 Incidence (per 100,000)

16 14 12

AML

10 8 6 4 2

80–84 85+

70–74 75–79

65–69

0 0–4 5–9 10–14 15–19 20–24 25–29 30–34 35–39 40–44 45–49 50–54 55–59 60–64

2216

Age (yr)

Figure 104-1 • Age-related incidence of acute myeloid leukemia (AML). The incidence is relatively stable until the age of 30 years and then increases dramatically.

Etiologic Agents and Associations Viruses A clear association of human T-cell leukemia virus infection with an adult form of T-cell leukemia has been recognized. No relationship between any viral infection and the development of AML, however, has been confirmed.

Carcinogens Heavy benzene exposure is associated with the development of aplastic anemia, myelodysplasia, and AML.4 Most studies examining the issue find a small but consistent increase in AML among cigarette smokers.5 Survivors of the atomic bomb explosions of Hiroshima and Nagasaki in 1945 demonstrated an increased risk of all leukemias except CLL, which began as early as 1.5 years after the explosions, peaked at approximately 7 years, and returned to baseline by 1970.6 Other evidence that ionizing radiation is leukemogenic comes from the 10-fold increased risk of AML among persons who received radiation treatment for ankylosing spondylitis in the 1930s and the 1940s.7

Treatment-Related Acute Myeloid Leukemia With the increasing use of irradiation and chemotherapy to treat malignancies, the incidence of treatment-related AML has grown. It is estimated that perhaps 6% to 10% of cases of AML are treatmentrelated.8 These can be grouped in several relatively distinct syndromes. AML developing after exposure to alkylating agents has a latency of 5 to 6 years, often first appears as an MDS, and frequently is associated with chromosomal abnormalities involving the long arm of chromosome 5 or 7. This association was first appreciated in the 1970s in patients who had previously received alkylating agents with or without irradiation as treatment for lymphoma.9 Alkylating agent– induced AML has been noted in patients who received nitrogen mustard for treatment of Hodgkin’s disease, in women in whom melphalan or cyclophosphamide was used to treat ovarian cancer, and in patients whose treatment for colorectal cancer included chloroethylnitrosourea.10–15 All alkylating agents probably are leukemogenic, with risk increasing with cumulative dose.16 Treatment with topoisomerase II inhibitors also increases the risk for development of AML. In contrast with the leukemias seen after exposure to alkylating agents, these leukemias develop relatively rapidly (often within 2 years), generally are not preceded by a myelodysplastic phase, and frequently are associated with chromosomal rearrangements involving 11q23, the locus for MLL (the mixed-

lineage leukemia gene), or 21q22.17 The epipodophyllotoxins etoposide and teniposide fall in this category; with these drugs, the risk of developing secondary AML appears to be dose-related and may increase when methotrexate or cisplatin also is administered.18,19 The anthracyclines also are topoisomerase II inhibitors, and their use— particularly when they are given with cyclophosphamide in doseintensive regimens—has been associated with the subsequent development of AML.20 Patients with non-Hodgkin’s lymphoma treated using autologous HCT appear to be at increased risk for the development of secondary AML, with cumulative incidence rates as high as 15% reported in some series.21,22 Leukemias typical of previous exposure to both an alkylating agent and a topoisomerase II inhibitor have been reported. Recent registry data demonstrate that the risk of MDS or AML after autologous HCT is predicted largely by the type and intensity of chemotherapy received by the patient before transplantation. This observation raises some questions about the exact contribution of the transplantation procedure itself to the development of secondary AML.23 Bimolane, a dioxopiperazine derivative used for the treatment of psoriasis, has been associated with the development of APL. Not all secondary leukemias fall into the discrete categories described here. Secondary leukemias with inv16, t(9;22), and abnormalities involving 3q21 have been reported.24 Not every leukemia developing in patients who have received radiation therapy or chemotherapy is necessarily due to that therapy. In fact, a significant elevation in risk for AML has been observed among patients with a previous malignancy treated only with surgery, suggesting a genetic or other predisposition.25

Ethnic Differences APL has been reported to be more common among Hispanic populations in the Los Angeles area than in the general population.26 A similar increased incidence has been reported in Spain.27

Familial Clustering The concordance rate of leukemia in identical twins is virtually 100% if leukemia develops in one twin before the first year of life, but the rate then declines with age.28 In a large comprehensive study of proband effects in Utah, using data from 125,000 patients with cancer, the relative risk of leukemia among first-degree relatives of probands with leukemia was 5.69, strongly suggesting that complex genetic factors may influence the development of leukemia in later life.29 Several single-gene leukemia syndromes also have been described, including an autosomal recessive syndrome of childhoodonset myelodysplasia with monosomy 7 and a familial syndrome of erythroleukemia.30,31 A familial syndrome of an aspirin-like platelet disorder with thrombocytopenia and propensity for the development of AML is due to germline mutations in the RUNX-1 (formerly AML-1) gene.32 Nearly all of these autosomal dominant leukemia syndromes (with the exception of the RUNX-1 syndrome) demonstrate anticipation with declining age at onset with each generation.

Constitutional Chromosomal Abnormalities Children with trisomy 21 (Down syndrome) have an increased risk for development of leukemia, with M7 AML seen in early childhood and ALL predominating in later years. Trisomy 8 mosaicism is a rare constitutional abnormality with features of mental retardation, multiple developmental defects, and an increased incidence of myeloid leukemias.

Genetic Syndromes Associated with Acute Myeloid Leukemia Several DNA-repair syndromes are associated with an increased incidence of AML. Bloom’s syndrome is a disorder of autosomal recessive inheritance resulting from mutations in the gene encoding a DNA helicase at 15q21.1 and is characterized by growth retardation, characteristic facial appearance, immunodeficiency, and, in 25% of

Acute Myeloid Leukemia in Adults • CHAPTER 104

patients, hematologic malignancies including AML.33 Ataxia-telangiectasia, inherited as an autosomal recessive trait, is due to mutations in the ATM gene at 11q22–23, which results in deficiencies in the G1-S checkpoint. Features of this disorder may include progressive cerebellar ataxia, telangiectatic skin lesions, and malignancies that more often are of lymphoid than of myeloid origin.34 Fanconi’s anemia is a syndrome of autosomal recessive inheritance characterized by pancytopenia and a variety of developmental disorders that include skeletal abnormalities (most notably, hypoplastic thumbs) and short stature. Exposure of cells from these patients to mitomycin C or diepoxybutane results in excess chromosome breaks. In almost 50% of patients with Fanconi’s anemia, myelodysplasia or AML develops by the age of 40 if death from other causes does not occur first.35 As indicated by chromosome complementation studies, at least eight different genes can result in this syndrome. The tumor suppressor gene syndromes Li-Fraumeni syndrome and neurofibromatosis 1 are associated with an increased risk of AML, but the exact extent of increased risk is unclear. Several congenital cytopenia syndromes are associated with a definite increased risk of AML: Blackfan-Diamond syndrome is characterized by congenital hypoplastic anemia, growth retardation, and a definite increase in AML. Severe congenital neutropenia, also sometimes called Kostmann’s syndrome, results in myelodysplasia or AML in 10% to 20% of affected persons who do not first succumb to infection.36 Schwachman syndrome is a disease of autosomal recessive inheritance characterized by pancreatic insufficiency, moderate dwarfism, and a hematologic picture resembling that in Fanconi’s anemia.37

TUMOR BIOLOGY Pathophysiology Clonality AML is a clonal disorder, with all leukemic cells in a given patient descending from a common progenitor. The initial proof of the clonality of AML came from studies of the disease in females who were heterozygotic for the X-linked glucose-6-phosphate dehydrogenase (G6PD) isoenzymes. In normal heterozygotic women, because of random X chromosome inactivation, any single blood cell will express one or the other isoenzyme, and hematopoietic cells overall will be a 50–50 mix. Leukemia cells in G6PD-heterozygotic females, however, were found in every case to be all of one isoenzyme or the other, indicating their origin from a single precursor.38 With the development of methods to detect X-chromosome-linked DNA polymorphisms on a broader scale, it has since become possible to assess the clonality of leukemia in virtually any female patient. Such studies have demonstrated differing patterns of clonal involvement among patients so that in some (generally younger) patients, only the frankly myeloid leukemic blasts are clonal, whereas in other (often older) patients, normal-appearing monocytes, platelets, and red cell precursors also may be of clonal origin.39 Studies of clonality also have given the surprising result that some patients in whom treatment achieves complete remission with recovery of entirely normal-looking hematopoiesis—including loss of a leukemic chromosome marker—may still have clonal hematopoiesis, a result consistent with the hypothesis of a multistep pathogenesis for AML.40,41

Cell of Origin The clonal nature of AML suggests that there is a leukemic stem cell capable of both self-renewal and proliferation. Identification of the AML stem cell is of considerable interest both for aiding in our understanding of the disease and because this cell would represent the ideal target of therapy. Recognition of cases of AML with blasts of various degrees of differentiation has given rise to two general hypotheses. In one model, progenitor cells at various levels of commitment and differentiation all are susceptible to transformation,

leading to considerable heterogeneity in AML stem cells. In an alternative model, only very undifferentiated hematopoietic stem cells are capable of being transformed, but based on the particular mutations involved, some degree of further commitment and differentiation of the leukemic cell is possible. Recent studies attempting to identify the AML stem cell based on the cell’s ability to transfer human leukemia to an immunodeficient (NOD-SCID) mouse are more consistent with the latter hypothesis. In human AML, when patients are tested to determine which fraction of cells is able to initiate leukemia in the NOD-SCID mouse, it is only the primitive CD34++CD38 fraction that is able to do so, regardless of the differentiative stage of leukemic blasts.42,43 The leukemias that subsequently develop in the animals have the same level of differentiation as for the leukemias from which the CD34++CD38 blasts are derived, suggesting that these leukemic clones are capable of some genetically determined degree of differentiation. The CD34++CD38 AML stem cells are at the same level of differentiation as for the normal hematopoietic stem cell capable of engrafting NOD-SCID mice and are rare cells among the leukemic mass, with a frequency of 0.2 per 106 to 100 per 106 cells. APL could represent an exception to this general model, and APL blasts do not easily engraft in NOD-SCID mice.43

Cell Kinetics Available data suggest that it is the persistence rather than the speed of proliferation that leads to the outgrowth of AML. Only a small fraction of leukemic cells are in cycle at any given time, and the cell cycle duration is, in fact, longer than that of normal hematopoietic cells.44 An unfortunate but instructive case is that of a woman with CML who, after an ablative preparative regimen, received a marrow transplant from her HLA-matched brother. Although the brother’s routine pretransplantation workup was negative, the transplanted marrow was later found to contain 38% AML blasts with t(1;5). In a somewhat surprising turn of events, engraftment with normal male hematopoiesis occurred, and the patient did not show evidence of her brother’s disorder until 6 months after transplantation.45

Marrow Failure Although the persistent growth of the AML clone leads to marrow failure at least in part by physically crowding out normal progenitors, other mechanisms of suppression of normal marrow probably exist. Frequently, peripheral blood counts start to fall weeks or months before the appearance of leukemic blasts in the marrow, and cases of hypoplastic AML are not uncommon. The mechanisms by which suppression of normal hematopoiesis occurs are not well understood.

Molecular Pathology The identification of recurrent chromosomal abnormalities—which can include translocations, point mutations, and gene duplications in AML—followed by the cloning of many of the involved genes has provided important insights into the pathogenesis of the disorder. The number of recurrent abnormalities so far identified is in the hundreds, a fact that would make it seem almost futile to attempt to make sense of such a wide range of abnormalities. With further investigation, however, it is becoming clear that many of these abnormalities tend to affect a limited number of transcriptional or signal transduction pathways. Of those abnormalities that have been studied extensively, most are pro-oncogenic and are not simply innocent bystanders in the leukemic process. Only a few of these abnormalities are both necessary and sufficient to cause leukemia in murine models, however—suggesting that multiple mutations are required for development of overt leukemia. A particularly useful model argues that most cases of AML have one mutation that results in differentiation blockade together with a second mutation that results in inappropriate proliferation.46 Some of the more common and better understood molecular categories of AML are described next.

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Part III: Specific Malignancies

Core Binding Factor Translocations

Inv(16)

Core binding factor (CBF) is a heterodimeric transcription factor made up of two subunits, CBFα (also known as RUNX-1 or AML1) and CBFβ. CBF plays an important role in the transcriptional activation of a number of genes required for normal hematopoietic differentiation (Fig. 104-2). A number of leukemias are associated with translocations or mutations that involve the components of CBF.

Inv(16)(p13;q22) and t(16;16)(p13;q22) both result in the fusion of the CBFβ gene at 16q22 to the smooth muscle myosin heavy chain gene (MYH11) at 16p1349 (see Fig. 104-2). As in the case of t(8;21), the resultant fusion protein acts as a dominant-negative regulator of transcription. CBFβ/MYH11 knockin mice have a phenotype identical to those noted for RUNX-1 knockouts and RUNX-1/MTG8 knockins. AML with involvement of 16q22 accounts for approximately 9% of adult AML cases, is associated with a unique myelomonocytic morphology, and, like t(8;21) leukemias, carries a favorable prognosis.

RUNX-1/MTG8 The t(8;21) abnormality seen in approximately 8% of cases of adult AML results in the fusion of the RUNX-1 (CBFα) gene on chromosome 21 to the MTG8 (formerly ETO) gene on chromosome 8.47 The RUNX-1/MTG8 fusion protein acts as a dominant negative inhibitor of the wild-type RUNX-1 gene, meaning that presence of the RUNX-1/MTG8 fusion protein blocks the ability of the wildtype RUNX-1 from the remaining nontranslocated chromosome to activate appropriately the transcription required for normal hematopoietic differentiation48 (see Fig. 104-2). The RUNX-1 “knockout” mouse and the RUNX-1/MTG8 “knockin” mouse have identical phenotypes, with embryonic death at day 11.5 and a characteristic pattern of central nervous system (CNS) hemorrhage and lack of hematopoiesis. AML characterized by t(8;21) is associated with a favorable prognosis.

CBFα

Target gene

CBFβ

A

TEL-RUNX-1 In up to 25% of cases of pediatric pre-B-cell ALL, a t(12;21) abnormality fusing the TEL gene with the RUNX-1 gene can be found50 (see Fig. 104-2).The resultant fusion protein appears to function as a dominant-negative regulator of transcription. In general, this translocation is associated with a favorable prognosis in childhood ALL.

Point Mutations Point mutations in the RUNX-1 gene occur in 3% to 5% of sporadic cases of adult AML.51,52 In addition, as noted earlier, inherited point mutations resulting in RUNX-1 haploinsufficiency are found in families with familial platelet disorder and a high prevalence of subsequent AML.32 The fact that AML does not always develop in these patients, or does so only after years, suggests that subsequent mutational events are required. The mechanisms by which t(8;21), inv(16), and t(12;21) fusion proteins exert their dominant-negative effects are only now becoming understood. Recent data suggest that segments of the fusion protein recruit nuclear co-repressor (NCoR)–histone deacetylase (HD) complexes in a matter analogous to that seen in APL, as is discussed shortly. The specific pattern of transcriptional suppression probably differs for each fusion product, as suggested by the somewhat different phenotypes associated with each.

Retinoic Acid Receptor-α Gene Translocations CBFα

ETO

Target gene

CBFβ

B

CBFα CBFβ

Target gene SMMHC

C

CBFα

TEL Target gene

CBFβ

D Figure 104-2 • A, CBFα and CBFβ form a heterodimeric transcription factor that regulates a spectrum of genes important in hematopoiesis, including those for IL-3, GM-CSF, and others. B, If instead of normal CBFα, the fusion product CBFα-ETO (now CBFα-MTG8) dimerizes with CBFβ, the transcription factor does not function, and target genes are not transcribed. C, The fusion product CBFβ-SMMHC is a dominant-negative regulator of gene transcription. D, Analogous to the situation with CBFα-ETO, the fusion product CBFα-TEL is a dominant-negative regulator of normal CBF function. CBF, core binding factor; GM-CSF, granulocyte macrophage colony stimulating factor; IL-3, interleukin-3.

APL, which accounts for approximately 8% of cases of adult AML, is almost always associated with t(15;17)(q22;q11.2), a translocation that fuses the promyelocytic leukemia (PML) gene on chromosome 15 to the retinoic acid receptor-α (RARa ) gene on chromosome 17. The resultant PML/RARα fusion product acts as a dominant-negative inhibitor of normal PML function and of the function of RXRα, an important heterodimeric partner of PML53 (Fig. 104-3). PML/ RARα recruits a nuclear co-repressor (NCoR) and the molecules sin3 and HD. HD deacetylates histones, a process that in turn inhibits binding of transcription factors, thereby inhibiting the expression of genes required for hematopoietic differentiation. The unique activity of ATRA in APL appears to be explained by its ability to bind to the PML/RARα fusion protein, changing its configuration and releasing the attached nuclear co-repressor (see Fig. 104-3). This then allows subsequent transcription and gene expression. Transgenic expression of the PML/RARα fusion protein in mice results in APL in a fraction of animals after a latency period of some months. The relatively long latency and incomplete penetration suggest that, as with many other leukemias, multiple mutations are required for the full development of overt APL. A number of other translocations, including t(5;17) and t(11;17), involve the RARα gene and result in the APL phenotype. These leukemias generally are unresponsive to ATRA because clinically achievable concentrations of the drug do not result in release of the NCoR-HD complex.

C/EBPa Gene Mutations Transcription factor CCAAT/enhancer-binding protein-α (C/EBPα) is mutated in 6% to 10% of patients with AML.54 C/EBPα is required

Acute Myeloid Leukemia in Adults • CHAPTER 104

RAR

NCoR HD

Target gene

PML

A NCoR HD

ATRA RAR

Target gene

PML

B Figure 104-3 • A, The abnormal fusion product PML-RARα binds a nuclear co-repressor (NCoR)-histone deacetylase (HD) complex. This deacetylates histones in the region, leading to inhibition of transcription. B, When ATRA binds to RAR, a change in confirmation leads to release of the NCoR-HD complex, acetylation of histones, and resumption of transcription. ATRA, all-trans-retinoic acid; RAR-PML, retinoic acid receptorpromyelocytic leukemia protein.

for normal granulocyte differentiation, and c/ebpα-null mice lack neutrophils and eosinophils. In humans, C/EBPα gene mutations presumably result in abnormal DNA binding and loss of normal myeloid differentiation. AML cases characterized by C/EBPα gene mutations tend to demonstrate M1 or M2 morphology, with intermediate-risk cytogenetics and a favorable clinical outcome.

Mixed-Lineage Leukemia Mutations Most nonrandom chromosomal abnormalities are associated with specific lineages or subtypes of leukemia, but abnormalities involving the mixed-lineage leukemia gene (MLL) located on 11q23 are exceptions, with many partner genes and many forms of hematologic malignancy including ALL, AML, and lymphoma. Altogether, translocations involving 11q23 account for approximately 7% of adult AML cases, and among these, t(9;11)(p22;q23), associated with acute monoblastic leukemia, is the most common.55,56 The translocation fuses MLL with AF9, and leukemia invariably develops in mice with this fusion gene knockin.57 Other MLL translocations seen in AML include t(6;11), t(10;11), t(11;17), and t(11;19). It also has been reported that as many as 10% of patients with AML and normal cytogenetics have tandem duplications of MLL.58 The human MLL gene has considerable homology with the Drosophila trithorax gene, a complex gene that regulates the transcription of other genes necessary for normal Drosophila development. The exact function of MLL in vertebrates is not entirely understood, but its structure suggests capability of minor groove DNA binding. Gene disruption experiments have shown that MLL positively regulates homeodomain (Hox) genes in mice and thus, like the trithorax gene, is required for normal development. Knockout mice die as embryos and demonstrate reduced hematopoiesis, suggesting that the gene has, as one of its functions, a broad effect on early hematopoiesis.59–61 A current hypothesis is that MML fusion proteins result in increased expression of HOX genes, with increased self-renewal of affected hematopoietic progenitors.

phosphorylation, and then subsequent phosphorylation activation of adaptive proteins (including GRB-2), which in turn activate Ras and other proteins. FLT3 is mutated in 30% to 35% of patients with AML.62,63 A majority of these are internal tandem duplications, but approximately one fourth of the mutations are in the form of point mutations. Both forms of mutations are activating. When inserted into murine cell lines, these mutations result in factor-independent growth.64 Retroviral transmission of these mutations into mouse marrow is not, by itself, sufficient to cause overt AML but does lead to a myeloproliferative phenotype.65 In clinical studies, the incidence of FLT3 mutations in AML appears to increase with age and to be associated with high white blood cell (WBC) counts at diagnosis and poorer clinical outcome.63,66,67 The negative impact of FLT3 mutations apprears to increase with increasing size of the duplication and with higher allelic ratios of mutated to wild-type genes in leukemic blasts.68,69 Clinical trials of small-molecule inhibitors of FLT3 are ongoing. Mutations in other receptor tyrosine kinase genes also are sometimes seen in AML. Point mutations in FMS have been reported in 10% to 20% of cases.70 Point mutations, deletions, or insertions of KIT also have been reported in a small percentage of patients.71 Mutations in one or another receptor tyrosine kinase are found in almost half of all AML cases.

RAS Mutations Ras is a monomeric guanosine diphosphate-binding protein activated by various tyrosine kinases. Activation of Ras has multiple and varied effects, which, depending on the target cell and its particular state, can result in proliferation, transformation, or differentiation. RAS mutations have been identified in 15% to 30% of cases of AML.72,73 In most cases, these mutations result in prevention of hydrolysis of Ras guanosine triphosphate (GTP), effectively keeping Ras in the “on” position. Thus, therapies to inhibit Ras function have been developed. To function normally, newly transcribed Ras must have a farnesyl or geranylgeranyl lipid attached, and for this reason farnesyl transferase inhibitors have been explored as therapeutic agents in AML.74

NPM1 Mutations NPM1 encodes an abundant nucleolar phosphoprotein with multiple hypothesized functions. Heterozygous mutations usually involving the C-terminus at exon 12 have been detected in approximately 30% of cases of AML. Mutations in NPM1 are seen more frequently in AML cases with monocytic differentiation, lack of CD34, normal cytogenetics, and FLT3 mutations. Among patients with normal cytogenetics, presence of an NPM1 mutation appears to be associated with a better prognosis, particularly among those without an FLT3 mutation.75,76

Mutations Involving 5q, 7q, and 20q AML evolving from myelodysplasia or developing from exposure to alkylating agent therapy frequently is associated with partial or complete loss of chromosomes 5, 7, and 20. The frequent loss of 5q, 7q, or 20q has led to the hypothesis that a classic tumor suppressor gene may exist in these areas. With classic tumor suppressor genes such as RB, when one allele is deleted, a mutation in the second results in disease. Despite considerable efforts to identify such genes, however, no classic tumor suppressor of AML in these regions has been reported.

Tyrosine Kinase Receptor Mutations

PATHOLOGY

FLT1, FLT3, FMS, KIT, and PDGF are members of a family of genes encoding receptor tyrosine kinases, each with an extracellular ligand– binding domain, transmembrane and juxtamembrane domains, and an intracellular domain with tyrosine kinase activity. In general, ligand binding with the receptor causes receptor dimerization, auto-

Histopathologic Features The diagnosis of AML generally is made by the examination of wellprepared peripheral blood and bone marrow specimens. For more than 3 decades, the French-American-British (FAB) system was used

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A

B

C

D

E

F

G

H

Figure 104-4 • The morphologic spectrum for the acute myeloid leukemias (AMLs) in bone marrow aspirates (A-G) and a marrow biopsy specimen (H). A, Acute myeloblastic leukemia with minimal (FAB AML-M0) or no (FAB AML-M1) maturation. The cells are myeloblasts with dispersed chromatin and variable amounts of agranular cytoplasm. Some display mediumsized, poorly defined nucleoli. B, Acute myeloblastic leukemia with maturation (FAB AML-M2). Some of the blasts contain azurophilic granules, and promyelocytes are evident. More mature neutrophils were present in other fields. Note the Auer rod (arrow). C, Acute promyelocytic leukemia (FAB AML-M3). All of these cells are promyelocytes containing coarse cytoplasmic granules, which sometimes obscure the nuclei. D, Acute myelomonocytic leukemia (FAB AML-M4). Promonocytes with indented nuclei are present with myeloblasts. The dense nuclear staining is unusual. E, Acute monoblastic leukemia (FAB AML-M5a). These characteristic monoblasts have round nuclei with delicate chromatin and prominent nucleoli. Cytoplasm is abundant. Nonspecific esterase staining was intense (not shown). F, Acute monocytic leukemia (FAB AML-M5b). Most of the cells in this field are promonocytes. Monoblasts and an abnormal monocyte also are present. G, Acute erythroid leukemia (FAB AML-M6). Dysplastic multinucleated erythroid precursors with megaloblastoid nuclei are present. H, Acute megakaryoblastic leukemia (FAB AML-M7). In this marrow biopsy specimen, large and small blasts and atypical megakaryocytes can be seen. FAB, French-American-British (Co-operative Group classification subtype).

to describe and classify AML, and according to this system, a finding of 30% blasts in marrow or peripheral blood was required to make the diagnosis.77 More recently, a WHO classification suggests that a finding of 20% blasts is sufficient for this diagnosis.78 AML blasts can be placed into the following categories on the basis of their appearance (Fig. 104-4): • Minimally differentiated AML (FAB M0) blasts are nondescript. Without immunophenotyping, it is very difficult to identify these cells as being of myeloid origin.

• AML with differentiation (FAB M1) defines cases with sparse cytoplasmic granules, only occasional Auer rods, and positive myeloperoxidase staining. • AML with maturation (FAB M2) is more clearly myeloid in origin, with increased cytoplasmic granules, clear myeloperoxidase positivity, and the frequent presence of Auer rods. • APL (FAB M3) is characterized by intense cytoplasmic granulation that often obscures the nucleus. A microgranular variant exists with marked nuclear folding and only subtle cytoplasmic granulations. In both subtypes, blasts stain intensely with Sudan black or myeloperoxidase. M3 AML is invariably associated with t(15;17) or one of its variants. • AML with myelomonocytic differentiation (FAB M4) often is characterized by dysplastic features, such as hypogranular cytoplasm and nuclear hyposegmentation. One subset associated with inv(16)(p13q22) is characterized by increased eosinophilia. In M4 AML, blasts stain positively with both myeloperoxidase and nonspecific esterase. Diagnosis of M4 AML is further strengthened by immunophenotyping demonstrating both myeloid and monocytic antigens. • Acute monocytic leukemia (FAB M5) is characterized by blasts with folded nuclei and abundant cytoplasm that stains positively with nonspecific esterase but is myeloperoxidase-negative. • AML (FAB M6), acute erythroid leukemia, may be associated with a variable appearance but usually is accompanied by dysplastic erythroid elements that, on occasion, can become the predominant cell type. • The diagnosis of acute megakaryocytic leukemia (FAB M7) requires that more than 30% of blasts be of the megakaryocytic lineage. These blasts often display clumping, multinucleation, and cytoplasmic blebbing, but immunophenotyping is usually required to make the diagnosis. Although in the past, considerable time and effort went into categorizing AML cases among these morphologic categories, morphology in fact has almost no significance once cytogenetic and (to a lesser extent) immunophenotypic information is taken into consideration.

Immunophenotype AML cases can be categorized according to the combinations of myeloid-associated antigens displayed on the surface of the malignant blast. In undifferentiated AML cases, including FAB M0 cases, expression of CD34, CD117, and CD33 is characteristic, but the blasts tend not to express CD65s. In more mature AML types, including most cases of FAB M1 and M2, expression of CD34, CD33, CD13, and CD65s is seen. In leukemias associated with t(8;21), often with M2 morphology, an immunophenotype similar to that with other M2 AML types is present, but expression of the NK marker CD56 and the B lymphoid marker CD19 also is seen. In APLs, blasts uniquely stain strongly with CD15s and weakly with CD15. In addition, they usually do not express CD34 or HLA-DR. In acute myelomonocytic and monocytic leukemias, blasts express CD14, the prototypical monocytic antigen. Early myeloid markers, including CD34 and CD117, generally are absent. Myelomonocytic leukemias associated with inv(16) blasts frequently express the T-cell antigen, CD2. In most acute erythroid leukemias, blasts fail to express early myeloid markers (e.g., CD34) but do express CD36 and CD71 and often express blood group H antigen, the precursor to ABO. In acute megakaryocytic leukemias, blasts react with antibodies to CD41a/ CD61 (GPIIb/IIIa). Mature platelets sometimes can adhere to the surface of M5 AML blasts, so that they appear as in M7 leukemias. In true M7 AML, however, expression of CD14 does not occur.

Cytogenetics Cytogenetic analysis of human leukemias has been absolutely central to the identification of the genetic events involved in leukemogenesis.

Acute Myeloid Leukemia in Adults • CHAPTER 104

In addition, cytogenetics has emerged as by far the single most important diagnostic factor in AML. Conventional cytogenetics involves the staining of metaphase cells and thus requires dividing cells. Because malignant cells in the marrow are more frequent and have a higher mitotic rate, marrow, rather than peripheral blood, is the preferred source for cytogenetic analysis. Cells usually are cultured for 24 hours, with arrest by shortterm incubation with colchicine; then, 20 metaphases typically are analyzed. The abnormalities detected include changes in chromosome number, gains or losses of portions of chromosomes, and reciprocal exchange of genetic material either between two or more chromosomes (translocations) or within a single chromosome (inversions). Two other molecular techniques sometimes are used. Fluorescence in situ hybridization (FISH) techniques involve hybridization of single-stranded DNA probes to homologous single-stranded sequences in chromosomes of metaphase or interphase cells. FISH has the advantage of being able to analyze large numbers of dividing (metaphase FISH) or nondividing (interphase FISH) cells with relatively little effort. Only those abnormalities targeted by the specific probe being applied will be detected, however. Thus, FISH is very useful for monitoring the disappearance or reappearance of a specific translocation—for example, t(9;22) in CML—but is not a substitute for conventional cytogenetics for initial evaluation of AML. Polymerase chain reaction (PCR) is a method capable of amplifying selected regions of DNA through repeated cycles of DNA synthesis, denaturation, and hybridization. To use PCR analysis, the specific gene sequences to be amplified must be known. Standardized PCR assays for several of the more common fusion gene transcripts have been developed and are proving useful for monitoring minimal residual disease. A listing of the most common cytogenetic abnormalities seen in adult AML is provided in Table 104-1. These abnormalities can be categorized according to underlying tumor biology and also prognostic significance (Fig. 104-5). Table 104-1 organizes these abnormalities according to biologic subgroups. Thus, t(8;21), t(16;21), inv(16), and t(16;16) all belong to the core binding factor leukemias. The abnormalities t(4;11), t(9;11), and del(11)(q23) constitute most of the MLL family of AML types. Leukemias involving the RARα gene include t(15;17), t(11;17), and t(5;17), whereas t(6;9) involves the fusion of the DEK and CAN genes. The EVI 1 gene is involved in inv(3) and t(3;3). Monosomy or interstitial deletions of chromosomes 5, 7, 17, and 20 are typical of AML evolving from MDS or developing after previous alkylating agent exposure. Trisomy 8 is quite common in AML and can appear as a sole abnormality or in combination with other abnormalities. By itself, trisomy 8 does not appear to influence prognosis, but when present it often is accompanied by other unfavorable risk cytogenetic abnormalities.79 Trisomy 11, 13, and 21 also often are seen in AML. A number of studies have analyzed the outcomes in patients with AML according to cytogenetics and have demonstrated that both complete remission rates and duration are strongly associated with pretreatment cytogenetics. In general, patients can be categorized as having a favorable, intermediate, or unfavorable cytogenetic risk status. Two of the largest prospective studies of adult AML were published by the Medical Research Council (MRC) and the Southwest Oncology Group (SWOG).55,56 These studies concerned adults younger than 60 years of age with newly diagnosed AML treated using contemporary chemotherapy and transplantation approaches. As noted in Table 104-2, the two groups of patients reached very similar categorizations, with CBF and RARα leukemias defining the group with favorable risk status; “normal” leukemias and trisomy 8, the intermediate-risk group; and abnormalities of chromosomes 5 and 7 and complex abnormalities, the poor-risk group. Some controversy remains about whether 11q23 leukemias should be considered to reflect intermediate or unfavorable risk status. Overall, as noted in Table 104-2, 85% to 90% of favorable-risk, 75% to 80%

Table 104-1

Cytogenetic Abnormalities in Acute Myeloid Leukemia

Abnormality

Incidence (%)*

Core binding factor translocations t(8;21) inv(16) or t(16;16) Retinoic acid receptor translocations

8 9 10

t(15;17) Mixed-lineage leukemia translocations t(9;11)

2

t(10;11)

1

Other MLL translocations

3

Trisomies +8

9

+21

3

Other trisomies

6

Deletions −5 (5q−)

6

−7 (7q−)

8

−9 (9q−)

3

Complex†

10

Other

17

None—normal

40

*All patients with a specific abnormality are considered, whether or not an additional cytogenetic change is present. Thus, because some patients are counted twice, the total incidence is greater than 100%. † Complex is defined as a clone with at least five abnormalities.

of intermediate-risk, and 55% to 60% of poor-risk patients are predicted to achieve complete remission. Survival at 5 years also is strongly associated with risk group, with 55% to 65% of good-risk, 38% to 41% of intermediate-risk, and only 11% to 15% of poor-risk patients predicted to be alive. Recent studies suggest that patients with no cytogenetic abnormalities and therefore in the intermediaterisk category can be further categorized as having a better or worse prognosis on the basis of NPM1 and FLT3 mutational status, with improved outcomes seen in those with NPM1 mutation but without mutation of FLT3.75,76 As discussed later under “Primary Treatment,” some of these outcomes are dependent on the particular treatment used. For example, the favorable outcomes seen in CBF leukemias may particularly depend on the use of high-dose cytarabine in the treatment regimen, whereas the use of allogeneic transplantation may overcome to some extent the impact of unfavorable risk cytogenetics.55,80

CLASSIFICATION The FAB classification schema for AML is presented in Table 104-3.77 This system, which has been in use for several decades, relies totally on morphology and is of only limited therapeutic or prognostic usefulness. More recently, the WHO has offered an alternative schema78 (Table 104-4). This schema comprises the following subgroups: AML with the most common recurrent genetic abnormalities, AML types that evolve from MDS, AML types that are clearly therapy related, and AML that does not fall into any of the other three categories, for which the system resorts to a morphologic categorization similar to the previous FAB system.

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A

B

C Figure 104-5 • Common cytogenetic abnormalities in adult acute myeloid leukemia. A, Red arrows mark the regions of chromosome breakage and rejoining. B, M2 subtype: diagrammatic systematized description of the structural aberration t(8;21). C, M3 subtype: acute promyelocytic leukemia (APL): Systematized description of the structural aberration t(15;17). (Courtesy of Prof. L.M. Secker-Walker.)

CLINICAL MANIFESTATIONS The initial clinical manifestations of AML usually are nonspecific and relate to the diminished production of normal blood cells. The onset most often is insidious over the course of several weeks to months, and it is not uncommon for a patient to be seen several times before a blood count is finally taken and the diagnosis of leukemia is suspected. Most patients complain of a brief, virus-like illness with fatigue and malaise. Some patients present with a chief complaint of easy bruising, and occasionally, a nonhealing skin wound brings the patient to a doctor’s attention. Anemia is present at diagnosis in most

patients, causing fatigue, pallor, headache, and, in the predisposed patient, angina. Thrombocytopenia usually is present, and when asked, approximately one third of patients note easy bruising, bleeding gums, epistaxis, or other evidence of bleeding at diagnosis. Approximately one third of patients with AML have significant infections (most often of bacterial origin) when the diagnosis is finally made. In addition to suppressing normal blood production, leukemia can infiltrate normal organs. Diffuse bone tenderness is a finding in approximately 25% of patients. Chloromas, which are local collections of blasts, can manifest as rubbery, fast-growing, soft-tissue

Table 104-2 Impact of Cytogenetics on Complete Response and Survival in Acute Myeloid Leukemia* Risk Status with Specific Cytogenetic Patterns

INCIDENCE (%)

CR RATES (%)

5-YEAR SURVIVAL RATE (%)

SWOG

MRC

SWOG

MRC

SWOG

MRC

20

23

84

91

55

65

46

66

76

86

38

41

30

10

55

63

11

14

4

—

54

—

24

—

Favorable inv(16), t(16;16), t(8;21), t(15;17) Intermediate normal, +8, +6, −y Unfavorable del5q, −5, del7q, −7, complex Unknown risk

CR, complete response; MRC, Medical Research Council; SWOG, Southwest Oncology Group. *The SWOG study includes only adults and does not exclude secondary AML, whereas the MRC data refer to both children and adults and exclude cases of secondary AML. In the SWOG data, 11q23 is defined as unfavorable, whereas in the MRC data, 11q23 is considered intermediate risk.

Acute Myeloid Leukemia in Adults • CHAPTER 104

Table 104-3 FAB Classification of Acute Myeloid Leukemia Subtype

Definition

M0: acute undifferentiated leukemia

≥30% blasts 30% blasts plus hypergranular promyelocytes

ACUTE MYELOID LEUKEMIA WITH MULTILINEAGE DYSPLASIA Following MDS or MDS/MPD Without antecedent MDS or MDS/MPD, but with dysplasia in at least 50% of cells in two or more myeloid lineages

Monocytosis

ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES, THERAPY-RELATED

>30% myeloblasts + monoblasts + promonocytes

Alkylating agent/irradiation-related type

>20% nonspecific esterase-positive

M7: acute megakaryocytic leukemia

Acute myeloid leukemia with abnormal bone marrow eosinophils and inv(16)(p13q22) or t(16;16)(q13;q22), CBFβ/MYH11

Acute myeloid leukemia with 11q23 (MLL) abnormalities

>20% myeloperoxidase-positive

M6: acute erythroid leukemia

Acute myeloid leukemia with t(8;21)(q22;q22), AML1/ETO

>30% blasts

Intense myeloperoxidase positivity

M5: acute monoblastic leukemia

ACUTE MYELOID LEUKEMIA WITH RECURRENT GENETIC ABNORMALITIES

Acute promyelocytic leukemia with t(15;17)(q22q12), PML/RARα and variants

>10% myeloid cells mature beyond blast stage

M4: acute myelomonocytic leukemia

World Health Organization Classification of Acute Myeloid Leukemia

30% myeloblasts + monoblasts + promonocytes

ACUTE MYELOID LEUKEMIA, NOT OTHERWISE CATEGORIZED

80% nonspecific esterase-positive

Acute myeloid leukemia, minimally differentiated

≥30% of nonerythroid cells are myeloblasts

Acute myeloid leukemia without maturation

>50% erythroid elements

Acute myelomonocytic leukemia

>30% blasts (myeloblasts + megakaryoblasts)

Acute monoblastic leukemia

>30% megakaryocytic elements defined by immunophenotyping or electron microscopy

Acute myeloid leukemia with maturation

Acute erythroid leukemia (erythroid/myeloid and pure erythroleukemia) Acute megakaryocytic leukemia Acute basophilic leukemia Acute panmyelosis with myelofibrosis Myeloid sarcoma

masses. Gingival hyperplasia due to leukemic infiltration of the gums sometimes is seen, particularly with M5 AML (Fig. 104-6). Leukemia sometimes infiltrates the skin, resulting in a raised, nonpruritic rash termed leukemia cutis (Fig. 104-7). Uncommonly, an occasional patient may present with meningeal signs or cranial neuropathies (most often affecting cranial nerve IV or VII) due to infiltration of the CNS with leukemia.

Figure 104-6 • Leukemic infiltration of the gums results in their expansion and thickening, with partial covering of the teeth.

MDS, myelodysplastic syndrome; MLL, mixed-lineage leukemia; MPD, myeloproliferative disease. Data from ref. 78.

LABORATORY MANIFESTATIONS Peripheral blood counts are abnormal at diagnosis in virtually every case of AML. Most patients have a normochromic, normocytic anemia. Most also are thrombocytopenic, with 50% of patients having less than 50,000 platelets/mm3 and 25% having below 20,000/mm3. Most patients are granulocytopenic, but the total WBC count is more variable. Approximately 25% have very high WBC counts (greater than 50,000/mm3), approximately 25% have low WBC counts (less than 5000/mm3), and the remainder are in between. Blasts usually can be seen in peripheral blood smears. Bone marrow examination generally reveals a hypercellular marrow containing 20% to 100% blast cells largely replacing the normal marrow. The morphologic, immunologic, and cytogenetic characteristics of AML are described in earlier sections of this chapter. The partial thromboplastin and prothrombin times can be prolonged, and in APL, reduced fibrinogen and other evidence of disseminated intravascular coagulation (DIC) are not infrequent. Results

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prevent uric acid nephropathy. Ideally, stable venous access should be established by placement of a Hickman or similar catheter. The diagnosis of leukemia causes a profound shock to the patient and family and has far-reaching implications. Thus, in addition to stabilizing the patient medically, many practitioners find it valuable to have a formalized conference at which the patient and family can be instructed about the nature of leukemia, the immediate plans for therapy, and the likely consequences of treatment.

Management of Emergencies

A

B

Figure 104-7 • Acute myeloid leukemia: M5 subtype. A, Multiple, raised, erythematous skin lesions caused by leukemic infiltration. B, Close-up view of nodular skin lesion. (From Hoffbrand AV, Pettit JE: Color Atlas of Clinical Hematology, 3rd ed. St. Louis, Mosby, 2000.)

of blood chemistry studies usually are normal, although in patients presenting with very aggressive and advanced disease, some evidence of tumor lysis syndrome at presentation is not uncommon, with hyperkalemia, hyperphosphatemia, hyperuricemia, hypocalcemia, increased lactate dehydrogenase, and renal insufficiency. This syndrome more often manifests shortly after therapy is initiated and can be rapidly fatal if it goes untreated. Occasional patients with monocytic leukemia may have tumor infiltration of the kidneys sufficient to cause renal impairment. Lumbar puncture will reveal unsuspected involvement with leukemia in approximately 2% of patients.81

DIFFERENTIAL DIAGNOSIS The diagnosis of AML usually is straightforward. The distinction between AML and advanced MDS is made according to the percentage of blasts and often is arbitrary, with little clinical relevance. Distinguishing AML from ALL can virtually always be accomplished using immunophenotyping. CML in myeloid blast crisis can mimic AML, but the presence of the Philadelphia chromosome, splenomegaly, and myeloid cells at all levels of differentiation distinguish CML from AML. Other small round cell neoplasms can infiltrate the marrow, sometimes mimicking leukemia, but immunologic markers easily differentiate between the two conditions. Leukemoid reactions sometimes are seen in infections such as tuberculosis, but the proportion of blasts in the marrow in nonmalignant diseases virtually never reaches the 20% to 30% required for a diagnosis of AML. Infectious mononucleosis and other viral infections sometimes can resemble ALL but are almost never confused with AML.

PRIMARY TREATMENT Advances in chemotherapy, HCT, and supportive care now enable many patients with AML to be cured. These therapeutic measures are complex, however, so they are best conducted at centers with appropriate experience and support services. Leukemia often is a rapidly progressive disease; accordingly specific therapy should be initiated soon after diagnosis—usually within 72 hours. Before therapy is initiated, acute hemorrhage and infection should be brought under control if at all possible. Patients should be hydrated and given allopurinol, 100 to 200 mg orally three times a day, to

Any of number of treatable emergencies may require management before specific antileukemic therapy can begin. Severe bleeding from thrombocytopenia usually can be controlled with platelet transfusions. DIC typically is associated with a diagnosis of APL. This coagulopathy rapidly abates with the institution of ATRA therapy, so many of the measures used in the past to attempt control of DIC (i.e., low-dose heparin, fresh-frozen plasma, and fibrinogen) are now no longer needed. In patients with fever and granulocytopenia, blood specimens should be obtained for culture, but meanwhile, broadspectrum antibiotic therapy should be started on an empirical basis. Very high WBC counts may constitute early evidence of tumor lysis syndrome; in such instances, the patient should be hydrated, placed on allopurinol to prevent further uric acid production, and given acetazolamide (500 mg daily) to alkalinize the urine. Patients presenting with very high WBC counts (greater than 100,000/mm3) also are at risk for hemorrhage or microinfarctions of small vessels, presumably due to leukostasis. Lung involvement can result in pulmonary infiltrates and hypoxia; CNS leukostasis can lead to mental status changes, seizures, and sudden death. Pulmonary or CNS leukostasis represents a medical emergency requiring intravenous hydration and measures to lower the blast count immediately. Although oral hydroxyurea often is used, whether it lowers counts any faster than intravenous cyclophosphamide, daunorubicin, or cytarabine is unknown. Leukapheresis is of short-term benefit.82 Patients with CNS symptoms should be given whole-brain irradiation emergently. Leukostasis has been associated with the expression of the adhesion molecule CD14 on malignant blasts; this may explain why the syndrome is almost never seen in lymphoid leukemias, in which this antigen is lacking.83

Remission Induction General Principles Without therapy, AML is a rapidly fatal disease; therefore, prompt initiation of antileukemic therapy is appropriate for a majority of patients. Some patients, however, may have a more smoldering variant of AML, often arising from a previous MDS. If such patients are elderly or have other serious medical problems, supportive care measures without attempts at remission induction or less intense chemotherapy designed to slow the progression of disease may be appropriate. The large majority of patients, however, should receive combination chemotherapy in an effort to eradicate the bulk of leukemic cells and allow the regrowth of normal marrow, resulting in a complete remission. Induction chemotherapy generally is given at relatively high doses and is followed by a period of significant pancytopenia before recovery of normal hematopoiesis. As discussed later on, most regimens include 3 days of an anthracycline and 7 days of cytarabine. The usual practice is to check the marrow status at day 14 after initiation of induction and then, if residual leukemic cells remain, to give a second course of therapy. Sometimes it is difficult to distinguish between residual leukemic cells and early recovery of normal hematopoiesis. In these cases, it is advisable to reassess marrow status in a few days. According to International Working Group recommendations, a morphologic complete remission (i.e., “complete response”) requires less than 5% blasts with recovery of peripheral counts to an absolute neutrophil count greater than 1000 mL

Acute Myeloid Leukemia in Adults • CHAPTER 104

and a platelet count greater than 100,000 mL.84,85 Complete remission does not imply eradication of the disease, and with newer, more sensitive technologies, leukemic cells can sometimes be detected in patients with a morphologic complete remission. If only induction therapy is given, disease will recur in essentially every patient. In an effort to consistently define the reasons for failure of induction chemotherapy, the International Working Group suggests that patients be categorized as those with resistant leukemia, those who die from complications of aplasia, and those who die but in whom information is insufficient to determine if the cause is persistent leukemia or failure of marrow recovery.84 Some studies of remission induction conducted in the 1970s suggested that the reasons for treatment failure tended to differ between younger and older patients, with relatively few patients younger than 55 to 60 years of age dying of early treatment-related complications but with a much higher incidence of this problem among older persons. Accordingly, more recently, separate studies have been conducted for younger and older patients.

Remission Induction in Younger Patients For more than 2 decades, standard induction therapy for patients with AML who are younger than 60 years of age generally has included 3 days of an anthracycline and 7 days of cytarabine. Four general questions dominated previous clinical trials of induction therapy: 1. 2. 3. 4.

What are the best type and dose of anthracycline? What are the best dose and schedule of cytarabine? Should additional chemotherapeutic agents be added? Is there a role for hematopoietic growth factors?

Daunorubicin, 45 mg/m2 for 3 days, generally has been viewed as the standard anthracycline component of therapy. Four randomized trials have compared idarubicin, 12 mg/m2 for 3 days, with daunorubicin, 45 mg/m2 for 3 days, both given with standard-dose cytarabine.86–89 The complete response rates were higher with idarubicin in three of the four trials, particularly in patients younger than 50. A problem with these studies is that idarubicin and daunorubicin were not compared at equitoxic doses. For example, the degree of myelosuppression during consolidation was considerably greater with idarubicin. No prospective randomized trial has yet been completed comparing daunorubicin in a dose of 45 mg/m2 with daunorubicin at 60 or 70 mg/m2—doses that have been shown to be well tolerated by patients younger than 60 years. Nor have randomized trials been reported comparing idarubicin to the higher-dose daunorubicin regimens. On the other hand, sequential trials from both the Southwest Oncology Group (SWOG) and the Eastern Cooperative Oncology Group (ECOG) suggest that in patients below age 60, complete response rates are higher with higher doses of daunorubicin (i.e., 60 or 70 mg/m2 for 3 days) than with lower doses (i.e., 45 mg/kg/m2 for 3 days).90–93 Cytarabine generally is given intravenously at a dose of 100 to 200 mg/m2 per day by bolus or by continuous infusion. Much higher doses of cytarabine are tolerable, and two prospective randomized trials compared a combination of daunorubicin and standard cytarabine with daunorubicin and cytarabine at 2 g/m2 per day for 6 days.91–94 In neither study was the complete response rate increased, although in the SWOG study, patients in the high-dose induction group tended to have an improved disease-free survival. The use of high-dose cytarabine is associated with more frequent and severe toxicities including more nausea, vomiting, and conjunctivitis. In an occasional patient, a disabling cerebellar toxicity may develop. Whether the addition of a third drug to the standard daunorubicin-plus-cytarabine regimen is beneficial is uncertain. Some regimens add 6-thioguanine, but no randomized trial exists showing a benefit. The addition of etoposide was studied by the Australian Leukemia Study Group and did not increase complete response rates but seemed to prolong disease-free survival without benefiting overall survival.95

Because profound myelosuppression always follows administration of induction chemotherapy, a large number of trials have asked whether administration of a myeloid growth factor immediately after completion of induction chemotherapy might hasten marrow recovery, thereby preventing serious and potentially lethal infections and improving complete response rates.96–100 In general, these studies found that administration of a myeloid growth factor after completion of induction chemotherapy accelerates subsequent myeloid recovery. In only a minority of studies, however, did this accelerated recovery result in fewer documented infections, and in only the rare study was the complete response rate or survival affected. In those studies in which addition of growth factor was assessed, the dollars saved by shorter hospitalizations with the use of these factors were approximately balanced by the cost of the agent.101,102 Administration of hematopoietic growth factors simultaneous with chemotherapy in an effort to induce cycling of leukemia and thus sensitize cells to treatment has yielded mixed results.103 In summary, standard induction therapy for younger patients with AML continues to be 3 days of an anthracycline dosed at an intensity approximately equal to that of daunorubicin 60 mg/m2 per day and 7 days of cytarabine. There is a lack of convincing evidence that alternatives in the dosing of cytarabine, inclusion of other chemotherapeutic agents, or the addition of hematopoietic growth factors consistently improves complete response rates or prolongs survival. Despite optimal therapy, as many as 30% of younger adults fail to achieve a complete remission with initial induction therapy—some because they die of treatment complications, others because they have resistant leukemia. Allogeneic HCT can cure 15% to 20% of patients in whom initial induction attempts fail, but the logistics of identifying a donor and initiating transplantation in a timely manner for such patients often are challenging. To facilitate this process, HLA typing should be sought in all younger patients with AML and siblings shortly after diagnosis, rather than waiting until induction has failed and only a very narrow window of opportunity remains for potentially curative therapy.

Remission Induction in Older Patients The advantages seen with more intensive anthracycline dosing—daunorubicin doses above 458 mg/m2 for 3 days or the equivalent—generally have been restricted to younger patients. For patients older than 60 years, most experts suggest limiting the anthracycline to a dose equivalent to daunorubicin 45 mg/m2 for 3 days, although clinical trials are under way to test more intensive dosing. As in younger patients, no clear advantage has been observed for one anthracycline over another when relatively equitoxic doses are used. A recent ECOG study compared daunorubicin with mitoxantrone or idarubicin (all given with standard-dose cytarabine) and found no advantage for any single treatment regimen.93 As with younger patients, no evidence has been found for an advantage of high-dose cytarabine or for the addition of further chemotherapeutic agents to the induction regimen for older patients. Many of the studies of the addition of hematopoietic growth factors to AML induction have been restricted to older patients, but, as with younger patients, the advantages of the addition of growth factor appear to be limited to faster hematopoietic recovery and fewer days with neutropenic fever, but with no consistent improvement in complete response rates or overall survival. In virtually every study conducted to date, the complete response rate drops as the age of patients increases. Although some of this effect could be due to a diminished ability of patients to tolerate therapy and to a tendency of physicians to reduce doses in older patients, even when identical doses of drugs are used and toxic deaths are censored, the incidence of remission failures increases with age. AML among older patients is much more likely to evolve from a myelodysplastic syndrome, to be accompanied by unfavorable-risk cytogenetics, and to be associated with expression of the multidrug resistance gene.104–106 All three of these have been found to be independent risk

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factors mitigating against the likelihood of achieving a complete remission. Thus, in a recent SWOG study of remission induction in patients with AML who are older than 55 years of age, the overall complete response rate with use of a standard preparative regimen was 45%. If patients had none of these three factors, their complete response rate was 81%, whereas if all three factors were present, the complete response rate was less than 15%.104 Similar results have been reported by a group of investigators at the M.D. Anderson Cancer Center.107

Postremission Therapy General Principles If no further therapy is given after remission is achieved, all patients will inevitably relapse and do so rapidly (on average in about 4 months), demonstrating the need for further therapy.108 Three types of postremission therapy are in general use: chemotherapy, autologous HCT, and allogeneic HCT.

Postremission Chemotherapy Postremission chemotherapy usually consists of several cycles of combination chemotherapy given at doses similar to those used for induction. This form of therapy often is termed consolidation when given within a few months of induction or late intensification if given after a greater delay. Some trials also have explored the use of low-dose “maintenance” chemotherapy. Most contemporary protocols include several consolidation cycles of high-dose cytarabine. The rationale for high-dose cytarabine came from studies suggesting that at higher doses, the drug is able to saturate deaminating enzymes resulting in production of higher levels of the active intracellular metabolite ARA-CTP, which in turn leads to enhanced inhibition of DNA synthesis.109 Early phase I and II trials suggested that high-dose cytarabine regimens were tolerated and could result in complete remissions in patients with relapsed AML.110,111 These observations led to the use of high-dose cytarabine for consolidation therapy after initial remission induction, with several phase II studies reporting sustained remissions in 30% to 40% of adults.112,113 Ultimately, several large randomized trials have explored the value of high-dose cytarabine for postremission therapy in adult cases of AML.91,114 The Cancer and Leukemia Group B (CALGB) randomly assigned 596 patients in complete remission to receive four courses of cytarabine at one of three doses: 1. 100 mg/m2/day by continuous infusion for 5 days 2. 400 mg/m2/day by continuous infusion for 5 days 3. 3 g/m2 as a 3-hour intravenous infusion twice daily on days 1, 3, and 5 High rates of CNS toxicity were observed among patients older than 60 years, and subsequent randomizations were limited to younger patients. At 3 years, disease-free survival rates were 21% in the 100-mg group, 25% in the 400-mg group, and 39% in the 3-g group. This trial established three or four doses of cytarabine at 3 g every 12 hours on days 1, 3, and 5 as among the most widely used consolidation regimens for younger patients with AML in first remission. Subsequent analyses have demonstrated that the advantage achieved with the highest-dose regimen was restricted to patients with favorable cytogenetics; thus, the intermediate-dose regimen may be as appropriate for patients with intermediate or unfavorable cytogenetics.80 A SWOG trial randomized patients to receive conventional or high-dose cytarabine during both induction and consolidation.91 The best result (52% 4-year survival rate) was seen among patients who received the high-dose cytarabine regimen, a result consistent with those of the CALGB study. A number of other multiagent postremission chemotherapy regimens have been developed. For example, the MRC reported on a regimen that uses as consolidation a cycle of standard anthracycline

plus cytarabine; a cycle of MACE combining amsacrine, conventional-dose cytarabine plus etoposide; and a cycle of high-dose cytarabine plus mitoxantrone. The reported results looked generally similar to those reported by CALGB and SWOG.115 No large randomized trials have been conducted comparing the various more commonly used consolidation regimens. These regimens, which include repetitive cycles of high-dose therapy, generally are inappropriate for patients older than 60 years of age. Most older patients are given several cycles of combination chemotherapy at moderate dosing, such as 2 days of daunorubicin plus 5 days of conventional-dose cytarabine. Almost no data are available suggesting superiority of any particular consolidation regimen for older patients with AML. With the increasing acceptance of short-term intensive consolidation chemotherapy as the standard for younger patients with AML, the concept of low-dose maintenance has fallen into disuse. Data from randomized trials in both younger and older patients, however, demonstrate that maintenance therapy can prolong the duration of first remission, although an impact on overall survival has not been seen.90,116,117

Autologous Hematopoietic Cell Transplantation The principles underlying the concept of autologous HCT and the general technique are outlined in Chapter 32 and are not repeated here. Based on encouraging results in patients in second or subsequent remission, a number of single-center phase II trials of autologous HCT for AML in first remission were conducted and reported in the mid-1980s.118–120 These small trials provided encouraging results, leading to wider use of the technique. Registry data describing results in hundreds of patients soon became available and suggested leukemia-free survival rates of approximately 45% at 5 years.121 In an effort to minimize the possible impact of treatment selection bias, several large prospective randomized trials have been conducted in which adults in first remission with matched siblings have been assigned to allogeneic transplantation, whereas those without have been randomized to receive either autologous HCT or postremission chemotherapy. Relapse rates were reduced in most trials with the use of autologous transplantation compared with chemotherapy.115,122–124 In several of these studies, autologous transplantation resulted in an improvement in disease-free survival, whereas in the others, it did not. Which patients may best benefit from autologous HCT in first remission is considered in the ensuing discussion, after the presentation of data related to studies of allogeneic transplantation. A large number of questions exist about how best to conduct autologous HCT for AML. The most commonly used preparative regimens are combinations of busulfan plus cyclophosphamide, busulfan plus etoposide, or cyclophosphamide plus total body irradiation (TBI), but few randomized trials have been conducted.125,126 Registry data suggest relative equivalence among regimens. Although gene-marking studies have provided unequivocal evidence that occult tumor cells in remission marrow can contribute to the risk of relapse, no comparative clinical trials have been published to confirm that the methods of ex vivo purging are of any clinical benefit.127,128 The prospective studies that show the greatest benefit of autologous HCT used it after three or four cycles of intensive therapy, whereas the studies that showed the least benefit applied the treatment almost immediately after induction. Because these were randomized trials, effects of patient selection on lead time bias should have been minimized, suggesting that autologous transplantation is of greatest differential benefit if applied after consolidation therapy, rather than as a substitute for it.

Allogeneic Hematopoietic Cell Transplantation The initial application of allogeneic HCT to treat AML was published by Thomas and colleagues129 in 1977, when they reported on

Acute Myeloid Leukemia in Adults • CHAPTER 104

54 patients with recurrent or refractory AML treated with TBIcontaining regimens and allogeneic HCT. At the time of the report, seven of these patients were alive in remission, and a subsequent follow-up more than 13 years later showed that 6 of the 54 remained alive and disease-free.130 In 1979, Thomas and associates131 published the initial results of a trial of allogeneic HCT for patients in first remission, reporting for a small group of patients a 5-year disease-free survival rate in excess of 50%. Similar results were soon reported by other investigators as well.132,133 The results reported in these small, uncontrolled, single-institution series were far superior to anything achieved at the time with conventional chemotherapy, but the potential impact of patient selection bias was unknown. Accordingly, these small, single-institution series were soon followed by a substantial number of single-institution or group trials comparing allogeneic transplantation for patients for whom donors were available with conventional chemotherapy for patients without donors. Studies published in the mid-1980s from the Royal Marsden, Seattle, UCLA, and Genoa all showed a markedly diminished risk of relapse with transplantation, a higher risk of treatment-related death with transplantation, and, in all four studies, improved disease-free survival with transplantation.134–137 Since the mid-1980s, of course, significant changes have occurred in both chemotherapy and the practice of allogeneic HCT, so comparisons of these techniques continue to be made. A recent metaanalysis included all studies published since 1995 that compared allogeneic HCT with chemotherapy or autologous HCT using an intention-to-treat analysis. The meta-analysis comprised 3100 subjects and demonstrated an overall survival advantage for allogeneic transplantation with a summary hazard ratio of 1.15. The survival advantage was greatest for patients with high-risk leukemia (hazard ratio, 1.39) but was lost in patients with favorable-risk cytogenetics (hazard ratio, 0.9).138 The general technique of allogeneic HCT is described in Chapter 32 and is not repeated here, except for the following few points specifically relevant to AML: Postremission consolidation chemotherapy before allogeneic HCT for AML in first remission does not improve outcome compared with proceeding directly to transplantation after achievement of first remission.139 The optimal preparative regimen for transplantation of AML in first complete remission is arguable. One prospective randomized trial demonstrates superiority of cyclophosphamide plus TBI over busulfan plus cyclophosphamide.140 A subsequent large registry study, however, found equivalence between the two approaches.125 Although bone marrow has been the usual source of stem cells, three recent randomized trials have shown faster engraftment with the use of granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood without increasing acute graft-versus-host disease (GVHD).141–143 In all three studies, a trend toward more chronic GVHD was observed with the use of peripheral blood, but in two of three trials, overall survival was improved with the use of peripheral blood as a source of stem cells. The combination of cyclosporine plus methotrexate is the most commonly used form of GVHD prophylaxis. Although some encouraging phase I and II studies of T-cell depletion have been published, no randomized trials have demonstrated an advantage of T-cell depletion for patients with AML in first remission.144 Recent trials using unrelated donors have shown results approaching those achieved with fully matched siblings.

Selection of Appropriate Postremission Therapy Postremission chemotherapy, autologous transplantation, and allogeneic transplantation all represent viable treatment options for the younger patient with AML in first remission. Opinions vary among experts, but in general, most would recommend allogeneic transplantation using a matched sibling or an unrelated donor for patients with AML and unfavorable-risk cytogenetics. For patients with favorablerisk cytogenetics, most experts would recommend consolidation che-

motherapy with repetitive cycles of high-dose cytarabine. Opinions regarding management of patients with intermediate-risk cytogenetics are more varied and may be influenced by subtle risk factors such as the age of the patient, the WBC count at diagnosis, and NPM1 and FLT3 mutation status, with transplantation being favored for patients with higher-risk disease. The foregoing recommendations are consistent with those of the National Comprehensive Cancer Network.145 For patients older than 60 years of age, conventional myeloablative transplantation normally is not used, although recent studies of nonmyeloablative transplantation for patients older than 60 are showing promise.146 The advantages of autologous transplantation seen in randomized trials were restricted to patients younger than 60. With older patients, as with all patients with AML, participation in well-designed clinical trials is appropriate and should be pursued actively.

TREATMENT OF RECURRENT ACUTE MYELOID LEUKEMIA General Principles Allogeneic or autologous HCT is the only therapy shown to be curative in a substantial proportion of patients with recurrent AML. For those patients who are transplantation candidates, who are found to be in early first relapse, and who have a previously identified source of stem cells, it may be appropriate to proceed directly to transplantation.147,148 For all other patients, an initial trial of chemotherapy in an attempt to obtain a second remission is appropriate (Box 104-1).

Reinduction Chemotherapy A number of large observational studies of reinduction chemotherapy have been published over the last 2 decades.149–153 In general, complete response rates have ranged from 30% to 50%, and the mortality rates associated with reinduction have been 15% to 25%. Three prognostic factors have consistently been identified with an improved outcome: younger age, favorable cytogenetic risk group, and longer duration of first remission.

Box 104-1.

MANAGEMENT OF NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA IN PATIENTS YOUNGER THAN 60 YEARS OF AGE

Induction Daunorubicin, 60 mg/m2/day for 3 days (or idarubicin, 12 mg/m2/day for 3 days), plus cytarabine, 200 mg/m2/day for 7 days, is the standard induction regimen.

Postremission Favorable risk: Cytarabine, 3 g/m2 over 3 hours every 12 hours, is given on days 1, 3, and 5 for four courses; autologous stem cells are stored if no HLA-matched sibling donor is available. Intermediate risk: If an HLA-matched sibling is available, allogeneic transplantation is indicated. If no HLA-matched sibling is available, cytarabine, 3 g/m2 over 3 hours, every 12 hours on days 1, 3, and 5, is given for two courses, followed by autologous transplantation. Unfavorable risk: If an HLA-matched sibling or HLA-matched unrelated donor is available, allogeneic transplantation is performed. If no such donor is available, management is the same as for intermediate-risk disease. HLA, human leukocyte antigen.

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A limited number of randomized trials have been conducted in this group of patients. Vogler154 found that adding etoposide to highdose cytarabine was of no advantage, whereas Karanes155 demonstrated a benefit of adding mitoxantrone to high-dose cytarabine. List and associates156 conducted a randomized trial testing whether the addition of cyclosporine to a regimen of high-dose cytarabine plus infusional daunorubicin would be of benefit. This study was based on the high incidence of multidrug resistance in recurrent AML and the ability of cyclosporine at clinically achievable levels to reverse drug resistance. In this randomized trial, the investigators found a drug resistance reduced incidence of resistant leukemia with the addition of cyclosporine and a resulting improvement in both disease-free and overall survival. Gemtuzumab ozogamicin combines a humanized anti-CD33 antibody with the potent antitumor agent calicheamicin. Gemtuzumab ozogamicin was developed on the basis of the observation that CD33 is expressed in virtually all cases of AML but not by normal hematopoietic stem cells or in nonhematopoietic tissues, and on the conclusion that by targeting CD33, a less toxic, effective therapeutic might result. Phase I studies showed saturation of CD33 antigenic sites at 9 mg/m2 and clearing of leukemic blasts in many patients at this dose.157 Subsequent phase II trials showed a complete response rate of 30% with less toxicity than might be expected with aggressive combination chemotherapy.158 On the basis of this result, gemtuzumab ozogamicin was approved by the U.S. Food and Drug Administration (FDA) for the treatment of recurrent AML in older patients. A large number of agents are in clinical trials including FLT-3 inhibitors, farnesyl transferase inhibitors, HD inhibitors, antiangiogenic agents, inducers of apoptosis, and deoxyadenosine analogs, among others.

Hematopoietic Cell Transplantation for Recurrent Disease Approximately 30% of patients with AML who undergo transplantation of marrow from matched siblings for untreated first relapse can expect to become long-term disease-free survivors.159–162 Autologous transplantation using marrow previously stored in first remission has resulted in a 26% 5-year disease-free survival rate.161 These outcomes are not markedly less than what might be expected for transplantation in second remission, so for those patients in early relapse with an identified source of stem cells, immediate stem cell transplantation is a reasonable option. A majority of patients will require reinduction, however. For those patients who achieve a second remission, have a matched sibling, and are younger than 55 years, allogeneic transplantation is the preferred form of therapy, and cure can be expected in 35% of cases.162 Although the published experience is less extensive, similar cure rates have been reported for older patients undergoing reduced-intensity allogeneic HCT.146 For patients without matched siblings, either autologous or matched unrelated donor transplantation should be considered. No randomized trials have been conducted comparing either approach to further chemotherapy or the two approaches to one another. In a retrospective case-control study by the European Bone Marrow Transplant Group, no statistically significant difference in disease-free survival or overall survival rate was found between autologous and matched unrelated-donor transplantation approaches.163 Without further outcome data, the decision between autologous versus unrelated-donor transplantation for AML in second remission is difficult, but an allogeneic approach may be appropriate for younger patients with poor-risk disease characteristics (e.g., a short remission duration and unfavorable cytogenetics), whereas autologous transplantation is a good strategy for older patients with more favorable disease characteristics, including long duration of first remission. The results of transplantation for patients in whom reinduction cannnot be achieved are less favorable, and long-term survival can be

expected in only 10% to 20% of patients undergoing allogeneic transplantation for refractory AML.164

TREATMENT OF ACUTE PROMYELOCYTIC LEUKEMIA APL is distinguished by both the t(15;17) translocation and a unique pattern of drug sensitivity demanding a different treatment strategy from those for other categories of AML. Specifically, APL is particularly sensitive to anthracyclines and to all-trans-retinoic acid (ATRA), and with appropriate use of both drugs, a high percentage of APL patients can expect to be cured165–167 (Box 104-2).

Initial Therapy A number of studies have been conducted in the attempt to define the best ways to incorporate high-dose anthracyclines plus ATRA into the management of newly diagnosed patients with APL. The use of ATRA as a single agent for induction results in complete response rates as high as those achieved with conventional chemotherapy regimens including anthracycline, and in improved overall survival.168 A European APL study has since shown that concurrent administration of ATRA with the chemotherapy regimen results in an improvement in overall event-free survival (84% versus 77% at 2 years).169 Combining chemotherapy and ATRA during induction has the added benefit of reducing the incidence of the retinoic acid syndrome (to be discussed shortly) from 25% down to less than 10%, and this is now considered standard therapy. Consolidation chemotherapy for APL generally involves giving repeated cycles of a regimen consisting of an anthracycline and ATRA. The optimal number of cycles of consolidation is unknown. A clear role is recognized for maintenance therapy in APL.170 In a large North American Intergroup study, patients were randomly assigned to receive maintenance therapy with daily ATRA or to observation. Those who received ATRA had an improved disease-free survival.168,171 Because ATRA induces enzymes that enhance its

Box 104-2.

MANAGEMENT OF ACUTE PROMYELOCYTIC LEUKEMIA

Newly Diagnosed Disease Induction ATRA, 45 mg/m2/day until CR is achieved, plus daunorubicin, 60 mg/ m2/day for 3 days, and cytarabine, 200 mg/m2/day for 7 days, is the standard induction regimen. Consolidation Two cycles of the following regimen are given: ATRA, 45 mg/m2/day for 7 days, and daunorubicin, 50 mg/m2/day for 3 days. Maintenance ATRA, 45 mg/m2/day for 15 days every 3 months, plus 6-MP, 100 mg/ m2/day, and MTX, 10 mg/m2/week for 2 years, is the maintenance regimen.

Recurrent Disease Induction Arsenic trioxide, 0.15 mg/kg daily, is given until a second CR is achieved. Consolidation Autologous transplantation is indicated if PCR assay-negative stem cells are available; otherwise, allogeneic transplantation is performed if a suitable donor is available. ATRA, all-trans-retinoic acid; CR, complete response; 6-MP, 6-mercaptopurine; MTX, methotrexate; PCR, polymerase chain reaction.

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metabolism, many investigators have suggested that intermittent ATRA therapy may be advantageous. The European APL 93 trial randomized patients to receive intermittent ATRA, 6-mercaptopurine plus methotrexate, or a combination of intermittent ATRA plus the combination chemotherapy. The patients in the treatment group receiving maintenance ATRA plus chemotherapy had the best overall survival.169 With current management, the prognosis for patients with APL has improved remarkably from what it was in the 1980s. For example, in the North American Intergroup Trial, the 5-year survival rate for patients randomized to receive ATRA induction and maintenance therapies was 69%.168,171 Both age and WBC count at diagnosis have prognostic importance, with best results seen in patients younger than 55 years and those presenting with a WBC count less than 10,000/mm3.172 Because of the excellent outcome with current regimens, transplantation has no generally accepted role during first remission of APL.

Treatment of Recurrent Acute Promyelocytic Leukemia In addition to its unique sensitivity to ATRA, APL also is remarkably sensitive to treatment with arsenic trioxide, as first reported by investigators from China.173,174 A more recent multicenter study has reported an 85% complete response rate among patients with recurrent APL using arsenic trioxide.175 The most important forms of toxicity include prolongation of the QT interval and a syndrome essentially identical to the retinoic acid syndrome, to be discussed shortly. The QT prolongation requires careful monitoring, because two recent reports have documented sudden deaths associated with the use of arsenic trioxide.176,177 Thus, metabolic abnormalities that also prolong the QT interval, such as hypokalemia, hypophosphatemia, and hypomagnesemia, should be corrected before therapy is initiated. Currently, reinduction with arsenic trioxide generally is considered as the initial treatment of choice for patients with APL in whom first-line therapy has failed, particularly if that failure occurred within 12 months of therapy with ATRA. Data are limited regarding the expected duration of second remission induced with arsenic trioxide. Although some patients may remain in remission for some time if given arsenic plus chemotherapy, most experts would recommend either autologous or allogeneic HCT for patients with APL in second remission. Sanz and associates178 presented results from the European Blood and Marrow Transplant Group for patients with APL in second remission and reported overall survival rates of 58% for allogeneic transplantation and 40% for autologous transplantation. The choice of autologous or allogeneic HCT may be influenced by the status of the autologous stem cell source. In a small but provocative study, Meloni reported on 15 patients with APL in second remission undergoing autologous transplantation. Only one of eight patients who received PCR assaynegative marrow subsequently relapsed, whereas all seven who received PCR assay-positive marrow did so.179

Retinoic Acid Syndrome After therapy with ATRA, a proportion of patients with APL exhibit a syndrome manifested by fever, weight gain, respiratory distress, pulmonary infiltrates and effusions, episodic hypotension, and renal failure.180 When ATRA is used as a single agent, this syndrome can be seen in as many as 25% of cases. Risk factors for the development of the syndrome are not obvious, but the simultaneous administration of chemotherapy during induction has seemed to diminish the risk of its development. The mortality rate for this syndrome, when first described, was about 30%, but with the recognition that the syndrome responds dramatically to the institution of dexamethasone, mortality rates have fallen to less than 5%. The observation that an identical syndrome can be seen with arsenic trioxide suggests that the

syndrome probably is associated with the differentiation of APL cells and is inaccurately named.

Supportive Care The treatment of AML is accompanied by a substantial number of complications and thus is best conducted at a center experienced in the management of these sometimes complex cases. During the granulocytopenic period after intensive induction and consolidation chemotherapy, most patients become febrile, and bacterial infections can be documented in roughly 50%. Gram-positive organisms (e.g., Staphylococcus epidermidis) and gram-negative enteric organisms (e.g., Escherichia coli, Klebsiella/Aerobacter) are common, but the experience can vary by medical center. Reactivation of herpes simplex infections can add to mucositis, and fungal infections can develop in patients on antibiotics. Thus, we generally recommend the following oral antimicrobial prophylactic regimen before initiation of chemotherapy: ciprofloxacin, 500 mg every 12 hours; fluconazole, 200 mg daily; and acyclovir, 800 mg every 12 hours. Patients who become febrile while neutropenic should be started on broad-spectrum antibiotics, such as monotherapy with imipenem, or a combination of an antipseudomonal penicillin and a third-generation cephalosporin. If patients are persistently febrile after 72 hours of broad-spectrum antibiotic therapy, additional antibiotics or antifungal coverage should be considered. The choice should be dictated by the unique clinical circumstances of the patient. Patients should continue on broad-spectrum antibiotics until recovery of granulocyte counts and defervescence are obtained. In patients with APL, hemoglobin should be maintained above 8 to 10 g/dL, and platelet counts should be kept above 10,000/mm3 in the absence of symptoms and above 20,000/mm3 in the presence of fever or hypertension. Many patients may become candidates for HCT, so if patients are cytomegalovirus (CMV)-seronegative at the start of induction, every effort should be made to ensure that they continue to receive either CMV-seronegative or filtered blood products. Malnutrition can be a significant problem, particularly in the older, frailer patient. Early institution of hyperalimentation should be considered if oral intake becomes inadequate.

MONITORING RESPONSE TO THERAPY Substantial effort has been made to develop assays of leukemic burden that are more sensitive than the admittedly gross estimates afforded by morphologic examination.181 Such assays, if sensitive, specific, and predictive of outcome, could be used to guide therapy so that, as one example, a patient in first remission could be taken to transplantation if the response to initial chemotherapy predicted a high probability of relapse, or the patient could be spared the toxicities of transplantation if the extent of response to initial therapy was so favorable as to predict a high probability of cure. Efforts to develop such assays have, to date, focused on PCR-based methods of detection of leukemia-specific translocations and multidimensional flow cytometry. The major attraction of PCR-based assays is their high level of sensitivity and specificity. In AML, however, no universal translocations have been recognized, so separate studies, each involving only a small subset of AML cases, have been required. The most encouraging results have come from the study of APL, in which PCR-based assays for t(15;17) conducted after induction and consolidation were predictive of outcome.182,183 The results with other translocations have been more perplexing. PCR-based assays for t(8;21) do not appear to predict relapse, and many patients show persisting positive PCR assays while remaining in complete remission for years after completing all therapy. Miyamoto and colleagues184 studied 18 patients off therapy for 1 to 12 years, all of whom remained PCR assay-positive for the AML/ETO chimeric transcript. The positive

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signal could be found in CD34+ marrow cells, and analyses of the PCR assay-positive colonies demonstrated them to be clonal. These results help explain why, at present, no easy PCR-based methods are available to monitor response to therapy in most patients with AML. An alternative approach to monitoring response to therapy is the use of multidimensional flow cytometry. This approach is not nearly as sensitive as PCR analysis, which detects 1 cell per 103 to 104. Flow cytometry does have the advantage that it is broadly applicable in virtually all cases of AML. Two different studies, one in pediatric AML and a second in adult AML, suggest that a flow-based assessment of the initial remission marrow sample adds information beyond that of routine prognostic factors regarding which patients are likely to relapse within the first several years after diagnosis.185,186

FUTURE DIRECTIONS IN TREATMENT With increased understanding of the molecular events involved in the development of AML, the number of potential therapeutic targets has grown. These targets can be divided into three general categories: 1. Those that are the immediate consequences of the mutational events leading to AML 2. Those that are the adaptive changes a leukemic cell must make to stay alive given the initial mutational event 3. Those that immunologically distinguish AML cells from normal hematopoietic stem cells The most common mutations in AML involve FLT-3, and because these mutations are activating, clinical trials are exploring the use of a number of inhibitors of FLT-3 tyrosine kinase, including CEP-701, PKC-412, SU11248, and CT53518. Ras molecules are downstream from tyrosine kinase receptors, so they themselves can be mutated in AML. Because farnesylation is required for RAS function, trials of several farnesyl transferase inhibitors (including R11597 and BMS214662) have been conducted. The CBF and retinoic acid

receptor translocations result in recruitment of HDs, which are thought to inhibit transcription, thereby contributing to the malignant phenotype. Thus, a number of HD inhibitors (including phenylbutyrate, trichostatin, depsipeptide, and MS-275) are being studied in AML. The mutational event or events giving rise to AML could require the cell to make other adaptive changes not required of normal cells in order to survive. Such responses might be particularly required during moments of cell stress. One example is Bcl-2, which is overexpressed in almost all AML samples compared with normal marrow and may be further overexpressed when cells are exposed to chemotherapy. Thus, agents that inhibit Bcl-2, such as Bcl-2 antisense, are being studied alone and with chemotherapy for AML. Cell surface antigens are being increasingly explored as targets for antibody- and cellular-based therapies. As noted earlier, the antiCD33 calicheamicin conjugate gemtuzumab ozogamicin is an active agent for the treatment of recurrent AML. Recent studies combining it with standard-dose daunorubicin and cytarabine as initial induction therapy have reported encouraging results, with 85% and 86% complete response rates, respectively, in two phase II trials.187,188 Studies using antibodies to target radionuclides to marrow in efforts to develop improved preparative regimens for transplantation also are yielding encouraging outcomes.189 The markedly lower rates of disease recurrence after allogeneic transplantation compared with identical-twin transplantation have encouraged further research into the development of cell-based immunotherapies. Some of these approaches, such as the further development of nonablative allogeneic transplants, attempt to make use of polymorphic minor histocompatibility differences between donor and host. Other investigators are exploring the possibility of targeting T cells to either mutational fusion proteins, such as the RUNX-1/MTG-8 protein, or to overexpressed self-antigens, such as PR3 and WT1. This long list of therapies currently in development offer great hope that the outcomes of treatment for patients with AML will continue to improve.

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