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2015v1.0
Abeloff’s
CLINICAL
ONCOLOGY
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Abeloff’s
CLINICAL
ONCOLOGY
SIXTH EDITION
JOHN E. NIEDERHUBER, MD
Executive Vice President, Inova Health System
President and CEO, Genomics and Bioinformatics Research Institute
Fairfax, Virginia;
Professor, Department of Public Health Sciences
Member, Center for Public Health Genomics
University of Virginia School of Medicine
Charlottesville, Virginia;
Adjunct Professor, Oncology and Surgery
The Johns Hopkins University School of Medicine
Deputy Director
Johns Hopkins Clinical Research Network
Baltimore, Maryland

JAMES O. ARMITAGE, MD JAMES H. DOROSHOW, MD


Joe Shapiro Professor of Medicine Bethesda, Maryland
University of Nebraska Medical Center
Omaha, Nebraska

MICHAEL B. KASTAN, MD, PhD JOEL E.TEPPER, MD


Executive Director, Duke Cancer Institute Hector MacLean Distinguished Professor of
William and Jane Shingleton Professor, Cancer Research
Pharmacology and Cancer Biology Department of Radiation Oncology
Professor of Pediatrics UNC Lineberger Comprehensive Cancer Center
Duke University School of Medicine University of North Carolina School of Medicine
Durham, North Carolina Chapel Hill, North Carolina
ABELOFF’S CLINICAL ONCOLOGY, SIXTH EDITION ISBN: 978-0-323-47674-4

Copyright © 2020 by Elsevier, Inc. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording, or any information storage and retrieval system, without
permission in writing from the publisher. Details on how to seek permission, further information about the
Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance
Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.

This book and the individual contributions contained in it are protected under copyright by the Publisher
(other than as may be noted herein).

Notices

Knowledge and best practice in this field are constantly changing. As new research and experience
broaden our understanding, changes in research methods, professional practices, or medical treatment
may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating
and using any information, methods, compounds, or experiments described herein. In using such
information or methods they should be mindful of their own safety and the safety of others, including
parties for whom they have a professional responsibility.
With respect to any drug or pharmaceutical products identified, readers are advised to check the
most current information provided (i) on procedures featured or (ii) by the manufacturer of each product
to be administered, to verify the recommended dose or formula, the method and duration of
administration, and contraindications. It is the responsibility of practitioners, relying on their own
experience and knowledge of their patients, to make diagnoses, to determine dosages and the best
treatment for each individual patient, and to take all appropriate safety precautions.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors,
assume any liability for any injury and/or damage to persons or property as a matter of products liability,
negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas
contained in the material herein.

Previous editions copyrighted 2014, 2008, 2004, 2000, and 1995.

Library of Congress Control Number: 2018953655

Executive Content Strategist: Robin Carter


Content Development Manager: Laura Schmidt
Publishing Services Manager: Catherine Jackson
Senior Project Manager: Amanda Mincher
Design Direction: Bridget Hoette

Printed in China

Last digit is the print number: 9 8 7 6 5 4 3 2 1

1600 John F. Kennedy Blvd.


Ste 1600
Philadelphia, PA 19103-2899
To my son, Matthew, and my wife, Kathy, who have and continue to make sacrifices so that I might pursue my passions in
medicine and research. To my colleagues at the National Cancer Institute, University of Virginia, Johns Hopkins, and across the
country, whose selfless dedication to patient care and cancer research is truly an inspiration to all. To the many students who have
trained with me over the years, to my patients, and to my colleagues at the Inova Translational Medicine Institute, who have given
me the opportunity to have this tremendously rewarding career. Lastly, to Tracey, and to Marty, who, in memory, inspire all who
knew them to work a little harder each day toward the elimination of the pain and suffering from this disease.
JOHN E. NIEDERHUBER, MD

To my wife, Nancy, for her love and support over 49 ½ years.


JAMES O. ARMITAGE, MD

To my wife, Robin Winkler Doroshow, MD, my classmate and greatest supporter, for her love, dedication, and commitment and
for the remarkable joy and caring she brings to her patients and to all around her. To my remarkable daughter, Deborah Doroshow,
MD, PhD, who is completing her training for a career in academic oncology; my fondest hope is that you will enjoy sharing with
and learning from those you help as much as I have. To my patients and colleagues at the City of Hope and the National Cancer
Institute who have all contributed so much of themselves to my continuing education as a physician and investigator, please accept
my appreciation and utmost gratitude.
JAMES H. DOROSHOW, MD

To my wife, Kathy, and my sons, Benjamin, Nathaniel, and Jonathan. You are the lights of my life. I also acknowledge all of my
mentors, colleagues, and patients, who have taught me so much. A special note of gratitude goes to Marty Abeloff, a mentor and an
inspiring role model for career and for life.
MICHAEL B. KASTAN, MD, PhD

To my wife, Laurie, who has been my soul mate for many years and has constantly reminded me of life’s priorities. To my family
including my daughters, Miriam and Abigail, and my grandchildren, Zekariah, Zohar, Samuel, Marcelo, Jonah, and Aurelio. They
have been an inspiration. To my many teachers through the years who have helped define and foster my professional career, but
especially Herman Suit and Eli Glatstein.
JOEL E. TEPPER, MD
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Memoriam

Martin D. Abeloff, MD (1942-2007)

Martin D. Abeloff, a founding editor of Clinical Oncology, dedicated that would be as valuable to the practicing oncologist as to the primary
his life to caring for patients with cancer and to teaching his art to care physician and physicians-in-training. The first edition of Clinical
fellows, residents, and students. He was a brilliant and caring clinician, Oncology was published in 1995 to a gratifying response. It is now
an extremely effective leader, and a beloved mentor to many trainees established as a cornerstone reference for those caring for patients
and young faculty. with cancer.
Marty was born on April 4, 1942, in Shenandoah, Pennsylvania. He In the sixth edition, we continue Marty’s vision for an ever better,
received his BA from The Johns Hopkins University in 1963 and his unique, and accessible text so that future generations of oncologists
MD from The Johns Hopkins University School of Medicine in 1966. will remember his inspiration and leadership.
He spent the next year as an intern at the University of Chicago Hospitals The editors again dedicate this text, which is already a recognized
and Clinics. His legacy in medicine was established on his return to tangible aspect of his legacy in medicine, as a living memorial to him.
Baltimore in 1971 as a fellow in clinical oncology. He would spend the Abeloff ’s Clinical Oncology will continue to serve as a reminder to all
rest of his career at The Johns Hopkins Hospital, achieving the rank of its users of this extraordinary person and exemplary physician who
Professor of Medicine in 1990. At various times, he served as the fel- went before them.
lowship training program director, chief of medical oncology, clinical
director of the cancer center, oncologist in chief at The Johns Hopkins John E. Niederhuber, MD
Hospital, and in 1992, was appointed the second director of The Johns James O. Armitage, MD
Hopkins Oncology Center, later renamed, thanks to Marty’s efforts, the James H. Doroshow, MD
Sidney Kimmel Comprehensive Cancer Center. Michael B. Kastan, MD, PhD
It was during his time as cancer center director that Marty brought Joel E. Tepper, MD
to life the idea of a comprehensive, user-friendly textbook of oncology

vii
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Contributors

James L. Abbruzzese, MD, FACP, FASCO, DSc (hon) Dara L. Aisner, MD, PhD
Duke Cancer Institute Distinguished Professor of Medical Oncology Associate Professor of Pathology
Chief, Division of Medical Oncology, Department of Medicine CU Anschutz Medical Campus
Associate Director for Clinical Research, Duke Cancer Institute University of Colorado
Duke University Medical Center Aurora, Colorado
Durham, North Carolina
Michelle Alonso-Basanta, MD, PhD
Omar Abdel-Wahab, MD Helene Blum Assistant Professor
Associate Attending Department of Radiation Oncology
Department of Medicine University of Pennsylvania
Leukemia Service Philadelphia, Pennsylvania
Memorial Sloan Kettering Cancer Center
New York, New York Jesus Anampa, MD, MS
Assistant Professor
Ghassan K. Abou-Alfa, MD Department of Oncology
Attending Physician Montefiore Medical Center
Memorial Sloan Kettering Cancer Center Albert Einstein College of Medicine
Professor of Medicine Bronx, New York
Weill Cornell Medicine
New York, New York Megan E. Anderson, MD
Assistant Professor
Janet L. Abrahm, MD Department of Orthopaedic Surgery
Professor of Medicine Harvard Medical School
Harvard Medical School Attending Orthopedic Surgeon
Member, Division of Palliative Care Department of Orthopedic Surgery
Psychosocial Oncology and Palliative Care Boston Children’s Hospital
Dana-Farber Cancer Institute Attending Orthopedic Surgeon
Boston, Massachusetts Department of Orthopedic Surgery
Beth Israel Deaconess Medical Center
Jeffrey S. Abrams, MD Boston, Massachusetts
Associate Director, Cancer Therapy Evaluation Program
Division of Cancer Treatment and Diagnosis Emmanuel S. Antonarakis, MD
National Cancer Institute Associate Professor of Oncology
Rockville, Maryland Sidney Kimmel Comprehensive Cancer Center
Johns Hopkins University School of Medicine
Jeremy S. Abramson, MD, MMSc Baltimore, Maryland
Director, Center for Lymphoma
Hematology/Oncology Richard Aplenc, MD, PhD
Massachusetts General Hospital Department of Pediatrics
Assistant Professor Section Chief, Hematologic Malignancies
Department of Medicine Chief Clinical Research Officer
Harvard Medical School Children’s Hospital of Philadelphia
Boston, Massachusetts Philadelphia, Pennsylvania

ix
x Contributors

Frederick R. Appelbaum, MD Karen Basen-Engquist, PhD, MPH


Executive Vice President and Deputy Director Professor of Behavioral Science
Fred Hutchinson Cancer Research Center University of Texas MD Anderson Cancer Center
Professor Houston, Texas
Division of Medical Oncology
University of Washington Lynda Kwon Beaupin, MD
Seattle, Washington Director, Adolescent and Young Adult Program
Roswell Park Cancer Institute
Luiz H. Araujo, MD, PhD Buffalo, New York
Scientific Director
COI Institute for Research and Education Ross S. Berkowitz, MD
Brazilian National Cancer Institute William H. Baker Professor of Gynecology
Rio de Janeiro, Brazil Department of Obstetrics and Gynecology
Harvard Medical School
Ammar Asban, MD Director of Gynecologic Oncology
Surgical Resident Department of Obstetrics and Gynecology
Department of Surgery Brigham and Women’s Hospital
University of Alabama at Birmingham Boston, Massachusetts
Birmingham, Alabama
Donald A. Berry, PhD
Edward Ashwood, MD Professor of Biostatistics
President and CEO Department of Biostatistics
ARUP Laboratories The University of Texas MD Anderson Cancer Center
Professor of Pathology Houston, Texas
University of Utah
Salt Lake City, Utah Therese Bevers, MD
Professor of Clinical Cancer Prevention
Farrukh T. Awan, MD, MS Medical Director, Cancer Prevention Center
Associate Professor of Medicine The University of Texas MD Anderson Cancer Center
Hematology Houston, Texas
The Ohio State University
Columbus, Ohio John F. Boggess, MD
Professor of Obstetrics and Gynecology
Juliet L. Aylward, MD University of North Carolina
Associate Professor of Dermatology Chapel Hill, North Carolina
University of Wisconsin School of Medicine and Public Health
Madison, Wisconsin Julie R. Brahmer, MD, MSc
Professor of Oncology
Arjun V. Balar, MD Department of Oncology
Associate Professor of Medicine Johns Hopkins Kimmel Cancer Center
Division of Hematology/Oncology Baltimore, Maryland
Director, Genitourinary Cancers Program
New York University Perlmutter Cancer Center Janet Brown, MD, FRCP, MSc, MBBS, BSc
New York University Langone Medical Center Professor
New York, New York Academic Unit of Clinical Oncology, Oncology, and Metabolism
Weston Park Hospital
Courtney J. Balentine, MD University of Sheffield
Assistant Professor of Surgery Sheffield, United Kingdom
Dallas VA Hospital
University of Texas Southwestern Karen Brown, MD
Dallas, Texas Attending Physician
Memorial Sloan Kettering Cancer Center
Stefan K. Barta, MD, MS, MRCP(UK) Professor of Clinical Radiology
Associate Professor Weill Medical College at Cornell University
Hematology and Oncology New York, New York
Fox Chase Cancer Center
Philadelphia, Pennsylvania Powel Brown, MD, PhD
Professor and Chairman
Nancy Bartlett, MD Clinical Cancer Prevention
Professor of Medical Oncology The University of Texas MD Anderson Cancer Center
Washington University School of Medicine Houston, Texas
St. Louis, Missouri
Contributors xi

Ilene Browner, MD Stephen J. Chanock, MD


Assistant Professor Director
Department of Oncology and Division of Geriatric Medicine Division of Cancer Epidemiology and Genetics
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins National Cancer Institute
and Johns Hopkins Bayview Bethesda, Maryland
The Johns Hopkins University
Baltimore, Maryland Claudia I. Chapuy, MD
St. Elizabeth’s Medical Center
Paul A. Bunn, MD Dana-Farber Cancer Institute
Distinguished Professor of Medical Oncology Boston, Massachusetts
CU Anschutz Medical Campus
University of Colorado Vikash P. Chauhan, PhD
Aurora, Colorado Massachusetts Institute of Technology
Boston, Massachusetts
William R. Burns, MD
Assistant Professor of Surgery Herbert Chen, MD, FACS
University of Michigan Health System Chairman and Fay Fletcher Kerner Endowed Chair
Ann Arbor, Michigan Department of Surgery
University of Alabama at Birmingham
John C. Byrd, MD Surgeon-in-Chief
Professor of Internal Medicine–Hematology University of Alabama at Birmingham Health System
The Ohio State University Birmingham, Alabama
Columbus, Ohio
Ronald C. Chen, MD, MPH
Karen Cadoo, MD Associate Professor
Attending Medical Oncologist Department of Radiation Oncology
Gynecologic Medical Oncology and Clinical Genetic Services University of North Carolina at Chapel Hill
Memorial Sloan Kettering Cancer Center Chapel Hill, North Carolina
Weill Cornell Medical College
New York, New York Nai-Kong V. Cheung, MD, PhD
Enid A. Haupt Endowed Chair in Pediatric Oncology
David P. Carbone, MD, PhD Department of Pediatrics
Professor of Medicine Memorial Sloan-Kettering Cancer Center
Director, James Thoracic Center New York, New York
James Cancer Center
The Ohio State University Medical Center Jennifer H. Choe, MD, PhD
Columbus, Ohio Medical Instructor
Division of Medical Oncology
H. Ballentine Carter, MD Duke Cancer Institute
Professor of Urology Durham, North Carolina
Johns Hopkins University School of Medicine
Baltimore, Maryland Michaele C. Christian, MD
Cancer Therapy Evaluation Program (Retired)
Jorge J. Castillo, MD National Cancer Institute
Physician Rockville, Maryland
Hematologic Malignancies
Dana-Farber Cancer Institute Paul M. Cinciripini, PhD
Assistant Professor Professor and Chair of Behavioral Science
Harvard Medical School The University of Texas MD Anderson Cancer Center
Boston, Massachusetts Houston, Texas

Alfred E. Chang, MD Michael F. Clarke, MD


Hugh Cabot Professor of Surgery Professor of Medicine
University of Michigan Health System Division of Oncology
Ann Arbor, Michigan Stanford School of Medicine
Palo Alto, California
Eric Chang, MD, FASTRO
Professor and Chair of Radiation Oncology Robert E. Coleman, MBBS, MD
Keck School of Medicine of USC Academic Unit of Clinical Oncology
Los Angeles, California Weston Park Hospital
University of Sheffield
Sheffield, United Kingdom
xii Contributors

Robert L. Coleman, MD Jeffrey Crawford, MD


Professor and Executive Director, Cancer Network Research Professor of Medicine
Department of Gynecologic Oncology and Reproductive Medicine Division of Medical Oncology
The University of Texas MD Anderson Cancer Center Duke Cancer Institute
Houston, Texas Durham, North Carolina

Adriana M. Coletta, PhD, RD Kristy Crooks, PhD


Department of Behavioral Science Assistant Professor of Pathology
Center for Energy Balance in Cancer Prevention and Survivorship CU Anschutz Medical Campus
The University of Texas MD Anderson Cancer Center University of Colorado
Houston, Texas Aurora, Colorado

Jerry M. Collins, PhD Daniel J. Culkin, MD


Associate Director Professor
Division of Cancer Treatment and Diagnosis Department of Urology
National Cancer Institute University of Oklahoma Health Sciences Center
Bethesda, Maryland Oklahoma City, Oklahoma

Jean M. Connors, MD Brian G. Czito, MD


Hematology Division Professor of Radiation Oncology
Brigham and Women’s Hospital Duke University Medical Center
Dana-Farber Cancer Institute Durham, North Carolina
Harvard Medical School
Boston, Massachusetts Piero Dalerba, MD
Assistant Professor of Pathology and Cell Biology
Michael Cools, MD Assistant Professor of Medicine
Department of Neurosurgery Division of Digestive and Liver Diseases
University of North Carolina Columbia University College of Physicians and Surgeons
Chapel Hill, North Carolina New York, New York

Kevin R. Coombes, PhD Josep Dalmau, MD, PhD


Professor of Biomedical Informatics ICREA Research Professor
The Ohio State University Hospital Clínic/Institut d’Investigació Biomèdica August Pi i Sunyer
Columbus, Ohio (IDIBAPS)
Barcelona, Spain
Jorge Cortes, MD Adjunct Professor Neurology
The University of Texas MD Anderson Cancer Center University of Pennsylvania
Houston, Texas Philadelphia, Pennsylvania

Mauro W. Costa, MSc, PhD Mai Dang, MD, PhD


Research Scientist Instructor in Neurology
The Jackson Laboratory Children’s Hospital of Philadelphia
Bar Harbor, Maine Philadelphia, Pennsylvania

Anne Covey, MD Michael D’Angelica, MD


Attending Physician Attending Physician
Memorial Sloan Kettering Cancer Center Memorial Sloan Kettering Cancer Center
Professor of Radiology Professor of Surgery
Weill Medical College at Cornell University Weill Medical College at Cornell University
New York, New York New York, New York

Kenneth H. Cowan, MD, PhD Kurtis D. Davies, PhD


Director, Fred and Pamela Buffett Cancer Center Assistant Professor of Pathology
University of Nebraska Medical Center CU Anschutz Medical Campus
Omaha, Nebraska University of Colorado
Aurora, Colorado
Christopher H. Crane, MD
Vice Chairman
Attending Physician
Memorial Sloan Kettering Cancer Center
New York, New York
Contributors xiii

Myrtle Davis, DVM, PhD James H. Doroshow, MD


Chief, Toxicology and Pharmacology Branch Bethesda, Maryland
Division of Drug Treatment and Diagnosis
National Cancer Institute Jay F. Dorsey, MD, PhD
National Institutes of Health Associate Professor of Radiation Oncology
Bethesda, Maryland University of Pennsylvania
Philadelphia, Pennsylvania
Nicolas Dea, MD, MSc, FRCSC
Spinal Neurosurgeon Marianne Dubard-Gault, MD, MS
Clinical Associate Professor Medical Genetics Fellow
Department of Surgery Department of Medicine
Vancouver General Hospital Memorial Sloan Kettering Cancer Center
University of British Columbia New York, New York
Vancouver, British-Columbia, Canada
Steven G. DuBois, MD, MS
Ana De Jesus-Acosta, MD Associate Professor
Assistant Professor of Oncology Department of Pediatrics
Sidney Kimmel Comprehensive Cancer Center Harvard Medical School
The Johns Hopkins University School of Medicine Attending Physician
Baltimore, Maryland Department of Pediatrics
Boston Children’s Hospital
Angelo M. DeMarzo, MD, PhD Dana Farber Cancer Institute
Professor of Pathology Boston, Massachusetts
Johns Hopkins University School of Medicine
Baltimore, Maryland Dan G. Duda, PhD, DMD
Associate Professor
Theodore L. DeWeese, MD Harvard Medical School
Professor and Director of Radiation Oncology and Molecular Boston, Massachusetts
Radiation Sciences
Johns Hopkins University School of Medicine Malcolm Dunlop, MD
Baltimore, Maryland MRC Institute of Genetics and Molecular Medicine
The University of Edinburgh
Maximilian Diehn, MD, PhD Western General Hospital
Associate Professor of Radiation Oncology Edinburgh, United Kingdom
Stanford University
Palo Alto, California Linda R. Duska, MD
University of Virginia Health System
Subba R. Digumarthy, MD Emily Couric Clinical Cancer Center
Massachusetts General Hospital Charlottesville, Virginia
Boston, Massachusetts
Madeleine Duvic, MD
Angela Dispenzieri, MD Professor and Deputy Chairman
Professor of Medicine and Laboratory Medicine Department of Dermatology
Mayo Clinic The University of Texas MD Anderson Cancer Center
Rochester, Minnesota Houston, Texas

Khanh T. Do, MD Imane El Dika, MD


Assistant Professor of Medicine Assistant Attending Physician
Harvard Medical School Memorial Sloan Kettering Cancer Center
Medical Oncology Instructor of Medicine
Dana-Farber Cancer Institute Weill Medical College at Cornell University
Boston, Massachusetts New York, New York

Konstantin Dobrenkov, MD Hashem El-Serag, MD, MPH


Clinical Director, Oncology Margaret M. and Albert B. Alkek Chair of the Department
Merck & Company, Inc. of Medicine
Kenilworth, New Jersey Professor of Gastroenterology and Hepatology
Baylor College of Medicine
Jeffrey S. Dome, MD, PhD Houston, Texas
Vice President, Center for Cancer and Blood Disorders
Children’s National Medical Center
Washington, D.C.
xiv Contributors

Jeffrey M. Engelmann, PhD Debra L. Friedman, MD, MS


Assistant Professor of Psychiatry and Behavioral Medicine Vanderbilt-Ingram Cancer Center
Medical College of Wisconsin Nashville, Tennessee
Milwaukee, Wisconsin
Arian F. Fuller, Jr., MD
David S. Ettinger, MD, FACP, FCCP Winchester Hospital
Alex Grass Professor of Oncology North Reading Medical
The Sidney Kimmel Comprehensive Cancer Center at Johns North Reading, Massachusetts
Hopkins Hospital
The Johns Hopkins University Lorenzo Galluzzi, PhD
Baltimore, Maryland Assistant Professor of Cell Biology in Radiation Oncology
Weill Cornell Medical College
Lola A. Fashoyin-Aje, MD, MPH New York, New York
Medical Officer
Office of Hematology and Oncology Products Mark C. Gebhardt, MD
Center for Drug Evaluation and Research Frederick W. and Jane M. Ilfeld Professor of Orthopaedic Surgery
U.S. Food and Drug Administration Harvard Medical School
Silver Spring, Maryland Surgeon-in-Chief
Department of Orthopedic Surgery
Eric R. Fearon, MD, PhD Beth Israel Deaconess Medical Center
Maisel Professor of Oncology Orthopedic Surgeon
Professor of Internal Medicine Department of Orthopedics
University of Michigan Medical School Children’s Hospital
Ann Arbor, Michigan Boston, Massachusetts

James M. Ford, MD Daniel J. George, MD


Professor of Medicine, Pediatrics, and Genetics Professor of Medicine
Division of Oncology and Medical Genetics Duke University Medical Center
Stanford University School of Medicine Durham, North Carolina
Stanford, California
Mark B. Geyer, MD
Wilbur A. Franklin, MD Assistant Attending
Professor Emeritus of Pathology Department of Medicine
CU Anschutz Medical Campus Leukemia Service and Cellular Therapeutics Center
University of Colorado Memorial Sloan Kettering Cancer Center
Aurora, Colorado Instructor in Medicine
Joan and Sanford I. Weill Department of Medicine
Phoebe E. Freer, MD Weill Cornell Medical College
Associate Professor New York, New York
Radiology and Imaging Sciences
University of Utah Hospitals/Huntsman Cancer Institute Amato J. Giaccia, PhD
Salt Lake City, Utah Jack, Lulu, and Sam Willson Professor of Cancer Biology
Department of Radiation Oncology
Boris Freidlin, PhD Stanford University School of Medicine
Division of Cancer Treatment and Diagnosis Stanford, California
National Cancer Institute
Bethesda, Maryland Mark R. Gilbert, MD
Senior Investigator and Chief
Alison G. Freifeld, MD Neuro-Oncology Branch
Professor of Internal Medicine National Cancer Institute
Infectious Diseases Division Bethesda, Maryland
University of Nebraska Medical Center
Omaha, Nebraska Whitney Goldner, MD
Associate Professor of Internal Medicine
Terence W. Friedlander, MD Division of Diabetes, Endocrinology, and Metabolism
Associate Clinical Professor University of Nebraska Medical Center
Medicine Omaha, Nebraska
UCSF Medical Center
San Francisco, California
Contributors xv

Donald P. Goldstein, MD Missak Haigentz, MD


Professor of Obstetrics, Gynecology, and Reproductive Biology Montefiore Medical Center
Harvard Medical School Bronx, New York
Senior Scientist
Department of Obstetrics and Gynecology John D. Hainsworth, MD
Brigham and Women’s Hospital Chief Scientific Officer
Boston, Massachusetts Sarah Cannon Research Institute
Nashville, Tennessee
Annekathryn Goodman, MD
Massachusetts General Hospital Benjamin E. Haithcock, MD
Boston, Massachusetts Associate Professor of Surgery
University of North Carolina at Chapel Hill
Karyn A. Goodman, MD, MS Chapel Hill, North Carolina
Professor of Radiation Oncology
Grohne Chair in Clinical Cancer Research Christopher L. Hallemeier, MD
University of Colorado School of Medicine Assistant Professor of Radiation Oncology
Aurora, Colorado Mayo Clinic
Rochester, Minnesota
Kathleen Gordon, MD
Medical Director of Ophthalmology Samir Hanash, MD, PhD
IQVIA Evelyn & Sol Rubenstein Distinguished Chair for Cancer Prevention
Co-Chair Professor of Clinical Cancer Prevention
IQIVA Ophthalmology Center of Excellence The University of Texas MD Anderson Cancer Center
Clinical Associate Professor of Ophthalmology Houston, Texas
University of North Carolina at Chapel Hill
Chapel Hill, North Carolina Aphrothiti J. Hanrahan, PhD
Assistant Lab Member
Laura Graeff-Armas, MD, MS Human Oncology and Pathogenesis Program
Associate Professor of Internal Medicine Memorial Sloan Kettering Cancer Center
Division of Diabetes, Endocrine and Metabolism New York, New York
University of Nebraska Medical Center
Omaha, Nebraska James Harding, MD
Assistant Attending Physician
Alexander J. Greenstein, MD, MPH Memorial Sloan Kettering Cancer Center
Associate Professor of Surgery Assistant Professor of Medicine
Icahn School of Medicine at Mount Sinai Weill Medical College at Cornell University
New York, New York New York, New York

Stuart A. Grossman, MD Michael R. Harrison, MD


Professor of Oncology, Medicine, and Neurosurgery Assistant Professor of Medicine
The Sidney Kimmel Comprehensive Cancer Center at Johns Division of Medical Oncology
Hopkins Medicine Duke Cancer Institute
The Johns Hopkins University Durham, North Carolina
Baltimore, Maryland
Muneer G. Hasham, PhD
Stephan Grupp, MD, PhD Research Scientist
Section Chief, Cellular Therapy and Transplant The Jackson Laboratory
Director, Cancer Immunotherapy Frontier Program Bar Harbor, Maine
CCCR Director of Translational Research
Children’s Hospital of Philadelphia Ernest Hawk, MD, MPH
Philadelphia, Pennsylvania Boone Pickens Distinguished Chair for Early Prevention of Cancer
Vice President and Division Head
Arjun Gupta, MD Division of Cancer Prevention and Population Sciences
Assistant Instructor The University of Texas MD Anderson Cancer Center
Department of Internal Medicine Houston, Texas
University of Texas Southwestern Medical Center
Dallas, Texas Jonathan Hayman, MD
Department of Internal Medicine
Irfanullah Haider, MD, MBA Johns Hopkins Bayview Medical Center
Breast Imaging Baltimore, Maryland
Brigham and Women’s Hospital
Boston, Massachussetts
xvi Contributors

Jonathan E. Heinlen, MD Clifford A. Hudis, MD


Assistant Professor Chief Executive Officer
Department of Urology American Society of Clinical Oncology
University of Oklahoma Health Sciences Center Alexandria, Virginia
Oklahoma City, Oklahoma
Stephen P. Hunger, MD
N. Lynn Henry, MD, PhD Jeffrey E. Perelman Distinguished Chair
Associate Professor Department of Pediatrics
Internal Medicine Chief, Division of Oncology
University of Utah Pediatrics
Salt Lake City, Utah Children’s Hospital of Philadelphia
Philadelphia, Pennsylvania
Joseph Herman, MD †
Professor and Division Head ad-interim Arti Hurria, MD
Department of Radiation Oncology Professor
The University of Texas MD Anderson Cancer Center Department of Medical Oncology and Therapeutics Research
Houston, Texas City of Hope Comprehensive Cancer Center
Duarte, California
Brian P. Hobbs, PhD
Associate Staff David H. Ilson, MD, PhD
Quantitative Health Sciences and The Taussig Cancer Institute Attending Physician
Cleveland Clinic Gastrointestinal Oncology Service
Cleveland, Ohio Department of Medicine
Memorial Sloan-Kettering Cancer Center
Ingunn Holen, BSc, MSc, PhD New York, New York
Oncology
University of Sheffield Annie Im, MD
Sheffield, United Kingdom Assistant Professor of Medicine
Department of Hematology and Oncology
Leora Horn, MD, MSc UPMC Hillman Cancer Center
Associate Professor of Medicine Pittsburgh, Pennsylvania
Medicine–Hematology Oncology
Vanderbilt University
Gopa Iyer, MD
Assistant Attending Physician, Genitourinary Oncology Service
Nashville, Tennessee
Department of Medicine
Memorial Sloan Kettering Cancer Center
Neil S. Horowitz, MD
New York, New York
Department of Obstetrics and Gynecology
Division of Gynecologic Oncology
Elizabeth M. Jaffee, MD
Brigham and Women’s Hospital
The Dana and Albert “Cubby” Broccoli Professor of Oncology
Dana Farber Cancer Institute
Deputy Director, Sidney Kimmel Comprehensive Cancer Center at
Boston, Massachusetts
Johns Hopkins
Johns Hopkins University School of Medicine
Steven M. Horwitz, MD Baltimore, Maryland
Associate Attending
Department of Medicine, Lymphoma Service Reshma Jagsi, MD, DPhil
Memorial Sloan Kettering Cancer Center Professor and Deputy Chair
Assistant Professor of Clinical Medicine Radiation Oncology
Weill-Cornell Medical College University of Michigan
New York, New York Ann Arbor, Michigan

Odette Houghton, MD Rakesh K. Jain, PhD


Associate Professor A.W. Cook Professor of Tumor Biology
Department of Ophthalmology Department of Radiation Oncology
Mayo Clinic Harvard Medical School
Scottsdale, Arizona Director
E.L. Steele Laboratory for Tumor Biology
Scott C. Howard, MD, MSc Department of Radiation Oncology
Professor of Acute and Tertiary Care Massachusetts General Hospital
University of Tennessee Health Sciences Center Boston, Massachusetts
Memphis, Tennessee

Deceased.
Contributors xvii

William Jarnagin, MD, FACS Hagop Kantarjian, MD


Winchester Hospital The University of Texas MD Anderson Cancer Center
North Reading Medical Houston, Texas
North Reading, Massachusetts
Giorgos Karakousis, MD
Aminah Jatoi, MD Assistant Professor of Surgery
Professor of Oncology University of Pennsylvania
Mayo Clinic Philadelphia, Pennsylvania
Rochester, Minnesota
Maher Karam-Hage, MD
Anuja Jhingran, MD Professor of Behavioral Science
The University of Texas MD Anderson Cancer Center The University of Texas MD Anderson Cancer Center
Houston, Texas Houston, Texas

David H. Johnson, MD Nadine M. Kaskas, MD


Chairman, Department of Internal Medicine Resident Physician
University of Texas Southwestern Medical School Department of Dermatology
Dallas, Texas The Warren Alpert Medical School of Brown University
Providence, Rhode Island
Brian Johnston, MD
Royal Victoria Hospital Michael B. Kastan, MD, PhD
Belfast, United Kingdom Executive Director, Duke Cancer Institute
Director, Cancer Immunotherapy Frontier Program

Patrick G. Johnston, MD William and Jane Shingleton Professor, Pharmacology and
Center for Cancer Research and Cell Biology Cancer Biology
School of Medicine, Dentistry, and Biomedical Sciences Professor of Pediatrics
Queen’s University Belfast Duke University School of Medicine
Belfast, United Kingdom Durham, North Carolina

Kevin D. Judy, MD Nora Katabi, MD


Professor of Neurosurgery Department of Pathology
Thomas Jefferson University Memorial Sloan-Kettering Cancer Center
Jefferson Medical College New York, New York
Philadelphia, Pennsylvania
Daniel R. Kaul, MD
Lisa A. Kachnic, MD Associate Professor of Infectious Disease
Professor and Chair of Radiation Oncology University of Michigan
Vanderbilt University Medical Center Ann Arbor, Michigan
Nashville, Tennessee
Scott R. Kelley, MD, FACS, FASCRS
Orit Kaidar-Person, MD Assistant Professor of Surgery
Ramban Medical Center Division of Colon and Rectal Surgery
Haifa, Israel Mayo Clinic
Rochester, Minnesota
Sanjeeva Kalva, MD, RPVI, FSIR
Chief, Interventional Radiology Nancy Kemeny, MD
Associate Professor of Radiology Attending Physician
University of Texas Southwestern Medical Center Memorial Sloan Kettering Cancer Center
Dallas, Texas Professor of Medicine
Weill Medical College at Cornell University
Deborah Y. Kamin, RN, MS, PhD New York, New York
Vice President
Policy and Advocacy Erin E. Kent, PhD, MS
American Society of Clinical Oncology Scientific Advisor
Alexandria, Virginia Outcomes Research Branch
Healthcare Delivery Research Program
Division of Cancer Control and Population Sciences
National Cancer Institute
Rockville, Maryland
ICF, Inc.
Fairfax, Virginia

Deceased.
xviii Contributors

Oliver Kepp, PhD Daniel A. Laheru, MD


Metabolomics and Cell Biology Platforms Ian T. MacMillan Professorship in Clinical Pancreatic Research
Gustave Roussy Cancer Campus Department of Medical Oncology
Villejuif, France The Johns Hopkins University School of Medicine
Baltimore, Maryland
Simon Khagi, MD
Assistant Professor Paul F. Lambert, PhD
University of North Carolina School of Medicine Professor of Oncology
Lineberger Comprehensive Cancer Center University of Wisconsin
Chapel Hill, North Carolina Madison, Wisconsin

Joshua E. Kilgore, MD Mark Lawler, PhD


Division of Gynecologic Oncology Chair in Translational Cancer Genomics
University of North Carolina School of Medicine Centre for Cancer Research and Cell Biology
Chapel Hill, North Carolina School of Medicine, Dentistry and Biomedical Sciences
Queen’s University Belfast
D. Nathan Kim, MD, PhD Belfast, United Kingdom
Associate Professor
Department of Radiation Oncology Jennifer G. Le-Rademacher, PhD
University of Texas Southwestern Medical Center Associate Professor of Biostatistics
Dallas, Texas Health Sciences Research
Associate Professor of Oncology
Bette K. Kleinschmidt-DeMasters, MD Mayo Clinic
Professor of Neurology, Neurosurgery, and Pathology Rochester, Minnesota
CU Anschutz Medical Campus
University of Colorado John Y.K. Lee, MD
Aurora, Colorado Associate Professor of Neurosurgery
University of Pennsylvania
Edward L. Korn, PhD Philadelphia, Pennsylvania
Biometric Research Program
National Cancer Institute Nancy Y. Lee, MD
Bethesda, Maryland Department of Radiation Oncology
Memorial Sloan-Kettering Cancer Center
Guido Kroemer, MD, PhD New York, New York
Team 11, Centre de Recherche des Cordeliers
Paris, France Susanna L. Lee, MD, PhD
Massachusetts General Hospital
Geoffrey Y. Ku, MD Boston, Massachusetts
Assistant Attending Physician
Gastrointestinal Oncology Service Jonathan E. Leeman, MD
Department of Medicine Department of Radiation Oncology
Memorial Sloan Kettering Cancer Center Memorial Sloan-Kettering Cancer Center
New York, New York New York, New York

Shivaani Kummar, MD Andreas Linkermann, MD


Professor of Medicine Department of Internal Medicine III
Director, Phase 1 Clinical Research Program Division of Nephrology
Stanford University University Hospital Carl Gustav Carus at the Technische
Palo Alto, California Universität Dresden
Dresden, Germany
Bonnie Ky, MD, MSCE
Assistant Professor of Medicine and Epidemiology Jinsong Liu, MD, PhD
Division of Cardiovascular Medicine Professor of Pathology
Senior Scholar The University of Texas MD Anderson Cancer Center
Center for Clinical Epidemiology and Biostatistics Houston, Texas
University of Pennsylvania School of Medicine
Philadelphia, Pennsylvania Simon Lo, MD, FACR
Professor and Vice Chair for Strategic Planning
Department of Radiation Oncology
Professor of Neurological Surgery
University of Washington School of Medicine
Seattle, Washington
Contributors xix

Jason W. Locasale, PhD Amit Maity, MD


Associate Professor University of Pennsylvania
Department of Pharmacology and Cancer Biology Philadelphia, Pennsylvania
Duke University School of Medicine
Durham, North Carolina Neil Majithia, MD
Mayo Clinic
Charles L. Loprinzi, MD Rochester, Minnesota
Regis Professor of Breast Cancer Research
Department of Oncology Marcos Malumbres, PhD
Mayo Clinic Group Leader
Rochester, Minnesota Cell Division and Cancer
Spanish National Cancer Research Center (CNIO)
Maeve Lowery, MD Madrid, Spain
Professor of Translational Cancer Medicine
Trinity College Karen Colbert Maresso, MPH
Dublin, Ireland Program Director
Division of Cancer Prevention and Population Sciences
Emmy Ludwig, MD The University of Texas MD Anderson Cancer Center
Associate Attending Physician Houston, Texas
Memorial Sloan Kettering Cancer Center
Associate Professor of Medicine John D. Martin, PhD
Weill Medical College at Cornell University The University of Tokyo
New York, New York Tokyo, Japan

Matthew A. Lunning, DO Koji Matsuo, MD, PhD


Associate Professor of Internal Medicine Assistant Professor of Obstetrics and Gynecology
University of Nebraska Medical Center University of Southern California
Omaha, Nebraska Los Angeles, California

Robert A. Lustig, MD Natalie H. Matthews, MD


Professor of Clinical Radiation Oncology Department of Dermatology
University of Pennsylvania The Warren Alpert Medical School of Brown University
Philadelphia, Pennsylvania Providence, Rhode Island

Mitchell Machtay, MD Lauren Mauro, MD


University Hospitals Cleveland Medical Center Assistant Professor of Medicine
Case-Western Reserve University School of Medicine George Washington University School of Medicine
Cleveland, Ohio Washington, D.C.

Crystal Mackall, MD R. Samuel Mayer, MD, MEHP


Endowed Professor of Pediatrics and Medicine Associate Professor and Vice Chair for Education
Stanford University Physical Medicine and Rehabilitation
Director The Johns Hopkins University School of Medicine
Stanford Center for Cancer Cell Therapy Medical Director, Cancer Rehabilitation Program
Director Physical Medicine and Rehabilitation
Parker Institute for Cancer Immunotherapy at Stanford The Johns Hopkins Hospital
Associate Director Baltimore, Maryland
Stanford Cancer Institute
Stanford, California Worta McCaskill-Stevens, MD
Chief, Community Oncology and Prevention Trials Research Group
David A. Mahvi, MD Division of Cancer Prevention
General Surgery Resident National Cancer Institute
Brigham and Women’s Hospital Rockville, Maryland
Boston, Massachusetts
Megan A. McNamara, MD
David M. Mahvi, MD Assistant Professor of Medicine
Professor of Surgery Department of Medical Oncology
Chief, Surgical Oncology Duke University Medical Center
Medical University of South Carolina Durham, North Carolina
Charleston, South Carolina
xx Contributors

Neha Mehta-Shah, MD Jarushka Naidoo, MB, BCH


Assistant Professor Assistant Professor of Oncology
Washington University The Sidney Kimmel Comprehensive Cancer Center at Johns
St. Louis, Missouri Hopkins Hospital
Baltimore, Maryland
Robert E. Merritt, MD
Director, Thoracic Surgery Amol Narang, MD
James Cancer Center Assistant Professor of Radiation Oncology
The Ohio State University Medical Johns Hopkins University School of Medicine
Columbus, Ohio Baltimore, Maryland

Matthew I. Milowsky, MD Heidi Nelson, MD, FACS, FASCRS


Professor of Medicine Professor of Surgery
Division of Hematology/Oncology Division of Colon and Rectal Surgery
UNC Lineberger Comprehensive Cancer Center Mayo Clinic
Chapel Hill, North Carolina Rochester, Minnesota

Lori M. Minasian, MD William G. Nelson, MD, PhD


Deputy Director Professor and Director
Division of Cancer Prevention Sidney Kimmel Comprehensive Cancer Center
National Cancer Institute Johns Hopkins University School of Medicine
National Institutes of Health Baltimore, Maryland
Bethesda, Maryland
Suzanne Nesbit, PharmD, BCPS, CPE
Tara C. Mitchell, MD Clinical Pharmacy Specialist, Pain Management
Assistant Professor of Medicine Research Associate, Department of Oncology
University of Pennsylvania The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins
Philadelphia, Pennsylvania The Johns Hopkins Hospital
Baltimore, Maryland
Demytra Mitsis, MD
Medical Oncology and Hematology Fellow Mark Niglas, MD, FRCPC
Department of Medicine Clinical Fellow
Roswell Park Cancer Institute Department of Radiation Oncology
Buffalo, New York Sunnybrook Health Sciences Centre
Toronto, Ontario, Canada
Michelle Mollica, PhD, MPH, RN
Program Director Tracey O’Connor, MD
Division of Cancer Control and Population Sciences Associate Professor of Oncology
Healthcare Delivery Research Program Department of Medicine
National Cancer Institute Roswell Park Cancer Institute
Bethesda, Maryland Buffalo, New York

Margaret Mooney, MD Kenneth Offit, MD, MPH


Branch Chief, Clinical Investigations Branch Chief, Clinical Genetics Service
Cancer Therapy Evaluation Program Robert and Kate Niehaus Chair in Inherited Cancer Genomics
Division of Cancer Treatment and Diagnosis Memorial Sloan Kettering Cancer Center
National Cancer Institute New York, New York
Rockville, Maryland
Mihaela Onciu, MD
Farah Moustafa, MD Medical Director
Department of Dermatology OncoMetrix Laboratories
The Warren Alpert Medical School of Brown University Poplar Healthcare
Providence, Rhode Island Memphis, Tennessee

Lida Nabati, MD
Instructor in Medicine
Harvard Medical School
Senior Physician
Dana-Farber Cancer Institute
Boston, Massachusetts
Contributors xxi

Eileen M. O’Reilly, MD Steven Z. Pavletic, MD, MS


Winthrop Rockefeller Chair in Medical Oncology Head, Graft-Versus-Host Disease and Autoimmunity Section
Section Head Hepatopancreaticobiliary & Neuroendocrine Cancers Experimental Transplantation and Immunology Branch
Gastrointestinal Oncology Service National Cancer Institute
Associate Director Bethesda, Maryland
David M. Rubenstein Center for Pancreatic Cancer Research
Attending Physician, Member Peter C. Phillips, MD
Memorial Sloan Kettering Cancer Center Professor of Neurology and Oncology
Professor of Medicine The Children’s Hospital of Philadelphia
Weill Medical College at Cornell University Philadelphia, Pennsylvania
New York, New York
Miriam D. Post, MD
Elaine A. Ostrander, PhD Associate Professor of Pathology
Chief and Distinguished Investigator CU Anschutz Medical Campus
Cancer Genetics and Comparative Genomics Branch University of Colorado
National Human Genome Research Institute Aurora, Colorado
Bethesda, Maryland
Amy A. Pruitt, MD
Lisa Pappas-Taffer, MD University of Pennsylvania
Assistant Professor of Clinical Dermatology Philadelphia, Pennsylvania
University of Pennsylvania
Philadelphia, Pennsylvania Christiane Querfeld, MD, PhD
Chief, Division of Dermatology
Drew Pardoll, MD, PhD Director, Cutaneous Lymphoma Program
Director, Bloomberg~Kimmel Institute for Cancer Immunotherapy Assistant Professor of Dermatology
Sidney Kimmel Comprehensive Cancer Center City of Hope
Johns Hopkins School of Medicine Duarte, California
Baltimore, Maryland
Vance A. Rabius, PhD
Jae H. Park, MD Research Director, Tobacco Treatment Program
Assistant Attending The University of Texas MD Anderson Cancer Center
Department of Medicine Houston, Texas
Leukemia Service and Cellular Therapeutics Center
Memorial Sloan Kettering Cancer Center S. Vincent Rajkumar, MD
Assistant Professor of Medicine Edward W. and Betty Knight Scripps Professor of Medicine
Joan and Sanford I. Weill Department of Medicine Division of Hematology
Weill Cornell Medical College Mayo Clinic
New York, New York Rochester, Minnesota

Anery Patel, MD Mohammad O. Ramadan, MD


Clinical Instructor Assistant Professor
Department of Internal Medicine Department of Urology
Division of Diabetes, Endocrine, and Metabolism University of Oklahoma Health Sciences Center
University of Nebraska Medical Center Oklahoma City, Oklahoma
Omaha, Nebraska
Erinn B. Rankin, PhD
Anish J. Patel, MD Assistant Professor of Radiation Oncology, Obstetrics,
Assistant Professor of Endocrinology and Gynecology
Department of Endocrinology Stanford University School of Medicine
University of Alabama at Birmingham Stanford, California
Birmingham, Alabama
Sushanth Reddy, MD
Steven R. Patierno, PhD Assistant Professor of Surgery
Deputy Director Department of Surgery
Duke Cancer Institute University of Alabama at Birmingham
Professor of Medicine Birmingham, Alabama
Professor of Pharmacology and Cancer Biology
Professor of Community and Family Medicine
Duke University Medical Center
Durham, North Carolina
xxii Contributors

Michael A. Reid, PhD Nadia Rosenthal, PhD


Postdoctoral Fellow Scientific Director
Department of Pharmacology and Cancer Biology The Jackson Laboratory
Duke University School of Medicine Bar Harbor, Maine
Durham, North Carolina Chair, Cardiovascular Science
National Heart and Lung Institute
Scott Reznik, MD Imperial College London
Associate Professor London, United Kingdom
Department of Cardiothoracic Surgery
University of Texas Southwestern Medical Center Meredith Ross, MD
Dallas, Texas Fellow
Department of Internal Medicine
Tina Rizack, MD, MPH Division of Diabetes, Endocrinology, and Metabolism
Hematologist/Oncologist University of Nebraska Medical Center
South County Health Omaha, Nebraska
Clinical Assistant Professor of Internal Medicine and Obstetrics
& Gynecology Julia H. Rowland, PhD
Alpert Medical School of Brown University Director, Office of Cancer Survivorship
Providence, Rhode Island Division of Cancer Control and Population Sciences
National Cancer Institute
Jason D. Robinson, PhD Rockville, Maryland
Associate Professor of Behavioral Science
The University of Texas MD Anderson Cancer Center Anthony H. Russell, MD
Houston, Texas Massachusetts General Hospital
Boston, Massachusetts
Leslie Robinson-Bostom, MD
Senior Attending Michael S. Sabel, MD, FACS
Department of Dermatology Associate Professor
The Warren Alpert Medical School of Brown University Surgery
Providence, Rhode Island University of Michigan
Ann Arbor, Michigan
Carlos Rodriguez-Galindo, MD
Departments of Global Pediatric Medicine and Oncology Arjun Sahgal, MD, FRCPC
St. Jude Children’s Research Hospital Professor of Radiation Oncology and Surgery
Memphis, Tennessee Deputy Chief, Department of Radiation Oncology
Sunnybrook Health Sciences Center
Paul B. Romesser, MD University of Toronto Faculty of Medicine
Department of Radiation Oncology Toronto, Ontario
Memorial Sloan-Kettering Cancer Center
New York, New York Ryan D. Salinas, MD
Resident Physician
Steven T. Rosen, MD Department of Neurosurgery
Provost & Chief Scientific Officer University of Pennsylvania
Director, Comprehensive Cancer Center and Beckman Philadelphia, Pennsylvania
Research Institute
Irell & Manella Cancer Center Director’s Distinguished Chair Erin E. Salo-Mullen, MS, MPH, CGC
Helen & Morgan Chu Director’s Chair, Beckman Research Institute Senior Genetic Counselor
City of Hope Clinical Genetics Service
Duarte, California Department of Medicine
Memorial Sloan Kettering Cancer Center
Myrna R. Rosenfeld, MD, PhD New York, New York
Senior Investigator, Neuroimmunology
Institut d’Investigació Biomèdica August Pi i Sunyer (IDIBAPS) Manuel Salto-Tellez, MD
Barcelona, Spain Center for Cancer Research and Cell Biology
Adjunct Professor Neurology School of Medicine, Dentistry, and Biomedical Sciences
University of Pennsylvania Queen’s University Belfast
Philadelphia, Pennsylvania Belfast, United Kingdom
Contributors xxiii

Sydney M. Sanderson, BS Konstantin Shilo, MD


PhD Candidate Department of Pathology
Department of Pharmacology and Cancer Biology James Cancer Center
Duke University School of Medicine The Ohio State University Medical
Durham, North Carolina Columbus, Ohio

John T. Sandlund, MD Eric Small, MD


Member Professor of Medicine
Department of Oncology University of California, San Francisco
St. Jude Children’s Research Hospital San Francisco, California
Professor of Pediatrics
University of Tennessee College of Medicine Angela B. Smith, MD, MS, FACS
Memphis, Tennessee Associate Professor of Urology
Department of Urology
Victor M. Santana, MD University of North Carolina
Department of Oncology Chapel Hill, North Carolina
St. Jude Children’s Research Hospital
Memphis, Tennessee Stephen N. Snow, MD
Professor of Dermatology
Michelle Savage, MD Northwest Permanente
Department of Clinical Cancer Prevention Portland, Oregon
The University of Texas MD Anderson Cancer Center
Houston, Texas David B. Solit, MD
Geoffrey Beene Chair
Eric C. Schreiber, PhD Director, Center for Molecular Oncology
Associate Professor Member, Human Oncology and Pathogenesis Program
Department of Radiation Oncology Attending Physician, Genitourinary Oncology Service, Department
University of North Carolina School of Medicine of Medicine
Chapel Hill, North Carolina Memorial Sloan Kettering Cancer Center
New York, New York
Lynn Schuchter, MD
C. Willard Robinson Professor of Hematology and Oncology Anil K. Sood, MD
Professor of Medicine Professor and Vice Chair
University of Pennsylvania Department of Gynecologic Oncology and Reproductive Medicine
Philadelphia, Pennsylvania The University of Texas MD Anderson Cancer Center
Houston, Texas
Liora Schultz, MD
Department of Pediatric Oncology, Division of Oncology Enrique Soto-Perez-de-Celis, MD
Stanford University International Fellow
Stanford, California Department of Medical Oncology and Therapeutics Research
City of Hope
Michael V. Seiden, MD, PhD Duarte, California
Texas Oncology Researcher in Medical Science
The Woodlands, Texas Cancer Care in the Elderly Clinic
Department of Geriatrics
Morgan M. Sellers, MD, MS Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran
Icahn School of Medicine at Mount Sinai Mexico City, Mexico
New York, New York
Joseph A. Sparano, MD
Payal D. Shah, MD Associate Chairman
Assistant Professor Department of Oncology
Medicine Montefiore Medical Center
University of Pennsylvania Professor of Medicine and Women’s Health
Philadelphia, Pennsylvania Department of Medicine and Oncology
Albert Einstein College of Medicine
Jinru Shia, MD Bronx, New York
Member and Attending Pathologist
Memorial Sloan Kettering Cancer Center Vladimir S. Spiegelman, MD, PhD
Professor of Pathology and Laboratory Medicine Professor of Pediatrics and Pharmacology
Weill Medical College at Cornell University Department of Pediatrics
New York, New York Pennsylvania State University College of Medicine
Hershey, Pennsylvania
xxiv Contributors

Sheri L. Spunt, MD, MBA James E. Talmadge, PhD


Endowed Professor of Pediatric Cancer Professor of Pathology and Microbiology
Department of Pediatrics University of Nebraska Medical Center
Division of Hematology/Oncology Omaha, Nebraska
Stanford University School of Medicine
Stanford, California David T. Teachey, MD
Department of Pediatrics
Zsofia K. Stadler, MD Divisions of Hematology and Oncology
Assistant Attending Physician Children’s Hospital of Philadelphia
Department of Medicine Philadelphia, Pennsylvania
Memorial Sloan Kettering Cancer Center
Assistant Professor of Medicine Catalina V. Teba, MD
Weill Cornell Medical College University Hospitals Cleveland Medical Center
New York, New York Case-Western Reserve University School of Medicine
Cleveland, Ohio
David P. Steensma, MD
Institute Physician Ayalew Tefferi, MD
Department of Medical Oncology Department of Hematology
Dana-Farber Cancer Institute Mayo Clinic
Associate Professor of Medicine Rochester, Minnesota
Harvard Medical School
Boston, Massachusetts Bin Tean Teh, MD, PhD
Professor
Richard M. Stone, MD Division of Medical Sciences
Professor of Medicine National Cancer Centre Singapore
Harvard Medical School Professor, Cancer and Stem Cell Biology Program
Chief of Staff Duke–NUS Medical School
Department of Medical Oncology Singapore
Dana-Farber Cancer Institute
Boston, Massachusetts Joyce M.C. Teng, MD, PhD
Associate Professor of Dermatology and Pediatrics
Steven Kent Stranne, MD, JD Stanford of Medicine
Polsinelli PC Stanford University
Washington, D.C. Palo Alto, California

Kelly Stratton, MD Joel E. Tepper, MD


Assistant Professor Hector MacLean Distinguished Professor of Cancer Research
Department of Urology Department of Radiation Oncology
University of Oklahoma Health Sciences Center University of North Carolina School of Medicine
Oklahoma City, Oklahoma University of North Carolina Lineberger Comprehensive
Cancer Center
Bill Sugden, PhD Chapel Hill, North Carolina
Professor of Oncology
University of Wisconsin Premal H. Thaker, MD
Madison, Wisconsin Professor in Gynecologic Oncology
Division of Obstetrics and Gynecology
Andrew M. Swanson, MD Washington University School of Medicine
Assistant Professor of Dermatology St. Louis, Missouri
University of Wisconsin School of Medicine and Public Health
Madison, Wisconsin Aaron P. Thrift, PhD
Assistant Professor
Martin S. Tallman, MD Dan L. Duncan Comprehensive Cancer Center
Chief, Leukemia Service Department of Medicine, Gastroenterology Section
Memorial Sloan-Kettering Cancer Center Baylor College of Medicine
Professor of Medicine Houston, Texas
Joan and Sanford I. Weill Department of Medicine
Weill Cornell Medical College Arthur-Quan Tran, MD
New York, New York Division of Gynecologic Oncology
University of North Carolina School of Medicine
Chapel Hill, North Carolina
Contributors xxv

Grace Triska, MS Richard L. Wahl, MD


Washington University School of Medicine Elizabeth Mallinckrodt Professor and Director
St. Louis, Missouri Mallinckrodt Institute of Radiology
Washington University School of Medicine
Donald Trump, MD, FACP St. Louis, Missouri
Chief Executive Officer and Executive Director
Inova Schar Cancer Institute Michael F. Walsh, MD, FAAP, FACMG, DABMG
Falls Church, Virginia Assistant Member
Department of Pediatrics and Medicine
Kenneth Tsai, MD, PhD Divisions of Solid Tumor and Clinical Genetics
Associate Member Memorial Sloan Kettering Cancer Center
Anatomic Pathology and Tumor Biology New York City, New York
H. Lee Moffitt Cancer Center and Research Institute
Tampa, Florida Thomas Wang, MD
Professor of Surgery
Chia-Lin Tseng, MD, FRCPC Department of Surgery
Assistant Professor University of Alabama at Birmingham
Radiation Oncologist Birmingham, Alabama
Sunnybrook Health Sciences Centre
Toronto, Ontario, Canada Jared Weiss, MD
Associate Professor of Medicine
Diane Tseng, MD, PhD Section Chief of Thoracic and Head/Neck Oncology
Department of Medicine Division of Hematology and Oncology
Division of Oncology University of North Carolina at Chapel Hill
Stanford University Chapel Hill, North Carolina
Stanford, California
Irving L. Weissman, MD
Sandra Van Schaeybroeck, MD Director, Institute for Stem Cell Biology and Regenerative Medicine
Center for Cancer Research and Cell Biology Director, Stanford Ludwig Center for Cancer Stem Cell Research
School of Medicine, Dentistry, and Biomedical Sciences and Medicine
Queen’s University Belfast Stanford University
Belfast, United Kingdom Palo Alto, California

Brian A. Van Tine, MD, PhD Shannon N. Westin, MD, MPH


Associate Professor Associate Professor of Gynecologic Oncology and
Internal Medicine Reproductive Medicine
Washington University in Saint Louis The University of Texas MD Anderson Cancer Center
St. Louis, Missouri Houston, Texas

Erin R. Vanness, MD Jeffrey D. White, MD


Associate Professor of Dermatology Associate Director, Office of Cancer Complementary and
University of Wisconsin School of Medicine and Public Health Alternative Medicine
Madison, Wisconsin Division of Cancer Treatment and Diagnosis
National Cancer Institute
Gauri Varadhachary, MD Bethesda, Maryland
Professor
Medical Director, Gastrointestinal Center Richard Wilson, MD
Executive Medical Director, Ambulatory Operations Center for Cancer Research and Cell Biology
Department of Gastrointestinal Medical Oncology School of Medicine, Dentistry, and Biomedical Sciences
The University of Texas MD Anderson Cancer Center Queen’s University Belfast
Houston, Texas Belfast, United Kingdom

Marileila Varella-Garcia, PhD Richard J. Wong, MD


Professor of Medicine and Medical Oncology Department of Surgery
CU Anschutz Medical Campus Memorial Sloan-Kettering Cancer Center
University of Colorado New York, New York
Aurora, Colorado
xxvi Contributors

Gary S. Wood, MD Timothy Zagar, MD


Professor and Chair of Dermatology Northeastern Radiation Oncology
University of Wisconsin School of Medicine and Public Health Glens Falls, New York
Middleton VA Medical Center
Madison, Wisconsin Elaine M. Zeman, PhD
Associate Professor
Yaohui G. Xu, MD, PhD Department of Radiation Oncology
Associate Professor of Dermatology University of North Carolina School of Medicine
University of Wisconsin School of Medicine and Public Health Chapel Hill, North Carolina
Madison, Wisconsin
Tian Zhang, MD
Meng Xu-Welliver, MD, PhD Assistant Professor of Medicine
Associate Professor of Radiation Oncology Duke University Medical Center
James Cancer Center Durham, North Carolina
The Ohio State University Medical Center
Columbus, Ohio James A. Zwiebel, MD
Cancer Therapy Evaluation Program (Retired)
Shlomit Yust-Katz, MD National Cancer Institute
Professor Rockville, Maryland
Davidoff Cancer Center
Rabin Medical Center
Petah Tikva, Israel
Preface

New insights into whole genome sequence variations and the genomic useful to students and trainees, experts in the various disciplines of
structural alterations associated with cancer, including their downstream oncology, and as a reference text for physicians from other medical
effects on protein structure and function, are helping us to define specific disciplines and the various staff who regularly care for patients with
communication pathway changes that drive cancer initiation, progression, cancer. It is our hope that readers will find this scholarly textbook
metastasis, and resistance. We have learned that each individual and properly balanced between the disciplines of science, clinical medicine,
each tumor may be unique. Individual physiognomies in terms of path and humanism and that it will serve them well in their efforts to prevent,
of progression and unique cellular communication pathway alterations diagnose, and effectively treat their patients suffering from cancer.
are continuing to define the nature of specific cancers and offer greater The multidisciplinary nature of cancer care is, and will continue to
opportunities for the development of highly prescriptive intervention(s). be, reflected in our editors. Experts in cancer biology, surgical oncology,
In addition, we have a much greater understanding of the relationship pediatric oncology, radiation oncology, medical oncology, and hema-
of the host’s tissues, the patient’s immune system, and the broad tumor tologic malignancies directed the development of the content. Reflecting
microenvironment, to the process of tumor development and progression the multispecialty approach necessary for optimal care of patients, the
and their impact on tumor control. This new body of knowledge on majority of chapters are the joint product of several of these disciplines.
how the body’s immune system and the tumor’s microenvironment Engaging the very best subject matter authorities was a guiding principle
are altered to support disease growth, invasion, and distant spread is for the editors and we are deeply indebted to our outstanding authors
providing opportunities for the development of novel therapeutic who, in a most diligent and thoughtful way, have brought their knowledge
interventions. There is exciting new evidence to support the presence and skills to the sixth edition of Abeloff ’s Clinical Oncology.
of a special subclass of cells within the tumor that has properties of
“stemness,” which places them in the key role of maintaining tumor
ACKNOWLEDGMENTS
growth and tumor spread. The cumulative effect of these advances—
where certain cancers can be prevented and where others will be detected This sixth edition represents a highly collaborative and dynamic effort
earlier and controlled—promises to be transformative in our effort to between the editors and Elsevier. We are greatly indebted to Laura
conquer cancer. Schmidt, Kathleen Schlom, and Kristi Batchelor for their creative input
The sixth edition of Abeloff ’s Clinical Oncology incorporates the and guidance and for turning the principles behind this text into a
exciting advances in basic, translational, clinical, and epidemiologic reality. Finally, we want to express our gratitude to our many contributing
oncology. Each chapter begins with a summary highlighting the key authors for their dedication to this project, their generosity of time,
points and comprises a critical analysis of the literature and updated and, of course, their very valuable friendship.
clinical studies—authors present their own opinions in specially identified
boxes and algorithms. John E. Niederhuber, MD
Despite significant progress, the diagnosis of cancer remains devastat- James O. Armitage, MD
ing to patients and their families. Our goal is to provide a reference James H. Doroshow, MD
textbook that is the most useful, understandable, attractive, and thorough Michael B. Kastan, MD, PhD
in presenting the principles of clinical oncology. It is meant to be equally Joel E. Tepper, MD

xxvii
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Contents

12 Viruses and Human Cancer,  165


Part I Science and Clinical Oncology Paul F. Lambert and Bill Sugden
Section A Biology and Cancer
13 Genetic Factors: Hereditary Cancer Predisposition
Syndromes, 180
1 Molecular Tools in Cancer Research,  2 Michael F. Walsh, Karen Cadoo,
Mauro W. Costa, Muneer G. Hasham, Erin E. Salo-Mullen, Marianne Dubard-Gault,
and Nadia Rosenthal Zsofia K. Stadler, and Kenneth Offit

2 Intracellular Signaling, 24 14 Genetic and Epigenetic Alterations in


Aphrothiti J. Hanrahan, Gopa Iyer, Cancer, 209
and David B. Solit Bin Tean Teh and Eric R. Fearon

3 Cellular Microenvironment and Metastases,  47 Section C Diagnosis of Cancer


Erinn B. Rankin and Amato J. Giaccia
15 Pathology, Biomarkers, and Molecular
4 Control of the Cell Cycle,  56 Diagnostics, 225
Marcos Malumbres Wilbur A. Franklin, Dara L. Aisner, Kurtis D. Davies,
Kristy Crooks, Miriam D. Post, Bette K.
5 Pathophysiology of Cancer Cell Death,  74 Kleinschmidt-DeMasters, Edward Ashwood,
Lorenzo Galluzzi, Andreas Linkermann, Oliver Kepp, Paul A. Bunn, and Marileila Varella-Garcia
and Guido Kroemer
16 Imaging, 254
6 Cancer Immunology, 84 Richard L. Wahl
Diane Tseng, Liora Schultz, Drew Pardoll,
and Crystal Mackall Section D Clinical Trials

7 Stem Cells, Cell Differentiation, and Cancer,  97 17 Biostatistics and Bioinformatics in Clinical
Piero Dalerba, Maximilian Diehn, Irving L. Weissman, Trials, 284
and Michael F. Clarke Brian P. Hobbs, Donald A. Berry,
and Kevin R. Coombes
8 Tumor Microenvironment: Vascular and
Extravascular Compartment,  108 18 Clinical Trial Designs in Oncology,  296
Rakesh K. Jain, John D. Martin, Vikash P. Chauhan, Edward L. Korn and Boris Freidlin
and Dan G. Duda 19 Structures Supporting Cancer Clinical Trials,  308
Jeffrey S. Abrams, Margaret Mooney,
9 Cancer Metabolism, 127 James A. Zwiebel, Worta McCaskill-Stevens,
Michael A. Reid, Sydney M. Sanderson, Michaele C. Christian, and James H. Doroshow
and Jason W. Locasale
20 Oncology and Health Care Policy,  317
Section B Genesis of Cancer Steven Kent Stranne, Clifford A. Hudis,
and Deborah Y. Kamin
10 Environmental Factors,  139
Steven R. Patierno Section E Prevention and Early Detection
11 DNA Damage Response Pathways and 21 Discovery and Characterization of Cancer Genetic
Cancer, 154 Susceptibility Alleles,  323
James M. Ford and Michael B. Kastan Stephen J. Chanock and Elaine A. Ostrander

xxix
xxx Contents

22 Lifestyle and Cancer Prevention,  337 Section B Symptom Management


Karen Basen-Engquist, Powel Brown,
Adriana M. Coletta, Michelle Savage, 35 Hypercalcemia, 565
Karen Colbert Maresso, and Ernest Hawk Anery Patel, Laura Graeff-Armas, Meredith Ross,
and Whitney Goldner
23 Screening and Early Detection,  375
Therese Bevers, Hashem El-Serag, Samir Hanash, 36 Tumor Lysis Syndrome,  572
Aaron P. Thrift, Kenneth Tsai, Scott C. Howard
Karen Colbert Maresso, and Ernest Hawk
37 Cancer-Related Pain,  581
24 Nicotine Dependence: Current Treatments and Suzanne Nesbit, Ilene Browner,
Future Directions,  399 and Stuart A. Grossman
Jeffrey M. Engelmann, Maher Karam-Hage,
Vance A. Rabius, Jason D. Robinson, 38 Cancer Cachexia,  593
and Paul M. Cinciripini Jennifer G. Le-Rademacher and Aminah Jatoi

39 Nausea and Vomiting,  598


Section F Treatment John D. Hainsworth
25 Cancer Pharmacology,  411 Section C Treatment Complications
Jerry M. Collins
40 Oral Complications,  607
26 Therapeutic Targeting of Cancer Cells: Era of Neil Majithia, Christopher L. Hallemeier,
Molecularly Targeted Agents,  420 and Charles L. Loprinzi
Khanh T. Do and Shivaani Kummar
41 Dermatologic Toxicities of Anticancer
27 Basics of Radiation Therapy,  431 Therapy, 621
Elaine M. Zeman, Eric C. Schreiber, Natalie H. Matthews, Farah Moustafa,
and Joel E. Tepper Nadine M. Kaskas, Leslie Robinson-Bostom,
and Lisa Pappas-Taffer
28 Hematopoietic Stem Cell Transplantation,  461
Annie Im and Steven Z. Pavletic 42 Cardiovascular Effects of Cancer Therapy,  649
Lori M. Minasian, Myrtle Davis, and Bonnie Ky
29 Gene Therapy in Oncology,  470
James E. Talmadge and Kenneth H. Cowan 43 Reproductive Complications,  665
Demytra Mitsis, Lynda Kwon Beaupin,
30 Therapeutic Antibodies and Immunologic and Tracey O’Connor
Conjugates, 486
Konstantin Dobrenkov and Nai-Kong V. Cheung 44 Paraneoplastic Neurologic Syndromes,  676
Josep Dalmau and Myrna R. Rosenfeld
31 Complementary and Alternative Medicine,  500
Jeffrey D. White 45 Neurologic Complications,  688
Shlomit Yust-Katz, Simon Khagi,
and Mark R. Gilbert

46 Endocrine Complications,  707


Part II Problems Common to Cancer Donald Trump
and Therapy 47 Pulmonary Complications of Anticancer
Section A Hematologic Problems and Infections Treatment, 715
Mitchell Machtay and Catalina V. Teba

32 Disorders of Blood Cell Production in Clinical Section D Posttreatment Considerations


Oncology, 514
Jennifer H. Choe and Jeffrey Crawford 48 Rehabilitation of Individuals With Cancer,  725
R. Samuel Mayer
33 Diagnosis, Treatment, and Prevention of
Cancer-Associated Thrombosis,  523 49 Survivorship, 732
Claudia I. Chapuy and Jean M. Connors Julia H. Rowland, Michelle Mollica, and Erin E. Kent

34 Infection in the Patient With Cancer,  544 50 Second Malignant Neoplasms,  741
Alison G. Freifeld and Daniel R. Kaul Debra L. Friedman
Contents xxxi

51 Caring for Patients at the End of Life,  751 Section B Head, Neck, and Eye
Lida Nabati and Janet L. Abrahm
64 Ocular Tumors,  968
Section E Local Effects of Cancer and Its Metastasis Odette Houghton and Kathleen Gordon

52 Acute Abdomen, Bowel Obstruction, 65 Cancer of the Head and Neck,  999
and Fistula,  764 Jonathan E. Leeman, Nora Katabi,
William R. Burns and Alfred E. Chang Richard J. Wong, Nancy Y. Lee,
and Paul B. Romesser
53 Superior Vena Cava Syndrome,  775
Arjun Gupta, D. Nathan Kim, Sanjeeva Kalva, Section C Skin
Scott Reznik, and David H. Johnson
66 Melanoma, 1034
54 Spinal Cord Compression,  786 Tara C. Mitchell, Giorgos Karakousis,
Mark Niglas, Chia-Lin Tseng, Nicolas Dea,
and Lynn Schuchter
Eric Chang, Simon Lo, and Arjun Sahgal

55 Brain Metastases and Neoplastic Meningitis,  794 67 Nonmelanoma Skin Cancers: Basal Cell and
Orit Kaidar-Person, Michael Cools, Squamous Cell Carcinomas,  1052
and Timothy Zagar Yaohui G. Xu, Juliet L. Aylward,
Andrew M. Swanson, Vladimir S. Spiegelman,
56 Bone Metastases,  809 Erin R. Vanness, Joyce M.C. Teng,
Robert E. Coleman, Janet Brown, and Ingunn Holen Stephen N. Snow, and Gary S. Wood

57 Lung Metastases,  831 Section D Endocrine


Jonathan Hayman, Jarushka Naidoo,
and David S. Ettinger 68 Cancer of the Endocrine System,  1074
Ammar Asban, Anish J. Patel, Sushanth Reddy,
58 Liver Metastases,  846 Thomas Wang, Courtney J. Balentine,
David A. Mahvi and David M. Mahvi and Herbert Chen

59 Malignancy-Related Effusions,  863


Lola A. Fashoyin-Aje and Julie R. Brahmer
Section E Thoracic

69 Cancer of the Lung: Non–Small Cell Lung Cancer


Section F Special Populations and Small Cell Lung Cancer,  1108
Luiz H. Araujo, Leora Horn, Robert E. Merritt,
60 Cancer in the Elderly: Biology, Prevention, Konstantin Shilo, Meng Xu-Welliver,
and Treatment,  874 and David P. Carbone
Enrique Soto-Perez-de-Celis and Arti Hurria†

61 Special Issues in Pregnancy,  882 70 Diseases of the Pleura and Mediastinum,  1159
Orit Kaidar-Person, Timothy Zagar,
Tina Rizack and Jorge J. Castillo
Benjamin E. Haithcock, and Jared Weiss
62 Human Immunodeficiency Virus (HIV) Infection
and Cancer,  894 71 Cancer of the Esophagus,  1174
Jesus Anampa, Stefan K. Barta, Missak Haigentz, Geoffrey Y. Ku and David H. Ilson
and Joseph A. Sparano
Section F Gastrointestinal

Part III Specific Malignancies 72 Cancer of the Stomach,  1197


Section A Central Nervous System Geoffrey Y. Ku and David H. Ilson

73 Cancer of the Small Bowel,  1211


63 Cancer of the Central Nervous System,  906 Morgan M. Sellers and Alexander J. Greenstein
Jay F. Dorsey, Ryan D. Salinas, Mai Dang,
Michelle Alonso-Basanta, Kevin D. Judy, 74 Colorectal Cancer,  1219
Amit Maity, Robert A. Lustig, John Y.K. Lee, Mark Lawler, Brian Johnston,
Peter C. Phillips, and Amy A. Pruitt Sandra Van Schaeybroeck, Manuel Salto-Tellez,
Richard Wilson, Malcolm Dunlop,

Deceased. and †Patrick G. Johnston
xxxii Contents

75 Cancer of the Rectum,  1281 87 Gestational Trophoblastic Disease,  1544


Scott R. Kelley and Heidi Nelson Donald P. Goldstein, Ross S. Berkowitz,
and Neil S. Horowitz
76 Cancer of the Anal Canal,  1300
Karyn A. Goodman, Lisa A. Kachnic, 88 Cancer of the Breast,  1560
and Brian G. Czito N. Lynn Henry, Payal D. Shah, Irfanullah Haider,
Phoebe E. Freer, Reshma Jagsi,
77 Liver and Bile Duct Cancer,  1314 and Michael S. Sabel
Ghassan K. Abou-Alfa, William Jarnagin,
Imane El Dika, Michael D’Angelica, Maeve Lowery, Section I Sarcomas
Karen Brown, Emmy Ludwig, Nancy Kemeny,
Anne Covey, Christopher H. Crane, James Harding, 89 Sarcomas of Bone,  1604
Jinru Shia, and Eileen M. O’Reilly Megan E. Anderson, Steven G. DuBois,
and Mark C. Gebhardt
78 Carcinoma of the Pancreas,  1342
Ana De Jesus-Acosta, Amol Narang, 90 Sarcomas of Soft Tissue,  1655
Lauren Mauro, Joseph Herman, Brian A. Van Tine
Elizabeth M. Jaffee, and Daniel A. Laheru
Section J Cancer of Undefined Site of Origin
Section G Genitourinary
91 Carcinoma of Unknown Primary,  1694
79 Cancer of the Kidney,  1361 Gauri Varadhachary and James L. Abbruzzese
Megan A. McNamara, Tian Zhang,
Michael R. Harrison, and Daniel J. George Section K Pediatrics
80 Carcinoma of the Bladder,  1382 92 Pediatric Solid Tumors,  1703
Angela B. Smith, Arjun V. Balar, Jeffrey S. Dome, Carlos Rodriguez-Galindo,
Matthew I. Milowsky, and Ronald C. Chen Sheri L. Spunt, and Victor M. Santana

81 Prostate Cancer,  1401 93 Childhood Leukemia,  1748


William G. Nelson, Emmanuel S. Stephen P. Hunger, David T. Teachey,
Antonarakis, H. Ballentine Carter, Stephan Grupp, and Richard Aplenc
Angelo M. DeMarzo and Theodore L. DeWeese
94 Childhood Lymphoma,  1765
82 Cancer of the Penis,  1433 John T. Sandlund and Mihaela Onciu
Jonathan E. Heinlen, Mohammad O. Ramadan,
Kelly Stratton, and Daniel J. Culkin Section L Hematological

83 Testicular Cancer,  1442 95 Acute Leukemias in Adults,  1783


Terence W. Friedlander and Eric Small Frederick R. Appelbaum

Section H Gynecological 96 Myelodysplastic Syndromes,  1798


David P. Steensma and Richard M. Stone
84 Cancers of the Cervix, Vulva, and Vagina,  1468 97 Myeloproliferative Neoplasms,  1821
Anuja Jhingran, Anthony H. Russell, Ayalew Tefferi
Michael V. Seiden, Linda R. Duska,
Annekathryn Goodman, Susanna L. Lee, 98 Chronic Myeloid Leukemia,  1836
Subba R. Digumarthy, and Arlan F. Fuller, Jr. Hagop Kantarjian and Jorge Cortes

85 Uterine Cancer,  1508 99 Chronic Lymphocytic Leukemia,  1850


John F. Boggess, Joshua E. Kilgore, Farrukh T. Awan and John C. Byrd
and Arthur-Quan Tran
100 Hairy Cell Leukemia,  1872
86 Carcinoma of the Ovaries and Fallopian Mark B. Geyer, Omar Abdel-Wahab,
Tubes, 1525 Martin S. Tallman, and Jae H. Park
Robert L. Coleman, Jinsong Liu, Koji Matsuo,
Premal H. Thaker, Shannon N. Westin, 101 Multiple Myeloma and Related Disorders,  1884
and Anil K. Sood S. Vincent Rajkumar and Angela Dispenzieri
Contents xxxiii

102 Hodgkin Lymphoma,  1911 105 Adult T-Cell Leukemia/Lymphoma,  1965


Nancy Bartlett and Grace Triska Matthew A. Lunning, Neha Mehta-Shah,
and Steven M. Horwitz
103 Non-Hodgkin Lymphomas,  1926
Jeremy S. Abramson Index 1975

104 Cutaneous T-Cell Lymphoma and Cutaneous


B-Cell Lymphoma,  1948
Christiane Querfeld, Steven T. Rosen,
and Madeleine Duvic
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P A R T
I

SCIENCE AND CLINICAL ONCOLOGY


A. BIOLOGY AND CANCER 13. Genetic Factors: Hereditary 22. Lifestyle and Cancer Prevention
Cancer Predisposition
1. Molecular Tools in Cancer 23. Screening and Early Detection
Syndromes
Research
24. Nicotine Dependence: Current
14. Genetic and Epigenetic
2. Intracellular Signaling Treatments and Future
Alterations in Cancer
Directions
3. Cellular Microenvironment and
Metastases C. DIAGNOSIS OF CANCER
F. TREATMENT
15. Pathology, Biomarkers, and
4. Control of the Cell Cycle 25. Cancer Pharmacology
Molecular Diagnostics
5. Pathophysiology of Cancer 26. Therapeutic Targeting of
16. Imaging
Cell Death Cancer Cells: Era of
D. CLINICAL TRIALS Molecularly Targeted Agents
6. Cancer Immunology
17. Biostatistics and Bioinformatics 27. Basics of Radiation Therapy
7. Stem Cells, Cell Differentiation, in Clinical Trials
and Cancer 28. Hematopoietic Stem Cell
18. Clinical Trial Designs in Transplantation
8. Tumor Microenvironment: Oncology
Vascular and Extravascular 29. Gene Therapy in Oncology
Compartment 19. Structures Supporting Cancer
Clinical Trials 30. Therapeutic Antibodies and
9. Cancer Metabolism Immunologic Conjugates
20. Oncology and Health Care
B. GENESIS OF CANCER Policy 31. Complementary and Alternative
Medicine
10. Environmental Factors
E. PREVENTION AND EARLY
11. DNA Damage Response DETECTION
Pathways and Cancer 21. Discovery and Characterization
12. Viruses and Human of Cancer Genetic
Cancer Susceptibility Alleles
A. BIOLOGY AND CANCER
1  Molecular Tools in Cancer Research
Mauro W. Costa, Muneer G. Hasham, and Nadia Rosenthal

S UMMARY OF K EY P OI N T S
• Our understanding and treatment of growth and differentiation has techniques, including whole-genome
cancer have always relied heavily on revolutionized the diagnosis, analysis, expression profiling, and
parallel developments in biologic prognosis, and treatment of refined genetic manipulation in and
research. Molecular biology provides malignant disorders. use of genetically diverse animal
the basic tools to study genes • This introductory chapter relates models, providing the conceptual
involved with cancer growth patterns basic principles of molecular biology and technical background necessary
and tumor suppression. An to emerging perspectives on the to grasp the central principles and
advanced understanding of the origin and progression of cancer and new methods of current cancer
molecular processes governing cell explains newly developed laboratory research.

Since the last edition of this book was published, advances in our genetic changes confer selective advantages on tumor cell clones by
understanding of the basic mechanisms of cancer have continued to disrupting control of cell proliferation. The identification of specific
inform and refine clinical approaches to prevention and therapy. New mutations that characterize a tumor cell has proved invaluable for
prognostic and predictive markers derived from molecular biology analyzing the neoplastic progression and remission of the disease. The
can now pinpoint specific genetic changes in particular tumors or emergence of cancer cells is a byproduct of the necessity for continuous
detect occult malignant cells in normal tissues, leading to improved cell division and DNA replication to maintain organ functionality
technologies for tumor screening and early detection. Diagnostic throughout the life cycle.
approaches have expanded from morphologic criteria and single-gene The highly heterogeneous nature of tumors, each composed of
analysis to whole-genome technologies and single-cell genomics multiple cell types, led to the formulation of the “cancer stem cell”
imported from other biologic disciplines. A new systemic vision of hypothesis, which posits that only a subpopulation of cancer cells is
cancer is emerging, in which the importance of individual mutation able to maintain self-renewal, unlimited growth, and capacity for
has been superseded by an appreciation for higher-order organization differentiation into other, more specialized cancer cell types. Cancer
and individual genetic background, disrupted by complex interactions stem cells display bona fide stem cell markers, in contrast to other
of disease-associated factors and gene-environmental parameters that cancer cells present in the tumor, which do not have tumorigenic
affect tumor cell behavior. Results from these cross-disciplinary potential. In fact, fewer than 1 in 10,000 cells present in human acute
investigations underscore the complexity of carcinogenesis and have myeloid leukemia are capable of reinitiating a new tumor when
profoundly influenced the design of strategies for both cancer prevention transplanted into animals. Cancer stem cells have been identified in
and advanced cancer therapy. many solid tumors in the brain, colon, ovaries, prostate, and pancreas,
This overview will serve as a foundation of conceptual and technical suggesting that more effective cancer therapies would target these
information for understanding the exciting new advances in cancer self-renewing cells, rather than the tumor as a whole. The cancer stem
research described in subsequent chapters. Since the discovery of cell concept differs from the original clonal evolution hypothesis,
oncogenes, which provided the first concrete evidence of cancer’s which states that every cell in a tumor mass is capable of self-renewal
genetic basis, applications of advanced molecular techniques and and differentiation, and suggests that detecting and targeting subtle
instrumentation have yielded new insights into normal cell biology. genetic and epigenetic differences that distinguish cancer stem cells
A basic fluency in molecular biology and genetics has become a necessary may provide a more effective avenue to intervention in disease
prerequisite for clinical oncologists because many of the new diagnostic progression.
and prognostic tools currently in use rely on these fundamental Heterogeneity can also arise as a result of stochastic mutational
principles of gene, protein, and cell function. events that lead to cancer progression. Clastogenic insults to the genome,
or genomic instability due to aberrant gene regulation, could lead to
OUR UNSTABLE HEREDITY loss of heterozygosity (LOH) of tumor suppressor genes such as TP53,
RB1, or BRCA, and can also lead to tumor heterogeneity and change
Cancer genetics has classically relied on the candidate-gene approach, in disease progression. Furthermore, activation of DNA or RNA editing
detecting acquired or inherited changes in specific genetic loci accu- enzymes in tumors could lead to kataegis, a DNA hypermutation
mulated in a single cell, which then proliferates to produce a tumor process, and increase tumor heterogeneity. Although there are molecular
composed of its identical clonal progeny. During the early steps of biology tools currently available to detect aberrant but stable genomes,
tumor formation, mutations that lead to an intrinsic genetic instability the later processes that lead to genomic instability make diagnosis
allow additional deleterious genetic alterations to accumulate. These and prognosis more challenging.

2
Molecular Tools in Cancer Research  •  CHAPTER 1 3

DETECTING CANCER MUTATIONS The functional unit of inherited information in DNA, the gene,
is most often represented by a discrete section of sequence necessary
Methods for mutation detection all rely on the manipulation of DNA, to encode a particular protein structure. Gene expression is initiated
the basic building block of heredity in the cell. DNA consists of two by forming a copy of the gene, messenger RNA (mRNA), constructed
long strands of polynucleotides that twist around each other clockwise base by base from the DNA template by a polymerase enzyme. Once
in a double helix (Fig. 1.1). Nucleic acid bases attached to the sugar transcribed, an mRNA transcript is modified and the processed product
groups of each strand face each other within the helix, perpendicular is transported out of the nucleus. In the cytoplasm, proteins are
to its axis. These comprise only four bases: the purines adenine and then synthesized, or translated, in macromolecular complexes called
guanine (A and G) and the pyrimidines cytosine and thymine (C and ribosomes that read the mRNA sequence and convert the nucleic acid
T). During assembly of the double helix, stable pairings of nucleotides code, based on three-base segments or codons, into a 20–amino acid
from either strand are made between A and T, or between G and C. code to form the corresponding protein.
Each base pair forms one of the billions of rungs in the long, unbroken Although these canonic processes drive gene expression in all normal
ladder of DNA forming a chromosome. cells, cancer cells defy the rules. For instance, uracils, which are found

Genes Cell nucleus


containing
23 pairs of
chromosomes

DNA
strand
Chromosomes
Sugar

Cytosine Bases
thymine

Bases

Adenine and
guanine
Phosphate
group P

H
H
C H
H
H C
H
P
H
H
P
H
C
H
H CH2
P P
H
CH2
H
CH2
H
P P
H
CH2
H CH2

Figure 1.1  •  DNA structure. Deoxyribonucleic acid (DNA) 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, composed of sugar (deoxyribose) and phosphate molecules,
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. (From http://www.terrapsych.com/dna.jpg.)
4 Part I: Science and Clinical Oncology

on RNA, can be detected in the DNA of cancer cells because of their is shared by all members of a species, the recent sequencing of thousands
high mutation rates. Paradoxically, these deviations from the norm of individual human genomes has given rise to the new field of
allow the development of molecular biology tools to better diagnose genomics, providing us with new tools to reveal the more subtle
and predict tumor progression. variations that arise between individuals. These variations are critical,
both as a natural engine driving heterogeneity within a species, and
GENERATING DIVERSITY WITH as a source of predisposition to cancer types. The most common forms
ALTERNATE SPLICING of human genetic variations, or alleles, arise as single-nucleotide
polymorphisms (SNPs). Because these allelic dissimilarities are abundant,
In higher organisms, most protein coding gene sequences are interrupted inherited, and dispersed throughout the genome, SNPs can be used
by stretches of noncoding DNA sequences, called introns. In the to track racial diversity, personal traits, and susceptibility to common
nucleus, these introns are removed after mRNA transcription to produce forms of cancer (Fig. 1.3). Commercial entities have developed tools
a continuous chain of coding sequences, or exons, that subsequently that can detect thousands of SNPs with relatively little sample material.
undergo translation into protein. The splicing process requires absolute Platforms such as MegaMUGA or GigaMUGA can allow mammalian
precision because the deletion or addition of a single nucleotide at genetic mapping that can aid in a number of diagnoses and can
the splice junction would throw the three-base coding sequence out distinguish between predictive and prognostic markers.
of frame, or lead to exon skipping or addition, creating abnormal How do SNPs arise between individuals? One source of variation
proteins. in DNA sequence derives from deviations in the strict base-pairing
The dramatic increase in genetic complexity conferred by alternate rule underlying the structure, storage, retrieval, and transfer of genetic
RNA splicing is underscored by the multiple splice patterns of many information. The duplicated genetic information in the two strands
medically relevant genes, in which different combinations of exons of DNA not only permits the repair of a damaged coding sequence
are chosen for the final mRNA transcript, such that one gene can but also forms the basis for the replication of DNA. During cell
encode many different proteins (Fig. 1.2). The choice of protein isoform division, polymerase enzymes unwind the DNA strands and copy
to be expressed from a gene with multiple splicing possibilities is a them, using the base sequences as a template for constructing a new
decision that can be perturbed in disease. Errors in splicing mechanisms helix so that the dividing cell passes its entire genetic content on to
have been associated with a large group of cancers. These include its progeny. Errors in this process are rare, and person-to-person
mutations in the oncogene p53 in more then 12 different types of differences comprise only about 0.1% of the human genome. SNPs
cancer, mutL homolog 1 protein (MLH1) mutation in hereditary are inherited if they occur in the germline. Many genetically inherited
nonpolyposis colorectal cancer, and several transcription factors and variations occur in regions that do not encode protein or alter the
cell signaling and membrane proteins. When mutations in the splicing regulation of nearby genes. Given the disruptive effects even subtle
site lead to insertion of novel sequences in the mRNA, the encoded genetic changes may have on cell function, it is important to distinguish
protein can be used as a potential clinical marker, as seen for the SNPs that represent true mutations from benign polymorphisms.
transcription factor NSFR in small cell lung cancer. Owing to their Our ability to monitor hundreds of thousands of SNPs simultane-
unique expression in cancer cells, these markers can be further explored ously is one of the most important advances in modern medical genetics.
as new cancer-specific therapeutic targets. Relatively simple genotyping technologies for SNP detection rely largely
on the polymerase chain reaction (PCR). In procedures that use this
GENOMICS OF CANCER reaction, two chemically synthesized single-stranded DNA fragments,
or primers, are designed to match chromosomal DNA sequences
The complete set of DNA sequences carried on all the chromosomes flanking the segment in which an SNP is positioned. With the addition
is known as the genome. Although the general map of the genome of nucleotide building blocks and a heat-stable DNA polymerase, the

Exon Exon Exon Exon Exon


Gene 1 2 3 4 5

1 2 3 4 5
RNA

Alternative splicing

RNA 1 2 3 4 5 1 2 4 5 1 2 3 5

Translation Translation Translation

Protein A Protein B Protein C

Figure 1.2  •  RNA splicing. Alternate splicing produces multiple related proteins, or isoforms, from a single gene. (From Guttmacher AE, Collins F.
Genomic medicine—a primer. N Engl J Med. 2002;347:1512–1520.)
Molecular Tools in Cancer Research  •  CHAPTER 1 5

Primary tumor Metastasis SNP density


11p15.5
11p15.4
11p15.3
11p15.2
11p15.1
11p14.3
11p14.2
11p14.1
11p13
11p12
Tens to hundreds
11p11.2
of changes between >10 million SNPs 11p11.12
primary and secondary between individuals 11p11.11
tumors 11q11
11q12.1
11q12.2
11q12.3
11q13.1
11q13.2
11q13.3
Primary tumor Metastasis 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

Figure 1.3  •  Determining cancer susceptibility with single-nucleotide polymorphisms (SNPs). Millions of 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 the tissues of the body, it might be
possible to distinguish differences in metastatic versus nonmetastatic tumors and in nontumor tissues before they ever happen to develop a solid tumor. (From
Hunter K. Host genetics influence tumour metastasis. Nat Rev Cancer. 2006;6:141–146.)

primer pairs, or amplicons, initiate synthesis of new DNA strands, base sequence that carries unique protein coding information. Other
using the chromosomal material as a template. Each successive copying noncoding DNA sequences are used for directing the transcription
cycle, initiated by “melting” the resulting double-stranded products of neighboring genes, through complex regulatory circuits involving
with heat, doubles the number of DNA segments in the reaction (Fig. protein binding and modification of the DNA itself, or shifting of
1.4). The technique is exceptionally sensitive; millions of identical its chromosomal packaging. Although genomic instability is generally
DNA copies can be generated in a matter of hours with PCR by using considered a consequence of tumor formation rather than the initial
a single DNA molecule as the starting material. trigger of cancer, the loss, gain, or rearrangement of chromosomal
Other novel methods for large-scale SNP detection include single- segments through deletion or translocation is a common form of
nucleotide primer extension, allele-specific hybridization, oligonucleotide neoplastic mutation, as protein coding segments from different genes
ligation assay, and invasive signal amplification, which detect poly- are combined or regulatory sequences are brought into new proximity
morphisms directly from genomic DNA without the requirement of to genes they do not normally control, as seen in chronic myeloid
PCR amplification. The International HapMap Project was established leukemia (CML). In CML, recombination events lead to the fusion
with the objective of identifying those variations (commonly thought of BCR and ABL genes (Philadelphia chromosome). This results in
to be on the order of 10 million in our genome) in the human popula- constitutive activation of the fused gene, leading to loss of proliferative
tion. This project is already in its third phase (HapMap3), now control in myeloid cells and consequently cancer. Gross changes in
including both SNPs and copy number variations observed in 1184 DNA arrangement can be detected by cytogenetic analysis of chro-
samples from 11 different human populations. Regardless of the method mosomal features on metaphase spreads. Although fluorescence in
used to characterize them, the collective SNPs in a selected genomic situ hybridization (FISH) provides greater resolution by localizing
region characterize a haplotype, or specific combination of alleles at specific chromosomal DNA sequences corresponding to fluorescently
multiple linked genetic loci along a chromosome that are inherited labeled probes (Fig. 1.5), and can be used to track specific alterations
together. in chromosomal structure where known genes are involved, spectral
Even when the SNPs within a given haplotype are not directly karyotyping (SKY) is a powerful and more general tool that could
involved in a disease, they provide markers for clonality and for the aid diagnosis of cancer genomes. With each fluorescently labeled
loss or rearrangement of specific chromosomal segments in growing chromosome assigned a specific color, translocations and additions
tumors. In the human nucleus, each of the 23 tightly compacted are revealed as multicolored chromosomes, or large deletions as pieces
chromosomes has a characteristic size and structure, and a distinctive of missing chromosomes.
6 Part I: Science and Clinical Oncology

Cycle 1 Cycle 2 Cycle 3 The plethora of data arising 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
Separation of strands

Separation of strands

Separation of strands
with equivalently sophisticated and rigorous analytic methodologies
for the true genetic cause of cancer to be teased out from our variable
and often unstable heredity.
Primers
Sequence
to be BUILDING GENE LIBRARIES
amplified
The engineering of genes by recombinant DNA technology evolved
from methods 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
Primers Heat Heat Heat 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
Figure 1.4  •  Amplification of DNA by polymerase chain reaction (PCR). from a patient sample with PCR, but the classic approach is still
The DNA sequence to be amplified is selected by primers, which are short, applied to the construction of recombinant DNA libraries.
synthetic oligonucleotides that correspond to sequences flanking the DNA to To be useful, a DNA library must be as complete as possible, with
be amplified. After an excess of primers is added to the DNA, together with recombinant members, or clones, sufficiently numerous to include
a heat-stable DNA polymerase, the strands of both the genomic DNA and all the sequences in an individual genome. For certain kinds of gene-
the primers are separated by heating and allowed to cool. A heat-stable
polymerase elongates the primers on either strand, thus generating two new,
linkage analysis that require long, uninterrupted stretches of DNA,
identical double-stranded DNA molecules and doubling the number of DNA special vectors, such as bacterial or yeast artificial chromosomes, can
fragments. Each cycle takes just a few minutes and doubles the number of carry foreign DNA fragments of enormous lengths. Chromosomal
copies of the original DNA fragment. segments represented in genomic DNA libraries can contain the

9 9 22 22

9 der(9) 22 der(22)

Figure 1.5  •  Detection of chromosomal translocations. Fluorescence in situ hybridization (FISH) 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). (Modified
from Varella-Garcia M. Molecular cytogenetics in solid tumors: laboratorial tool for diagnosis, prognosis, and therapy. Oncologist. 2003;8:45–58.)
Molecular Tools in Cancer Research  •  CHAPTER 1 7

structure of an entire gene, including the information that regulates The interaction between protein and DNA is increasingly
its expression, and formed the starting material for sequencing of the used to identify transcription factor binding sites in a regulatory
human genome. region. Whereas electrophoretic mobility shift assays (EMSAs), or
Many cancer-associated genes were originally identified through DNA footprinting, were once standard techniques for determining
use of partial DNA libraries, which contain only the DNA sequences protein-DNA interactions, emerging genome-wide technologies, such
transcribed by a particular tissue or type of cell. The starting mate- as chromatin immunoprecipitation on microarray chip (ChIP-chip)
rial in this case is mRNA. For cloning purposes, the enzyme reverse and chromatin immunoprecipitation on sequencing (ChIP-seq), are
transcriptase can convert mRNA into complementary DNA (cDNA). revolutionizing the way in which we see the interaction of a transcription
The number of clones in a cDNA library is much smaller than factor complex with virtually all of its potential genomic targets in
in a genomic library because a cDNA library represents only the a particular cell state. These strategies involve the use of candidate
genes expressed by the tissue of interest and contains exclusively the protein–specific antibodies to pull down DNA targets regulated by
coding portion of genes. For this particular reason, this technique them. These targets are further identified with the use of microarray
has now become obsolete for organisms whose genome has now ChIP-chip or next generation sequencing ChIP-seq technologies
been fully sequenced. New advances in PCR chemistry allowed (see Fig. 1.14).
for the direct cloning of increasingly larger cDNA fragments with Our appreciation of oncogenic perturbations, by mutation of
high specificity and low error rates. Highly accurate PCR technol- regulatory protein coding genes or by loss of controlled signaling by
ogy, coupled with the constantly evolving generation of genomic cell cycle switches or in the target sequences these proteins recognize,
sequence maps in humans and model organisms, has exponentially has recently extended to include posttranslational modifications that
expanded the availability of candidate genes to be tested in cancer control protein activity, such as phosphorylation, ubiquitylation, and
biology. SUMOylation. Tumor-associated changes in these modifications
underscore the multiple levels of control necessary to ensure correct
LOSING CONTROL OF THE GENOME gene expression that is so central to the normal function of the cell.

Mutations that lead to oncogenic transformation of a cell invariably EPIGENETICS AND CANCER
affect the expression of its genetic information that specifies functional
products, either RNA molecules or proteins used for various cellular Epigenetics refers to general control of gene expression that is inherited
functions. The primary level of gene control is the transcription of during cell division, although not part of the DNA sequence itself.
DNA into RNA. Gene regulation, or the control of RNA synthesis, Epigenetic regulation involves changes in chromatin, a higher-order
represents a complex process that is itself a frequent target of neoplastic building block of chromosomes that wraps DNA into coils with
mutation. scaffolding proteins such as histones. Histones are a necessary com-
DNA regulatory sequences do not encode a product. However, ponent of chromosomal compaction, but also play a critical role in
without them a cell could not coordinate the expression of the hundreds gene accessibility (Fig. 1.7). Active genetic loci are associated with
of thousands of genes in its nucleus, select only certain genes for loosely configured euchromatin, whereas silent loci are condensed in
expression, and activate or repress them in response to precise internal heterochromatin. The state of chromatin configuration (euchromatin
or external signals. These control centers of the genome contain binding or heterochromatin) both controls and is controlled by patterns of
sites for multiple proteins, called transcription factors, which interact histone modifications such as methylation and acetylation on specific
to form regulatory networks controlling gene transcription. Their DNA sequences. This pattern relates the underlying genetic information
function can be altered by signals that induce modifications such as to its higher-order structure that determines whether a particular gene
phosphorylation, or by interactions with other regulators such as steroid regulatory element is available to transcription factors (on or off status).
hormones. Many of the cell’s responses to a wide variety of external These epigenetic modifications of the nuclear environment that
stimuli, such as neurotransmitters, antigens, cytokines, and growth determine the accessibility of a gene can persist during cell division,
factors, are mediated through transcription factors binding to DNA because inherited epigenetic patterns provide permanent marks for
regulatory sequences. altered chromatin configuration in daughter cells. The pattern of
Certain regulatory DNA sequences common to many genes are modifications generated by the epigenetic code rivals the complexity
positioned upstream of the transcription start site (Fig. 1.6). Collectively of the DNA code itself.
called the “promoter” of a gene, these proximal sequences comprise The accessibility of genomic regions can favor mutations. Enzymes
binding sites for the RNA polymerase and its numerous cofactors. such as the APOBEC family exploit this accessibility to induce C to
Whereas the position of the promoter with regard to the transcription U mutation, which is then converted to T or staggered single-strand
start site is relatively inflexible, other DNA regulatory elements, known breaks. If not rectified, these point mutations or breaks can lead to
as enhancers, occur in unpredictable locations, often at a considerable hypermutations. Kataegis is an example wherein such hypermutation
distance from the genes they control. Some transcription factors bind occurs on the BRCA locus, generating neoplasia.
to particular regions of enhancers and drive their associated genes in Research has linked rearrangement of chromatin and associated
many types of cells, whereas others, active in only a limited variety DNA methylation with the inactivation of tumor suppressor genes and
of cells, maintain a tissue-specific pattern of gene expression. Enhancers neoplastic transformation. Defects that could lead to cancer involve
are often responsible for the aberrant expression of genes induced by perturbations in the “epigenotype” of a particular locus, through the
chromosomal translocation associated with specific forms of cancer: silencing of normally active genes or activation of normally silent
a normally quiescent gene promoting cell growth that is dislocated genes, associated with changes in DNA methylation, histone modifica-
to a position near a strong enhancer may be activated inappropriately, tion, and chromatin proteins (Fig. 1.8). Changes in the number or
resulting in loss of growth control. density of heterochromatin proteins associated with cancer-related
Enhancers and promoters have been assigned specific roles by means genes such as EZH2, or of euchromatic proteins such as trithorax in
of cell culture assays or in transgenic animals in which putative regula- leukemia, can also be associated with abnormal patterns of methyla-
tory DNA sequences are linked to test or “reporter” genes, and are tion in gene promoter regions and with higher-order chromosomal
examined for their ability to activate expression of the reporter gene structures that are only beginning to be understood. Finally, it is
in response to the appropriate signals. Through assessment of the increasingly evident that interactions among the “epigenome,” the
effects of deleting, adding, or changing DNA sequences within the genome, and the environment are common targets for mutation
regulatory element, the precise nucleotides that are critical for recogni- and can have profound effects on the gene expression readout of a
tion by transcription factors can be determined. cancer cell.
8 Part I: Science and Clinical Oncology

Gene structure
Exon 1 Exon 2 Exon 3
Enhancer Promoter Intron 1 Intron 2

TATAA GT AG GT AG AATAAA

Transcription Gene expression


factors

RNA polymerase
Exon 1 Exon 2 Exon 3

Transcription
Transcription-initiation pre-
complex 5' 3' mRNA
Transcript processing

RNA-clipping enzyme

AAUAAA

5' cap

PolyA tail
AAAA...

Adenosine-adding
Intron lariat enzyme (terminal
transferase)
Splicing
Nucleus AAAA...

Spliceosome

Processed
transcript mRNA
Cytoplasm AAAA...

Translation into protein

Figure 1.6  •  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 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 (bp) 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. (From Rosenthal
N. Regulation of gene expression. N Engl J Med. 1994;331:931–932.)
Molecular Tools in Cancer Research  •  CHAPTER 1 9

Nucleosome

DNA

The solenoid

Figure 1.7  •  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 connected by a thread of free
linker DNA. Each nucleosome, together with its linker, packages about 200 bp (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 Jakob Waterborg. www.umkc.edu/sbs/waterborg/chromat/chromatn.html. Copyright 1998 Jakob Waterborg.)

Gene X Gene X
X

Gene Y
X

Gene Y

A Normal B Epigenetic lesions


Figure 1.8  •  Gene accessibility through epigenetics. Illustration depicts known and possible defects in the epigenome that could lead to disease. (A) Gene
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). Gene 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.
10 Part I: Science and Clinical Oncology

PROFILING TUMORS underlying tumor progression by following the changes in a tumor


cell’s transcriptional landscape.
Monitoring global gene expression patterns of cells represents one of With reliance on two-color fluorescence-based microarray technology
the latest breakthroughs in developing a molecular taxonomy of cancer. (DNA microarray), simultaneous evaluation of thousands of gene
Although classic blotting and probe hybridization techniques (Northern transcripts and their relative expression can provide a snapshot of the
blot) are still a reliable way to monitor expression of individual genes, “transcriptome,” the full complement of RNA transcripts produced
these techniques have limitations, such as unequal hybridization at a specific time during the progression of malignancy.
efficiency of individual probes, sensitivity for low copy or small Transcriptional profiling with microarrays typically involves screens
transcripts, and difficulty in detecting multiple RNAs simultaneously of mRNA expression from two sources (such as tumor and normal
or in simultaneously analyzing a large number of targets. For cancer cells), using cDNA or oligonucleotide libraries that are arranged in
studies, it is important to be able to compare the expression pattern extremely high density on microchips. These are probed with a mixture
of all known RNAs, including noncoding RNAs, between cancer cells of fluorescently tagged cDNAs generated from the tumor and normal
and normal cells. Thus new genome-wide analytic techniques are the samples, which results in differential staining of each gene spot. The
state-of-the-art choice to detect mRNA expression profiles at a single relative intensity of the two different colors reflects the RNA expres-
point in time or cell state. Genome-wide profiling of gene expression sion level of each gene; this is analyzed with a laser confocal scanner
in tumors delivers an unprecedented view into the biologic processes (Fig. 1.9). With microarrays, single genes that constitute diagnostic,

Tumors
Reference Tumor
RNA RNA

Genes

Statistical
cDNA
analysis
Hybridization
of probe to
microarray Multidimensional-scaling plot

A B C

Donor Recipient
paraffin block paraffin block
D E Tissue microarray

Figure 1.9  •  Microarray-based expression profiling of 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 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 upregulated in the tumors appear red, whereas 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 to occur 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 relations 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 in order 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.)
Molecular Tools in Cancer Research  •  CHAPTER 1 11

prognostic, or therapeutically relevant markers can be systematically technique relies on the generation of small fragments of cDNA from
monitored. Alternatively, the entire set of expressed genes can be any RNA sample, followed by sequencing of these expressed tags from
collectively analyzed through use of powerful statistical methods to one end (single-end sequencing) or both ends (pair-end sequencing),
classify tumors according to their transcriptional profile. Microarray resulting in fragments of 30 to 400 base pairs (bp). The resulting
analysis has already dramatically improved our ability to explore the sequences can be then mapped against the known reference genome
genetic changes associated with cancer etiology and development and or transcriptome of a certain species. Unlike microarray analysis of
is providing new tools for disease diagnosis and prognostic assessment. preselected gene sets, RNA-seq allows the unbiased identification of
For example, DNA microarray analysis of multiple primary breast all genes, or even the presence of different isoforms, expressed in the
tumor transcriptomes has revealed reproducible signature expression of sample, allowing a comprehensive comparison of transcript levels
70 associated genes. These markers have been recently cleared by the between normal and cancer cells.
US Food and Drug Administration (FDA) for PCR–based diagnostics The technologies just described can also be applied to the analysis
showing that expression analysis of a relative small gene group can of noncoding RNA species. In addition to the 20,000 protein coding
predict the prognosis of early stage breast cancers. When applied on a transcripts used to classify a wide variety of human tumors, hundreds
larger scale, these assays can predict response to chemotherapy, or opti- if not thousands of small, noncoding interference RNA species, with
mize pharmaceutical intervention by targeting therapeutic approaches critical functions in multiple biologic processes, have been discovered;
to specific patient populations and ultimately to individualized therapy. many of these RNA species are directly or indirectly involved in the
A novel high-throughput approach for global transcriptome analysis control of cell proliferation. Known as microRNAs (miRNAs), these
has been made possible by advances in strategies that allow mass short transcripts arise from primary genome-encoded transcripts of
sequencing of DNA fragments. With this technique, called RNA-seq, variable sizes that are processed into 70- to 100-nucleotide hairpin-
it is now possible to obtain a comprehensive and unbiased analysis shaped precursors, which are processed into mature miRNAs of 21 to
of all mRNA transcripts present in cells or tissues. (Fig. 1.10). The 23 bp RNA molecules (Fig. 1.11). miRNAs function by base-pairing

2x poly (A) RPKM RPKM


selection
Brain 0.0 0.0

Liver 0.0 0.0

25-bp
reads
Add standards
and shatter RNA Muscle 0.5 50.1

Make cDNA
and sequence Muscle splices

1 kb
Map 25-bp tags RNA-Seq
onto genome graph
Myf5
Myf6
Conservation

25-bpm Uniquely mappable


Calculate splices
transcript
Repeating elements by RepeatMasker
prevalence
Myf6
Conservation 20 kb
Uniquely map.
2 RPKM 1 RPKM 1 RPKM RepeatMasker
A B C

Figure 1.10  •  Methods for high-throughput transcriptome analyses. (A) Schematics of regular protocol for RNA-seq sample preparation, showing poly-A
tail specific mRNA isolation followed by fragmentation of RNA into smaller regions, further used for cDNA conversion. Polymerase chain reaction (PCR)
fragments are then tethered by adaptors, sequenced by synthesis, and aligned to the reference genome or transcriptome to calculate relative prevalence of
mRNAs (RPKM). (B) Target fragments can be used to map exon-intron boundaries and thus infer present and quantify different mRNA isoforms in the
sample of interest, as shown for the muscle specific gene Myf6 in this example. (C) Data generated with this method can also be compared with analysis of
other tissues or samples, allowing assessment of relative quantification of targets, as exemplified here for a highly specific gene (red peaks) for muscle samples.
(From Mortazavi A, Williams BA, et al. Mapping and quantifying mammalian transcriptomes by RNA-seq. Nat Methods. 2008;5:621–628.)
12 Part I: Science and Clinical Oncology

Protein-coding gene MicroRNA gene

Transcription of pri-microRNA
Transcription
of mRNA

Pri-microRNA
OR

Drosha

DGCR8
Processing
Nucleus
of pri-microRNAs
into pre-microRNA

Pre-microRNA Ran-GTP

Exportin 5
Transport of
pre-microRNAs into
the cytoplasm

Processing of
Dicer pre-microRNA into
small RNA duplexes
Loqs/TRBP
Cytoplasm
RISC

||||||||||||||||||||
||||
|||
|||
|||
||| ||||||||||||||||||||||||||||| An
||| |||||||||
||| ||||||
||| |||| |||||
|||| |||||
|||||||||

Delivery of
RISC-microRNA
complex

mRNA degradation Translational repression

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 homologue of the
human immunodeficiency virus transactivating response RNA-binding protein. 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. (Modified from Chen CZ. New Eng J Med. 2005;353:1768–71.)

with target mRNAs to inhibit translation and/or promote mRNA suppression. The usefulness of monitoring the expression of miRNAs
degradation. In the context of cancer, miRNAs may act in concert with in human cancer is just now being explored, but preliminary findings
other effectors such as p53 to inhibit inappropriate cell proliferation. reveal an extraordinary level of diversity in miRNA expression across
A global decrease in miRNA levels is often observed in human cancers, cancers, and the large amount of diagnostic information encoded
indicating that small RNAs may have an intrinsic function in tumor in a relatively small number of miRNAs. Significant technologic
Molecular Tools in Cancer Research  •  CHAPTER 1 13

advances facilitating the profiling of the miRNA expression patterns CANCER PROTEOME
in normal and cancer tissues hint at the unexpected greater reliability of
miRNA expression signatures than the respective signatures of protein The term proteome describes the entire complement of proteins expressed
coding genes in classifying cancer types. Along with their potential by the genome of a cell, tissue, or organism. More specifically, it is
diagnostic value, miRNAs are also being tested for their prognostic used to describe the set of all the expressed proteins at a given time
use in predicting clinical behaviors of cancer patients. point in a defined setting, such as a tumor. Like RNA transcription,
Because probe specificity in miRNA microarray analysis is prob- the synthesis of proteins is a highly regulated process that contributes
lematic owing to the small target size, hybridization can be performed to the specific proteome of a particular cell and can be perturbed in
first in solution, and then quantified with multicolor flow sorting. diseases such as cancer.
Real-time PCR can also be used to quantify specific miRNA sets, or Advances in protein analytic techniques over the last decades have
to capture a more detailed picture of their changing expression profiles progressed to the point that even small numbers of specific proteins
in tumor progression. Identification of the miRNAs involved in tumor expressed in tissues can be used to predict the prognosis of a cancer.
pathogenesis and elucidation of their action in a specific cancer will The improvement of protein-based assays has made it possible to
be the next necessary steps for their manipulation in a therapeutic identify and examine the expression of most proteins, and to envision
setting. large-scale protein analysis on the level of gene-based screens. Various
Advances in this field have revealed that miRNAs are also involved systematic methodologies have contributed to the current explosion
in cancer initiation and progression, and specific modulation of of information on the proteome. These are now being compared for
such RNAs may serve as a therapeutic strategy. Inhibition of key their suitability as platforms for the generation of databases on protein
miRNAs using antagomirs (a class of chemically modified anti-miRNA structural features, interaction maps, activity profiles, and regulatory
oligonucleotides) has been effective in suppressing tumor growth in modifications.
mouse models. It remains to be seen if these results can be extended to The yeast two-hybrid system has been a popular genetics-based
treatment of cancer in the clinic, but interference with miRNA function approach for detecting protein-protein interactions inside a cell (Fig.
is an attractive new tool for the development of cancer therapies. 1.12). One protein fused to the DNA binding domain (bait) and a

DNA-binding domain
fused to protein A A

A Promoter Reporter gene

Activator region fused to


protein B
B

B Promoter Reporter gene


Figure 1.12  •  Exploring protein-protein interactions with the yeast two-hybrid
system. Two-hybrid technology exploits the fact that transcriptional activators are Activator region fused to
modular in nature. Two physically distinct functional domains are necessary to get protein B
transcription: a DNA-binding domain that binds to the DNA of the promoter and DNA-binding domain
an activation domain that binds to the basal transcription apparatus and activates fused to protein A A B Transcription
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. C Promoter Reporter gene
(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 96 Bait strains 1 Prey strain
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 yeast extract
peptone dextrose (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 Replicate velvet
new replica velvet, and the impression is lifted with a plate containing diploid Diploids
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 YPD
interacts with the bait at that location. (C from http://www.nature.com/…/journal/
v403/n6770/full/403601a0_r.html. D from Bartel PL, Fields S, eds. The Yeast Replicate velvet
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. D X-Gal
genetics.wayne.edu/finlab/YTHnetworks.html.)
14 Part I: Science and Clinical Oncology

different protein fused to the activation domain of a transcriptional as phosphorylation, SUMOylation, or ubiquitination. These LC-MS/
activator (prey) are expressed together in yeast cells. If the bait MS systems, such as the iTRAQ, allow for a more precise and indi-
and prey interact, transcription of a reported gene is induced and vidualized diagnosis of cancer.
detected typically by a color reaction that reflects the transactiva- Monoclonal antibodies (mAbs) have been a cornerstone of protein
tion of the reporter gene, and by proxy, the interaction of the two analysis in cancer research, and more recently have risen to prominence
test proteins. The method can also be used for large-scale protein as cancer therapeutics based on their exquisite specificity for protein
interactions, determination of RNA-protein interactions, and protein- targets and their potent interference with protein function. Novel
ligand binding. strategies have been developed that target not only antigens highly
As a complementary proteomics tool, mass spectrometry (MS) is expressed in cancer cells but also to enhance the innate immune
an accurate mass measurement of charged peptides isolated by two- response against cancer cells. These antibodies can act via several
dimensional gel electrophoresis, producing a mass-to-charge ratio of mechanisms, including antibody-dependent cellular cytotoxicity
charged samples under vacuum that can be used to determine the (ADCC), complement-mediated cytotoxicity (CMC), and antibody-
sequence identity of peptides. Combined with a specific proteolytic dependent cellular phagocytosis (ADCP) (Fig. 1.13). Laboratory mice
cleavage step, mass spectroscopy can be used for peptide mass mapping. have been the animal model of choice for generating a ready source
Automation of this process has made mass spectroscopy the analytic of diverse, high-affinity and high-specificity mAbs; however, the use
tool of choice for many proteomics projects. For diagnostic purposes, of rodent antibodies as therapeutic agents has been restricted by the
liquid chromatography and mass spectrometry (LC-MS/MS) have inherent immunogenicity of mouse proteins in a human setting. The
been combined to detect not only a single–amino acid change in the more recent application of transgenic mouse technology to introduce
whole proteome, but also posttranslational protein modification such variable regions encoded by human sequences into the corresponding

Signal Bispecific
BiTE
perturbation Toxin
cytotoxicity

cytotoxic cells
Direct

Nonrestricted
activation of
Radionuclide

Drug
TrioMab

Tumor cell
CMC death
MAC

inhibitory signaling
Fc-mediated immune
effector engagement

Blockade of
ADCC

Phagocytosis

ADCP

APC Helper T cell Cytotoxic T cell


IC uptake MHC class II presentation MHC class I cross-presentation
Introduction of adaptive immune responses

Antigen BiTE MHC TCR T cell


class I Phagocytic
APC
Monoclonal Immunotoxin MHC Fc receptor Tumor cell
antibody class II
Perforin and
Bispecific Compliment Innate granzymes
CD3 KIR
antibody (C1q) effector

Figure 1.13  •  Mechanisms for antibody-based therapies used against cancer cells. Multiple current approaches involve direct cytotoxicity, Fc-mediated
immune effector engagement, nonrestricted activation of cytotoxic T cells, and blockade of inhibitory signaling. The diverse spectrum of action of these therapies
will allow the inclusion of various anticancer targets in the near future (From Weiner LM, Murray JC, Shuptrine CW. Antibody-based immunotherapy of
cancer. Cell. 2012;148:1081–1084.)
Molecular Tools in Cancer Research  •  CHAPTER 1 15

mouse immunoglobulin genes has enabled the generation of “human- being developed that have increased specificity toward individualized
ized” therapeutic mAbs with reduced immunogenicity. In addition, cancers.
bispecific antibodies (bsAbs) with dual affinity for tumor antigens, From an epigenetic perspective, new techniques are enabling the
such as TriomAb, have been shown to effectively kill tumor cells by genome-wide characterization of protein-DNA interactions that can
inducing memory T-cell protective immunity. In addition to the uncover novel transcription factor targets, histone modifications,
expected use of mAbs directed at extracellular epitopes (protein regions and DNA methylation patterns within a cancer cell. Combining
recognized by the antibody), evidence from mouse models has raised chromatin immunoprecipitation (ChIP) with microarray (ChIP-chip)
the possibility of using antibodies targeting intracellular epitopes for allows genome-wide screening for the binding position of protein
anticancer therapies. Targeting such antigens would enrich immuno- factors to their gene targets. In ChIP-chip assays or ChIP-seq, a
therapy, allowing the use of tumor-specific intracellular mediators of cross-linking reagent is applied in vivo to proteins associated with
cell survival and proliferation. Numerous mAb-based agents are currently DNA in the nucleus, which then can be coimmunoprecipitated
in trial or in use as therapeutics for cancer, and the potential for with specific antibodies to the protein under analysis. The bound
further optimization of mAbs through genetic engineering promises DNA and appropriate controls are then fluorescently labeled and
to open new avenues for in vivo therapy. applied to microscopic slides for microarray analysis, or directly
A recent advancement in mAb-based cancer therapy is the generation sequenced, rendering a simultaneous profile of all the binding posi-
of chimeric antigen receptor (CAR) T lymphocytes to target tumors tions of specific proteins in the cancer cell’s genome (Fig. 1.14). The
in vivo. These are effector T-lymphocytes engineered to express a mAb global profiling of promoter occupancy of specific cancers, wherein
that recognizes specific groups of cancer cells. The receptors are chimeric, protein-DNA interaction profiles discriminate patients with tumors
composed of engineered molecules from diverse sources. The first from those presenting different clinical outcomes, is a promising
generation of CAR-modified T cells (CAR T cells) showed success in predictive method.
preclinical trials and have entered phase I clinical trials in ovarian After a decade of development, proteomics is still primarily a
cancer, neuroblastoma, and various types of leukemia and lymphoma. basic research activity, yet in the near future this technology is likely
Newer generations of these therapeutic lymphocytes are currently to have a profound impact on medicine. By defining the collective

Analysis and visualization Peak calling Alignment

Chr1:13456-13486
Chr1:24323-24293
2
Chr1:45678-45708 Sequencing
Chr1:54321-54351
Information content

1.5
Chr1:55679-55709
1
etc...
0.5
ChlP-Seq 0
1 2 3 4 5 6 7 8 9 10
Position

Library
construction

Antibody
Binding sites

TF TF

105
4
P < 0.001
log2 enrichment

20Kb

104
Cy3 intensity

0
–1
1000
ChlP-on-chip 200bp
Binding sites

ChlP replicate 1
100
Peak

ChlP replicate 2
Peak
10
10 100 1000 104 105
Cy5 intensity

Binding site identification Array data analysis Genomic arrays

Figure 1.14  •  Methods for unbiased identification of transcription factor binding sites. Chromatin immunoprecipitation on sequencing (ChIP-seq) and
chromatin immunoprecipitation on microarray chip (ChIP-chip) can provide location, isolation, and identification of the DNA sequences occupied by specific
DNA-binding proteins in cells. Proteins capable of DNA interactions are targeted with specific antibodies. DNA and the associated proteins are cross-linked;
DNA is fragmented into 150 to 500 bp and immunoprecipitated. After reversion of the cross-link, DNA is isolated and either mass-sequenced (ChIP-seq)
or used as probes in a genomic array (ChIP-chip), and binding sites occupied by the proteins can be identified in the genome. These binding sites may indicate
functions of various transcriptional regulators and help identify their target genes during development and disease progression. The types of functional elements
identified with these techniques include promoters, enhancers, repressor and silencing elements, insulators, boundary elements, and sequences that control
DNA replication. (From Kim TH, Ren B. Annu Rev Genomics Hum Genet. 2006;7:81–102 and Liu et al. BMC Biol. 2010;8:56.)
16 Part I: Science and Clinical Oncology

Human Drosophila Yeast C. elegans Arabidopsis

Transcriptional Virus-host Metabolic Protein-protein Disease


regulatory network network network interaction network
Alzheimer’s
disease

Hypertension
Pseudohypo-
Atherosclerosis aldosteronism
Asthma

Figure 1.15  •  Interactome networks and human disease. Networks are integrated sources of information obtained from biochemical, molecular, proteomic,
and other high-throughput analyses. Different networks can be obtained for each organism, organ, or cell. In the first instance, central regulatory “nodes”
identify important components in the network. These networks and their data can then be integrated and compared with healthy and disease models, allowing
an integrative view of events that is much more powerful than isolated networks. (Modified from Vidal M, Cusick ME, Barabási A. Interactome networks
and human disease. Cell. 2011;144:986–998.)

protein-protein interactions in a cancer cell (its “interactome”), recently, PDX models have been further optimized with the use of
functional relationships between disease-promoting genes may be humanized host mice that are modified to contain human immune
revealed that provide novel candidates for intervention (Fig. 1.15). systems.
Networks of disorder-gene associations are already being built that
offer a platform for describing all known phenotype and disease-gene TRANSGENIC MODELS OF CANCER
associations, often indicating the common genetic origin of many
diseases. A precise diagnosis of cancer through use of proteomics Integrating an oncogene that causes malignancy into the genome of
can be envisioned, based on highly discriminating patterns of a mouse without altering the mouse’s own genes generates a transgenic,
proteins in easily accessible patient samples. Proteomics informa- cancer-prone mouse that transmits this trait to its offspring with a
tion also promises to provide sophisticated mathematical models of dominant pattern of inheritance. The technology for producing
the molecular events underlying a process as complex as neoplastic transgenic mice joins recombinant DNA methodology with standard
transformation, which will capture the dynamics of the disease with techniques that are used today by in vitro fertilization clinics, relying
unprecedented power. on the understanding of mammalian reproduction and the development
of protocols to harvest, manipulate, and reimplant eggs and early
MODELING CANCER IN VIVO embryos (Fig. 1.17). The transgene is constructed so that the gene
product will be expressed under appropriate spatial and temporal
Once the mechanistic underpinnings of a particular cancer have been control. In addition to all the standard signals necessary for efficient
described, creating an animal model to test that mechanism becomes transcription and translation of the gene, transgenes contain a promoter,
critical to understanding the pathophysiology and to design therapeutic or regulatory region, that drives transcription in either a ubiquitous
strategies for treatment. Advances in manipulation of the mouse genome or a tissue-restricted pattern. This requires an extensive knowledge of
have resulted in more sophisticated models of human cancer. These genetic regulation in the target cells. A recent advance that circumvents
methodologies can circumvent embryonic death by targeted alteration this requirement involves embedding the transgene inside another
of gene expression only after a critical period in development, and gene locus that is expressed in the desired pattern. Held in a bacterial
reduce the complexity of gene functional analysis by restricting its artificial chromosome (BAC) for easier manipulation, this long stretch
pattern of activation. Inducible gene expression or silencing also allows of DNA surrounding the host gene is likely to carry all the necessary
acute, as opposed to chronic, effects to be assessed. Although species regulatory information to guarantee a predictable expression pattern
differences in tumor susceptibility and disease remission exist between of the introduced transgene.
mouse and man, the tools for genetic manipulation in mouse are The transgene DNA is then injected into the male pronucleus of
superior to those in other mammals, and useful information about a fertilized mouse egg, obtained from a female mouse in which
the function of oncogenes can be gained by targeted expression of hyperovulation has been hormonally induced. The injected eggs are
mutant protein products in mouse tissues. cultured to the two-cell stage and then implanted in the oviduct of
A major hurdle in generation of clinically relevant mouse models another recipient female mouse. Transgenic pups are identified by the
to develop cancer treatments stems from the lack of patient tailoring. presence of the transgene in their genomic DNA (obtained from the
Cancer cells present a highly heterogeneous population that varies tip of the tail and analyzed with PCR assay). Typically, several copies
with the genetic makeup of the individual patient. This shortcoming of the transgene are incorporated in a head-to-tail orientation into a
has been addressed with the advent of patient-specific avatars, also single random site in the mouse genome. About 30% percent of the
known as personalized mouse models or patient-derived xenograft resulting pups will have integrated the transgene into their germline
(PDX) models (Fig. 1.16). Implanting patient biopsy specimens into DNA and constitute the founders of the transgenic lines. RNA analysis
immunodeficient mice allows growth of the tumor, generating in vivo of their progeny determines the level of transgene expression, and
precision models without further in vitro manipulation of the tumor whether the transgene is being expressed in the desired location or at
tissue. These models show great promise for designing treatment and the appropriate time. Given the variability in transgene number and
drug tests that should best target the patient-specific tumor. Most chromosomal location, transgene expression patterns and levels can
Molecular Tools in Cancer Research  •  CHAPTER 1 17

A B
Figure 1.16  •  Mouse avatar (PDX) models. (A) Patient-derived xenograft (PDX) mice are generated by implanting patient tumors into immunodeficient/
humanized mice. The tumors can then be propagated for several passages in fresh mice for a number of generations. (B) Usually, after the third generation
the tumors can be isolated and characterized for further study. These mice can potentially be used for patient drug-specific testing and molecular characterization,
therefore allowing for personalization of cancer treatment. (From http://www.the-scientist.com/?articles.view/articleNo/42470/title/My-Mighty-Mouse/.)

diverge considerably among different founder lines carrying the same The general scheme involves two mouse lines, one carrying the
transgene. recombinase either as a transgene driven by inducible regulatory
In general, transgenesis is optimal for modeling oncogenic mutations elements or knocked into one allele of a gene expressed in the desired
that cause a gain of function, producing disease even when they occur tissue. The other mouse line harbors a modified gene target including
in only one of a gene’s two alleles. For example, an activating mutation recognition sequences. Mating the two lines results in progeny carrying
in a growth factor that causes abnormal cell proliferation can be both the target gene and the recombinase, which interacts with the
mimicked by introducing a transgenic version of the mutated growth target gene only in the desired tissue.
factor gene under the control of an appropriate regulatory sequence A popular conditional methodology is based on the activation of
for expression in the tissue of interest. The relative susceptibility of nuclear hormone receptors to control gene expression. Two current
such a transgenic mouse to tumorigenesis can help distinguish between systems involve activation of a mammalian estrogen receptor, estrogen
a primary and secondary role of the mutant factor, and established analogue 4-hydroxy-tamoxifen, or an insect hormone receptor with
lines of these animals can be used for testing new therapeutic the corresponding ligand ecdysone. Although several variations on
protocols. these hormone-receptor systems are currently in use, the underlying
principle is the same. The Cre recombinase gene, or another regulatory
CONDITIONAL CONTROL OF protein, such as a transcription factor, is fused with the ligand-binding
ONCOGENE ACTIVATION domain (LBD) from a nuclear hormone receptor protein. The resulting
chimeric transgene is placed under the control of a promoter that
The genetic construction of cancer-prone transgenic mice with the directs expression to the tissue of interest, and transgenic animals are
capacity to induce oncogene expression in vivo provides a new avenue generated. In the absence of the hormone or an analogue, the fusion
to modeling the role of oncogenes in tumor generation and mainte- protein accumulates in the desired tissue but is rendered inactive
nance. This technology relies on conditional mutagenesis. Producing through its association with resident heat shock proteins. Hormone,
conditional mutations in mice requires a DNA recombinase enzyme administered either systemically or topically, binds to the LBD moiety
that does not recognize any mouse sequence, but rather targets short, of the fusion protein, dissociates it from the heat shock protein, and
foreign recognition sequences to catalyze recombination between them. allows the transcriptional regulatory component to find its natural
By strategic placement of these recognition sequences in appropriate DNA targets and promote lox-P mediated recombination, or in the
orientations either beside or within a mouse gene, the recombination case of an inducible transcription factor, activate expression of the
results in deletion, insertion, inversion, or translocation of associated corresponding genes. If the LBD is fused to a recombinase, administra-
genomic DNA (Fig. 1.18). Two recombinase systems are currently in tion of hormone leads to the rearrangement of target sequences. This
use: the Cre-loxP system from bacteriophage P1, and the Flp-FRT reaction is not reversible, but lends additional temporal control over
system from yeast. The 34 bp loxP or FRT recognition sequences do recombinase-based mutation. If the LBD is fused to a transcription
not occur in the mouse genome, and both Cre and Flp recombinases factor, removal of hormone leads to inactivation of the fusion protein
function autonomously, without the need for cofactors. Cre- or Flp- and gene downregulation.
mediated recombination is not distance or cell-type dependent, and Another inducible method in use is the tetracycline (tet) regula-
can occur in proliferating or differentiated tissues. tory system. In the classic design (tTA or tet-off ), a fusion protein
18 Part I: Science and Clinical Oncology

Figure 1.17  •  Generation of transgenic mice. The transgene containing


3' Flanking the DNA sequences necessary for the expression of a functional protein is
Promoter 5' UT Coding region 2' UT
region injected into the male (larger) pronucleus of uncleaved fertilized eggs through
Transgene
a micropipette. The early embryos are then transferred into the reproductive
tract of a mouse 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
Collection of fertilized eggs from different numbers of transgenes at different integration sites (and thus express
a superovulated donor mouse various amounts of the protein of interest) are usually studied. UT, Untranslated.
(From Schuldiner AR. Transgenic animals. N Engl J Med. 1996;334:653–655.)

Cell type specific


promoter Cre loxP Target gene loxP

X
Injection
of transgene into
male pronucleus of
uncleaved fertilized egg

Transfer of early embryos into reproductive


tract of a pseudopregnant mouse Cre Cre

A Special cell type All other cells

CMV-β actin
– promoter
– βgeo 3PA EGFP

+ loxP loxP
+ Cre

CMV-β actin
promoter
+ EGFP
Assay of genomic DNA from tails of founder
animals for incorporation of the transgene B loxP

Figure 1.18  •  Conditional mutagenesis schemes. (A) Two mouse lines are
required for conditional gene deletion: first, a conventional transgenic mouse
line with Cre targeted to a specific tissue or cell type; and second, 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 those cells
expressing 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
Sequential matings to determine control of the cytomegalovirus enhancer/chicken actin promoter. Expression
germline integration is widespread, with 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
Study of phenotype green fluorescent protein expression in tissues expressing 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 Kay-Uwe Wagner,
National Institutes of Health; B from Novak A, Guo C, Yang W, Nagy A,
Lobe CG. Z/EG, a double reporter mouse line that expresses enhanced green
fluorescent protein upon Cre-mediated excision. Genesis. 2000;28:147–155.)
Molecular Tools in Cancer Research  •  CHAPTER 1 19

combining a bacterial tet repressor and a viral transactivation domain Overlapping genetic functions can also be defined by crossbreeding
drives expression of the target transgene by binding to upstream tet mice with mutations in different genes. In this way it is possible to
operator sequences flanking the transgene transcription start site. In the study the combinatorial effects of oncogene and tumor suppressor
presence of the antibiotic inducer, the fusion protein is dissociated from gene mutations.
the operator sequences, inactivating the transgene. In a complementary Several caveats are important in considering the use of knockout
design, called reverse tetracycline-controlled transactivator (rtTA or technology in modeling cancer. Most knockouts generate loss-of-
tet-on), structural modification of the tet repressor makes the antibiotic function (null) germline mutations. Inactivation of widely expressed
an active requirement for binding of the fusion protein to the operator genes with multiple functions may have complex phenotypes. Con-
sequences, such that its administration activates transgene expression at versely, if the functions of two genes overlap, a mutation in one of
any time during the life span of the mouse, whereas withdrawal results in the genes may not produce an abnormal phenotype, owing to compensa-
downregulation of the gene. It is important that the transgene integrate tion by the unaltered partner.
into a genomic locus that permits proper tTA or rtTA regulation so that Perhaps the greatest drawback of conventional knockout technology
the system exhibits minimal “intrinsic leakiness” and good antibiotic derives from the disruption of gene function at the earliest stage
responsiveness. of its expression. If the gene has a vital developmental role, the
Conditional expression systems have already been developed to identification of functions later in development can be occluded.
generate hematopoietic, leukemogenic, and lymphomagenic mutations Therefore, although the generation of a null mutation is an excel-
in the mouse, as well as solid tumors. These inducible cancer models lent starting point for analysis, it is far from being functionally
can be exploited to identify oncogenic signals that influence host-tumor exhaustive. For these reasons, conditional mutagenesis is the emerg-
interactions, to establish the role of a given oncogenic lesion in advanced ing method of choice for the elucidation of the gene functions
tumors, and to evaluate therapies targeted toward cancer-causing that exert pleiotropic effects in a variety of cell types and tissues
mutations. Potential clinical application of inducible systems include throughout the life of the animal, which is particularly relevant
targeting virally delivered transgene expression to malignant tissues for the generation of mouse models of adult-onset diseases such
by the use of specific inducible regulatory elements, restricting the as cancer.
expression of transgenes exclusively to affected tissues, and increasing Use of recombinase-mediated gene mutation as described earlier
the therapeutic index of the vectors, particularly in the context of for conditional transgenesis, conditional knockout mutations can be
solid tumors. In all cases, a basic knowledge of the specific mutations designed to disrupt the function of a target gene in a specific tissue
involved in the molecular genetics of malignancies is required because (spatial control) and/or life stage (temporal control). Depending on
it is often unclear that the causal mutation underlying the genesis of the design of the experiment, recombinase action can delete an entire
neoplasia continues to play a central role in the progression to the gene, remove blocking sequences to induce gene expression, or rearrange
fully transformed state. This is particularly important in modeling chromosomal segments. With the advent of recent internationally
cancers characterized by genetic plasticity, wherein drug resistance can coordinated systematic mutagenesis programs aiming to place a
arise subsequent to primary tumor formation. conditional inactivating mutation in each of the 20,000 genes in the
mouse genome, the possibilities for modeling cancer are limited only
MODELS OF RECESSIVE GENE MUTATIONS by a researcher’s choice of the gene loci to test. The constantly evolving
IN CANCER techniques for gene manipulation in vivo constitute a major advance
in cancer research.
In contrast to dominantly acting oncogenes, recessive genetic disorders, Genetically modified mice are of great value in dissecting the
such as loss-of-function mutations in tumor suppressor genes, require pathogenesis of many tumor types. In some knockout studies, the
both copies (alleles) of a gene to be inactivated. The methods needed phenotype of the mutated gene is anticipated by prior knowledge
to produce animal models of recessive genetic disease differ from those of the gene’s function. However, unexpected mutant phenotypes
used in studying dominant traits. Gene knockout technology has been may help clarify the mechanism of the underlying neoplasia.
developed to generate mice wherein one allele of an endogenous gene Pharmacologic manipulation of transgenic, knockout, diversified
is removed or altered in a heritable pattern (Fig. 1.19). Gene disruption animal models of cancer will prove useful in screening therapeutic
or replacement is first engineered in pluripotential cells, termed agents with potential for study in clinical trials. Therapy involving
embryonic stem cells (ESCs), which are genetically altered by introduc- gene or cell replacement can be also tested in genetically engineered
tion of a replacement gene that is inactive or mutant. disease models.
To reduce random integration of the foreign DNA, the replacement
gene is embedded into a long stretch DNA from its native locus in EXPLOITING MOUSE DIVERSITY FOR
the mouse, which targets the recombination event to the homologous CANCER RESEARCH
position in the ESC genome. Inclusion of selectable markers along
with the replacement gene allows selection of the cells in which A novel in vivo tool has emerged that aids in understanding the etiology
homologous recombination has taken place. Site-specific recombinase of cancers, by more accurately reflecting the broad genetic variability
systems combined with gene targeting techniques in ESCs can also in the human population. Cancer research performed with mice has
be used to induce recessive single point mutations or site-specific largely focused on a few individual highly inbred strains with limited
chromosomal rearrangements in a tissue- and time-restricted pattern. genetic diversity, which would equate to single individuals in the
In a variation on this theme called knockin, a foreign gene, such as population. Yet drugs designed to treat one individual are often not
one encoding a marker or a mutated gene, can be placed in the locus effective in other patients. The Collaborative Cross (CC) was created
of an endogenous gene. The engineered ESCs are then microinjected to provide mouse models that better represent the diversity seen in
into the cavity of an intact mouse blastocyst sufficiently early in gestation natural human populations while still retaining the broad power of
that they can, in principle, populate all the tissues of the developing genetic analysis seen in mice. The CC resource is a large panel of
chimeric embryo. This is rarely the case, so contribution of ESCs to recombinant inbred (RI) strains generated by randomly mixing the
the resulting animal is most often assessed with use of ESCs and genetic diversity of eight extant inbred mouse lines, and can be used
blastocysts whose genes for coat color differ. to test the impact of treatments in a diverse genetic pool akin to the
If the ESCs contribute to the germ cells of the founder mouse, human population (Fig. 1.20). A related resource, the Diversity Outcross
their entire haploid genome can be passed on to subsequent generations. (DO), offers higher mapping resolution by randomized outcrossing
Through mating together of subsequent progeny of the founder mouse, of partially inbred CC strains, which segregates the same allelic variants
both alleles of the mutated gene can be passed to a single animal. but embeds them in a distinct population architecture in which each
20 Part I: Science and Clinical Oncology

Embryonic stem cell

Tumor suppressor gene

5' Homologous 3' Homologous


region region
Intron
Cellular
gene

Embryonic stem
Plasmid
cell culture pgk-neo pgk-tk DNA
Knockout
vector
Homologous
recombination
Cellular gene
replaced

Injection of embryonic stem Implantation of chimeric


cells into host blastocyst blastocyst in foster mother
Selection by neomycin and ganciclovir

Germline offspring Chimeric offspring

Figure 1.19  •  Gene knockout strategies. 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). 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 homozygous for tumor suppressor deficiency. (Modified from Mazjoub JA, Muglia LJ. Knockout mice. N Engl J Med. 1996;334:904–906.)
Molecular Tools in Cancer Research  •  CHAPTER 1 21

Founder strains Diversity Outbred (DO) formation and holds great promise for improved treatment of
A/J
human cancer.
Chemotherapy still has numerous side effects. As a number of
C57BL/6J
Outbreeding oncologists may testify, patients sometimes forgo treatment because
129S1/SvImJ of its toxic nature. In a number of cases of chronic lymphocytic
leukemia, if the symptoms of the disease are “under control,” treatment
NOD/ShiLtJ is not prescribed. Therefore it is important to find alleviation strategies
NZO/H1LtJ Collaborative Cross (CC) and cures that minimize the side effects. The goal should be to cure
Inbreeding the patient without devastating the person. One future avenue, apart
CAST/EiJ from finding treatment regimens that reduce side effects, is the discovery
PWK/PhJ of chemotherapies with minimal side effects. The nature of several
generations of chemotherapy was to attack cellular processes, such as
WSB/EiJ DNA replication, metabolism, and cell division, with brute force, and
A by trial and error find a balance between alleviating the neoplastic
growth and not interfering with the normal processes. It may be time
to rethink that premise. We now see examples wherein a less potent
chemotherapy elicits a similar effect on a cancer as a potent one,
although the less potent compound may take longer to achieve its
effect. However, because of its low potency, the side effects are minimal.
Therefore, in the future, the success of a therapy needs to be measured
B not only in terms of how quickly a patient is cancer free, but how
well the patient is during and after the treatment. With emerging
Figure 1.20  •  Generation and characteristics of Collaborative Cross (CC) technologies we will be able to fine-tune current successful therapies
and Diversity Outbred (DO) mice. (A) Each CC line originates from intercrosses so that treatment is not so burdensome.
obtained from eight founder lines. Individual unique independent line is In any field of medicine, resistance to the therapy occurs. Evolution-
inbred for at least 15 generations, creating individual CC lines. Together, those ary processes show that predation leads to natural selection of species
lines represent a much broader genetic variation when compared with the
parental lines and therefore are a better representation of natural populations with mutations that avoid their elimination. Similarly, in cancers,
but contain a high degree of homozygosity. Random mating from early 144 during the predation—chemotherapy—clones arise that become
CC crosses leads to a highly genetic heterogeneous outbred population that resistant to the therapy. With emerging technologies such as single-cell
more closely resembles the diversity found in human populations. (B) Images deep sequencing, we will be able to not only detect the rare subclone
of mice representing CC lines. (From Centre for Diabetes Research, Harry that could give rise to resistance, but also predict the probability of
Perkins Institute of Medical Research, Nedlands 6009, W.A Australia” and the development of resistance by sequencing certain markers. For
“School of Medical and Health Sciences, Edith Cowan University, Joondalup instance, clones with mutations in DNA repair pathways have a higher
W.A. 6027 Australia.) probability for genomic instability, which is a precursor for the rise
of resistant clones.
While we pursue new technologies to diagnose patients and
understand the molecular nature of the cancers, an emerging trend
will be to reexamine previous formulated hypotheses or treatments
that could not be tackled before because of the lack of technologies
animal has a high degree of heterozygosity and carries a unique or resources. For instance, it has been long thought that genetic variation
combination of alleles. plays an important role in cancer treatment. This premise was observed
These diversified mice have been shown to be a powerful tool in in human clinical trials, wherein conditions in some populations were
determining the etiology of cancers. In a recent study, analysis of mice refractory to certain treatments. In the future, we would be able to
generated by crossing CC strains with a mouse line carrying a mutated predict the extent to which a treatment would either work or fail in
tumor suppressor APCmin showed that colon cancer frequency and certain population by using diverse mice, and to map alleles that
spectrum varied predictably with genetic background. Identification confer resistance or susceptibility of a tumor before reaching human
of these genetic modifiers of cancer genesis or suppression would clinical trials. Another example is processes that lead to neoplasticity,
inform the design of novel mouse cancer models, potentially yielding such as LOH, which have been studied in cell lines that are homo-
genetic markers that can predict human cancers. geneous in nature. Attempts to use primary cells were not possible
because of polyclonality of cells and the short life span of tumor cells
FUTURE VIEW in vitro.
With all the aforementioned technologies, we now can, and
Recent discoveries that cancer stem cells share essential signaling nodes need to, reexamine older hypotheses with the appropriate type of
with normal stem cells suggest that targeting critical steps in these primary cells. We will probably uncover novel processes of cancer
pathways may lead to improved alternative cancer therapies. However, that were masked, and resolve decades-old controversies and
every human tumor is subtly different. We are now fast approaching competing hypotheses, leading to better understanding and cures
a new era in medicine: creating “tailored” treatments to individual for cancer.
tumors by obtaining integrative “personal omics profiles” (Fig. 1.21). In the future, fields of medicine will continue to marry and
The generation of novel mouse strains that represent the diversity merge; this has been exemplified in numerous examples, some
seen in human populations will likely lead to a more tailored approach mentioned in this chapter—for instance, the use of viruses to deliver
in understanding cancer diversity and therefore will allow the develop- chemotherapy, or the use of engineered T cells to attack cancer. As
ment of more efficient treatments. emerging molecular tools uncover novel processes in different fields
Understanding of underlying molecular biologic principles of of medicine, the cross talk between oncology and these other fields
malignancy, with pathophysiologic consequences, will generate an will continue, enabling discovery of new avenues to alleviate or even
invaluable resource for resolution of complex genetics of tumor cure cancers.
22 Part I: Science and Clinical Oncology

SAMPLE TYPE METHOD ANALYSES

Whole-genome
Variant calling/phasing
sequencing
Heteroallelic and variant
expression
Whole-transcriptome
PBMC sequencing RNA editing
(mRNA and miRNA)
Quantitative differential

Integrated personal OMICS


expression & dynamics

Variant confirmation in
Proteome profiling
RNA and protein

Untargeted proteome Quantitative differential


profiling expression and dynamics

Targeted proteome
Quantitative expression
profiling (cytokines)

Serum Metabolome profiling Dynamics

AutoAntibodyome Differential reactivity


profiling

Medical/lab tests Glucose, HbA1c, CRP,


Telomere length
A

3 4

5
2
Serpina 1
E366K
RNA edits
6

Heteroallelic SNVs
1

Protein-downregulated
7

(HRV vs healthy)
Protein-upregulated
Y

(HRV vs healthy)
RNA-downregulated
8

(HRV vs healthy)
X

RNA-upregulated
(HRV vs healthy)
Indels
9
22

SV-duplications
21

SV-deletions
20

10

Chr. ideogram
19

18
11
17 14 Chr. number
16 12
B 15
14 13

Figure 1.21  •  Integrative personal omics profiles (iPOP). (A) Integrative analysis of iPOP experimental design, indicating tissues and techniques analyzed
in healthy and diseased individuals. (B) Circos plot summarizing iPOP. From outer to inner rings: chromosome ideogram; genomic data (pale blue ring),
structural variants (deletions [blue tiles], duplications [red tiles]), indels (green triangles); transcriptomic data (yellow ring); proteomic data (light purple ring).
(Modified from Chen R, Mias GI, et al. Personal omics profiling reveals dynamic molecular and medical phenotypes. Cell. 2012;148:1293–1307.)
Molecular Tools in Cancer Research  •  CHAPTER 1 23

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human disease. Nature. 2007;447:433–440. Targets, and Therapeutics. USA: Oxford University 2007;9:993–999.
Frese KK, Tuveson DA. Maximizing mouse cancer models. Press; 2005. Wu J, Smith LT, Plass C, Huang TH. ChIP-chip comes
Nat Rev Cancer. 2007;7:654–658. Svenson KL, Gatti DM, Valdar W, Welsh CE, Cheng R, of age for genome-wide functional analysis. Cancer
Goh KI, Cusick ME, Valle D, Childs B, Vidal M, Barabasi Chelser EJ, et al. High resolution genetic mapping Res. 2006;66:6899–6902.
AL. The human disease network. Proc Natl Acad Sci using the mouse diversity outbred population. Genetics.
USA. 2007;104:8685–8690. 2012;190:437–447.
2  Intracellular Signaling
Aphrothiti J. Hanrahan, Gopa Iyer, and David B. Solit

S UMMARY OF K EY P OI N T S
• Ligand binding and activation of growth and survival, a phenomenon the current inability to directly inhibit
cell surface and internal receptors known as oncogene addiction. some oncogenic proteins (i.e.,
trigger the activation and/or • Drugs that selectively target mutated mutant KRAS), the development of
suppression of signaling cascades proteins critical for the maintenance drug resistance, technical hurdles
that regulate diverse cellular of the transformed phenotype have posed by limited tissue availability
processes including cell growth, shown unprecedented clinical for prospective molecular
proliferation, survival, and invasion, activity in genetically defined cancer characterizing, and intratumoral and
among others. subsets. lesion-to-lesion genomic
• Multiple nodes within these • Precision medicine refers to the use heterogeneity.
intracellular signaling networks are of genetic and epigenetic information • Routine genomic analysis of tumors
genetically and epigenetically altered unique to an individual cancer or tumor-derived cell free DNA in
in human cancers, leading to patient to develop treatment plasma is now a component of
constitutive pathway activation or regimens that target the driver standard care in an increasing
suppression. oncogenes and tumor suppressors number of cancer types, with the
• Some cancers are dependent on responsible for tumor progression. results used to guide treatment
genomic alterations in oncogenes or Potential challenges to the selection.
tumor suppressor genes for their application of this approach include

The underlying basis of the cancer phenotype is deregulated cell RECEPTOR TYROSINE KINASE SIGNALING
growth, which stems from two main hallmarks of cancer: uncon-
trolled proliferation, and loss of programmed cell death (enhanced The receptor tyrosine kinases (RTKs) comprise a family of
survival). In normal cells, these processes are tightly controlled transmembrane (TM) cell surface receptors that transduce
through integration of signaling cascades that translate extracellular extracellular signals internally to promote growth and survival and/
and intracellular cues into specific output responses. These signaling or to regulate other cellular phenotypes.2,3 Members of this protein
pathways are often initiated on binding of ligand to the extracellular family share a similar modular domain structure. Growth factors
domain of a receptor, followed by recruitment of adaptor proteins bind to the extracellular ligand-binding domain of RTKs and
or kinases that activate an intracellular cascading network of protein induce dimerization of two receptor monomers, juxtaposing the
and lipid intermediaries that ultimately produce a cellular response. intracellular tyrosine kinase domains of each monomer.4 This results
In normal cells, the specificity, amplitude, and duration of signaling in transphosphorylation of tyrosine residues within the cytoplasmic
are tightly regulated, and these constraints are often abrogated in domains of the RTK dimer. Following transphosphorylation, a variety
human cancers. of intracellular proteins are recruited to the activated RTK through
Investigation of the signal transduction pathways that regulate normal Src homology 2 (SH2) domains that recognize the phosphotyrosine
cellular functions has revealed that key components of these networks plus a specific amino acid sequence motif C-terminal to the tyrosine
are commonly altered in cancer cells by mutation, amplification and residues.5,6
deletion, chromosomal translocation, overexpression, or epigenetic Over 117 SH2 domains have been characterized, each with unique
silencing. These alterations lead to activation or suppression of phosphotyrosine sequence specificities.7 Each domain is part of a larger
signaling cascades that underlie the various hallmarks of the cancer adaptor protein involved in transducing extracellular signals to activate,
phenotype. This chapter reviews the major signal transduction cascades, or in some cases suppress, specific intracellular signaling cascades. Thus
with a focus on those that are frequently altered in human cancers. the complement of signaling pathways that a given RTK regulates is
Individual sections highlight signaling intermediaries that have been dictated by the profile of phosphorylated tyrosine residues plus flanking
validated as drug targets in patients with cancer. Table 2.1 summarizes amino acids within their intracellular domains.8,9 However, more than
actionable gene-level and mutation-level alterations in cancer and one adaptor protein can often recognize individual context-dependent
the drugs that are currently approved by the US Food and Drug phosphotyrosine motifs within an RTK, underscoring how this system
Administration (FDA) for treatment, that are recommended standard is designed to provide both specificity and diversity of intracellular
of care biomarkers, or that have promising clinical or preclinical signaling.
efficacy.1 Text continued on p. 29

24
Intracellular Signaling  •  CHAPTER 2 25

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer
Gene Variant Cancer Type Drug Evidencea
ABL1 BCR-ABL1 fusion ALL Dasatinib 1
Imatinib 1
CML Dasatinib 1
Imatinib 1
Nilotinib 1
AKT1 E17K Breast AZD5363 3
Ovarian AZD5363 3
All Tumors ARQ 751 4
ALK Fusions NSCLC Alectinib 1
Ceritinib 1
Crizotinib 1
Oncogenic mutations NSCLC Brigatinib 1
Fusions Soft tissue sarcoma Ceritinib 2
Crizotinib 2
L1196M, L1196Q NSCLC Brigatinib 3
R1275Q Embryonal tumor Crizotinib 4
ARAF S214A Histiocytosis Sorafenib 3
S214C NSCLC Sorafenib 3
ATM N2875K, R3008C, truncating mutations Prostate cancer Olaparib 4
BRAF V600D, V600E, V600G, V600K, V600M, V600R Melanoma Cobimetinib + vemurafenib 1
Dabrafenib 1
Dabrafenib + trametinib 1
Vemurafenib 1
Histiocytosis Vemurafenib 2
NSCLC Dabrafenib 2
Dabrafenib + trametinib 2
Vemurafenib 2
Colorectal Binimetinib + cetuximab + 3
encorafenib
Panitumumab + vemurafenib 3
Colorectal Fluorouracil + radiation + 4
trametinib
V600E, V600K Melanoma Trametinib 1
Fusions Ovarian Paclitaxel + selumetinib 3
K601E Melanoma Trametinib 3
L597Q, L597R, L597S, L597V Melanoma Trametinib 3
D594E, D594N, G466V, G469A, G469V, G596C Melanoma Trametinib 4
KIAA1549-BRAF Fusion Soft tissue sarcoma Sorafenib + temsirolimus 4
L597Q, L597V Melanoma BGB659 4
BRCA1 Oncogenic mutations Ovarian Niraparib 1
Rucaparib 1
Olaparib 2
BRCA2 Oncogenic mutations Ovarian Niraparib 1
Rucaparib 1
Olaparib 2
CDK4 Amplification Dedifferentiated liposarcoma Abemaciclib, palbociclib 2
Well-differentiated Abemaciclib, palbociclib 2
liposarcoma
CDKN2A Oncogenic mutations Breast Letrozole + palbociclib 4
Esophagogastric Palbociclib 4
EGFR 729_761del, 729_761indel, L858R NSCLC Afatinib 1
Erlotinib 1
Gefitinib 1
Osimertinib 4
E709_T710delinsD, E709K, G719A, G719C, G719D, G719S, NSCLC Afatinib, erlotinib, gefitinib 1
A763_Y764insFQEA, L747P, A750P, A763_Y764insFQEA,
L833V, L861Q, L861R, S768I, EGFR-KDD
T790M NSCLC Osimertinib 1
762_823ins NSCLC EGF816 4
762_823ins, G719A, L861R, S768I NSCLC AP32788 4

Continued
26 Part I: Science and Clinical Oncology

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d
Gene Variant Cancer Type Drug Evidencea
ERBB2 Amplification Breast Ado-trastuzumab emtansine 1
Lapatinib 1
Lapatinib + trastuzumab 1
Pertuzumab + trastuzumab 1
Trastuzumab 1
Esophagogastric Trastuzumab 1
Breast Neratinib 3
Oncogenic mutations Breast Neratinib 3
V659E NSCLC Lapatinib 3
E770_K831indel, E770_K831ins NSCLC AP32788 4
ERCC2 Oncogenic mutations Bladder Cisplatin 3
ESR1 Oncogenic mutations Breast AZD9496, fulvestrant 3
D538G, Y537S Breast GDC-0810 4
EZH2 Oncogenic mutations Diffuse large B-cell GSK126 4
lymphoma Tazemetostat 4
FGFR1 Amplification Lung squamous cell AZD4547 3
carcinoma Debio1347 3
BCR-FGFR1 fusion Leukemia Ponatinib 4
FGFR2 Fusions Adrenocortical carcinoma Debio1347 3
JNJ-42756493 3
Bladder Debio1347 3
JNJ-42756493 3
Cholangiocarcinoma BGJ398 3
Debio1347 3
Endometrial Debio1347 3
JNJ-42756493 3
FGFR3 Fusions Adrenocortical carcinoma Debio1347 3
JNJ-42756493 3
Bladder Debio1347 3
JNJ-42756493 3
Glioma Debio1347 3
JNJ-42756493 3
G370C, G380R, K650E, K650M, K650N, K650Q, K650R, K650T, Bladder Debio1347 3
R248C, S249C, S371C, Y373C JNJ-42756493 3
Breast Debio1347 4
FLT3 Y572_Y630ins AML Sorafenib 3
IDH1 R132C, R132G, R132H, R132Q, R132S AML AG-120 3
All tumors BAY1436032 4
CB-839 4
IDH2 R140Q, R172G, R172K, R172M, R172S All liquid tumors AG-221 3
JAK2 PCM1-JAK2 fusion Leukemia Ruxolitinib 3
KIT 449_514mut, 550_592mut, A502_Y503dup, D579del, D820G, GIST Sunitinib 1
E554_K558del, H697Y, K550_W557del, K558delinsNP, L576P, Thymic tumor Sunitinib 2
P551_M552del, V555_L576del, V560D, V560del, V654A
449_514mut, 550_592mut, D419del, D579del, E554_I571del, GIST Imatinib 1
E554_K558del, E554_V559del, F522C, I563_L576del, I653T,
K550_W557del, K558N, K558_E562del, K558_V559del,
K558delinsNP, K642E, L576P, M541L, M552_W557del,
N564_Y578del, N822H, N822Y, P573_D579del, P577_
W582delinsPYD, P838L, Q556_K558del, T417_D419delinsI,
T417_D419delinsRG, T574insTQLPYD, V530I, V555_L576del,
V555_V559del, V559C, V559D, V559G, V559_V560del,
V559del, V560D, V560G, V560del, V569_L576del, W557G,
W557R, W557_K558del, Y553N, Y553_K558del, Y570H, Y578C
449_514mut, 550_592mut, D820G, D820Y, K550_W557del, GIST Regorafenib 1
K558delinsNP, N822K, V560D
D816F, D816Y, D820G, D820Y, L576P, N822I, V559D, V560G, GIST Dasatinib 2
W557_K558del
D816V, D820A, D820G, D820Y, K642E, L576P, V555_L576del, GIST Nilotinib 2
V559C, V559D, V654A, W557_K558del
D820A, D820E, D820G, D820Y, K642E, N505I, P577_D579del, GIST Sorafenib 2
V559D, W557_K558del Thymic tumor Sorafenib 2
K642E, L576P, V559A Melanoma Imatinib 2
Intracellular Signaling  •  CHAPTER 2 27

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d
Gene Variant Cancer Type Drug Evidencea
KRAS Wild type Colorectal Cetuximab 1
Panitumumab 1
Regorafenib 1
Cabozantinib + 4
panitumumab
Panitumumab + regorafenib 4
Pembrolizumab 4
Oncogenic mutations All tumors alpelisib + binimetinib 4
Cobimetinib + GDC-0994 4
Colorectal Atezolizumab + cobimetinib 4
Fluorouracil + radiation 4
therapy + trametinib
NSCLC Abemaciclib, PD0325901 + 4
palbociclib, palbociclib,
ribociclib, ribociclib +
trametinib
Binimetinib + erlotinib 4
Binimetinib, selumetinib, 4
trametinib
Docetaxel + trametinib 4
MAP2K1 Oncogenic mutations Histiocytic disorder Cobimetinib, selumetinib, 3
trametinib
Low-grade serous ovarian Cobimetinib, selumetinib, 3
trametinib
Melanoma Cobimetinib, selumetinib, 3
trametinib
NSCLC Cobimetinib, selumetinib, 3
trametinib
MDM2 Amplification Liposarcoma DS-3032b 3
RG7112 3
SAR405838 4
MET 963_D1010splice, 981_1028splice, X1006_splice, X1007_ NSCLC Crizotinib 2
splice, X1008_splice, X1009_splice, X1010_splice, X963_ Cabozantinib 3
splice Capmatinib 3
Amplification NSCLC Crizotinib 2
RCC Cabozantinib 2
D1010H, D1010N, D1010Y NSCLC Crizotinib 2
Cabozatinib 3
Capmatinib 3
MTOR E2014K Bladder Everolimus 3
C1483F, F1888L, L2230V, S2215F, T1977K RCC (clear cell) Everolimus, rapamycin, 4
temsirolimus
NF1 Oncogenic mutations Glioblastoma Trametinib 4
Melanoma Trametinib 4
Neurofibroma Binimetinib 4
Neurofibroma PLX3397 4
NRAS Oncogenic mutations Colorectal Atezolizumab + cobimetinib 3
Melanoma Binimetinib 3
Binimetinib + ribociclib 3
Thyroid Radioiodine uptake therapy 3
+ selumetinib
Colorectal Fluorouracil + radiation 4
therapy + trametinib
NTRK1 Fusions All tumors Larotrectinib 3
Salivary gland Entrectinib 3
NTRK2 Fusions Salivary gland Entrectinib 3
Larotrectinib 3
NTRK3 Fusions Salivary gland Entrectinib 3
Larotrectinib 3

Continued
28 Part I: Science and Clinical Oncology

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d
Gene Variant Cancer Type Drug Evidencea
PDGFRA FIP1L1-PDGFRA fusion Leukemia Imatinib 1
Fusions Myelodysplasia Imatinib 1
Myeloproliferative Neoplasm Imatinib 1
560_561insER, A633T, C450_K451insMIEWMI, C456_N468del, GIST Imatinib 2
C456_R481del, D568N, D842I, D842_H845del, D842_
M844del, D846Y, E311_K312del, G853D, H650Q, H845Y,
H845_N848delinsP, I843del, N659K, N659R, N659S, N848K,
P577S, Q579R, R748G, R841K, S566_E571delinsR, S584L,
V469A, V536E, V544_L545insAVLVLLVIVIISLI, V561A, V561D,
V561_I562insER, V658A, W559_R560del, Y375_K455del,
Y555C, Y849C, Y849S
D842V GIST Dasatinib 2
PDGFRB Fusions Dermatofibrosarcoma Imatinib 1
Protuberans
Myelodysplasia Imatinib 1
Myeloproliferative neoplasm Imatinib 1
PIK3CA Oncogenic mutations Breast Alpelisib 3
Alpelisib + fulvestrant 3
Buparlisib 3
Buparlisib + fulvestrant 3
Copanlisib 3
Fulvestrant + taselisib 3
GDC-0077 3
Serabelisib 3
Taselisib 3
Alpelisib + everolimus 4
Alpelisib + letrozole, Alpelisib 4
+ letrozole + ribociclib
Alpelisib + LJM716 + 4
trastuzumab
Alpelisib + olaparib, 4
buparlisib + olaparib
AZD5363 + fulvestrant 4
AZD8835 + fulvestrant 4
MLN0128 + serabelisib 4
All tumors ARQ 751 4
AZD5363 + olaparib 4
GDC-0077 4
Endometrial Alpelisib + fulvestrant 4
Buparlisib + fulvestrant 4
Fulvestrant + taselisib 4
Ovarian Alpelisib + fulvestrant 4
Buparlisib + fulvestrant 4
Fulvestrant + taselisib 4
PTCH1 Truncating mutations Embryonal tumor Sonidegib 3
Skin cancer, nonmelanoma Sonidegib 3
Vismodegib 3
PTEN Oncogenic mutations All tumors ARQ 751 4
AZD5363 + olaparib 4
AZD8186 4
Gedatolisib + palbociclib 4
GSK2636771 4
LY3023414 4
Endometrial Olaparib 4
Prostate Enzalutamide + LY3023414 4
RAF1 S257L Lung adenocarcinoma Sorafenib 4
RET Fusions NSCLC Cabozantinib 2
Vandetanib 3
ROS1 Fusions NSCLC Crizotinib 1
D2033N Cabozantinib 3
Intracellular Signaling  •  CHAPTER 2 29

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d
Gene Variant Cancer Type Drug Evidencea
TSC1 Oncogenic mutations CNS Everolimus 2
RCC Everolimus 2
TSC2 Oncogenic mutations CNS Everolimus 2

a
Levels of evidence:
• Level 1: FDA-recognized biomarker predictive of response to an FDA-approved drug in this indication
• Level 2: Standard care biomarker predictive of response to an FDA-approved drug in this indication or another indication; including those recommended by NCCN, but
not FDA recognized as standard of care
• Level 3: Evidence of clinical activity in this indication, or another indication
• Level 4: Preclinical or biologic evidence of activity
ALL, Acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; CNS, central nervous system; FDA, US Food and Drug Administration;
GIST, gastrointestinal stromal tumor; NCCN, National Comprehensive Cancer Network; NSCLC, non-small cell lung cancer; RCC, renal cell carcinoma.
Data modified from oncokb.org1; Chakravarty D, Gao J, Phillips S Kundra R, Zhang H, Wang J. OncoKB: a precision oncology knowledge base. JCO Precis Oncol. Published
online May 16, 2017.

Recruitment of signaling intermediaries to the plasma membrane epigen.19–21 Growth factor binding promotes either homodimerization
facilitates their interaction with membrane-bound proteins responsible or heterodimerization with other HER family members, followed by
for stimulating a diverse array of downstream pathways (Fig. 2.1). As transphosphorylation.22 A ligand for HER2 has not yet been identified;
an example, the lipid kinase phosphatidylinositol 3-kinase (PI3 kinase), instead, HER2 is activated through heterodimer formation with one
described in more detail in a later section, recognizes and binds to a of the other three ligand-bound receptors.23 Notably, HER2 is the
pattern of phosphorylated tyrosine residues present within multiple preferred dimerization partner for EGFR, and EGFR-HER2 heterodi-
activated RTKs through the SH2 domain located in its p85 regulatory mers are more stable than EGFR homodimers, remaining at the cell
subunit. Binding of the p85 regulatory subunit in turn results in surface for a longer duration and undergoing endocytosis at a lower
activation of its kinase activity. Approximately 20 classes of RTKs rate than EGFR homodimers.24,25 Furthermore, HER2 reduces the
have been defined based on growth factor specificity. This section will dissociation rate of EGF from EGFR, allowing for a more sustained
focus on those RTK classes for which specific cancer therapies exist period of EGF-induced signaling.19 The EGFR and HER2 components
or are in development. of the EGFR-HER2 heterodimer are also more likely to be recycled
back to the cell surface than EGFR homodimers, which are more
Epidermal Growth Factor Receptor Signaling readily targeted for degradation.26 In addition, HER2-HER3 het-
erodimers possess the most potent mitogenic activity among the
Historically, the growth factors that stimulate RTKs were first heterodimer and homodimer HER kinase combinations.27 In contrast
discovered, followed by the structural and functional characterization to the other HER kinase family members, HER3 does not have intrinsic
of the RTKs themselves.10,11 Epidermal growth factor (EGF) was kinase activity and preferentially forms heterodimers with HER2.28
initially purified from mouse submaxillary glands in 1962 by Stanley The ligands for HER3 and HER4 are the neuregulins, including
Cohen and was found to stimulate premature eyelid opening and heregulin.
incisor eruption, phenotypes that suggested a role for EGF in the A number of tumor types frequently exhibit alterations within the
regulation of cellular proliferation.12 In 1978, the epidermal growth EGFR family of RTKs.21 Sustained activation of these pathways can
factor receptor (EGFR) was identified as the cell surface binding result in oncogene- or pathway-addicted tumors, and selective HER
site for EGF.13 Over the next several years, tyrosine phosphorylation kinase inhibitors are now a component of the standard treatment of
was identified in cells, followed by the discovery that the viral Src several malignancies. Alterations that affect RTK activity include
oncogene, which induces transformation of cells in vitro, is itself a mutations that result in constitutive activation of the tyrosine kinase;
tyrosine kinase, underscoring the potential importance of tyrosine overexpression of the receptor, often due to gene amplification; and
phosphorylation for oncogenesis.14,15 Once the complete sequence elevated levels of RTK ligands that stimulate signaling. EGFR mutations
of the EGFR protein was elucidated in the 1980s,16 the amino acid are found in 10% to 25% of non–small cell lung cancers (NSCLCs),
sequence of the receptor cytoplasmic domain was found to be similar with variation in the frequency of such alterations influenced by
to Src, suggesting that EGFR also possessed tyrosine phosphorylation ethnicity and geographic location; in-frame microdeletions in exon
activity. The connection between RTK activation and oncogenesis was 19 and point mutations in exon 21 (most commonly L858R or L861Q)
further solidified when the amino acid sequence of EGFR was found or exon 18 (G719X) comprise over 80% of these alterations.29–31 In
to be homologous to the avian erythroblastosis virus erbB oncogene, glioblastoma multiform (GBM), EGFR mutations, indels (including
which, when infected into chicken red blood cell precursors, is sufficient the EGFRvIII variant in which exons 2 to 7 of the extracellular domain
to induce erythroleukemia.17,18 The erbB oncogene encodes a TM are deleted, generating a ligand-independent, activated protein),
protein that lacks the extracellular ligand binding domain of EGFR amplification, splice variants, and rearrangements occur in 57% of
but possesses a cytoplasmic kinase domain that, when expressed in cells, tumors.32–34 However, because of the heterogeneity of GBM tumors,
can signal in a growth factor–independent manner. Subsequent studies targeting EGFR is complicated; EGFR alteration is often concurrent
have since identified within human cancers numerous alterations of with amplification or mutation of another RTK such as PDGFR,
EGFR and other RTKs that enhance proliferation without the need MET, or FGFR, or the presence of EGFRvIII on extrachromosomal
for growth factor stimulation. DNA, or activation of IDH1.35–38 Overexpression of wild-type EGFR
The EGFR class of RTKs comprises four receptor proteins encoded as a result of gene amplification has been observed in NSCLC and
by four genes (in parentheses): EGFR (ERBB1), HER2/Neu (ERBB2), breast, gastric, colorectal, and head and neck cancers, and less commonly
HER3 (ERBB3), and HER4 (ERBB4). EGFR binds to and is activated in other tumor types.39–41
by a number of ligands, including EGF, transforming growth factor–α Up to 30% of breast cancers display overexpression of HER2,
(TGF-α), HB-EGF, amphiregulin, betacellulin, epiregulin, and which is an unfavorable prognostic factor, and therapy for these
30 Part I: Science and Clinical Oncology

Growth Factor Ligand oncogene


tumor suppressor
Receptor Tyrosine Kinase

P Shc
Ras-GDP NF1
P Grb2 Trafficking/
Sos RalGDS RalA/B
proliferation
Ras-GTP PLCε
Vermurafenib
Dabrafenib
Raf p85
P P TIAM1
MEKK1/NF-κB PIK3CA-
p110α
Trametinib P P
Selumetinib MEK
Cobimetinib
CDC42/RAC AKT

SCH772984 P P
DUSPs NF- κB/actin
BVD-523 ERK
DEL-22379 mTORC1
P
p90RSK
translation
NF- κB, Myt-1, GSK3, PP-1

P
Fos/Jun/etc. Proliferation/growth

Negative feedback

Figure 2.1  •  Canonical Ras/MAPK signaling pathway. Ras proteins cycle between GDP-bound inactive and GTP-bound active states. Ras is often activated
in response to ligand-specific binding to its cognate receptor. Ras can also be activated via intracellular cross talk. This schematic depicts the classic Ras/MAPK
signal transduction cascade. Growth factor stimulation induces receptor tyrosine kinase (RTK) dimerization and autophosphorylation of tyrosine residues
located within the intracellular domain of the receptor. These phosphorylated tyrosine residues serve as docking sites for scaffold proteins that facilitate activation
of intracellular signaling cascades. For example, the adaptor protein Grb2, via its SH3 domain, recruits the Ras GEF (guanine-nucleotide exchange factor)
SOS. Colocalization of SOS and Ras facilitates substitution of GTP for GDP and thus Ras activation. Active GTP-bound Ras binds and recruits the Raf (A-,
B-, and C-Raf ) serine/threonine kinases to the plasma membrane and facilitates their activation. Active Raf in turn phosphorylates and activates MEK, which
in turn phosphorylates and activates ERK. ERK phosphorylates substrates in the cytoplasm (p90RSK) and in the nucleus (Jun, Fos, Ets-2, Elk-1, CREB1,
AP-1, ATF-2, among others), which regulate cell proliferation and survival. Ras interacts with more than 20 effector proteins, including the p110α subunit
of PI3 kinase, RasGDS, PLCε, and TIAM1, which in sum control transcription, translation, vesicular trafficking, cell cycle progression, cytoskeletal changes,
metabolic processes, immune inflammatory responses, and survival. Induction of Ras signaling also upregulates negative feedback elements that inhibit the
pathway (e.g., DUSPs and SPROUTYs/SPREDs). Several proteins within the Ras signaling cascade are proto-oncogenes (green) and tumor suppressors (red)
that are mutated, amplified, or deleted in many cancers. A number of selective inhibitors of Ras effectors have been tested as anticancer therapies. Examples
include kinase inhibitors, which selectively target B-Raf and its downstream effectors MEK and ERK (see red boxes).

ERBB2-amplified breast cancers is now distinct from that of breast of KRAS wild-type colorectal and head and neck cancers.51–56 Pani-
cancers with normal HER2 expression levels.21,42 ERBB2 amplification tumumab, another human monoclonal anti-EGFR antibody, is also
is also a driving event in gastric and to a lesser extent in bladder, approved for KRAS wild-type metastatic colorectal cancer.56
endometrial, and cervical cancer.43–45 More recently, activating mutations The first-generation reversible EGFR tyrosine kinase inhibitors
and in-frame insertions/indels in ERBB2 were found to occur in 1% gefitinib and erlotinib, as well as the second-generation irreversible
to 2% of all cancer patients, most commonly in patients with bladder inhibitor afatinib, are FDA approved for the treatment of NSCLC,
cancer.44 Mutations in ERBB2 localize to either the extracellular domain, with greatest efficacy in patients with EGFR mutations or in-frame
where they are presumed to promote dimer formation, or the kinase deletions.57–60 A second site mutation in EGFR (T790M) is a common
domain.46,47 In addition, activating mutations in ERBB3 have also mechanism of acquired resistance to first generation EGFR inhibitors.
been identified in bladder, colon, and gastric cancers.48,49 Osimertinib (AZD9291), a third-generation EGFR inhibitor, is highly
Numerous targeted agents have been developed that selectively active in patients with NSCLC in which resistance is mediated by the
inhibit EGFR-induced signaling (see Table 2.1 and Fig. 2.2).21,50 EGFR T790M mutation and is now FDA approved for this indica-
Cetuximab, a chimeric monoclonal antibody that binds to the tion.61,62 The development of osimertinib and the fourth-generation
extracellular domain of EGFR and competitively inhibits ligand binding, EGFR inhibitor EAI04563 highlights how studies of acquired resistance
thereby preventing receptor activation, is approved for the treatment can lead to the rational development of more effective kinase inhibitors.
Intracellular Signaling  •  CHAPTER 2 31

XL147
Buparlisib Growth Factor Ligand
Alpelisib (α-specific) EGFR:Cetuximab
AZD5363 AZD8186 (β-specific) EGFR/HER2:Lapa­nib,
Copanlisib (α/δ-specific) Receptor
Afureser­b Trastuzumab
Idelalisib (δ-specific) Tyrosine
Kinase

EGFR:gefi­nib, erlo­nib,
PIP2/3 P
PIP2 P osimer­nib
AKT P PI3K-
p85
IRS1 EGFR/HER2:lapa­nib,
P P P
PTEN p110α afa­nib, nera­nib
P
Other: ima­nib, alec­nib,
PIP3 Ras-
IκB PDK1 larotrec­nib, etc
P GTP
mTORC2 SGK CDC42/RAC
Bad
P Dual PI3K-mTOR inhibitors:
P RHEB-GDP NF-κB/ac­n
NF-κB dactolisib, voxtalisib
GSK3β apoptosis TSC1
TSC2

RHEB-GTP
P P
Cyclin
D1 mTOR inhibitors:
PRAS40 mTORC1 rapamycin, everolimus,
temsirolimus, AZD8055, RapaLink
degrada­on
P P
4EBP1 p70S6K
P
Protein transla­on S6

FOXO1/4 Arrest/Apoptosis
oncogene
tumor suppressor

prolifera­on and survival


Figure 2.2  •  PI3K/mTOR signaling pathway. The PI3 kinase family proteins are lipid kinases that transduce signals from receptor tyrosine kinases (RTK)
and G protein–coupled receptors to intracellular cascades that control proliferation, survival, and other cellular phenotypes. As an example, growth factor
binding causes receptor dimerization and subsequent phosphorylation of tyrosine residues in the intracellular domain of the receptor. These tyrosine phosphoryla-
tion sites serve as docking sites for the p85 regulatory subunit of PI3 kinase and adaptor proteins such as IRS-1 in the case of signaling induced by the insulin/
IGF1 receptors. This results in allosteric activation of the catalytic subunit of PI3 kinase, which converts PIP2 to PIP3. PIP3 recruits PDK1 and AKT to the
membrane via their pleckstrin homology (PH) domains. Colocalization of PDK1 and AKT results in phosphorylation (on threonine 308) and activation of
AKT. Phosphorylation of AKT on Ser473 by mTORC2 is required for full activation of AKT. Activated AKT phosphorylates several effectors, including
GSK3β, Bad, PRAS40, IκB, the FOXO1/4 transcription factors, and TSC2. AKT phosphorylation of TSC2, which is bound to TSC1, inhibits the GTPase
function of this complex, thereby allowing activation of Rheb and subsequent activation of mTORC1. In turn, mTORC1 phosphorylates p70S6 kinase
(p70S6K) and 4EBP1, an inhibitor of the eIF4E component of the cap-dependent translation initiation complex. p70S6K and mTOR also function to negatively
regulate the pathway by initiating the phosphorylation and inhibition of IRS-1. PI3 kinases can also signal to other effectors such as Rac/CDC42 and the
serum-glucocorticoid kinase (SGK) family to promote cellular survival, motility, and cytoskeletal rearrangement. Many components of the PI3 kinase signaling
pathway are mutationally altered in cancer (oncogenes in green, tumor suppressors in red). A variety of compounds have been developed that selectively inhibit
PI3 kinase signaling components. US Food and Drug Administration (FDA)–approved drugs and novel inhibitors in clinical testing that target RTKs, PI3
kinase, mTOR kinase, and AKT are highlighted in the red boxes.

ERBB2 amplification strongly correlates with HER2 protein Although the introduction of trastuzumab has resulted in a sig-
overexpression, and the presence of either marker predicts for trastu- nificant improvement in the survival of patients with HER2-
zumab response in certain cancers.64 Trastuzumab, a humanized antibody overexpressing breast cancers, drug resistance remains a major clinical
that binds to the extracellular domain of HER2, has been FDA approved problem. Potential resistance mechanisms include concomitant
for the treatment of breast65–71 and esophagogastric72 cancers displaying overexpression of other HER kinase family members and/or ligands,
HER2 overexpression. In patients with breast and gastric cancers, PTEN loss, and the expression of a truncated HER2 protein lacking
trastuzumab has modest activity when administered as single- the extracellular antibody binding site.79 Additional HER2-directed
agent therapy and is most commonly used in combination with agents include the tyrosine kinase inhibitors lapatinib and neratinib
chemotherapy.72–74 The combination of docetaxel, trastuzumab, and (see Table 2.1). Lapatinib is FDA approved for use in combination
pertuzumab,75 an antibody that binds to a different HER2 epitope with capecitabine in patients with HER2-overexpressing advanced or
(the dimerization domain) than trastuzumab and results in impaired metastatic breast cancer that has progressed on prior therapy with
dimer formation, is also approved for breast cancer.76–78 trastuzumab and certain classes of chemotherapy.80 The combination
32 Part I: Science and Clinical Oncology

of lapatinib and trastuzumab is also FDA approved in HER2-amplified alectinib and ceritinib that are either more potent or more selective
breast cancer.81–83 Lapatinib also received accelerated approval for use for ALK have significant clinical activity in patients with acquired
in combination with the aromatase inhibitor letrozole.84 Clinical efficacy resistance to crizotinib and are FDA approved for this indication.116,117
has been reported with lapatinib in HER2-mutant NSCLC.85,86 The In addition, brigatinib, a dual inhibitor of ALK and EGFR, was
irreversible pan-HER kinase inhibitor neratinib has shown promising granted accelerated FDA approval in 2017 for patients with metastatic
clinical activity in patients with ERBB2-amplified and ERBB2-mutant NSCLC and in patients with ALK alterations who progressed on
breast tumors, but also other cancer types.46,87–93 crizotinib.118

Insulin, Insulin-Like Growth Factor-1 Receptor Platelet-Derived Growth Factor Receptor, KIT,
Signaling, ALK, and ROS1 and FLT-3 Signaling
The insulin and insulin-like growth factor 1 (IGF1) receptor family Platelet-derived growth factor (PDGF) is the ligand for PDGFRs,
is dysregulated in multiple malignancies.94 The insulin receptor exists which stimulate the proliferation and migration of mesenchymal cells,
as two isoforms encoded by splice variants of the same gene.95 Each such as oligodendrocyte precursors, vascular smooth muscle cells, and
isoform can dimerize with the other (forming hybrid dimers) or with pericytes during embryonic development.119 PDGF signaling is also
itself.96 The IGF1 receptor (IGF1R) can dimerize with either of the implicated in organ development, including lung and intestinal epithelial
insulin receptor isoforms or with itself, resulting in six different dimer folding and glomerular capillary tuft formation. Furthermore, PDGFs
combinations.96 The insulin receptor is stimulated by insulin or promote angiogenesis, wound healing, and erythropoiesis.120 Aberrations
insulin-like growth factor 2 (IGF2), whereas IGF1R can be activated in the PDGFR pathway result in uncontrolled proliferation and
by either IGF1 or IGF2. Both of these latter ligands can stimulate enhanced angiogenesis.
IGF1R in an autocrine fashion or can be elaborated from distant Four isoforms of PDGF have been identified: PDGFA, PDGFB,
sites.97,98 Circulating IGF binding proteins have a similar affinity for PDGFC, and PDGFD.121 These isoforms are activated by proteolytic
IGF1 and 2 as IGF1R does and therefore compete for binding to cleavage and assemble into five homodimeric or heterodimeric com-
both ligands, thus titrating the amount of free ligand available for binations that bind to and stimulate either PDGFRα or PDGFRβ.
IGF1R stimulation.99 IGF binding protein proteases provide an PDGFRα homodimers inhibit chemotaxis, whereas PDGFRβ
additional mechanism for controlling ligand levels by increasing the homodimers and α/β heterodimers stimulate chemotaxis within
half-life of free ligand available for receptor binding.100 fibroblasts and smooth muscle cells.122 Angiogenic endothelial cells
After ligand binding, IGF1R dimerizes and undergoes transphos- recruit PDGFRβ-positive pericytes to cover blood channels and aid
phorylation, leading to activation of downstream signaling pathways, in their maturation and stabilization through secretion of PDGFβ.123
including both the Ras-Raf-MAPK and the PI3 kinase-AKT-mTOR Following dimerization and transphosphorylation, PDGFRs activate
cascades (see individual sections later in the chapter; see also Fig. signal transduction pathways through recruitment of adaptor proteins
2.1).101 Specifically, insulin receptor substrate 1 (IRS1) binds to a containing SH2 domains, most notably the Grb2 protein, which in
phosphotyrosine motif on IGF1R via its SH2 domains and is phos- turn binds the guanine nucleotide exchange factor (GEF) Sos, which
phorylated by IGF1R.102 It subsequently recruits PI3 kinase to the subsequently activates Ras.124,125 In addition, phosphorylated tyrosine
plasma membrane, which converts phosphatidylinositol-4,5-bisphosphate residues serve as docking sites for SH2 domain–containing kinases,
(PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3), subsequently including PI3 kinase, phospholipase-Cγ, and Src, as well as the tyrosine
resulting in AKT and mTOR pathway activation. phosphatase SHP2 and the STAT transcription factor family.125
Activating mutations of IGF1R do not appear to be common in Alterations in PDGFR signaling in cancer include excess autocrine
human cancer. However, amplification of the IGF1R gene locus has secretion of PDGF (glioblastoma, sarcomas), gain-of-function mutations
been identified in some colon, pancreas, and lung cancers. Sarcomas that cause constitutive tyrosine kinase activation (GISTs),126 transloca-
often exhibit either increased expression of the IGF1 and IGF2 ligands tion of either the PDGF or PDGFR gene (dermatofibrosarcoma
or decreased IGFBP-3 expression (Ewing sarcoma), which results in protruberans, chronic myelomonocytic leukemia, hypereosinophilic
increased IGF1 levels in the tumor microenvironment.103 Gastrointestinal syndrome),127–129 and PDGFR gene amplification (glioblastoma).130
stromal tumors (GISTs) lacking c-KIT and platelet-derived growth PDGFRα mutations are found in approximately 10% of KIT wild-type
factor receptor (PDGFR) mutations also commonly harbor IGF1R GISTs and are sensitive to imatinib, a tyrosine kinase inhibitor of
amplification.104 AMG479, a monoclonal human antibody targeting KIT, BCR-ABL, and PDGFRs, which is standard of care in this setting
IGF1R, has shown promising antitumor activity in patients with (see Table 2.1).122,126,131 The D842V mutation comprises approximately
Ewing sarcoma.103 two-thirds of PDGFRα activating mutations, and confers resistance
Activating kinase domain point mutations and gene rearrange- to imatinib. Notably, the second-generation inhibitor dasatinib is
ments of the insulin receptor family members anaplastic lymphoma effective in preclinical models of imatinib-resistant GIST.126,132,133
kinase (ALK) and ROS1 play driving roles in many cancers, most Dermatofibrosarcoma protuberans is a rare, low-grade cutaneous
notably lymphomas, neuroblastoma, NSCLC, and thyroid cancer.105–108 sarcoma that harbors a chromosome 17;22 translocation that fuses
Chromosomal translocations involving ALK and at least 22 5′ fusion portions of the COL1A1 (collagen 1A1) gene and PDGFB, resulting
partners have been identified,108 which dictate spatial and temporal in overexpression of PDGF-β and subsequent stimulation of PDGFR
expression of the ALK fusions, and likely their function and tumorigenic signaling.122 Twenty other fusions partners have been identified in
potential.105 In NSCLC, the EML4 gene is the preferred translocation PDGFβ rearrangements, including ETV6 and EBF1. Imatinib has
partner, resulting in the expression of an EML4-ALK fusion protein shown significant benefit in patients with recurrent or metastatic
in 4% to 6% of patients.109,110 Notably, EML4-ALK fusions are found dermatofibrosarcoma protuberans, myelodysplasia, and myeloprolifera-
in a mutually exclusive pattern with EGFR kinase domain mutations, tive neoplasms and is FDA approved for these indications.134–138
suggesting that they have overlapping downstream effects. ROS1 gene The KIT gene is a member of the type III RTK family, which
rearrangements are also found in a minority of NSCLC patients with includes PDGFR and FLT3 (see later).139–141 It was first identified as
binding partners including SLC34A2 and CD74.106,111,112 Crizotinib, the human homologue of the viral oncogene v-Kit responsible for the
an inhibitor of the ALK, ROS1, and MET tyrosine kinases (see Table Hardy-Zuckerman IV feline sarcoma virus.142 Mutation of KIT or its
2.1), is now FDA approved for use in NSCLC patients with ALK ligand, stem cell factor (SCF),143–146 in mice induces coat color
or ROS1 fusions (see Table 2.1),113–115 although acquired resistance abnormalities (“white spotting”), anemia, and mast cell deficiencies,
mutations in ALK (of note, C1156Y and the gatekeeper mutation suggesting that it plays a role in hematopoiesis and melanogenesis.147,148
L1196M) commonly develop. Newer ALK inhibitors including Furthermore, the KIT protein was discovered as a cell surface receptor
Intracellular Signaling  •  CHAPTER 2 33

in acute myeloid leukemia (AML).149 KIT expression is mainly restricted factors, including tissue-specific FGF ligand and receptor expression, the
to mast cells, hematopoietic cells, germ cells, melanocytes, and the presence of cell surface molecules that facilitate the interaction between
interstitial cells of Cajal (ICCs) in the gut.150,151 SCF and KIT integrate individual FGF ligands and receptors, and the differential binding
signals that lead to mitogen-activated protein kinase (MAPK), PI3K, capability of the ligands themselves for specific FGFRs.178 Subsequent
and SRC pathway activation, and mediate critical survival and prolifera- stimulation of the tyrosine kinase domain leads to phosphorylation
tion cues to distinct hematopoietic lineages, including the bone marrow and activation of multiple downstream signaling proteins in the same
and progenitor cells. manner as described earlier for other RTKs. Unique to the FGFR
Hot-spot mutations in exons 9 and 11 of KIT have been identified signaling complex is FGFR substrate 2 (FRS2), an adaptor protein
in several tumor types, including GIST, melanomas, and germ cell that binds to specific phosphotyrosines on the intracellular domain
tumors.146,152–156 In GIST, 85% of tumors have activating KIT mutations of active FGFR dimers.179 FRS2 is itself phosphorylated by FGFRs
that drive the transformation of precursors of ICCs.153 Imatinib157–161 and serves as a docking site for the Grb2-Sos adaptor complex, which
and sunitinib158,162,163 inhibit KIT and PDGFR, among other kinases, activates the Ras/Raf/MAPK pathway. Phosphorylated FRS2 also
and are FDA approved for use in patients with KIT mutant GIST recruits Grb2-associated binding protein 1 (GAB1), which activates
(see Table 2.1). Regorafenib is approved for patients with imatinib- or PI3 kinase. In addition, phospholipase-Cγ binds to phosphorylated
sunitinib-refractory GIST.164 Imatinib has also been shown to induce FGFR dimers via an SH2 domain, leading to its activation and the
tumor regression in patients with KIT-mutant or KIT-amplified cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) to form inositol
melanoma.165,166 Second site mutations in KIT, typically in exon 17, 1,4,5-triphosphate (IP3) and diacylglycerol (DAG).
are a mechanism of acquired resistance to imatinib therapy in patients Germline mutations of the FGFR genes are the basis of a spectrum
with GIST.167 Novel agents that retain activity in the setting of an of skeletal developmental disorders that are thought to derive from
exon 17 KIT mutation are now in clinical testing (clinical trial premature differentiation and growth restriction of chondrocytes
NCT02401815).168 New areas of investigation into mutant KIT resulting from dysregulated FGFR pathway activation.180,181 FGFR
therapies in GIST include blocking mutant KIT subcellular localization signaling is dysregulated in cancer by multiple mechanisms includ-
to the Golgi150 and combination of FGFR3 and KIT inhibitors to ing mutational or translocation-induced activation of FGFRs, gene
quench pathway cross talk.169 amplification of receptors, and abnormal ligand regulation.182 For
The FMS-like tyrosine kinase 3 receptor (FLT3), a third member example, autocrine and paracrine FGF ligand secretion with resultant
of the RTK class that includes PDGFR and KIT, is involved in the pathway activation has been reported to occur in a subset of melanomas
development of normal hematopoietic cells. It contains an extracel- and prostate cancers, respectively.183,184 FGFR1 amplification occurs in
lular region composed of five immunoglobulin (Ig) domains, TM approximately 17% of squamous cell lung cancers and 6% of small cell
and juxtamembrane domains, and two cytoplasmic tyrosine kinase lung cancers (SCLCs).185 Approximately 10% of diffuse-type, aggressive
domains that transmit proliferative signals through the RAS/MAPK, gastric cancers display FGFR2 gene amplification, and cell lines with
PI3K/AKT, and STAT5 pathways.170 Two main FLT3 alterations are this amplification show ligand-independent pathway activation and
common in hematopoietic malignancies, namely in approximately sensitivity to selective FGFR inhibitors.186,187 Whereas FGFR1 mutations
30% of AMLs.171 First, internal tandem duplication (ITD) within are rather rare, FGFR2 mutations are found in approximately 10%
exons 14 and 15 of the FLT3 gene (FLT-ITD) interferes with the of endometrial cancers.188,189 FGFR3 mutations occur in up to 75%
negative regulatory function of the juxtamembrane segment.172 This of non–muscle invasive bladder cancers and 15% of patients with
duplication results in ligand-independent activation of FLT3 and is advanced urothelial tumors.44,179,189,190 Activating mutations within
associated with a poor prognosis in patients with AML. Second, kinase FGFR3 result in constitutive receptor dimerization and subsequent
domain mutations at or near D835 in the activation loop of FLT3 signaling. Unlike EGFR-activating mutations, which predominantly
disrupt autoinhibitory interactions and render the kinase open and affect the tyrosine kinase domain of the receptor, FGFR3 mutations
active.173 The clinical activity of FLT3 inhibitors has been modest to are commonly located within the extracellular domain (R248, S249)
date, although responses appear to be more common in patients with and TM segment (G370, Y373) and promote ligand-independent
FLT3/ITD AML.171 TAK-659, a reversible dual Syk/Flt inhibitor, receptor dimerization through formation of an aberrant disulfide bridge
showed early clinical activity in numerous lymphoma subtypes and between two receptor monomers.191 Chromosomal rearrangement of
AML.174,175 Sorafenib has been shown in preclinical in vitro studies, FGFRs have been identified using next-generation sequencing. Up to
mouse models, and in a phase I study of AML patients to reduce 15% of multiple myelomas harbor an intergenic 4;14 translocation
leukemia burden and block signaling selectively in FLT3-ITD versus between the FGFR3 gene and the Ig heavy chain locus, which places
FLT-wt settings.176 Interesting to note, resistance to FLT3 inhibition in FGFR3 expression under the highly active heavy chain promoter.192,193
such patients is associated with selection for secondary mutations within More recently, translocations involving FGFR2 have been reported
the tyrosine kinase domain of FLT3, suggesting a central role of FLT3 in in cholangiocarcinoma and more rarely in other cancers, whereas
AML pathogenesis.171 FGFR3 fusions are most common in glioblastoma and bladder
cancers but also are found rarely in other solid tumor types.194,195
Fibroblast Growth Factor Receptor Signaling The FGFR3-TACC3 constitutively active fusion protein has been
characterized to induce aneuploidy by disrupting proper chromosomal
Fibroblast growth factor receptors (highly conserved FGFR1, FGFR2, segregation.196
FGFR3, and FGFR4; and FGFRL1/FGFR5, which lacks a kinase Multiple FGFR inhibitors are currently being tested in early-phase
domain) comprise a family of RTKs that regulate cell proliferation, clinical trials, but the majority of these compounds are multitargeted
differentiation, and migration as well as selective apoptosis during tyrosine kinase inhibitors, many of which also potently inhibit members
embryogenesis. The FGFRs are composed of an extracellular ligand- of the VEGFR and PDGFR families. The close structural similarity
binding domain, a hydrophobic TM region, and an intracellular tyrosine between these RTKs has made development of FGFR-selective inhibitors
kinase domain.177 The extracellular domain is organized into three Ig challenging, although several such drugs are now in early clinical testing,
domains; differential splicing of the second half of the third Ig domain such as BGJ398, AZD4547, JNJ-42756493, and Debio1347 (see Table
dictates tissue-specific expression of the receptor. 2.1).179,182,197–200 On-target hyperphosphatemia resulting from FGFR1
Fibroblast growth factors (FGFs) are protein ligands that bind to inhibition is a primary toxicity with this class of agents, suggesting that
the extracellular domain of the FGFRs in combination with specific the development of isoform-selective FGFR inhibitors may be a more
heparan sulfate glycosaminoglycans inducing FGFR dimerization and rational approach for patients whose tumors are driven by mutations
transphosphorylation of intracellular tyrosine residues. Eighteen FGFs or translocations in FGFR2 and FGFR3. FGFRs are located on the
have been identified, and specificity for FGFRs is based on numerous cell surface and thus may also be susceptible to monoclonal antibody
34 Part I: Science and Clinical Oncology

mediated inhibition similar to trastuzumab-mediated inhibition of growth factors 1 and 2.211 VEGF-A has four isoforms produced by
HER2. FGFR ligand traps are also in development.182 alternative gene splicing, with the 165–amino acid length isoform
playing a central role in tumor angiogenesis.212 Specifically, VEGF-A
RET Signaling enhances vascular permeability and stimulates endothelial cell
proliferation, resulting in new blood vessel formation. Vascular
The RET (Rearranged During Transfection) gene encodes three endothelial growth factor receptors (VEGFR1 to VEGFR3) are
alternatively spliced isoforms (RET9, RET43, and RET51) which are RTKs that possess a modular structure consisting of an extracellular
TM RTKs that contain four cadherin-like extracellular repeats important domain with seven Ig-like regions, a TM domain, and an intracellular
for dimerization, a cysteine-rich juxtamembrane region critical for tyrosine kinase domain.211 VEGF-A, VEGF-B, and placental growth
ligand binding and conformation, and an intracellular kinase domain.201 factor all bind VEGFR1 (also known as FLT1), but the exact role
RET9 and RET51 are highly conserved in all vertebrates and play of VEGFR1 in tumor angiogenesis has yet to be fully elucidated. In
important roles in the normal development and maintenance of many some settings it may act as a decoy receptor that prevents ligand-
tissues, including the kidney, spermatogonial stem cells, and the enteric mediated stimulation of VEGFR2 (also known as FLK1/KDR).213
nervous system.201–203 RET is expressed predominantly on the surface VEGFR2 has been implicated in the development of vasculature
of neural crest tissues, and glial-derived neurotrophic factors (GDNFs) during development and is considered the primary receptor through
such as neurturin, artemin, and persephin serve as ligands for RET. which VEGF exerts its angiogenic effects in endothelial cells.213,214
GDNFs initially bind to their cognate coreceptors, the GDNF receptors Binding of ligand to VEGFR2 results in receptor dimerization and
(GFPα1 to GFPα4), on the cell surface, which recruits RET into transphosphorylation followed by activation of multiple mitogenic
lipid raft membrane domains and causes conformational changes via signal transduction cascades.215,216 More recently, VEGF has been
the cadherin-like moieties, dimer formation, and then subsequent implicated in many angiogenesis-independent roles including regula-
transphosphorylation of tyrosine residues and kinase activation.204 tion of immune cells in the tumor microenvironment, fibroblasts
Y1062 is the common docking site for all three RET isoforms and in the tumor stroma, and cancer stem cells.217 VEGF can also bind
serves to recruit many adapters including SHC1, FRS2, IRS1/2, DOK, and signal through a class of TM glycoprotein coreceptors called
and JNK.201 Phosphorylation of Y752 and Y928 binds STAT3, whereas neuropilins (NRP1 and NRP2), which are found on tumor cells
other phosphorylated residues are recognized by Src, resulting in and can signal along many oncogenic axes including Hedgehog
activation of focal adhesion kinase (FAK), which promotes cell migration and JNK.217
and metastatic spread. In addition, the MAP kinase, PI3 kinase/AKT, The complex network of cross talk among VEGFs, VEGFRs, and
and phospholipase-Cγ pathways can be activated by RET to promote canonic oncogenic pathways makes VEGF and VEGFR critical but
cellular proliferation and survival.204 elusive targets in cancer therapy. Targeted therapies that inhibit VEGF
Germline loss-of-function RET mutations occur in Hirschsprung signaling include antibodies that bind circulating ligand and RTK
and CAKUT (congenital anomalies of the kidney and urinary tract) inhibitors. The humanized monoclonal antibody bevacizumab binds
disease, which causes abnormalities of the developing gut and kidneys, to free VEGF, thereby preventing its association with VEGFRs. This
respectively. Conversely, germline activating RET mutations are the antibody has been FDA approved for use in combination with che-
basis for the multiple endocrine neoplasia type 2 (MEN2) syndromes. motherapy for patients with several cancers, including metastatic
Patients with MEN2 develop familial medullary thyroid carcinomas colorectal218 and nonsquamous NSCLCs.211,219 Bevacizumab also has
and other cancers.205 MEN2A is mainly driven by mutations in six activity in patients with glioblastoma220 and metastatic renal cell
cysteine resides in the RET extracellular domain (C609, C611, C618, carcinoma, where it is often used in combination with interferon-α
C620, C630, and C634), whereas the kinase domain mutations M918T (IFN-α).221 In addition, ramucirumab is a VEGFR2-directed antibody
or A883F are associated with MEN2B. Sporadic medullary thyroid that has received FDA approval with or without chemotherapy in
carcinomas are much more common, and up to 60% of such tumors several cancers.222
harbor somatic mutations in RET, notably G691S, which are thought Sorafenib, sunitinib, pazopanib, and axitinib are multitargeted
to be a driver alteration in this disease.206 Furthermore, RET gene tyrosine kinase inhibitors with nanomolar potency for VEGFR2.
rearrangements with numerous fusion partners, including CCDC6 Sunitinib is used in the treatment of patients with metastatic renal
and NCOA4, termed RET-PTC1 and RET-PTC3, respectively, occur cell carcinoma, GISTs, and pancreatic neuroendocrine tumors.223
in 20% to 40% of papillary thyroid carcinomas (PTCs) and often Sorafenib has been approved for the treatment of liver and renal cell
occur as a consequence of high doses of radiation.207 cancers.224,225 Although these agents inhibit multiple kinases, their
RET inhibitors have shown significant antitumor activity in patients antitumor effects have been attributed primarily to their antiangiogenic
with medullary thyroid cancer. Vandetanib, an oral inhibitor of RET, activity. More recently, the tyrosine kinase inhibitor pazopanib was
EGFR, and VEGFR, is FDA approved for the treatment of patients approved for the initial treatment of metastatic renal cell carcinoma
with advanced medullary thyroid cancer.208 A randomized, placebo- and in cytokine-pretreated patients,226 and axitinib227 was approved
controlled phase III study of cabozantinib, an oral, multitargeted TKI in the second-line setting following failure of prior systemic therapy.
that inhibits RET, VEGFR2, and MET, was also recently conducted Lenvatinib, a multitargeted RTK inhibitor that inhibits VEGFR1,
in patients with unresectable, locally advanced, or metastatic medullary VEGFR2, and VEGFR3, has received recent FDA approval for both
thyroid carcinoma.209 This trial documented a statistically significant thyroid cancer (as monotherapy)228 and renal cell carcinoma in combina-
improvement in median progression-free survival with cabozantinib tion with everolimus.229
as compared with placebo (11.2 months versus 4.2 months in placebo Despite widespread activity in preclinical models, antiangiogenic
arm, P < .0001). More recently, cabozantinib was FDA approved for therapies have shown disappointing activity in several tumor types.
the treatment of renal cell carcinomas that progressed on antiangiogenic A number of resistance mechanisms have been hypothesized to
therapy, although the activity of cabozantinib in this context may not explain the lack of broader clinical activity, including the activation
be attributable to its inhibition of RET. Several RET inhibitors have, of redundant signaling pathways that promote angiogenesis; the
however, shown promising clinical activity in patients with NSCLC recruitment by tumors of bone marrow–derived endothelial progenitor
treated with RET fusions (see Table 2.1).210 cells; increased pericyte density around existing blood vessels, which
enhances vascular growth and survival; and the ability of tumor cells
Vascular Endothelial Growth Factor Signaling to invade surrounding stroma to co-opt additional blood supply.230
A better understanding of these resistance mechanisms may lead
Six vascular endothelial growth factor (VEGF) ligands have been to the development of more effective antiangiogenic therapies in
identified: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental the future.
Intracellular Signaling  •  CHAPTER 2 35

Hepatocyte Growth Factor Receptor Signaling Tropomyosin Receptor Kinases/Neurotrophic


Tyrosine Kinase
The hepatocyte growth factor receptor (HGFR or MET) is encoded
by the MET gene.231,232 Both MET and its ligand hepatocyte growth First cloned as an oncogenic fusion partner of the tropomyosin receptor
factor/scatter factor (HGF/SF) are expressed as immature precursors kinases and subsequently characterized for their role in neural dif-
that require proteolytic cleavage.233 The MET extracellular domain ferentiation and survival in the peripheral and central nervous systems,
consists of an alpha subunit connected by a disulfide bridge to a TM the TRKA/B/C family of RTKs, encoded by the neurotrophic tyrosine
beta subunit, and contains a Sema domain, a PSI domain, and four kinase (NTRK) genes 1 to 3 (NTRK1/2/3), respectively, integrate
IPT domains.234,235 The intracellular portion of the receptor contains ligand stimulation from nerve growth factor, brain-derived neurotrophic
a juxtamembrane region that harbors a serine residue (Ser 975) that factor, neurotrophins 3 to 6 with downstream activation of PI3K,
inhibits RTK activity on phosphorylation, as well as a tyrosine kinase phospholipase-C (PLC)-gamma, and MAPK signaling.257–261 Chro-
domain with Y1234 and Y1235 acting as key sites of autophosphoryla- mosomal rearrangements involving the tropomyosin receptor kinases
tion required for activation.235 A tyrosine residue at position 1003, (TrkA/NTRK1, TrkB/NTRK2, and TrkC/NTRK3) were recently shown
proximal to the tyrosine kinase domain, serves as an interaction site to occur at high frequency in several rare cancer types including
for the ubiquitin ligase CBL, which marks the receptor for endocytosis mammary secretory carcinoma of the breast and congenital-infantile
and degradation.236,237 C-terminal residues Y1349 and Y1356 represent fibrosarcoma.262–264 NTRK fusions also occur at low frequency in a
docking sites for adaptor proteins.238 On binding of HGF/SF to the broad range of more common adult solid tumors.260,265 These NTRK
extracellular portion of MET, receptor dimerization occurs, followed fusions induce ligand-independent constitutive kinase activity, resulting
by transphosphorylation. A number of adaptor proteins then bind in upregulation of canonic downstream signaling pathways involved
to phosphorylated tyrosine residues, including Grb2 and GAB1, in growth and survival. TPM3, LMNA, MPRIP, TRIM24, ETV6, and
phospholipase-C (PLC), and SRC, which promotes the activation of PPL, among others, have been identified as fusions partners with the
the MAP kinase and PI3 kinase/AKT signaling pathways.239,240 MET NTRK genes.266,267 Dramatic and durable clinical responses have recently
can also activate RAC1/CDC42 and p21-activated kinase (PAK1), both been reported in patients with NTRK fusions, with first- and second-
of which regulate cytoskeletal proteins and integrin expression and generation TRK inhibitors such as larotrectinib (LOXO-101),
activation, and thus cell migration.238,239 MET also plays an important entrectinib, and LOXO-195 (see Table 2.1).268–273 Notably, clinical
role in driving epithelial-to-mesenchymal transition (EMT) cell migra- activity was observed in both adult and pediatric patients and was
tion during embryo development, and organ regeneration.238,241 independent of site of tumor origin.264
Dysregulation of MET signaling can occur through multiple
mechanisms, including activating point mutations (often in the kinase G PROTEIN–COUPLED RECEPTOR SIGNALING
domain; prevalent in lung cancer), exon skipping events, receptor
overexpression, and upregulation of HGF, which can activate MET in G protein–coupled receptors (GPCRs) are seven TM domain–containing
an autocrine and/or paracrine manner.238,240,242–246 Germline mutations proteins that transduce ligand-specific signals across the plasma
of MET are found in patients with hereditary papillary renal cell membrane to mediate numerous physiologic processes including sensory
carcinomas, and MET overexpression is observed in a significant perception, immunologic responses, neurotransmission, weight regula-
proportion of sporadic papillary cancers as well as collecting duct tion, and cardiovascular activity.274 GPCRs also regulate basic cellular
carcinomas.247 Multiple other malignancies exhibit aberrations in functions including growth, motility, differentiation, and gene transcrip-
MET signaling, including lung, breast, pancreatic, colon, and gastric tion. The GPCR family comprises more than 800 receptors, which
cancers. Amplification of MET is associated with a worse prognosis are the targets of over 30% of all FDA-approved drugs, although few
in lung and gastric cancers, whereas expression of MET or HGF is to date have found a role as anticancer therapies.275,276 Given that
an unfavorable prognostic biomarker in liver, kidney, colorectal, and GPCRs activate many of the signaling cascades that are deregulated
gastric cancers.248 in human cancer, it is not surprising that studies have implicated
Recurrent somatic splice site alterations involving MET exon 14 GPCRs in cancer initiation and progression.277–279
(METex14) have been identified in lung cancer. These mutations GPCRs can be categorized into five or six families, depending on
result in exon skipping, loss of the juxtamembrane CBL E3-ubiquitin the nomenclature used.280 In a more recent phylogenetic classification,
ligase-binding site, diminished receptor turnover, and ultimately, the five major families are represented by the acronym GRAFS:
MET activation.243,249,250 These exon 14 MET mutations are mutually Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, and Secretin.281
exclusive with activating mutations in EGFR and KRAS as well as The Rhodopsin family of receptors (also referred to as class A GPCRs)
ALK, ROS1, and RET fusions, and treatment of patients with exon 14 is the largest class, with more than 670 members. Crystallization of
MET splice variants with a MET kinase inhibitor is now considered the bovine rhodopsin receptor in 2000 provided the first high-resolution
to be a standard treatment option (see Table 2.1).210 Several RTKs insight into the structure of GPCRs.282 In general, Rhodopsin family
have also been shown to activate MET, including EGFR, HER2, and receptors have short N-termini. Included in this family are the α
IGF1R. For example, EGFR activation can stimulate MET signaling, group (histamine, dopamine, serotonin, adrenoceptors, and muscarinic,
and resistance to EGFR inhibitors in some lung cancers has been prostanoid, and cannabinoid receptors), the β group (endothelin,
shown to stem from coactivation of MET in the setting of gene gonadotropin-releasing hormone, and neuropeptide Y), the γ group
amplification.251 (opioid, somatostatin, and angiotensin), and the δ group (P2RYs,
Inhibitors of MET signaling have been in development for a number glycoprotein-binding FSHR/TSHR/LHCGR, PARs, and olfactory
of years. Therapeutic strategies for targeting MET activation in cancer receptors).283
patients include antibodies that target the extracellular domain of the The 15 Secretin receptors (class B) all have conserved cysteines in
receptor, antibodies that bind to and thus sequester circulating HGF, the first and second extracellular loop, and most have three cysteine
and small-molecule tyrosine kinase inhibitors that selectively target bridges in the N-termini. This class includes the calcitonin-like,
MET or are multi-kinase MET inhibitors.248 Durable responses to corticotrophin-releasing hormone, glucagon-like, gastric inhibitory
crizotinib and cabozantinib, multikinase MET inhibitors, have been polypeptide, growth hormone–releasing hormone, adenylate cyclase–
reported in patients with NSCLC with MET splice mutants or MET activating polypeptide, parathyroid hormone, secretin, and vasoactive
amplification (see Table 2.1).252,253 Cabozantinib is also now standard intestinal peptide receptors.284 The Adhesion family (also included in
of care in MET-amplified renal cell carcinoma.254,255 Combination class B, according to another classification system) has distinctly long
therapies to combat MET reactivation after EGFR kinase inhibitor N-termini, and only 3 of 33 receptors in the family have known
therapy suggest an improvement in progression-free survival.256 ligands (epidermal growth factor-like module containing mucin-like
36 Part I: Science and Clinical Oncology

receptors EMR2, EMR, EMR4).285 The Glutamate receptor family stimulates G protein–regulated inwardly rectifying K+ channels adenylyl
(class C) binds ligands in their N-termini, which have a complex cyclase (types II, IV, VII), PLCβ, PI3Kγ, Src, and GPCR kinases
two-domain folded structure bridged by disulfide bonds. Common (GRKs; see later). It inhibits adenylyl cyclase (type I), some Ca+
receptors in this family of 22 include the glutamate, GABAB, calcium- channels, and calmodulin; stabilizes Gα in the GDP-bound, inactive
sensing, sweet and umami taste (TAS1R1–TAS1R3), and GPCR6 state; and helps specify coupling to the proper Gα member.274,295,296,300
receptors.286,287 The Frizzled receptors (FZD1–FZD10 and SMO; see Gα signaling is switched off by a family of GTPase activating
later for in depth discussion of signaling) bind Wnt ligands in the proteins called Regulators of G-protein Signaling, or RGS proteins,
extracellular domain region containing nine conserved cysteine resi- which enhance the intrinsic rate of GTP hydrolysis by greater than
dues.288 The Taste2 receptors (25 members of T2R, bitter taste) have 1000-fold.312,313 Some RGS proteins enhance signaling through
varying sequence homologies that likely allow the sensing of thousands RhoGEFs and act as scaffolds for other signaling cascades (e.g., tethering
of distinct bitter tastes.289,290 Overall, GPCRs share the seven–TM to Raf and MEK2). The GPCR effectors PKA and PKC also contribute
domain structure, but have different regions of conservation. All of to receptor inhibition by phosphorylating their cognate-activated
the families include orphan receptors, which are related by sequence GPCR and thereby uncoupling and inactivating Gαs/Gαq in a classic
and structure but have no identified ligand to date. negative feedback loop.314,315
All GPCRs have seven α-helical domains that weave through the GPCRs can also function via G protein–independent mechanisms
plasma membrane and are interconnected by flexible extracellular and by binding to the arrestin family of cytosolic adapter proteins.316,317
intracellular segments, thus engendering the synonyms serpentine or GPCR-coupled arrestins have pleiotropic cellular roles including (1)
heptahelical receptors. The specificity of biologic response initiated by dampening G-protein signaling by scaffolding enzymes that degrade
each GPCR depends on (1) ligand recognition; (2) the distinct structure G-protein second messengers; (2) desensitizing receptors by binding
of each receptor class and subclass; and (3) ligand-directed binding GPCR kinase (GRK)–phosphorylated GPCRs and sterically blocking
of specific cytosolic enzymes and adapters that initiate a plethora of access to further Gα subunits; (3) mediating GPCR trafficking and
intracellular signaling cascades (general and cancer-specific examples endocytosis to clathrin-coated pits; and (4) acting as a scaffold for
are discussed further later). The GPCR Network was created in 2010 multiple MAP kinase cascades. For example, MEK1 is engaged by a
to tackle the challenge of delineating the structure and function of tethered complex of Raf-1, ERK2, and β-arrestin to facilitate mitogenic
this diverse class of receptors.291 signaling.318,319
Operationally, ligand binding on the GPCR extracellular surface GPCRs are well established drug targets for antihistamine, antacid,
induces a conformational change in the receptor, mainly via TM cardiovascular, and antipsychotic drugs; pain suppressants; and
helices TM3, TM5, and TM6, which creates a deep pocket in the antihypertension therapies.275,276 Although less well appreciated as drug
intracellular face of the receptor.292–294 This cleft enables binding and targets in cancer, there is increasing evidence that dysregulated GPCR
activation of heterotrimeric G proteins, which consist of an inactive, signaling contributes to cancer initiation and progression. For example,
GDP-bound Gα subunit and a Gβγ-subunit dimer, which act as a in 1986 the wild-type MAS1 gene, which encodes the MAS GPCR,
molecular switch.295,296 Activated GPCRs promote GDP for GTP was reported to induce transformation by coupling to the small G
exchange on the Gα nucleotide binding site.297 This GTP-bound Gα protein Rac.320,321 Large-scale deep sequencing efforts have revealed
dissociates from Gβγ and the receptor, and then both activated subunits that GPCR mutations occur in approximately 20% of all cancers.
go on to initiate signaling cascades. There are four members of the Receptors for thyroid-stimulating hormone (TSHR), Hedgehog
Gα family, Gαs, Gαi/o, Gαq/11, and Gα12/13, which can be further (Smoothened receptor [SMO]), glutamate (GRM), the adhesion family,
subtyped and can each stimulate several downstream effectors. Moreover, lysophosphatidic acid (LPA), and sphingosine-1-phosphate (SIP) are
each GPCR can couple to multiple Gα family members, thus generating the most frequent GPCRs altered in cancer.277,312,313,322 G proteins
a complex pattern of intracellular signaling. themselves are also mutated in cancer. In particular, recurrent mutations
Classic GPCR activation of Gαs stimulates adenylyl cyclase, which in GNAS (which encodes Gαs) have been identified in thyroid and
generates the second messenger 3′-5′-cyclic adenosine monophosphate pituitary tumors, as well as mutations in GNAQ and GNA11 in
(cAMP).298,299 cAMP activates multiple downstream effectors including melanoma of the eye and skin, respectively.322 Hot spot resides that
cAMP-gated ion channels; Epac, a GEF for Rap1/2 (which functions disrupt GTPase activity and render the protein constitutively active
in cell adhesion and junction formation); and protein kinase A have been identified in GNAS at positions R201 and Q227, in GNAQ
(PKA).274,295,300–303 cAMP binds to PKA regulatory subunits, releasing at R183, and in GNA11 at Q209. Numerous inhibitors of GPCR
catalytic subunits and triggering the activation of cytosolic and signaling are also being studied in the clinic including BKT-140/
nuclear substrates, including the transcription factor CREB (cAMP BL-8040 in pancreatic adenocarcinoma and blood cancers (target:
response element binding protein), which can induce proliferation and CXCR4 receptor), and CXCR2 ligands (target: CXCR2 receptor).278
differentiation, among other phenotypes, depending on cell origin.304–306 Detailed later are a few specific examples of GPCRs that have been
Gαs also activates the Src tyrosine kinase and the GTPase activity of shown to play a role in cancer initiation and/or progression.
tubulin.295,300 GPCR-coupled Gαi/o typically works in opposition to A connection between GPCRs and EGFR signaling has been
Gαs by inhibiting adenylyl cyclase and decreasing cAMP levels.274,307 established in both normal cell physiology and in colon, lung, breast,
In addition, some Gαi/o isoforms can signal to K+ and Ca+ channels, ovarian, prostate, and head and neck cancer development.323–326
increase cGMP phosphodiesterases, interact with Rap1GAP1, Ligand-bound GPCRs activate Src, PKC, Ca+ channels, and PKA
and cross talk to the MAP kinase pathway (as described later in intermediaries, which stimulate proteolytic cleavage and release of
the chapter).274 membrane-tethered growth factors that bind and thereby transactivate
Members of the Gαq/11 family activate phospholipase-Cβ, which EGFR. Specifically, estrogen binding to the GPCR GRP30 facilitates
catalyzes the hydrolysis of PIP2 to yield IP3 and DAG.308 IP3 mobilizes matrix metalloproteinase–2 (MMP2) and MMP9-mediated cleavage of
calcium from intracellular stores, whereas DAG activates some isoforms the growth factor precursor pro-heparin-binding-EGF (pro-HB-EGF),
of protein kinase C (PKC).309,310 Both Gαq/11 and Gα12/13 activate a thus initiating HB-EGF–mediated EGFR transactivation.324 Through
variety of RhoGEFs (p115-RhoGEF, PDZ-RhoGEF, LARG, Lbc, a related mechanism, LPA-, SIP- and thrombin-activated GPCRs
AKAP-Lbc) and thus regulate Rho activity (mainly RhoA) and its transactivate EGFR in breast cancer cells via growth factor shedding
contribution to actin stress fiber formation, cell shape and polarity, cell of tumor necrosis factor–α (TNF-α) through the action of TACE/
adhesion and migration, gene transcription, and cell cycle progression.311 ADAM17 zinc-dependent proteases.326 Thrombin-mediated N-terminal
In addition, Gα12/13 activates the Na+/H− exchanger, inducible nitric cleavage and activation of proteinase-activated receptor 1 (PAR1), which
oxide synthase, phospholipase D, E-cadherin, radixin, and protein can act through EGFR, has been found to promote metastasis and
phosphatase 5. Gβγ signaling is equally complex, as the active dimer invasiveness in melanoma, breast, colon, and prostate cancers.278,279
Intracellular Signaling  •  CHAPTER 2 37

Intriguingly, MMP1 was found to function similarly to thrombin adenomatous polyposis (FAP).357 Efforts to develop selective inhibitors
in activating PAR1 and promoting breast cancer tumorigenesis and of Wnt signaling are ongoing and will be aided by current endeavors
invasion.327,328 This cross talk between GPCRs and EGFR provides to crystallize members of the Wnt cascade.354,358 Of note, nonsteroidal
a rationale for the combinatorial use of GPCR agonists/antagonists antiinflammatory drugs (NSAIDs) have shown some promise in
and EGFR inhibitors in patients with EGFR-driven lung and modulating Wnt signaling, likely by inhibiting the Wnt-output
colorectal cancers. gene COX-2 or by enhancing E-cadherin signaling.349,359 COX-2
Aberrant GPCR signaling through Gα12/13 also contributes to inhibitors have shown efficacy in reducing the risk of polyps in patients
tumorigenesis by enhancing cancer cell migration, invasion, angio- with FAP.358,360
genesis, and metastasis.329 Ligand-activated LPA, PAR1, SIP, throm-
boxane A2 (TP), CXC chemokine (CXCR4), and prostaglandin E2 CYTOKINE RECEPTOR SIGNALING
(PGE2) receptors couple to Gα12/13 and RhoGEFs to hyperactivate
RhoA (see earlier), which elicits these progression-associated phenotypes Cytokines are protein and glycoprotein ligands secreted by immune
in glioma, melanoma, lung, breast, and ovarian cancers.278,279 Over- cells; they initiate diverse and often opposing effects based on target
expression of Gα12/13 and RhoA in breast, prostate, and colon cancers cell lineage. Processes regulated by cytokine signaling include cell
also promotes metastasis by decreasing cell adhesion.311 RhoGEF proliferation, differentiation, survival, inflammation, angiogenesis,
inhibitors; RhoGTPase inhibitors; inhibitors of prenylation, which antiviral activity, and modulation of immune function. Cytokines
could indirectly impair proper Rho localization (statins, farnesyl/ signal in an autocrine and/or paracrine fashion and can be subclassified
geranylgeranyl transferase inhibitors); and inhibitors of kinases by protein structure into four families, which total over 100 members:
downstream of Rho (ROCK, LIMK, MRCK, PAK) have all been hematopoietins, IFNs, chemokines, and the TNF superfamily.
developed and tested in biochemical, cell line, and mouse experiments. The hematopoietin family consists of interleukins (IL-1 to IL-31),
However, a clear benefit to the use of such compounds in cancer growth hormone, prolactin, erythropoietin, thrombopoietin, leptin,
patients has yet to be established.330 granulocyte colony-stimulating factor and granulocyte-macrophage
Two other prominent examples of dysregulated GPCR signaling colony-stimulating factor, and a few others.361 The majority bind to
in human cancer are the Hedgehog/Smoothened and Wnt/Frizzled either class I or II cytokine receptors, which are single TM glycoproteins
signaling pathways.277,278,331 Both pathways, along with Notch, also that lack kinase activity and diverge in their extracellular domains in
play critical roles in cell fate and have been implicated in the underlying order to specify ligand binding.362,363 Class I receptors function as a
pathway cross talk that is key to cancer stem cells.332 Secreted Hedgehog cluster of two or three subunits that each have two sets of conserved
(Hh) ligands (first identified based on their roles in normal development cysteine pairs and a WSXWS motif in their external domains. Class
and stem cell homeostasis, with Sonic Hedgehog [SHH] being the II receptors lack the WSXWS motif and one of the class I conserved
most ubiquitous) bind to the 12-pass TM receptor Patched (PTCH), cysteine pairs, but contain conserved proline, tryptophan, and an
which relieves its repression of the GPCR Smoothened (SMO).333,334 additional two conserved cysteine residues. Ligand binding causes
Activated Smoothened couples to Gαi and Gα12, which regulate the aggregation of the γ-chain (also called γc, CD132) which is common
glioma-associated oncogene homologue (GLI) transcription factors, to many cytokines, and the β-chain (also known as IL-2Rβ, IL-15Rβ,
which in turn regulate proliferative, survival, and differentiation signals or CD122).364,365 In the case of specific cytokines, such as IL-2,
involving cyclin D1, myc, BCL2 and the Forkhead transcription factors, association with a third subunit, the α chain (also called IL-2Rα,
to name a few.335,336 Mutations in SHH, PTCH, and SMO are found CD25, or Tac antigen), allows for high-affinity ligand binding.366,367
in patients with inherited and sporadic basal cell carcinomas337,338 and The IFN family members are divided into type I, II, and III
ameloblastomas,339 whereas overexpression of Hh ligands has been classes.368,369 Most IFNs are type I and can be further subtyped.370 All
shown to result in hyperactivation of the pathway in breast, colon, type I IFNs bind the type I IFN receptor, which consists of two
prostate, and pancreatic ductal adenocarcinomas331,340 Vismodegib and subunits (IFNAR1 and IFNAR2). The sole type II IFN, IFN-γ, binds
sonidegib, both selective Smoothened receptor inhibitors, are FDA the type II IFN receptor, which is composed of the IFNGR1 and
approved for the treatment of advanced basal cell carcinomas (see IFNGR2 subunits. Both types of IFN receptors belong to the class
Table 2.1).341–344 Several other Hh/SMO inhibitors are currently being II cytokine receptor family. The IFN family also includes a third
tested in patients with cancer, mostly for advanced and/or metastatic branch, the IFN-like molecules, IFN-λ1 (IL-29), IFN-λ2 (IL-28A),
solid tumors.278,345,346 and IFN-λ3 (IL-28B), which display some structural overlap with
The secreted Wnt glycolipoprotein ligands activate the single ILs and the antiviral properties of IFNs, and bind a distinct receptor
TM low-density lipoprotein–related coreceptors LRP5/6 and the made up of IFNLR1/IL-28Rα and IL-10Rβ.371 Given that cytokine
GPCR-like TM protein Frizzled (Fz), which are phosphorylated and receptors are promiscuous and bind multiple cytokine ligands, there
likely couple to Gαq and Gαo, respectively, to activate the cytoplasmic is a high degree of redundancy in the output profiles of individual
scaffold Dishevelled.347–349 Dishevelled in turn inhibits the β-catenin cytokines. This redundancy serves to amplify and sustain signaling
degradation complex (which consists of APC, axin, CKIα and GSK-3β, downstream of these transitory stimuli.
and the E3 ubiquitin ligase β-TrCP).350 This results in accumulation Hematopoietin or IFN binding induces oligomerization of cytokine
of β-catenin, which translocates to the nucleus where it induces TCF/ receptor subunits and autophosphorylation and activation of the JAK
LEF-mediated transcription of genes important for cell differentiation (Janus activated kinase) family of intracellular tyrosine kinases (TYK2,
and proliferation, including myc, cyclin D1, VEGF, FGF4/18, E-cadherin, JAK1–JAK3), which are constitutively bound to box I and box II
COX-2, and members of the Wnt cascade itself.349,351,352 Noncanonic α-helix motifs in the receptor cytoplasmic tail.372 Activated JAK proteins
Wnt/Fz pathways include signaling to the transcription factors phosphorylate tyrosine residues on the cytokine receptor chains, which
NFAT via Gαq/i/Gβγ/PLC/PKC/Ca+ (Wnt-calcium pathway) and classically bind the STAT (signal transducer and activator of transcrip-
AP1 via Rho/Rac/JNK (planar cell polarity pathway).277,353 These tion) family of transcription factors (STAT1–STAT6).361,373 Docking
pathways regulate cell polarity and migration and are implicated in of STATs facilitates their phosphorylation by JAKs, which in turn
cancer metastasis.353 Aberrations in canonical Wnt signaling promote causes STAT dimerization, nuclear translocation, and alterations in
tumorigenesis in melanoma and colon, liver, ovarian, and prostate gene transcription.374 There is also cross talk between STAT signaling
cancers.354,355 Specifically, loss-of-function mutations or truncations and the nuclear factor–κB (NF-κB) and SMAD signaling pathways.375
in APC and AXIN1/2 and gain-of-functions mutations in β-catenin STATs are also activated by RTKs, SRC, and ABL, and STAT activation
are found in almost all colorectal cancers, with APC alteration found also plays a role in transformation initiated by these oncogenes.376
in over 85%.349,356 Germline mutations in the APC gene are also JAK/STAT activity is regulated by several posttranslational modifications
the basis for the inherited cancer predisposition syndrome familial as well as by the SOCS (suppressor of cytokine signaling) and PIAS
38 Part I: Science and Clinical Oncology

(protein inhibitor of activated STAT) proteins.375 In addition to response.383 JNK1 is upregulated in hepatocellular carcinoma and
JAK-STAT signaling, cytokine receptors can transduce messages through prostate cancer, whereas p38α activity is either lost (in hepatocellular
LCK and SYK (Src-family kinases), through BCL-2, and via PI3 carcinoma) or activated in numerous cancers, and targeted inhibitors
kinase/AKT and Ras/Raf mediated upregulation of Fos- and Jun- are being developed.385,397
dependent transcription.376,377
The 29-member TNF superfamily of receptors (TNFSFR1) and SERINE/THREONINE RECEPTOR SIGNALING
their corresponding 19 ligands (TNFSF) induce inflammation in
addition to prosurvival and proapoptotic phenotypes.378,379 TNF ligands Receptor serine/threonine kinases are exemplified by the TGF-β type
are TM proteins that function as membrane-integrated or cleaved, I and II receptors.398,399 Ligands for these receptors include the TGF-β
soluble trimers that bind and activate preformed, single-TM TNF superfamily, which comprises the TGF-β1-3 isoforms, activins, inhibins,
receptor trimers on the cell surface.380 Conserved cysteine residues in Nodal, and Lefty, and the more distantly related bone morphogenic
the TNF receptor external domains dictate ligand binding, and the proteins (BMPs), growth and differentiation factors (GDFs) and
presence of death domains (DDs), TRAF-interacting motifs (TIMs), müllerian inhibitory substance (MIS). TGF-β ligands regulate a diverse
or neither (decoy) dictate downstream signaling. For example, on array of physiologic processes including growth, proliferation, survival,
apoptotic stimuli, ligand activation of the TNF-R1 and DR3 receptors hormone release, and differentiation.398,400 They thus have a central
recruits the adaptor TRADD (TNFR-associated death domain) via role in embryonic patterning, tissue development, and morphogenesis.
DDs, which in turn binds to FADD (Fas-associated protein with Signaling mediated by TGF-β, the namesake and most studied ligand
death domain).378,379,381 FADD binds procaspase-8 and procaspase-10 of the superfamily, will be used to exemplify the general structure of
via death effector domains (DEDs), and induces their cleavage to the serine/threonine kinase cascades.
form active enzymes, which cleave caspase-3 and induce apoptosis. TGF-β is ubiquitously expressed and requires a multistep maturation
TRADD binding is also capable of inducing the intrinsic apoptotic and secretion process to be functional and bioavailable. Initially
cascade (mitochondrial release of ROS, cytochrome C, and Bax, which translated as an immature proprotein, the prodomain (called latency-
leads to caspase-9 and caspase-3 activation and apoptosis).378,382 associated protein [LAP]) is cleaved and noncovalently bound to the
Under proliferative stimuli, TNF-α–dependent activation of remaining mature form of TGF-β.401 Covalently bound mature, active
TNF-R1/TRADD and TNF-R2 converges on the recruitment of dimers are further bound to latent TGF-β binding protein (LTBP)
TRAF2 (TNFR-associated factor 2). TRAF2 binding sequentially such that this complex is sequestered by LTBP binding to the extracel-
recruits the RIP (receptor interacting protein), TAK1 (TGF-β activated lular matrix (ECM) until appropriate signals initiate matrix metal-
kinase 1), and IκB kinase (IKK) trimer; this functions to degrade loproteinase, plasmin, or thrombin-dependent cleavage of LTBP and
IκB-α (inhibitor of NF-κB-α) and in turn facilitates the activation subsequent release of active TGF-β dimers.401,402 TGF-β dimers bind
and translocation of NF-κB.378,379 NF-κB in turn induces mediators constitutively active, single TM, homodimeric TGF-β type II receptors
of inflammation and cytoprotective phenotypes.383 Alternatively, TAK1 (TβRII). Once bound by ligand, TβRII forms a complex with TGF-β
can signal to MKK3/6 (MAP kinase kinase 3/6) and to two MAP type I (TβRI) receptor homodimers, thus creating a receptor hetero-
kinases, ERK (proproliferation) and p38α (to activate the transcription tetramer.398,400 TβRII receptors phosphorylate and activate the intrinsic
factor AP-1 [activator protein-1]).384 Upstream, TRAF2 can additionally kinase activity of TβRI, which in turn phosphorylates serine residues
trigger MEKK1 (MAP/ERK kinase kinase 1), MKK7, and JNK (c-Jun in the C-terminal–SSXS motif of receptor-activated SMAD proteins
activating kinase), the recruitment of which also converges to activate (R-SMAD2 and R-SMAD3 for TGF-β).403 Activated R-SMAD2 and
AP-1 and thus regulate proliferation and survival.385–387 There is an R-SMAD3 then form a heterotrimer with SMAD4, which then localizes
important avenue of cross talk between the TNF-directed NF-κB and to the nucleus, where it both binds DNA and partners with other
JNK pathways, the balance of which ultimately decides cell fate.388 transcription factors (i.e., Forkhead, homeobox, zinc-finger, AP-Ets,
In cancer, dysregulation of cytokine signaling promotes chronic and bHLH family transcription factors) and cofactors (e.g., p300,
inflammatory signals and prevents the immune system from attacking CBP).404,405 These SMAD-containing complexes then target selective
cancer cells. Unfortunately, because of their pleiotropic effects, which promoter elements with high affinity, leading to the induction or
are often cell type and microenvironment specific, therapeutic strategies repression of hundreds of genes, depending on cell context. For example,
that modulate cytokine signaling have demonstrated only modest TGF-β induces the expression of 4EBP1 and the cyclin-dependent
clinical activity in patients with cancer. For example, the recombinant kinase inhibitors INK4B and p21 and represses Myc to elicit growth
human IFN-α2a mimetic, Roferon-A, has been shown to inhibit inhibitory effects.406 It also activates DAPK (death-associated protein
tumor growth in patients with melanoma and hairy cell leukemia, kinase), GADD45β (growth arrest and DNA damage-inducible 45β),
but such treatments have now been supplanted by more active thera- and BIM to promote apoptosis and PDGF in smooth muscle cells
pies.389 Moreover, focal delivery of TNF-α to limbs affected by soft to enhance proliferation, among other effects.407 TGF-β also signals
tissue sarcomas and melanomas through isolated limb perfusion through many non-SMAD effectors including Shc, which can result
techniques has been more effective than systemic use, which is in enhancement of Ras/ERK signaling, and TRAF6, which activates
toxic.390,391 Proapoptotic, anti-TRAIL therapies, such as mapatumumab, the TAK1/MKK3&6/JNK/p38 cascades.408 TGF-β, through indirect
are also in clinical testing.381 mediators, can also activate Src, Rho, and PI3 kinase, and through
Downstream components of the cytokine signaling cascades are pathway cross talk the Wnt, Hedgehog, and Notch cascades.409 The
also being explored as targets for drug development. Gain-of-function outcome of TGF-β–directed transcriptional responses depends on
mutations in the JAK2 (hot spot V617F) and MPL genes, the latter access of TGF-β to particular signaling receptors and SMAD complexes,
of which encodes the thrombopoietin receptor, are common in the availability of transcription factors, and the epigenetic status of
myeloproliferative neoplasms, and the selective JAK1/2 kinase inhibitor the cell.410
ruxolitinib has been approved for this indication (see Table 2.1).392–394 Alterations in TGF-β signaling are common in cancer.407,411,412
STAT3 is frequently hyperactivated in cancer, because it is a downstream For example, mutational inactivation of TβRII is seen in colorectal
effector of both cytokine receptors and mutated and amplified tyrosine cancers with microsatellite instability.413 Mutations and loss of SMAD4
kinases.376,395 Constitutive activation of STAT5 and STAT6 plays a expression are common in colorectal, pancreas, and head and neck
critical role in BCR-Abl–driven chronic myelogenous leukemia (CML), cancers.414,415 Conversely, increased TGF-β expression occurs in breast,
and IL-13–driven lymphomas and leukemias.376 On the basis of these prostate, and colorectal cancers and has been associated with cancer
findings, direct inhibitors of JAK and STAT are currently in develop- progression and the development of metastases.407 TGF-β has also
ment.374,396 Inhibition of NF-κB has been shown to induce apoptosis been shown to play a role in maintaining the tumor-initiating cell
in leukemias and lymphomas and enhance chemotherapy and radiation or cancer stem cell population in gliomas, leukemias, and breast
Intracellular Signaling  •  CHAPTER 2 39

cancer.407 It has been proposed that TGF-β signaling promotes clinical trials.422,434–436 Newer avenues of drug development include
invasion and metastasis by promoting EMT.406 Interesting to note, monoclonal antibodies targeting Notch receptors or ligands.437
in pancreatic ductal adenocarcinoma, wherein loss of SMAD4 is
common, TGF-β can play a novel tumor suppressor role by inducing NUCLEAR HORMONE RECEPTOR SIGNALING
a lethal EMT through the lineage and progenitor regulators Klf5
and Sox4.416 The nuclear hormone superfamily is composed of hormone receptors
Intense efforts are underway to develop therapeutic strategies that and orphan receptors (receptors for which no ligand has yet been
inhibit the tumorigenic properties of TGF-β signaling. These include identified). The nuclear hormone receptors are characterized structurally
the development of selective TGFβRI inhibitors, TGF-β blocking by a ligand binding domain, a DNA binding domain, and a hinge
antibodies, soluble TGF-β antisense therapies, and selective kinase region that connects the ligand and DNA binding domains.438 Nuclear
inhibitors.411,417,418 The development of inhibitors of TGF-β signaling hormone receptors are classified into four subtypes based on ligand
has, however, been confounded by the ability of TGF-β to both promote specificity: steroid, retinoid X receptor (RXR), monomeric or tethered
and suppress tumor progression in a context-specific manner.419,420 orphan receptors, and dimeric orphan receptors.439 The steroid receptor
ligands include estrogen, progesterone, androgen, and growth hormone.
NOTCH RECEPTOR SIGNALING Ligand binding occurs in the cytoplasm and results in receptor
homodimerization followed by nuclear translocation. Once translocated
The mammalian Notch receptors, Notch1 to Notch4, are single-pass into the nucleus, the ligand-bound receptor acts as a transcription
TM receptors that are functionally unique with regard to receptor factor that modulates the expression of several downstream proteins
processing and signal activation and transduction.421–424 Notch receptors through binding to steroid response elements, which are conserved
are translated as immature receptors that undergo S1 cleavage by nucleotide sequences within the regulatory regions of genes.440 In
furin-like convertase during Golgi trafficking.425 This generates two contrast to the steroid receptors, the RXR receptors form heterodimeric
subunits that are retethered at the cell membrane by noncovalent complexes with other partners, including the retinoic acid receptor,
bonds in the heterodimerization domain.423,426 The extracellular domain the thyroid hormone receptor, and vitamin D receptors.
subunit consists of ligand-binding EGF-like repeats, a heterodimer Hormone receptor blockade is a cornerstone in the treatment of
domain, three negative regulatory LIN12 and Notch repeats (LNRs) estrogen receptor (ER)– and progesterone receptor–expressing breast
containing numerous cysteines, and a hydrophobic TM-interacting cancers. The selective estrogen receptor modulator (SERM) tamoxifen
region. The other subunit contains the TM domain and the intracellular competes with estradiol for binding to ER. Notably, tamoxifen binding
domain, which has ankyrin repeats and a RAM domain central to results in ER dimerization, nuclear translocation, and receptor binding
the Notch receptor’s unusual activity as a direct transcription factor, to estrogen response elements in the promoter regions of estradiol-target
and a PEST motif important for the quick termination of Notch genes. It is thought that the ER/tamoxifen complex recruits transcrip-
activity and ubiquitin-mediated degradation. tional corepressors, in contrast to ER/estradiol binding, which recruits
Delta-like ligand (DLL1, DLL3, DLL4) and Jagged (JAG1 and transcriptional coactivators.440,441 Although tamoxifen has antiprolifera-
JAG2) are the five TM-protein ligands for the Notch receptors.427 A tive effects in ER-expressing breast cancer cells, it causes hypertrophy
Notch receptor on one cell binds to DLL or JAG ligand on an adjacent and neoplastic transformation of endometrial tissue, likely as a result
cell. This results in a change in Notch receptor conformation, which of cell type and context-specific recruitment of transcriptional coactiva-
facilitates a series of proteolytic cleavages required for receptor activation. tors that are differentially expressed in these tissues.440 Recently,
First, the ADAM17/TACE (a disintegrin and metalloprotease-17/ mutations in the ESR1 gene, which encodes the ER, have been shown
TNF-α converting enzyme) metalloprotease performs S2 cleavage of to be a common mechanism of acquired resistance to hormonal therapy
the extracellular domain.428,429 This allows subsequent S3 cleavage of in patients with breast cancer.442 Retrospective studies suggest that
the TM domain by the γ-secretase complex and release of the Notch patients with ESR1-mutant breast cancer may have more durable
intracellular domain (NICD).430 The active NICD translocates to the responses to the selective estrogen receptor downregulator (SERD)
nucleus, where it binds to and converts the repressor complex CSL fulvestrant than those treated with the aromatase inhibitor exemestane
(CBF1, Suppressor of Hairless, Lag-1) into a transcriptional activator (see Table 2.1).442,443
that recruits coactivators including the Mastermind-like family proteins Dihydrotestosterone is the primary ligand of the androgen receptor
and p300. NICD target genes include the HES (hairy enhancer of (AR). AR blockade by antiandrogens such as bicalutamide is a com-
split) and HRT/HEY (hair-related transcription factor) transcriptional monly used therapeutic modality for patients with locally advanced
repressors, as well as cyclin D1, myc, p21, NF-κB, and the Notch or metastatic prostate cancer. Bicalutamide competes with dihydrotes-
receptors and ligands themselves, among others.422,426,431 Notch is also tosterone for binding to cytoplasmic AR.444 The mechanism by which
heavily involved in cross talk with other pathways, including RTKs, bicalutamide-bound AR inhibits androgen-dependent gene transcription
PI3 kinase, Ras/ERK, JAK/STAT, Wnt, Hedgehog, TGF-β/SMAD, is unclear but may involve the recruitment of transcriptional corepressors
and p53. Overall, Notch plays a role in proliferation, cell fate determina- as well as histone modifications that lead to tighter chromatin binding
tion in development, and survival. and therefore reduced access to promoter regions by transcription
Notch’s causal role in tumorigenesis was established on its discovery factors. Enzalutamide, a nonsteroidal small-molecule antagonist of
in 1991, when a translocation event in T-cell acute lymphoblastic the AR that inhibits AR nuclear translocation and DNA binding is
leukemia or lymphoma (T-ALL) was identified that fused the T-cell FDA approved for the treatment of patients with metastatic prostate
receptor-β promoter/enhancer elements to Notch, generating a cancer that has progressed following treatment with bicalutamide and
truncated form of Notch1 that was constitutively nuclear and active.432 medical castration (see Table 2.1).445 Abiraterone acetate, an irreversible
Activating NOTCH1 mutations have since been found to occur in inhibitor of CYP17A1 that enables intratumoral and adrenal androgen
approximately 60% of T-ALL patients.433 Subsequent studies have depletion, also has significant clinical activity in patients with castrate-
determined that Notch and its ligands have both oncogenic and tumor resistant prostate cancer.446
suppressor properties in several hematologic and solid tumor
malignancies, including melanoma, glioblastoma, and breast, lung, INTEGRIN RECEPTOR SIGNALING
colorectal, and pancreatic cancers, depending on cellular context.423,426
Thus far, efforts to develop inhibitors of Notch signaling have focused The integrin receptor family regulates cell adhesion, migration, invasion,
primarily on inhibiting cleavage, and thus activation of Notch. and cell survival.447,448 Integrin receptors are heterodimeric molecules
Specifically, selective γ-secretase inhibitors (GSIs), such as MK-0752, consisting of combinations of alpha and beta subunits. Each combina-
PF03084014, and BMS-906024, are currently being tested in early-stage tion dictates the spectrum of ECM components to which these receptors
40 Part I: Science and Clinical Oncology

bind. Once ligated to the ECM, the receptors recruit multiple proteins ubiquitously, whereas the tissue distribution of the latter six is more
to the cell membrane, including cytoskeletal molecules such as paxillin restricted.467 Together, SRC family kinases have pleiotropic roles in
and vinculin that form focal adhesions to ECM components.449 Unlike cellular proliferation, apoptosis, differentiation, motility, adhesion,
the RTK family, integrin receptors do not possess intrinsic kinase angiogenesis, and immunity.468,469
activity but rather promote signaling by facilitating the activation of SRC is by far the most intensively studied family member and
kinases such as Src or FAK.450 Integrins are also unique in that they was the first gene observed to have oncogenic potential.470 Peyton
participate in bidirectional signaling. “Inside-out” signaling occurs Rous was awarded the Nobel Prize for a series of experiments showing
when intracellular adapters, of which talin-1 and kindlin-1/2/3 are that a transmissible factor was present in avian sarcomas capable of
the best known activators, trigger a conformational change of the initiating tumors in recipient birds. Five decades later the viral oncogene
cytoplasmic tails of the alpha and beta subunits, which is transduced v-Src was identified as the oncogenic factor in the Rous sarcoma
to the extracellular component of the receptor, resulting in increased virus.471–473 Bishop and Varmus later showed that v-Src was a mutant
affinity for portions of the ECM.451 Conversely, “outside-in” signaling form of the cellular proto-oncogene c-Src, and Hunter and colleagues
involves binding of ligand to integrins, which stimulate the activation showed that its transformative capacity was dependent on its tyrosine
of multiple intracellular signaling pathways.452 Collectively, the term kinase activity.14,15,474
integrin adhesome is used to describe the site at which integrins initiate SRC is regulated in a number of ways. First, SRC has a myristoylation
contact with the ECM and other cells and recruit all of the underlying site in its N-terminus that is necessary for membrane localization and
intracellular machinery (e.g., cytoskeleton, scaffolds, signaling adapters— that promotes its interaction with nearby membrane-bound effectors.475
over 200 intrinsic and transient components) to generate diverse types The SH2 and SH3 domains facilitate protein-protein interactions and
of adhesions (e.g., focal complexes, focal adhesions, fibrillar adhesions, conformational changes in the protein. Inactive SRC is maintained
podosomes, invadopodia).453 in a closed conformation with phosphorylated Y530 (mediated by
Integrins are expressed on cancer cells and have been shown to CSK, C-terminal SRC, and Csk homology kinases) interacting with
promote disease progression.447,450,454 Integrins are also present on the folded-over SH2 domain.476 The closed, inactive confirmation
stromal cells, including pericytes (which promote endothelial cell of SRC is further stabilized by proline-rich segments of the kinase
growth and proliferation and thus angiogenesis) and fibroblasts, where domain associating with the SH3 domain.477 SRC activation requires
they influence the surrounding microenvironment and thus indirectly dephosphorylation of Y530, likely by PTPα/γ/1β (protein tyrosine
stimulate tumor growth and proliferation. For example, vascular cell phosphatase α/γ/1β) or SHP1/2 (SH-containing phosphatases), which
adhesion molecule 1 (VCAM1) is expressed on pericytes and binds allows the kinase to assume an open conformation.466,478 Autophos-
to the integrin receptor α4β1, which is found on the surface of phorylation of Y419 in the activation loop of the kinase domain also
endothelial cells, resulting in pericyte recruitment to sites of vascular promotes full activity,479 whereas binding of FAK and CRK-associated
maturation.455 Integrin signaling also plays a role in the activation of substrate (CAS) to the SH2 domain induces SRC activation and links
matrix metalloproteinase 2,456 which promotes cell invasion, and has SRC signaling to the regulation of focal adhesion, actin reorganization,
been shown to regulate cyclin D and cyclin-dependent kinase inhibitor and migratory phenotypes.480,481 SRC is a downstream mediator of
expression, thereby controlling cell cycle progression.457 Finally, integrin numerous receptor families including RTKs, integrin receptors, hormone
receptor activation can lead to increased secretion of growth factors, receptors, cytokine receptors, and GCPRs and promotes signaling
which then stimulate tumor invasion through autocrine and paracrine through the PI3 kinase/AKT, Ras/MAP kinase, and JAK/STAT cascades,
mechanisms.456 among others.466,467,469,478,482–486 More than two decades of research
Tumors that express integrin receptors include melanomas, glio- have uncovered numerous SRC substrates, including p85-cortactin,
blastomas, and breast cancers. In melanoma, the αvβ3 and α5β1 p110-AFAP1, p130Cas, p125FAK, and p120-catenin.487
integrin receptors promote vertical growth and metastatic spread to Although mutations in SRC are rare in human cancers, SRC is
lymph nodes.458,459 In glioblastoma, αvβ3 and αvβ5 are expressed frequently activated as a consequence of other mutational events in
mainly at the edge of tumors, suggesting a role in tumor invasion.460 colorectal, breast, esophageal, gastric, pancreatic, hepatocellular, ovarian,
The expression of the α6β4 and αvβ3 integrin receptors in breast and lung cancers.466 In colorectal and hepatocellular carcinomas, SRC
cancer is associated with higher grade and tumor size,461 the development activation occurs in the setting of concomitant loss of CSK.488–490 Newer
of bone metastases,462 and decreased survival.463 To date, drug develop- signaling discoveries have identified roles for SRC in promoting tissue
ment has targeted three integrins—αIIbβ3, α4β1, and α4β7—in the repair after intestinal inflammatory injury, as seen in inflammatory bowel
context of platelet activation and blood clotting, multiple sclerosis, diseases and colorectal cancer, via the IL-6 cytokine coreceptor gp130
and inflammatory bowel disease.451 Clinical trials of monoclonal and YAP.491 Furthermore, norepinephrine-mediated, β-adrenergic/PKA
antibodies that target integrin receptors are underway in several cancer activation of SRC has recently been shown to enhance tumor cell
types. For example, etaracizumab, a humanized monoclonal antibody migration, invasion, and growth.492 The tyrosine kinase inhibitor
targeting αvβ3 integrin, is being tested in solid tumors and has shown dasatinib, which is used in the treatment of CML and Philadelphia
activity in patients with metastatic melanoma.464 Cilengitide, an chromosome–positive acute lymphoblastic leukemia (ALL), inhibits
inhibitor of the αvβ3 and αvβ5 integrins, inhibited angiogenesis and SRC family kinases, in addition to BCR-ABL, KIT, Ephrin A2 receptor,
tumor cell proliferation in preclinical studies; however, a phase III and PDGFR (see Table 2.1).493–495 Additional dual SRC/ABL and SRC
trial in combination with temozolomide and radiation in patients selective inhibitors are approved in CML (bosutinib, ponatinib; see
with glioblastoma did not result in improved outcomes compared later section on ABL signaling) or are in clinical testing, including
with chemoradiation alone.456,465 saracatinib, XL-228, KX2-391, and DCC2036. Most have shown
limited single-agent activity and are being developed as combination
NON–RECEPTOR TYROSINE therapies.469,478,484–486
KINASE SIGNALING
ABL Signaling
SRC Signaling
The ABL gene was first identified in 1980 as the oncogene responsible
The SRC family of intracellular, non-RTK proteins is composed of for driving the Abelson murine leukemia virus, and later was found
11 members (SRC, FYN, YES, Blk, Yrk, Frk/Rak, Fgr, Hck, Lck, to be part of the translocations found in many types of human leu-
Srm, and Lyn).466 They share common structural features including kemias.496 ABL1 and ABL2 isoforms have both overlapping and
the so-called SRC-homology (SH) domains 1 to 4. The SH1 domain divergent functions. ABL is found in an autoinhibited conformation
includes the kinase domain. Only SRC, FYN, and YES are expressed dictated by clamping of the N-terminal hydrophobic region, SH3
Intracellular Signaling  •  CHAPTER 2 41

and SH2 domains onto the C-lobe of the kinase domain.497 Inter- neurofibromin (NF1), GAP1IP4BP, and CAPRI, negatively regulate
molecular interactions with adaptors such as RIN1 or phosphorylation RAS activity and thus function as tumor suppressors.514,518
by SRC, among others, promote stabilization of the open, active form Binding of GTP to RAS induces conformational changes in the
of ABL 23842626. The ABL tyrosine kinase is found in both the switch I (loop 2 residues 30–38) and switch II (helix 2 and loop 4
cytoplasm and the nucleus, and its function varies based on subcellular residues 60–76) domains, which facilitate the association of RAS with
localization.498 Cytoplasmic ABL has been implicated in G1/S check- regulators and downstream effectors.531,532 RAS directly interacts with
point regulation and interaction with actin on C-terminal binding over 20 effector proteins, of which the RAF kinases, PI3 kinases, and
sites,499 whereas nuclear ABL inhibits binding of the DNA repair RALGDS are the best characterized (see Fig. 2.1). The canonic RAS/
protein Rad51 to sites of DNA damage.500,501 Activated ABL kinases RAF/MEK/ERK (classic MAP kinase) cascade is by far the most
are now recognized to play an important role in tumor initiation by extensively characterized RAS effector pathway. This prototype of a
disrupting cell polarity and by promoting invasion and metastasis by three-tiered kinase signaling cascade exemplifies numerous RAS-
regulating invadopodia.502 dependent mitogen-activated protein kinase (MAPK) cascades that
In cancer, translocation of the ABL gene on chromosome 9 with respond to diverse signals, including cell stress and cytokine signaling
the breakpoint cluster region (BCR) gene on chromosome 22 results (see section on cytokines for details of the JNK and p38 pathways).533
in the expression of a BCR-ABL fusion protein.503 This translocation, The RAF protein family (which represent the top-tier MAPK kinase
the Philadelphia chromosome, is found in almost all CMLs and kinases [MAPKKKs], or MEK kinases [MEKKs]) is composed of
represents the pathognomonic molecular lesion in this disease. three differentially expressed isoforms, A-RAF, B-RAF, and C-RAF
BCR-ABL translocations are also found in approximately one-third (RAF-1).534,535 RAF, via its RAS binding domain (RBD) and cysteine
of acute lymphoblastic leukemias (ALLs).504,505 In addition, at least rich domain (CRD), interacts with GTP-bound Ras.536–539 Binding
eight other fusion partners of ABL have been discovered.502 The of RAF to GTP-bound RAS results in RAF localization to the plasma
realization that the proliferation and survival of CML cells is critically membrane and its subsequent phosphorylation and activation.540 The
dependent on the ABL fusion protein led to the development of mechanisms of RAF-1 and B-RAF phosphorylation and activation
imatinib, an inhibitor of the ABL tyrosine kinase.505 To date, there are distinct and are derived from the summation of signaling inputs
are five FDA-approved inhibitors for CML: imatinib, dasatinib, from small G proteins (RAS, RAC, CDC42, RAP-1), kinases (activating
bosutinib, nilotinib, and ponatinib (see Table 2.1).506 Moreover, imatinib inputs: SRC/PAK/PKC, inhibitory inputs: PKA/AKT/SGK), isoform
and dasatinib are approved for treatment of ALL.507–511 A secondary homodimerization and heterodimerization, phosphatases (PP2, PP2A),
mutation in the gatekeeper site T315I is the most common reason scaffolding proteins (KSR, RKIP, HSP90, and so on) and cofactors
for acquired resistance to ABL kinase inhibitors. Combination of (14-3-3), with phosphorylation events regulating critical aspects of
ATP-competitive and allosteric inhibitors such as GNF-5 may be a RAF activation.535,541 In brief, RAS binding to RAF-1 releases 14-3-3,
novel strategy to combat resistance.512 a negative regulator that binds to basally phosphorylated residues
S259 and S261 and sequesters RAF-1 in the cytosol in a closed,
RAS/MAP KINASE PATHWAY SIGNALING inactive conformation. Liberation from 14-3-3 exposes the PKA/
AKT/SGK-dependent inhibitory phosphorylation on S259, facilitating
First identified over 30 years ago as the oncogenes responsible for the its dephosphorylation by protein phosphatase 2A.542–546 Loss of this
transforming potential of the Harvey and Kirsten murine sarcoma inhibitory phosphorylation primes RAF-1 for RAS, PAK, SRC, growth
retroviruses (Ha-MSV and Ki-MSV), RAS proteins are guanine factor, and integrin-stimulated activating phosphorylations on S338,
nucleotide binding proteins.513–517 Ras proteins have intrinsic GTPase Y341, T491, and S494.547–551 A-RAF is activated similarly to RAF-1.
activity and cycle between inactive GDP- and active GTP-bound Notably, B-RAF activation requires fewer steps owing to its constitutive
states. GDP/GTP exchange thus allows Ras proteins to function as phosphorylation at S445, a site analogous to S338 in RAF-1.541,552
binary molecular switches. Binding to RAS-GTP stimulates B-RAF phosphorylation at critical
In the human genome, there are three RAS genes, which encode residues in its activation loop, T599 and S602 (analogous to T491
four homologous proteins (HRas, NRas, and the alternative splice and S494 in RAF-1).553,554 The B-RAF isoform is the most potent
variants KRAS4A and KRAS4B) with highly conserved N-terminal activator of ERK pathway output.535,552,554
and variable C-terminal regions. Following stimulation of cells by Activated Raf proteins bind and phosphorylate MEK1 and MEK2
serum growth factors, cytokines, hormones, and neurotransmitters, (mitogen-activated protein kinase/extracellular signal-regulated kinase
Ras undergoes a series of C-terminal (C186AAX) posttranslational kinases 1 and 2, or MAPKK, or MAP2K) on serines 217 and 221.555
modifications that result in its localization to specific membrane Activated MEK, a dual-specificity threonine/tyrosine kinase, in turn
microdomains.518 Membrane localization is required for the transforming phosphorylates MAPK/ERK-1/2 (mitogen-activated protein kinases/
properties of Ras, because mutation of Ras at C186 results in cytosolic extracellular signal-regulated kinases 1 and 2) on threonine 183 and
localization and protein inactivation whereas Ras activity can be rescued tyrosine 185, inducing a conformational change, activation, and disso-
by myristoylation, which promotes membrane localization.519–521 GEFs ciation from MEK.556 Activated ERK then phosphorylates substrates in
promote Ras activation by binding to GDP-bound Ras and facilitating the cytosol (e.g., p90RSK) and in the nucleus (such as the transcription
the release of GDP and the binding of GTP. SOS1, RAS-GRF (dual factors ELK-1, ETS-2, FOS, JUN, ATF-2, AP-1, MYC, CREB1), which
specificity GEFs for RAS and RAC), and RAS-GRP (stimulated by promote proliferation and survival.557,558 Raf-1 has also been shown
DAG/phorbol esters and Ca+) are the mostly highly characterized to mediate suppression of apoptosis through non–MEK-dependent
RAS-GEFs.522–524 Using RTK stimulation as an example, ligand binding interactions with ASK1, MST2, and MEKK1/NF-κB.559
induces RTK dimerization and autophosphorylation of tyrosine residues RAS/RAF signaling triggers numerous regulatory mechanisms,
in the receptor cytoplasmic tail. Phosphotyrosine docking sites recruit including classic negative feedback loops, which serve to attenuate
scaffold proteins such as SHC and permit interaction with the SH2 pathway output.560 The Sprouty and Sprouty-related EVH1-domain-
domains of adaptor proteins such as Grb2.525 Grb2 in turn recruits containing protein (Spred) proteins (encoded by the SPRY1–4 and
SOS1 via its SRC homology 3 (SH3) domain, thereby positioning SPRED1–4 genes, respectively) inhibit the cascade at the level of RAS
SOS near membrane-anchored RAS (see Fig. 2.1).525–529 SOS1 in turn and RAF activation.561–564 The dual-specificity phosphatases (MKPs/
activates RAS via its CDC25 homology (RASGEF) domain and DUSPs) dephosphorylate MAP kinases, including ERK.565,566 In
N-terminal RAS exchanger motif (REM or RASGEFN domain). addition, increased pathway activity induces ERK-dependent negative
Ras inactivation is catalyzed by GTPase-activating proteins phosphorylations on B-RAF (at S151, S750, T401 and T753) and
(GAPs), which enhance the intrinsic GTPase activity of Ras proteins on RAF-1 (at S29, S43, S289, S296, S301 and S642) that abrogate
by 100,000-fold.530 RAS GAPs, which include p120-RASGAP, interactions with RAS and homodimer and heterodimer formation.567,568
42 Part I: Science and Clinical Oncology

RAS signaling is further regulated by RKIP (RAF kinase-inhibitory emerged as a mechanism of acquired resistance to RAF inhibition.594,595
protein), which disrupts the RAF-1/MEK interaction and IMP (impedes Hot spots include mutations at MEK1 residues F53, K57, and P124
mitogenic-signal propagation), which represses KSR-dependent scaf- and at MEK2 residue F57, which is paralogous to MEK1 F53. ERK
folding of RAF/MEK, among other functions.534,569–571 amplification and mutation, although equally rare (approximately 1%
In its active, GTP-bound state, RAS alternatively binds to the of all tumors), may also emerge as mechanisms of resistance to upstream
p110α catalytic subunit of class I PI3 kinases,572,573 causing activation MAPK inhibition, including alteration at the hot spot residue E322.596
of its lipid kinase activity and thereby generating PIP3, which in turn Given the high prevalence of RAS pathway alterations in human
stimulates the proproliferative and prosurvival kinases PDK1 and cancers, significant attention has been directed toward the development
AKT (see section on PI3 kinase signaling for details). RAS-dependent of selective inhibitors of this pathway.560,580,597,598 To date, clinically
activation of PI3 kinase can further stimulate RAC, a RHO family effective direct inhibitors of oncogenic RAS have yet to be identified.
GTPase involved in regulation of actin and NF-κB.513,574,575 A third The inability to directly target RAS has been attributed to the high
major class of RAS effectors is the group of GEFs for RALA and RALB, affinity of the RAS-GTP interaction. Extensive efforts were thus directed
namely RALGDS, RGL, and RGL2.576–578 These RAL exchange factors toward inhibiting the posttranslation modifications required for RAS
stimulate phospholipase D and the CDC42/RAC-GAP RAL binding activation. Specifically, inhibitors of the enzyme farnesyltransferase,
protein 1 (RALBP1) and inhibit Forkhead transcription factors to which regulates RAS localization, were tested in randomized phase III
regulate transcription, vesicular trafficking, and cell cycle progression. trials but were found to be inactive.599 The failure of farnesyltransferase
Several additional RAS effectors have been identified. These include inhibitors in KRAS-mutant tumors was predicted by the preclinical
(1) PLCε, which generates IP3 and DAG and regulates calcium release observation that geranylgeranyl modification can substitute for farne-
and PKC activation; (2) T-cell lymphoma invasion and metastasis-1 sylation in targeting KRAS and NRAS to the plasma membrane.600,601
(TIAM1), which facilitates actin reorganization via RAC; (3) AF6, Small molecules that irreversibly bind to the mutant cysteine in tumors
which contributes to cytoskeletal changes; (4) RIN1, which regulates with G12C K-RAS have been developed.602 Because this class of
endocytosis; and (5) RASSF and NORE1, which have been shown compounds is selective for the G12C mutant, they are a promising
to regulate apoptosis and cell cycle progression.513,518 Many other novel approach for a subset of patients with KRAS-mutant tumors.
nondirect connections allow RAS to affect the cellular microenviron- As an alternative, extensive efforts have focused on the development
ment, metabolic signaling, autophagy, inflammation, and immune of selective inhibitors of key kinase effectors of RAS transformation.
responses. Overall, the complex effects of RAS activation result in a The selective RAF inhibitors vemurafenib and dabrafenib induce tumor
diversity of context-dependent phenotypes ranging from cell prolifera- regressions in most patients with BRAF V600E mutant melanoma
tion to cell death that are influenced by a wide variety of extracellular and are FDA approved for this indication (see Table 2.1).603–612 Notably,
stimuli and intricately woven layers of regulation. these agents selectively inhibit RAF signaling in BRAF-mutant tumors
The potent oncogenic effects of RAS signaling are highlighted by and are thus inactive in RAS-mutant tumors.613 Several mechanisms
the high prevalence of mutations in the RAS genes and its proximal of resistance to RAF inhibitors have emerged, including BRAF V600E
downstream effectors.518,579–581 RAS mutations are found in approxi- splice variants or amplification; loss or mutation of NF1; parallel
mately 30% of all human tumors, the majority of which (85%) occur pathway activation of RTKs; mutation of PI3K/AKT components;
in the KRAS isoform. KRAS is frequently mutated in pancreatic cancers mutations in NRAS, RAF1, and MEK1/2; and amplification of
(58%), colorectal and biliary tree cancers (33 and 31%), and non–small MITF.592,614 Sorafenib, a multikinase inhibitor of RAF, VEGFR2, and
cell lung adenocarcinomas (17%). Mutations in HRAS are most PDGFRβ, has been shown to have some clinical efficacy in ARAF-
common in low-grade bladder cancers (11%), whereas mutation in mutant histiocytosis and NSCLC.588,589,615
NRAS is a frequent event in melanoma (18%) and biliary tree cancers The selective MEK inhibitor trametinib is also FDA approved
(11%). Mutations that lock RAS in its GTP-bound state confer for use in melanomas with BRAF mutation (see Table 2.1).605,616–619
oncogenic potential. Point (missense) mutations in residues 12, 13, Furthermore, the combination of a RAF and a MEK inhibitor has
61 (exons 2 and 3) generate a constitutively active RAS oncoprotein been shown to have greater activity than Raf inhibitor monotherapy
by abrogating intrinsic GTPase activity and inhibiting GAP binding.582 in both preclinical models and in patients with melanoma, including
Other mutations, including those at residues 117 and 146, contribute the combinations of vemurafenib plus cobimetinib620 and dabrafenib
to RAS activation by increasing GDP-to-GTP exchange.583,584 Heritable plus trametinib.605,619,621,622 MEK inhibition is also emerging as a
germline mutations of several RAS/MAPK pathway components have strategy to target NRAS-mutant melanoma,623 colorectal624 and thyroid
been shown to be the underlying cause of the so-called RASopathies cancers,625 NF1-mutant melanoma,587 GBM,626 and neuroblastoma.627
neurofibromatosis type 1 (NF1); Noonan, Costello, and cardiofacio- MEK inhibition in combination with immunotherapy, chemotherapy,
cutaneous syndromes; and other developmental disorders.579 More radiation, and PI3K and CDK4/6 inhibitors is being investigated
recently, deep sequencing efforts have identified recurrent somatic in KRAS-mutant tumors including colorectal and lung cancer (see
mutations in the RAS-GAP NF1 in glioblastoma585 and melanoma,586,587 Table 2.1).624,628 Furthermore, clinical evidence of activity of the MEK
as well as in A-RAF (hot spot at S214) in histiocytosis and lung inhibitors cobimetinib, selumetinib, and trametinib has been revealed
cancer588–590 and in RAF-1 (hot spot S257).588 Mutations in BRAF in cases and preclinical reports of MEK1-mutant histiocytosis,589
are also common in human cancer and typically occur in a mutually low-grade serous ovarian cancer,629 NSCLC,594 and melanoma.630
exclusive pattern with RAS mutations. BRAF mutations have been Alternative strategies for inhibiting MAP kinase signaling include
identified in approximately 8% of all cancers, most notably in melanoma HSP90 inhibitors that induce the degradation of RAF1 and mutant
(50%–60%) and in papillary thyroid (30%–50%), biliary tree (14%), BRAF.631 Drug development of kinase- and dimerization-targeted ERK
colorectal (10%), ovarian (12%), and lung cancers (3%) and hairy inhibitors (SCH772984, BVD-523, DEL-22379) is also underway and
cell leukemia (100%).534,535,559,579,591,592 A single valine to glutamic acid may provide a downstream alternative when upstream RAF or MEK
substitution at residue 600 (V600E) accounts for more than 80% of inhibitors fail.632–635 Combinatorial approaches are also being actively
all BRAF mutations and renders BRAF an active monomer in settings pursued, given the modest activity of MEK inhibitors in patients
of low RAS activity, that is sensitive to RAF inhibition. Other non- with RAS-mutant tumors and the frequent co-occurrence of RAS and
V600E BRAF mutants have been recently identified and characterized PI3 kinase pathway alterations in multiple cancer types.636–638 Given
to function as RAS-independent dimers that are insensitive to current the recent success of immunotherapy in many cancers, preclinical
RAF inhibitors, such as vemurafenib, that only effectively inhibit evidence supports the triple combination of BRAF and MEK inhibitors
mutant monomers.593 Activating mutations in MEK1 (MAP2K1) and with immunotherapy in BRAF V600E mutant melanoma, despite
MEK2 (MAP2K2) are also present in approximately 1% of human substantial liver toxicities described with initial trials with combined
tumors, more prominently in melanoma and lung cancer, and have treatment of vemurafenib and ipilimumab.639 In addition, elevated
Intracellular Signaling  •  CHAPTER 2 43

antitumor immune responses in mouse models of triple negative AKT-mediated antiapoptotic effects occur through phosphorylation
breast cancer following combined MEK inhibition and immuno- and inhibition of Bad, which negatively regulates the antiapoptotic
therapy suggest that hyperactivate MEK signaling may contribute to protein Bcl-xL; caspase-9, a proapoptotic protease; and the FOXO1
immune evasion.640 transcription factor which regulates the expression of proapoptotic
Given the prominent role of activated ERK in initiating the genes.660–662 AKT also controls cell survival by upregulating NF-κB
transcription and translation of cell cycle components such as cyclin activity through phosphorylation and activation of Iκb kinase, which
D1, inhibitors of cell cycle kinase components have emerged as marks Iκb, an inhibitor of NF-κB, for degradation.663,664 Once released
an alternative strategy to abrogate the output of MAPK signaling. from IκB, NF-κB translocates into the nucleus, where it regulates a
CDK4, a critical serine/threonine kinase that functions in an active multitude of genes involved in cell survival. AKT also phosphorylates
complex with cyclin D to phosphorylate RB and stimulate E2F and activates Mdm2, an E3 ubiquitin ligase that binds to the pro-
release and S phase entry, is amplified in liposarcoma. Palbociclib, apoptotic tumor suppressor p53 and directs it for proteasomal
an inhibitor of CDK4, is FDA approved for use in postmenopausal degradation.665
women with ER+, HER2− metastatic breast cancer; a second drug, AKT-independent effectors of PI3 kinase activation have also been
abemaciclib, has also show efficacy in this same patient population identified and likely play important roles in the development and
(see Table 2.1).641–644 progression of some cancers. These include CDC42 and Rac1, which
are involved in motility and reorganization of the cytoskeleton, and
PI3 KINASE/AKT/MTOR PATHWAY SIGNALING the serum glucocorticoid kinase (SGK) family of serine/threonine
kinases, which promote cell survival.666,667
The PI3 kinase/AKT/mTOR pathway is a key regulator of growth Many of the canonic functions of AKT with regard to cell growth
factor–mediated proliferation and survival.645 Several extracellular and proliferation are mediated through the mTOR pathway. mTOR
growth factors stimulate PI3 kinase by binding to their cognate RTKs is a serine/threonine kinase and a member of the phosphatidylino-
or GPCRs.646 Activated PI3 kinase phosphorylates the 3-OH group sitol kinase-related kinase family.668 mTOR is a component of two
of the inositol ring of phosphatidylinositol, catalyzing the conversion complexes, the rapamycin-sensitive mTORC1 and the rapamycin-
of PIP2 to PIP3. PIP3 then binds to the pleckstrin homology domain insensitive mTORC2 complexes.669 Within mTORC1, mTOR
(PH domain) of multiple proteins, facilitating their recruitment to the associates with Raptor (regulatory associated protein of mTOR) and
plasma membrane and thus regulating their function (see Fig. 2.2). mLST8, whereas the mTORC2 complex consists of mTOR, Rictor
PI3 kinases are grouped into class I, II, and III kinases based on (rapamycin insensitive companion of mTOR), SIN1, and mLST8.670
their structure and substrate preferences, although class II and III In addition to its role in activating AKT as described earlier, mTORC2
kinases have been much less studied. Class I PI3 kinases are subdivided also controls cytoskeletal changes through regulation of paxillin, Rho,
into two groups, IA and IB. Class IA comprises the PIK3CA, PIK3CB, Rac, and PKCα.671
and PIK3CD genes, which encode the catalytic p110α, p110β, and mTORC1 activation is regulated in part by AKT phosphorylation
p110δ subunits, respectively. These p110 components heterodimerize of TSC2. TSC2 forms a heterodimeric complex with TSC1 that acts
with a regulatory subunit (p85), of which there are five isoforms and as a GTPase-activating protein (GAP) for the small GTPase Rheb,
splice variants encoded by the PIK3R1-3 genes. Together, the p110/ causing accumulation of inactive Rheb-GDP.672,673 TSC2 phosphoryla-
p85 complex functions primarily in the generation of PIP3.647,648 The tion results in suppression of this GAP activity and subsequent activation
PIK3CG gene encodes the class IB p110γ isoform, which couples and accumulation of Rheb-GTP, which then activates mTORC1.
with p101 (PIK3R5) or p87 (PIK3R6) regulatory subunits. Notably, mTORC1 serves as a central nexus for integration of multiple extracel-
p110δ/γ catalytic subunits have specialized expression patterns mainly lular signals, including oxygen and amino acid levels, growth factors,
in leukocytes, whereas p110α/β are ubiquitous.649 Analogous to the and stress. Based on these input signals, mTORC1 activity influences
activation of the Ras pathway detailed previously, the p85 regulatory cellular growth, metabolism, protein synthesis, and cell cycle progres-
subunit of PI3 kinase associates with phosphorylated tyrosine residues sion. One such example is the energy sensing mechanism of the cell
located on the intracellular domains of RTKs through an SH2 domain, comprised of LKB1 and AMPK.674 Increasing levels of AMP, a marker
resulting in allosteric activation of the p110 catalytic subunit. PI3 of decreased nutrient levels, results in AMPK phosphorylation and
kinases can also be activated indirectly through the adaptor protein activation by LKB1. AMPK phosphorylates TSC2, which, as described
Grb2 following its association with the scaffolding protein GAB1. earlier, inhibits mTORC1 activity, leading to downregulation of protein
PI3 kinase pathway activity is negatively regulated by the tumor synthesis and cell growth in response to low nutrient levels.675 In part,
suppressor PTEN (phosphatase and tensin homolog), which is a dual mTORC1 activation regulates these phenotypes via phosphorylation
lipid and protein phosphatase that dephosphorylates PIP3, converting and activation of p70S6 kinase and inhibition of 4EBP1. The former
it back to PIP2.650,651 protein stimulates mRNA translation, whereas the latter inhibits
The best characterized effectors of PI3 kinase are the three members translation of mRNA transcripts with a 5′ cap.676
of the serine/threonine kinase AKT family (AKT1, AKT2, and AKT3). Both mTORC1 and S6 kinase also participate in a negative feedback
Both AKT and phosphoinositide-dependent kinase 1 (PDK1) are loop in which both proteins activate insulin receptor substrate 1 (IRS1),
recruited by PIP3 to the plasma membrane via their PH domain, which results in inhibition of insulin-mediated PI3 kinase activation.677
where AKT is phosphorylated at Thr308 by PDK1, resulting in its AKT can also activate mTORC1 in a TSC2-independent manner via
activation.652–654 Phosphorylation at a second residue, Ser473, by mTOR phosphorylation of PRAS40, a protein that interacts with mTORC1.
complex 2 (mTORC2) further enhances AKT activity.655 Activated The mechanisms responsible for PI3 kinase pathway activation in
AKT promotes cellular proliferation, survival, and other phenotypes cancer are diverse and include activating mutations and amplification
through activation of multiple downstream effectors. Proliferative of PIK3CA, AKT1, AKT2, and AKT3, and mTOR; deletion or loss
effects are regulated through phosphorylation and inhibition of GSK3β, of PTEN expression or function; mutation in PIK3R1; loss of TSC1
which phosphorylates cyclin D1 and marks it for degradation; FOXO4, or TSC2 function; RAS mutation; and dysregulated growth factor
a transcription factor that regulates the expression of the CDK inhibitor receptor and integrin activation, as outlined later.645,648,678 In human
p27; and p21, a second CDK inhibitor that, on phosphorylation, tumors, activation of PI3 kinase is frequently a direct consequence
translocates from the nucleus to the cytoplasm, where it regulates cell of dysregulated RTK signaling secondary to mutation, amplification,
survival.656–659 In sum, these actions promote the expression of cyclin or ligand overexpression. For example, ERBB2 amplification in breast
D1, which drives progression of cells through the cell cycle and the and gastric cancer results in AKT activation.27,678 Similarly, kinase
downregulation of cyclin-dependent kinase inhibitors that inhibit cell domain mutations of EGFR induce constitutive AKT activation in
cycle progression. lung cancers and glioblastomas, and AKT activity in these tumors
44 Part I: Science and Clinical Oncology

is critical for EGFR-mediating transformation.637,679 Oncogenic Temsirolimus and everolimus, analogues of rapamycin that inhibit
Ras mutations also activate PI3 kinase, and PI3 kinase activation is the mTORC1 complex, are FDA approved for use in patients with
required for Ras-mediated tumorigenesis in genetically engineered renal cell carcinomas, and everolimus is FDA approved for the treatment
mouse models.572,680–682 of patients with pancreatic neuroendocrine tumors (see Table 2.1).705–708
Mutations in the PIK3CA gene, which encodes the p110α catalytic Everolimus also has significant clinical activity in patients with sub-
subunit, are frequently observed in tumors of the colon, breast, brain, ependymal giant cell astrocytomas that arise in the setting of tuberous
stomach, and ovary and other cancers.648,678,683 The most frequent are sclerosis, an inherited cancer-predisposition syndrome resulting from
E542K and E545K, located in the helical domain (exon 9), and germline mutations in the TSC1 and TSC2 genes.709,710 In metastatic
H1047R (exon 20), located in the kinase domain.684 All three mutants renal cell carcinoma, patients that benefited from treatment with
demonstrate increased lipid kinase activity, induce phosphorylation everolimus more commonly harbored TSC1/2 or mTOR mutations
of AKT and its downstream effectors, and can transform chicken (see Table 2.1).711
embryo fibroblasts.685 Exon 9 helical domain mutations block the A complete response to everolimus has been reported in a patient
inhibitory interaction between the p85 regulatory subunit and the with metastatic bladder cancer that harbored loss-of-function mutations
p110 subunit and result in constitutive kinase activation.686 Exon 20 in TSC1 and NF2, suggesting that such agents can induce significant
catalytic domain mutations result in constitutive kinase activation.678 antitumor responses in genetically selected patients.712 Additional
PIK3CA mutations commonly co-occur with KRAS mutations, and responses to everolimus were observed in TSC1-mutant tumors but
ERBB2 amplification in colon and breast cancers, respectively, and not to the degree of the complete responder, indicating that coaltered
expression of mutant PI3 kinase in breast cell lines as well as fibroblasts genes likely confer resistance to mTOR inhibitory therapy even in
causes neoplastic transformation.687 Unlike wild-type p110α which the setting of a canonic predictive biomarker of response. In sum,
lacks oncogenic potential, wild-type overexpression of the other three mTOR inhibitors are likely most effective when used in genetically
isoforms can induce transformation of cultured cells.688 PIK3R1 encodes defined patient populations, with the majority of patients requiring
the p85 regulatory subunit of PI3 kinase. Alterations in within this combination therapies because of the presence of coaltered genes. As
gene have also been reported in multiple cancers, including glioblas- an example of the latter, everolimus in combination with exemestane
tomas and colorectal, endometrial, and ovarian cancers.585,689 A recurrent was FDA approved for the treatment of advanced hormone receptor–
PIK3R1 mutation, PIK3R1(R348∗), has been shown to have neo- positive, HER2− breast cancer.713
morphic functions resulting in activation of MEK and JNK and Resistance to mTOR inhibition is thought to derive from multiple
sensitivity to inhibitors of these effector kinases.690 Furthermore, partial mechanisms, including activation of parallel signaling networks such
loss of the PIK3R1 gene product p85α was demonstrated to increase as the MAP kinase pathway.714 Furthermore, inhibiting mTORC1
the proportion of p110α/p85 heterodimers bound to active receptors, can preferentially lead to an increase in mTORC2 signaling, and, as
indicating that targeting p110α-selective inhibitors may be effective described earlier, mTORC2 enhances AKT activation via phosphoryla-
in the setting of PIK3R1 loss.691 tion of the serine 473 residue.715 As an alternative approach, potent
Loss of PTEN function is the most frequently observed PI3 kinase/ mTOR kinase inhibitors are being developed, including RapaLink-1,
AKT pathway alteration in human malignancies and is common in and have shown significant preclinical activity in cell lines resistant
tumors of the prostate, breast, ovary, lung, colon, and bladder as well to rapamycin and in in vivo models of glioblastoma.716,717
as melanomas and glioblastomas.678 Loss of PTEN function in tumors
is mediated by a diversity of mechanisms including mutation, deletion, TRANSLATIONAL IMPLICATIONS
posttranslational modification, and promoter hypermethylation.678
Dysregulated expression of microRNAs that target the 3′-untranslated As outlined earlier, mutational and epigenetic alterations induce
region of PTEN has also been shown to induce cell survival and constitutive activation of a broad array of signaling pathways in human
cisplatin resistance.692 tumors. In some instances, the growth and survival of tumor cells
As AKT activation enhances proliferation and suppresses apoptosis, have been shown to be dependent on a single signaling pathway
its activation would be predicted to have strong oncogenic func- activated by a mutated oncogene or tumor suppressor, a phenomenon
tion. Indeed, AKT was initially identified as a proto-oncogene in referred to as oncogene addiction.718 In such instances, targeted inhibitors
the mouse leukemia virus AKT8.693 A recurring hot spot mutation of such pathways have demonstrated unprecedented clinical activity
in the PH-domain of AKT1 (E17K) occurs with low frequency in in molecularly defined subsets of patients (see Table 2.1). Examples
breast, colorectal, bladder, endometrial, and ovarian cancers.694–696 include imatinib in patients with CML, erlotinib in patients with
This mutation results in constitutive localization to the plasma EGFR-mutant NSCLC, and vemurafenib in patients with BRAF-mutant
membrane without the need for PIP3 recruitment.684 Amplification melanoma.30,505,511,603,612,719 Despite these dramatic successes, the majority
of the AKT2 gene has been reported in ovarian and pancreatic cancers, of cancer patients have yet to benefit from this approach. Potential
and gain-of-function AKT3 mutations have been reported to occur in explanations for this lack of benefit include the redundant regulation
melanoma.697 of key downstream mediators of transformation by multiple signaling
Given the significant proportion of malignancies that harbor muta- pathways, the lack of specificity of the drug for the driver alteration,
tions in the PI3 kinase/AKT/mTOR pathway, a concerted effort is and intrinsic and acquired drug resistance due to second site mutations
ongoing to identify selective inhibitors of various PI3 kinase pathway in the target gene or by other mechanisms. Progress in this field has
components. These agents can be categorized as pan-selective or selec- also been delayed by the continued practice of performing clinical
tive PI3 kinase inhibitors, dual PI3 kinase/mTOR kinase inhibitors, trials of targeted inhibitors in unselected patient populations.
AKT inhibitors, and mTOR inhibitors. Both isoform-specific and Recent advances in sequencing methodology have made it feasible
pan-selective inhibitors of PI3 kinase are being explored in several to now prospectively sequence all patients with advanced cancer with
clinical trials across cancers. Most notably, the PI3Kδ inhibitor idelalisib the goal of identifying potentially “actionable” genomic alterations.720
is FDA approved for non-Hodgkin lymphoma and certain types of Such prospective sequencing efforts have highlighted several challenges
leukemia.698,699 Promising clinical responses have also been reported that have slowed the application of targeted inhibitors in cancer patients.
with α-selective PI3 kinase inhibitors in patients with several cancer As one example, most mutations, even in well-characterized cancer
types, most notably breast cancer (see Table 2.1).700–703 Although clinical genes, are likely inert passenger mutations. Furthermore, not all
activity with AKT inhibitors has been modest in patients, a pan-cancer activating mutations respond similarly to targeted inhibitors. For
basket trial of the ATP-competitive pan-AKT kinase inhibitor AZD5363 example, exon 19 EGFR deletions are sensitive to the EGFR kinase
demonstrated significant clinical responses in patients with AKT1 E17K inhibitor erlotinib, whereas exon 20 insertions are intrinsically
mutant tumors.704 resistant.721,722 Given the large number of somatic mutations present
Intracellular Signaling  •  CHAPTER 2 45

in each human tumor, and the variable biologic and clinical significance respond to vemurafenib, whereas colorectal tumors with the same
of individual mutant alleles, there is an urgent need for clinical support mutation are intrinsically resistant. Resistance in the latter results from
tools that will aid clinicians in interpreting molecular tumor profiling rapid adaptation of the cancer cell resulting in activation of EGFR.724
and guiding treatment selection. This phenomenon of adaptive resistance, defined as a rapid reactivation
A second challenge is that many oncogenic alterations are rare. To of parallel signaling pathways after relief of negative feedback signals,
address this challenge, novel clinical trial designs have been formulated likely abrogates the effects of selective pathway inhibitors in many
to test the efficacy of targeted inhibitors in patients with defined cancer types.592,725,726 Because significant drug development efforts are
molecular events independent of the primary site of disease. Eligibility currently focused on the development of targeted inhibitors of
for these “basket” studies is based on the presence of a particular oncogene-activated signaling pathways, a detailed understanding of
molecular alteration (e.g., BRAF V600E, AKT1 E17K, or an NTRK normal physiologic pathways and their dysregulation in cancer will
fusion) rather than site of tumor origin.268,273,704,723 Although promising be critical for the optimal development and clinical application of
results from such trials highlight the importance of tumor mutational targeted kinase inhibitors.
profile in dictating drug sensitivity, lineage-specific differences also
play a role in determining clinical response to inhibitors of activated The complete reference list is available online at
signaling pathways. As an example, most BRAF V600E melanomas ExpertConsult.com.

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implications of targeting AKT signaling in mela- subependymal giant cell astrocytomas associated with 724. Prahallad A, et al. Unresponsiveness of colon cancer
noma. Enzyme Res. 2011;2011:327923. tuberous sclerosis complex (EXIST-1): a multicentre, to BRAF(V600E) inhibition through feedback
698. Rosenthal A. Small molecule inhibitors in chronic randomised, placebo-controlled phase 3 trial. Lancet. activation of EGFR. Nature. 2012;483:100–103.
lymphocytic lymphoma and B cell non-Hodgkin 2013;381:125–132. 725. Camidge DR, Pao W, Sequist LV. Acquired resistance
lymphoma. Curr Hematol Malig Rep. 2017;12: 711. Kwiatkowski DJ, et al. Mutations in TSC1, TSC2, to TKIs in solid tumours: learning from lung cancer.
207–216. and MTOR are associated with response to rapalogs Nat Rev Clin Oncol. 2014;11:473–481.
699. Miller BW, et al. FDA approval: idelalisib mono- in patients with metastatic renal cell carcinoma. Clin 726. Holohan C, Van Schaeybroeck S, Longley DB,
therapy for the treatment of patients with follicular Cancer Res. 2016;22:2445–2452. Johnston PG. Cancer drug resistance: an evolv-
lymphoma and small lymphocytic lymphoma. Clin 712. Iyer G, et al. Genome sequencing identifies a basis ing paradigm. Nat Rev Cancer. 2013;13:714–
Cancer Res. 2015;21:1525–1529. for everolimus sensitivity. Science. 2012;338:221. 726.
46.e12 Part I: Science and Clinical Oncology

SELF-ASSESSMENT REVIEW QUESTIONS ANSWERS


1. Next-generation sequencing has helped reveal numerous unusual 1. (b) PTEN is a tumor suppressor of the AKT pathway and
genomic events, including chromosomal translocations. Which would not be a likely candidate fusion partner, because
of the following genes is NOT found as oncogenic fusion truncating mutations or deletions confer loss of PTEN function.
partners in chromosomal re-arrangements? All of the other genes listed have been found in patients with a
a. ABL variety of fusions partners. BCR-ABL, the Philadelphia
b. PTEN chromosome, which is found in nearly all cases of chronic
c. PDGFRβ myelogenous leukemias (CML) and one-third of ALLs, is
d. ALK the classic example of an oncogenic fusion driving cancer.
e. NTRK EML4-ALK fusions play a driving role in non–small cell lung
2. Which of the following is true of Hedgehog (SHH) signaling cancer (NSCLC). COL1A1-PDGFRβ fusions occur in
and targeted therapies? dermatofibrosarcoma protruberans, a rare sarcoma.
a. Secreted hedgehog ligands bind Smoothened receptors, which TPM3-TRKA fusions were first identified in colon cancer.
causes receptor dimerization and autophosphorylation of 2. (c)  The Smoothened receptor is a GPCR, not a receptor
intracellular tyrosine residues. tyrosine kinase, and therefore it does not get activated by
b. Erlotinib and gefitinib are FDA approved Smoothened dimerization and transphosphorylation. Vismodegib and
receptor inhibitors. sonidegib are FDA-approved smoothened receptor inhibitors.
c. Sonic hedgehog (SHH) binds to the receptor Patched Mutations in SHH, PTCH, and SMO are found in patients
(PTCH), which relieves its repression of Smoothened with inherited and sporadic basal cell carcinomas. Smoothened
(SMO). receptors couple to the G proteins Gαi and Gα12.
d. Mutations in SHH, PTCH, and SMO are found in patients 3. (d)  No direct inhibitors of Ras are available for clinical use.
with small cell lung cancer. Farnesyltransferase inhibitors were tested but were found to be
e. Smoothened receptors couple directly to actin. ineffective in tumors in which Ras mutations are common,
3. The Ras/MAP kinase and PI3 kinase/mTOR signaling cascades likely because of the ability of geranylgeranyl modifications to
are parallel but interconnected pathways that are frequently substitute for farnesylation in effectively targeting Ras to the
mutated in human cancer. Which of the following has NOT membrane. New drug development of allosteric inhibitors that
been an effective approach in targeting these pathways? specifically target KRAS G12C is underway. Trametinib is an
a. Allosteric inhibitors of MEK kinase FDA-approved allosteric inhibitor of MEK. Rapamycin and its
b. Small-molecule inhibitors and kinase of mTOR analogues, as well as novel kinase inhibitors such as RapaLink,
c. Monoclonal antibodies that bind to EGFR are efficacious mTOR inhibitors. Cetuximab and panitumumab
d. Farnesyltransferase inhibitors of Ras are anti-EGFR monoclonal antibody therapies. Vemurafenib,
e. Kinase inhibitors of mutant BRAF an ATP-competitive inhibitor of BRAF, is approved for the
4. Acquired resistance commonly occurs after treatment with treatment of patients with BRAF V600E mutant melanoma.
targeted inhibitors. Which of the following are known 4. (e)  Several mechanisms of resistance to kinase inhibitors have
mechanisms of resistance? emerged. For example, patients with BRAF V600E mutations
a. Parallel pathway upregulation that are treated with the specific RAF inhibitor vemurafenib
b. Gatekeeper mutation often develop resistance mediated by BRAF V600E splice
c. Amplification of specific RTKs variants or amplification; loss or mutation of NF1; parallel
d. Alternative splicing or amplification of the driver oncogene pathway activation of RTKs; mutation of PI3K/AKT
e. All of the above components; mutations in NRAS, RAF1 and MAP2K1/2
5. Which of the following statements is FALSE? (MEK1/2); and amplification of MITF.
a. Chromosomal rearrangement of BCR and ALK to generate 5. (a)  Translocation of the ABL gene on chromosome 9 with the
the Philadelphia chromosome is the driving event in CML. breakpoint cluster region (BCR) gene on chromosome 22 results
b. Recurrent somatic splice site alterations involving MET exon in the expression of a BCR-ABL fusion protein, called the
14 (METex14) have been identified in lung cancer. Philadelphia chromosome. This event is the pathognomonic
c. Integrin receptors perform inside-out and outside-in signaling molecular lesion in almost all chronic myelogenous leukemias
at the integrin adhesome. (CML). It is also found in approximately one-third of acute
d. ESR1 mutations are a common mechanism of acquired lymphoblastic leukemias (ALLs). The ABL tyrosine kinase
resistance to hormonal therapy in patients with breast cancer. inhibitor imatinib, as well as four other ABL inhibitors, are
e. Extracellular and transmembrane FGFR3 mutations are FDA approved in CML; imatinib and dasatinib are approved
recurrent in noninvasive and high-grade invasive bladder for ALL.
cancer.
Cellular Microenvironment and Metastases 3 
Erinn B. Rankin and Amato J. Giaccia

S UMMARY OF K EY P OI NT S
• Metastases are responsible for more the distant tissue; dormancy; and blood flow and by tissue-specific
than 90% of all cancer-related reactivation and proliferation in the factors.
deaths. new tissue microenvironment. • Primary tumors possess stem cells
• Gene dysregulation, the tumor • Colonization of metastatic tumor that can recapitulate the tumor from
microenvironment, and host cells cells requires the ability to a single cell, and a subset of these
drive the metastatic spread of tumor metabolically adapt, develop cancer stem cells may inherently
cells. angiogenesis, overcome dormancy, possess altered gene expression
• Metastasis can be subdivided into and proliferate in a foreign tissue. changes with increased metastatic
invasion and migration from the • The formation of a premetastatic potential.
primary tumor; intravasation into niche is essential for the growth of • Antimetastatic therapy will likely
the vasculature; dissemination extravasating metastatic tumor require the targeted inhibition of
and survival in the circulation; cells. many pathways that control
extravasation from the vasculature; • Organ specificity of tumor proliferation, invasion, angiogenesis,
survival and metabolic adaptation in metastases is determined both by and immune evasion.

One of the most important challenges in clinical oncology is the the tumor microenvironment such as hypoxia and extracellular vesicles
prevention and treatment of metastatic disease. With advances in promote metastatic phenotypes within these cell types.
surgical techniques and conventional and targeted therapies, localized
disease is effectively managed in the clinic. However, metastatic disease Cancer Stem Cells
is the primary cause of cancer-related deaths. Tumor metastasis involves
tumor cell invasion and migration from the primary tumor, intravasation The traditional view of tumors with a relatively homogeneous
into the vasculature, dissemination and survival in the circulation, population of tumorigenic cells has been significantly revised with
extravasation into distant tissues, survival and metabolic adaptation the isolation and characterization of cancer stem cells. Stem cells are
in the distant tissue, dormancy, reactivation, and overt colonization primal cells that retain the ability to renew themselves through cell
to form a new macroscopic tumor at a distant site.1 This process is division and can differentiate into a wide range of specialized cell
highly inefficient; it has been estimated that less than 0.01% of tumor types. Cells with stem cell properties have been identified in a variety
cells that enter the circulation develop into metastases. Despite this of solid tumors including colon, breast, head and neck, and pancreatic
inefficiency, metastases are responsible for more than 90% of all tumors, glioblastomas, medulloblastomas, and melanoma.3 This rare
cancer-related deaths. Understanding the biology and vulnerabilities subpopulation of tumor cells exhibits enhanced tumor-initiating
of metastatic tumor cells is of critical importance to improve overall potential: the ability to self-renew and differentiate into multiple
survival rates in cancer patients. With little evidence to support cell types.
mutations in “metastasis genes” as drivers of metastasis, current data The origin of cancer stem cells remains unclear and may differ
suggest that microenvironmental factors as well as epigenetic changes among tumor types. It has been hypothesized that cancer stem cells
may play key roles in metastasis. This chapter describes the cellular arise from either transformed resident stem and/or progenitor cells,
and molecular traits driving tumor metastasis and addresses how the or may represent a dedifferentiated epithelial tumor cell. In intestinal
tumor microenvironment influences this process. Most important, it cancer, mouse modeling studies have identified intestinal stem cells as
discusses how this knowledge can be translated into current and future the cells of origin. For example, intestinal stem cell–specific deletion
cancer therapies. of adenomatous polyposis coli (APC) leads to rapid cellular transforma-
tion with uncontrolled cellular growth and solid tumor formation.
TUMOR MICROENVIRONMENT AND In contrast, deletion of APC in short-lived transit-amplifying cells
METASTASIS was not sufficient to induce long-term tumor growth.4 Stem cell
properties can also be acquired by tumors cells through epithelial-
Although cellular intrinsic traits acquired by tumor cells are required mesenchymal transition (EMT). Induction of EMT in immortalized
for successful metastatic colonization, cellular and molecular factors human mammary epithelial cells was sufficient to induce the expression
within the tumor microenvironment significantly contribute to meta- of stem cell markers, enhance self-renewal, and increase the number
static progression. The tumor microenvironment contains a number of tumor-initiating cells.5 Although the molecular mechanisms that
of cell types that promote tumor progression, including cancer stem drive the cancer stem cell phenotype in tumor cells remains largely
cells, angiogenic vascular cells, infiltrating immune cells, and cancer- unknown, a study showed that the transcription factors Slug and
associated fibroblasts (CAFs).2 In addition, extracellular factors within Sox9 drive EMT and the cancer stem cell phenotype in breast cancer

47
48 Part I: Science and Clinical Oncology

cells.6 Future studies are eagerly awaited to further delineate the


intracellular signaling pathways driving the cancer stem cell phenotype I, TSP-1
ICAM IL-3
ctin, 3, C
in vivo. sel
e D2
, P- io n 48
The role of cancer stem cells in multistage tumor progression, B3 sat n Intr
KF ava atio ava
particularly with respect to metastasis, is poorly defined. Given that PF Intr avas cy sat
tr n ion
metastasis is an infrequent event that is achieved by only a small Ex orma
D
portion of cancer cells that reach distant sites, it has been hypothesized

GF
that cancer stem cells may be responsible for metastatic disease. Evidence Endothelial Pericyte

s, VEGF, CCLI8, E

MMP
to support this hypothesis has been generated in models of breast and

Extravasation

Premetastatic nic
Intravasation
pancreatic cancer. In the MMTV-PyMT model of breast cancer,

s, ALOX5, HGF, T
Invasion

Intravasation
Malanchi and colleagues observed that only the CD90+ CD24+

Invasion
population of cancer stem cells isolated from primary breast tumors Macrophage TME Neutrophil

contained cells with the ability to form metastases in the lung when

MMP
introduced into the tail vein.7 Furthermore, this study identified the

he
extracellular matrix protein, periostin, produced by fibroblasts and Platelet Fibroblast

GF
stromal tumor cells, as a critical factor to maintain the cancer stem on

-B
lati Pre
me
ircu on
cell phenotype and metastatic potential in these cells.7 In human n c
al i sat
i Intr tasta
tic
breast cancers, single-cell analysis of early-stage metastatic cells
av
rviv ava n rin
s CX Inv asati niche
Su Extr vasio asi on
demonstrated that these samples express distinct stemlike, EMT, eg
In t CL on
,in I2,
tin
prosurvival, and dormancy-associated gene expression signatures
GM
lec -CS
-se F, P
compared with metastatic cells from high-burden tissues.8 Moreover,
, P
TGF-B OSTN

transplanted stemlike metastatic cells from low-burden tissues exhibited


tumor-initiating capacity and were able to differentiate into luminal-like Figure 3.1  •  Multiple cellular components of the tumor microenvironment
cancer cells.8 Similarly in pancreatic cancer, lineage-tracing studies (TME) contribute to metastasis. Key cell types and mechanisms of action are
showed that circulating pancreatic tumor cells exhibit EMT and cancer shown.
stem cell properties, and initiate tumor formation. A critical role for
inflammation to maintain the ability of cancer stem cells to metastasize
was observed in this study.9 In addition, the extracellular protein
tenascin C (TNC), expressed by stem cell niches and cancer cells, Adhesion to the endothelium is an initial step in extravasation
was also found to promote stem cell signaling and lung metastases in that is followed by transendothelial cell migration.13 Tumor cell adhesion
breast cancer cells.10 Collectively, these data strongly implicate the to the endothelium requires the expression of cognate ligands and
tumor microenvironment in promoting the cancer stem cell phenotype receptors by cancer cells and endothelial cells.13 A variety of ligand-
and the initiation of tumor metastasis. receptor interactions have been shown to contribute to tumor
extravasation, including interactions with selectins, integrins, cadherins,
Angiogenic Vascular Cells CD44, and immunoglobulin (Ig) superfamily receptors (for a review,
see Reymond and colleagues13). For example, endothelial cell P- and
Tumor angiogenesis facilitates the hematogenous spread of metastatic E-selectins bind to tumor cells through cell-cell adhesion molecules
tumors. The aberrant production of proangiogenic factors by tumor such as integrins and CD44.14–19 Studies have suggested a role for the
cells results in malformed and irregular tumor blood vessels that often immunosuppressive cytokine interleukin (IL)-35 in facilitating cancer
contain breaks in their lining that facilitate tumor cell intravasation cell adhesion to endothelial cells. IL-35 was found to be highly expressed
and dissemination. Endothelial cells and pericytes are important by human pancreatic cancer cells in which it promoted ICAM1
structural and functional components of the tumor and distant tissue expression to facilitate endothelial cell adhesion and transendothelial
vasculature, and both have been considered as potential targets in migration via an ICAM1-fibrogen-ICAM1 bridge.20
metastatic therapy.11 Finally, endothelial cells within in the perivascular niche of the
bone marrow have been implicated in promoting tumor cell dormancy.
Endothelial Cells Similar to hematopoietic stem cells, dormant cancer cells have been
Endothelial cells play an active role in tumor cell intravasation, found to be localized within perivascular niches of the bone marrow
extravasation, and dormancy (Fig. 3.1). Studies have shown that microenvironment.21 Within stable microvasculature niches, endothelial
metabolic reprogramming of tumor endothelial cells toward a glycolytic cells promote tumor cell quiescence through the production of
phenotype contributes to the early stages of metastasis by promoting thrombospondin-1 (TSP1)21. In contrast, endothelial cells isolated
an abnormal tumor vasculature, tumor intravasation, and dissemination. from sprouting neovasculature promote tumor proliferation through
Cantelmo and colleagues performed mRNA sequencing analysis of the production of tumor-promoting factors including transforming
tumor-associated endothelial cells and discovered that tumor endothelial growth factor–β1 (TGF-β1) and periostin from sprouting endothelial
cells expressed a hyperglycolytic signature compared with normal cell tips.21
endothelial cells.12 The glycolytic phenotype within tumor-associated
endothelial cells could be inhibited through genetic and pharmacologic Pericytes
inhibition of the glycolytic enzyme 6-phosphofructo-2-kinase/fructose- Similar to endothelial cells, pericytes are thought to play a protumori-
2,6-bisphosphatase-3 (PFKFB3). Moreover, PFKFB3 inhibition in genic role by supporting blood vessel maturation and function (see
endothelial cells was sufficient to normalize the tumor vasculature Fig. 3.1). Pericytes promote endothelial cell survival and stabilize the
and significantly reduce tumor cell intravasation and metastasis.12 tumor vasculature.22 In addition, pericytes can directly facilitate distant
There are multiple mechanisms by which PFKFB3 inhibition in metastasis by promoting tumor cell intravasation through the upregula-
endothelial cells may have impaired metastatic dissemination, including tion of the transmembrane receptor endosialin (CD248).23 Studies
enhancing the integrity of the endothelial cell barrier, improving vessel have demonstrated that pericytes can also promote tumor cell intravasa-
maturation and pericyte coverage, and inhibiting cancer cell adhesion tion and metastasis through the IL-33–dependent recruitment of
molecules. These findings suggest that targeting glucose metabolism macrophages.24 Therefore it has been proposed that targeting pericytes
in tumor endothelial cells may offer therapeutic benefit for anticancer alone or in combination with endothelial cells may be an effective
therapy. strategy in the treatment of cancer.24,25 However, clinical data indicate
Cellular Microenvironment and Metastases  •  CHAPTER 3 49

that low pericyte coverage is associated with metastasis and poor circulating tumor cells within DNA meshes at distant tissues, (2)
prognosis in a number of cancers.25 A recent study demonstrated that promoting vascular permeability and the premetastatic niche, and (3)
depletion of pericytes suppressed tumor growth and enhanced metastasis stimulating tumor cell invasion and proliferation at distant tissue
of murine models of breast cancer.25 Enhanced metastasis was associated sites.36–38 Important to note, digesting NETs with DNase I is sufficient
with increased levels of tumor hypoxia, EMT, and Met receptor to reduce metastasis in multiple metastatic tumor models, suggesting
activation.25 These findings indicate that pericyte coverage may be that therapeutic targeting of NETs may prevent metastasis.36,37
important to stabilize the tumor vasculature and prevent the hypoxic
selection of aggressive tumor cells (see later discussion of hypoxia and Platelets
metastasis). These findings raise concerns when considering antipericyte Platelets are the second most abundant cell type in the blood and
treatments in cancer therapy, especially in the setting of metastasis. play an important role in hemostasis, thrombosis, inflammation, and
vascular biology.39 Platelets are among the first cell types that interact
Immune Cells with cancer cells in the circulation.20 Tumor cells release platelet
coagulation factors such as thrombin to promote platelet aggregation
Most solid tumors contain immune infiltrates derived from both the around circulating tumor cells. Indeed, increased blood coagulation
myeloid and lymphoid lineages that can have both positive and negative is often observed in cancer patients as a result of elevated levels of
effects on metastasis.2 This section focuses on immune cells that thromboplastin, procoagulant A, and phosphatidylserine produced
stimulate metastatic progression. by tumor cells.40,41 Platelets facilitate metastasis through multiple
mechanisms (see Fig. 3.1). Platelets are thought to form a physical
Macrophages barrier around disseminated cancer cells, protecting them from shear
Studies have linked a unique subset of macrophages, termed tumor- stress and lysis mediated by natural killer cells.42 In addition, platelets
associated macrophages (TAMs), to tumor progression and metastasis. can facilitate tumor cell extravasation by enhancing tumor cell adhesion
Clinically, the presence of excessive macrophages is associated with to endothelial cells and by promoting endothelial cell permeability.43–46
poor prognosis in the majority of patients with solid tumors including Finally, platelets have been shown to directly stimulate cancer cell
breast, prostate, cervix, bladder, endometrial, and kidney cancer.26–28 EMT and invasion through the release of TGF-β.47 Therapeutic
TAMs express a variety of growth factors and cytokines that promote inhibition of platelet binding to cancer cells through the disruption
tumor angiogenesis, invasion, intravasation, metastasis, and immune of α6β1 integrin/ADAM9 receptor may be an effective therapeutic
suppression (see Fig. 3.1)28,29. For example, macrophages within the strategy to target platelet-mediated metastasis in vivo.48
primary tumor contribute to tumor cell invasion and extravasation
by releasing a variety of proteases including serine, cysteine, and Fibroblasts and Mesenchymal Stem Cells
metalloproteases that remodel the extracellular matrix to enhance
tumor cell invasion and migration (for a review, see Cho and col- Accumulating evidence supports a critical role for CAFs in tumor
leagues2). Perivascular macrophages are thought to play a particularly progression and metastasis (see Fig. 3.1). Clinically it was observed
important role in tumor cell intravasation by promoting vascular that epigenetic changes and mutations commonly found in cancer
permeability. In preclinical models of breast cancer, genetic ablation cells, such as p53 and PTEN mutations, can also be found in cancer-
of vascular endothelial growth factor (VEGF) from macrophages was associated stroma.49,50 These studies provided early evidence to suggest
sufficient to reduce transient vascular permeability, leading to reduced that alterations in the stroma may contribute to tumor progression.
numbers of circulating tumor cells.30 Moreover, macrophages can Gene expression profiling studies of CAFs in human specimens and
promote tumor cell invasion through the release of chemokines and murine tumor models revealed an “activated” proinflammatory gene
cytokines including CCL18 and epidermal growth factor (EGF; for signature.51 Among the cytokines produced by CAFs, the chemokine
a review, see Ruffell and colleagues28). CXCL12 is of particular interest, given its role in driving tumor cell
Many questions remain regarding the factors that control the biologic migration and recruitment of endothelial progenitor cells expressing
activities of TAMs within the tumor microenvironment. TAMs localize the CXCR4 receptor. Consistent with these data, an important role
to regions of hypoxia, and thus it has been hypothesized that the for CAFs in promoting tumor invasion has been observed in several
hypoxic microenvironment may influence the unique characteristics murine models of cancer.51 In breast cancer, Hu and colleagues identified
and gene expression profiles of TAMs. Indeed, TAMs express high a role for CAFs in the transition of mammary ductal carcinoma in
levels of the hypoxia inducible transcription factor (HIF) HIF-2, which situ (DCIS) to invasive carcinoma. Coinjection of fibroblasts with
has been found to be an independent prognostic factor of outcome.31 DCIS cells resulted in invasive carcinomas, whereas injection of DCIS
When exposed to hypoxia, macrophages upregulate the expression of cells alone was only sufficient to induce mammary DCIS.52 CAFs
mitogenic and proangiogenic cytokines.32 Experimentally, deletion of promote tumor cell invasion by establishing invasion-permissive tracks
myeloid HIF-2 reduced TAM infiltration into tumors and impeded in the extracellular matrix through the secretion of factors that induce
tumor proliferation and growth.32 Thus, targeting TAMs and CD11b matrix remodeling.51,53 CAFs can also support tumor cell survival and
cells may be a viable mechanism for antimetastatic therapies. migration through the release of extracellular vesicles carrying annexin
A6/LDL receptor–related protein/thrombospondin 1 (ANXA6/LRP1/
Neutrophils TSP1) complexes.54 In addition to promotion of tumor invasion,
Neutrophils represent 50% to 75% of the total circulating leukocytes supportive roles for CAFs in the maintenance of cancer stem cell
in blood. They play an important role in inflammation and are early signaling and the establishment of the premetastatic niche have been
responders to pathogens including bacteria, fungi, and viruses. found.51 Further characterization of the distinct subsets of CAFs relevant
Neutrophils mediate direct antimicrobial activities through the release to metastasis in various tumor types is needed. In addition, further
of enzymes and toxic factors, the generation of reactive oxygen species, elucidation of the interplay between tumors and the stroma is warranted
and the release of nuclear material into extracellular traps called and may reveal novel strategies in the treatment of metastatic disease.
neutrophil extracellular traps (NETs).33 Studies have suggested an important role for a subset of CAFs,
Although neutrophils can inhibit the metastatic seeding of dis- mesenchymal stem cells (MSCs), in tumor progression and metastasis.
seminated tumor cells under some conditions, accumulating evidence Although MSCs are commonly isolated in CAF preparations, MSCs
suggests a prometastatic role for neutrophils (see Fig. 3.1).34–37 Study are functionally distinct in that they are able to undergo multipotent
findings have suggested that that metastatic cancer cells promote the differentiation. MSCs have been identified as important components
formation of NETs through the release of G-CSF.37 NETs may promote of the tumor stroma that promote the progression of ovarian, colon,
metastasis through multiple mechanisms including (1) trapping and pancreatic cancers.55–57 Cancer-associated MSCs have recently
50 Part I: Science and Clinical Oncology

been shown to promote pancreatic cancer cell proliferation, inva- exosome content and shedding. The shedding of exosomes from tumor
sion, and transendothelial cell migration through the production cells is increased under hypoxic conditions. In breast cancer cells,
of the cytokine granulocyte-macrophage colony-stimulating factor hypoxia promotes microvesicle shedding through the HIF-dependent
(GM-CSF).55 These findings suggest that inhibition of tumor- regulation of the small GTPase RAB22A, a protein that is localized
MSC cross talk may be a therapeutic strategy for the treatment of to budding microvesicles and promotes metastasis.74 Hypoxia also
pancreatic cancer. plays an important role in regulating exosome content and function.
Clinically, the levels of hypoxia-regulated proteins in plasma exosomes
Extracellular Vesicles are significantly higher in glioblastoma multiforme (GBM) patients
compared with healthy control patients.75 Moreover, exosomes derived
Extracellular vesicles are emerging as key factors that promote metastasis. from hypoxic GBM cells are potent inducers of angiogenesis compared
Studies have shown that tumor and stromal cells secrete small vesicles with exosomes isolated from normoxic GBM cells.75 Given that
that contain a variety of bioactive molecules such as proteins, lipids, extracellular vesicles can be detected in patient biologic fluids and
RNA, and DNA that can promote intercellular signaling and tumor contribute to cancer progression, there is great interest in use of
progression. Extracellular vesicles can be derived from either the exosomes as diagnostic biomarkers and therapeutic targets in cancer.
endosome (exosome) or the plasma membrane (microvesicle) to mediate
intercellular signaling with tumor and stromal cells within the local Hypoxia
and distant microenvironments. Clinically, extracellular vesicles and
their cargo have been associated with tumor progression. The concentra- Hypoxia is a potent microenvironmental factor driving metastatic
tions of exosomes have been found to be increased in the peripheral tumor progression. Clinically, hypoxia is associated with metastasis
blood of patients with ovarian, breast, and pancreatic cancers compared and poor survival in variety of cancer patients.76–79 Hypoxia selects
with healthy control patients (for a review, see Becker and colleagues58). for cells with low apoptotic potential and increases genomic instability,
Specific nucleic acid and protein expression signatures within exosomes allowing for rapid mutational adaptations.80–82 In addition, hypoxia
have also been associated with tumor progression and metastasis. For directly increases the expression of genes involved in glycolysis,
example, exosomal miR-141 expression in the serum of prostate cancer angiogenesis, cell survival, invasion, immune suppression, the cancer
patients is significantly higher in metastatic prostate patients compared stem cell phenotype, and metastasis. All of these changes allow cells
with patients with localized disease.59 An exosomal protein signature to adapt to oxygen-deprived conditions and permit cells to escape
containing the melanoma-specific protein tyrosinase-related protein-2 these conditions by establishing new blood supplies or by physically
(TYRP2), very late antigen 4 (VLA-4), HSP-70, and the MET moving from an oxygen-poor environment to an oxygen-rich
oncoprotein has been associated with metastasis in melanoma patients.60 environment.
In pancreatic cancer, the level of circulating glypican-1–positive The primary molecular mediators of hypoxic signaling are HIF-1
exosomes correlates with poor patient survival.61 and HIF-2. In the presence of oxygen, the alpha subunits of HIF-1
Functionally, extracellular vesicles have been shown to play an and HIF-2 are rapidly degraded through the cooperative actions of
important role in the tumor microenvironment and promote metastasis prolyl hydroxylase (PHD) enzymes PHD1, PHD2, and PHD3, and
through a variety of mechanisms including tumor immune suppression, the E3 ubiquitin ligase substrate recognition component VHL.83,84
invasion, angiogenesis, and the premetastatic niche (for a review, see Under hypoxia, HIF-1 and HIF-2 are stabilized and activate the
Kalluri and colleagues62). For example, melanoma cells promote immune expression of genes containing hypoxia response elements (HREs).76
evasion through the release of FasL-bearing microvesicles that trigger Over 200 genes that allow cells to survive and adapt to low oxygen
Fas-dependent apoptosis of lymphoid cells.63 Tumor-derived exosomes tensions are activated in response to HIF-1 and HIF-2.
can also directly promote the immunosuppressive phenotype of Consistent with the association between hypoxia and metastasis,
myeloid-derived suppressor cells through the activation of STAT3 HIF-1 and HIF-2 are highly expressed in primary tumors and
signaling in these cells.64 Tumor-derived exosomes promote an invasive metastases. Immunohistochemical analysis of human cancer specimens
phenotype by activating stromal cells to produce matrix metallo­ indicates that the majority of primary tumors and metastases have
proteinases (MMPs) such as MMP1.65 In addition, tumor-derived increased HIF-1 and/or HIF-2 protein compared with the normal
extracellular vesicles can directly promote the invasive capacity of adjacent tissue.76–78 Moreover, increased HIF expression is often
nonmetastatic cells at local and distant sites by transferring mRNAs associated with increased patient mortality.85 Experimentally, overexpres-
involved in migration and metastasis.66 Conversely, stromal microvesicles sion of HIF in tumor cells promotes metastasis,86 whereas inactivation
have also been shown to promote tumor cell invasion and metastasis. of HIF significantly decreases the metastatic potential of metastatic
Fibroblast-secreted exosomes promote breast cancer cell migration tumor cells.87–89 These clinical and experimental findings indicate an
through the activation of planar cell polarity (PCP) signaling.67 important role for HIF in metastatic tumor progression.
Astrocyte-mediated transfer of exosomal PTEN-targeting microRNAs HIFs influence multiple steps within the metastatic cascade
leads to PTEN loss in brain metastatic tumor cells that can support (Fig. 3.2). HIF-1 and HIF-2 activate the early stages of metastasis in
metastatic outgrowth through mechanisms involving the increased the primary tumor by promoting EMT, the cancer stem cell phenotype,
expression of the chemokine CCL2 and adaptive metastatic outgrowth.68 invasion, and migration (for a review, see Rankin and Giaccia79). HIFs
Exosome-mediated transfer of miR-105 by breast cancer cells promotes promote invasion and migration through multiple mechanisms. First,
vascular leakiness and metastasis by disrupting endothelial cell tight HIF regulates the E- to N-cadherin switch during EMT phenotype
junctions.69 Tumor-derived exosomes may also play a key role in through the direct activation of E-cadherin repressors including Twist1,
establishing a premetastatic niche at distant sites by activating signaling Zeb1/2, Snail.86,90,91 HIF signaling has also been shown to indirectly
within organ-specific cells to recruit bone marrow–derived macrophages, promote EMT through the activation of Notch, TGF-β, integrin-linked
activate Src phosphorylation and proinflammatory S100 gene expression, kinase (ILK), and the receptor tyrosine kinase AXL (for a review, see
and increase nutrient availability.70–72 Rankin and Giaccia79). Second, HIF directly upregulates the expression
The factors that regulate extracellular vesicle formation and content of multiple factors driving cellular invasion and migration. Most
in cancer remain poorly understood. Ostrowski and colleagues used notably, HIF drives the expression of the extracellular matrix protein
an RNA interference screen to identify factors important in exosome LOX.92 Increased LOX expression correlates with decreased distant
secretion in cancer cells. These studies revealed an important role for metastasis-free survival and overall survival in patients with breast and
the Rab GTPases Rab27a and Rab27b and the Rab27 effectors Slp4 head and neck cancer.92 In addition, LOX activation promotes the
and Slac2b in exosome secretion.73 Studies have implicated hypoxia invasive and metastatic potential of breast cancer cells. Erler and
as an important microenvironmental factor that can influence both colleagues demonstrated that genetic and pharmacologic inhibition
Cellular Microenvironment and Metastases  •  CHAPTER 3 51

Intravasation/
Invasion/migration Premetastatic niche Distant growth
extravasation

BMDC

Fibroblast
SNAIL AXL ANGPTL44 LOX
VEGF HK1/2
TWIST CDCP1 L1CAM LOXL2
ANGPT1/2 LDHA
ZEB1/2 ZEB1/2 VEGF LOXL4
PDFG PDK1
MMPs MET UPAR SDF-1
PIGF CKB
LOX PLOD1/2 MMPS VEGF
PAI-1 ENO1
CTGF AKAP12 SDF-1/CXCR4 CXCL12
TIE-2 ALDHA
CCR5 RAB222 VCAM1 CCL2
PGK1 GLUT-1/3
PGF PLUAR ICAM1 EXOSOMES

Figure 3.2  •  Hypoxia inducible transcription factor (HIF) signaling regulates multiple steps within the metastatic cascade. The key steps of the metastatic
cascade and target genes by which HIF signaling regulates these processes are shown.

of LOX in metastatic breast cancer is sufficient to prevent hypoxia- proliferation of endothelial cells and supports pericytes.29 VEGF-A is
induced cell invasion and metastasis. These findings indicate that a well-established HIF target and is significantly induced by HIF
LOX is a promising therapeutic target for the treatment of metastatic signaling in both primary tumors and metastases.102 In summary, HIF
disease. affects multiple aspects of tumor metastasis, indicating that therapeutic
HIF signaling also facilitates tumor cell intravasation and extravasa- targeting of HIF may be an effective strategy to selectively inhibit
tion from the vasculature. HIF activity in tumor cells results in the multiple aspects of metastasis.
release of factors that modulate endothelial-endothelial cell and
endothelial-tumor cell interactions. The upregulation of angiopoietin-
like 4 (ANGPTL4) by HIF promotes tumor cell motility and PATTERNS OF METASTASIS
intravasation of tumor cells through blood vessels.88 Simultaneously, Seed and Soil Hypothesis
HIF strengthens tumor cell–endothelial cell interactions through the
activation of L1 cell adhesion molecule (L1CAM).88 Another mechanism The patterns of colonization cannot solely be explained by the circula-
by which HIF promotes tumor cell intravasation and extravasation tory, lymphatic, and transcelomic routes described earlier. The propensity
is through the activation of genes that control vascular permeability. for certain tumors to seed in particular organs was first discussed as
Hypoxic induction of VEGF, angiopoietin 2, MMPs, and UPAR the “seed and soil” theory by Paget in 1889.103 For example, prostate
cooperatively act to destabilize the vascular wall and allow for tumor cancer often metastasizes to the bones, colon cancer has a tendency
cell entry.93 to metastasize to the liver, and the primary site for ovarian metastasis
Third, HIF activity in the primary tumor is involved in the forma- is the omentum.104 Colonization is an extremely inefficient process
tion of the metastatic niche. As mentioned earlier, priming the pre- that is heavily dependent on the interactions between “seeding” tumor
metastatic site for the recruitment and survival of metastatic tumor cells and the “soil” microenvironment of the secondary site. Many
cells is a critical step in successful metastatic colonization. In breast factors including formation of a premetastatic niche and interactions
cancer cells, HIF activity results in the upregulation and release of between circulating tumor cells and the distant microenvironment
LOX and LOX-like proteins (LOXL2 and LOXL4). These proteins determine patterns of colonization.
recruit bone marrow–derived cells (BMDCs) into the lung and prime
the lung for metastatic colonization.87,92,94 BMDCs produce chemokines Premetastatic Niche
that recruit tumor cells and stimulate blood vessel invasion.95–98 Studies
have also demonstrated a role for LOX in the establishment of the Over the past decade, studies have convincingly shown that formation
premetastatic niche in the bone wherein tumor-secreted LOX is both of a premetastatic niche is essential for the growth of extravasating
necessary and sufficient to induce osteolytic bone lesions and cortical metastatic tumor cells.98 Soluble factors and BMDCs are recruited to
bone loss in mice before the arrival of tumor cells.99 Another mechanism the distant site before the arrival of tumor cells to establish a permissive
by which HIF signaling controls the directional migration of metastatic environment for malignant colonization. The mechanisms by which
tumor cell sites is through the upregulation of SDF1/CXCR4 signal- the premetastatic niche is formed remain largely unknown. However,
ing.100,101 Stromal cells within target tissue sites produce stromal derived recent studies have shown that factors secreted by the primary tumor
factor-1 (SDF1), which recruits cancer cells expressing the receptor are directly involved in establishing a permissive ECM environment
CXCR4.29 Although these studies have indicated an important cellular and in recruiting BMDCs to the distant site.
intrinsic role for hypoxia and HIF signaling in the primary tumor, BMDCs expressing VEGFR1, c-kit, CD133, and CD134 have been
future studies are eagerly awaited to elucidate the role of HIF signaling detected at distant sites and increase angiogenesis at the premetastatic
in tumor support cells. sites. Targeted inhibition of VEGFR1 prevented niche formation and
Finally, HIF promotes the late stages of metastasis at a distant site subsequent metastatic progression. This tissue preconditioning may thus
by stimulating angiogenesis. As in the primary tumor, angiogenesis represent a key step that could be targeted therapeutically, although
is a critical step for metastatic tumor growth. VEGF-A is a proangiogenic studies with anti-VEGF therapy have failed to show significant benefit
factor produced by tumor cells that stimulates the recruitment and in preventing metastatic growth for long periods of time. The role of
52 Part I: Science and Clinical Oncology

hematopoietic progenitor cells and other BMDCs in tumor progression demonstrated different abilities to metastasize to the bone, lung, or
is reviewed by Kaplan and colleagues.105 adrenal medulla. These studies indicated that there are particular
An additional function of the premetastatic niche is to guide requirements for circulating tumor cells to colonize specific organs.
metastases to specific organs. Kaplan and coworkers demonstrated that The factors contributing to tissue specific colonization include cellular
injection of secreted factors collected from cancer cells that metastasize intrinsic factors within the disseminated tumor cells and specific factors
to multiple organs could permit cancer cells that only metastasize within the tissue microenvironment. Some of the key cellular intrinsic
to the lung when grown as subcutaneous tumors in mice to display molecules determining organ-specific metastasis have been identified
widespread metastasis through governing BMDC accumulation.98 and are briefly discussed in the following section. In addition, we are
Elevated fibronectin expression by fibroblasts and fibroblast-like cells beginning to unravel the mechanisms by which infiltrating tumor
resident at premetastatic sites seems to be a critical factor in the develop- cells “educate” the normal tissue stroma at distant sites to support
ment of the premetastatic niche. The key tumor-secreted factors that metastatic growth. Elucidation of the complex signaling networks that
determine metastatic sites and mediate premetastatic niche formation exist between tumor cells and their tissue microenvironment offers
have yet to be identified, although a role for tumor necrosis factor–α new opportunities for targeted therapy against cancer.
(TNF-α), TGF-β, and VEGF-α pathways has been demonstrated.106
MMPs may also play an important role in this process. For example, Metastases to the Bone
VEGFR1 signaling has been shown to be required for premetastatic
induction of MMP-9 expression in endothelial cells and macrophages There are two types of bone metastases: osteoblastic and osteolytic.112
of the lungs by distant primary tumors.107 This is thought to make Osteoblastic metastases are observed in patients with advanced prostate
the lung microenvironment more compliant for invasion of metastasiz- cancer. Both the differentiation of osteoblastic precursors and the
ing cells. This concept is supported by the finding that pericyte activity of osteoblast cells are stimulated by tumor and microenviron-
recruitment and angiogenesis are not observed in tumor-bearing mice mental signals such as bone morphogenetic protein (BMP), fibroblast
with MMP-9 knockout bone marrow cells.108 Furthermore, stromal- growth factor receptors (FGFRs), and insulin-like growth factor 1
derived MMP-2 and MMP-9 have also been shown to contribute to receptor (IGF1R).113 Runx-2 is a key transcription factor that regulates
establishment and growth of metastases.109 Thus, whereas there is the differentiation of osteoblasts and osteoblastic precursor cells, and
evidence that MMPs play multiple roles in metastases, clinical trials represents a potential new target for inhibiting osteoblastic metastases
with MMP inhibitors have failed to show significant efficacy. In large by preventing osteoblastic precursor differentiation.114 In contrast,
part, this has been due to unexpected normal tissue toxicities and osteolytic metastases are observed in patients with breast cancer or
conflicting roles in metastases. multiple myeloma, and in these patients interactions between tumor
cells and the bone microenvironment result in bone resorption and
Organ Specificity metastatic growth as a result of the unique interplay between osteoblasts
and osteoclasts (Fig. 3.3).112,115 Parathyroid hormone–related peptide
The organ distribution of metastases from a primary tumor is not (PTHrP) secreted by the tumor cells stimulates osteoblasts to produce
random. Minn and colleagues used bioluminescence imaging to receptor activator of nuclear factor–κB (RANK) ligand (RANKL).
reveal patterns of metastasis formation by human breast cancer cells Consequently, bone-resorbing osteoclast cells are activated by RANKL
in mice.110 They also showed that individual cells from the pleural when it binds to the RANK receptor. Activated osteoclasts upregulate
effusion of a breast cancer patient showed distinct patterns of organ- MMPs, which degrade the bone matrix–releasing growth factors such
specific metastasis.111 Single-cell progenies derived from this population as TGF-β, IGFs, platelet-derived growth factor (PDGF), fibroblast

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Figure 3.3  •  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 peptide (PTHrP), secreted by the tumor cells, stimulates osteoblasts to produce
receptor activator of nuclear factor–κB (RANK) ligand (RANKL). Bone-resorbing osteoclast cells are activated by RANKL when it binds to the RANK receptor.
The activated osteoclasts upregulate matrix metalloproteinases (MMPs) that degrade the bone matrix–releasing growth factors such as transforming growth
factor–β (TGF-β), insulin-like growth factors (IGFs), platelet-derived growth factor (PDGF), fibroblast growth factors (FGFs), and bone morphogenetic proteins
(BMPs). These factors stimulate tumor cells to release PTHrP, thus completing the vicious cycle. (Modified from Steeg PS. Tumor metastasis: mechanistic
insights and clinical challenges. Nat Med. 2006;12:895–904.)
Cellular Microenvironment and Metastases  •  CHAPTER 3 53

growth factors (FGFs), and BMP.112,116,117 These factors stimulate tumor that interactions between breast cancer stem cells and the lung stroma
cells to release PTHrP, thus restarting this pathway of bone resorption. are necessary for metastatic colonization. The ECM components TNC
Gene profiling has identified other important mediators of osteoclastic and periostin (POSTN) have recently been shown to be essential
bone metastases including CXCR4, MMP-1, CTGF, and osteopontin.118 factors required for tumor initiating cells to form metastases in the
Tumor cells additionally induce osteoclast formation by overexpressing lung.7,10,142 Malanchi and colleagues showed that tumor-initiating cells
ILs such as IL-8 and IL-11, and by downregulating macrophage induce POSTN expression in lung myofibroblasts to initiate coloniza-
colony-stimulating factor.119,120 All of these factors represent new targets tion and maintain their stem cell phenotype.7 Similarly, Oskarsson
for metastases, although the importance of each factor in osteoclastic and colleagues demonstrated a role for the extracellular protein TNC
bone metastases requires further clarification. in supporting the breast cancer cell stem cell phenotype and metastases
in the lung.10 Interesting to note, previous studies showed that TNC
Metastases to the Brain and POSTN form complexes together with collagen type I and
fibronectin in the ECM. It was shown that POSTN promotes the
Brain metastases are most commonly observed in patients with breast incorporation of TNC into the ECM to strengthen the ECM archi-
cancer, lung cancer, and melanoma. Vascular access to the brain is tecture, suggesting that these factors may act through similar pathways
strictly regulated by the blood-brain barrier, an endothelial layer in promoting cancer stem cell metastasis.143 In support of this notion,
surrounding the brain, connected by tight junctions and further lined maintenance of the cancer stem cell phenotype by TNC and POSTN
by a basement membrane, pericytes, and astrocytes.121 Macromolecules was mediated at least in part through the induction of WNT signaling.
are not usually able to traverse the blood-brain barrier, and it remains These studies suggest that targeting signaling pathways mediated
unclear how tumor cells are able to penetrate the blood-brain barrier. through the ECM may be an effective strategy against cancer stem
However, once the tumor cells are within the brain parenchyma, glial cell–driven metastasis.
cells permit establishment and growth of metastases by secreting
chemokines, cytokines, and growth factors.122 Other neurotransmitter Metastases to the Liver
hormones in the brain such as norepinephrine have also been reported
to increase tumor cell motility and metastatic spread.123 Liver metastases are observed in patients with breast, lung, and
Little is known about the key factors that determine colonization pancreatic cancers. However, liver metastases are most commonly
of the brain, mostly because there is a lack of good in vivo models of found in patients with metastatic colorectal cancer, because the liver
brain metastasis. Overexpression of