Leibel and Phillips Textbook of Radiation Oncology - E-Book
3688 pages
English

Vous pourrez modifier la taille du texte de cet ouvrage

Leibel and Phillips Textbook of Radiation Oncology - E-Book

-

Obtenez un accès à la bibliothèque pour le consulter en ligne
En savoir plus
3688 pages
English

Vous pourrez modifier la taille du texte de cet ouvrage

Obtenez un accès à la bibliothèque pour le consulter en ligne
En savoir plus

Description

Stay on top of the latest scientific and therapeutic advances with the new edition of Leibel and Phillips Textbook of Radiation Oncology. Dr. Theodore L. Phillips, in collaboration with two new authors, Drs. Richard Hoppe and Mack Roach, offers a multidisciplinary look at the presentation of uniform treatment philosophies for cancer patients emphasizing the "treat for cure" philosophy. You can also explore the implementation of new imaging techniques to locate and treat tumors, new molecularly targeted therapies, and new types of treatment delivery.

  • Supplement your reading with online access to the complete contents of the book, a downloadable image library, and more at expertconsult.com.
  • Gather step-by-step techniques for assessing and implementing radiotherapeutic options with this comprehensive, full-color, clinically oriented text.
  • Review the basic principles behind the selection and application of radiation as a treatment modality, including radiobiology, radiation physics, immobilization and simulation, high dose rate, and more.
  • Use new imaging techniques to anatomically locate tumors before and during treatment.
  • Apply multidisciplinary treatments with advice from experts in medical, surgical, and radiation oncology.
  • Explore new treatment options such as proton therapy, which can facilitate precise tumor-targeting and reduce damage to healthy tissue and organs.
  • Stay on the edge of technology with new chapters on IGRT, DNA damage and repair, and molecularly targeted therapies.

Sujets

Informations

Publié par
Date de parution 09 septembre 2010
Nombre de lectures 0
EAN13 9781437737752
Langue English
Poids de l'ouvrage 6 Mo

Informations légales : prix de location à la page 0,0872€. Cette information est donnée uniquement à titre indicatif conformément à la législation en vigueur.

Exrait

  • Supplement your reading with online access to the complete contents of the book, a downloadable image library, and more at expertconsult.com.
  • Gather step-by-step techniques for assessing and implementing radiotherapeutic options with this comprehensive, full-color, clinically oriented text.
  • Review the basic principles behind the selection and application of radiation as a treatment modality, including radiobiology, radiation physics, immobilization and simulation, high dose rate, and more.
  • Use new imaging techniques to anatomically locate tumors before and during treatment.
  • Apply multidisciplinary treatments with advice from experts in medical, surgical, and radiation oncology.
  • Explore new treatment options such as proton therapy, which can facilitate precise tumor-targeting and reduce damage to healthy tissue and organs.
  • Stay on the edge of technology with new chapters on IGRT, DNA damage and repair, and molecularly targeted therapies.

" />

Leibel and Phillips Textbook of Radiation Oncology
Third Edition

Richard T. Hoppe, MD, FACR, FASTRO
Henry S. Kaplan—Harry Lebeson Professor in Cancer Biology and Chair, Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California

Theodore Locke Phillips, MD, FACR, FASTRO
Professor of Radiation Oncology, Department of Radiation Oncology, University of Arizona, Tucson, Arizona;, University of California, Davis, Sacramento, California

Mack Roach, III, MD, FACR
Professor and Chairman, Department of Radiation Oncology, University of California, San Francisco, San Francisco, California
Saunders
Front Matter

Leibel and Phillips Textbook of Radiation Oncology
THIRD EDITION
Richard T. Hoppe, MD, FACR, FASTRO
Henry S. Kaplan–Harry Lebeson Professor in Cancer Biology and Chair
Department of Radiation Oncology
Stanford University School of Medicine
Stanford, California
Theodore Locke Phillips, MD, FACR, FASTRO
Professor of Radiation Oncology
Department of Radiation Oncology
University of Arizona
Tucson, Arizona;
University of California, Davis
Sacramento, California
Mack Roach, III, MD, FACR
Professor and Chairman
Department of Radiation Oncology
University of California, San Francisco
San Francisco, California
With 1623 illustrations
Copyright

1600 John F. Kennedy Blvd.
Ste 1800
Philadelphia, PA 19103-2899
LEIBEL AND PHILLIPS TEXTBOOK OF RADIATION ONCOLOGY ISBN: 978-1-4160-5897-7
Copyright © 2010, 2004, 1998 by Saunders, an imprint of Elsevier Inc.
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.
Library of Congress Cataloging-in-Publication Data
Leibel and Phillips textbook of radiation oncology / [edited by] Theodore Locke Phillips, Richard T. Hoppe, Mack Roach, III.—3rd ed.
p. ; cm.
Other title: Textbook of radiation oncology
Rev. ed. of: Textbook of radiation oncology / [edited by] Steven A. Leibel, Theodore L. Phillips. 2nd ed. c2004.
Includes bibliographical references and index.
ISBN 978-1-4160-5897-7 (hardcover : alk. paper) 1. Cancer–Radiotherapy. 2. Medical physics. 3. Tumors–Radiography. 4. Radioisotope brachytherapy. I. Phillips, Theodore L. II. Hoppe, R. (Richard) III. Roach, Mack. IV. Leibel, Steven A. V. Textbook of radiation oncology. VI. Title: Textbook of radiation oncology.
[DNLM: 1. Neoplasms–radiotherapy. 2. Health Physics–methods. 3. Radiation Oncology–methods. QZ 269 L525 2010]
RC271.R3T45 2004
616.99′40642–dc22
2010017535
Acquisitions Editor: Dolores Meloni
Senior Developmental Editor: Janice M. Gaillard
Publishing Services Manager: Catherine Jackson
Senior Project Manager: Rachel E. McMullen
Design Direction: Louis Forgione
Printed in China
Last digit is the print number: 9 8 7 6 5 4 3 2 1
Contributors

Andre Abitbol, MD, Associate Director Department of Radiation Oncology Baptist Hospital Miami, Florida

David H. Abramson, MD, Chief, Ophthalmic Oncology Service Department of Surgery Memorial Sloan-Kettering Cancer Center New York, New York

Ranjana Advani, MD, Associate Professor of Medicine Division of Oncology Stanford University Medical Center Stanford, California

Mohammed Ahmed, MBBS, Kurnool Medical College Kurnool, Andhrapradesh, India

Oguz Akin, MD, Assistant Professor of Radiology, Weill Medical College of Cornell University Attending Radiologist, Memorial Sloan-Kettering Cancer Center Department of Radiology Memorial Sloan-Kettering Cancer Center New York, New York

Kaled M. Alektiar, MD, Associate Attending Radiation Oncologist Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Michael Alvarado, MD, Assistant Professor of Surgery Department of Surgery University of California, San Francisco San Francisco, California

Howard I. Amols, PhD, Chief, Clinical Physics Service Medical Physics Memorial Sloan Kettering Cancer Center New York, New York

John G. Armstrong, MD, FRCPI, Professor of Radiation Oncology Consultant Radiation Oncologist & Director of Research St. Luke’s Institute of Cancer Research St. Luke’s Hospital Dublin, Ireland

Barbara L. Asselin, MD, Associate Professor of Pediatrics and Oncology Department of Pediatrics University of Rochester School of Medicine Golisano Children’s Hospital at URMC Rochester, New York

Igor J. Barani, MD, Assistant Professor In-Residence Department of Radiation Oncology University of California, San Francisco San Francisco, California

Christopher A. Barker, MD, Resident Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Luc Beaulieu, PhD, Associate Professor and Head of Medical Physics Research Department of Physics, Physics Engineering and Optics Université Laval Department of Radiation Oncology Centre Hospitalier Universitaire de Québec Québec, Canada

Joel S. Bedford, DPhil, Professor Environmental and Radiological Health Sciences Colorado State University Fort Collins, Colorado

Adrian C. Begg, PhD, Professor, Senior Scientist, Group Leader Experimental Therapy The Netherlands Cancer Institute Amsterdam, The Netherlands

Søren M. Bentzen, PhD, DSc, Professor of Human Oncology; Medical Physics; Biostatistics and Medical Informatics Department of Human Oncology University of Wisconsin School of Medicine and Public Health Madison, Wisconsin

Alison Bevan, MD, PhD, Attending Radiation Oncologist Radiation Oncology Centers Radiological Associates of Sacramento Sacramento, California

Luc M. Bidaut, PhD, Director, Image Processing and Visualization Lab Imaging Physics University of Texas, M.D. Anderson Cancer Center Houston, Texas

Eleanor A. Blakely, PhD, Senior Staff Biophysicist Life Sciences Division Lawrence Berkeley National Laboratory Berkeley, California

J. Martin Brown, DPhil, Professor Radiation Oncology Stanford University Stanford, California

Chandra M. Burman, PhD, Attending Physicist Department of Medical Physics Memorial Sloan-Kettering Cancer Center New York, New York

Oren Cahlon, MD, Attending Physician Princeton Radiation Oncology Center Jamesburg, New Jersey

Matthew D. Callister, MD, Assistant Professor of Oncology Department of Radiation Oncology, Mayo Clinic Arizona Scottsdale, Arizona

Peter Carroll, MD, MPH, Professor and Chair Department of Urology University of California, San Francisco Comprehensive Cancer Center San Francisco, California

Joseph R. Castro, MD, FACR, Professor of Radiation Oncology, Emeritus Department of Radiation Oncology University of California Medical Center San Francisco, California

Daniel T. Chang, MD, Assistant Professor Department of Radiation Oncology Stanford University Stanford, California

Susan M. Chang, MD, Professor, Director of Division of Neuro-Oncology Department of Neurological Surgery University of California, San Francisco San Francisco, California

Devron H. Char, MD, President, Tumori Foundation, Clinical Professor Department of Ophthalmology Stanford University San Francisco, California

Allen M. Chen, MD, Assistant Professor in Residence, Director of Research Department of Radiation Oncology University of California, Davis, Cancer Center Sacramento, California

Andy Chen, MD, PhD, Assistant Professor Center for Hematologic Malignancies Oregon Health & Science University Portland, Oregon

Chien Peter Chen, MD, PhD, Department of Radiation Oncology University of California San Francisco San Francisco, California

Dennis S. Chi, MD, Associate Professor Gynecology Service, Department of Surgery Memorial Sloan-Kettering Cancer Center New York, New York

Prakash Chinnaiyan, MD, Assistant Member Department of Radiation Oncology and Experimental Therapeutics H. Lee Moffitt Cancer Center and Research Institute Tampa, Florida

Robert W. Cho, MD, Instructor of Pediatric Stem Cell Transplantation Department of Pediatrics Stanford University Stanford, California

Walter H. Choi, MD, Attending Physician Department of Radiation Oncology Beth Israel Medical Center New York, New York

Lanceford M. Chong, MD, MPH, Medical Director Department of Radiation Oncology Western Regional Medical Center Cancer Treatment Centers of America Phoenix, Arizona

Orlo H. Clark, MD, FACS, Professor of Surgery Department of Surgery University of California, San Francisco San Francisco, California

Michael F. Clarke, MD, Associate Director Karel and Avice Beekhuis Professorship in Cancer Biology Professor of Medicine (Oncology) Stanford Institute for Stem Cell & Regenerative Medicine Stanford University School of Medicine Palo Alto, California

Fergus V. Coakley, MD, Professor of Radiology, Vice Chair of Clinical Services Abdominal Imaging Section Chief Department of Radiology and Biomedical Imaging University of California, San Francisco San Francisco, California

A. Dimitrios Colevas, MD, Associate Professor of Medicine Stanford University Medical Center Palo Alto, California

Louis S. Constine, MD, FASTRO, Professor of Radiation Oncology and Pediatrics Vice Chair, Department of Radiation Oncology James P. Wilmot Cancer Center University of Rochester Medical Center Rochester, New York

Steven Coutre, MD, Associate Professor of Medicine, Hematology Department of Medicine Stanford University School of Medicine Stanford, California

Bruce Culliney, MD, Attending in Medicine Division of Hematology/Oncology Beth Israel Medical Center; Assistant Professor of Medicine Albert Einstein College of Medicine New York, New York

Inder K. Daftari, PhD, Sr. Physicist Department of Radiation Oncology University of California, San Francisco San Francisco, California

Sally J. DeNardo, MD, Emeritus Professor of Internal Medicine (Oncology) and Radiology (Nuclear Medicine) Division of Hematology and Oncology Section of Radiodiagnosis and Therapy Department of Internal Medicine University of California, Davis School of Medicine Sacramento, California

Maximilian Diehn, MD, PhD, Assistant Professor Department of Radiation Oncology and Institute for Stem Cell Biology & Regenerative Medicine Stanford University Stanford, California

Paul J. Donald, MD, FRCS(C), Professor and Vice Chair-Director Center for Skull Base Surgery, Director, Head and Neck Oncologic Skull Base and Microvascular Surgery Fellowship Department of Otolaryngology–Head and Neck Surgery University of California, Davis Sacramento, California

Ira J. Dunkel, MD, Associate Attending Physician Department of Pediatrics Memorial Sloan-Kettering Cancer Center New York, New York

Mark Dunphy, DO, Assistant Attending Physician Department of Radiology Memorial Sloan-Kettering Cancer Center New York, New York

Linda R. Duska, MD, Associate Professor, Gynecologic Oncology Fellowship Director Department of Obstetrics and Gynecology Division of Gynecologic Oncology University of Virginia Charlottesville, Virginia

Sharon C. Dutton, MD, MPH, Attending Radiation Oncologist Radiation Oncology Centers Radiological Associates of Sacramento Sacramento, California

Michael S.B. Edwards, MD, FAAP, FACS, Lucile Packard Endowed Professor of Neurosurgery and Pediatrics Co-Director Center for Children’s Brain Tumors Director Regional Pediatric Neurosurgery Lucile Packard Children’s Hospital Stanford University Medical Center Stanford, California

Diana L. Farmer, MD, Professor of Surgery, Pediatrics, and Obstetrics, Gynecology & Reproductive Sciences Chief, Division of Pediatric Surgery Vice Chair, Department of Surgery Surgeon-in-Chief, UCSF Children’s Hospital Department of Surgery University of California, San Francisco School of Medicine San Francisco, California

Edith J. Filion, MD, FRCPC, Department of Radiation Oncology Centre Hospitalier de l’Université de Montréal Montreal, Quebec, Canada

Nancy J. Fischbein, MD, Associate Professor of Radiology and, by courtesy, Otolaryngology-Head and Neck Surgery, Neurology, and Neurosurgery Department of Radiology Stanford University Medical Center Stanford, California

George A. Fisher, MD, PhD, Associate Professor of Medicine (Oncology) Stanford Cancer Center Stanford, California

Paul Graham Fisher, MD, Professor Neurology and Pediatrics, and by courtesy, Neurosurgery and Human Biology The Beirne Family Professor of Pediatric Neuro-Oncology Chief, Division of Child Neurology Stanford University and Lucile Packard Children’s Hospital Palo Alto, California

James M. Ford, MD, Associate Professor of Medicine and Genetics Division of Oncology Stanford University School of Medicine Stanford, California

Barbara Fowble, MD, FACR, FASTRO, Clinical Professor Health Sciences Department of Radiation Oncology Helen Diller Family Comprehensive Cancer Center University of California, San Francisco Stanford, California

Jennifer M. Fu, MD, Solano Dermatology Associates El Cerrito, California; Volunteer Clinical Faculty Department of Dermatology University of California, San Francisco San Francisco, California

Karen K. Fu, MD, Professor Emeritus Department of Radiation Oncology University of California, San Francisco San Francisco, California

Zvi Y. Fuks, MD, Member Memorial Sloan-Kettering Cancer Center New York, New York

Ignacio Azinovic Gamo, MD, PhD, Chief of Radiation Oncology Plataforma de Oncología USP Hospital San Jaime Torrevieja, Alicante, Spain

Kristen N. Ganjoo, MD, Assistant Professor of Medicine Department of Medical Oncology Stanford University Stanford, California

Amato J. Giaccia, PhD, Jack, Lulu and Sam Willson Professor of Cancer Biology Department of Radiation Oncology Stanford University School of Medicine Stanford, California

Iris C. Gibbs, MD, Associate Professor Department of Radiation Oncology Stanford University School of Medicine Stanford, California

Michael T. Gillin, PhD, Deputy Chair and Chief of Clinical Services Radiation Physics University of Texas, M.D. Anderson Cancer Center Houston, Texas

Michelle S. Ginsberg, MD, Director, Thoracic Imaging Department of Radiology Memorial Sloan-Kettering Cancer Center; Associate Professor of Radiology Weill Medical College of Cornell University New York, New York

Brian J. Goldsmith, MD, Attending Radiation Oncologist Radiation Oncology Centers Radiological Associates of Sacramento Sacramento, California

Daniel R. Gomez, MD, Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Karyn Goodman, MD, MS, Assistant Attending Radiation Oncologist Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Alexander R. Gottschalk, MD, PhD, Associate Professor, Department of Radiation Oncology Director of the CyberKnife Radiosurgery Program University of California, San Francisco Helen Diller Family Comprehensive Cancer Center San Francisco, California

Edward E. Graves, PhD, Assistant Professor Department of Radiation Oncology Stanford University Stanford, California

Sheryl Green, MB, Bch, Assistant Professor Department of Radiation Oncology Mount Sinai Medical Center New York, New York

Roy C. Grekin, MD, Professor of Dermatology Department of Dermatology University of California, San Francisco San Francisco, California

Ravinder K. Grewal, MD, Assistant Professor of Radiology Weill Medical College of Cornell University; Assistant Attending Physician Division of Nuclear Medicine Department of Radiology Memorial Sloan-Kettering Cancer Center, New York New York, New York

Leonard L. Gunderson, MD, MS, Getz Family Professor Department of Radiation Oncology Mayo Clinic Cancer Center–Arizona Scottsdale, Arizona

Philip H. Gutin, MD, Chairman, Department of Neurosurgery Fred Lebow Chair in Neurooncology; Executive Co-Director MSKCC Brain Tumor Center Memorial Sloan-Kettering Cancer Center New York, New York

Daphne A. Haas-Kogan, MD, Professor Departments of Radiation Oncology and Neurosurgery; Vice-Chair and Program Director Departments of Radiation Oncology and Neurosurgery University of California, San Francisco San Francisco, California

Michael G. Haddock, MD, Associate Professor of Radiation Oncology Department of Radiation Oncology Mayo Clinic Rochester, Minnesota

Ester M. Hammond, PhD, The Gray Institute for Radiation Oncology and Biology University of Oxford Oxford, United Kingdom

Paul M. Harari, MD, Jack Fowler Professor and Chairman Department of Human Oncology University of Wisconsin School of Medicine Madison, Wisconsin

Louis B. Harrison, MD, FASTRO, Clinical Director Continuum Cancer Centers of New York; The Gerald J. Friedman Chair of Radiation Oncology Beth Israel Medical Center, St. Luke’s and Roosevelt Hospitals; Professor of Radiation Oncology Albert Einstein College of Medicine New York, New York

Lauren C. Harshman, MD, Instructor of Medicine Department of Medicine Division of Oncology Stanford University School of Medicine Stanford, California

Melanie G. Gephart Hayden, MD, MAS, Resident Neurosurgery Stanford University Hospital and Clinics Stanford, California

Russell W. Hinerman, MD, Associate Professor Dept of Radiation Oncology University of Florida Gainesville, Florida

Alice Y. Ho, MD, MBA, Assistant Attending Radiation Oncologist Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Richard T. Hoppe, MD, FACR, FASTRO, Henry S. Kaplan–Harry Lebeson Professor in Cancer Biology and Chair Department of Radiation Oncology Stanford University School of Medicine Stanford, California

Sandra J. Horning, MD, Professor Emeritus Department of Medicine/Oncology Stanford University Stanford, California

Hedvig Hricak, MD, PhD, Drhc, Chairman Radiology Memorial Sloan-Kettering Cancer Center New York, New York

Annie Hsu, PhD, Clinical Instructor Department of Radiation Oncology Stanford University Stanford, California

I-Chow Joe Hsu, MD, Associate Professor and Vice Chair Department of Radiation Oncology University of California, San Francisco San Francisco, California

Kenneth S. Hu, MD, Attending Physician Department of Radiation Oncology Beth Israel Medical Center; Associate Professor Albert Einstein College of Medicine New York, New York

Melissa M. Hudson, MD, Director, Cancer Survivorship Division Department of Oncology St. Jude Children’s Research Hospital Memphis, Tennessee

John L. Humm, PhD, Attending Physicist Department of Medical Physics Memorial Sloan-Kettering Cancer Center New York, New York

Peter Johannet, MD, Chief of Plastic Surgery VA Palo Alto Healthcare System Palo Alto, California

Michael J. Kaplan, MD, Professor and Chief, Head and Neck Surgery Department of Otolaryngology Head & Neck Surgery Stanford University School of Medicine Stanford, California

Daniel S. Kapp, PhD, MD, Professor Department of Radiation Oncology Stanford University School of Medicine Stanford, California

Mohammed Kashani-Sabet, MD, Senior Scientist and Director, Melanoma Program California Pacific Medical Research Institute and California Pacific Medical Center San Francisco, California

Laurence Katznelson, MD, Associate Professor Medical Director, Pituitary Center Department of Neurosurgery and Medicine Stanford University Stanford, California

Noah D. Kauff, MD, Director of Ovarian Cancer Screening and Prevention Gynecology and Clinical Genetics Services Memorial Sloan-Kettering Cancer Center New York, New York

Paul J. Keall, PhD, Associate Professor Department of Radiation Oncology Stanford University Stanford, California

Youn H. Kim, MD, Professor Department of Dermatology Director, Multidisciplinary Cutaneous Lymphoma Program Stanford Cancer Center Stanford, California

Christopher R. King, PhD, MD, Associate Professor of Radiation Oncology & Urology Department of Radiation Oncology Institute of Urologic Oncology University of California, Los Angeles School of Medicine Los Angeles, California

Susan J. Knox, PhD, MD, Associate Professor Department of Radiation Oncology Stanford University School of Medicine Stanford, California

Cameron J. Koch, PhD, Professor Radiation Oncology University of Pennsylvania Philadelphia, Pennsylvania

Marisa M. Kollmeier, MD, Assistant Attending Radiation Oncologist Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Albert Koong, MD, PhD, Associate Professor Radiation Oncology Stanford University School of Medicine Stanford, California

Lee M. Krug, MD, Associate Attending Physician Department of Medicine, Thoracic Oncology Service Memorial Sloan-Kettering Cancer Center New York, New York

Pamela L. Kunz, MD, Assistant Professor Department: Medicine (Oncology) Stanford University Medical School Stanford, California

Michael P. La Quaglia, MD, FACS, FAAP, FRCS (Ed Hon), Chief, Pediatric Surgical Service Department of Surgery Memorial Sloan-Kettering Cancer Center Affiliated with Weill Medical College of Cornell University New York, New York

David A. Larson, MD, PhD, Professor Department of Radiation Oncology University of California San Francisco San Francisco, California; Co-director Gamma Knife Center Washington Hospital Fremont, California

Steven Larson, MD, FACNP, FACR, Donna & Benjamin M. Rosen Chair in Radiology Chief of Nuclear Medicine Service Department of Radiology Memorial Sloan-Kettering Cancer Center New York, New York

Edward R. Laws, MD, FACS, Director, Pituitary/Neuroendocrine Center Neurosurgery Brigham & Women’s Hospital, Harvard University Boston, Massachusetts

Quynh-Thu Le, MD, Professor Department of Radiation Oncology Stanford University Stanford, California

Andrew K. Lee, MD, MPH, Associate Professor Department of Radiation Oncology M.D. Anderson Cancer Center Houston, Texas

Nancy Lee, MD, Associate Attending Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Gloria C. Li, PhD, Member and Attending Biophysicist Departments of Radiation Oncology and Medical Physics Memorial Sloan-Kettering Cancer Center New York, New York

Patricia Lillis-Hearne, MD, MHA, COL, Director, Armed Forces Radiobiology Research Institute Uniformed Services University Bethesda, Maryland

C. Clifton Ling, PhD, Attending Physicist Medical Physics Memorial Sloan-Kettering Cancer Center New York, New York

David E. Linstadt, MD, Medical Director, Auburn Radiation Oncology Center Radiological Associates of Sacramento Auburn, California

Jay S. Loeffler, MD, FACR, Herman and Joan Suit Professor and Chair Department of Radiation Oncology Massachusetts General Hospital and Harvard Medical School Boston, Massachusetts

Billy W. Loo, Jr., MD, PhD, Assistant Professor and Thoracic Radiation Oncology Program Leader Department of Radiation Oncology Stanford University and Cancer Center Stanford, California

Thomas LoSasso, PhD, Attending Physicist Department of Medical Physics Memorial Sloan-Kettering Cancer Center New York, New York

Gikas S. Mageras, PhD, Attending Physicist and Chief, Computer Service Department of Medical Physics Memorial Sloan-Kettering Cancer Center New York, New York

Lawrence Margolis, MD, Professor Emeritus Radiation Oncology University of California, San Francisco San Francisco, California

Brian P. Marr, MD, Assistant Attending Ophthalmic Oncology Department Memorial Sloan-Kettering Cancer Center New York, New York

Katherine K. Matthay, MD, Mildred V. Strouss Professor of Translational Research Chief Pediatric Hematology-Oncology Department of Pediatrics University of California, San Francisco School of Medicine San Francisco, California

Sean M. McBride, MD, Resident Harvard Radiation Oncology Program Massachusetts General Hospital 100 Blossom Street, Cox-3 Boston, Massachusetts

Beryl McCormick, MD, FACR, Attending and Member Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Michael W. McDermott, MD, Professor in Residence Departments of Neurosurgery, Radiation Oncology & Otolaryngology, Robert & Ruth Halperin Chair in Meningioma Research University of California, San Francisco San Francisco, California

Michelle Melisko, MD, Assistant Clinical Professor Department of Medicine University of California, San Francisco San Francisco, California

Karine Michaud, MD, Clinical Instructor Neurosurgery University of California, San Francisco San Francisco, California

Robert C. Miller, MD, Associate Professor of Radiation Oncology Department of Radiation Oncology Mayo Clinic Rochester, Minnesota

Bruce D. Minsky, MD, Associate Dean and Professor of Radiation and Cellular Oncology Chief Quality Officer University of Chicago Medical Center Chicago, Illinois

Kavita K. Mishra, MD, MPH, Assistant Professor Department of Radiation Oncology University of California, San Francisco San Francisco, California

Radhe Mohan, PhD, Professor and Chairman Department of Radiation Physics M.D. Anderson Cancer Center Houston, Texas

Robert J. Myerson, MD, PhD, Professor of Radiation Oncology Dept of Radiation Oncology Washington University School of Medicine St. Louis, Missouri

Subir Nag, MD, FACR, FACRO, Director of Brachytherapy Services Department of Radiation Oncology Kaiser Permanente Medical Center Santa Clara, California

Jean L. Nakamura, MD, Assistant Professor Department of Radiation Oncology University of California, San Francisco San Francisco, California

Ashwatha Narayana, MD, Associate Professor Department of Radiation Oncology and Neurosurgery New York University Medical Center New York, New York

Dattatreyudu Nori, MD, FACR, FACRO, FASTRO, Professor and Chairman Department of Radiation Oncology New York Presbyterian Hospital–Weill Cornell Medical College New York, New York; Director of the Cancer Center New York Hospital, Queens Flushing, New York

Jeffrey A. Norton, MD, Professor of Surgery Department of Surgery Stanford University Stanford, California

Colin G. Orton, PhD, Professor Emeritus Wayne State University Detroit, Michigan

Matthew B. Parliament, MD, Director of Radiation Oncology Department of Oncology Cross Cancer Institute and University of Alberta Edmonton, Alberta, Canada

Paula L. Petti, PhD, Director of Gamma Knife Physics Taylor McAdam Bell Neuroscience Institute Washington Hospital Healthcare System Fremont, California

Theodore Locke Phillips, MD, FACR, FASTRO, Professor of Radiation Oncology Department of Radiation Oncology University of Arizona Tucson, Arizona; University of California, Davis Sacramento, California

Carlos E. Pineda, MD, Resident Department of Surgery Stanford University School of Medicine Stanford, California

Isabel M. Pires, PhD, The Gray Institute for Radiation Oncology and Biology University of Oxford Oxford, United Kingdom

Jean Pouliot, PhD, Professor and Vice Chair, Director of the Physics Division Department of Radiation Oncology University of California, San Francisco San Francisco, California

Joseph Presti, Jr., MD, Thomas A. Stamey Research Professor of Urology Department of Urology Stanford University School of Medicine Stanford, California

Jeanne M. Quivey, MD, Professor Irwin Mark Jacobs and Joan Klein Jacobs Endowed Chair in Head and Neck Cancer Department of Radiation Oncology University of California, San Francisco Comprehensive Cancer Center San Francisco, California

Andrew Quon, MD, Chief, Clinical PET/CT Department of Radiology Stanford University School of Medicine Stanford, California

Rachel Rabinovitch, MD, Professor Department of Radiation Oncology University of Colorado Denver Comprehensive Cancer Center Aurora, Colorado

Lawrence Recht, MD, Professor Neurology Stanford University School of Medicine Stanford, California

Sunil A. Reddy, MD, Staff Physician Department of Medical Oncology/Medicine Stanford Cancer Center Stanford, California

Andreas Rimner, MD, Assistant Professor, Weill Medical College of Cornell University, Assistant Attending Radiation Oncologist Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Mack Roach, III, MD, FACR, Professor and Chairman Department of Radiation Oncology University of California, San Francisco San Francisco, California

Jonathan E. Rosenberg, MD, Assistant Professor and Clinical Director Lank Center for Genitourinary Oncology Dana-Farber Cancer Institute, Harvard Medical School Boston, Massachusetts

Seth A. Rosenthal, MD, FACR, Attending Radiation Oncologist Radiation Oncology Centers Radiological Associates of Sacramento Sacramento, California

Kenneth E. Rosenzweig, MD, Associate Attending Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Lawrence N. Rothenberg, PhD, Member Emeritus Department of Medical Physics Memorial Sloan-Kettering Cancer Center; Adjunct Professor Department of Applied Physics and Applied Mathematics Columbia University New York, New York

Daniel Ruan, MD, Associate Surgeon Surgery Brigham and Women’s Hospital Boston, Massachusetts

Anthony H. Russell, MD, FACR, Associate Professor of Radiation Oncology Harvard Medical School; Chief, Gynecologic Radiation Oncology Service Massachusetts General Hospital Boston, Massachusetts

Janice Ryu, MD, FACR, Attending Radiation Oncologist Radiation Oncology Centers Radiological Associates of Sacramento Sacramento, California

Amy C. Schefler, MD, Chief resident, Vitreoretinal fellow Department of Ophthalmology Bascom Palmer Eye Institute Miami, Florida

Tracey E. Schefter, MD, Associate Professor Radiation Oncology University of Colorado Comprehensive Cancer Center Denver, Colorado

Daniela Schulz-Ertner, MD, Professor Department of Radiation Oncology Radiological Institute (MVZ) Markus Hospital Frankfurt, Germany

Karen D. Schupak, MD, Director, Regional Radiation Oncology Programs, Vice Chairman Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center Basking Ridge, New Jersey

Granger R. Scruggs, MD, Department of Radiation Oncology Baylor University Medical Center, Texas Oncology Dallas, Texas

Roy B. Sessions, MD, Attending Department of Head and Neck Surgery Ralph Johnson VA Medical Center Charleston, South Carolina

Dennis C. Shrieve, MD, Professor and Chair Department of Radiation Oncology University of Utah School of Medicine Salt Lake City, Utah

Eric J. Small, MD, Professor and Chief Division of Hematology and Oncology Department of Medicine University of California, San Francisco San Francisco, California

Penny K. Sneed, MD, Professor Department of Radiation Oncology University of California, San Francisco San Francisco, California

Marnee M. Spierer, MD, Assistant Professor Department of Radiation Oncology Montefiore Medical Center/Albert Einstein College of Medicine Bronx, New York

Sandy Srinivas, MD, Associate Professor of Oncology Department of Medicine Stanford University Palo Alto, California

Paul Stauffer, MSEE, CCE, Professor and Director Hyperthermia Physics Department of Radiation Oncology Department Duke University Medical Center Durham, North Carolina

Richard G. Stock, MD, Professor and Chair Department of Radiation Oncology Mount Sinai School of Medicine New York City, New York

Xiaorong Sun, MD, Associate Attending PET-CT Center Shandong Cancer Hospital and Institute Jinan, Shandong, P. R. China

Susan M. Swetter, MD, Professor of Dermatology Stanford University Medical Center/VA Palo Alto Health Care System Palo Alto, California

Patrick S. Swift, MD, Department of Radiation Oncology Alta Bates Comprehensive Cancer Center Berkeley, California

Margaret A. Tempero, MD, Doris and Donald Fisher Distinguished Professorship in Clinical Cancer Research Professor of Medicine, Division of Hematology and Oncology Director of Research Programs and Deputy Director, UCSF Helen Diller Family Comprehensive Cancer Center; Department of Medicine University of California, San Francisco San Francisco, California

Hirohiko Tsujii, MD, PhD, Executive Director National Institute of Radiological Sciences Chiba, Japan

Francesco Tuniz, MD, Radiosurgical Fellow Department of Neurosurgery Stanford University School of Medicine Stanford, California

Raul C. Urtasun, MD, FASTRO, Professor Emeritus Department of Oncology and Radiation Oncology University of Alberta Faculty of Medicine and Cross Cancer Institute, Edmonton, Alberta, Canada

Alan P. Venook, MD, Professor of Clinical Medicine Division of Hematology and Oncology Department of Medicine University of California, San Francisco San Francisco, California

Lynn J. Verhey, PhD, Professor Emeritus Department of Radiation Oncology University of California, San Francisco San Francisco, California

Raquel Wagman, MD, Attending Department of Radiation Oncology St. Barnabas Medical Center Livingston, New Jersey

Kent Wallner, MD, Radiation Oncologist Veteran Administration Puget Sound Health Care System and Group Health Cooperative Seattle, Washington

Robert Warren, MD, Chief, Surgical Oncology Department of Surgery University of California, San Francisco Comprehensive Cancer Center San Francisco, California

Irving L. Weissman, MD, Director and Virginia and D.K. Ludwig Professor for Clinical Investigation in Cancer Research Stanford Institute of Stem Cell Biology and Regenerative Medicine Stanford University Stanford, California

Mark L. Welton, MD, MHCM, FACS, FASCRS, Professor and Chief, Colon and Rectal Surgery Department of Surgery Stanford University, School of Medicine Stanford, California

Moody D. Wharam, Jr., MD, FACR, FASTRO, Professor of Radiation Oncology Department of Radiation Oncology and Molecular Radiation Sciences The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins The Johns Hopkins University School of Medicine Baltimore, Maryland

George David Wilson, PhD, Chief, Radiation Biology Department of Radiation Oncology William Beaumont Hospital Royal Oak, Michigan

Paul F. Wilson, PhD, Senior Staff Scientist Biosciences and Biotechnology Division Lawrence Livermore National Laboratory Livermore, California

Suzanne L. Wolden, MD, Associate Attending Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Shiao Y. Woo, MD, FACR, Professor Department of Radiation Oncology U.T. M.D. Anderson Cancer Center Houston, Texas

Ping Xia, PhD, Head of Medical Physics Department Radiation Oncology Cleveland Clinic Cleveland, Ohio

Lei Xing, PhD, Professor Department of Radiation Oncology Stanford University School of Medicine Stanford, California

Joachim Yahalom, MD, Member and Professor Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Yoshiya Yamada, MD, FRCPC, Associate Attending Radiation Oncologist Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Sue S. Yom, MD, PhD, Assistant Professor of Clinical Radiation Oncology University of California, San Francisco San Francisco, California

Michael J. Zelefsky, MD, Professor of Radiation Oncology Chief of Brachytherapy Service Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York
Foreword
This book is dedicated to Steven A. Leibel, MD, FACR, FASTRO, radiation oncologist extraordinaire, who died unexpectedly while vacationing with his wife, Margy, in Hawaii, on February 7, 2008. He had recently completed planning this third edition with Ted Phillips, his co-editor.
Steve was born in the Stanford-Lane Hospital in San Francisco in 1946. After growing up in the Bay Area, he went to Michigan State University for his undergraduate degree. Steve then returned to San Francisco, receiving his MD degree and completing his residency in radiation oncology at UCSF. At UCSF, he was influenced by Ted Phillips and the late Glenn Sheline to pursue a career in academic radiation oncology. Steve fulfilled his military commitment at the Bethesda Naval Hospital and then joined the faculty at Johns Hopkins with Dr. Stanley Order for two years. But the San Francisco Bay Area beckoned, and he returned to UCSF in 1980, where he spent the next 8 years, focusing his academic work on the treatment of gynecologic, liver, and brain tumors. He also headed the residency program and the brachytherapy service. In 1988, Steve was recruited to the Memorial Sloan-Kettering Cancer Center, where he joined Zvi Fuks as Vice-Chairman and Clinical Director of Radiation Oncology. In 1998, he became Chairman of the department.
Steve’s research focus throughout his career was the development and introduction of new technology to improve patient care. At MSKCC, he pioneered the use of IMRT, especially its application to the treatment of prostate cancer. He demonstrated that dose escalation was associated with increased likelihood of local control, leading the way toward IMRT becoming the standard of care for the radiation therapy of prostate cancer.
In 2004, Steve was recruited to be Medical Director of the Stanford Clinical Cancer Center and he was appointed as the John and Anne Doerr Professor. Steve played a key role in Stanford’s successful effort to receive National Cancer Institute Cancer Center designation. He successfully coordinated the activity of more than 350 professionals who worked in the Cancer Center and helped to further its prominence internationally in cancer research and care.
Steve provided significant service to the field of radiation oncology. He served as President and Chairman of the Board of ASTRO, a member of the Board of Chancellors of the ACR and Chair of its Commission on Cancer, a member and Chair of the Residency Review Committee for Radiation Oncology, and a Trustee and President of the American Board of Radiology. At the same time, Steve found time to co-edit the Textbook of Radiation Oncology and participate in the launch of the third edition. He was elected as a fellow in the American College of Radiology and was a member of the inaugural fellowship class of ASTRO. Steve’s accomplishments were recognized by the ASTRO Gold Medal and special alumni achievement awards from Johns Hopkins and Michigan State.

Over the years, Steve formed many close friendships with his colleagues, including many prominent surgeons, medical oncologists, diagnostic radiologists, and radiation oncologists, as well as numerous patients. He touched the lives of many who enjoyed his wit, his personality, his boundless energy, his warmth, his congeniality, his devotion to the task at hand, and his leadership. He is missed by all.

Richard T. Hoppe, MD, FACR, FASTRO

Larry E. Kun, MD, FACR, FASTRO

Theodore Locke Phillips, MD, FACR, FASTRO
Preface
This third edition of the Textbook of Radiation Oncology comes 6 years after the second edition published in 2004. Steve Leibel and Ted Phillips planned the third edition and selected the authors in 2007. Steve’s death in early 2008 required a rethinking of the editorial effort and Rich Hoppe and Mack Roach agreed to join Ted as editors. The text was renamed Leibel and Phillips Textbook of Radiation Oncology. A reorganization of the editing assignments was required, resulting in some delay.
True to the goals of the previous editions, the latest advances in radiation oncology were included in all of the site chapters, with expanded sections on emerging modalities and imaging. The importance of imaging in today’s radiation oncology practice cannot be overemphasized. Color illustrations were incorporated throughout to further stress imaging as fundamental to the specialty. Multimodality approaches to treatment are essential and to assure adequate surgical and medical oncologic input, co-authors from these specialties were included in most site chapters.
As in the past the biological and anatomic background needed to select the correct targets and doses is provided rather than a rote prescription of “classic” ports and plans. Biological and genetic factors are well covered to allow the reader to understand and explore further the huge literature on molecular changes in cancer and their impact on treatment and outcome.

Richard T. Hoppe, MD, FACR, FASTRO

Theodore Locke Phillips, MD, FACR, FASTRO

Mack Roach, III, MD, FACR
Acknowledgments
The task of assembling a major textbook of this size and scope requires the efforts of many people. We would like to thank Eva DeVos-Jaggs, our Coordinator and Executive Assistant to Steve Leibel during the first two editions, for her help in contacting, following, and identifying the authors and co-authors. Dr. Rich Hoppe was ably assisted by Patrice McNealy, Administrative Associate. Dr. Mack Roach was assisted by Michelle Rakin, Administrative Assistant. Alan Taniguchi, Special Projects Analyst and Clayton Akazawa, Treatment Planning Specialist at UCSF were of great help to Dr. Ted Phillips. The staff at Elsevier was indispensable, including Senior Acquisitions Editor Dolores Meloni, who never lost faith; Senior Developmental Editor Janice Gaillard, who kept us on the straight and narrow; and Senior Project Manager Rachel McMullen, who supervised the production. Finally, we would like to thank the authors and co-authors of the chapters for their outstanding work and devotion to the advancement of education in the field of radiation oncology.

Richard T. Hoppe, MD, FACR, FASTRO

Theodore Locke Phillips, MD, FACR, FASTRO

Mack Roach, III, MD, FACR
Table of Contents
Instructions for online access
Front Matter
Copyright
Contributors
Foreword
Preface
Acknowledgments
Section I: Radiation Biology and Physics
Chapter 1: Radiobiologic Principles
Chapter 2: Dna Damage and Repair
Chapter 3: Fractionation Effects in Clinical Practice
Chapter 4: Chemical Modifiers of Radiation Response
Chapter 5: Prediction of Radiation Response
Chapter 6: Radiotherapy and Chemotherapy
Chapter 7: Principles of Radiation Physics
Chapter 8: Imaging in Radiation Oncology
Chapter 9: Molecular Imaging and PET/CT
Chapter 10: Three-Dimensional Conformal Radiotherapy and Intensity-Modulated Radiotherapy
Chapter 11: Immobilization and Simulation
Chapter 12: Image-Guided Adaptive Radiotherapy
Chapter 13: Modern Principles of Brachytherapy Physics
Chapter 14: High Dose Rate Brachytherapy
Chapter 15: Total Body Irradiation
Chapter 16: Intraoperative Radiation Therapy
Section II: Imaging
Chapter 17: Head and Neck
Chapter 18: Thorax
Chapter 19: Abdomen
Chapter 20: Pelvis
Section III: Radiation Oncology
Part 1: Central Nervous System
Chapter 21: Central Nervous System Tumors
Chapter 22: Meningeal Tumors
Chapter 23: Skull-Base Tumors
Chapter 24: Pituitary Tumors
Chapter 25: Radiosurgery
Chapter 26: Spinal Cord Tumors
Part 2: Head and Neck Tumors
Chapter 27: Cancer of the Nasopharynx
Chapter 28: Cancer of the Oropharynx
Chapter 29: Cancer of the Oral Cavity
Chapter 30: Cancer of the Hypopharynx
Chapter 31: Cancer of the Larynx
Chapter 32: Tumors of the Salivary Glands
Chapter 33: Cancer of the Nasal Cavity and Paranasal Sinuses
Chapter 34: Cancer of the Thyroid
Part 3: Tumors of the Thorax
Chapter 35: Tumors of the Lung, Pleura, and Mediastinum
Part 4: Gastrointestinal Tumors
Chapter 36: Cancer of the Esophagus
Chapter 37: Cancer of the Stomach
Chapter 38: Cancer of the Pancreas
Chapter 39: Cancer of the Liver, Bile Duct, and Gallbladder
Chapter 40: Cancer of the Colon
Chapter 41: Cancer of the Rectum
Chapter 42: Cancer of the Anal Canal
Part 5: Urologic Tumors
Chapter 43: Cancer of the Kidney
Chapter 44: Cancer of the Bladder
Chapter 45: Cancer of the Prostate
Chapter 46: Cancer of the Testis
Chapter 47: Cancer of the Male Urethra and Penis
Part 6: Gynecologic Tumors
Chapter 48: Cancer of the Uterine Cervix
Chapter 49: Cancer of the Endometrium
Chapter 50: Cancer of the Ovary
Chapter 51: Cancer of the Vagina
Chapter 52: Cancer of the Vulva
Part 7: Pediatric Tumors
Chapter 53: Pediatric Central Nervous System Tumors
Chapter 54: Pediatric Leukemias and Lymphomas
Chapter 55: Pediatric Bone and Soft Tissue Tumors
Chapter 56: Neuroblastoma
Chapter 57: Wilms Tumor
Part 8: Cancer of the Breast
Chapter 58: Cancer of the Breast
Part 9: Adult Sarcomas
Chapter 59: Tumors of Bone and Soft Tissue
Part 10: Lymphomas
Chapter 60: Hodgkin’s Disease
Chapter 61: Non-Hodgkin’s Lymphomas
Chapter 62: Mycosis Fungoides
Chapter 63: Plasma Cell Tumors
Part 11: Ocular Cancer
Chapter 64: Uveal Melanoma
Chapter 65: Retinoblastoma
Part 12: Skin Cancer
Chapter 66: Cancer of the Skin
Chapter 67: Melanoma
Part 13: Benign Disease
Chapter 68: Benign Disease
Section IV: Emerging Radiation Modalities
Chapter 69: Proton Therapy
Chapter 70: Carbon Ion Radiotherapy
Chapter 71: Molecular Targeted Therapy
Chapter 72: Cancer Stem Cell Biology and Its Role in Radiotherapy
Chapter 73: Tumor-Targeted Radioisotope Therapy
Chapter 74: Hyperthermia
Chapter 75: Stereotactic Body Radiation Therapy
Index
Section I
Radiation Biology and Physics
1 Radiobiologic Principles

Paul F. Wilson, PhD, Joel S. Bedford, DPhil

Fundamental Radiobiology
X-rays and gamma rays interact with biologic material primarily by the Compton effect, producing energetic recoil electrons that traverse the cell and induce ionization events along their tracks by removing orbital electrons, either from critical molecules in the cell (direct effect) or from water molecules located 3 to 5 nm from the critical molecules (indirect effect) . 1 The direct and indirect effects produce highly reactive oxygen and nitrogen species (ROS/RNS, or free radicals), which subsequently diffuse and result in further biologic damage. 2 By the mid-1950s, studies on the various radiation syndromes in mammals following whole or partial body irradiation had led Quastler 3 to conclude that these biologic effects resulted directly from damage to the reproductive capacity of individual stem cells that were responsible for cell renewal in the irradiated tissues. Earlier work on cell killing in bacteria, yeast, and protozoa had found that single acute x-ray doses in the range of 10 to 100 Gy were required to destroy the reproductive integrity of approximately 50% of the cells. This finding led many investigators to erroneously conclude that a similar cell-killing process involving the loss of reproductive integrity was unlikely to account for tissue effects in mammals, including humans, because in mammals these effects were observed after a radiation dose of only a few Gy. If the radiosensitivities of mammalian cell were similar to the radiosensitivities displayed by microorganisms, hardly any mammalian cells at all would lose their reproductive integrity after a few Gy. However, Quastler was able to deduce that this conclusion must be incorrect and that the radiosensitivity of mammalian cells, with respect to loss of their reproductive integrity (that is, the ability to undergo multiple cell divisions to clonally repopulate a tissue), must be much greater than that of microorganisms and did in fact underlie those effects in tissues and tumors that are of interest for radiotherapy. Furthermore, it was recognized as early as 1906 that the manifestation of radiation-induced tissue injury was expressed much earlier and more severely in tissues with rapidly dividing cells than in those with slowly dividing cells. Cell death associated with dividing cells is called mitotic-linked death , and the increase in tissue response with an increase in mitotic activity is referred to as the law of Bergonie and Tribondeau . 4 Finally, for simplification of terminology, cell killing or cell lethality is defined as the loss of the cell’s reproductive integrity; and, conversely, cell survival is defined as the ability of the cell to undergo multiple cell divisions to form a colony after irradiation.
This chapter is devoted primarily to the cellular processes involved in mitotic-linked killing of mammalian cells and other ionizing radiation (IR)-induced cell death mechanisms. Considerable evidence supports the notion that DNA double strand breaks (DSBs), or a subset of DSBs, are the most important lesions mediating IR-induced cellular lethality. Several lines of evidence have led to this assertion. First, selectively irradiating different parts of the cell using charged particle microbeams or incorporated radionuclides showed that the most critical targets for cell inactivation reside in the nucleus. 1 , 5 - 8 Second, cells are very sensitive to killing by the induction of DSBs following incorporation of the thymidine analog 125 iododeoxyuridine ( 125 IdU) into the DNA, 6 , 8 which produces approximately one DSB (or several clustered DSBs) per decay depending on local chromatin topography. 9 - 11 Third, cells are radiosensitized for both cell killing and chromosomal aberration induction by incorporation of the thymidine analog bromodeoxyuridine (BrdU) into DNA, 12 , 13 with the increase in aberrations occurring only where the BrdU incorporates into DNA. 14 , 15 Fourth, and perhaps most importantly, cells that are genetically deficient in rejoining DNA DSBs are very hypersensitive to cell killing and chromosomal aberration induction by IR (discussed further later and in Chapter 2 ). Details of radiation chemistry, the enzymology of DNA repair, and a discussion of the ongoing studies of DNA repair genes are briefly discussed later and in more detail in other parts of this book, as well as in many reviews. 16 - 19 The mutagenic and carcinogenic effects of radiation, which can induce secondary neoplasms following radiotherapy (RT) of the primary tumor, are not discussed, nor are they important aspects of therapeutic gain, which is defined as achieving a greater effect of radiation on tumors than on surrounding normal tissues.

The First Survival Curves for Mammalian Cells
At about the same time Quastler was reaching the conclusions described in the preceding section, Puck and Marcus 20 , 21 made several important observations about the radiosensitivity of mammalian cells. First, they demonstrated that mammalian cells in culture are at least one or two orders of magnitude more sensitive to ionizing radiation than microorganisms, and that the doses sufficient to kill 50% of the animals (LD 50 ) from hematopoietic or intestinal damage might be killing upwards of 95% to 99% of the stem cells responsible for cell renewal in bone marrow and gut. Second, they showed that even though IR-sterilized cells could not proliferate to form macroscopic colonies, virtually all the cells they studied succeeded in completing at least one, often two to three, and sometimes even four to five abortive divisions before proliferation ceased for doses up to a few Gy, and these often formed giant cells that remained metabolically alive but were reproductively dead. Tolmach later showed that these nonclonogenic cells disappear from irradiated cultures with a half time of about a week. 22 Third, Puck and Marcus noted that functions other than reproductive capacity, such as the ability to metabolize nutrients or even the complex processes necessary to reproduce viruses, were largely unaffected by doses of even tens or hundreds of Gy. Fourth, they suggested that chromosomal aberrations induced by radiation were somehow responsible for the killing, based on, among other things, observations that these aberrations began to appear in some cells at the lowest doses at which killing first began to be measurable, and presumably at doses where the most vulnerable of cell processes would be affected.
Puck and Marcus published the first IR dose–response curve for mammalian cell killing for HeLa human cervical adenocarcinoma cells, 20 which is reproduced in Fig. 1-1 A . In general, to obtain such survival curves, single-cell suspensions are prepared and known numbers of cells are inoculated into cell culture dishes containing the appropriate growth medium. Adherent cells then settle to the bottom, where they attach firmly to the dish. Without irradiation, inoculation of 100 single cells into a dish will yield ≤100 macroscopic colonies, with each colony containing thousands of cells that divided from these single cells following incubation for 1 to 2 weeks. This fraction of colony-forming cells in the unirradiated or untreated cultures is known as the plating (or cloning ) efficiency (PE) and is often expressed as a percentage rather than as a fraction. Plating efficiencies for various tumor and normal cells are typically in the 40% to 90% range, although the system works reasonably well for plating efficiencies between 10% and 40%. Below 10%, the cells surviving without treatment may not be representative of the clonogenic population if the plating procedure itself either has sensitized the cells to IR or plating has selectively killed a subpopulation of cells, so caution should be exercised to rule out the influence of such artifacts. Irradiated cultures show reduced fractional survival values relative to unirradiated control cultures. For high doses of irradiation corresponding to survival values of 1% or less, no surviving colonies would be observed for 100 cells inoculated. Therefore, for high doses leading to low survival, the number of cells inoculated per dish is usually increased. The relative surviving fraction S is calculated as:

FIGURE 1-1 • X-ray or gamma ray dose–survival curves for mammalian cells. A, The first such survival curve (for HeLa cervical adenocarcinoma cells) reported in 1956 by Puck and Marcus. 20 B, A range of survival curves for other mammalian cells. The dashed lines encompass the range for wild-type cells of various origins. The steepest curves show a range typical of hypersensitive mutants, such as cells from patients with ataxia-telangiectasia (A-T). Note that in A, the dose is expressed in roentgens (R), which for the cells X-irradiated on glass, must be multiplied by ∼1.4 to obtain the dose in cGy.

where PE is the fraction (not percentage) of unirradiated cells that form colonies compared to the number of cells initially plated.
For large IR doses that yield low survival values or cultures with very low plating efficiencies for which large numbers of cells must be plated, appropriate controls using irradiated nonclonogenic feeder cells should be used to maintain a constant cell density and ensure that variations in survival because of differences in cell density in these cultures do not occur. 23 Note that the survival curve illustrated in Fig. 1-1 A , like many others, have a characteristic shoulder region at low doses; the reduction in survival per unit dose (i.e., the slope of the survival curve) is usually less than the approximately constant slope for survival values below about 0.1. Another interesting finding from these and subsequent studies was that cell populations derived from single cells surviving high radiation doses were not radioresistant, but had essentially the same radiosensitivity as the original parent populations. In fact, in many cases, populations derived from single cells surviving high doses of radiation have been found to be slightly more radiosensitive than the original population. 24 , 25 This finding may relate to the observation that progeny of cells surviving irradiation divide at a reduced rate and have genetic instabilities 26 , 27 and reduced plating efficiencies. 24
Subsequent to these initial survival studies that used cultured tumor cells, cells from human normal tissues were next to be studied, and other techniques and methods were devised to measure survival of both normal and tumor cells in in vivo experimental animal models. Hewitt and Wilson devised an ingenious dilution assay method to determine the dose response for cell killing in a transplantable murine lymphocytic leukemia. 28 They found that, on average, only two cells were necessary to induce a tumor in recipient mice. This corresponds to the inflection point on the titration curve relating the percent of injected animals that develop tumors to the number of cells injected per animal. The number, or dose , of cells for 50% tumor take is known as the TD 50 . Hewitt and Wilson reasoned that if a radiation treatment killed half of the cells, for example, then an injection of twice as many cells would be required to transplant the tumor. In other words, the inflection point or TD 50 of the titration curve would be shifted from two cells for an unirradiated tumor to four cells for an irradiated tumor in which half the cells were killed. Thus, a radiation dose survival curve could be constructed by dividing the TD 50 for unirradiated tumors (derived from unirradiated transplanted cells) by the TD 50 for tumor cells receiving various radiation doses before inoculation. The survival curve the authors obtained for mouse lymphocytic leukemia cells, 28 shown in Fig. 1-1 B , was similar to the curve Puck obtained with HeLa cells.
At about the same time the results of Hewitt and Wilson were published for mouse tumor cells in vivo, Till and McCulloch 29 reported an equally clever technique for assaying the survival of normal mouse hematopoietic cells. In mice (and humans), both the spleen and the bone marrow are major hematopoietic organs. The authors noticed that colonies arising from surviving cells could easily be seen as nodules in the spleen during necropsies of mice euthanized at various times after irradiation. For high IR doses, no such colonies could be seen; however, injection of nucleated bone marrow cells from unirradiated mice could rescue the irradiated mice if the doses were not too high, and some of the injected cells were also found to lodge in the spleen and form nodules. The number of these nodules was directly proportional to the number of cells injected. Thus, the spleens of the supralethally-irradiated mice acted as in vivo culture dishes in which colonies could be counted, and the injection of irradiated cells resulted in proportionally fewer colonies in the spleen. Survival curves constructed in this way using normal hematopoietic cells injected into mice were again similar to those for human HeLa cells and mouse leukemia cells, as also shown in Fig. 1-1 B . These hematopoietic cells responsible for generating spleen colonies have been loosely termed stem cells , although more recent work with cell lineages in the hematopoietic system suggest that such spleen colonies are likely generated by surviving hematopoietic progenitor cells. These are sometimes referred to as spleen colony forming units (CFUs) to avoid implying a precise knowledge of their origin.
These early studies (and several others immediately following) pointed to a general conclusion that the radiosensitivities of mammalian cells examined were quantitatively similar, at least of the same order of magnitude. Doses differed by only factors of about two to three to reach equitoxic survival levels for various cells studied, but no striking systematic differences were observed in relation for tumor cells versus normal cells or those derived from rodents or humans. The range of dose–survival responses for x-rays or gamma rays delivered at high dose rates (20 to 2000 cGy/min) is shown by the dashed curves in Fig. 1-1 B . It is noteworthy that although the majority of reports describing dose–survival responses for a wide variety of cells were carried out using in vitro (ex vivo) culture systems, the in vivo studies of Hewitt and Wilson and Till and McCulloch were not the only ones addressing the question of radiosensitivity of cells in more native in vivo tumor or normal tissue contexts. For example, Withers and coworkers, Gould and Clifton and coworkers, and others, have determined dose responses for in vivo IR-induced cell killing in a variety of tissues, including skin cells (D 0 ∼1.4 Gy), 30 crypt cells of the jejunum (D 0 ∼1.4 Gy), 31 , 32 testes spermatogonial stem cells (D 0 ∼1.7 Gy) ( 33 ), kidney tubule-regenerating cells (D 0 ∼1.5 Gy) ( 34 ), mammary ductal cells (D 0 ∼1.3 Gy), 35 and thyroid cells (D 0 ∼2 Gy). 36 At least for these cell types, the native tissue context does not seem to alter their radiosensitivity relative to typical mammalian cell radiosensitivities documented in vitro.
The previous observations, however, do not rule out potentially important roles for tumor or tissue-specific signaling factors and the local microenvironment in altering tissue IR responses (discussed by Barcellos-Hoff et al. 37 ). It was also recognized at a fairly early stage in the development of modern cellular radiobiology that there were consistent differences among cell types. Cells of lymphoid origin (normal or tumor) appear to be somewhat more radiosensitive than cells from other tissues. It is also important to note that virtually all of the early studies focused on single acute (high dose–rate) exposures, and radiosensitivity was judged largely by examination of survival curves in the 5 to 15 Gy range. In the past 25 years or so, it has become better appreciated that cellular responses in the first decade of survival are not only pertinent for standard multifraction radiotherapy regimens but also can reveal important systematic differences underlying cellular radiosensitivity, especially for cells exposed to low doses or low dose–rates. 38 The surviving fraction after 2 Gy or SF 2 is an index of radiosensitivity that has been widely used. 39 , 40 Recently though, the clinical use of single-fraction (or two to three large-fraction) high-dose radiotherapy referred to as stereotactic body radiotherapy (SBRT) 41 has received much attention and renewed interest in the survival responses of both normal and tumor cells following doses of 15 to 30+ Gy. 42 The role of tumor and normal tissue cell killing and therapeutic gain following SBRT are briefly discussed later in this chapter in the apoptosis section.

Definitions and Descriptions of Radiosensitivity: N’s, D 0 ’s, α’s and β’s
Typical IR cell survival curves are constructed with survival plotted on a log scale against dose on a linear scale (typically in cGy or Gy; where 1 Gy = 1 J/kg and 1 cGy = 1 rad), and are characterized by an initial shoulder followed by a linear (or relatively linear) portion at higher doses (see Fig. 1-1 ). Much effort, thought, and ingenuity in the early days of radiobiology have gone into devising mathematical models to describe the shapes of cell survival curves, based on certain assumptions including mechanisms of cell killing. With appropriate adjustment of various parameters, many mutually exclusive models of radiation action adequately fit any given set of experimental data, so curve fits by themselves tell us essentially nothing about underlying radiobiologic mechanisms. Still, they are useful, principally, in two ways. First, these mathematical descriptions of survival curves provide a very helpful way to catalog and compare radiosensitivities for different cells and different irradiation conditions in terms of these descriptive parameters, so long as one uses the same general mathematical description to fit the survival data. Second, these mathematical survival curve descriptions are useful for predicting the way cell survival may change for different radiation conditions, including changes in dose-rate or dose-fractionation, modification of the chemical environment during irradiation, or changes in radiation quality (linear energy transfer [LET]). Differences in survival may also occur because of alterations in the proportion of various subpopulations of cells that have different radiosensitivities (such as that observed as cells progress through the cell cycle, discussed later).
Historically, the mathematical descriptions of dose–survival curves most widely used for mammalian cells were borrowed from target theory , an analytical approach whose development began in the 1920s and whose aim was to deduce the size and number of critical targets in cells in which IR-induced damage led to a biologic effect. The need for such a target concept, in which damaging ionization events or ion clusters of ∼100 eV are deposited in critical structures such as nuclear DNA, stems from the fact that the amount of energy deposited by exposure to 10 Gy, which kills about 99% of the cells, would raise the temperature of the target material by only about 0.002° C. 1 Thus, the difference is that for IR exposures, relatively large amounts of energy are deposited in very small volumes (sufficient to break chemical bonds), whereas raising the temperature only increases the overall average kinetic energy of the molecules in the absorbing material slightly. It has been widely appreciated that a number of complicating factors make classical target theory largely impractical and inappropriate to describe the molecular mechanisms underlying cell killing. The original hit hypothesis and its development to the so-called target theory dealt mainly with numbers of hits or targets and their distribution among cells, and did not account for complications like heterogeneity of sensitivity within a cell population or to repair processes. More realistically, we can think of the distribution of events or hits that could be lethal and are not processed properly or are misrepaired to leave a lethal lesion some time after an exposure, rather that within milliseconds after exposure.
Although the complications effectively defeat the original aims of target theory, the mathematical dose–survival expressions from target theory reasonably fit observed dose–survival data and have been very useful for descriptive purposes. The simplest of these is of the form:
(1)
where S is the fraction of cells surviving a dose, D. This type of survival curve is illustrated in Fig. 1-2 A . In this expression, D 0 is the dose on the straight-line portion of the log-linear survival plot necessary to reduce the survival from some value S to (e −1  S) or (0.368 S). It is also sometimes called the mean lethal dose because it is the dose necessary to produce an average of one event per target in a cell containing N targets, all of which must be hit to kill the cell. If N–1 of these targets have already been hit, as would be the case in virtually all the cells not yet killed after a dose corresponding to survival values below about 0.1, an additional dose of D 0 would leave an average of one hit in the previously nonhit targets and would thus reduce the survival from S to (e −1  S). The number N, which in this case is the number of targets, can be obtained by extrapolating the high dose, low survival portion of the curve to its intercept on the ordinate at zero dose. This value is more appropriately referred to as the extrapolation number rather than the target number, since the ideal situation in which the two are the same is seldom if ever seen in practice. It is easy to see, however, that the extrapolation number is a measure of the size or width of the shoulder of the survival curve. Another measure is the quasithreshold dose or D q which is the intercept of the extrapolated high dose, low survival portion (below 0.1 survival) of the curve back to the dose axis drawn through the ordinate survival axis at a surviving fraction of 1.0. Another way to determine D q is to recognize that the equation for the high dose portion of the curve is approximated very closely by:

FIGURE 1-2 • Different mathematical expressions relating radiation dose to cell survival and the parameters commonly used to characterize cellular radiosensitivity. A, Illustration of a form of the so-called N–D 0 model and how N, D 0 , and D q are calculated or estimated from such a curve. B, Illustration of the α/β model in which cell killing occurs by either a single-event process or a double-event process such that the overall killing by either process is the product of the two, and the α/β ratio is the dose at which both processes contribute equally to the total killing, i.e., the dose corresponding to the intersection of the two upper curves. Note that the upper curve is survival for the α component only, the middle curve is for the β component only, and the lower curve is for both the α and β components.

Thus, for a surviving fraction of 1.0 corresponding to the Dq dose:
(2)
To actually determine the D 0 from the straight-line portion of the curve fitted to a set of data, for practical purposes it is much easier to divide the dose (D 10 ) necessary to reduce survival by a factor of 10 (e.g., from ∼0.1 to 0.01) by 2.3 (where loge10 = 2.3) than to work out directly the dose necessary on the strait line portion of the dose versus log survival plot to reduce the survival from some value f to a value of e –1 (f) or 0.368 (f). Thus, log e 0.1 = −D 10 /D 0 ; −2.3 = −D 10 /D 0 , and D 0 = D 10 /2.3. Also, D 0 values cannot be estimated very well for data extending over only one log of survival (to 0.1 or above) unless the curve has an extrapolation number very near 1.0. Furthermore, for comparisons of radiosensitivities of different cells or different radiation conditions, survival data over the same dose range are often used for the comparisons, either as estimates of D 0 or as ratios of survival for a given dose, when in fact, comparisons of D 0 values should be made over the same survival range. However, if one curve extends over only one log or decade of survival while the other extends over three logs, for example, the comparison should be focused on the first decade for both curves, ignoring the second and third decades; in this case, the comparison can be the ratio of doses, called a dose modifying factor for isosurvival, or survival values for 2 Gy for example.
By far the main drawback for using the so-called simple multitarget expression ( equation 1 ) is the fact that it usually gives a very poor fit to survival data in the shoulder region, that is, in upper part of the first decade of survival. This is a problem because, as already mentioned, the first decade (in the shoulder region) is the region of the curve that is of special interest for radiotherapy delivered in multiple 2-Gy fractions. The shape of the single acute dose survival curve below about the 0.1 survival fraction is of little interest for this purpose, though for single high dose fraction radiotherapy it certainly is of interest. The multitarget-type curve has zero slope at zero dose, and data indicates that survival curves with large shoulders do not fit the multitarget expression well at low doses (below 2 to 3 Gy). As well as providing better consistency for other considerations such as dose-rate and fractionation effects, better curve fits are obtained by adding a single-hit component to the expression, giving:
(3)
where 1/D 1 + 1/D n = 1/D 0 and D 0 is the reciprocal slope of the curve as defined earlier and N is the extrapolation number. This expression provides a good fit to practically any set of data. It retains the N – D 0 convention as an index of radiosensitivity and, as mentioned earlier is also able to handle analyses involving changes in survival with dose rate, LET, and other factors affecting responses.
Another mathematical approach to the description of dose–survival relationships which lends itself to analyses involving changes in dose rate and or other modifying factors, and which has other appealing features, is the so-called α–β model. In this model, survival follows the expression shown in Fig. 1-2 B :
(4)
One of the several appealing features mentioned earlier is that this model readily derives from a mechanism of cell killing that we now know to be largely correct, although for some cells, additional mechanisms such as apoptosis also contribute to cell killing (see later). The mathematics amounts to a simple description of the dose response for the formation of chromosomal aberrations, rather than some abstract targets of obscure identity and behavior. The model derives from the fact that cells die largely as a result of loss of genetic information in the progeny of cells bearing certain chromosomal aberrations induced by radiation. This is not just one of a number of plausible mechanisms of cell killing, but there is a large body of evidence to support it. 21 , 43 - 48 Not only has quantitative agreement been found between chromosome aberration induction and cell killing, but the strongest possible evidence is that provided by Revell and associates, 49 who showed that virtually every diploid cell of the types they studied irradiated in G1 and reaching the first mitosis with a fragment-generating (micronucleus-generating) aberration failed to form a colony, while every irradiated cell reaching the first mitosis without such an aberration did form a colony. Virtually all the cells they studied did reach the first mitosis. If these aberrations and the putative lethal events were unrelated in a cause-and-effect sense, and some other unseen lesions with the same radiosensitivity for its production were actually the real lethal lesion, then some cells with aberrations should have formed colonies while some cells without aberrations should not. The latter was not observed. As already mentioned other mechanisms of cell killing such as apoptosis, may contribute to cell killing to a greater or lesser extent depending on the circumstances (mainly the cell type). For some agents and particular hormonal induction processes, apoptosis may account for all of the cell death; but for exposure to ionizing radiation, most cells die principally as a result of the production of certain chromosomal aberrations. Apoptosis, when it is involved does not nullify the lethal effects of these aberrations in cells bearing them, but only adds to the overall lethality when it is involved.
Most chromosomal aberrations are exchange types requiring two chromosome breaks for their formation, with one event or electron track producing one break and another independent event or second electron track producing the other, or in some proportion of the cases, with one electron track producing both breaks ( Fig. 1-3 ). When two independent events must occur in close proximity in space and time, the frequency of such double events increases as the square of the total number of separate single events. This is analogous to second order or bimolecular reaction kinetics in chemistry. The single break events are, of course, directly proportional to the radiation dose, so the exchanges from a coincidence of independent events will increase as the square of the dose. Although when two breaks occur close enough together to form an exchange, they do not necessarily do so. Instead, they can restitute by joining the way they were, or they may fail to rejoin, which would result in a terminal deletion. In fact, exchange-type aberrations are much more frequent than terminal deletions in human cells. The most interesting events are those that result from the closely spaced breaks that mis-rejoin to form either a symmetrical (nonlethal) or an asymmetrical (usually lethal) exchange. When an exchange occurs between two chromosomes, the exchange is symmetrical if the exchange does not join the two centromeres together and is asymmetrical if the two centromeres are joined together. Thus, the initial break-pair forms what may be termed a potentially lethal break-pair or simply a potentially lethal lesion. This will be discussed in more detail in connection with a certain kind of repair process. As mentioned earlier, the yield of these independently produced lethal aberrations per cell, Y 2 , increases as the square of the dose D, or

FIGURE 1-3 • The α/β description of chromosome aberration production Y = αD + βD 2 where Y is the average chromosome aberration yield per cell and D is the dose. The single-track α component of chromosome-type asymmetric inter change production (dicentrics) is shown on the left , where a single electron track produces a break-pair in each of two different chromosomes and (in some proportion of the cases) these subsequently rejoin asymmetrically to produce a dicentric and acentric fragment. The same general type of exchange process occurring in the same chromosome can produce asymmetrical chromosome-type intra changes yielding interstitial deletions. These types of exchanges can also occur as a result of two independent electron tracks, as shown on the right . After high doses, for which the two-hit β component predominates, most of the aberrations seen in the first mitosis after G0/G1 irradiation are of these types. After low doses or dose rates for which the one-hit α component predominates, there is an increase in the proportion of aberrations that are terminal deletions, in which breaks simply fail to rejoin. In human cells, however, very few terminal deletions are observed compared to exchange types, even after low doses of IR.

where β is the proportionality constant relating to the fraction of potentially lethal break-pairs that are actually converted to lethal aberrations under a given condition. It is also possible, however, that two breaks could be produced along a single electron track. For example perhaps this would be more likely near the end of an electron track where the number of ionizations per unit track length (LET) increases. 1 In any case, the yield of such two-break single-track events would simply increase in direct proportion to dose D, rather than as the square of the dose so the yield Y 1 , would be

where α is a proportionality constant. The total yield of lethal aberrations per cell Y would then be the sum of Y 1 + Y 2 so
(5)
The total aberration yield per cell Y is of course only an average; it does not mean that every cell has exactly Y aberrations. But if aberration induction occurs independently in cells and all cells have the same sensitivity, the probability that a cell will have exactly n = 0, 1, 2, 3, etc. lethal events can be calculated, because such processes are well described by the Poisson distribution that states that

where P x is the probability that exactly x events will occur for a given trial when the mean number of events over a large number of trials is µ. In our case µ = Y, and the cell must have no lethal events (aberrations) to survive, so x = 0 and the probability of exactly no events is:
(6)
and since for large numbers of cells or trials, P 0 → S (the fraction surviving), and Y = αD+βD 2 , we can write which is equation (4) displayed earlier and plotted in Fig. 1-2 B . Curves of the form shown in Fig. 1-2 B fit experimental data very well, especially over the first and second decades of survival, but it is also the case that the curve slope continues to increase for higher doses so the curve fit is not always good in the higher dose region. This is an issue in situations where it may be desirable to estimate cell survival for high dose single fraction radiotherapy, but we will return to this later. In addition, recent studies have shown that a substantial fraction of aberrations for doses above 3 to 4 Gy, are complex , meaning they involve 3 or more breaks in 2 or more chromosomes. 50 - 53 The ways in which these form, and the impact on the mathematical description of dose responses from a mechanistic standpoint are not presently known, but there is still no doubt that the increase in lethal asymmetrical exchange aberrations closely follows a linear-quadratic function of dose. It has been argued that one flaw of this linear-quadratic model is that it gives a survival curve that continuously bends downward at higher and higher doses, whereas, very good experimental data for cell survival over 5 or more decades of cell killing tend to show a straight line on a log linear plot for the whole range of survivals below about 0.1. In fact, this is not a very good argument against the connection between aberrations and cell killing, because these lethal aberrations do not actually increase as αD+βD 2 at doses much higher than about 8 to 10 Gy; that is, they tend to saturate and approach linearity at high doses (at least up to 15 to 20 Gy).
The earlier arguments for model descriptions that best serve the purposes described have been well recognized and addressed over the past decade or so. These have been well described by several authors and the interested reader may find the descriptions and discussions of Dale, 54 Carlone and colleagues, 55 Guerrero and Li, 56 and Sachs and colleagues 57 are of particular interest. For most purposes, the α/β description is probably the best basic starting model to use, but because many workers still use the N – D 0 description, we have included it in this chapter as well.

Cellular Processing of Radiation Damage
Cells are able to process radiation damage and are largely able to repair the molecular lesions that can lead to chromosomal aberrations and cell death. In some instances, however, the repair may be incomplete, or misrepair may occur. As we mentioned previously, most lethal chromosome aberrations are exchanges (in human and other types of cells, mostly asymmetric intra changes yielding interstitial deletions or inter changes yielding dicentrics with an acentric fragment). Two events must occur close together for such exchanges to occur, not only in space but also in time. Cells are able to repair or rejoin chromosome breaks, so if the entire radiation dose is delivered over a period of a few seconds or minutes, all the breaks that are ever going to be produced will occur together, or for practical purposes, nearly simultaneously. After such a dose given in a short period ( acute dose), there would be a certain number of break-pairs, each produced by two independent electron tracks spaced close enough together for a possible exchange. However, if the same total dose were delivered over a period of several days, for example at a low dose rate, then fewer of these independent two-track break-pairs that could form potential exchanges will occur simultaneously. Even though breaks may occur in the same proximity in the cell nucleus, the break-pairs would never (or rarely) exist together, because the first break would have rejoined and disappeared long before the second break of the pair needed for an exchange ever arrived. Thus, the βD 2 component is very dose rate–dependent. There would, of course, still be the same number of break-pairs produced by the single-track mechanism; therefore, this αD component of cell killing is dose rate–independent and also important for mediating biologic effects of very low acute doses since the higher LET-like track ends of recoil electrons may well produce most of the lethal damage. 1 These concepts are illustrated again by reference to Fig. 1-3 . Note that the formation of complex aberrations, which involve ≥3 breaks on two or more chromosomes, requires some refinement of the mechanistic description, but this does not alter the established fact that aberrations for the most part underlie cell killing in fibroblasts and epithelial cells, and at least up to doses of 8 to 10 Gy, these increase as a linear-quadratic function of X-ray or gamma ray dose.
Local chromatin topography (compaction status, epigenetic and histone modifications, accessory proteins, etc.) plays a major role in the accessibility of various repair proteins to DSBs and therefore modulates aberration induction. Evidence for this comes from the observation that both IR and restriction endonuclease-induced DSBs occur preferentially in G-light chromosome bands, i.e., euchromatic, more open regions of active gene transcription. 58 - 64 Similarly, a study by Barrios and coworkers who examined cells from radiotherapy patients found that the preponderance of exchange points of radiation induced interchanges occurred in G-light bands. 65 It is known that the heterochromatic inactive supernumerary X chromosomes of individuals with Kleinfelter syndrome are resistant to IR-induced chromosome interchange formation. 66 , 67 Recently, a study by Cowell et al. demonstrated γ-H2AX foci generated at sites of IR-induced DSBs form preferentially in euchromatic regions of the interphase nucleus. 68 The next topics for discussion are the well-studied cellular repair process known as sublethal damage repair (SLDR) and potentially lethal damage repair (PLDR). These are terms that are operationally defined .

Sublethal Damage Repair
In 1957, Jacobson presented some of the first evidence that X-irradiated cells (chlamydomonas) sustain sublethal damage (SLD) that can be repaired. 69 Jacobson showed that a radiation dose delivered in two separate fractions separated in time gave a higher survival than if the dose was given in one single fraction. The first such study of the survival of mammalian cells by Elkind and Sutton (1959) compared single-fraction acute dose radiation survival of V79 Chinese hamster cells to that following doses split into two separate fractions and delivery times. 70 In 1960, Elkind and Sutton 71 extended these observations and reasoned that the shoulder on survival curves for mammalian cells (or any other cells with similar survival curves having shoulders) by itself indicates that a damage accumulation process must be involved in cell killing. 25 Some arguments have arisen over this point because notions of repair saturation have been suggested, but even in this instance, the putative saturation will still require damage accumulation. It also follows from this argument that cells surviving a radiation dose high enough that the survival would be off the shoulder (survival <0.1) must be sublethally damaged. In other words, cells have accumulated damage that makes them more susceptible to killing by the next IR dose than if they had not received any radiation. For this reason, such damage was termed sublethal damage or SLD. 25 , 71 The question they then asked was, if a population of sublethally damaged cells surviving one dose were allowed to incubate for various periods, would the cells be able to repair this sublethal damage? If repair of all the sublethal damage occurred, a dose–survival curve determined for these surviving cells should have a shoulder similar to cells that had never been irradiated. If no repair occurred, the dose–survival response should be a simple exponential decrease continuing at the survival level corresponding to the initial dose and along the same curve as for the original cell population.
As illustrated in Fig. 1-4 A , the shoulder of the survival curve indeed returned, indicating that the cells had recovered from their sublethal injury and implying that the sublethal damage had been literally repaired , so the cells were in this respect “restored to their original condition.” In further studies, Elkind and coworkers found that this shoulder returned with the half time for SLDR being of the order of half an hour. 25 However, as discussed in the next section on cell cycle effects, a complete return of the shoulder does not necessarily mean that all sublethal damage has been repaired. Many other workers have obtained similar results both for cells in vitro and in vivo, and repair half times range from about 0.5 to 1.5 hours. Typically, the way such studies are carried out is by the split dose technique, in which changes in survival are measured as a function of incubation time between two equal doses. The technique is illustrated in Fig. 1-4 B . Generally, for an in vitro experiment, a number of identical cultures would be set up, all of which have the same number of cells that receive the same total dose, 2D; the only difference is that for some cultures, the dose would be given all at once, although for others, the dose would be given in two fractions of D, separated by various periods of time between the doses.

FIGURE 1-4 • Illustration of sublethal damage (SLD) and its repair (SLDR) (A), and the rate of SLDR (B). A, A single-dose dose–response curve (lower curve) and another curve obtained by first delivering a dose of 5 Gy and then incubating the cells (in this case 8 hours) before determining survival for irradiated cells that survived the first dose. If these cells returned to their initial preirradiation state, they should respond (as they do) along the upper curve from 5 to 10 Gy, whereas, if no change occurred during incubation in the cells surviving the first dose (i.e., no recovery from sublethal damage), the response would continue along the lower curve from 5 to 10 Gy. The increase in survival after the total dose of 10 Gy compared to the two 5 Gy radiation doses amounts to a factor of ∼5 in this case. B, The rate at which this increase in survival occurs shown as a function of time between the two IR doses. The complex curve for cycling cells results from the fact that there is a large difference in the radiosensitivity of cells depending on their position in the cell cycle (see text and Fig. 1-5 ). For noncycling cells, this cell cycle–related complexity is not observed.
The concept of sublethal damage and SLDR is readily apparent in the induction of fragment-producing exchange-type aberrations. One break by a single electron track is sublethal; by itself it almost always rejoins and is not lethal. After a certain dose, at which the survival is less than about 0.1, surviving cells will have many such single (sublethal) breaks and will, therefore, be more susceptible to killing by a further dose than if they had no previous irradiation. With incubation before a second dose, these sublethal single breaks will gradually disappear, and if the second dose is given much later, the survival of the cells will be much the same as if the cells had never been irradiated. Sublethal damage, then is damage which exists in surviving cells, by itself is not lethal, and is evident in the way it influences the response of cells to a second radiation dose. The repair of sublethal lesions is responsible for the dose-rate and dose-fractionation effects that are so important as a fundamental process in radiotherapy.
According to this view, two breaks produced by a single electron track can also lead to a lethal exchange, and the number of such events is strictly proportional to the total dose; that is, it is dose rate–independent. The contribution of this single-event two-break process to the total lethal damage is given by the αD term of equation 5 . It is only the βD 2 term that governs dose-rate or dose-fractionation effects related to SLDR. In the simple case of the split dose experiment described earlier, if the total dose 2D is given all at once, the lethal aberration yield will be:
(7)
If the total dose 2D is given as two doses of D separated by a time sufficient to allow complete repair of sublethal damage, the yield of lethal aberrations will be:
(8)
Notice that the αD terms in equations 7 and 8 are the same, but the βD 2 terms differ by a factor of 2, which translates to a survival for the split dose treatment (using equations 6 , 7 , and 8 ) that is higher by a factor of than for the single 2D dose. For many small doses, or in the limit of continuous irradiation at a sufficiently low dose rate, the βD 2 term disappears altogether, and the only remaining damage is that from the αD component. By the same arguments, a dose-rate reduction factor depending on the rate of rejoining of breaks and their rate of formation can be applied to the βD 2 or repairable component of damage. This was first done by Lea 72 for the yield of chromosomal aberrations for doses delivered over different periods of time, and by Kellerer and Rossi 73 in their theory of dual radiation action. Variations on this same theme 54 , 74 , 75 as well as other approaches 76 also have been described. As mentioned earlier, the recent work of Guerrero and Li 56 ; of Carlone et al. 55 ; and of Sachs et al. 57 are particularly pertinent. The general case for different dose rates derived by Dale 54 is:
(9)
where
(10)
and R = dose rate in Gy/hr, µ = repair constant in hr −1 (with the half time for repair equal to 0.693/µ), and T = total duration of irradiation in hours. For very low dose rates of ∼0.3 Gy/hr or less, S = e − αD .
There has been a great deal of discussion in recent years about the relative radiosensitivity and repair capacities of cells whose damage leads to early versus late effects in normal tissues, or effects in tumor versus late normal tissue damage. This discussion generally has been placed in the context of the α/β cell survival expression. 77 The point in question is how the responses of these tissues change with dose fractionation and dose rate. Generally, there appears to be a greater dose fractionation sparing effect with late-responding normal tissues than with early-responding tissues. In terms of α, β, and SLDR we have discussed, the radiosensitivity of the cells whose damage underlies late effects has a larger β component relative to the α component than the radiosensitivity of tumor cells or cells for early effects. Put in another way, the α/β ratio is smaller for late effects than for early effects. The α/β ratio gives an index of the proportion of damage subject to the dose fractionation sparing effect. If α were zero, the ratio would be zero, and all the damage would be reparable; therefore, for low enough dose rates or a sufficiently low dose per fraction, the killing effect of radiation would disappear altogether. As β approaches zero, the ratio increases without bound, so no dose rate or fractionation effect at all would be seen; in fact, the survival curve would be linear without a shoulder and would be described by the α component only. Furthermore, as illustrated in Fig. 1-2 B , the α/β ratio corresponds to the dose for which there is an equal contribution to the damage from both the α and β components. Thus, when



The relationship between survival and dose (using values of α and β in equation 4 ) has been used to convert one fractionation scheme to another fractionation scheme that is calculated to give the same amount of radiation damage as the original scheme. 78 For example, n 1 fractions of dose d 1 /fraction can be converted into n 2 fractions of dose d 2 /fraction with the following equation:
(11)

Variation in Radiosensitivity Through the Cell Cycle
So far we have only briefly mentioned an important factor complicating the interpretation of dose fractionation and dose-rate effects, namely the observation that the radiosensitivity of cells changes through the cell cycle. This phenomenon was first discovered by Terasima and Tolmach 79 and has been studied extensively by many others. 12 , 80 - 84 Fig. 1-5 shows dose–survival curves for synchronous cells irradiated at different stages of the cell cycle. Generally, cells in late G2 and mitosis are most sensitive, cells in mid-to-late S phase (the portion of the cell cycle when DNA is replicated) and early G2 are most resistant. Often, cells around the G1/S border (late G1/early S) are sensitive, and cells in mid-G1 are more resistant. The effect of this differential cell cycle radiosensitivity when asynchronous, randomly dividing cell populations are studied in dose fractionation experiments is that the first dose selectively kills the more radiosensitive G1 and G2 cells in the population. The surviving S phase cells continue to repair sublethal damage and begin to progress towards the more sensitive G2 and G1 phases in the cell cycle, following release from a dose-dependent S-phase delay. Both phenomena affect the way in which these cells will respond to the second dose. For example, after the first dose, the selective survival of the more radioresistant S phase cells, reflected by a large shoulder on the survival curve, complicates the interpretation of whether or not recovery of sublethal damage is complete. For complete recovery, the shoulder of the survival curve for the second dose should be larger than the shoulder for the first dose delivered to the original asynchronous cells (consisting of both radiosensitive G1 and G2 phase cells and radioresistant S phase cells). 85 Because the rate of SLDR is usually faster than cell cycle progression, SLDR leads to the rapid early increase in survival in split-dose experiments. By contrast, cell cycle progression leads to a gradual decrease in survival over time (via cell cycle redistribution or reassortment), but is eventually followed by an increase in survival as the semisynchronous surviving population divides (repopulates) (see Fig. 1-4 B ). These three processes have been referred to by Withers as three of his four R’s of radiotherapy, that is, repair, redistribution, and repopulation (along with the fourth R, reoxygenation , discussed later). 86

FIGURE 1-5 • The cell cycle dependence of radiosensitivity. The figure shows typical survival curves for cells irradiated in various phases of the cell cycle. Also shown is the survival curve for an asynchronously dividing log phase culture, which is composed of a mixture of cells with their sensitivities indicated. In this case, the curve labeled asynchronous mixture is a composite curve for a cell population consisting of 15% of cells in late-G1/early-S , 50% in mid-S , 25% in late-S/early-G2 , and 10% of cells in late-G2/M . This curve for asynchronous cells looks fairly homogeneous, and it would be difficult to deduce from a simple inspection of the curve that, in fact, it is derived from a cell population composed of subpopulations with very different radiosensitivities.
Cell cycle dependent radiosensitivity and progression of cells in the cycle after irradiation can be important factors, both in comparing the radiosensitivities of different cell types and in determining the responses of cell populations in normal tissues and tumors when doses are fractionated or delivered continuously at low dose rates. 38 , 81 , 87 - 89 For example, accumulation of cells in the radiosensitive phase at the end of G2 can, in certain instances, overshadow the dose-rate effect due to repair, such that the effect per unit dose actually increases rather than decreases with a reduction in dose rate. 88 In general, irradiated cells have the greatest cell cycle delay during G2, with approximately 1 to 2 min per cGy for cells irradiated in late S and G2 25 and about one third less delay during G2 when they are irradiated in G1 or early S. 90 The delay from G1 into S is usually only an hour or two, even for doses as high as 6 Gy, 90 but appears to increase in cells that express wild-type Tp 53 (discussed later). 91 The accumulation of cells in G1 that is observed approximately 16 hours after irradiation in cells expressing wild-type Tp 53 is also associated with accumulation of cells in G2, 92 and this reduction in the number of cells in the radioresistant S phase should cause a reduction in the shoulder of the survival curve for a second dose delivered ∼16 hr after the first dose (see Fig. 1-4 ). A simple illustration of effects of heterogeneous populations on radiation survival curves, with explanatory equations, was presented many years ago, 93 and has been discussed and applied in practice on other occasions. 25 , 94
Finally, it is extremely important to consider the overall distribution of cells in the cell cycle when comparing the radiosensitivities of different cell lines and other IR-related biologic endpoints. For example, if two cell lines, line A and B, had identical radiosensitivities, but the line A culture was primarily distributed in G1 at the time of irradiation, while the line B culture had its cells primarily distributed in S or was dividing asynchronously, a comparison of the radiation responses of cell lines A and B would lead to the erroneous conclusion that line A is more radiosensitive than line B (compare G1 with asynchronous or S cells in Fig. 1-5 ). This important concept of intrinsic cellular and differential cell cycle-phase radiosensitivity prompted Steel, McMillan, and Peacock 95 to suggest a fifth “R” for radiation therapy, in addition to Wither’s original 4 R’s. 86

Potentially Lethal Damage Repair
Cell survival also can be altered by the incubation conditions of cultures after irradiation. Many years before the work with mammalian cells, experiments showed that holding bacteria for a time after irradiation in a buffered salts solution and then plating for the survival assay gave a much higher survival than if the cells were kept in full nutrient broth after irradiation. 96 This phenomenon is referred to as liquid holding recovery. Similarly, if mammalian cells are held in suboptimal growth conditions for various times after irradiation, such as by treating cells with metabolic inhibitors, 97 balanced salts solution, 25 , 98 reduced temperatures, 44 , 99 or maintaining cultures in G0/G1 contact inhibition 48 , 100 or under serum starvation, 101 their survival is greatly enhanced. This is illustrated in Fig. 1-6 for holding cultures in plateau phase. 102

FIGURE 1-6 • A, The effect of potentially lethal damage (PLD) and its repair (PLDR) on the dose–survival responses of cells for immediate versus delayed subculture. B, Survival increase due to PLDR after 6 Gy as a function of time of delay of subculture. PLD is operationally different from SLD. PLD is damage that is either expressed or repaired depending on postirradiation conditions. Cells that may survive for a dose given under one condition but may not survive the same dose under another condition. In contrast to SLDR, the increase in survival resulting from PLDR is not caused by a change in the response of cells surviving one dose before a second dose, but, in this particular example, but rather it shifts the balance between damage repair and misrepair or fixation in favor of increased repair when cells are kept in density-inhibited plateau phase (delayed subculture) rather than being immediately subcultured after irradiation.
In Fig. 1-6 A , survival curves are shown for apparently normal primary human fibroblasts irradiated as plateau-phase G0/G1 cultures and then either subcultured immediately or 24 hours later to assess survival by single-cell colony formation. In Fig. 1-6 B , the rate of PLDR is reflected by an increase in survival as a function of the time delay between irradiation and subculture for the survival assay for cultures receiving the same dose. The interpretation of such observations was that radiation produced lesions that were potentially lethal , and that under one set of incubation conditions, a damage repair process operating in opposition to a damage fixation process resulted in a certain fraction of these lesions being converted to nonlethal lesions (via repair and restitution) while the remaining fraction was fixed into lethal lesions that killed the cells (via an improper misrepair event or a lack or repair). 103 As postirradiation conditions are changed, they may become more favorable to the repair processes, less favorable to the fixation processes, or both, so that the final result would be a higher proportion of the potentially lethal lesions being converted into nonlethal lesions. From the perspective of chromosomal aberration formation, a highly quantitative correlation between cell survival and the yield of aberrations is observed following manipulation of postirradiation culture conditions. 48 One set of incubation conditions may favor the restitution of breaks as opposed to exchange, while for another set, restitution is less favored. 44 , 104 The reason for this phenomenon is not known, although we know that certain suboptimal growth conditions, such as holding cultures in plateau-phase confluency, favor restitution. Also, treatment with anisotonic salts, which inhibits PLDR and SLDR, 105 , 106 greatly affects chromatin structure, which could alter the proximity of lesions, such as DSBs and clustered DNA damage lesions, 107 in some way to affect the fixation and repair processes involved in aberration formation. 104 , 108 , 109
Numerous reports of postirradiation colony formation ability, mitotic and PCC-induced chromosomal aberration induction, gel electrophoresis and γ-H2AX foci kinetics over the last 40 years have documented similar kinetics of PLD repair, 48 , 85 , 100 , 110 - 117 with the majority (≥90%) of PLD repaired by 6 hours. Repair of PLD has been noted in most cell lines examined (except ATM −/− and BRCA1 −/− cells), both normal and malignant in origin, making clinical use of chemical inhibitors of PLD processes ineffective for cancer treatment purposes. 118 - 120 Weichselbaum et al. proposed a link between intrinsic PLD repair capability and the radiocurability of tumors, 121 but further work 119 could not substantiate such a correlation. Although the degree of cellular PLD response may be of little prognostic value in the radiotherapy clinic, such variations may be of great importance for the study of the responsible molecular repair mechanisms. For example, Schwartz et al., 122 Franken et al., 123 and Alsbeih et al. 124 recently showed PLD repair to be a Tp53-dependent process. Functional inactivation of Tp53 by HPV-16 infection or direct mutation of Tp53 in human and isogenic mouse cell lines significantly moderated the increases in survival following 24 hours of confluent holding. However, since Tp53 gene inactivation is a classic feature of transformed and tumor cells, the observation that many tumor cells demonstrate PLD repair indicates other (Tp53-independent) pathways in these cells must also contribute to this response.

Chemical Modification of Radiation Damage
Details on modification of radiation damage by chemical sensitizers such as oxygen or other electron-affinic agents or chemical protectors such as sulfhydryl amines or thiophosphates are provided elsewhere in this volume, but it is appropriate to mention briefly the way such agents act to modify the parameters of dose–response relationships discussed earlier in this chapter. Most of these agents act as dose-modifying agents and alter the dose response curve by a parameter termed the dose modification factor (DMF). This is called a synergistic type of interaction between the modifying agent and IR, which is distinctly different from an additive type of interaction that only reduces the shoulder of the survival curve. For an independent type of interaction, the overall shape of the IR survival curve is unaffected, but is shifted downward by an amount equal to the reduction in survival (the DMF) resulting from treatment with the modifying agent. 125 In fact for these types of independent interactions, following treatment of synchronous populations or asynchronous populations with minimal variation is modifying agent-induced cytotoxicity through the cell cycle, the net survival for the combination of both agents is simply the IR survival multiplied by survival for the dose of modifying agent employed (see Dewey et al., 12 and Dewey 125 for these definitions and discussion of complications caused by cell cycle variations in sensitivities to the two agents).
Oxygen is the classical example of a synergistic type of interaction with IR. Oxygen sensitizes cells to IR by reacting directly with damaged target radicals to fix them or render them less capable of repair by a competing, fast hydrogen-donating reaction, which restores the damaged target molecules to their original condition. Other cellular species, such as reactive nitrogen species (RNS), can also act in a way similar to oxygen by causing damage fixation (not repair). When the concentration of oxygen is reduced, for example to only about 1% to 2% of the concentration of O 2 in air (159 mm Hg), fewer oxidative radicals are present to fix the damage, so increased fast repair by hydrogen donation occurs. Oxygen, therefore, simply increases the yield of fixed target radical species, producing essentially the same effects observed by increasing the low LET radiation dose without the presence of oxygen. This oxygen effect disappears with increasing LET, and oxygen must be present during irradiation or within a few milliseconds after irradiation to produce radiosensitization. The role played by oxygen in metabolic respiratory processes has nothing to do with its radiosensitizing effect. The oxygen effect is the same whether the irradiation occurs at 37° C or at 0° C, at which point oxygen-dependent cellular respiration is greatly slowed.
When a sensitizing agent like oxygen acts as a simple dose modifying agent—in this case, the DMF is ∼3.0 for fully oxygenated versus anoxic conditions (this is the maximum oxygen enhancement ratio or OER)—then, only about one third the IR dose or D/3 is required to produce the same level of effect produced by the dose D in the absence of oxygen. The effect on the mathematical dose–response relationship and survival curve is then easy to calculate, as shown below. For the N – D 0 expression, if the survival under anoxic conditions S N is

then under fully oxygenated conditions the dose D would effectively be multiplied by a DMF of 3, so

As can be seen by multiplying both the numerator and denominator of the exponent by , so that

the oxygen effect effectively reduces the D 0 by a factor of 3 without changing the extrapolation number N. A similar calculation applies for the N, D 1 , D n –based expression in equation (3) .
For the α/β survival expression, if the survival under anoxic conditions S N is

the survival under fully oxygenated conditions is therefore

or

Oxygen in this case has the effect of increasing the anoxic α constant by a factor of three and the anoxic β constant by a factor of 9.
This threefold increase in dose required for a given amount of IR-induced cell killing under hypoxic conditions compared with irradiation under oxygenated conditions is thought to play an important role in radiotherapy. 126 - 128 Many tumors contain a significant fraction of hypoxic and therefore radioresistant cells compared to the well-oxygenated cells in surrounding normal tissue. For a single acute dose of IR, the dose required to kill about 90% of the tumor cells (e.g., a survival fraction of 10 −13 for a tumor with 10 12 clonogenic tumor cells) would have to be about three times larger than it would be if all of the cells in the tumor were well oxygenated. However, the situation is not this bleak, because in most tumors, considerable reoxygenation occurs during fractionated radiotherapy. This reoxygenation effect is Wither’s fourth “R” of radiotherapy, which follows the three “R’s” mentioned previously, i.e., repair, redistribution, and repopulation . 86 It should also be noted that not all tumor cells are likely to be equally clonogenic and capable of regenerating a tumor (often referred to as tumor or cancer stem cells); this will lower the total number of cells in a tumor that require inactivation. 129 , 130
Similar calculations can be made for radioprotective compounds that act as dose modifying agents, except that to calculate the biological effect in the presence of the protector, IR doses delivered in the absence of the protector must be divided by the DMF. Cysteine has been shown to act as a dose modifying radioprotector with a DMF of about 1.5 to 2, which results in an effective reduction of α by and β by . 131 , 132

Radiation Quality, Relative Biologic Effectiveness, and Linear Energy Transfer
Radiation quality has been known for a long time to affect biologic responses to ionizing radiation. Densely ionizing radiations such as alpha particles and heavy charged particles (HCP) are more effective in killing mammalian cells than sparsely ionizing X-rays or gamma rays. The relative biologic effectiveness (RBE) of radiations with different qualities (i.e., different linear energy transfer or LET values) is defined as the ratio of doses of two different types of radiation that are required to produce the same degree of the same biologic effect. X-rays generated at 250 kV p have been widely used in radiobiologic studies, so they became the standard used for RBE comparisons and calculations. The RBE of a test radiation in question is then defined as:

Naturally, other irradiation conditions such as oxygenation levels, dose-rate, and so on, should be equal.
An example of the dose–survival curves for mammalian cells for radiations of different LET is shown in Fig. 1-7 . The LET is, to a first approximation, a track-averaged description of the density of ionizations along the ionization track in irradiated material and increases as the LET increases. Actually, more sophisticated microdosimetric and nanodosimetric quantities (such as lineal energy) are used for precisely describing radiation quality, but for our purposes, LET is a sufficient tool. Low-LET ionizing radiations such as β particles and secondary electron recoils from X-rays and γ-rays deposit energy stochastically in materials by liberating secondary electrons with tortuous track structures and have LET values on the order of 0.3 to 1 keV/µm (β particles are primary electrons released from radioactive decay processes of unstable radioactive nuclei). High-LET ionizing radiations such as alpha particles, nuclear fragment recoils from high-energy neutron interactions, and other HCP such as constituents of galactic cosmic radiation (GCR) deposit their energy along more direct tracks producing nuclear (i.e., atomic) fragments and scattering secondary high-energy delta-ray electrons with combined LET values that may approach 1000 keV/µm or more. 1 It should be noted that protons used in radiotherapy have an LET of ∼1.0 keV/µm, similar to x-rays and gamma rays. 133 , 134 Protons have the primary advantage of improved dose distribution compared to photon and electron beams, however the risks of low dose scattered neutron exposures in the surrounding normal tissues remains to be determined and addressed. 135

FIGURE 1-7 • Dose–response curves for radiations with different linear energy transfer (LET) values. The relative biologic effectiveness (RBE) of neutrons or alpha particles relative to the x-rays is defined as a ratio of doses required to produce the same level of biological effect, with the numerator of the ratio being the x-ray dose and the denominator being the dose of the other radiation type being compared.
The relationship between RBE and LET can be illustrated in terms of cell killing caused by the induction of chromosomal aberrations. For low LET x-rays or gamma rays, the distance between ionizations or ion clusters along an individual recoil electron track is almost always greater than the minimum interaction distance for two chromosome breaks to interact to form an exchange (∼0.2 to 0.5 µm 136 , 137 ). Therefore, two or more electron tracks are usually necessary to form an exchange, and most of the ionizations that contribute to the total dose are “wasted” because they are unlikely ever to be involved in generating a potential aberration-producing break-pair. As the LET increases, a peak in RBE is eventually reached at which an optimum spacing of ionizations occurs for producing break-pairs with maximum efficiency. 1 This optimum RBE also probably includes a repair component, since the higher density of damage also appears to influence the processing and repair of DNA damage and also the complexity of IR-induced chromosomal aberrations. 107 , 138 - 140 Depending on the biologic endpoint measured and other factors such as total dose and dose-rate, RBE values from ∼1 for low-LET radiations to >100 for high-LET radiations have been calculated. Maximum RBEs of 1.2 to 1.6 for DNA DSB induction 138 and PCC-induced interphase chromosome breaks 141 and ∼3 to 10 for chromosomal aberrations and cell killing occur at an LET of ∼100 keV/µm. 1 With further increase in LET, the RBE subsequently declines, because more energy is being deposited than is necessary to produce the given biologic effect, so although this wasted energy contributes to increasing the dose, the efficiency per unit dose decreases.
The relationship between RBE and LET is shown in Fig. 1-8 for different biologic endpoints. Especially relevant to radiotherapy with heavy ions, the oxygen enhancement ratio (OER) for cell killing decreases from a maximum of ∼3 for low LET radiation to near unity as the LET increases to ∼100 keV/µm and above. 1 This probably occurs because the high LET track traversals induce so much localized damage in the DNA and other sensitive cellular targets that the additional contribution from ROS/RNS-mediated damage becomes relatively unimportant, or it has also been suggested that recombination of these free radicals occurs within the track volume before diffusion. 142 Not only does an increase in LET cause an increase in the relative effectiveness per unit dose (up to a maximum), but an increase in LET also decreases repair of DNA damage, SLDR, PLDR, and often the sensitizing and protecting effect of chemical modifiers. 138 - 146 Effectively, the dose response for high LET radiations, like alpha particles and heavy ions, is totally dominated by a dose rate– and repair-independent α component of the expression . Whereas the value of α for x-rays or gamma rays might be of the order of 0.2 Gy −1 , the α parameter for 100 keV/µm alpha particles might be as much as 10 times higher, or ∼2.0 Gy −1 , with the β parameter close to zero.

FIGURE 1-8 • A semiquantitative illustration of the relationship between RBE and LET for different biologic damage endpoints. Except for DNA base damage and single strand break induction (SSBs), the RBE versus LET curves are qualitatively similar, peaking at ∼100 to 200 keV/µm for cell killing, chromosomal aberrations (Chrom Aberr), initial interphase chromosome breaks scored in prematurely condensed chromosomes (PCC breaks), and DNA double strand breaks (DSBs). The peak RBEs are much higher for cell killing and chromosomal aberrations than for initial PCC chromosome breaks or DNA DSBs, presumably due to an additional component of high LET-induced DNA damage that is refractory to repair. This repair component would not influence measurements of initial DSBs or PCC breaks after irradiation. The decline in RBE for LET over 200 keV/µm is the result of wasted energy (see text). For base damage and SSBs, the RBE decreases continuously with increasing LET over the entire range, again because of wasted energy.

Ionizing Radiation–Sensitive Mutants
A review of the molecular processes involved in the repair of DNA damage, entitled “DNA Damage and Repair,” is available in Chapter 2 of this volume, and other excellent reviews are available in the literature. 16 , 17 , 19 Ionizing radiations are nearly unique in that a high relative proportion of the prompt or initial DNA damage they produce are DSBs, at least compared to other agents like alkylating agents, H 2 O 2 , and UV-C irradiation. 2 The few other agents that also produce a high proportion of prompt DSBs relative to other lesions include the bleomycins, neocarzinostatin and restriction endonucleases. DSBs are produced endogenously during V(D)J recombination, meiosis, and chromosome decatenation after DNA replication, 147 , 148 but here we will focus on the phenotypic effects of lesions of principal concern following IR exposure. The following discussion is largely focused on important characteristics of radiosensitive cells with limited discussion of their sensitivity other exogenous or endogenous DNA damaging agents or the underlying molecular repair processes. We have also not discussed nontargeted effects of low dose IR (≤10 cGy), such as adaptive responses, bystander effects, and low dose hyperradiosensitivity (HRS), for which several excellent reviews are available. 149 - 153 Although these would likely be of relatively minor consequence in the context of the radiation therapy of tumors using higher IR doses, their role and risks in normal tissue responses in low dose–exposed regions beyond the treatment window is an active area of radiobiologic research.
Our current understanding of DNA repair processes in prokaryotic (e.g., Escherichia coli ) or lower eukaryotic microorganisms (e.g., budding yeast Saccharomyces cerevisiae ) following treatment with agents such as ionizing or nonionizing radiations, or genotoxic chemical agents has been based primarily on the availability and study of mutants that are hypersensitive to such agents. 154 Progress in this area for mammalian cells was initially very slow and depended on recognition of naturally occurring mutants including human patients affected by various radiosensitivity syndromes, until the development and application of strategies for producing and isolating mutants generated by chemical mutagenesis. 155 - 158 More recently, progress has gradually accelerated, both because increasing numbers of individuals with radiosensitive phenotypes have been recognized, and also the strategies for producing radiosensitive mutants (namely gene targeting approaches 159 , 160 ) have improved. These approaches include the generation of specific gene knockouts, gene silencing or knock-downs (e.g., using RNA interference or RNAi approaches 94 ) and other reverse genetic techniques that allow one to examine the effects of alterations of single genes and their protein products using cells of the same genetic (isogenic) background (also obviating the need to study genetically unstable tumor cells). These latter approaches, however, require knowledge of a candidate gene to test, whereas other techniques such as replica plating can uncover mutants in unknown genes.
In the 1960’s, Gotoff and coworkers reported that patients with ataxia-telangiectasia (A-T) undergoing radiation treatment for cancer uniformly showed “untoward responses” to X-irradiation. 161 Taylor and his colleagues later showed that cells from such patients are very hypersensitive to ionizing radiation. 162 , 163 A-T results from autosomal recessive gene mutation in ATM (ataxia-telangiectasia mutated), located on chromosome 11q22–23, which was cloned in 1995. 164 The 9168 bp ATM gene spans 66 exons stretching over 160 kb and encodes a 3056 amino acid, 350-kD serine/threonine protein kinase that plays a pivotal role in orchestrating cellular responses to IR-induced damage including the initial signaling of damage, the activation of DNA repair, and the execution of cell death mechanisms. 16 , 163 , 165 The complexity of the ATM-regulated damage network is illustrated by a recent report from Matsuoka et al. that documented over 900 phosphorylation site targets for ATM in over 700 proteins. 166 The phenotype for homozygous recessive ATM −/− individuals includes a progressive neurodegeneration, hypersensitivity to ionizing radiation, immune deficiencies, and a predisposition to cancer. 163 The defect in such individuals appears to involve not only DNA repair but signal transduction and checkpoint pathways as well (discussed later). ATM +/− heterozygotes appear clinically normal, and cellular responses to radiation are near normal, although there are suggestions that heterozygotes may have an increased incidence of breast cancer, 167 and several reports show distinct, though mild hypersensitivity to ionizing radiation 117 , 168 - 170 ). Genetic mutations of two of the three members of the MRN (Mre11-Rad50-Nbs1) complex are associated with clinical conditions: NBN (Nbs1 or nibrin) with Nijmegen breakage syndrome (NBS) 171 - 173 and MRE11 with A-T-like disorder (ATLD). 174 , 175 Cells derived from these patients share similar, but not identical, phenotypes with cells derived from A-T patients (e.g., radioresistant or unscheduled DNA synthesis, chromosomal and telomere instability, checkpoint defects, and hypersensitivity to DNA-damaging agents). 17 , 171 - 175 Other genetic diseases subsequently found to be associated with ionizing radiation sensitivity include Ligase 4 (LIG4) syndrome involving DNA ligase IV, radiosensitive SCID (RS-SCID) involving the Artemis protein, and ATR-Seckel syndrome involving the ataxia-telangiectasia and Rad3-related serine/threonine protein kinase ATR. 17 , 176 - 179
The first radiosensitive mammalian cell line, the mouse lymphoma cell line L5178Y-S (LY-S), was reported in 1961 by Alexander. 180 The responses of LY-S cells and the parental LY-R cells to a variety of genotoxic agents are detailed in a recent pair of reviews by Szumiel. 181 , 182 LY-S cells appear to be deficient in nonhomologous end joining (NHEJ), although the responsible mutation remains to be identified. 183 Many more radiosensitive mutants of rodent origin were isolated during chemical mutagenesis screens during the past 30 years and several reviews detailing the derivation and characterization of these lines are available (see Collins 184 and Thacker and Zdzienicka 185 and references therein). More recently, groups employing gene-targeting techniques have generated isogenic cell lines with specified repair gene deletions 186 , 187 ; this provides an advantage over chemically mutagenized mutant lines that likely harbor additional unknown mutations. Several mutant cell lines deficient in the NHEJ proteins derived from mouse and Chinese hamster cells (primarily derived from V79 lung fibroblast and CHO ovarian epithelial cells) have been phenotypically characterized. Likewise, mutants of the homologous recombinational repair, Fanconi anemia, and IR-responsive checkpoint signaling pathways have been generated and characterized, and are generally only slightly radiosensitive compared to the hypersensitive A-T and NHEJ mutant cells. HRR and FA-deficient cells, however, are hypersensitive to cell killing by other DNA damaging agents such as alkylating agents, DNA interstrand crosslinking agents and UV-C radiation. 186 , 187 Radiosensitive mouse strains have also been identified and studied, including the NHEJ-deficient scid and BALB/c strains that bear defects in the gene encoding DNA-PKcs (Prkdc/Xrcc7) . 188 - 192
All of these mutant cells are hypersensitive to IR to some degree with respect to both cell killing and induction of chromosomal aberrations, and have provided extensive insights into the underlying molecular processes that mediate DNA repair, cell cycle checkpoint induction and cell survival after irradiation. The dose–survival response for some radiosensitive mutants is shown in Fig. 1-1 B where the x-ray or gamma ray D 0 is in the 0.4 to 0.5 Gy range compared to values of 1.5 to 2.0 Gy for the corresponding wild-type cells. Coupled with the fact that the dose response curves for the radiosensitive mutants also generally show a greatly reduced shoulder, the relative radiosensitivities measured by the ratio of doses to yield a 10% level of survival is around fourfold to fivefold and can be even greater for lower doses and higher survival levels. All the genetic and epigenetic factors controlling radiosensitivity are by no means known, so there is much yet to learn about factors controlling radiosensitivity and how we might manipulate them for therapeutic gain; this discussion, however, is beyond the scope of this introductory chapter. In the future, such knowledge should be extremely useful for the radiation oncologist in the clinic. At the very least, it expands our (limited) scientific understanding of the prognostic factors involved in the treatment of patients with cancer, might result in molecular strategies targeted to IR-responsive pathways, and could be used prospectively to tailor customized patient RT regimens (assuming laboratory methods for accurately determining individual radiosensitivity can be developed given the large degree of inherent genetic variation in the human population).

Mechanisms of Ionizing Radiation–Induced Cell Death
Radiation-induced cell death mechanisms are influenced to varying degrees by cell and tissue-type-specific factors. In addition to mitotic linked death that results from genetic loss from acentric fragment-generating chromosome aberrations in the progeny of irradiated cells, other processes that may mediate IR-mediated cell killing include permanent cell cycle arrest, mitotic catastrophe, apoptosis, autophagy, and necrosis. As mentioned previously, cells of hematopoietic origin are prone to IR-induced apoptosis. Cells of the stroma (fibroblasts and endothelial cells) and parenchyma (epithelial cells) are less susceptible to apoptosis, but rather tend to undergo IR-induced mitotic cell death, mitotic catastrophe, or experience Tp53-mediated permanent cell cycle arrest or premature senescence while maintaining continued (and potentially altered) metabolic and biochemical signaling activities. 1 , 193 - 199 Each of these IR-induced cell death mechanisms can collectively contribute, in varying and often interrelated ways (depending on cell type), to the overall loss of clonogenic capacity following irradiation. It is not surprising then that it has been difficult to obtain strong correlations between tumor cell survival after irradiation (as measured by standard in vitro clonogenic assays) and the induction of non-mitotic linked cell death mechanisms, such as apoptosis. The failure to obtain such strong correlations implies that these various cell death mechanisms collectively influence cellular lethality.
Both cytoprotective (growth) and cytotoxic (stress) pathways are induced following IR and anticancer drug exposures. The severity of cellular damage can influence a particular outcome by tipping the balance of the cellular response in either direction. These responses can be mediated by subtle changes in protein levels or intracellular localization, a number of protein posttranslational modifications (such as phosphorylation and ubiquitination), or, in the case of tumor cells, altered or novel protein activities endowed by genetic mutation. Many intracellular signaling pathways are activated by IR exposure upon initial sensing by the cell’s damage sensors. Intercellular communication and signaling in the tissue microenvironment may also modulate IR-induced cell killing, overall tumor and normal tissue responses, and the risks of early and late effects, although the quantitative influence of these factors in the microenvironment has not been elucidated. 37 We have sought to introduce some of the pertinent molecular interactions underlying these modes of cell death since many novel cancer treatment strategies are being engineered to specifically target and exploit these pathways, both as standalone treatments and as agents to maximize the efficacy of more traditional radiotherapy and chemotherapy treatment regimens.

Mitotic Cell Death
As discussed previously, the generation of lethal chromosome aberrations leads to an irreversible loss of proliferative capacity within only two to three cell generations because of the loss of critical genetic information on acentric fragments. These acentric fragments, which may contain hundreds of genes, often fail to segregate properly to daughter cells during mitosis. These fragments generate micronuclei in daughter cells that not only perturb the proper expression of accompanying genes, but more importantly, they are subsequently lost during successive rounds of cell division. Therefore, in most cell types, IR-induced cell killing is greatly influenced by the induction, signaling and repair of double-stranded DNA damage. This includes DSBs and clustered DNA damage formed immediately after IR exposure 2 , 107 and those that can be generated from subsequent repair processes, since the intermediate steps of DNA repair often create aberrant DNA structures that could be considered damage lesions if left unattended by the DNA repair machinery. DSBs are also an important lesion underlying cell killing by chemotherapeutic drug exposures, but many require DNA replication to achieve maximum efficacy. 19 , 148 As such, DSBs are considered the determinant lesion dictating tumor cell survival and the underlying lesion responsible for the induction of chromosome aberrations, chromosomal instability, mutagenesis, and carcinogenesis in normal tissues following IR exposure. 200 - 204 It is very important to consider not only the immediate killing of cells in tumors or normal tissues with rapid turnover, but also those cells in normal tissues that do not proliferate as rapidly where late effects may occur sometimes a decade or more after treatment. Generally, cell turnover in these late responding normal tissues is very slow, and stem cells that repopulate these tissues and maintain tissue homeostasis are called upon to divide more infrequently. This is why proliferative or mitotic-linked cell death is also considered an underlying cause of dose-limiting late effects, as opposed to some delayed signal transduction mechanism in which some signal , triggered years earlier during treatment, reemerges later for reasons unrelated to cell proliferation.
Before DSB repair, IR-induced DSBs are detected by one of a (ever-growing) family of DSB sensor and mediator proteins. To date, these sensor proteins include the Ku70/80 heterodimer, the MRN complex, Tp53-binding protein 1 (53BP1), mediator of the DNA damage checkpoint-1 (MDC1/NFBD1), the Rad9-Rad1-Hus1 or 9-1-1 complex, BRCA1, and DNA and RNA polymerases that become stalled on sites of DNA damage. 16 , 204 - 211 These sensor proteins transmit damage signals to the transducer protein kinases ATM, ATR, and DNA-PK, 16 , 212 members of the phophatidyinositol-3 kinase (PI3K)-like kinase (PIKK) family that target protein serine and threonine residues for phosphorylation rather than lipids. These proteins mediate the phosphorylation of the histone variant H2AX on serine 139, forming γ-H2AX foci at sites of DSBs in a dose-dependent manner 213 , 214 ; an example of these IR-induced foci in human fibroblasts can be seen in Fig. 1-9 . Such foci serve to recruit additional DSB signaling and repair proteins to the site of damage, the activities of which are likewise modulated by subsequent phosphorylations by ATM, ATR and DNA-PK. 213 - 215 ATR and ATM also phosphorylate the downstream effector kinases CHK1 and CHK2, respectively; these proteins induce cell cycle checkpoints that prevent cells with DNA damage from undergoing DNA replication or entering mitosis. Extensive and partially overlapping autoregulatory feedback mechanisms exist for every step of these pathways, and although these pathways are lesion-specific and dose-dependent in initiation, they converge on several common critical cellular response pathways. 16 , 204 , 210 , 212 , 215 , 216

FIGURE 1-9 • G0/G1-phase primary human fibroblasts irradiated with 0 cGy (A), 50 cGy (B), 100 cGy (C), or 200 cGy of gamma rays (D), fixed 30 minutes after irradiation, and stained with fluorescent antibodies for two key DSB signaling proteins. These panels demonstrate the colocalization of phosphorylated γ-H2AX foci (pS139, in green) and phosphorylated ATM foci (pS1981, in red) at sites of IR-induced DSBs (cell nuclei in blue are counterstained with 4,6-diamidino-2-phenylindole [DAPI]).
As discussed in Chapter 2 , the two main pathways for DSB repair in human cells, NHEJ and homologous recombinational repair (HRR), operate in a cell cycle phase-dependent manner. 16 , 82 - 84 NHEJ is the primary DSB repair pathway throughout the mammalian cell cycle, while HRR contributes to DSB repair in duplicated S and G2-phase chromosomes by using the sister chromatid as a template for accurate repair. HRR activity is a major factor underlying the increased radioresistance observed in S-phase cells compared to the other cell cycle phases. 82 - 84 NHEJ operates in S and G2-phase, possibly with increased fidelity, 217 implying these two major DSB repair pathways are not mutually exclusive during the cell cycle. Interchromosomal HRR between homologous or heterologous chromosomes in G1-phase chromosomes can be detected but occurs at such a low frequency (∼10 −6 ) to be of biologic importance. 218 - 220 NHEJ-mediated repair of radiation-induced DSBs may result in small deletions or nucleotide changes at the repair junction at low frequencies because of exonucleolytic processing. 58 , 221 , 222 Such error-prone DSB repair through NHEJ-mediated pathways is thought to constitute the predominant source of potentially mutagenic lesions associated with radiation exposure and carcinogenesis, and is thought only to be tolerated in mammalian cells because of the high percentage of non–protein coding DNA sequences in mammalian genomes. 223
Following DSB sensing and signaling, rejoining or restitution occurs most of the time, but in a certain proportion of the cases, interaction with a nearby DSB leads to mis-rejoining or chromosome exchange formation. As discussed earlier, about half of these exchanges are lethal in diploid cells irradiated in G0/G1, because half are asymmetrical and generate acentric chromosome fragments that lead to large losses of genetic information in the progeny of cells. A-T cells and many other radiosensitive mutant cells show a large increase in aberration production per unit dose relative to wild-type cells. 102 , 224 For A-T fibroblast cells irradiated in G0/G1, this increase in chromosomal aberration yield results from a much larger proportion, relative to normal cells, of initial chromosome breaks that rejoin incorrectly to yield an excess number of acentric fragment-generating chromosome aberrations. 102 This was studied by inducing premature chromosome condensation (PCC) 225 in human fibroblasts and measuring the number of excess PCC fragments in cultures held in G0/G1 quiescence for up to 46 hours after irradiation before the initiation of PCC. 102 The results for A-T versus normal cells are shown in Fig. 1-10 . The initial number of chromosome breaks is apparently the same for normal and A-T cells, although there is some uncertainty in estimating the number immediately after irradiation since the cell fusion and PCC process itself requires 15 to 20 minutes at 37° C. The rate of rejoining also is the same, but the residual number of excess fragments after 24 or even 46 hours while the cells remained in G1 is much higher for A-T cells than for normal cells. This difference fully accounts for the difference in radiosensitivity with respect to cell killing, and develops while the cells are in a noncycling G0-state. 48 Specifically, the number of fragments observed in G0/G1 after repair (PLDR) was complete was the same as the number of aberrations observed in metaphase after the cells had traversed S phase and G2. Furthermore, one lethal aberration per cell was observed for both A-T cells and normal cells when survival was reduced to 37% by irradiation with 0.7 Gy and 4 Gy, respectively. Similar techniques have been used by many other investigators to study genotoxic agent-induced chromosome breakage (clastogenicity) and rejoining of chromosomes. 226 - 228

FIGURE 1-10 • The initial breakage and rejoining of G0/G1-phase human chromosomes in apparently normal and ataxia-telangiectasia (A-T) cells after an X-ray dose of 6 Gy. The initial break frequency and the rate of rejoining were determined by holding the cells in plateau phase in G0/G1 and allowing them to recover for various times before the chromosomes were condensed by fusing the G0/G1 cells with mitotic inducer cells (PCC). Note that the initial break frequency is the same for normal cells (bottom curve) and A-T cells (top curve), but the residual frequency of excess chromosome fragments is much higher for A-T cells. The magnitude of this difference quantitatively accounts for the difference in sensitivity to cell killing between normal and A-T cells, and develops during a period when the cells are in a noncycling state. The difference is unrelated to IR-induced G1 delay or repair occurring in S or G2-phase cells (see text). Apoptosis occurring before the cells entering mitosis does not appear to contribute significantly to the killing of the cells.
(From Cornforth MN and Bedford JS: On the nature of a defect in cells from individuals with ataxia-telangiectasia, Science 227:1589–1591, 1985.)
Therefore, the increased radiosensitivity of A-T cells is not dependent on the failure of irradiated A-T cells to arrest in G1, which would result in the cells having less time to repair DNA damage before they enter S phase. In fact, the hypothesis that Tp53-induced G1 arrest enhances radioresistance 91 could not apply for most asynchronous populations in which the overall radiosensitivity is determined by the fraction of radioresistant S-phase cells, not cells in G1 (see Fig. 1-5 ). Finally, the suggestion that radiation-induced apoptosis is principally responsible for A-T fibroblast cells being radiosensitive 229 is unlikely, because practically all of the cells irradiated in G1 enter the first mitosis without the appearance of apoptotic cells. 102 The possibility that the loss of chromosome fragments in subsequent generations following their initial production in A-T cells might result in a (Tp53- independent ) delayed apoptotic death has been investigated by several groups. Some have also reported minimal induction of apoptosis at later times (48 hours) after irradiation 230 while others have reported significantly increased rates of apoptosis in A-T fibroblasts (12% to 35%) compared to apparently normal fibroblasts (∼2%). 231

Permanent G1-Phase Cell Cycle Arrest
The loss of proliferative capacity associated with mitotic cell death was initially considered the primary or perhaps the sole process responsible for IR-induced cell killing. Other cell death mechanisms such as apoptosis and permanent cell cycle arrest initially did not receive as much attention in the earlier days of radiobiology research. This may have been a result of a heavy reliance on the use of a limited number of cell lines such as HeLa, Chinese hamster cell lines like CHO and V79, and several mouse cell lines, most (if not all) of which were tumor or transformed cells and were Tp53-deficient (in other words, cells that are genetically abnormal). As discussed previously, the ∼1 : 1 correlation between lethal IR-induced chromosome aberrations and cell survival documented in AG01522 human fibroblasts in the Cornforth and Bedford study 48 (see Fig. 1-10 ) suggested that the majority of IR-induced cell killing was due to the induction of lethal CAs. But there is evidence that even in normal fibroblasts, which are not generally prone to IR-induced apoptosis, there can be contributions to cell killing from processes such as permanent G1-phase arrest. 230 - 234
Many reports have now demonstrated that human fibroblasts and epithelial cells undergo a dose-dependent G1-phase arrest. 235 This arrest was shown to be Tp53-dependent. A recent report by Borgmann et al. 236 that examined several Tp53 -wild-type apparently normal human fibroblast strains concluded that more lethal events per cell occurred than chromosome aberrations per cell, whereas in cells known to be Tp53 -deficient, one lethal event per cell corresponded to one lethal chromosome aberration per cell. Whether there is any connection between the presence of chromosome aberrations and the sensing and signaling pathways that trigger G1 delay or permanent G1 arrest in Tp53 -proficient cells is not known. As many cells and tissues in the body are quiescent or slowly cycling, a large fraction of them are in the G0 or an extended G1 stage of the cell cycle, 194 IR-induced permanent G1 arrest of cells and senescence, a state in which cells no longer proliferate but remain metabolically active, is likely to contribute to the extent of acute or late effects, as well as the slow or more rapid turnover of cells that die from early or late mitotic cell death.
IR exposure of cells in the G0/G1 stage of the cell cycle results in the rapid activation of ATM and DNA-PK and the stabilization of Tp53 via phosphorylation. 237 - 239 The primary mediator of the Tp53-dependent G1 delay is CDKN1A (p21 Waf1/Cip1 ), a potent inhibitor of cyclin D–CDK4/6 and cyclin E–CDK2 complexes that function to drive the retinoblastoma protein (pRb)-mediated G1-S transition. 232 Tp53 is found in low levels in cells because of steady-state ubiquitin-targeted degradation mediated by its negative regulator, MDM2. 240 , 241 Tp53 is activated by phosphorylation and inhibition of MDM2 binding, and upon stabilization, Tp53 functions as a tetrameric transcription factor that induces the expression of a number of cell cycle control and repair genes that function to halt further cell cycle progression and initiate DNA repair or apoptosis (discussed in a following section). 92 , 237 , 242 - 244 A number of additional posttranslational modifications 241 , 244 , 245 also contribute to the Tp53-mediated DNA damage response regulatory network. Overall, stabilization of Tp53 followed by higher CDKN1A expression results in the accumulation of hypophosphorylated pRb and prevents transition into S phase.
The Tp53 gene is located on human chromosome 17p13.1 and consists of 11 exons that encode a 393 amino acid 53-kDa protein. 241 The Tp53 protein includes a number of functional domains involved in transactivation, DNA binding (both Tp53-consensus sequences and damaged DNA sequences), oligomerization (tetramerization), and regulation of protein function and cellular distribution. Over 20,000 mutations have been identified in Tp53 in the majority of human cancer types, 80% of which occur in the DNA binding domain and result in loss of the Tp53-DNA major and minor groove contacts (per the International Agency for Research on Cancer who maintains an extensive Tp53 mutation database at http://www-p53.iarc.fr [release 13, November 2008] 246 ). The functionality of wild-type Tp53 protein can be affected by either loss of both wild-type alleles (such as the loss-of-heterozygosity [LOH] observed in tumor cells from Li-Fraumeni syndrome patients) or the mutation of the wild-type allele. Mutant Tp53 alleles can sometimes result in the expression of mutant Tp53 protein with potentially deregulated activity; loss-of-function mutations in certain domains resulting in a dominant-negative effect; or gain-of-function mutations endowing novel protein function, altered protein–protein interactions or subcellular localization. 241
The effect on Tp53 mutations on cellular radiosensitivity is not completely clear, but an increasing body of evidence suggests that Tp53 mutation generally results in increased radioresistance. 233 , 234 , 247 The radioresistant phenotype was first observed in fibroblasts derived from patients with Li-Fraumeni syndrome, a rare autosomal dominant syndrome caused by Tp53 heterozygosity and characterized by soft tissue sarcomas appearing in infancy or childhood and a high incidence of familial clustering of early-onset (<45 years) sarcomas, breast cancers, brain tumors, and adrenocortical carcinomas. 248 - 250 The list of radioresistant Tp53 -mutant or deficient cells has grown to include a large number of human and rodent normal cell types infected with viruses that inactivate Tp53 function, transformed cells, and tumor cells. 124 , 233 , 234 , 251 Many studies examining the correlation of Tp53 mutation in human tumors and the efficacy of radiotherapy have reported decreased local control of tumors harboring mutations in Tp53 , 233 , 234 , 247 a testament to the critical cellular functions that this protein mediates.
Retinoblastoma protein-mediated pathways play critical roles in regulating properly timed cell cycle transitions, most notably the G1 to S phase transition; differentiation programs by inducing tissue-specific gene expression and permanent withdrawal from the cell cycle; and they participate in Tp53-mediated apoptotic responses after DNA damage. 252 - 254 As such, the Rb1 locus also presents an attractive target for deregulation as evidenced by the gene’s mutation in ∼80% of sporadic human cancers. 255 Oncoproteins encoded by various DNA tumor viruses target pRb and disrupt its function, and have proven to be invaluable tools in the study of the mechanisms of oncogenic transformation. 252 Amplification of the cyclin-dependent kinase genes CDK4 and CDK6 or their cyclin partners CCND1–3 (cyclin D1–D3), or loss of CDKN2A (p16 Ink4a ), a specific CDK4/6 inhibitor, through direct deletion or by promoter silencing provide yet other routes of pRb pathway disruption. 253 , 256 , 257
The 4.7-kb mRNA transcript of the Rb1 gene encodes a 110-kDa nuclear phosphoprotein composed of 928 amino acids denoted pRb. 258 The pRb protein is a member of the so-called pocket protein family that also includes the proteins p107 and p130. The primary function of these pocket proteins is to bind these E2F/DP1 transcription factor family members in transcriptional repressor complexes. 259 - 262 A direct role for E2F-1 in DNA repair is also becoming apparent. 261 , 262 The activity of the pRb protein is controlled by its phosphorylation status, which provides a sensitive regulatory mechanism for the G1 to S-phase transition. 263 The active form of pRb is hypophosphorylated and functions in the early G1-phase to bind members of the E2F/DP1 transcription factor family and a multitude of other interacting proteins. This prevents the premature transcription of E2F target genes involved in the onset of S phase and the initiation of DNA synthesis. 264 The DP1 and DP2 proteins function as transcriptional repressors, providing the basis of an autoregulatory loop that eventually halts transcription of the E2F-responsive genes as cells complete DNA synthesis and prepare to enter G2 phase. Essentially all pRb-associated interacting proteins fail to bind hyperphosphorylated pRb, and phosphorylation site mutants of pRb are potent inhibitors of cell cycle progression and transcription in virtually all cell types studied. 252 Rb1 heterozygosity is not clearly associated with obvious changes in radiosensitivity in vitro 38 , 265 ; homozygous deletion of Rb1 in mice is an embryonic lethal condition. 266 - 268

Mitotic Catastrophe
Mitotic catastrophe results from aberrant mitosis and can produce giant, multinucleated aneuploid cells that remain metabolically active. Mitotic catastrophe is associated with deficiencies of the G2 and mitotic spindle checkpoints that function to limit the abnormal division of cells with damaged DNA and chromosomes. 195 , 196 , 269 Efficient regulation of the G2 and mitotic phase transitions ensures that cells have completely and accurately duplicated their genome and other essential cellular components (required proteins and organelles, etc.) for partitioning into daughter cells. Radiation exposure in the G2-phase of the cell cycle engages a checkpoint to delay mitosis and induce repair processes to administer to defects in the DNA, chromosomes and spindle apparatus. 270 - 272 Certain forms of DNA damage, such as base/nucleotide damage and inter/intrastrand crosslinks, may fail to elicit a G2-phase checkpoint delay and may instead induce an S-phase checkpoint delay in the following cell cycle when lesions are encountered by the DNA replication machinery. 273 , 274 Chemical treatments that do not induce prompt DSBs, e.g., camptothecin (a topoisomerase-I inhibitor), and sublethal fluences of UV-C radiation are not effective at inducing G2-phase delay, however treatment with etoposide, bleomycins, and topoisomerase-II inhibitors that do produce DSBs are effective at inducing a dose-dependent G2 delay. 275
The primary regulator of the G2 phase transition is the maturation-promoting factor (MPF), a complex composed of the CDK1 kinase and cyclin B1. The activity of this kinase complex is controlled by levels of cyclin B1 and cyclin A, another competing CDK1 partner and the phosphorylation status of CDK1. 276 , 277 To initiate passage into mitosis, the CDC25C phosphatase must dephosphorylate inhibitory CDK1 phosphorylations for full activation of the complex. Substrates of the activated MPF kinase include histones, nuclear lamins, cytoskeletal intermediate filaments and microtubule-associated proteins, and other structural proteins necessary for nuclear envelope breakdown and chromosome alignment. 278 As the cell progresses through mitosis, MPF also activates the anaphase promoting complex (APC/C Cdc20 ) or cyclosome , whose E3-like ubiquitin ligase function is necessary for chromatid separation at anaphase and proper orchestration of the final stages of mitosis. 279 , 280
The PIKKs ATM and ATR (ATM/Rad3-related) provide alternative, and partially overlapping, pathways for G2-phase delay. 243 , 270 ATM (with the assistance of BRCA1) and ATR phosphorylate and activate the protein kinases CHK2 and CHK1, respectively, and both these downstream kinases phosphorylate CDC25C. 281 - 284 This inhibitory phosphorylation promotes the sequestration of CDC25C by the 14-3-3 proteins and its export out of the nucleus, displacing CDC25C from the site of its activity. 283 , 284 CHK1 has also been shown to phosphorylate WEE1/MYT1 after irradiation, promoting the inhibitory phosphorylation of CDK1. 276 The timing of these two complementary pathways is illustrated in Xu et al. in which cells irradiated in G2 were first delayed in an ATM-dependent, but dose-independent, manner from entering mitosis. 285 Cells in late S/early G2-phase at the time of irradiation were later delayed at the G2-M transition point in an ATM-independent, but now dose-dependent, manner by the parallel ATR-mediated pathway (see also DeSimone et al. 243 ). In addition to the ATM-CHK2-CDC25C damage response pathway, ATM also functions to phosphorylate and stabilize Tp53, providing another route to G2-phase delay through CDKN1A induction. 92 , 243 , 270 , 286
Originally, A-T cells were thought to experience a much-shortened G2-phase delay. 271 , 287 , 288 Experiments using tritiated thymidine or BrdU-labeling and flow cytometry techniques later revealed that ATM -deficient cells, following an initial failure to block late G2-phase cells from entering mitosis, later experience a second and considerably longer G2-phase delay in the proportion of late-S/early-G2 cells. 272 , 289 - 291 presumably mediated through this redundant ATR-mediated mechanism. The timing of these two events may underlie the argument of an abrogated or extended G2-phase delay in A-T cells, depending on the exact experimental conditions used to measure the delay. Cre-mediated deletion of a floxed (flanked by lox sites) ATR gene in human cells or siRNA knockdown of the ATR-interacting protein (ATRIP) abrogates IR-induced G2-phase delay and causes cell death. 292 This implies an essential role for the ATR–CHK1–Cdc25C pathway in maintenance of the G2-phase delay, perhaps in temporal continuation of its role as the cardinal mediator of the replication and intra-S phase checkpoints. 293
Cells that experience a G2-phase delay may eventually enter into mitosis after moderate doses of DNA damage before repair is complete, a phenomenon known as adaptation . 274 Often such cells will fail in critical stages of mitosis and suffer mitotic catastrophe, most notably the final stage of karyokinesis (nuclear cleavage) and cytokinesis (cellular cleavage) which results in giant cells reforming a single nuclear envelope with tetraploid DNA content and double the normal G1 chromosome number. 294 Agents that interfere with the mechanisms of the G2-phase delay, such as caffeine and okadaic acid, increase the radiosensitivity of treated cells. 295 Caffeine, originally thought to target the ATM and ATR kinases, more probably inhibits an as-yet-unidentified HRR protein. 296 , 297 Cortez demonstrated caffeine treatment results in hyper phosphorylation of the ATM and ATR kinases and their downstream kinase effectors, CHK1 and CHK2. 298 Interestingly, caffeine was shown to be ineffective in reducing radiation-induced G2-phase delay in normal human fibroblasts, in sharp contrast to human tumor cell lines. 299 , 300 These findings suggest possible redundant (and apparently caffeine- in sensitive) pathways of G2-phase delay induction in normal cells and disruption of these pathways in tumor cells. 243
Cells irradiated directly in mitosis may suffer a mitotic checkpoint delay if damage is induced before the metaphase-anaphase transition point. 301 A spindle-assembly checkpoint is engaged if chromosomes are not properly aligned on the metaphase plate because of disruption of chromosome or mitotic spindle integrity (e.g., through the use of the spindle “poisons”), insufficient spindle tension and attachment to the centromeric kinetochore protein complexes, or absence of proper kinetochore function. 196 , 274 The progress of mitotic spindle formation also feeds into Tp53 regulatory pathways through the casein kinases, 302 , 303 which are required for the faithful segregation of chromosomes and are intimately associated with microtubules and centrosomes. Mitotic spindle proteins, such as the MAD2 and BUB1/BUBR1 proteins, and chromosomal passenger proteins, such as Survivin and the Aurora kinases, likewise monitor the integrity of the mitotic spindle and can signal for mitotic checkpoint arrest. 195 , 196 , 279 , 304 , 305
Similar to the G2-M checkpoint, cells can undergo anaphase checkpoint adaptation, a process known as mitotic slippage , and enter into anaphase prematurely with damaged or misaligned chromosomes. 195 , 196 Damage induced after the anaphase transition is insufficient to induce a mitotic checkpoint delay, and cells complete cytokinesis and karyokinesis unimpeded. 301 , 306 While IR exposure during mitosis may engage the mitotic checkpoint if cells are irradiated in early prophase, few if any chromosome aberrations are seen in the irradiated mitotic cells at that time. 307 This is likely due to the fact that the mitotic chromosomes are highly condensed and compacted (i.e., DSBs remain tightly bound in this chromatin state). The high degree of chromatin compaction in mitotic cells results in significantly higher rates of DNA single- and double-strand break induction, which underlies the increased radiosensitivity of mitotic cells compared to G1-phase cells. 109 , 308 - 310 An additional Tp53 and CDKN1A-dependent checkpoint at the following G1–S checkpoint, the tetraploidy checkpoint , is thought to exist to prevent such cells from undertaking an additional round of DNA replication. 274 , 311 , 312 Tetraploid fibroblasts may form metabolically active giant cells that may remain viable in the culture for up to a week, 22 but engagement of this checkpoint in lymphoblasts usually results in apoptosis. However, as with the G2-M and anaphase checkpoints, this tetraploidy checkpoint is also capable of becoming adapted , especially in Rb1 − / − and Tp53 −/− mutant cells. 274 , 294 , 311 - 313 Tetraploid cells in general are no more radiosensitive than their euploid counterparts; although burdened with twice the load of aberrations per unit IR dose, they are likely better able to tolerate otherwise lethal chromosome fragment losses due to the redundancy of essential genes. 200 , 314
Tumor cells that survive an abnormal mitosis and undergo asymmetrical cell division may generate genomically unstable aneuploid cells that may be potentially more tumorigenic, 195 , 196 Higher levels of persistent DNA damage in these cells also may contribute to further genomic instability. 315 , 316 Mitotic catastrophe provides a means to halt the further division of such cells and importantly, evidence of IR-induced mitotic catastrophe has been observed in irradiated solid tumors. 247 , 317 Studies have also shown that cells undergoing mitotic catastrophe may subsequently die by apoptosis and mitotic cell death, suggesting that mitotic catastrophe may not be a specific cell death program but precedes other modes of cell death. 195 , 318 Proteins such as Survivin present attractive targets for therapeutic modulation since its deficiency induces mitotic catastrophe in both BCL-2 and Tp53-dependent and independent manners. 318 - 320 Targeting Survivin and other G2-M and spindle checkpoint pathways that increase cell death by mitotic catastrophe, in combination with treatments that promote apoptosis and other types of cell death, could dramatically improve therapeutic gain after IR or anticancer drug treatment in solid tumors. 320

Apoptosis
Apoptosis is a well-defined form of programmed cell death (type I)—a tightly regulated ATP- and Tp53-dependent process that targets individual cells for complete disintegration while preventing tissue inflammatory responses. 317 , 321 , 322 Apoptosis is critical to the development and maintenance of proper tissue homeostasis, by balancing mitotic cell proliferation and by limiting the growth and clearing damaged or unwanted cells. Apoptosis is a mode of early cell death observed in cells exposed to IR or anticancer drugs, and can sometimes be a useful prognostic indicator of tumor response after treatment. It has been suggested that resistance to apoptosis may play a more critical role in the etiology and treatment of hematological malignancies, compared to solid tumors. 317 Evasion of the apoptotic program is considered one of the cardinal features of tumor cells, 315 and acquired resistance to apoptosis in tumor cells during cancer therapy because of mutation of Tp53 , loss or defects in proapoptotic proteins, or overexpression of antiapoptotic proteins is generally associated with poorer prognosis 247 , 317
Apoptotic cells are characterized by cell membrane blebbing, cytoplasmic and nuclear condensation and fragmentation, and nuclear pyknosis, phenotypes easily detectable by several in vitro and in vivo assays. 317 , 321 , 322 One of the initial characteristic effects of apoptosis is cellular dehydration resulting in cytoplasmic shrinkage. Membrane and organelle integrity remains mostly intact in the initial phases of apoptosis, other than depolarization of mitochondria. Mitochondria also cluster around the nucleus, 323 possibly providing additional energy for subsequent chromosome condensation and cleavage of DNA into a characteristic 200-bp DNA ladder of nucleosomal fragments. Finally, the nucleus and cytosol fragment into membrane-bound apoptotic bodies that are phagocytosed by surrounding cells and macrophages, preventing an inflammatory response commonly seen during “accidental” cell death, or necrosis. Several laboratory assays for detecting apoptotic cells are available including gel electrophoresis methods to detect apoptotic DNA laddering; terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to label DNA strand breaks; flow cytometry (FACS) analysis of DNA content or caspase-3 and annexin V–stained cells (annexin V binds phosphatidylserine exposed on the outer membrane after leaflet switching); and various light and electron microscopy methods. Several in vivo methods for detecting apoptosis are currently available or in development, and include the use of 99m Tc-radiolabeled annexin V, imaging, scintigraphy (TAVS), and caspase cleavage assays. 324 , 325
At least two distinct molecular pathways for the initiation of apoptosis after IR exposure have been identified. 317 , 321 , 322 Both these pathways converge in the activation of caspase executioner cascades by several mitochondria-dependent and independent pathways to complete the cell death program. The first apoptosis signaling pathway, the extrinsic pathway, is initiated by the sensing of membrane damage, which results in: (1) the activation of membrane death receptors, such as Fas, tumor necrosis factor (TNF), and the TNF-related apoptosis-inducing ligand (TRAIL) receptors; and (2) the generation of ceramide. 326 The second apoptosis signaling pathway, the intrinsic pathway, is an intracellular pathway that can be initiated by growth factor withdrawal, oncogene activation, or the sensing of oxidative or IR-induced DNA damage. DNA damage signaling results in Tp53 stabilization/activation and the expression of a number of Tp53-inducible genes, including the genes encoding several members of the Bcl-2/Bax family, 327 - 329 the death receptors, 328 , 330 , 331 and many others. 332 Tp53 also functions to repress the expression of the antiapoptotic protein Bcl-2. 327 , 329
IR exposure induces activation of Fas, TNF and TRAIL receptor-mediated signaling pathways at the cell membrane that is indistinguishable from ligand binding. This then results in the PKCδ-mediated phosphorylation and activation of acid sphingomyelinase (ASMase). 333 which subsequently cleaves sphingomyelin in the membrane to form the potent second messenger, ceramide. 326 Ceramide promotes the activation of the RAS and JNK/SAPK stress pathways that results in c-jun activation and further cytokine (Fas-ligand, TNFα, TRAIL) production, producing an autoregulatory feedback loop. Ceramide also inhibits the PLCγ/PI3K growth pathway signaling. These receptors also stimulate caspase-8 and caspase-10 activation through the recruitment of FADD (Fas-associated death domain) and TRADD (TNFR-associated death domain) adaptor molecules, and the formation of death-inducing signaling complexes (DISC). 334 These caspases also function to proteolytically cleave and activate downstream caspases and the protein Bid, whose proapoptotic C-terminal fragment tBid translocates to mitochondria and activates the proapoptotic Bax. 335 Activation of the death receptors also prompts the dephosphorylation of the proapoptotic protein Bad by Ca 2+ -dependent calcineurin 336 or the protein phosphatases PP1 and PP2A, 337 and inhibits AKT/MAPK-mediated phosphorylation of Bad. 338 This allows Bad to translocate from the cytosol to the mitochondria and counter the inhibitory effects of the antiapoptotic protein complexes located there.
The members of the Bcl-2/Bax family represent both proapoptotic and antiapoptotic proteins with at least one of the four conserved Bcl-2 homology domains (BH1-BH4) that tightly regulate the release of cytochrome c from mitochondria. 329 , 339 They are divided into three groups, and include both cytoplasmic and mitochondrial and ER/nuclear membrane-bound proteins. The first group includes membrane-bound members that inhibit apoptosis and contain BH1-4 domains, such as Bcl-2, Bcl-x L , Bcl-w, and Mcl-1. The second group consists of the proapoptotic proteins Bax and Bak, predominantly membrane-bound proteins with BH1-BH3 domains. The final group consists of cytoplasmic apoptosis promoters, such as Bid, Bim, Bad, Noxa and Puma that contain the BH3 domain only and activate Bax and Bak. The stoichiometry of these family members dictates the mitochondrial response. If sufficient numbers of proapoptotic members can escape inhibition by antiapoptotic members, Bax and Bak can oligomerize to form membrane channels and interact with the voltage-dependent anion channel (VDAC) and the adenine nucleotide transporter (ANT) to open a permeability transition pore (PTP) resulting in cytochrome c release from the mitochondria, activation of the caspase cascade, and the eventual depolarization of the mitochondria as an influx of water bursts the outer mitochondrial membrane. 340 , 341 This releases additional cytochrome c, ROS/RNS, and ions from the intermembrane space, along with several procaspases (2, 3, and 9) and members of the inhibitor of apoptosis (IAP) family of caspase inhibitors. 342 These IAP proteins are themselves inhibited by proteins such as Smac/DIABLO 343 , 344 and Omi/Htra2. 345 which thus promotes caspase activation and apoptosis.
As the primary executioners of the apoptotic program, the cysteinyl aspartate–specific proteinases or caspases irreversibly cleave a number of key substrates within the cell. 346 , 347 Caspases are manufactured in a procaspase form whose activation requires removal of the pro domain and subsequent dimerization. Initially, caspases-8 and 10 are activated by trimerization of the death receptors and they function primarily to activate downstream caspases and liberation of further cytochrome c from mitochondria. Cytosolic cytochrome c results in the formation of the apoptosome —a complex formed by procaspase-9 and an adaptor molecule Apaf-1. Upon cytochrome c and dATP binding, Apaf-1 releases the apoptosome complex from its regulator Bcl-2 and caspase-9 is activated by autocatalytic cleavage or cleavage by other caspases, such as caspase-8. 348 As downstream executioner caspases (caspases-3, 4, 6, 7) are activated, at least 100 documented cytoplasmic and nuclear targets are cleaved. Activation of caspase-activated DNAse (DFF40/CAD) and endonuclease G is responsible for the characteristic 200-bp ladder observed in apoptotic cells. 349 Other caspase substrates include structural proteins, signaling proteins, and various DNA repair, replication, and transcription proteins. The ultimate result of the caspase cascade is evident morphologically as the cell blebs its contents into apoptotic bodies characterized by the exposure of phosphatidylserine on their outer membrane (a result of membrane leaflet switching) and is subsequently phagocytosed by neighboring cells or macrophages.
The time-course of IR-induced apoptosis in vitro and in vivo is illustrated in Fig. 1-11 (from Dewey et al. 350 with data from Olive et al. 351 and Stephens et al. 352 The upper 4 and 7.5 Gy curves represent the apoptotic responses of TK6 lymphoblastoid cells in culture, which both reach a peak and plateau at approximately 20 hours. 351 The lower 2.5 and 25 Gy curves represent the apoptotic responses of OCa-I murine ovarian carcinoma cells in vivo 352 ; in this case, the peak apoptotic response is observed approximately 3 to 5 hours after irradiation, presumably because of clearance of apoptotic cells by neighboring cells and the immune system. In Fig. 1-12 A (also from Dewey et al. 350 with data from Olive et al. 351 ), the dose response for the induction of apoptosis 24 to 36 hours following irradiation in TK6 cells, as measured by the single-cell Comet assay, shows a relatively linear response below 5 to 7 Gy followed by a plateau of ∼90% for 10 to 15 Gy (suggesting that ∼10% of cells were resistant to apoptosis even at these doses). Also shown is the apoptotic fraction (AP) of ∼3% for apoptosis-resistant Tp53 -deficient CHO cells following 15 Gy. Using the same data set, Fig. 1-12 B demonstrates the relative contribution of apoptosis to overall cell killing. The rightmost curve 100–AP is the dose response for the percentage of cells surviving apoptosis derived from A, and the dashed curve Sa × 100 is the calculated survival of the remaining apoptosis-susceptible cells. 350 Compared to the measured clonogenic survival of TK6 cells from this study, line S × 100 , it is evident that only a small fraction of the reduction in overall proliferative capacity of these cultures was due to apoptosis. A subsequent report by Schwartz et al. reported an identical dose response for apoptosis induction in TK6 cells (and minimal apoptosis in Tp53 -mutant WI-L2-NS cells, a closely related line derived from the same individual), and also reported chromosome aberration frequencies that correlated significantly with overall cell killing in these lines. 353 Collectively these results suggest that although apoptosis is an important early cellular response to IR, it likely contributes only a small fraction to overall cell killing compared to mitotic linked death, even in apoptosis-susceptible cells. 350

FIGURE 1-11 • Percent of apoptotic cells in vitro and in vivo plotted against time after irradiation. Solid symbols are for TK6 human lymphoblastoid cells in vitro assayed by the Comet assay. Open symbols are for the murine ovarian carcinoma (OCa-I) in vivo. The assay of the number of apoptotic bodies per 100 OCa-I cells was performed by microscopic examination of H&E-stained sections. 352
(Data from Olive PL, Frazer G, and Banath JP: Radiation-induced apoptosis measured in TK6 human B lymphoblast cells using the comet assay, Radiat Res 136:130–136, 1993; Stephens LC, Hunter NR, Ang KK, et al: Development of apoptosis in irradiated murine tumors as a function of time and dose, Radiat Res 135:75,80, 1993; Dewey WC, Ling CC, and Meyn RE: Radiation-induced apoptosis: relevance to radiotherapy, Int J Radiat Oncol Biol Phys 33:781–796, 1995.)

FIGURE 1-12 • A, Percentage of apoptotic cells (AP) plotted against dose for in vitro TK6 lymphoblastoid cells scored 24 to 36 hours after irradiation, as shown in Fig. 1-11 . For comparison, data are shown for apoptosis-resistant Tp53-defective CHO cells that were treated identically. 351 B, Data in A were converted into the percent of the total population that survived apoptosis, 100–AP, and the survival curve for the remaining apoptosis-susceptible cells Sa × 100 was calculated by Dewey et al. 350 The actual clonogenic survival curve obtained for TK6 cells, 351 labeled S × 100, is also shown. It can be seen from comparing the Sa and S curves that the overall loss of proliferative capacity following irradiation due to apoptosis only contributes a small portion to killing in these cells. 350
(From Dewey WC, Ling CC, and Meyn RE: Radiation-induced apoptosis: relevance to radiotherapy, Int J Radiat Oncol Biol Phys 33:781–796, 1995.)
Increasing evidence suggests that stereotactic body radiation therapy (SBRT) regimens of a single or few multiple high-dose IR fractions may exploit the apoptotic response of endothelial cells of the tumor vasculature 354 to achieve a higher therapeutic ratio than would be calculated from tumor cell survival parameters alone. 42 The success of SBRT stems from recent advances in high-resolution image guidance and high-precision beam delivery technologies to accurately target and treat the tumor volume, while limiting exposure to surrounding normal tissues. 41 As recently discussed by Brown and Koong, although the benefits of the four R’s that apply to fractionated RT regimens are not as relevant for single dose SBRT exposures, it has nevertheless been shown to result in very attractive responses in several tumor types (see Brown and Koong 42 and references therein). Although standard RT doses of ∼2 Gy do not result in significant endothelial cell apoptosis in the tumor vasculature and thus normally does not contribute greatly to tumor control, Garcia-Barros et al. demonstrated that this response becomes important for doses of 8 to 10 Gy and higher (doses typical of SBRT). 354 In this study, 15 to 20 Gy treatments of xenografted MCA/129 sarcomas and B16F1 melanomas resulted in significant microvasculature apoptosis and tumor control in wild-type mice, but these responses were not observed in apoptosis-resistant Bax −/− or asmase −/− knockout mice. These results suggest that chemical agents that promote endothelial cell apoptosis may result in even higher therapeutic gain if given in conjunction with SBRT.

Autophagy and Autophagic Cell Death
Autophagic cell death is another programmed cell death mechanism (type II) distinct from apoptosis that is induced by hypoxia, glucose deprivation, and other conditions routinely encountered in the tumor microenvironment. 321 , 355 , 356 Autophagy is defined as the turnover and catabolic degradation of proteins and organelles in lysosomal vesicles and is thought to be a method for cells to shift intracellular nutrient allocation (e.g., amino acids) to support essential metabolic activities. However, cells may not specifically undergo autophagic cell death after anticancer treatments, but rather use autophagy as a prosurvival mechanism to recycle damaged organelles or increase intracellular nutrient availability to support renewed tumor cell proliferation. Cells die from autophagic cell death when the balance of protein degradation via autophagy exceeds protein synthesis. 357 Like apoptosis, autophagic cell death is an ATP-dependent process that does not generate an inflammatory response, but unlike apoptosis, it is generally considered a late response following IR and anticancer drug treatment. 322 Apoptosis and autophagic cell death are not mutually exclusive modes of cell death, as evidence of both has been observed in the same tissue and even in the same cells. 356 , 358
Autophagy begins with the formation of the autophagosome , a double membrane-bound vesicle that engulfs portions of the cytosol and organelles and subsequently matures upon fusion with lysosomes and other endolytic cell vesicles to form an autolysosome . 321 , 355 - 357 , 359 Levels of autophagy in tumor cells tend to be correlated with the stages of tumor progression. Kondo et al. (2005) has suggested that inhibition of autophagy in early stage tumors experiencing high levels of cell proliferation would facilitate tumor growth by reducing the rate of protein degradation and promote the accumulation of genotoxic ROS/RNS and further genomic instability. 356 Levels of autophagy may increase in later stages of tumor progression in poorly vascularized portions of tumors experiencing nutrient deprivation and hypoxia. Several types of cancer cell lines including breast and colon cancer, melanoma, hepatoma, and malignant glioma cells undergo autophagy following nutrient deprivation (see Kondo et al. 356 and references therein).
Although the molecular mechanisms of autophagy are not as well defined as those mediating apoptosis, genetic analysis has identified more than 30 genes in yeast and several corresponding mammalian homologues. 321 , 355 , 356 These genes encode autophagy-associated (Atg) proteins that mediate two main pathways for autophagosome formation, and membrane-based signaling by both Class 1 and 3 PI3Ks has been shown to play a prominent role in both pathways. 359 Cellular components targeted for degradation by the lysosomal hydrolases of the autolysosome are first tagged with the ubiquitin-like conjugates Atg12 and Atg8/LC3 (microtubule-associated protein 1 light chain 3). 359 , 360 In one pathway, signaling by Class 1 PI3Ks activates an AKT-PKB-mTOR signaling cascade 361 that serves to inhibit autophagy. The AKT/PKB pathway is down regulated via dephosphorylation by the PTEN/MMAC1 phosphatase, 362 overexpression of which enhances autophagy. 363 In the second pathway, signaling by Class 3 PI3Ks, which promote trafficking of lysosomal enzymes from the trans-Golgi network to the lysosome, stimulates autophagy by regulating autophagosome formation. 364
Cells undergoing autophagic cell death can be identified by the presence of autophagic vacuoles (AVs, alternatively termed acidic vesicular organelles or AVOs) and enlargement and degradation of the endoplasmic reticulum (ER) and Golgi apparatus. The identification of autophagic cells in the laboratory is currently more difficult than identifying apoptotic cells, and is at present limited to in vitro or ex vivo samples. Autophagic cells can be identified by transmission electron microscopy, 358 a time-consuming technique that requires a highly trained and skilled microscopist, although more rapid in vitro and in vivo biochemical and immunohistochemical methods are being introduced. 324 , 356 Caspases are not activated during autophagic cell death, unlike apoptosis, and the characteristic 200-bp ladder and nuclear fragmentation are also not observed. Immunohistochemical staining using antibodies against the LC3 protein and green fluorescent protein (GFP)-tagged LC3 constructs have been developed to monitor autophagosomes in vitro and in vivo. 324 , 356
In a report by Paglin et al., 365 5 to 15 Gy IR exposures of MCF-7 breast cancer cells, LNCaP prostate carcinoma cells, and LoVo colon adenocarcinoma cells induced autophagy in vitro. In the same study, inhibition of autophagy using the H + -ATPase inhibitor bafilomycin A1 in MCF-7 cells, which may prevent the fusion of autophagosomes and lysosomes, resulted in increased apoptosis. 365 Inhibition of autophagy by treatment of MDA-MB-231 and MCF-7 breast cancer cells with RAD001 (everolimus, a rapamycin analog) resulted in radiosensitization and significantly increased levels of apoptosis in 3 Gy-exposed samples, as measured by a caspase-3 cleavage assay. 366 Similar results were observed in DU145 and PTEN-deficient PC-3 prostate cancer cells treated with RAD001. 367 Doses of 2 Gy induced autophagic cell death and mTOR-dependent phosphorylation of p70 S6K , a molecular marker for autophagy induction, in DNA-PK-deficient M059J malignant glioma cells, but not in DNA-PK-expressing M059K cells, suggesting crosstalk between NHEJ, the PI3K-AKT-mTOR signaling pathway, and autophagy mediators. 368 Several anticancer drugs have been shown to induce autophagy without inducing apoptosis in tumor cell lines, including staurosporine, TNFα, and tamoxifen treatments in MCF-7 cells. 369 - 371
Human tumor samples and cell lines have been documented with mutations in autophagy-related signaling pathways. 361 , 362 , 372 For example, haploinsufficiency (allelic deletion) of the beclin gene BECN1 on chromosome 17q21 has been documented in several breast cancer cell lines and tumor samples 372 and also in ovarian and prostate tumors. 356 Transfection of BECN1 -expressing constructs into MCF-7 breast cancer cells resulted in increased autophagy and decreased tumorigenicity in nude mice. 372 In addition, overexpression of beclin in MCF-7 cells lowers the incidence of tumor formation in nude mouse xenografts. 372 Heterozygous BECN1 +/− mice show an increased incidence of lung cancer, hepatocellular carcinoma, and lymphoma, 373 , 374 suggesting that Beclin1 functions as a dosage-dependent tumor suppressor. Beclin was initially identified as a Bcl-2-interacting protein, which suggests the likelihood of molecular crosstalk between the apoptotic and autophagic cell death pathways. While much work remains to be done to better understand the role of autophagy and autophagic cell death in tumor progression and cell survival following IR or chemotherapeutic agent treatment, future targeted drugs or synthetic lethal gene therapy approaches may be especially useful for tumors that have acquired resistance to antiapoptotic drugs. 375

Necrosis
Compared to apoptosis, necrosis (type III cell death) is considered a mode of unprogrammed or “accidental” cell death generated by tissue trauma and can provoke an inflammatory response. Necrosis is an ATP-independent cell death mechanism characterized by osmotic swelling of cell membranes and organelles and is observed after high doses of IR and anticancer drugs. 321 , 322 While nuclear fragmentation is also observed in necrotic cells, its random nature results in a smear of DNA using gel electrophoresis compared to the typical CAD-mediated 200-bp ladder seen in apoptotic cells. Following the loss of membrane integrity, the release of intracellular contents from necrotic cells can provoke a tissue inflammatory response.
Necrosis can be induced through Fas ligand and TNFα-mediated activation of the membrane-bound death receptors, suggesting that necrosis may not necessarily be an uncontrolled event. 376 The activity of the RIP1 (receptor-interacting protein 1) kinase can modulate the induction of necrosis after Fas ligand treatment in Jurkat cells. 377 Single-stranded DNA damage induced by IR, higher RIP1-mediated mitochondrial ROS/RNS output, or treatment with DNA alkylating agents that is sensed by the DNA repair protein poly-ADP ribose polymerase 1 (PARP-1) may also activate necrosis. 378 , 379 PARP-1 has been shown to induce translocation of the apoptosis-inducing factor (AIF) protein from the mitochondria to the cell nucleus and promote caspase-independent cell death. 380
The extent of the role of necrosis in tumorigenesis and in local tumor control after IR or anticancer drug treatments remains to be identified, but it remains an attractive target since necrotic tumor cells may stimulate an antitumor immune response. 381 Much of the difficulty in establishing a role for necrosis in tumor treatment is a lack of specific markers or experimental methods that specifically induce necrosis in vitro and in vivo, without likewise inducing alternative modes of cell death. Screening for caspase-cleaved (apoptotic) or uncleaved (necrotic/nonapoptotic) forms of cytokeratin-18 (CK18) in patient serum samples has allowed some discrimination of the relative contributions of these modes of cell death after treatment, 382 although prognostic value of this assay for radiotherapy remains unclear. 383 The relative role of necrosis in tumor control following RT remains to be determined, but it is clear that some degree of necrotic cell death likely occurs after IR exposure, especially after high dose per fraction regimens such as SBRT.

Acknowledgments
This work was supported in part by grants NNX07AP85G and NAG9-1569 from the National Aeronautics and Space Administration (Bedford JS); grant CA112566-01A1 from the National Cancer Institute (LHT/Wilson PF); and grants DE-FG02-07ER64350 (Bedford JS) and FWP SCW-0543 (LHT/Wilson PF) from the US Department of Energy Low Dose Radiation Research Program. A portion of this work was performed under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344.

References

1 Hall E, Giaccia A: Radiobiology for the radiologist, Philadelphia, 2006, Lippincott Williams & Wilkins.
2 Ward JF. Complexity of damage produced by ionizing radiation. Cold Spring Harb Symp Quant Biol . 2000;65:377-382.
3 Quastler H. The nature of intestinal radiation death. Radiat Res . 1956;4:303-320.
4 Bergonie J, Tribondeau L. Intrepretation de quelques resultats de la radiotherapie et essai de fixation d’une technique rationnelle. Compt Rend Acad Sci . 1906;143:983-985.
5 Cole A, Humphrey RM, Dewey WC. Low-voltage electron beam irradiation of normal and 5-bromouridine deoxyriboside-treated L-P59 mouse fibroblast cells in vitro. Nature . 1963;199:780-782.
6 Hofer KG, Harris CR, Smith JM. Radiotoxicity of intracellular 67 Ga, 125 I and 3 H. Nuclear versus cytoplasmic radiation effects in murine L1210 leukaemia. Int J Radiat Biol Relat Stud Phys Chem Med . 1975;28:225-241.
7 Munro TR. The site of the target region for radiation-induced mitotic delay in cultured mammalian cells. Radiat Res . 1970;44:747-757.
8 Warters RL, Hofer KG. Radionuclide toxicity in cultured mammalian cells: elucidation of the primary site for radiation-induced division delay. Radiat Res . 1977;69:348-358.
9 Sedelnikova OA, Rogakou EP, Panyutin IG, et al. Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma-H2AX antibody. Radiat Res . 2002;158:486-492.
10 Yasui LS. GammaH2AX foci induced by gamma rays and 125 IdU decay. Int J Radiat Biol . 2004;80:895-903.
11 Elmroth K, Stenerlow B. DNA-incorporated 125 I induces more than one double-strand break per decay in mammalian cells. Radiat Res . 2005;163:369-373.
12 Dewey WC, Stone LE, Miller HH, et al. Radiosensitization with 5-bromodeoxyuridine of Chinese hamster cells x-irradiated during different phases of the cell cycle. Radiat Res . 1971;47:672-688.
13 Dewey WC, Sedita BA, Humphrey RM. Radiosensitization of X chromosome of Chinese hamster cells related to incorporation of 5-bromodeoxyuridine. Science . 1966;152:519-521.
14 Cecchini S, Girouard S, Huels MA, et al. Single-strand-specific radiosensitization of DNA by bromodeoxyuridine. Radiat Res . 2004;162:604-615.
15 Dextraze ME, Wagner JR, Hunting DJ. 5-Bromodeoxyuridine radiosensitization: conformation-dependent DNA damage. Biochemistry . 2007;46:9089-9097.
16 Thompson L, Limoli C. Origin, recognition, signaling, and repair of DNA double-strand breaks in mammalian cells. In: Caldecott KW, editor. Eukaryotic DNA Damage Surveillance and Repair . Philadelphia: Kluwer Academic/Plenum Publishers; 2004:107-145.
17 O’Driscoll M, Jeggo PA. The role of double-strand break repair—insights from human genetics. Nat Rev Genet . 2006;7:45-54.
18 Bailey SM, Bedford JS. Studies on chromosome aberration induction: what can they tell us about DNA repair? DNA Repair (Amst) . 2006;5:1171-1181.
19 Helleday T, Lo J, van Gent DC, et al. DNA double-strand break repair: from mechanistic understanding to cancer treatment. DNA Repair (Amst) . 2007;6:923-935.
20 Puck TT, Marcus PI. Action of x-rays on mammalian cells. J Exp Med . 1956;103:653-666.
21 Puck TT. Action of radiation on mammalian cells III. Relationship between reproductive death and induction of chromosome anomalies by x-irradiation of euploid human cells in vitro. Proc Natl Acad Sci USA . 1958;44:772-780.
22 Tolmach LJ. Growth patterns in x-irradiated HeLa cells. Ann N Y Acad Sci . 1961;95:743-757.
23 Highfield DP, Holahan EV, Holahan PK, et al. Hyperthermic survival of Chinese hamster ovary cells as a function of cellular population density at the time of plating. Radiat Res . 1984;97:139-153.
24 Sinclair WK. X-ray-induced heritable damage (small-colony formation) in cultured mammalian cells. Radiat Res . 1964;21:584-611.
25 Elkind M, Whitmore G. The Radiobiology of Cultured Mammalian Cells . New York: Gordon and Breach; 1967.
26 Marder BA, Morgan WF. Delayed chromosomal instability induced by DNA damage. Mol Cell Biol . 1993;13:6667-6677.
27 Kadhim MA, Macdonald DA, Goodhead DT, et al. Transmission of chromosomal instability after plutonium alpha-particle irradiation. Nature . 1992;355:738-740.
28 Hewitt HB, Wilson CW. A survival curve for mammalian leukaemia cells irradiated in vivo (implications for the treatment of mouse leukaemia by whole-body irradiation). Br J Cancer . 1959;13:69-75.
29 Till JE, McCulloch EA. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res . 1961;14:213-222.
30 Withers HR. The dose–survival relationship for irradiation of epithelial cells of mouse skin. Br J Radiol . 1967;40:187-194.
31 Withers HR, Elkind MM. Dose–survival characteristics of epithelial cells of mouse intestinal mucosa. Radiology . 1968;91:998-1000.
32 Thames HDJr, Withers R, Mason KA, et al. Dose–survival characteristics of mouse jejunal crypt cells. Int J Radiat Oncol Biol Phys . 1981;7:1591-1597.
33 Withers HR, Hunter N, Barkley HTJr, et al. Radiation survival and regeneration characteristics of spermatogenic stem cells of mouse testis. Radiat Res . 1974;57:88-103.
34 Withers HR, Mason KA, Thames HDJr. Late radiation response of kidney assayed by tubule-cell survival. Br J Radiol . 1986;59:587-595.
35 Gould MN, Clifton KH. The survival of rat mammary gland cells following irradiation in vivo under different endocrinological conditions. Int J Radiat Oncol Biol Phys . 1978;4:629-632.
36 Mulcahy RT, Gould MN, Clifton KH. The survival of thyroid cells: in vivo irradiation and in situ repair. Radiat Res . 1980;84:523-528.
37 Barcellos-Hoff MH, Park C, Wright EG. Radiation and the microenvironment—tumorigenesis and therapy. Nat Rev Cancer . 2005;5:867-875.
38 Wilson PF, Nagasawa H, Warner CL, et al. Radiation sensitivity of primary fibroblasts from hereditary retinoblastoma family members and some apparently normal controls: colony formation ability during continuous low-dose-rate gamma irradiation. Radiat Res . 2008;169:483-494.
39 Fertil B, Malaise EP. Inherent cellular radiosensitivity as a basic concept for human tumor radiotherapy. Int J Radiat Oncol Biol Phys . 1981;7:621-629.
40 Deacon J, Peckham MJ, Steel GG. The radioresponsiveness of human tumours and the initial slope of the cell survival curve. Radiother Oncol . 1984;2:317-323.
41 Chang BK, Timmerman RD. Stereotactic body radiation therapy: a comprehensive review. Am J Clin Oncol . 2007;30:637-644.
42 Brown JM, Koong AC. High-dose single-fraction radiotherapy: exploiting a new biology? Int J Radiat Oncol Biol Phys . 2008;71:324-325.
43 Joshi GP, Nelson WJ, Revell SH, et al. X-ray-induced chromosome damage in live mammalian cells, and improved measurements of its effects on their colony-forming ability. Int J Radiat Biol Relat Stud Phys Chem Med . 1982;41:161-181.
44 Dewey WC, Miller HH, Leeper DB. Chromosomal aberrations and mortality of x-irradiated mammalian cells: emphasis on repair. Proc Natl Acad Sci USA . 1971;68:667-671.
45 Dewey WC, Furman SC, Miller HH. Comparison of lethality and chromosomal damage induced by x-rays in synchronized Chinese hamster cells in vitro. Radiat Res . 1970;43:561-581.
46 Bedford JS, Mitchell JB, Griggs HG, et al. Radiation-induced cellular reproductive death and chromosome aberrations. Radiat Res . 1978;76:573-586.
47 Carrano AV. Chromosome aberrations and radiation-induced cell death. II. Predicted and observed cell survival. Mutat Res . 1973;17:355-366.
48 Cornforth MN, Bedford JS. A quantitative comparison of potentially lethal damage repair and the rejoining of interphase chromosome breaks in low passage normal human fibroblasts. Radiat Res . 1987;111:385-405.
49 Revell SH. Relationships between chromosome damage and cell death. In: Ishihara T, Sasaki MS, editors. Radiation-Induced Chromosome Damage in Man . New York: Alan R. Liss, Inc.; 1983:215-233.
50 Brown JM, Kovacs MS. Visualization of nonreciprocal chromosome exchanges in irradiated human fibroblasts by fluorescence in situ hybridization. Radiat Res . 1993;136:71-76.
51 Brown JM, Evans JW, Kovacs MS. Mechanism of chromosome exchange formation in human fibroblasts: insights from “chromosome painting.”. Environ Mol Mutagen . 1993;22:218-224.
52 Cornforth MN. Analyzing radiation-induced complex chromosome rearrangements by combinatorial painting. Radiat Res . 2001;155:643-659.
53 Savage JR. Reflections and meditations upon complex chromosomal exchanges. Mutat Res . 2002;512:93-109.
54 Dale RG. The application of the linear-quadratic dose-effect equation to fractionated and protracted radiotherapy. Br J Radiol . 1985;58:515-528.
55 Carlone M, Wilkins D, Raaphorst P. The modified linear-quadratic model of Guerrero and Li can be derived from a mechanistic basis and exhibits linear-quadratic-linear behaviour. Phys Med Biol . 2005;50:L9-13.
56 Guerrero M, Li XA. Extending the linear-quadratic model for large fraction doses pertinent to stereotactic radiotherapy. Phys Med Biol . 2004;49:4825-4835.
57 Sachs RK, Hahnfeld P, Brenner DJ. The link between low-LET dose–response relations and the underlying kinetics of damage production/repair/misrepair. Int J Radiat Biol . 1997;72:351-374.
58 Forrester HB, Radford IR. Ionizing radiation-induced chromosomal rearrangements occur in transcriptionally active regions of the genome. Int J Radiat Biol . 2004;80:757-767.
59 Bauchinger M, Gotz G. Distribution of radiation induced lesions in human chromosomes and dose-effect relation analysed with G-banding. Radiat Environ Biophys . 1979;16:355-366.
60 Winegar RA, Preston RJ. The induction of chromosome aberrations by restriction endonucleases that produce blunt-end or cohesive-end double-strand breaks. Mutat Res . 1988;197:141-149.
61 Folle GA, Obe G. Intrachromosomal localization of breakpoints induced by the restriction endonucleases AluI and BamHI in Chinese hamster ovary cells treated in S phase of the cell cycle. Int J Radiat Biol . 1996;69:447-457.
62 Folle GA, Martinez-Lopez W, Boccardo E, et al. Localization of chromosome breakpoints: implication of the chromatin structure and nuclear architecture. Mutat Res . 1998;404:17-26.
63 Martinez-Lopez M, Folle GA, Cassina G, et al. Distribution of breakpoints induced by etoposide and x-rays along the CHO X chromosome. Cytogenet Genome Res . 2004;104:182-187.
64 Yunis JJ, Kuo MT, Saunders GF. Localization of sequences specifying messenger RNA to light-staining G-bands of human chromosomes. Chromosoma . 1977;61:335-344.
65 Barrios L, Miro R, Caballin MR, et al. Cytogenetic effects of radiotherapy. Breakpoint distribution in induced chromosome aberrations. Cancer Genet Cytogenet . 1989;41:61-70.
66 Muhlmann-Diaz MC, Bedford JS. Breakage of human chromosomes 4:19 and Y in G0 cells immediately after exposure to gamma-rays. Int J Radiat Biol . 1994;65:165-173.
67 Muhlmann-Diaz MC, Bedford JS. A comparison of radiation-induced aberrations in human cells involving early and late replicating X chromosomes. In: Obe G, Natarajan AT, editors. Chromosomal Alterations: Origin and Significance . New York: Springer-Verlag; 1994:125-131.
68 Cowell IG, Sunter NJ, Singh PB, et al. GammaH2AX foci form preferentially in euchromatin after ionising-radiation. PLoS ONE . 2007;2:e1057.
69 Jacobson BS. Evidence for recovery from x-ray damage in Chlamydomonas. Radiat Res . 1957;7:394-406.
70 Elkind MM, Sutton H. X-ray damage and recovery in mammalian cells in culture. Nature . 1959;184:1293-1295.
71 Elkind MM, Sutton H. Radiation response of mammalian cells grown in culture. I. Repair of x-ray damage in surviving Chinese hamster cells. Radiat Res . 1960;13:556-593.
72 Lea D. Actions of Radiations on Living Cells . New York: Cambridge University Press; 1955.
73 Kellerer AM, Rossi HH. The theory of dual radiation action. Curr Top Radiat Res Q . 1974;8:85-158.
74 Bedford JS, Cornforth MN. Relationship between the recovery from sublethal x-ray damage and the rejoining of chromosome breaks in normal human fibroblasts. Radiat Res . 1987;111:406-423.
75 Thames HDJr, Hendry J. Fractionation in Radiotherapy . London: Taylor & Francis; 1987.
76 Lajtha LG, Oliver R. Some radiobiological considerations in radiotherapy. Br J Radiol . 1961;34:252-257.
77 Thames HDJr, Withers HR, Peters LJ, et al. Changes in early and late radiation responses with altered dose fractionation: implications for dose-survival relationships. Int J Radiat Oncol Biol Phys . 1982;8:219-226.
78 Fowler JF. The linear-quadratic formula and progress in fractionated radiotherapy. Br J Radiol . 1989;62:679-694.
79 Terasima T, Tolmach LJ. X-ray sensitivity and DNA synthesis in synchronous populations of HeLa cells. Science . 1963;140:490-492.
80 Sinclair WK, Morton RA. Variations in x-ray response during the division cycle of partially synchronized chinese hamster cells in culture. Nature . 1963;199:1158-1160.
81 Bedford JS, Mitchell JB. Dose-rate effects in synchronous mammalian cells in culture. Radiat Res . 1973;54:316-327.
82 Rothkamm K, Kruger I, Thompson LH, et al. Pathways of DNA double-strand break repair during the mammalian cell cycle. Mol Cell Biol . 2003;23:5706-5715.
83 Hinz JM, Yamada NA, Salazar EP, et al. Influence of double-strand-break repair pathways on radiosensitivity throughout the cell cycle in CHO cells. DNA Repair (Amst) . 2005;4:782-792.
84 Tamulevicius P, Wang M, Iliakis G. Homology-directed repair is required for the development of radioresistance during S phase: interplay between double-strand break repair and checkpoint response. Radiat Res . 2007;167:1-11.
85 Winans LF, Dewey WC, Dettor CM. Repair of sublethal and potentially lethal x-ray damage in synchronous Chinese hamster cells. Radiat Res . 1972;52:333-351.
86 Withers HR. Four R’s of radiotherapy. In: Lett JT, Adler H, editors. Advances in radiation biology . New York: Academic Press, Inc; 1975:241-247.
87 Mitchell JB, Bedford JS. Dose-rate effects in synchronous mammalian cells in culture. II. A comparison of the life cycle of HeLa cells during continuous irradiation or multiple-dose fractionation. Radiat Res . 1977;71:547-560.
88 Mitchell JB, Bedford JS, Bailey SM. Dose-rate effects in mammalian cells in culture III. Comparison of cell killing and cell proliferation during continuous irradiation for six different cell lines. Radiat Res . 1979;79:537-551.
89 Zeman EM, Bedford JS. Changes in early and late effects with dose-per-fraction: alpha, beta, redistribution and repair. Int J Radiat Oncol Biol Phys . 1984;10:1039-1047.
90 Leeper DB, Schneiderman MH, Dewey WC. Radiation-induced cycle delay in synchronized Chinese hamster cells: comparison between DNA synthesis and division. Radiat Res . 1973;53:326-337.
91 Murnane JP, Schwartz JL. Cell checkpoint and radiosensitivity. Nature . 1993;365:22.
92 Kastan MB, Onyekwere O, Sidransky D, et al. Participation of p53 protein in the cellular response to DNA damage. Cancer Res . 1991;51:6304-6311.
93 Dewey WC, Cole A. Effects of heterogeneous populations on radiation survival curves. Nature . 1962;194:660-662.
94 Peng Y, Zhang Q, Nagasawa H, et al. Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation. Cancer Res . 2002;62:6400-6404.
95 Steel GG, McMillan TJ, Peacock KH. The 5Rs of radiobiology. Int J Radiat Biol . 1989;56:1045-1048.
96 Stapleton GE, Billen D, Hollaender A. Recovery of x-irradiated bacteria at suboptimal incubation temperatures. J Cell Physiol . 1953;41:345-357.
97 Phillips RA, Tolmach LJ. Repair of potentially lethal damage in x-irradiated HeLa cells. Radiat Res . 1966;29:413-432.
98 Belli JA, Shelton M. Potentially lethal radiation damage: repair by mammalian cells in culture. Science . 1969;165:490-492.
99 Whitmore GF, Gulyas S. Studies on recovery processes in mouse L cells. Natl Cancer Inst Monogr . 1967;24:141-156.
100 Little JB. Repair of sublethal and potentially lethal radiation damage in plateau phase cultures of human cells. Nature . 1969;224:804-806.
101 Mayne LV, Lehmann AR. Failure of RNA synthesis to recover after UV irradiation: an early defect in cells from individuals with Cockayne’s syndrome and xeroderma pigmentosum. Cancer Res . 1982;42:1473-1478.
102 Cornforth MN, Bedford JS. On the nature of a defect in cells from individuals with ataxia-telangiectasia. Science . 1985;227:1589-1591.
103 Iliakis G. Radiation-induced potentially lethal damage: DNA lesions susceptible to fixation. Int J Radiat Biol Relat Stud Phys Chem Med . 1988;53:541-584.
104 Cornforth MN, Bedford JS. Ionizing radiation damage and its early development in chromosomes. In: Lett JT, Adler H, editors. Advances in radiation biology . New York: Academic Press; 1993:423-496.
105 Dettor CM, Dewey WC, Winans LF, et al. Enhancement of x-ray damage in synchronous Chinese hamster cells by hypertonic treatments. Radiat Res . 1972;52:352-372.
106 Raaphorst GP, Dewey WC. Alterations in the radiosensitivity of CHO cells by anisotonic treatments: correlations between cell lethality and chromosomal aberrations. Radiat Res . 1979;79:403-416.
107 Sutherland BM, Bennett PV, Sidorkina O, et al. Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation. Proc Natl Acad Sci USA . 2000;97:103-108.
108 Dewey WC, Noel JS, Dettor CM. Changes in radiosensitivity and dispersion of chromatin during the cell cycle of synchronous Chinese hamster cells. Radiat Res . 1972;52:373-394.
109 Roos WP, Binder A, Bohm L. The influence of chromatin structure on initial DNA damage and radiosensitivity in CHO-K1 and xrs1 cells at low doses of irradiation 1–10 Gy. Radiat Environ Biophys. . 2002;41:199-206.
110 Malcolm AW, Little JB. Rapid recovery in plateau-phase mammalian cells. Radiat Res . 1979;80:38-48.
111 Cox R, Masson WK, Weichselbaum RR, et al. The repair of potentially lethal damage in x-irradiated cultures of normal and ataxia telangiectasia human fibroblasts. Int J Radiat Biol Relat Stud Phys Chem Med . 1981;39:357-365.
112 Thacker J, Stretch A. Recovery from lethal and mutagenic damage during postirradiation holding and low-dose-rate irradiations of cultured hamster cells. Radiat Res . 1983;96:380-392.
113 Astor MB, Morse TD. Rapid repair of potentially lethal damage in normal and ataxia telangiectasia cell lines. Int J Radiat Biol . 1989;55:631-639.
114 Nagasawa H, Chen DJ, Strniste GF. Response of x-ray-sensitive CHO mutant cells to gamma radiation. I. Effects of low dose rates and the process of repair of potentially lethal damage in G1 phase. Radiat Res . 1989;118:559-567.
115 Stackhouse MA, Bedford JS. An ionizing radiation-sensitive mutant of CHO cells: irs-20. II. Dose-rate effects and cellular recovery processes. Radiat Res . 1993;136:250-254.
116 Lobrich M, Kuhne M, Wetzel J, et al. Joining of correct and incorrect DNA double-strand break ends in normal human and ataxia telangiectasia fibroblasts. Genes Chromosomes Cancer . 2000;27:59-68.
117 Kato TA, Nagasawa H, Weil MM, et al. Levels of gamma-H2AX foci after low-dose-rate irradiation reveal a DNA DSB rejoining defect in cells from human ATM heterozygotes in two at families and in another apparently normal individual. Radiat Res . 2006;166:443-453.
118 Hall EJ, Marchese MJ, Astor MB, et al. Response of cells of human origin, normal and malignant, to acute and low dose rate irradiation. Int J Radiat Oncol Biol Phys . 1986;12:655-659.
119 Hall EJ, Marchese M, Hei TK, et al. Radiation response characteristics of human cells in vitro. Radiat Res . 1988;114:415-424.
120 Mamon HJ, Dahlberg W, Azzam EI, et al. Differing effects of breast cancer 1, early onset (BRCA1) and ataxia-telangiectasia mutated (ATM) mutations on cellular responses to ionizing radiation. Int J Radiat Biol . 2003;79:817-829.
121 Weichselbaum RR, Schmit A, Little JB. Cellular repair factors influencing radiocurability of human malignant tumours. Br J Cancer . 1982;45:10-16.
122 Schwartz JL, Jordan R, Kaufmann WK, et al. Evidence for the expression of radiation-induced potentially lethal damage being a p53-dependent process. Int J Radiat Biol . 2000;76:1037-1043.
123 Franken NA, van Bree C, ten Cate R, et al. Importance of TP53 and RB in the repair of potentially lethal damage and induction of color junctions after exposure to ionizing radiation. Radiat Res . 2002;158:707-714.
124 Alsbeih G, Torres M, Al-Harbi N, et al. Loss of wild-type Trp53 protein in mouse fibroblasts leads to increased radioresistance with consequent decrease in repair of potentially lethal damage. Radiat Res . 2004;161:185-192.
125 Dewey WC. In vitro systems: standardization of endpoints. Int J Radiat Oncol Biol Phys . 1979;5:1165-1174.
126 Brown JM, Wilson WR. Exploiting tumour hypoxia in cancer treatment. Nat Rev Cancer . 2004;4:437-447.
127 Brown JM. Tumor hypoxia in cancer therapy. Methods Enzymol . 2007;435:297-321.
128 Tatum JL, Kelloff GJ, Gillies RJ, et al. Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy. Int J Radiat Biol . 2006;82:699-757.
129 Diehn M, Clarke MF. Cancer stem cells and radiotherapy: new insights into tumor radioresistance. J Natl Cancer Inst . 2006;98:1755-1757.
130 Baumann M, Krause M, Hill R. Exploring the role of cancer stem cells in radioresistance. Nat Rev Cancer . 2008;8:545-554.
131 Maisin JR. Chemical protection against ionizing radiation. Adv Space Res . 1989;9:205-212.
132 Maisin JR, Albert C, Henry A. Reduction of short-term radiation lethality by biological response modifiers given alone or in association with other chemical protectors. Radiat Res . 1993;135:332-337.
133 Raju MR. Proton radiobiology, radiosurgery and radiotherapy. Int J Radiat Biol . 1995;67:237-259.
134 Gerweck LE, Kozin SV. Relative biological effectiveness of proton beams in clinical therapy. Radiother Oncol . 1999;50:135-142.
135 Brenner DJ, Hall EJ. Secondary neutrons in clinical proton radiotherapy: a charged issue. Radiother Oncol . 2008;86:165-170.
136 Nikiforova MN, Stringer JR, Blough R, et al. Proximity of chromosomal loci that participate in radiation-induced rearrangements in human cells. Science . 2000;290:138-141.
137 Aten JA, Stap J, Krawczyk PM, et al. Dynamics of DNA double-strand breaks revealed by clustering of damaged chromosome domains. Science . 2004;303:92-95.
138 Blocher D. DNA double-strand break repair determines the RBE of alpha-particles. Int J Radiat Biol . 1988;54:761-771.
139 Rydberg B, Lobrich M, Cooper PK. Repair of clustered DNA damage caused by high LET radiation in human fibroblasts. Phys Med . 1998;14(Suppl 1):24-28.
140 Anderson RM, Marsden SJ, Wright EG, et al. Complex chromosome aberrations in peripheral blood lymphocytes as a potential biomarker of exposure to high-LET alpha-particles. Int J Radiat Biol . 2000;76:31-42.
141 Bedford JS, Goodhead DT. Breakage of human interphase chromosomes by alpha particles and x-rays. Int J Radiat Biol . 1989;55:211-216.
142 Chapman JD, Doern SD, Reuvers AP, et al. Radioprotection by DMSO of mammalian cells exposed to x-rays and to heavy charged-particle beams. Radiat Environ Biophys . 1979;16:29-41.
143 Blakely EA, Chang PY, Lommel L. Cell-cycle-dependent recovery from heavy-ion damage in G1-phase cells. Radiat Res . 1985;Suppl 8:S145-S157.
144 Afzal SM, Ainsworth EJ. Radioprotection of mouse colony forming units—spleen against heavy-charged particle damage by WR 2721. Radiat Res . 1987;109:118-126.
145 Suzuki M, Kase Y, Kanai T, et al. Change in radiosensitivity with fractionated-dose irradiation of carbon-ion beams in five different human cell lines. Int J Radiat Oncol Biol Phys . 2000;48:251-258.
146 Bennett PV, Cutter NC, Sutherland BM. Split-dose exposures versus dual ion exposure in human cell neoplastic transformation. Radiat Environ Biophys . 2007;46:119-123.
147 Vilenchik MM, Knudson AG. Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer. Proc Natl Acad Sci USA . 2003;100:12871-12876.
148 Jeggo PA, Lobrich M. DNA double-strand breaks: their cellular and clinical impact? Oncogene . 2007;26:7717-7719.
149 Little JB. Genomic instability and bystander effects: a historical perspective. Oncogene . 2003;22:6978-6987.
150 Morgan WF. Non-targeted and delayed effects of exposure to ionizing radiation: I. Radiation-induced genomic instability and bystander effects in vitro. Radiat Res . 2003;159:567-580.
151 Morgan WF. Non-targeted and delayed effects of exposure to ionizing radiation: II. Radiation-induced genomic instability and bystander effects in vivo, clastogenic factors and transgenerational effects. Radiat Res . 2003;159:581-596.
152 Wolff S. The adaptive response in radiobiology: evolving insights and implications. Environ Health Perspect . 1998;106(Suppl 1):277-283.
153 Marples B, Collis SJ. Low-dose hyper-radiosensitivity: past, present, and future. Int J Radiat Oncol Biol Phys . 2008;70:1310-1318.
154 Friedberg EC. A brief history of the DNA repair field. Cell Res . 2008;18:3-7.
155 Thompson LH, Rubin JS, Cleaver JE, et al. A screening method for isolating DNA repair-deficient mutants of CHO cells. Somatic Cell Genet . 1980;6:391-405.
156 Busch DB, Cleaver JE, Glaser DA. Large-scale isolation of UV-sensitive clones of CHO cells. Somatic Cell Genet . 1980;6:407-418.
157 Jeggo PA, Kemp LM, Holliday R. The application of the microbial “tooth-pick” technique to somatic cell genetics, and its use in the isolation of x-ray sensitive mutants of Chinese hamster ovary cells. Biochimie . 1982;64:713-715.
158 Stamato TD, Weinstein R, Giaccia A, et al. Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell. Somatic Cell Genet . 1983;9:165-173.
159 Capecchi MR. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet . 2005;6:507-512.
160 Santiago Y, Chan E, Liu PQ, et al. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci USA . 2008;105:5809-5814.
161 Gotoff SP, Amirmokri E, Liebner EJ. Ataxia telangiectasia. Neoplasia, untoward response to x-irradiation, and tuberous sclerosis. Am J Dis Child . 1967;114:617-625.
162 Taylor AM, Harnden DG, Arlett CF, et al. Ataxia telangiectasia: a human mutation with abnormal radiation sensitivity. Nature . 1975;258:427-429.
163 Lavin MF. Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer. Nat Rev Mol Cell Biol . 2008;9:759-769.
164 Savitsky K, Bar-Shira A, Gilad S, et al. A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science . 1995;268:1749-1753.
165 Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature . 2003;421:499-506.
166 Matsuoka S, Ballif BA, Smogorzewska A, et al. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science . 2007;316:1160-1166.
167 Thompson D, Duedal S, Kirner J, et al. Cancer risks and mortality in heterozygous ATM mutation carriers. J Natl Cancer Inst . 2005;97:813-822.
168 Nagasawa H, Kraemer KH, Shiloh Y, et al. Detection of ataxia telangiectasia heterozygous cell lines by postirradiation cumulative labeling index: measurements with coded samples. Cancer Res . 1987;47:398-402.
169 Mitchell EL, Scott D. G2 chromosomal radiosensitivity in fibroblasts of ataxia-telangiectasia heterozygotes and a Li-Fraumeni syndrome patient with radioresistant cells. Int J Radiat Biol . 1997;72:435-438.
170 Tchirkov A, Bay JO, Pernin D, et al. Detection of heterozygous carriers of the ataxia-telangiectasia (ATM) gene by G2 phase chromosomal radiosensitivity of peripheral blood lymphocytes. Hum Genet . 1997;101:312-316.
171 Demuth I, Digweed M. The clinical manifestation of a defective response to DNA double-strand breaks as exemplified by Nijmegen breakage syndrome. Oncogene . 2007;26:7792-7798.
172 Carney JP, Maser RS, Olivares H, et al. The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell . 1998;93:477-486.
173 Varon R, Vissinga C, Platzer M, et al. Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome. Cell . 1998;93:467-476.
174 Taylor AM, Groom A, Byrd PJ. Ataxia-telangiectasia-like disorder (ATLD)—its clinical presentation and molecular basis. DNA Repair (Amst) . 2004;3:1219-1225.
175 Stewart GS, Maser RS, Stankovic T, et al. The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell . 1999;99:577-587.
176 Badie C, Goodhardt M, Waugh A, et al. A DNA double-strand break defective fibroblast cell line (180BR) derived from a radiosensitive patient represents a new mutant phenotype. Cancer Res . 1997;57:4600-4607.
177 O’Driscoll M, Cerosaletti KM, Girard PM, et al. DNA ligase IV mutations identified in patients exhibiting developmental delay and immunodeficiency. Mol Cell . 2001;8:1175-1185.
178 O’Driscoll M, Ruiz-Perez VL, Woods CG, et al. A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome. Nat Genet . 2003;33:497-501.
179 O’Driscoll M, Gennery AR, Seidel J, et al. An overview of three new disorders associated with genetic instability: LIG4 syndrome, RS-SCID and ATR-Seckel syndrome. DNA Repair (Amst) . 2004;3:1227-1235.
180 Alexander P. Mouse lymphoma cells with different radiosensitivities. Nature . 1961;192:572-573.
181 Szumiel I. L5178Y sublines: a look back from 40 years. Part 1: general characteristics. Int J Radiat Biol . 2005;81:339-352.
182 Szumiel I. L5178Y sublines: a look back from 40 years. Part 2: response to ionizing radiation. Int J Radiat Biol . 2005;81:353-365.
183 Brzoska K, Kruszewski M, Szumiel I. Nonhomologous end-joining deficiency of L5178Y-S cells is not associated with mutation in the ABCDE autophosphorylation cluster. Acta Biochim Pol . 2006;53:233-236.
184 Collins AR. Mutant rodent cell lines sensitive to ultraviolet light, ionizing radiation and cross-linking agents: a comprehensive survey of genetic and biochemical characteristics. Mutat Res . 1993;293:99-118.
185 Thacker J, Zdzienicka MZ. The XRCC genes: expanding roles in DNA double-strand break repair. DNA Repair (Amst) . 2004;3:1081-1090.
186 Hinz JM, Tebbs RS, Wilson PF, et al. Repression of mutagenesis by Rad51D-mediated homologous recombination. Nucleic Acids Res . 2006;34:1358-1368.
187 Tebbs RS, Hinz JM, Yamada NA, et al. New insights into the Fanconi anemia pathway from an isogenic FancG hamster CHO mutant. DNA Repair (Amst) . 2005;4:11-22.
188 Blunt T, Gell D, Fox M, et al. Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc Natl Acad Sci USA . 1996;93:10285-10290.
189 Danska JS, Holland DP, Mariathasan S, et al. Biochemical and genetic defects in the DNA-dependent protein kinase in murine scid lymphocytes. Mol Cell Biol . 1996;16:5507-5517.
190 Araki R, Fujimori A, Hamatani K, et al. Nonsense mutation at Tyr-4046 in the DNA-dependent protein kinase catalytic subunit of severe combined immune deficiency mice. Proc Natl Acad Sci USA . 1997;94:2438-2443.
191 Okayasu R, Suetomi K, Yu Y, et al. A deficiency in DNA repair and DNA-PKcs expression in the radiosensitive BALB/c mouse. Cancer Res . 2000;60:4342-4345.
192 Yu Y, Okayasu R, Weil MM, et al. Elevated breast cancer risk in irradiated BALB/c mice associates with unique functional polymorphism of the Prkdc (DNA-dependent protein kinase catalytic subunit) gene. Cancer Res . 2001;61:1820-1824.
193 Darzynkiewicz Z, Juan G, Li X, et al. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry . 1997;27:1-20.
194 Campisi J. Cancer and ageing: rival demons? Nat Rev Cancer . 2003;3:339-349.
195 Castedo M, Perfettini JL, Roumier T, et al. Cell death by mitotic catastrophe: a molecular definition. Oncogene . 2004;23:2825-2837.
196 Kops GJ, Weaver BA, Cleveland DW. On the road to cancer: aneuploidy and the mitotic checkpoint. Nat Rev Cancer . 2005;5:773-785.
197 Suzuki K, Mori I, Nakayama Y, et al. Radiation-induced senescence-like growth arrest requires TP53 function but not telomere shortening. Radiat Res . 2001;155:248-253.
198 Gorbunova V, Seluanov A, Pereira-Smith OM. Expression of human telomerase (hTERT) does not prevent stress-induced senescence in normal human fibroblasts but protects the cells from stress-induced apoptosis and necrosis. J Biol Chem . 2002;277:38540-38549.
199 Jones KR, Elmore LW, Jackson-Cook C, et al. p53-Dependent accelerated senescence induced by ionizing radiation in breast tumour cells. Int J Radiat Biol . 2005;81:445-458.
200 Bedford JS. Sublethal damage, potentially lethal damage, and chromosomal aberrations in mammalian cells exposed to ionizing radiations. Int J Radiat Oncol Biol Phys . 1991;21:1457-1469.
201 Little JB. Radiation-induced genomic instability. Int J Radiat Biol . 1998;74:663-671.
202 Hoeijmakers JH. Genome maintenance mechanisms for preventing cancer. Nature . 2001;411:366-374.
203 van Gent DC, Hoeijmakers JH, Kanaar R. Chromosomal stability and the DNA double-stranded break connection. Nat Rev Genet . 2001;2:196-206.
204 Khanna KK, Jackson SP. DNA double-strand breaks: signaling, repair and the cancer connection. Nat Genet . 2001;27:247-254.
205 Schultz LB, Chehab NH, Malikzay A, et al. p53 binding protein 1, (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J Cell Biol . 2000;151:1381-1390.
206 Venkitaraman AR. Cancer susceptibility and the functions of BRCA1 and BRCA2. Cell . 2002;108:171-182.
207 Shang YL, Bodero AJ, Chen PL. NFBD1, a novel nuclear protein with signature motifs of FHA and BRCT, and an internal 41-amino acid repeat sequence, is an early participant in DNA damage response. J Biol Chem . 2003;278:6323-6329.
208 Goldberg M, Stucki M, Falck J, et al. MDC1 is required for the intra-S-phase DNA damage checkpoint. Nature . 2003;421:952-956.
209 Parrilla-Castellar ER, Arlander SJ, Karnitz L. Dial 9-1-1 for DNA damage: the Rad9-Hus1-Rad1 (9–1-1) clamp complex. DNA Repair (Amst) . 2004;3:1009-1014.
210 Mochan TA, Venere M, DiTullio RAJr, et al. 53BP1 and NFBD1/MDC1-Nbs1 function in parallel interacting pathways activating ataxia-telangiectasia mutated (ATM) in response to DNA damage. Cancer Res . 2003;63:8586-8591.
211 D’Amours D, Jackson SP. The Mre11 complex: at the crossroads of dna repair and checkpoint signalling. Nat Rev Mol Cell Biol . 2002;3:317-327.
212 Bakkenist CJ, Kastan MB. Initiating cellular stress responses. Cell . 2004;118:9-17.
213 Rogakou EP, Pilch DR, Orr AH, et al. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem . 1998;273:5858-5868.
214 Rogakou EP, Boon C, Redon C, et al. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol . 1999;146:905-915.
215 Paull TT, Rogakou EP, Yamazaki V, et al. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol . 2000;10:886-895.
216 Thompson LH, Schild D. Recombinational DNA repair and human disease. Mutat Res . 2002;509:49-78.
217 Kruger I, Rothkamm K, Lobrich M. Enhanced fidelity for rejoining radiation-induced DNA double-strand breaks in the G2 phase of Chinese hamster ovary cells. Nucleic Acids Res . 2004;32:2677-2684.
218 Richardson C, Moynahan ME, Jasin M. Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations. Genes Dev . 1998;12:3831-3842.
219 Richardson C, Jasin M. Recombination between two chromosomes: implications for genomic integrity in mammalian cells. Cold Spring Harb Symp Quant Biol . 2000;65:553-560.
220 Sonoda E, Takata M, Yamashita YM, et al. Homologous DNA recombination in vertebrate cells. Proc Natl Acad Sci USA . 2001;98:8388-8394.
221 Taghian DG, Nickoloff JA. Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells. Mol Cell Biol . 1997;17:6386-6393.
222 Elliott B, Richardson C, Winderbaum J, et al. Gene conversion tracts from double-strand break repair in mammalian cells. Mol Cell Biol . 1998;18:93-101.
223 Sengupta S, Harris CC. p53: traffic cop at the crossroads of DNA repair and recombination. Nat Rev Mol Cell Biol . 2005;6:44-55.
224 Nagasawa H, Latt SA, Lalande ME, et al. Effects of X-irradiation on cell-cycle progression, induction of chromosomal aberrations and cell killing in ataxia telangiectasia (AT) fibroblasts. Mutat Res . 1985;148:71-82.
225 Hittelman WN, Rao PN. Premature chromosome condensation. I. Visualization of x-ray-induced chromosome damage in interphase cells. Mutat Res . 1974;23:251-258.
226 Waldren CA, Johnson RT. Analysis of interphase chromosome damage by means of premature chromosome condensation after x- and ultraviolet-irradiation. Proc Natl Acad Sci U S A . 1974;71:1137-1141.
227 Iliakis GE, Pantelias GE. Production and repair of chromosome damage in an x-ray sensitive CHO mutant visualized and analysed in interphase using the technique of premature chromosome condensation. Int J Radiat Biol . 1990;57:1213-1223.
228 Sasai K, Evans JW, Kovacs MS, et al. Prediction of human cell radiosensitivity: comparison of clonogenic assay with chromosome aberrations scored using premature chromosome condensation with fluorescence in situ hybridization. Int J Radiat Oncol Biol Phys . 1994;30:1127-1132.
229 Meyn MS, Strasfeld L, Allen C. Testing the role of p53 in the expression of genetic instability and apoptosis in ataxia-telangiectasia. Int J Radiat Biol . 1994;66:S141-S149.
230 Enns L, Barley RD, Paterson MC, et al. Radiosensitivity in ataxia telangiectasia fibroblasts is not associated with deregulated apoptosis. Radiat Res . 1998;150:11-16.
231 Zhang Y, Dimtchev A, Dritschilo A, et al. Ionizing radiation-induced apoptosis in ataxia-telangiectasia fibroblasts. Roles of caspase-9 and cellular inhibitor of apoptosis protein-1. J Biol Chem . 2001;276:28842-28848.
232 Di Leonardo A, Linke SP, Clarkin K, et al. DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts. Genes Dev . 1994;8:2540-2551.
233 Bristow RG, Benchimol S, Hill RP. The p53 gene as a modifier of intrinsic radiosensitivity: implications for radiotherapy. Radiother Oncol . 1996;40:197-223.
234 Cuddihy AR, Bristow RG. The p53 protein family and radiation sensitivity: yes or no? Cancer Metastasis Rev . 2004;23:237-257.
235 Little JB, Nagasawa H. Effect of confluent holding on potentially lethal damage repair, cell cycle progression, and chromosomal aberrations in human normal and ataxia-telangiectasia fibroblasts. Radiat Res . 1985;101:81-93.
236 Borgmann K, Dede M, Wrona A, et al. For X-irradiated normal human fibroblasts, only half of cell inactivation results from chromosomal damage. Int J Radiat Oncol Biol Phys . 2004;58:445-452.
237 Siliciano JD, Canman CE, Taya Y, et al. DNA damage induces phosphorylation of the amino terminus of p53. Genes Dev . 1997;11:3471-3481.
238 Chao C, Saito S, Anderson CW, et al. Phosphorylation of murine p53 at ser-18 regulates the p53 responses to DNA damage. Proc Natl Acad Sci USA . 2000;97:11936-11941.
239 Turenne GA, Paul P, Laflair L, et al. Activation of p53 transcriptional activity requires ATM’s kinase domain and multiple N-terminal serine residues of p53. Oncogene . 2001;20:5100-5110.
240 Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett . 1997;420:25-27.
241 Hainaut P, Hollstein M. p53 and human cancer: the first ten thousand mutations. Adv Cancer Res . 2000;77:81-137.
242 Lu X, Lane DP. Differential induction of transcriptionally active p53 following UV or ionizing radiation: defects in chromosome instability syndromes? Cell . 1993;75:765-778.
243 DeSimone JN, Bengtsson U, Wang X, et al. Complexity of the mechanisms of initiation and maintenance of DNA damage-induced G2-phase arrest and subsequent G1-phase arrest: TP53-dependent and TP53-independent roles. Radiat Res . 2003;159:72-85.
244 Fei P, El-Deiry WS. P53 and radiation responses. Oncogene . 2003;22:5774-5783.
245 Saito S, Yamaguchi H, Higashimoto Y, et al. Phosphorylation site interdependence of human p53 post-translational modifications in response to stress. J Biol Chem . 2003;278:37536-37544.
246 Petitjean A, Mathe E, Kato S, et al. Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Hum Mutat . 2007;28:622-629.
247 Gudkov AV, Komarova EA. The role of p53 in determining sensitivity to radiotherapy. Nat Rev Cancer . 2003;3:117-129.
248 Li FP, Fraumeni JFJr. Soft-tissue sarcomas, breast cancer, and other neoplasms. A familial syndrome? Ann Intern Med . 1969;71:747-752.
249 Blattner WA, McGuire DB, Mulvihill JJ, et al. Genealogy of cancer in a family. JAMA . 1979;241:259-261.
250 Malkin D, Li FP, Strong LC, et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science . 1990;250:1233-1238.
251 Bohnke A, Westphal F, Schmidt A, et al. Role of p53 mutations, protein function and DNA damage for the radiosensitivity of human tumour cells. Int J Radiat Biol . 2004;80:53-63.
252 Morris EJ, Dyson NJ. Retinoblastoma protein partners. Adv Cancer Res . 2001;82:1-54.
253 Weinberg RA. The retinoblastoma protein and cell cycle control. Cell . 1995;81:323-330.
254 Zheng L, Lee WH. Retinoblastoma tumor suppressor and genome stability. Adv Cancer Res . 2002;85:13-50.
255 Sherr CJ. Cancer cell cycles. Science . 1996;274:1672-1677.
256 Mittnacht S. Control of pRB phosphorylation. Curr Opin Genet Dev . 1998;8:21-27.
257 Sherr CJ. The INK4a/ARF network in tumour suppression. Nat Rev Mol Cell Biol . 2001;2:731-737.
258 Friend SH, Bernards R, Rogelj S, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature . 1986;323:643-646.
259 de Bruin A, Maiti B, Jakoi L, et al. Identification and characterization of E2F7, a novel mammalian E2F family member capable of blocking cellular proliferation. J Biol Chem . 2003;278:42041-42049.
260 Maiti B, Li J, de Bruin A, et al. Cloning and characterization of mouse E2F8, a novel mammalian E2F family member capable of blocking cellular proliferation. J Biol Chem . 2005;280:18211-18220.
261 Stevens C, La Thangue NB. The emerging role of E2F-1 in the DNA damage response and checkpoint control. DNA Repair (Amst) . 2004;3:1071-1079.
262 Genovese C, Trani D, Caputi M, et al. Cell cycle control and beyond: emerging roles for the retinoblastoma gene family. Oncogene . 2006;25:5201-5209.
263 Sherr CJ, McCormick F. The RB and p53 pathways in cancer. Cancer Cell . 2002;2:103-112.
264 Dyson N. The regulation of E2F by pRB-family proteins. Genes Dev . 1998;12:2245-2262.
265 Little JB, Nichols WW, Troilo P, et al. Radiation sensitivity of cell strains from families with genetic disorders predisposing to radiation-induced cancer. Cancer Res . 1989;49:4705-4714.
266 Lee EY, Chang CY, Hu N, et al. Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature . 1992;359:288-294.
267 Jacks T, Fazeli A, Schmitt EM, et al. Effects of an Rb mutation in the mouse. Nature . 1992;359:295-300.
268 Clarke AR, Maandag ER, van Roon M, et al. Requirement for a functional Rb-1 gene in murine development. Nature . 1992;359:328-330.
269 Vakifahmetoglu H, Olsson M, Zhivotovsky B. Death through a tragedy: mitotic catastrophe. Cell Death Differ . 2008;15:1153-1162.
270 Iliakis G, Wang Y, Guan J, et al. DNA damage checkpoint control in cells exposed to ionizing radiation. Oncogene . 2003;22:5834-5847.
271 Zampetti-Bosseler F, Scott D. Cell death, chromosome damage and mitotic delay in normal human, ataxia telangiectasia and retinoblastoma fibroblasts after x-irradiation. Int J Radiat Biol Relat Stud Phys Chem Med . 1981;39:547-558.
272 Nagasawa H, Keng P, Harley R, et al. Relationship between gamma-ray-induced G2/M delay and cellular radiosensitivity. Int J Radiat Biol . 1994;66:373-379.
273 Akkari YM, Bateman RL, Reifsteck CA, et al. DNA replication is required to elicit cellular responses to psoralen-induced DNA interstrand cross-links. Mol Cell Biol . 2000;20:8283-8289.
274 Andreassen PR, Lohez OD, Margolis RL. G2 and spindle assembly checkpoint adaptation, and tetraploidy arrest: implications for intrinsic and chemically induced genomic instability. Mutat Res . 2003;532:245-253.
275 Kaufmann WK, Kies PE. DNA signals for G2 checkpoint response in diploid human fibroblasts. Mutat Res . 1998;400:153-167.
276 Parker LL, Piwnica-Worms H. Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase. Science . 1992;257:1955-1957.
277 Liu F, Stanton JJ, Wu Z, et al. The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the endoplasmic reticulum and Golgi complex. Mol Cell Biol . 1997;17:571-583.
278 Ubersax JA, Woodbury EL. Quang PN, Targets of the cyclin-dependent kinase Cdk1. Nature . 2003;425:859-864.
279 van Leuken R, Clijsters L, Wolthuis R. To cell cycle, swing the APC/C. Biochim Biophys Acta . 2008;1786:49-59.
280 Sullivan M, Morgan DO. Finishing mitosis, one step at a time. Nat Rev Mol Cell Biol . 2007;8:894-903.
281 Liu Q, Guntuku S, Cui XS, et al. Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev . 2000;14:1448-1459.
282 Chaturvedi P, Eng WK, Zhu Y, et al. Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Oncogene . 1999;18:4047-4054.
283 Sanchez Y, Wong C, Thoma RS, et al. Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. Science . 1997;277:1497-1501.
284 Peng CY, Graves PR, Thoma RS, et al. Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. Science . 1997;277:1501-1505.
285 Xu B, Kim ST, Lim DS, et al. Two molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation. Mol Cell Biol . 2002;22:1049-1059.
286 Bunz F, Dutriaux A, Lengauer C, et al. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science . 1998;282:1497-1501.
287 Painter RB, Young BR. Radiosensitivity in ataxia-telangiectasia: a new explanation. Proc Natl Acad Sci USA . 1980;77:7315-7317.
288 Scott D, Spreadborough AR, Roberts SA. Radiation-induced G2 delay and spontaneous chromosome aberrations in ataxia-telangiectasia homozygotes and heterozygotes. Int J Radiat Biol . 1994;66:S157-163.
289 Ford MD, Martin L, Lavin MF. The effects of ionizing radiation on cell cycle progression in ataxia-telangiectasia. Mutat Res . 1984;125:115-122.
290 Bender MA, Rary JM, Kale RP. G2 chromosomal radiosensitivity in ataxia telangiectasia lymphocytes. Mutat Res . 1985;152:39-47.
291 Beamish H, Lavin MF. Radiosensitivity in ataxia-telangiectasia: anomalies in radiation-induced cell cycle delay. Int J Radiat Biol . 1994;65:175-184.
292 Cortez D, Guntuku S, Qin J, et al. ATR and ATRIP: partners in checkpoint signaling. Science . 2001;294:1713-1716.
293 Bartek J, Lukas C, Lukas J. Checking on DNA damage in S phase. Nat Rev Mol Cell Biol . 2004;5:792-804.
294 Borel F, Lohez OD, Lacroix FB, et al. Multiple centrosomes arise from tetraploidy checkpoint failure and mitotic centrosome clusters in p53 and RB pocket protein-compromised cells. Proc Natl Acad Sci USA . 2002;99:9819-9824.
295 Kaufmann WK, Heffernan TP, Beaulieu LM, et al. Caffeine and human DNA metabolism: the magic and the mystery. Mutat Res . 2003;532:85-102.
296 Wang X, Wang H, Iliakis G, et al. Caffeine-induced radiosensitization is independent of nonhomologous end joining of DNA double-strand breaks. Radiat Res . 2003;159:426-432.
297 Wang H, Wang X, Iliakis G, et al. Caffeine could not efficiently sensitize homologous recombination repair-deficient cells to ionizing radiation-induced killing. Radiat Res . 2003;159:420-425.
298 Cortez D. Caffeine inhibits checkpoint responses without inhibiting the ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases. J Biol Chem . 2003;278:37139-37145.
299 Ning S, Knox SJ. G2/M-phase arrest and death by apoptosis of HL60 cells irradiated with exponentially decreasing low-dose-rate gamma radiation. Radiat Res . 1999;151:659-669.
300 Jha MN, Bamburg JR, Bernstein BW, et al. Caffeine eliminates gamma-ray-induced G2-phase delay in human tumor cells but not in normal cells. Radiat Res . 2002;157:26-31.
301 Morrison C, Rieder CL. Chromosome damage and progression into and through mitosis in vertebrates. DNA Repair (Amst) . 2004;3:1133-1139.
302 Appel K, Wagner P, Boldyreff B, et al. Mapping of the interaction sites of the growth suppressor protein p53 with the regulatory beta-subunit of protein kinase CK2. Oncogene . 1995;11:1971-1978.
303 Sayed M, Pelech S, Wong C, et al. Protein kinase CK2 is involved in G2 arrest and apoptosis following spindle damage in epithelial cells. Oncogene . 2001;20:6994-7005.
304 Skoufias DA, Andreassen PR, Lacroix FB, et al. Mammalian mad2 and bub1/bubR1 recognize distinct spindle-attachment and kinetochore-tension checkpoints. Proc Natl Acad Sci USA . 2001;98:4492-4497.
305 Ruchaud S, Carmena M, Earnshaw WC. Chromosomal passengers: conducting cell division. Nat Rev Mol Cell Biol . 2007;8:798-812.
306 Mikhailov A, Cole RW, Rieder CL. DNA damage during mitosis in human cells delays the metaphase/anaphase transition via the spindle-assembly checkpoint. Curr Biol . 2002;12:1797-1806.
307 Evans JH. Chromosome aberrations induced by ionizing radiations. Int Rev Cytol . 1962;13:221-321.
308 Radford IR, Broadhurst S. Enhanced induction by X-irradiation of DNA double-strand breakage in mitotic as compared with S-phase V79 cells. Int J Radiat Biol Relat Stud Phys Chem Med . 1986;49:909-914.
309 Chapman JD, Stobbe CC, Gales T, et al. Condensed chromatin and cell inactivation by single-hit kinetics. Radiat Res . 1999;151:433-441.
310 Stobbe CC, Park SJ, Chapman JD. The radiation hypersensitivity of cells at mitosis. Int J Radiat Biol . 2002;78:1149-1157.
311 Lanni JS, Jacks T. Characterization of the p53-dependent postmitotic checkpoint following spindle disruption. Mol Cell Biol . 1998;18:1055-1064.
312 Andreassen PR, Lohez OD, Lacroix FB, et al. Tetraploid state induces p53-dependent arrest of nontransformed mammalian cells in G1. Mol Biol Cell . 2001;12:1315-1328.
313 Meraldi P, Honda R, Nigg EA. Aurora-A overexpression reveals tetraploidization as a major route to centrosome amplification in p53-/- cells. EMBO J . 2002;21:483-492.
314 Schwartz JL, Murnane J, Weichselbaum RR. The contribution of DNA ploidy to radiation sensitivity in human tumour cell lines. Br J Cancer . 1999;79:744-747.
315 Hanahan D, Weinberg RA. The hallmarks of cancer. Cell . 2000;100:57-70.
316 Kim GJ, Chandrasekaran K, Morgan WF. Mitochondrial dysfunction, persistently elevated levels of reactive oxygen species and radiation-induced genomic instability: a review. Mutagenesis . 2006;21:361-367.
317 Brown JM, Attardi LD. The role of apoptosis in cancer development and treatment response. Nat Rev Cancer . 2005;5:231-237.
318 Saito T, Hama S, Izumi H, et al. Centrosome amplification induced by survivin suppression enhances both chromosome instability and radiosensitivity in glioma cells. Br J Cancer . 2008;98:345-355.
319 Kumar P, Coltas IK, Kumar B, et al. Bcl-2 protects endothelial cells against gamma-radiation via a Raf-MEK-ERK-survivin signaling pathway that is independent of cytochrome c release. Cancer Res . 2007;67:1193-1202.
320 Capalbo G, Rodel C, Stauber RH, et al. The role of survivin for radiation therapy. Prognostic and predictive factor and therapeutic target. Strahlenther Onkol . 2007;183:593-599.
321 Okada H, Mak TW. Pathways of apoptotic and non-apoptotic death in tumour cells. Nat Rev Cancer . 2004;4:592-603.
322 Kim R, Emi M, Tanabe K, et al. The role of apoptotic or nonapoptotic cell death in determining cellular response to anticancer treatment. Eur J Surg Oncol . 2006;32:269-277.
323 Nechushtan A, Smith CL, Lamensdorf I, et al. Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J Cell Biol . 2001;153:1265-1276.
324 Verheij M. Clinical biomarkers and imaging for radiotherapy-induced cell death. Cancer Metastasis Rev . 2008;27:471-480.
325 Blankenberg FG. In vivo detection of apoptosis. J Nucl Med . 2008;49(Suppl 2):81S-95S.
326 Kolesnick R, Fuks Z. Radiation and ceramide-induced apoptosis. Oncogene . 2003;22:5897-5906.
327 Miyashita T, Krajewski S, Krajewska M, et al. Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene . 1994;9:1799-1805.
328 Yang JY, Xia W, Hu MC. Ionizing radiation activates expression of FOXO3a, Fas ligand, and Bim, and induces cell apoptosis. Int J Oncol . 2006;29:643-648.
329 Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer . 2002;2:647-656.
330 Sheikh MS, Burns TF, Huang Y, et al. p53-dependent and -independent regulation of the death receptor KILLER/DR5 gene expression in response to genotoxic stress and tumor necrosis factor alpha. Cancer Res . 1998;58:1593-1598.
331 Gong B, Almasan A. Apo2 ligand/TNF-related apoptosis-inducing ligand and death receptor 5 mediate the apoptotic signaling induced by ionizing radiation in leukemic cells. Cancer Res . 2000;60:5754-5760.
332 Marchetti F, Coleman MA, Jones IM, et al. Candidate protein biodosimeters of human exposure to ionizing radiation. Int J Radiat Biol . 2006;82:605-639.
333 Zeidan YH, Hannun YA. Activation of acid sphingomyelinase by protein kinase Cdelta-mediated phosphorylation. J Biol Chem . 2007;282:11549-11561.
334 Budihardjo I, Oliver H, Lutter M, et al. Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol . 1999;15:269-290.
335 Li H, Zhu H, Xu CJ, et al. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell . 1998;94:491-501.
336 Wang HG, Pathan N, Ethell IM, et al. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science . 1999;284:339-343.
337 Garcia A, Cayla X, Guergnon J, et al. A. Fleischer and A. Rebollo, Serine/threonine protein phosphatases PP1 and PP2A are key players in apoptosis. Biochimie . 2003;85:721-726.
338 Zundel W, Giaccia A. Inhibition of the anti-apoptotic PI(3)K/Akt/Bad pathway by stress. Genes Dev . 1998;12:1941-1946.
339 Hockenbery D, Nunez G, Milliman C, et al. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature . 1990;348:334-336.
340 Finucane DM, Bossy-Wetzel E, Waterhouse NJ, et al. Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL. J Biol Chem . 1999;274:2225-2233.
341 Kluck RM, Esposti MD, Perkins G, et al. The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol. J Cell Biol . 1999;147:809-822.
342 Roy N, Deveraux QL, Takahashi R, et al. The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases. EMBO J . 1997;16:6914-6925.
343 Du C, Fang M, Li Y, et al. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell . 2000;102:33-42.
344 Verhagen AM, Ekert PG, Pakusch M, et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell . 2000;102:43-53.
345 Hegde R, Srinivasula SM, Zhang Z, et al. Identification of Omi/HtrA2 as a mitochondrial apoptotic serine protease that disrupts inhibitor of apoptosis protein-caspase interaction. J Biol Chem . 2002;277:432-438.
346 Salvesen GS, Dixit VM. Caspases: intracellular signaling by proteolysis. Cell . 1997;91:443-446.
347 Thornberry NA, Lazebnik Y. Caspases: enemies within. Science . 1998;281:1312-1316.
348 Liu X, Kim CN, Yang J, et al. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell . 1996;86:147-157.
349 Widlak P, Garrard WT. Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G. J Cell Biochem . 2005;94:1078-1087.
350 Dewey WC, Ling CC, Meyn RE. Radiation-induced apoptosis: relevance to radiotherapy. Int J Radiat Oncol Biol Phys . 1995;33(4):781-796.
351 Olive PL, Frazer G, Banath JP. Radiation-induced apoptosis measured in TK6 human B lymphoblast cells using the comet assay. Radiat Res . 1993;136(1):130-136.
352 Stephens LC, Hunter NR, Ang KK, et al. Development of apoptosis in irradiated murine tumors as a function of time and dose. Radiat Res . 1993;135(1):75-80.
353 Schwartz JL, Jordan R, Sedita BA, et al. Different sensitivity to cell killing and chromosome mutation induction by gamma rays in two human lymphoblastoid cell lines derived from a single donor: possible role of apoptosis. Mutagenesis . 1995;10:227-233.
354 Garcia-Barros M, Paris F, Cordon-Cardo C, et al. Tumor response to radiotherapy regulated by endothelial cell apoptosis. Science . 2003;300:1155-1159.
355 Yorimitsu T, Klionsky DJ. Autophagy: molecular machinery for self-eating. Cell Death Differ . 2005;12(Suppl 2):1542-1552.
356 Kondo Y, Kanzawa T, Sawaya R, et al. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer . 2005;5:726-734.
357 Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene . 2004;23:2891-2906.
358 Searle J, Lawson TA, Abbott PJ, et al. An electron-microscope study of the mode of cell death induced by cancer-chemotherapeutic agents in populations of proliferating normal and neoplastic cells. J Pathol . 1975;116:129-138.
359 Xie Z, Klionsky DJ. Autophagosome formation: core machinery and adaptations. Nat Cell Biol . 2007;9:1102-1109.
360 Geng J, Klionsky DJ. The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. “Protein modifications: beyond the usual suspects” review series. EMBO Rep . 2008;9:859-864.
361 Rosner M, Hanneder M, Siegel N, et al. The mTOR pathway and its role in human genetic diseases. Mutat Res . 2008;659:284-292.
362 Steck PA, Pershouse MA, Jasser SA, et al. Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat Genet . 1997;15:356-362.
363 Arico S, Petiot A, Bauvy C, et al. The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the phosphatidylinositol 3-kinase/protein kinase B pathway. J Biol Chem . 2001;276:35243-35246.
364 Backer JM. The regulation and function of Class III PI3Ks: novel roles for Vps34. Biochem J . 2008;410:1-17.
365 Paglin S, Hollister T, Delohery T, et al. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res . 2001;61:439-444.
366 Albert JM, Kim KW, Cao C, et al. Targeting the Akt/mammalian target of rapamycin pathway for radiosensitization of breast cancer. Mol Cancer Ther . 2006;5:1183-1189.
367 Cao C, Subhawong T, Albert JM, et al. Inhibition of mammalian target of rapamycin or apoptotic pathway induces autophagy and radiosensitizes PTEN null prostate cancer cells. Cancer Res . 2006;66:10040-10047.
368 Daido S, Yamamoto A, Fujiwara K, et al. Inhibition of the DNA-dependent protein kinase catalytic subunit radiosensitizes malignant glioma cells by inducing autophagy. Cancer Res . 2005;65:4368-4375.
369 Bursch W, Ellinger A, Kienzl H, et al. Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy. Carcinogenesis . 1996;17:1595-1607.
370 Janicke RU, Sprengart ML, Wati MR, et al. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem . 1998;273:9357-9360.
371 Scarlatti F, Bauvy C, Ventruti A, et al. Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1. J Biol Chem . 2004;279:18384-18391.
372 Liang XH, Jackson S, Seaman M, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature . 1999;402:672-676.
373 Qu X, Yu J, Bhagat G, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest . 2003;112:1809-1820.
374 Yue Z, Jin S, Yang C, et al. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci U S A . 2003;100:15077-15082.
375 Moretti L, Yang ES, Kim KW, et al. Autophagy signaling in cancer and its potential as novel target to improve anticancer therapy. Drug Resist Updat . 2007;10:135-143.
376 Vercammen D, Brouckaert G, Denecker G, et al. Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways. J Exp Med . 1998;188:919-930.
377 Holler N, Zaru R, Micheau O, et al. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol . 2000;1:489-495.
378 Bernstein C, Bernstein H, Payne CM, et al. DNA repair/pro-apoptotic dual-role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis. Mutat Res . 2002;511:145-178.
379 Zong WX, Ditsworth D, Bauer DE, et al. Alkylating DNA damage stimulates a regulated form of necrotic cell death. Genes Dev . 2004;18:1272-1282.
380 Yu SW, Wang H, Poitras MF, et al. Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science . 2002;297:259-263.
381 de Bruin EC, Medema JP. Apoptosis and non-apoptotic deaths in cancer development and treatment response. Cancer Treat Rev . 2008;34:737-749.
382 Kramer G, Erdal H, Mertens HJ, et al. Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18. Cancer Res . 2004;64:1751-1756.
383 Debucquoy A, Libbrecht L, Roobrouck V, et al. Morphological features and molecular markers in rectal cancer from 95 patients included in the European Organisation for Research and Treatment of Cancer 22921 trial: prognostic value and effects of preoperative radio (chemo) therapy. Eur J Cancer . 2008;44:791-797.
2 Dna Damage and Repair

Ester M. Hammond, PhD, Isabel M. Pires, PhD, Amato J. Giaccia, PhD

Summary
Our aim in this chapter is to highlight the biologic effort expended by a cell in protecting its deoxyribonucleic acid (DNA). Cellular DNA is under constant attack from both exogenous and endogenous agents and it is crucial that DNA damage is repaired efficiently and with fidelity to maintain genomic integrity. Five major pathways exist to repair specific types of DNA damage: base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), nonhomologous end joining (NHEJ), and homologous recombination (HR). We discuss each of these in turn, and include an overview of the pathways and highlight the human syndromes or cancers caused by abrogation of the pathways.

Introduction
Preserving the integrity of the genome is of paramount importance to any organism. However, our DNA is under continual assault from both endogenous and exogenous damaging agents. These include the products of normal metabolism (reactive oxygen species [ROS]), ultraviolet (UV) and ionizing radiation, and genotoxic compounds such as those found in cigarettes. Inefficient or inadequate DNA repair can have dire consequences, which can lead to cell death, affect essential metabolic processes such as gene transcription, or result in the creation of a mutator phenotype. 1 Decreased efficiency of DNA repair pathways have been linked with cancer predisposition in humans. This is clearly illustrated by studies that show that mutations in DNA repair pathways are associated with cancer-predisposing syndromes in humans. Furthermore, the loss of the ability to effectively repair damaged DNA leads to an increase in genomic instability and can contribute to tumorigenesis. 2 DNA repair pathways are highly conserved throughout evolution, with many of the initial discoveries in the field having been made in Escherichia coli, but are still highly applicable to human cells. The purpose of this chapter is to give an overview of the five main repair pathways (BER, NER, MMR, NHEJ, and HR), and to highlight how mutations in these pathways affect tumor formation and response to therapy ( Fig. 2-1 ).

FIGURE 2-1 • The principle DNA damage repair mechanisms and examples of the initiating lesions. Also depicted are the number of genes involved in these processes and examples of genes relevant to each pathway that are known to be mutated in cancer. Each of these pathways is described in further detail in later sections of this chapter.

DNA Damage Response Pathways
There are numerous mechanisms for the repair of damaged DNA, which are determined in part by the nature of the damage to which they respond and the presence of functioning repair pathways. For example, in the absence of functional BER, a single-strand break (SSB) can form a DNA double-strand break (DSB), which will then be processed by HR. 3 In virtually all DNA repair pathways, DNA damage is detected by sensors that create a signal that is further transduced and amplified to initiate the repair pathway. The most critical DNA repair pathways are those that remove damaged bases that result as a consequence of cellular metabolism or genotoxic insult. BER, NER, and MMR pathways are involved in removing damaged bases and are discussed in the following text.

Base Excision Repair
The BER pathway has a central role to play in preserving genomic integrity. Alterations in BER signaling are predicted to predispose humans to cancer and have been associated with colorectal adenomas and colon cancer. In contrast to other DNA repair pathways discussed in this chapter, only recently has an association between loss of BER and a human disease syndrome been identified. Patients affected by this syndrome, termed E. coli mutY human homolog (MYH)–associated polyposis (MAP), develop large numbers of colonic polyps (in the tens to hundreds range) by the age of 40, and nearly 50% present with colon cancer. 4 The essential role of this pathway is supported by the finding that mice nullizygous for the genes involved were either lethal, indicating an absolute requirement for the gene product, or showed little to no phenotype because of a high degree of redundancy within the pathway. For example, loss of APE1, LIG1, LIG3, XRCC1, FEN1, and POLB all result in embryonic lethality, whereas deletion of NTH1, OGG1, UNG, AAAG , and MUTYH result in no discernible developmental defects. 5 The absolute requirement for BER stems from its ability to repair DNA damage from both exogenous and endogenous sources. The majority of damage to the base portion of each nucleotide is the result of the products of normal metabolism that oxidize or alkylate DNA, such as ROS. Included in these lesions is the formation of 8-oxo-7,8-dihdroguanine (8-oxoG), which has mutagenic properties and also blocks DNA replication and transcription. BER is also used by the cell to handle the high rate of depurination (loss of purines from the DNA), and it has been estimated that a human being loses approximately a trillion guanines from his or her DNA every hour. In addition, BER is also used to remove a large number of cytosines that become spontaneously deaminated to form uracil. The BER mechanism has been extensively characterized in both E. coli and mammals, and includes five stages, shown schematically in Fig. 2-2 . In brief, there is an initial recognition of the damaged base by DNA N-glycosylases. Different glycosylases recognize specific lesions, although there is considerable redundancy between them. The damaged base is then removed to give an apurinic/apyrimidinic (AP) site, which is processed by an AP-specific endonuclease (monofunctional glycosylase) or with an additional AP-lyase (bifunctional glycosylase), which leaves a single-strand interruption. These gaps are then filled by a DNA polymerase either with a single nucleotide (short patch) or polynucleotides (longer repair patch) before a ligation occurs. During BER, an SSB is formed as an intermediate. These SSBs are also generated by exogenous agents such as ionizing radiation and other oxidizing compounds. In response to ionizing radiation, BER effectively repairs the damage (SSB) although some additional enzymatic activities are thought to be required, such as PARP1. The control of BER has been recently described and indicates that BER proteins that are not actively involved in repair are ubiquitinated and degraded by the proteosome. 6

FIGURE 2-2 • Schematic representation of the base excision repair pathway. In this pathway, the first step is the recognition and removal of a damaged base by a DNA glycosylase. The subsequent steps depend on the initial base removal events and can correspond to short-patch or long-patch repair (single nucleotide or 2 to 10 nucleotides respectively). Upon generation of an apurinic/apyrimidinic (AP) site by AP endonuclease/lyase, the subsequent single strand gap is filled by DNA polymerases (polymerases β or δ/ε respectively for short patch or long patch), followed by a final ligation step.

Nucleotide Excision Repair
NER was discovered in the 1960s through elegant studies on the effects of UV irradiation on DNA synthesis and repair replication in bacteria. Since then it has been characterized extensively in mammals and has been described as the principle repair pathway for the removal of bulky adducts induced by UV radiation or other environmental carcinogens. 7 - 9 In contrast to the BER pathway, damage detected by the NER proteins is not done in a specific manner because there is no equivalent to DNA N-glycosylases involved in BER, described previously. It is hard to imagine that a specific glycosylase could evolve for each type of DNA damage, especially in an environment in which exogenous damaging agents are numerous and constantly attack epithelial cells in the skin. Consequently, NER has been described as a more flexible version of BER. The major lesions recognized by NER are bulky adducts that result from intrastrand crosslinks, UV-induced cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts. When these lesions are not removed, they distort the DNA helix and in doing so prevent DNA replication and transcription, which can result in the employment of the error-prone translesion synthesis repair pathway. The process of NER can be subdivided into two pathways: global genome repair (GG-NER) or global genomic repair (GGR), and transcription coupled repair-nucleotide excision (TC-NER) or transcription-coupled repair (TCR). 10 The process of GG-NER is genome wide (i.e., lesions can be removed from anywhere). In contrast, TC-NER only removes lesions in DNA strands of actively transcribed genes. When a DNA strand that is being actively transcribed becomes damaged, the ribonucleic acid (RNA) polymerase can block access to the site of damage and hence prevent DNA repair. TC-NER has evolved to prevent this by effectively removing the RNA polymerase from the site of damage to allow the repair proteins access. The mechanism of GG-NER and TC-NER differ only in the detection of the lesion; the subsequent pathway is the same. In general NER involves the recognition of the lesion, by proteins specific to GG-NER or TC-NER, followed by incision of the DNA strand near the damage, removal of the affected stretch of DNA, repair replication using the complementary strand as a template, and, finally, ligation to seal the 3′ end of the repair patch with the parental DNA ( Fig. 2-3 ). 11 , 12

FIGURE 2-3 • Schematic representation of the nucleotide excision repair (NER) pathway. The two subpathways of NER, global genome repair (GG-NER/GGR) and transcription-coupled repair (TC-NER/TCR) differ alone on the participants of the initial damage recognition step. GGR uses the XPC-XPE protein complexes, whereas in TCR the NER proteins are recruited by the stalled RNA polymerase in cooperation with ERCC8 and ERCC6. Following recognition, the lesion is demarked by binding of the transcription factor IIH (TFIIH) complex, XPA, and RPA. The TFIIH complex helicase function unwinds the DNA and generates an open stretch around the lesion, at which point the XPG and XPF-ERCC1 endonucleases make incisions at the 3′ and 5′ ends, respectively, releasing a 25-29 oligomer. The resulting gap is filled by the polymerases δ/ε aided by RCF and PCNA and the strand is finally ligated.
Three human syndromes that occur as a result of mutation of the NER pathways have been identified: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). 13 Many of the principle genes involved in NER are called XP, for example XPA, XPB, and so on. This is because they were identified in individuals suffering from XP. 14 XP results from mutation in any one of seven complementation groups: XPA, XPB (ERCC3), XPC, XPD (ERCC2), XPE (DDB2), XPF and XPG. Interestingly XP patients with mutations in XPC or XPE are deficient in GG-NER, whereas the remaining are deficient in both GG-NER and TC-NER. XP presents early in life as extreme sensitivity to the sun; freckles appear at a young age (1 year) even after short exposure to the sun, and significant pigmentation abnormalities are common. The eyes of XP patients are photosensitive and often exhibit eye damage as a result of constant UV-induced conjunctivitis and keratitis. This extreme sensitivity to UV translates to a 1 in 4000-fold increased risk of skin cancer in XP individuals, often appearing as early as age 10. 15 These malignancies are usually squamous and basal cell carcinomas, but melanomas have also been reported. Neurologic abnormalities also frequently occur, including peripheral neuropathy, sensorineural deafness, and progressive mental retardation. Similar to XP patients, those with CS are also very sensitive to UV, although they do not exhibit the increased risk of skin cancer. Instead CS patients have distinct developmental abnormalities, which include growth retardation (dwarfism), progressive cognitive impairment, skeletal abnormalities, severe mental retardation, cataracts, and retinitis pigmentosa. CS patients tend to die younger than the age of 20 (the average life span is 12 years) as a result of infectious or renal complications rather than cancer. The genetic determinants of CS are mutations in either the CSA or CSB genes, both of which are essential for recognition of DNA damage and initiation of the TC pathway of NER. CS individuals remain proficient in GG-NER. Finally, mutation of the XPB or XPD genes results in TTD. Again extreme sensitivity to the sun is a characteristic in some of these patients (approximately 50%), although they possess a low incidence of developing skin cancer. TTD individuals are similar to CS in presentation but have the additional symptom of brittle nails and hair; this is a result of reduced sulfur content in the component proteins. The differences and similarities between these three syndromes have proved intriguing, in that they all show UV sensitivity but only XP individuals have an increased risk of skin cancer. This has been attributed to the determination that loss of GG-NER is what predisposes individuals to skin cancer. In contrast, the developmental abnormalities associated with these syndromes (retardation, dwarfism, etc.) are the result of a loss of TC-NER. Given the essential role of NER in the removal of UV-induced damage it is not surprising that XP individuals succumb to skin cancer, but an increased risk of other malignancies would also be expected and this has not been noted. There have been reports documenting modest alteration in NER genes as a result of polymorphisms that contribute to the risk of solid tumors.

Mismatch Repair
During normal replication and recombination, mistakes can be made that result in the insertion of an incorrect nucleotide leading to a mismatch. These mismatches also occur as a result of base modifications. If mismatches escape surveillance of the proofreading capabilities of DNA polymerases, there is a permanent change in sequence or mutation. The MMR pathway functions to identify such mistakes and correct them before they are propagated further. MMR also functions to remove small loops (insertion–deletion loops [IDL]) in the DNA that arise as a consequence of spontaneous slippage-dependent misalignment between primer and template DNA strands. These occur particularly in highly repetitive microsatellite regions that, in the absence of effective MMR, show a large increase in the frequency of spontaneously occurring mutations (microsatellite instability [MSI]). 16 The process of MMR can be subdivided into four components: First, the mismatch must be identified by sensors that transduce a signal; second, MMR factors are recruited; third, the newly synthesized strand harboring the mismatch is identified and the incorrect or altered nucleotides are excised; and in the fourth stage, resynthesis and ligation of the excised tract occurs. MMR was first characterized in E. coli, which encodes MUT genes; homologues of these gene products have now been identified and extensively characterized in both yeast and humans. 17 See Fig. 2-4 for a schematic representation and an indication of the critical gene products. The significance of MMR was highlighted when it became apparent in the 1990s that hereditary nonpolyposis colorectal cancer (HNPCC) and sporadic MSI-positive colon cancers were caused by defects in MMR. HNPCC, also known as Lynch syndrome, affects 1 in 1000 people and is characterized by early-onset colorectal cancers that have a high level of MSI. Individuals with HNPCC have an 80% risk of colorectal cancer in their lifetime, as well as an increased risk of other malignancies such as endometrial (50% risk), ovarian, and gastric malignancies. In 70% to 80% of the germline mutations found in HNPCC, the mutations occur in the MLH1 or MLH2 genes. In 10% of cases there is a mutation in MSH6, and, in rare cases, PMS1, PMS2, MLH3, and EXO1. 18 Epigenetic silencing of the MMR genes has also been noted and shown to increase the mutation rate. In very rare cases, both copies of an MMR gene, such as MLH1, MSH2, MSH6, or PMS2 have been found mutated (homozygous germline mutations). 19 These individuals have a reduced life span and succumb to malignancy of hematologic or brain origin.

FIGURE 2-4 • Schematic representation of the mismatch repair (MMR) pathway. The MMR pathway is initiated by a recognition step of mismatched bases that involves the E. Coli hMutS orthologs MSH2-MSH6/MSH2-MSH3 complexes. These in turn recruit the MMR factors MLH1-PMS2, MLH1-PMS1, and MLH1-MLH3 (orthologs of MutL ), alongside the exonuclease EXO1 that catalyzes the excision step that follows. A gap-filling step by polymerases δ/ε, RCF, and PCNA is followed by a final ligation step.
The MMR proteins have been described as having a role to play in cell cycle checkpoints and apoptosis. Specifically, both hMutSα- and hMutLα-deficient cells exhibit defective S-phase checkpoint responses. 20 The proposed model behind this observation is that MMR proteins act as sensors of DNA damage and then recruit the checkpoint proteins and hence activate these signaling pathways. This is supported by the findings that hMutSα and hMutLα physically interact with ataxia-telangiectasia mutated (ATM) , ataxia-telangiectasia Rad3-related (ATR) , ATR-interacting protein (ATRIP) , c-Abl, and p73 in cells exposed to DNA-damage-inducing agents. Also supportive of this model is the wealth of data indicating that cells that become defective in MMR gain resistance to cytotoxic agents. Interestingly, patients with MSI-positive tumors have a greater chance of survival than those that do not, although whether this is as a result in differences in tumorigenesis or therapeutic response is unclear.

Double-Strand Break Repair
DSBs are essential during the development of the immune system and during meiosis to ensure genetic diversity. However, it has been estimated that a single unrepaired DNA DSB is lethal to a cell. DSBs are induced by ionizing radiation, chemotherapeutic agents such as bleomycin and doxorubicin, replication over an SSB during repair of intra-strand DNA cross-links, and by ROS. The repair of DSBs is essential for genomic integrity, and without it situations such as aneuploidy, deletions (loss of heterozygosity), and translocations occur, all of which can promote tumorigenesis. Many of the enzymes involved in DSB repair have been identified as a result of their roles in various cancer-prone human and animal disease syndromes, which will be discussed later. DSBs can be dealt with by one of two major repair pathways: HR and NHEJ.

Nonhomologous End-Joining
In contrast to HR, the NHEJ repair pathway does not make use of a template strand of DNA to maintain sequence fidelity during repair, but instead relies on short regions of microhomology (1-6 bp) ( Fig. 2-5 ). NHEJ is predominantly active in the G 1 phase of the cell cycle, but can function in all phases and is estimated to repair 90% of all DSBs in humans. NHEJ is also required for the development of both T- and B-cell repertoires. The essential role for NHEJ in the development of the immune system is highlighted by the finding that severe combined immunodeficient mice result from a loss of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), one of the principle players in NHEJ repair. DNA-PK is composed of a cs and the Ku70/80 heterodimer. In the presence of a DSB, DNA ends are recognized by the Ku70/80 heterodimer, which in turn recruits DNA-PKcs as well as other proteins involved in strand alignment. The XRCC4-ligase IV complex then seals the breaks (see Fig. 2-5 ). Unless the DSB is blunt-ended, several nucleotides can be either gained or lost during the process, resulting in genetic errors. Numerous elegant mouse models have shown that by disrupting NHEJ in vivo, there are significant effects on radiosensitivity, genomic instability, immunodeficiency, development, and cancer predisposition.

FIGURE 2-5 • Schematic representation of the nonhomologous end joining (NHEJ) double-strand break (DSB) repair pathway. The initial step of the core NHEJ pathway is the binding of the DNA ends at the DSB by the KU70/KU80 heterodimer. This complex then recruits and activates the cs of DNA-PK (DNA-PKcs), whose role is the juxtaposition of the two DNA ends. The DNA-PK complex then recruits the ligase complex (XRCC4/XLF-LIGIV) , which promotes the final ligation step.

Homologous Recombination
HR occurs predominantly in the S and G 2 phases of the cell cycle and is estimated to repair 10% of DSBs acquired by human cells. The process of HR, which takes place in cells with a 2N DNA content, uses the presence of a second copy of each sequence to ensure that DNA repair occurs with no loss or alteration in genomic sequence. The process of HR is shown schematically in Fig. 2-6 . In brief, DSBs are detected by the MRE11-NBS1-RAD50 (MRN) complex; the nuclease MRE11 processes the break to give a 3′ single-stranded DNA (ssDNA) tail. This region of ssDNA is then coated with replication protein A (RPA) to form a nucleoprotein filament. This filament then undergoes strand invasion (i.e., it invades the complementary sequence on a sister chromatid to form heteroduplex DNA). The genes involved in HR are numerous and include MRE11, NBS1, RAD50, BRCA1, BRCA2 , and RAD51 .

FIGURE 2-6 • Schematic representation of the homologous recombination (HR) double-strand break (DSB) repair pathway. HR is initiated by the recognition of the lesion and processing of the DSB DNA ends into 3′ DNA ssDNA tails, which are then coated by RPA forming a nucleoprotein filament. These initial steps are thought to be promoted by the MRN complex. Following this, specific HR proteins are recruited to the nucleoprotein filaments, such as RAD51, RAD52, BRCA1, and BRCA2. RAD51 then mediates the invasion of the homologous sequence of the sister chromatid, leading to formation of heterocomplex DNA Holliday junctions. This process requires, apart from RAD51, other HR factors such as RAD52, BRCA1, BRCA2, RAD54, and the RAD51 paralogs. The Holliday junctions are finally resolved into two DNA duplexes.

Syndromes Associated With a Failure in DNA Repair

Ataxia-Telangiectasia
The ATM gene has a critical role to play in DNA repair and is often referred to as sentinel kinase, meaning that it is one of the first kinases activated in response to DNA damage, and is responsible for the initial activation of repair signaling. ATM is a member of the PI3 kinase family and acts by phosphorylating serine or threonine residues that are followed by a glutamine residue (i.e., serine-glutamine or threonine-glutamine). Although the majority of ATM signaling has been described in response to agents that cause DSBs, more recent data is emerging to suggest that ATM may also respond to non-DNA damaging stresses. 21 , 22 Currently a model has been proposed that suggests ATM exists within the cell as an inactive dimer which, upon activation in response to a DSB and with the aid of the mediator proteins and complexes, undergoes autophosphorylation to become active monomers. These monomeric, phosphorylated ATM molecules then in turn phosphorylate many downstream targets. 23 However, mice bearing ATM mutated at the 1987 amino acid autophosphorylation site retain their ATM -mediated signaling pathways, 24 suggesting a difference between human and mouse ATM activation in response to DNA damage. The downstream targets of ATM include many proteins involved in DNA repair, for example, NBS1, MRE11, CHK2, H2AX, FANC D2 , and p53. Cells lacking ATM activity are extremely sensitive to DNA damage agents, fail to delay DNA replication, and continue to replicate in the presence of nonrepaired DNA (radio-resistant DNA synthesis). Normal tissue as well as cells derived from AT patients exhibit hypersensitivity to conventional doses of radiotherapy. AT patients have a 10% risk of malignancy, usually lymphoma or leukemia, which usually occurs at an early age. However, AT presents as a diverse group of symptoms prior to the formation of malignancies. These include progressive neurodegeneration, immune deficiency, ocular apraxia, and telangiectasia. Both Nijmegen breakage syndrome (NBS) and ataxia telangiectasia-like disorder show some overlap with AT patients, including defects in chromosome repair. 25 , 26 This has been attributed to the finding that the genes mutated in these syndromes ( NBS and MRE11, respectively) are both downstream targets of ATM and are also involved in the initial activation of ATM in response to DNA damage.

The p53 Tumor Suppressor Gene and Li-Fraumeni Syndrome
The p53 tumor suppressor gene, once dubbed the “guardian of the genome,” is the most mutated gene in human cancers. It has been estimated that it, or its downstream pathways, are mutated in more than 75% of cancers. The p53 gene encodes a protein that is at the heart of the cellular response to stress, including DNA damage, hypoxia, UV damage, and starvation. 27 In response to stress, the levels of p53 protein, which are maintained at low levels in unstressed cells, increases rapidly because of increased protein translation and stabilization. The function of p53 is primarily as a transcription factor, and as such it binds to a consensus sequence in a sequence specific manner to target genes to regulate their expression (recently reviewed in Riley, 2008 28 ). The number of p53 -activated and repressed genes is steadily increasing and, relevant to this chapter, include genes involved in cell cycle checkpoints, DNA repair, and apoptosis. Examples of genes that fall into these three categories are the cell-cycle inhibitory gene p21; the proapoptotic genes BAX and PERP; and the DNA repair genes DDB2, DDIT4, TRIM22, GADD45α, and XPC. More recently, there have been reports documenting a nontranscriptional role of p53, in particular in the induction of apoptosis at the mitochondria. 29 , 30 Included in this nontraditional role for p53 is also the ability to modulate BER though direct interactions with DNA polymerase beta and OGG1, HR through modulation of the recQ helicases, and NER through a direct interaction with XPB. 31 Consequently, in response to stress, p53 functions to arrest the cell cycle and signal to the repair pathways to restore genomic integrity unless the damage is too severe, in which case the cell is directed toward apoptosis. Without functional p53, cells are allowed to progress through the cell cycle before repair has taken place or escape apoptosis, and therefore accumulate genomic instability, which can contribute to tumorigenesis.
Li-Fraumeni syndrome is an autosomal-dominant inherited disorder, which is caused by mutation of the p53 gene in 70% to 85% of cases. Affected individuals inherit one mutated copy of the p53 gene and subsequently lose the remaining allele by mutation or loss of heterozygosity. Individuals with Li-Fraumeni syndrome usually present at an early age with a bone or soft tissue sarcoma, but may also develop breast cancer, a brain tumor, lymphoma, leukemia, and adrenocortical carcinoma. 32 - 34

Familial Breast Cancer, BRCA1 and BRCA2
The breast cancer susceptibility genes ( BRCA1 and BRCA2 ) are mutated in between 5% and 10% of all breast cancer cases and are also associated with familial ovarian cancers. Familial breast cancer is distinct from sporadic cases in that it has an earlier age of onset, an increased frequency of bilateral tumors, and can be associated with other tumor types within the affected family. However, familial and sporadic cases of breast cancer are indistinguishable at the histologic level and in their metastatic patterns. Interestingly, BRCA1 and BRCA2 mutations in sporadic cases of breast cancer are a rare occurrence. Both BRCA1 and BRCA2 have been associated with a plethora of cellular responses, including DNA damage, repair, cell cycle progression, transcription, ubiquitination, apoptosis; most recently BRCA1 and BRCA2 have been found to play a role in the determination of stem-cell fate. 35 - 38 The links between both BRCA1 and BRCA2 and the DNA repair pathways are numerous and extend beyond the HR repair pathway, although their role in HR is most characterized. Cells lacking either BRCA1 or BRCA2 have a high degree of genomic instability, which is highly indicative of the important role of HR in maintaining genomic integrity. This was highlighted more than 10 years ago by the finding that mice nullizygous for BRCA1 have the same phenotype as those null for the HR essential protein RAD51. 39 Functionally, BRCA1 acts as a sensor of DNA damage and replication stress and mediates HR through BRCA2. Biochemically, in response to DNA damaging agents, BRCA1 is phosphorylated at numerous sites by ATM, ATR, and CHK2, and subsequently associates with many other repair components. 40 , 41 These include MSH2, MSH6, ATM, RAD51, and the MRN complex. BRCA2 plays a more direct role in HR by interacting with RAD51 and facilitating the formation of aggregates of RAD51 at the sites of DNA breaks. 42 Therefore, in the absence of either BRCA1 or BRCA2, repair relies on the error-prone NHEJ pathways. The resulting accumulation of mutations undoubtedly contributes to tumorigenesis. BRCA1 has also been described as having a role to play in the regulation of genes involved in NER, including XPC and DDB2. 43

Fanconi Anemia
Fanconi anemia (FA) was first described by Fanconi in 1927 when he studied a family with three boys, all with reduced blood cell number (pancytopenia) and birth defects. Since this initial description, it has become clear that the phenotype of this disease is highly variable and as such is difficult to diagnosis clinically. FA patients are described as having an increased risk of acute myeloid leukemia; squamous cell carcinomas of the head, neck, and esophagus; gynecologic carcinomas; and liver tumors at a young age as well as a range of congenital anatomical abnormalities. The range of congenital abnormalities seen in 60% to 75% of FA patients include characteristic thumb malformations and abnormal skin pigmentation. 44 , 45 At the molecular level FA is diagnosed as an extreme sensitivity to agents that induce DNA crosslinks, for example mitomycin C, diepoxybutane, cisplatin, and to a lesser degree ionizing radiation. In contrast to the majority of familial cancer syndromes, FA is not the result of the mutation of a single gene; instead 13 genetic complementation groups have been identified to date. 46 , 47 Mutations in any one of these can cause FA. The FANC genes all encode proteins that are involved in the repair of DNA crosslinks. This repair mechanism is somewhat uncharacterized and seems to by a hybrid of both excision repair and DSB repair systems, in particular HR. Of the 13 FA genes that have been identified, 8 have been shown to form a nuclear complex in response to DNA damage, and there is evidence to suggest that this complex has ubiquitin-E3 ligase activity. 48 During the DNA damage response to crosslinking agents, this core nuclear complex monoubiquitinates FANCD2, 49 which then translocates to co-localize with proteins involved in HR such as BRCA1, 50 RAD51, 51 and BRCA2/FANCD1 52 in nuclear foci. Both the ATM 53 and ATR 54 kinases have been demonstrated to regulate the FANC/BRCA1 pathway in response to DNA damage and replication stress respectively. Recently, a critical role for the Fanconi pathway in the survival of neural stem and progenitor cells in both development and adult neurogenesis has been reported. 55 , 56

Homologs of RECQ: Bloom Syndrome, Werner Syndrome, and Rothmund-Thompson Syndrome
RECQ was first described in E. coli and has been extensively characterized in that system. 57 Since then, five human homologs of RECQ have been described, three of which have clearly been shown to be mutated in cancer-prone syndromes. 58 The RECQ homologs are DNA helicases that have two enzymatic activities: they unwind DNA 3′ to 5′ in an ATP -dependent manner 59 , 60 and also act 3′ to 5′ as an exonuclease. 61 The RECQ helicases have been described as the “guardians of the DNA replication fork” and are thought to act primarily at the interface between replication and recombination. 62 There is significant evidence to support this claim, which includes the following observations: First, cells with perturbed RECQ activity are extremely sensitive to agents such as hydroxyurea (Hu) and aphidicolin (APH), both of which inhibit replication. Second, even in the absence of agents such as APH and Hu, cells with RECQ mutations did not progress through S phase normally and instead accumulated abnormal DNA structures. Third, the activity and expression of the RECQ helicases is seen to peak in the S phase of the cell cycle, indicating this is the period in which they function. Finally, the helicase are localized to sites of DNA replication and also seem to protect replication forks during periods of fork stalling, and regulate fragile site stability. 63 With regard to human disease, the most well-studied human recQ helicases are Bloom (BLM) syndrome, Werner (WRN) syndrome and Rothmund-Thompson (RT) syndrome. Loss of the BLM helicase results in BLM syndrome, which is associated with a high incidence of cancer and in particular lymphomas, leukemias, and solid tumors of the gastrointestinal tract and breast. Skin disorders, sunlight sensitivity, dwarfism, immunodeficiency, male sterility, and elevated levels of sister chromatid exchange are also all associated with BLM syndrome. 64 , 65 Individuals with WRN syndrome also show an increased risk of cancers, but also characteristically demonstrate premature aging. This puts patients at risk for age-related diseases such as cancer, diabetes, osteoporosis, and atherosclerotic cardiovascular disease. Increased genomic instability is also manifested in WS, as high levels of chromosomal deletions and translocations. 66 - 68 Finally, RT syndrome, caused by a loss of the RECQ4 helicase, displays growth deficiency, photosensitivity with poikilodermatous skin changes, early graying and hair loss, juvenile cataracts, skeletal dysplasias, and a predisposition to malignancy, especially osteogenic sarcomas and chromosomal instability. 69 , 70

DNA Repair Deficiency: A Therapeutic Target
Loss, by either mutation or epigenetic silencing, of DNA repair pathways sensitizes cells to genotoxic agents such as chemotherapy and radiotherapy. The finding that in comparison with normal, nontumorigenic cells, rapidly proliferating cancer cells exhibit increased sensitivity to exogenously induced DNA damage and can be selectively killed forms the base of modern cancer therapies. Unfortunately, the treatment of human cancers is rarely this straightforward, as tumor cells acquire mutations in genes that control the cell-cycle and activate apoptotic programs. For these reasons, there is an effort to selectively kill tumor cells by exploiting deficient DNA repair. Examples include inhibitors of DNA-PK, ATM, and ERCC1 (reviewed in Helleday and colleagues, 2008 71 ; and Martin and colleagues, 2008 72 ). More recently, this concept of selectivity has been expanded to the targeting of specific repair pathways in tumor cells with specific genetic backgrounds. This approach is likely to receive more attention for cancer therapy, and at present is best illustrated by the use of poly (ADP-ribose) polymerase (PARP) inhibitors. PARP is involved in BER and specifically in the sensing of SSBs. 73 When an SSB goes unrepaired by BER, a DSB is formed after replication, which is repaired by HR. However in tumor cells that lack BRCA1 or BRCA2, HR is severely compromised, making them sensitive to PARP inhibition. It has been effectively demonstrated that BRCA1 - and BRCA2 -deficient cells are sensitive to PARP inhibition, whereas normal or heterozygote BRCA1 and BRCA2 cells are not. 74 , 75 The deficiency of HR in BRCA1 - and BRCA2 -deficient cells presents an ideal therapeutic window to treat cancer cells with PARP inhibitors, and to minimize the effect on the normal surrounding tissues. The success of PARP inhibitors is the result of the synthetic lethality of inhibiting two repair pathways, in this case BER and HR. Thus, although the loss of the HR repair pathway may be tolerated in BRCA1 - and BRCA2 -deficient cells, the loss of a second repair pathway BER, which alone may also be compatible with viability, cannot be tolerated in the HR-deficient background, and leads to cell death ( Fig. 2-7 ). 76 - 79

FIGURE 2-7 • Schematic representation of the synthetic lethality principle. Synthetic lethality occurs when individual loss of two genes ( A or B ) is viable but is lethal when in combination ( A and B ). This principle is exemplified with genes involved in DNA repair pathways, PARP (base excision repair) and BRCA1 (homologous recombination), as described in the text.

Conclusions
Recently it was shown that during the very early stages of tumorigenesis, DNA damage signaling is initiated by the activation of oncogenes. It was further proposed that at this stage tumor cells are kept in check by the activation of cell cycle checkpoints and in some cases apoptosis. These studies led to DNA damage signaling being described as acting as a barrier to tumorigenesis. 80 , 81 However, once the neoplastic cells take on a mutator phenotype, they start losing essential components of damage signaling and repair. These findings highlight just how reliant human cells are on effective means of both sensing and repairing DNA damage. Further study of DNA repair pathways will undoubtedly affect the way we treat, diagnose, and potentially prevent disease.

References

1 Sjoblom T, Jones S, Wood LD, et al. The consensus coding sequences of human breast and colorectal cancers. Science . 2006;314(5797):268-274.
2 Loeb LA, Loeb KR, Anderson JP. Multiple mutations and cancer. Proc Natl Acad Sci U S A . 2003;100(3):776-781.
3 Tutt AN, Lord CJ, McCabe N, et al. Exploiting the DNA repair defect in BRCA mutant cells in the design of new therapeutic strategies for cancer. Cold Spring Harb Symp Quant Biol . 2005;70:139-148.
4 Cheadle JP, Sampson JR. MUTYH-associated polyposis—from defect in base excision repair to clinical genetic testing. DNA Repair (Amst) . 2007;6(3):274-279.
5 Hakem R. DNA-damage repair; the good, the bad, and the ugly. EMBO J . 2008;27(4):589-605.
6 Parsons JL, Tait PS, Finch D, et al. CHIP-mediated degradation and DNA damage-dependent stabilization regulate base excision repair proteins. Mol Cell . 2008;29(4):477-487.
7 Boyce RP, Howard-Flanders P. Release of ultraviolet light-induced thymine dimers from DNA in E. Coli K-12. Proc Natl Acad Sci U S A . 1964;51:293-300.
8 Pettijohn D, Hanawalt P. Evidence for repair-replication of ultraviolet damaged DNA in bacteria. J Mol Biol . 1964;9:395-410.
9 Setlow RB, Carrier WL. The disappearance of thymine dimers from DNA: an error-correcting mechanism. Proc Natl Acad Sci U S A . 1964;51:226-231.
10 Hanawalt PC. Subpathways of nucleotide excision repair and their regulation. Oncogene . 2002;21(58):8949-8956.
11 de Laat WL, Jaspers NG, Hoeijmakers JH. Molecular mechanism of nucleotide excision repair. Genes Dev . 1999;13(7):768-785.
12 Wood RD. Nucleotide excision repair in mammalian cells. J Biol Chem . 1997;272(38):23465-23468.
13 Thoms KM, Baesecke J, Emmert B, et al. Functional DNA repair system analysis in haematopoietic progenitor cells using host cell reactivation. Scand J Clin Lab Invest . 2007;67(6):580-588.
14 Cleaver JE. Defective repair replication of DNA in xeroderma pigmentosum. Nature . 1968;218(5142):652-656.
15 Kraemer KH, Lee MM, Scotto J. DNA repair protects against cutaneous and internal neoplasia: evidence from xeroderma pigmentosum. Carcinogenesis . 1984;5(4):511-514.
16 Peltomaki P. Role of DNA mismatch repair defects in the pathogenesis of human cancer. J Clin Oncol . 2003;21(6):1174-1179.
17 Li GM. Mechanisms and functions of DNA mismatch repair. Cell Res . 2008;18(1):85-98.
18 Vasen HF. Review article: the Lynch syndrome (hereditary nonpolyposis colorectal cancer). Aliment Pharmacol Ther . 2007;26(Suppl 2):113-126.
19 Felton KE, Gilchrist DM, Andrew SE. Constitutive deficiency in DNA mismatch repair. Clin Genet . 2007;71(6):483-498.
20 Brown KD, Rathi A, Kamath R, et al. Nat Genet . 2003;33(1):80-84.
21 Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature . 2003;421(6922):499-506.
22 Hammond EM, Kaufmann MR, Giaccia AJ. Oxygen sensing and the DNA-damage response. Curr Opin Cell Biol . 2007;19(6):680-684.
23 Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature . 2003;421(6922):499-506.
24 Pellegrini M, Celeste A, Difilippantonio S, et al. Autophosphorylation at serine 1987 is dispensable for murine Atm activation in vivo. Nature . 2006;443(7108):222-225.
25 Carney JP, Maser RS, Olivares H, et al. The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell . 1998;93(3):477-486.
26 Stewart GS, Maser RS, Stankovic T, et al. The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell . 1999;99(6):577-587.
27 Giaccia AJ, Kastan MB. The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev . 1998;12(19):2973-2983.
28 Riley T, Sontag E, Chen P, et al. Transcriptional control of human p53-regulated genes. Nat Rev Mol Cell Biol . 2008;9(5):402-412.
29 Chipuk JE, Kuwana T, Bouchier-Hayes L, et al. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science . 2004;303(5660):1010-1014.
30 Mihara M, Erster S, Zaika A, et al. p53 has a direct apoptogenic role at the mitochondria. Mol Cell . 2003;11(3):577-590.
31 Chang YC, Jan KY, Cheng CA, et al. Direct involvement of the tumor suppressor p53 in nucleotide excision repair. DNA Repair (Amst) . 2008;7(5):751-761.
32 Frebourg T, Barbier N, Yan YX, et al. Germ-line p53 mutations in 15 families with Li-Fraumeni syndrome. Am J Hum Genet . 1995;56(3):608-615.
33 Malkin D, Li FP, Strong LC, et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science . 1990;250(4985):1233-1238.
34 Srivastava S, Zou ZQ, Pirollo K, et al. Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-Fraumeni syndrome. Nature . 1990;348(6303):747-749.
35 Hartman AR, Ford JM. BRCA1 induces DNA damage recognition factors and enhances nucleotide excision repair. Nat Genet . 2002;32(1):180-184.
36 Liu S, Ginestier C, Charafe-Jauffret E, et al. BRCA1 regulates human mammary stem/progenitor cell fate. Proc Natl Acad Sci U S A . 2008;105(5):1680-1685.
37 Smalley MJ, Reis-Filho JS, Ashworth A. BRCA1 and stem cells: tumour typecasting. Nat Cell Biol . 2008;10(4):377-379.
38 Zhang J, Powell SN. The role of the BRCA1 tumor suppressor in DNA double-strand break repair. Mol Cancer Res . 2005;3(10):531-539.
39 Lim DS, Hasty P. A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol Cell Biol . 1996;16(12):7133-7143.
40 Wang Y, Cortez D, Yazdi P, et al. BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev . 2000;14(8):927-939.
41 Xu B, Kim S, Kastan MB. Involvement of BRCA1 in S-phase and G(2)-phase checkpoints after ionizing irradiation. Mol Cell Biol . 2001;21(10):3445-3450.
42 Moynahan ME, Chiu JW, Koller BH, et al. BRCA1 controls homology-directed DNA repair. Mol Cell . 1999;4(4):511-518.
43 Hartman AR, Ford JM. BRCA1 and p53: compensatory roles in DNA repair. J Mol Med . 2003;81(11):700-707.
44 Alter BP. Cancer in Fanconi anemia, 1927–2001. Cancer . 2003;97(2):425-440.
45 D’Andrea AD, Grompe M. The Fanconi anaemia/BRCA pathway. Nat Rev Cancer . 2003;3(1):23-34.
46 Gurtan AM, D’Andrea AD. Dedicated to the core: understanding the Fanconi anemia complex. DNA Repair (Amst) . 2006;5(9–10):1119-1125.
47 Kennedy RD, D’Andrea AD. DNA repair pathways in clinical practice: lessons from pediatric cancer susceptibility syndromes. J Clin Oncol . 2006;24(23):3799-3808.
48 Grompe M, van de Vrugt H. The Fanconi family adds a fraternal twin. Dev Cell . 2007;12(5):661-662.
49 Wang X, D’Andrea AD. The interplay of Fanconi anemia proteins in the DNA damage response. DNA Repair (Amst) . 2004;3(8–9):1063-1069.
50 Garcia-Higuera I, Taniguchi T, Ganesan S, et al. Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol Cell . 2001;7(2):249-262.
51 Taniguchi T, Garcia-Higuera I, Andreassen PR, et al. S-phase-specific interaction of the Fanconi anemia protein, FANCD2, with BRCA1 and RAD51. Blood . 2002;100(7):2414-2420.
52 Wang X, Andreassen PR, D’Andrea AD. Functional interaction of monoubiquitinated FANCD2 and BRCA2/FANCD1 in chromatin. Mol Cell Biol . 2004;24(13):5850-5862.
53 Taniguchi T, Garcia-Higuera I, Andreassen PR, et al. S-phase-specific interaction of the Fanconi anemia protein, FANCD2, with BRCA1 and RAD51. Blood . 2002;100(7):2414-2420.
54 Wang X, D’Andrea AD. The interplay of Fanconi anemia proteins in the DNA damage response. DNA Repair (Amst) . 2004;3(8–9):1063-1069.
55 Sii-Felice K, Barroca V, Etienne O, et al. Role of Fanconi DNA repair pathway in neural stem cell homeostats. Cell Cycle . 2008;7(13):1911-1915.
56 Sii-Felice K, Etienne O, Hoffschir F, et al. Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors. EMBO J . 2008;27(5):770-781.
57 Nakayama H, Nakayama K, Nakayama R, et al. Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway. Mol Gen Genet . 1984;195(3):474-480.
58 Sharma S, Brosh RMJr. Human RECQ1 is a DNA damage responsive protein required for genotoxic stress resistance and suppression of sister chromatid exchanges. PLoS ONE . 2007;2(12):e1297.
59 Gray MD, Shen JC, Kamath-Loeb AS, et al. The Werner syndrome protein is a DNA helicase. Nat Genet . 1997;17(1):100-103.
60 Suzuki N, Shimamoto A, Imamura O, et al. DNA helicase activity in Werner’s syndrome gene product synthesized in a baculovirus system. Nucleic Acids Res . 1997;25(15):2973-2978.
61 Huang S, Li B, Gray MD, et al. The premature ageing syndrome protein, WRN, is a 3′→5′ exonuclease. Nat Genet . 1998;20(2):114-116.
62 Bachrati CZ, Hickson ID. RecQ helicases: guardian angels of the DNA replication fork. Chromosoma . 2008;117(3):219-233.
63 Pirzio LM, Pichierri P, Bignami M, et al. Werner syndrome helicase activity is essential in maintaining fragile site stability. J Cell Biol . 2008;180(2):305-314.
64 Ellis NA, German J. Molecular genetics of Bloom’s syndrome. Hum Mol Genet . 1996;5 Spec No:1457-1463.
65 Gruber SB, Ellis NA, Scott KK, et al. BLM heterozygosity and the risk of colorectal cancer. Science . 2002;297(5589):2013.
66 Oshima J. The Werner syndrome protein: an update. Bioessays . 2000;22(10):894-901.
67 Shen JC, Loeb LA. The Werner syndrome gene: the molecular basis of RecQ helicase-deficiency diseases. Trends Genet . 2000;16(5):213-220.
68 Yu CE, Oshima J, Fu YH, et al. Positional cloning of the Werner’s syndrome gene. Science . 1996;272(5259):258-262.
69 Kitao S, Shimamoto A, Goto M, et al. Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome. Nat Genet . 1999;22(1):82-84.
70 Wang LL, Gannavarapu A, Kozinetz CA, et al. Association between osteosarcoma and deleterious mutations in the RECQL4 gene in Rothmund-Thomson syndrome. J Natl Cancer Inst . 2003;95(9):669-674.
71 Helleday T, Petermann E, Lundin C, et al. DNA repair pathways as targets for cancer therapy. Nat Rev Cancer . 2008;8(3):193-204.
72 Martin SA, Lord CJ, Ashworth A. DNA repair deficiency as a therapeutic target in cancer. Curr Opin Genet Dev . 2008;18(1):80-86.
73 Hassa PO, Hottiger MO. The diverse biological roles of mammalian PARPS, a small but powerful family of poly-ADP-ribose polymerases. Front Biosci . 2008;13:3046-3082.
74 Bryant HE, Schultz N, Thomas HD, et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature . 2005;434(7035):913-917.
75 Farmer H, McCabe N, Lord CJ, et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature . 2005;434(7035):917-921.
76 Bryant HE, Schultz N, Thomas HD, et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature . 2005;434(7035):913-917.
77 Farmer H, McCabe N, Lord CJ, et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature . 2005;434(7035):917-921.
78 Hartwell LH, Szankasi P, Roberts CJ, et al. Integrating genetic approaches into the discovery of anticancer drugs. Science . 1997;278(5340):1064-1068.
79 Iorns E, Lord CJ, Turner N, Ashworth A. Utilizing RNA interference to enhance cancer drug discovery. Nat Rev Drug Discov . 2007;6(7):556-568.
80 Bartkova J, Horejsi Z, Koed K, et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature . 2005;434(7035):864-870.
81 Gorgoulis VG, Vassiliou LV, Karakaidos P, et al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature . 2005;434(7035):907-913.
3 Fractionation Effects in Clinical Practice

Søren M. Bentzen, PhD, DSc
Radiation dose fractionation —the temporal programming of radiation delivery—has been a fruitful field of clinical research throughout the history of radiation therapy and has been one of the main arenas for attempting to improve the therapeutic ratio. * With the further development and wider use of the linear-quadratic (LQ) bioeffect formula around 1980 and its success in explaining a flurry of clinical and experimental fractionation data during the next decade or so, many clinical radiation researchers believed that the main effects of dose fractionation on tumors and normal tissues were finally well understood; everything that could be discovered had been discovered, and it was only a question of time before the last outstanding problems were cleared away. Interestingly, the whole field of clinical fractionation research has undergone a veritable renaissance since then, and there seems to be even more research challenges and opportunities today than there were back in the 1980s and 1990s. This revived interest has been stimulated by the introduction of new treatment planning and delivery technologies during this period. In addition, the wider use of combined treatment modalities has reopened many questions relating to optimal dose fractionation. But more than anything, a large body of clinical and biologic research has challenged many of the dogmas taught just 10 or 15 years ago. Although much of the teaching of dose fractionation biology has traditionally relied on assumed parallelisms between humans and a variety of biologic model systems, the discussion can now largely be based on clinical data from case series or even from randomized controlled trials. In this chapter, results of randomized trials are cited whenever relevant, but, rather than tabulating a long string of individual trials, the focus of this chapter is on using and understanding the biologic insights derived from clinical studies.


Conventional and Altered Fractionation

The Origins of Conventional Fractionation
Within a few years after Röntgen’s discovery of x-rays in 1895, the first attempts were made at using these new rays in the treatment of benign and malignant disease and the first cures of human skin cancer with radiation therapy were reported independently by two Swedish physicians, Tage Sjögren 2 and Thor Stenbeck, 3 in 1899 and 1900. It became almost immediately clear that the biologic effects of a given physical absorbed dose depend strongly on the details of how this dose is delivered over time. As early as in 1902, Ludwig Teleky recommended dose fractionation as a means of reducing the level of late side effects in a discussion in the Royal Society of Physicians in Vienna. 4
With advances in x-ray technology, it became technically possible to deliver a large enough dose to produce a visible skin reaction in a single sitting. Biologic reasons for preferring short, intensive schedules were mainly speculative rather than based on empirical data, but these schedules won many supporters especially in Germany and Sweden. A few systematic experimental studies tried to compare single dose versus fractionated radiotherapy for various tissues. In 1927, Regaud and Ferroux showed that it was possible to sterilize a ram’s testis without excessive skin reactions using fractionated radiation but not using single doses, 5 experiments that have become part of radiotherapy folklore. Regaud proposed, controversially at the time of publication, that spermatogenesis could serve as a model of human cancers. We now know that the “inverse fractionation effect” he observed (i.e., a larger effect with decreasing dose per fraction [F]) is unique to the spermatogenic system. What Regaud did realize was that it is not the biologic effect of higher dose per fraction in itself than matters, but rather the differential effect of changes in dose per fraction for tumors and the relevant normal tissues. Jens Juul in Denmark experimented on transplantable murine tumors in the late 1920s and used skin as the relevant normal tissue in a series of studies based on the idea of treating to isotoxicity 6 and concluded that fractionated radiotherapy yielded a superior therapeutic index. For a more detailed account of these studies, see Bentzen and Thames. 7 The fractionation debate was finally settled by clinical observations and by the early 1930s there was a widespread consensus that curative radiation therapy should be delivered in multiple fractions (so-called fractionated-protracted radiotherapy) rather than as a large single dose. What is even today referred to as conventional fractionation originated from a series of systematic clinical studies conducted by Baclesse and Coutard between approximately 1910 and the mid-1920s in Paris. These studies aimed to mimic Regaud’s fractionated daily external radium treatments and succeeded in reproducing the good clinical outcome achieved by Regaud. It was this superior clinical outcome of the “Paris schedule” that convinced the doubters and at least temporarily created a consensus on dose fractionation. It is worth stipulating that conventional fractionation originated from a series of clinical experiments aiming at optimizing the local control of head and neck squamous cell carcinoma (HNSCC) while maintaining a tolerable level of skin reactions. This schedule influenced what became conventional fractionation in many other tumor sites. It is only relatively recently that clinical researchers have fully embraced the idea that fractionation should be optimized separately for each tumor type and for the most clinically relevant toxicities associated with treating that tumor.
As a result of strained resources in the wake of World War II, Ralston Patterson at the Christie Hospital in Manchester introduced a 3-week schedule delivering 52.5 or 55 Gy in 15 or 16 F. In the early 1950s, Patterson looked at the clinical outcome of these hypofractionated schedules and concluded that they warranted continued use even as the shortage of resources were partly relieved. A variant of the Manchester schedule was developed in Edinburgh in Scotland delivering therapy over 4 instead of 3 weeks. 7a The Manchester and Edinburgh schedules were adopted in many institutions, mainly in the British Commonwealth. These schedules coexisted—not always peacefully—with the standard fractionation schedule for decades, and it was only much later, with an improved quantitative understanding of dose-time-fractionation effects, that it became clear why these schedules produce near-equivalent outcomes to that of standard fractionation.

The Four “Rs” of Fractionated Radiotherapy in a Clinical Perspective
Withers 8 reviewed the radiobiologic basis of fractionated radiotherapy in 1975 and summarized the main rationale for dose fractionation in his now famous mnemonic referring to the four “Rs” of radiotherapy: repair, repopulation, redistribution, reoxygenation. All of these are biologic effects that occur in the time interval between dose fractions. They are characterized by the magnitude of the effects as well as by their kinetics, and it is the differential action of these in tumors and normal tissues that determine whether a change in dose fractionation will generate an improved therapeutic ratio. A fifth R, radiosensitivity, has been proposed 9 as a major factor determining radiotherapy outcome. However, this R is clearly of a different nature than the four Rs proposed by Withers as it does not take place in the interfraction interval. The most elegant experiments illustrating the four Rs of radiotherapy are in vitro or small animal studies, but these are not reviewed in this chapter. Instead, a brief summary is given of the status of the four Rs as deduced from clinical observations. A distinction is made between the observable clinical effect and the underlying radiobiologic interpretation.
Repair at the cellular level has traditionally been seen as a manifestation of sublethal damage repair. At the tissue (or clinical) level, the term recovery is preferred by some authors as a more phenomenologic description of the recovery from damage observed when splitting the same physical dose into fractions, without assuming a specific cellular or molecular mechanism behind this effect. The clinical manifestation of recovery is the decreased biologic effect resulting from delivering a constant physical absorbed dose with decreasing dose per fraction. Fig. 3-1 shows dose-response curves for the incidence of moderate and severe (G2+) subcutaneous fibrosis. Note that the doses are estimated at the relevant reference depth for subcutaneous fibrosis 10 ; in other words, they are not identical to the prescribed dose to the target delivered in 12 or 22 F. The dose, D 12 , corresponding to a 50% incidence of G2+ subcutaneous fibrosis is lower than the dose, D 22 , that would produce the same incidence in the 22 F group. The two doses are related by a mathematic relationship—the LQ model. This fractionation effect has been observed in countless studies, perhaps most clearly in clinical studies in which a larger dose per fraction has been introduced without any reduction in total dose. The classical example is the breast radiotherapy study by Montague 11 in which 35 to 40 Gy was delivered across 4 weeks, giving 5 F per week in 88 patients and 3 F per week without a total dose reduction in 30 patients. The incidence of late complication was much higher in the large dose-per-fraction group. More support comes from studies in which the dose per fraction has been increased, but although a reduction of total dose was implemented, this later turned out to be insufficient. This occurred, for example, in studies 12 , 13 using the nominal standard dose formula created by Ellis 14 as a guide for reducing the dose, which effectively led to an over-dosage of late normal-tissue endpoints.

FIGURE 3-1 • The fractionation effect. Dose-response curves fitted to clinical data on the incidence of moderate and severe subcutaneous fibrosis as a function of dose delivered in 12 (red) or 22 F (blue) in patients receiving postmastectomy radiotherapy. The dose is estimated at a tissue depth of 4.1 mm, not to be confused with the prescription dose. The doses D 12 (delivered in 12 F) and D 22 (delivered in 22 F) are associated with a 50% incidence of G2+ subcutaneous fibrosis (i.e., they are said to be isoeffective). Other incidence levels can also be used when defining isoeffective doses.
(Data from Bentzen et al., 10 which provides a more detailed discussion.)
In addition to the magnitude of recovery, the kinetics of recovery is important for biologic effect. A dramatic illustration of this is the study by Nguyen et al. 15 in which 39 patients received rapid hyperfractionated radiotherapy: 6 to 8 F of 0.9 Gy per day with a 2-hour interval between fractions were delivered five days a week for a total dose of 66 to 72 Gy. Radiotherapy was delivered in two series of 33 to 36 Gy separated by a 2- to 4-week rest interval. If the 2-hour interval had been sufficient for complete or near-complete recovery, this dose should have been relatively well-tolerated. Instead, after a minimum follow-up of 2 years, 70% of patients experienced late complications, and in 54% of cases these reactions were considered severe, causing death in 13% of patients.
Repopulation is clinically reflected in the influence of overall treatment time on local control of at least some tumor types (in which it is often called accelerated proliferation ) and on the incidence of some early normal tissue effects. This phenomenon is often referred to as the time factor, a term that has the advantage of referring to a clinically observable effect rather than to an underlying hypothetical cellular mechanism. Several studies tried to estimate the tumor time factor by comparing patients who completed therapy in the planned overall time versus patients who had protracted overall time resulting from unplanned treatment interruptions. 16 A strong case can be made, however, that patients with and without unplanned interruptions are not likely to present with comparable tumor and patient characteristics. 17 Much stronger evidence has subsequently been obtained from randomized controlled trials, at least for some tumor types.
Redistribution of the number of cells in various phases of the cell cycle is observed after irradiation of an asynchronous cell population caused by varying radiosensitivity in these phases. There is no directly observable clinical parallel to redistribution. It is interesting to note that the European Organisation for Research and Treatment of Cancer (EORTC) 22791 trial by Horiot et al. 18 used two fractions per day in an attempt to “catch” the more rapidly proliferating tumor cells that were hypothesized to have progressed into a more sensitive phase of the cell cycle at the time of the second daily fraction. 19 When this trial was completed, the data were widely interpreted in terms of a differential sensitivity to dose per fraction (i.e., as a manifestation of differences in repair or recovery capacity) as predicted by the LQ model. Redistribution is also of great interest in combining drugs with radiation and many cytotoxic drugs such as cisplatin, 5-fluorouracil (5-FU), and gemcitabine require cell-cycle progression to act as radiosensitizers. 20 Carefully timed administration of drugs and radiation has been proposed in an attempt to synchronize surviving tumor cell populations and then irradiate them in a sensitive phase of the cycle—an idea that so far has not worked convincingly in clinical trials, most likely because human cell populations in vivo are so heterogeneous in their cell kinetics parameters that synchrony is quickly lost in a population of patients.
Reoxygenation refers to the empirical observation that hypoxic regions in tumors may improve their oxygenation during fractionated radiotherapy. Traditionally, this has been viewed as a consequence of preferential killing by radiation of well-oxygenated cells that in turn leads to a lower metabolic consumption of oxygen, which again leads to improved oxygenation of the predominantly hypoxic cells surviving a dose fraction. The kinetics of reoxygenation is not well-studied in humans. However, a wide tumor-to-tumor variability in reoxygenation has been detected in human tumors by means of positron emission tomography scan using hypoxia-sensitive tracers. 21 Because many human tumors, in contrast to most normal tissues, contain hypoxic cell populations, reoxygenation provides further rationale for fractionated radiation therapy. Advances in hypoxia-related cancer research suggest that this rather simple model of hypoxia and its modification during fractionated radiotherapy is an over-simplification. Hypoxia is now seen as an integral aspect of malignant progression and as an active biologic process rather than the passive push of cells into oxygen deprivation. 22 Hypoxia-mediated radioresistance is also emerging as an actively controlled phenomenon, with the hypoxia-inducible factor–1α as a key player, and not just a direct consequence of the physicochemical absence of molecular oxygen. 23 - 25 From a clinical radiation research perspective, many strategies have been devised to modify tumor hypoxia, including treatment in hyperbaric oxygen, administration of electron-affinic oxygen mimetic drugs such as nitroimidazoles, 26 or hypoxic cytotoxins such as tirapazamine. 27 These have been plagued by logistic difficulties and unexpected toxicities, but more discouragingly have yielded modest improvements in tumor control. 28

The Linear-Quadratic Model

Mechanistic Background
The LQ model originated from work by Lea and Catcheside in Cambridge around the time of World War II. 28a In a series of published studies, they investigated the influence of dose and dose rate on chromosome damage in cells. Lea and colleagues developed a framework for interpreting their experimental data in terms of inactivation of discrete targets. 28b They hypothesized that inactivation could result from single or double “hits” and fitted an expression of the form α · D + β · D 2 to experimental data on the number of chromatid exchanges per cell as a function of dose. Curiously, they used the notations α and β for the two coefficients in their “linear-quadratic” equation. 28c Numerous investigators derived LQ bioeffect relationship under various assumptions; for example, Kellerer and Rossi 29 published an elaborate theory of dual radiation action in 1972. However, it was the 1976 paper by Douglas and Fowler, 30 in which they used the LQ model to fit data on skin reactions after fractionated radiotherapy, that stimulated the modern interest in the LQ model. The full effect plot devised by Douglas and Fowler facilitated the analysis of isoeffect data for any sign or symptom of radiation therapy without the need to know the underlying target cell survival curve. At the same time, the F e plot provided a simple graphic test of the fit of the LQ model to the data. A few years later, Thames and colleagues 31 realized that the numeric value of the ratio of the two model parameters, α/β, differed between early and late effects of radiation therapy, with values for early effects typically ranging from 8 to 12 Gy and for late effects between 1 and 5 Gy. Thames and colleagues hypothesized that the difference in α/β reflected differences in the curvature of the dose-survival curve of the putative target cells. The compelling link between the LQ model and the target cell hypothesis remained strong for many years, but this has come under increasing pressure recently.

Beyond the Target Cell Hypothesis
Advances in molecular pathology have considerably improved our understanding of radiation-induced pathologic conditions of late normal tissue effects in particular. 32 It has become clear that many late effects result from a powerful, sustained wound healing response at the tissue level rather than simple parenchymal cell loss. The biology of this “fibrogenic-atrophic” radiation response pathway has been unraveled in quite some detail during the preceding decade or so. A key switch in this response is the activation of the multifunctional, strongly profibrotic cytokine transforming growth factor (TGF)-β. Ionizing radiation is one of the few exogenous factors that can activate TGF-β directly. Thus the target cell hypothesis may not explain the main pathogenic pathways for late effects. This is in contrast to the radiation-induced pathologic conditions of early effects that typically occur in tissues with a hierarchical proliferative organization and depend on the relative depletion of the stem cell pool. 33 In the present context, this puts a question mark over the traditional explanation of the difference in α/β ratio between early and late effects, namely that this reflects differences in the shape of the (in vivo) cellular dose-survival curve for the putative target cells giving rise to the two types of radiation effects. At the time of writing, it is not clear what part of the TGF-β pathways determines the relatively high fractionation sensitivity of late effects. Suffice it to say that an overwhelming body of clinical and experimental data show that the dose adjustment required for maintaining a constant level of side effects when dose per fraction is changed is much larger for late than for early endpoints. This “postmodern” view of the LQ model simply acknowledges the empirical success of the model in estimating biologically equivalent doses when changing dose-fractionation—at least within a limited dose range—but does not presume any deeper mechanistic validity of the mathematic form of this model.

Pragmatic Derivation—Correction for Dose per Fraction
The LQ model can be derived based on a few general clinical observations. Assume that a total dose (D) delivered with dose per fraction (d) produces a specific biologic effect. Then the following four statements are supported by a large body of clinical and preclinical data:
1 A higher total dose, D + ΔD, delivered with dose per fraction d will result in an increased biologic effect.
2 Delivering the dose D with a higher dose per fraction, d + Δd, will result in an increased biologic effect.
3 There is an interaction between dose and dose per fraction such that the effect of a 1-Gy increase in total dose will be larger for a larger dose per fraction.
4 In the limit of a very low dose per fraction (or low dose rate in the case of continuous low-dose-rate irradiation) the biologic effect is proportional to total dose.
Assume further that there is a strictly increasing function (f) linking a given biologic dose to an observable clinical effect (E). The mathematic form of f is possibly very complex; however, the fact that it is strictly increasing means that a higher biologic dose produces an increased biologic effect. Mathematically, the simplest expression for the biologic dose that is consistent with points 1 through 4 is:
(Eq. 1)
Other functional forms could work as well, but this expression is as simple as it gets. Now we are ready to derive the isoeffect formula, referred to as the Withers formula. Two dose fractionation schedules, delivering a dose D 1 in dose per fraction d 1 , produces the same biologic effect as a dose D 2 delivered in dose per fraction d 2 , if and only if:
(Eq. 2)
This follows from the assumption that f is a strictly increasing function. We assume that the overall treatment time is identical in the two schedules and that redistribution and reoxygenation can be ignored. Equation 2 can easily be rearranged to yield:

Dividing by β on both sides of the equation yields:

Finally, we isolate D 1 on the left-hand side:
(Eq. 3)
which is the so-called Withers formula. 34 Note that there is only one parameter in this formula that needs to be estimated from clinical or experimental data: the α/β ratio. This formula describes the relationship between the two isoeffective dose in Fig. 5-1 ; or, alternatively, if we observe the shift in the dose-response curves, we can calculate the α/β ratio for the endpoint in question, subcutaneous fibrosis (in turns out to be α/β = 1.8 Gy 10 ). More precise estimates can be obtained using statistical techniques and these also allow estimation of the uncertainty of the α/β (as well as other model parameters) and allow correction for the latent period and censoring in patients who were alive and well at the last follow-up. 35 , 36 In other words, it is not necessary to know the separate values of α and β to estimate the biologically equivalent dose when changing from one fraction size to another. Up-to-date compilations of α/β values for experimental 37 and clinical 38 normal-tissue and tumor endpoints have been published recently.
For an endpoint with a known α/β ratio, Equation 3 can be applied to convert an arbitrary dose, D, delivered with dose per fraction, d, into an equivalent dose in 2-Gy fractions, (EQD 2 ). 38 This quantity is equivalent to the normalized total dose introduced by Withers et al. 34 :
(Eq. 4)
A simple numeric example follows: A simultaneous integrated boost plan is being considered for a patient with HNSCC. The plan will deliver 66 Gy in 22 F to the gross tumor volume. Assuming that α/β = 10 Gy for HNSCC, what is the equivalent dose in 2-Gy fractions, EQD 2 ? Inserting the known values in Equation 4 , we get:


Recovery Kinetics
Split-dose recovery was discovered in cell lines in vitro by Elkind and Sutton 39 in 1960. By varying the interval between two fractions (“splitting” the same physical dose in two), Elkind and Sutton were able to show how the two fractions had the same biologic effect as a single dose of the same size if the interval between fractions was very short, and how this effect asymptotically approached a certain minimum effect as the interval became very long. The same effect can be seen in clinical studies when the interval between fractions is varied, and this is also an important component in explaining the dose-rate effect in continuous irradiations. Mathematically, the effect of protracting therapy (i.e., delivering each dose fraction over longer and longer time) can be described by introducing the Lea-Catcheside factor (g), which can be seen as a factor modifying the effective fraction size:
(Eq. 5)
Under fairly simple assumptions, the g-function can be expressed in analytic form for many of the situations arising in clinical radiation oncology such as continuous irradiation or multiple fractions per day (MFD) with incomplete repair between fractions (see, for example, Joiner and Bentzen 37 ).

Correction for Overall Treatment Time
Empirically, the biologic effect of a given total dose delivered in a fixed number of fractions decreases with increasing overall treatment time (i.e., with increasing time between the first and last dose fraction). In case of tumor control, this effect is often referred to as the tumor time factor, and it is often interpreted as the result of tumor clonogen (cancer stem cell) proliferation in this time interval. Fig. 3-2 summarizes data from the Danish Head and Neck Cancer collaborative group (DAHANCA) comparing the outcome of fractionated radiotherapy delivered with 2-Gy fractions over 6 weeks and 10 weeks. 40 With longer overall treatment time, the tumor dose-control curve shifted to the right; in other words, an increased dose was required to maintain the same level of tumor control. In contrast, the dose-response curve for laryngeal edema did not change. This caused a narrowing of the therapeutic window. Assuming that laryngeal edema and local failure are statistically independent, 41 the probability of uncomplicated cure (P+) becomes TCPx (1 − normal tissue complication probability [NTCP]). The maximum value of P+ dropped from 78% for continuous course to 47% for split-course radiation therapy.

FIGURE 3-2 • The tumor time factor. Dose-response curves fitted to clinical data for tumor control and the incidence of laryngeal edema as a function of dose delivered as continuous course (overall treatment time 6 weeks) and split-course radiotherapy (overall treatment time 10 weeks, including a planned 3-week gap). Assuming that laryngeal edema and locoregional failure are statistically independent, the stippled green curve is the probability of uncomplicated cure (P+) originally proposed by Holthusen.
(Data from Overgaard et al. 40 )
A time factor is also seen for many early endpoints; again, this is thought to reflect the effect of repopulation of stem cells during treatment. A simple empirical adjustment for overall treatment time can be made as follows. Assume that a schedule delivers total dose D 2 with dose per fraction d 2 in an overall treatment time of T 2 days. First we correct for dose per fraction by calculating the EQD 2 and then we adjust for treatment time relative to an arbitrary reference duration of T 1 days by taking the difference between T 2 and T 1 and multiplying this quantity by the dose recovered per day, D prolif , a parameter estimated directly from clinical data:
(Eq. 6)
In other words, if total dose D 2 is delivered in T 2 days and T 2 is greater than T 1 , we will subtract a (positive) dose from the estimated equivalent dose, reflecting the fact that biologic effect is lost because of the prolonged treatment time. It is assumed that both T 1 and T 2 are sufficiently long for accelerated proliferation to occur at a constant rate. As this formula is likely to be used in the comparison of two fractionation schedules, the arbitrary choice of the length of the reference schedule (T 1 ) will cancel out.
The majority of empirical D prolif estimates are derived from HNSCC data, in which this parameter consistently across a large number of studies 38 , 42 comes out at approximately 0.65 Gy/day. This means that if a schedule is prolonged by, for example, 5 days, the estimated decrease in the EQD 2 is 5 days × 0.65 Gy/day = 3.25 Gy. Mechanistically, the cellular proliferative response to a cytotoxic insult is a complex phenomenon, 33, 43, 44 but this simple linear correction is likely to be a good approximation over a narrow range of treatment times, in the order of 1 to 2 weeks perhaps for a 6- or 7-week schedule. The numeric value of D prolif cited previously is estimated toward the end of treatment schedules with an overall treatment time of some 5 to 7 weeks. Proliferation before the start of radiotherapy corresponds to a much lower dose per day. This phenomenon is called accelerated proliferation (or accelerated repopulation ).
When does accelerated proliferation start? In their classic paper from 1988, Withers et al. 45 proposed that isoeffect doses for local control of HNSCC remained largely constant up until approximately 4 weeks of overall treatment time, after which accelerated proliferation would kick in and the dose lost per day because of proliferation would be approximately 0.6 to 0.7 Gy/day. The appearance of this biphasic relationship, dubbed the dog-leg, depended on the exact assumptions of the modeling performed by Withers et al. to pool data from multiple studies on the same graph, and some authors—including the present one—argued that these assumptions were unrealistic 46 and that the data could in reality not discriminate between the dog-leg shape and a straight line all the way down to very short schedules. A specific weakness of the available data was an absence of schedules treating in less than 4 weeks.
The continuous, hyperfractionated, accelerated radiotherapy (CHART) trial is very interesting in this context, as the experimental CHART arm finished radiotherapy in only 12 days. This provides a sensitive test for the existence of the “kink” on the dog-leg graph ( Fig. 3-3 ). As CHART employed 1.5-Gy fractions, we use Equation 4 with an assumed α/β of 10 Gy (this parameter should ideally be estimated from the data as well) to calculate the EQD 2 of the CHART arm: 51.75 Gy. From the reported hazard ratio (HR) derived from the 918 patients in the trial, we can calculate the apparent dose in 2-Gy fractions that would be isoeffective with 66 Gy in 45 days: the result is 51.3 Gy with 95% confidence limits 49.3 Gy and 53.3 Gy, plotted in Fig. 3-3 . This is considerably higher than the 44.6 Gy estimated from back-extrapolating the 66 Gy by 0.65 Gy/day all the way down to 12 days’ overall time. In other words, delivering dose in just 12 days is not nearly as effective as one would estimate if the dose recovered per day was constant all the way down to 12 days. *

FIGURE 3-3 • The isoeffective dose for tumor control is expected to increase with overall treatment time due to accelerated proliferation in head and neck squamous cell carcinoma (HNSCC): the “dog-leg.” Near-equivalence was observed between the conventional arm (66 Gy in 33 F over 45 days) and the accelerated arm (54 Gy in 36 F over 12 days) in the continuous, hyperfractionated, accelerated radiotherapy HNSCC trial. Using the 66 Gy in 33 F as the reference point, a linear back-extrapolation using D prolif = 0.65 Gy/day predicts that the isoeffective dose would follow the stippled line . The data point (with error bars depicting 95% confidence limits on the estimate) is the equivalent dose with 2-Gy fractions delivered in 12 days as estimated from the trial outcome data and this deviates significantly from the stippled line. This bi-phasic relatiohship is referred to as the “dog-leg.”

Thus, Withers turned out to be right: our current best estimates of the radiobiologic parameters for accelerated repopulation are almost exactly as derived in the 1988 paper. This has huge implications for how far accelerated fractionation schedules can be pushed. It is difficult from the available data to decide whether dose recovery with extended treatment time is truly a biphasic relationship or whether there are more phases. What is clear, however, is that a constant rate of proliferation corresponding to a recovered dose of D prolif = 0.6-0.7 Gy/day cannot be back-extrapolated all the way down to schedules of 1 or 2 weeks’ duration. The standard way to incorporate this phenomenon in the modeling of the overall time effect is to assume D prolif = 0 Gy/day up until a defined kick-off time (T k ) and then assume a constant D prolif thereafter (see Fig. 3-3 ). In practice, this means that if T 1 or T 2 (or both) is shorter than T k then this (or these) times are set equal to T k when entered into equation 6.

Alternative Formulations of the LQ Model
A couple of mathematically equivalent formulations of the LQ model have been used as alternatives to the EQD 2 formula. Among these, only the biologically effective dose (BED) formula has found any wider use. 47 The mathematic equivalence ensures that calculations made in any of these frameworks will produce the same end result and the choice between these formulations typically comes down to how this was first taught to an individual or a more subjective preference for one or the other of these methods. The BED formula and the language associated with its use are solidly anchored in the target cell hypothesis: “logs of cell kill,” “potential doubling times,” “repairable and irreparable damage,” “α- and β-component of damage”—in contrast to the EQD 2 formula, which is more naturally linked to clinical dose fractionation and observable clinical outcomes. Apart from this difference in conceptual context, the EQD 2 formula has the advantage that all doses calculated for parts or the whole of a fractionated radiotherapy schedules, are immediately recognized as “real” doses in 2-Gy fractions, a circumstance that reduces the risk of making numerical errors.

Range of Applicability of the LQ Model
Limitations to the range of dose per fraction in which the LQ model can be usefully applied spring in part from experimental data suggesting that the mathematic form of the LQ model may not be adequate at very low or high dose per fraction and in part from the statistical uncertainty of available parameter estimates. Fig. 3-4 shows the range (green) from approximately 1 Gy to approximately 5 Gy, in which the applicability of the LQ model has been supported by clinical data from a variety of normal-tissue endpoints and, to a smaller extent, tumor endpoints. This range could obviously depend on the endpoint in question and should be taken as a crude reminder of the fraction sizes in which we possibly could see deviations from the simple LQ formula, as briefly discussed in the following.

FIGURE 3-4 • Appropriate range of fraction sizes in which the linear equation (LQ) model is supported by clinical data is indicated in green. At doses lower than 1 Gy per fraction there are potential concerns—mainly supported by experimental data—that the LQ model could underestimate biologic effect. At high doses per fraction there are concerns that the LQ model might overestimate biologic effect.

Deviations From the LQ Model at Low Dose Per Fraction
Modern delivery technologies typically irradiate large normal-tissue volumes to relatively low doses. Some in vitro tumor cell survival curves display excess cell killing at low dose per fraction relative to that predicted from the LQ model. 48 This phenomenon is called low-dose hyper-radiosensitivity (HRS). Extensive studies have shown that HRS leads to apoptotic death of cells in the G2 phase of the cell cycle after doses of less than about 0.3 Gy. At higher doses a G2 checkpoint is activated that allows deoxyribonucleic acid repair to take place, which leads to increased cell survival, an effect referred to as induced radiation resistance . Dose planning studies have shown that if HRS plays a major role for the dose-limiting late side effects, this could reduce the benefit from intensity-modulated radiation therapy (IMRT) in some cases. 49 It remains an open question, however, whether this is actually the case. Under the target cell hypothesis, the expectation is that, because of the very low G2 phase fraction in the cell population involved in late effects, HRS is unlikely to be a main issue for late effects. It is not clear if this expectation can be extended to biologic damage-induction or damage-processing models. 32

Deviations From the LQ Model at High Dose Per Fraction
At the other extreme, there are in vitro and small animal data suggesting that the LQ model overestimates the effect of dose per fraction exceeding 8 to 10 Gy; for example, see Guerrero and Li. 50 This range of fraction sizes has become increasingly important with the clinical exploration of stereotactic body radiation therapy (SBRT), stereotactic radiation surgery, and intraoperative radiation therapy, as well as some schedules used in high-dose rate (HDR) brachytherapy.

Uncertainty of Radiobiologic Parameter Estimates
Numeric values of radiobiologic parameters for a specific tumor or normal-tissue endpoint should ideally be estimated empirically from clinical observations. Using generic values (e.g., α/β = 3 Gy for late effects) should be the last resort when no empirical estimates are available for the endpoint of interest. These clinical estimates, however, are associated with a statistical uncertainty, typically specified by the standard error of the estimate or the 95% confidence limits around the estimate. Fig. 3-5 shows an example for prostate cancer. Brenner et al. 51 estimated α/β at 1.1 Gy with 95% confidence limits 0.03 Gy and 4.1 Gy. Using α/β = 4.1 Gy, we can estimate the dose for a given fraction size that will be isoeffective with 76 Gy in 38 fractions ( red curve in Fig. 3-5 ). For example, for a fraction size of 5 Gy, we see that 5 Gy × 8 is expected to produce approximately the same effect as 76 Gy in 38 F. However, the interval from 0.03 Gy to 4.1 Gy includes with 95% probability the “true” value of α/β. Depending on what this “true” value actually is, the 40 Gy in 8 F produces a tumor effect equivalent to somewhere between 59 Gy and 97 Gy in 2-Gy fractions. A more precise titration of the equivalent dose in 8 F requires an improved α/β estimate, most likely to result from clinical trials using fraction sizes in the relevant range.

FIGURE 3-5 • Effect of uncertainty in α/β on the estimated equivalent dose in 2-Gy fractions is illustrated for prostate cancer. The red curve is the estimated dose as a function of fraction size for a hypofractionated regimen that is equivalent to 76 Gy in 38 F using Brenner et al.’s estimate, 51 α/β = 1.1 Gy with 95% confidence limits 0.03 Gy and 4.1 Gy. The stippled, vertical line indicates a dose per fraction of 5 Gy. The blue lines define the upper and lower 95% confidence limits on the estimated equivalent dose for the “true” tumor effect of the hypofractionated schedule resulting from the uncertainty in the α/β estimate.
Other fractionation-model parameters will also have an uncertainty associated with them; unfortunately, there are few published estimates and even fewer with adequate statistical estimates of their accuracy.

Altered Fractionation
In the wake of the wide interest in the LQ model, it was hypothesized that standard fractionation would not be optimal for some tumor types and some (especially late) toxicities. 52 There is no general consensus on the terminology used to describe classes of altered fractionation schedules. The definitions proposed by myself 53 have the advantage that any schedule can be classified using these terms:
Hyperfractionation: any schedule delivering a dose per fraction of less than 1.8 Gy
Accelerated fractionation: any schedule delivering radiation therapy with a rate of dose accumulation exceeding an EQD 2 of 10 Gy per week
Hypofractionation: any schedule delivering a dose per fraction larger than 2.2 Gy
Note that there is no consideration of total dose in any of these definitions, nor is there any requirement in terms of number of fractions per day or per week. Fig. 3-6 shows the dose per fraction and dose accumulation tested in the experimental arms of a large number of altered fractionation trials in HNSCC.

FIGURE 3-6 • Scatter plot of selected altered fractionation schedules tested against conventional fractionation in randomized controlled trials according to the dose per fraction employed and the rate of dose accumulation. These schedules explored the potential benefit from hyperfractionation and accelerated fractionation to a varying extent. The area of each circle is proportional to the total number of patients in the trial.

Fractionated Radiotherapy Alone or Combined With Other Modalities

Head and Neck Cancer

Hyperfractionation in Head and Neck Squamous Cell Carcinoma
The interest in hyperfractionation followed directly from the LQ model. As the fractionation sensitivity of at least some tumor types was assumed to be relatively low (i.e., characterized by α/β = 10 Gy) compared with clinically relevant (“dose limiting”) late side effects (thought to have typical α/β values of 2 or 3 Gy), then lowering the dose per fraction would spare late effects more than it would lead to a loss of tumor control. The interest concentrated on HNSCC versus late side–effects, but these rationales were extended to other tumor histologies as well.
As an example, the EORTC 22791 trial 54 gave 80.5 Gy delivered as 70 F of 1.15 Gy per fraction, 2 F per day, over 7 weeks as definitive therapy in patients with T 2 -T 3 oropharyngeal carcinomas. For a tumor with α/β = 10 Gy, the equivalent dose in 2-Gy fractions would be:

Whereas for late effects with an α/β ratio of, for example, 2 Gy, the equivalent dose in 2 Gy fractions would become:

Thus the equivalent dose delivered to the tumor would be 4.8 Gy (6.9%) higher than the 70 Gy in 35 F delivered in the control arm. At the same time, the equivalent dose in 2-Gy fractions for a late endpoint would be reduced by 6.6 Gy (9.4%). Applying estimates from the literature of the steepness of dose-response curves, * these changes in dose can be converted into expected changes in tumor control and incidence of neck fibrosis. For tumor control, the 6.9% higher dose is estimated to yield a 12% increase in control. This is in reasonable agreement with the observed 19% difference in local control in the two arms of the randomized controlled trial when the statistical uncertainty in the observed local control estimates are taken into account. 54 However, the expected 28% reduction in the incidence of neck fibrosis was not observed: there was no clear difference between the incidence of this endpoint in the two arms of the trial. This observation has been suggested to reflect incomplete recovery in the 6-hour interval between dose fractions in view of the long recovery halftimes estimated from the UK CHART HNSCC trial. 56


Accelerated Fractionation in Head and Neck Squamous Cell Carcinoma
The clinical radiobiologic rationale for accelerated fractionation in HNSCC was the observed loss of tumor control 57 when the same total dose was delivered as “split-course” therapy (i.e., extending the overall treatment time by including a planned gap in the schedule, typically of 2 to 3 weeks’ duration). The importance of overall treatment time in HNSCC was further supported by Withers’s “dog-leg” graph in which he plotted the dose required to control 50% of tumors as a function of overall treatment time and found a linear relationship (i.e., a constant dose lost per day of treatment time) at least after a lag time of 3 to 4 weeks. 45 Although the details of this analysis were questioned at the time of publication, 46 it became hugely influential. In hindsight, the conclusions of the paper by Withers and colleagues have been shown to be correct (see Fig. 3-3 ). The significant tumor time factor together with the expectation—later supported by clinical data—that the overall time factor would be negligible for late side-effects 58 provided a strong rationale for shortening the overall treatment time (i.e., increasing the dose delivered per week; see Fig. 3-2 ).
The cleanest test of accelerated fractionation in HNSCC was probably the trial conducted by DAHANCA in which the total dose of 66 to 68 Gy and the 2-Gy fraction size was kept identical in the two trial arms, but acceleration was achieved by delivering 6 F per week in the experimental arm versus the standard 5 F per week in the control arm, which shortened the overall treatment time by 7 days, from 46 to 39 days. 59 This roughly increased the rate of dose accumulation from 10 Gy per week to 12 Gy per week. Between January 1992 and December 1999, 1476 patients treated with primary radiotherapy alone were randomly allocated to the two trial arms. Using Equation 6 with D prolif = 0.65 Gy/day, it is estimated that the 7-days-shorter treatment time corresponds to a dose escalation of 7 days × 0.65 Gy/day = 4.6 Gy. This is equivalent to a 6.7 to 6.9% change in the total dose. Using the previously discussed approximation, we estimate that this should result in a 12% improvement in tumor control probability. This is in agreement with the improvement from 64% to 76% ( P = 0.0001) observed in the trial. 59 There was no statistically significant increase in late toxicity in the 6 F per week arm relative to the 5 F per week arm. Thus the DAHANCA trial provides direct evidence, without the possible confounding effect of differences in total dose or dose per fraction, for the importance of the overall time factor in HNSCC and that a therapeutic gain is achievable by treatment acceleration.

Current Status of Altered Fractionation in Head and Neck Squamous Cell Carcinoma
HNSCC is the most intensively studied tumor type with respect to its response to altered fractionation. As shown in Fig. 3-6 , a broad region of time- and dose-per-fraction space was almost systematically sampled by the cumulated research effort of a large number of trials conducted by investigators all over the world: a meta-analysis published in 2006 included data from 15 randomized controlled trials including 6515 patients with locally advanced HNSCC, mainly oropharyngeal or laryngeal carcinomas. 60 With a median follow-up of 6 years, altered fractionated radiotherapy provided an absolute 5-year survival benefit of 3.4% ( P = 0.003). The absolute benefit was 8% with hyperfractionated radiotherapy versus 2% with accelerated radiotherapy. 60 There are two limitations to this analysis. First of all, toxicity data were not analyzed because they could not be retrieved in a format suited for pooled analysis. Second, the use of standard meta-analysis techniques required a rather crude grouping of trials—with varying modeled biologic efficacy within each group—into broad categories, thereby averaging the treatment benefit over more and less effective schedules. In a way, one could argue that the whole is less than the sum of the parts in this case—that the information content in the highest quality trials exceeds that of the meta-analysis.
A head-to-head comparison of accelerated fractionation with hyperfractionation was conducted in the four-arm Radiation Therapy Oncology Group (RTOG) 9003 phase III trial. 61 Both altered fractionation schedules gave a significantly improved local control compared with conventional fractionation for a comparable incidence of late effects. The outcomes in the accelerated and hyperfractionated arms were similar and both agreed well with the expectations from radiobiologic modeling.
Perhaps the most impressive aspect of the experience with altered fractionation in HNSCC is how well the model predictions held up. The fact that these trials were conducted in the “low conformality” era has undoubtedly helped: parallel opposing fields or simple 90-degree wedged fields with margins based on the normal x-ray anatomy provided a high likelihood that the tumor was in the field and actually received the prescribed dose-fractionation schedule, with a near-uniform dose distribution. Local recurrences were typically seen in the high-dose region rather than as geographic misses.

Lessons from the Head and Neck Squamous Cell Carcinoma Fractionation Trials
The radiobiologic lessons from the very large HNSCC fractionation trials have been important and may briefly be summarized as follows:
1 Recovery kinetics between MFD is slower than originally inferred from small animal models, at least for the late normal-tissue endpoints. 56 This limits the therapeutic differential between late effects and tumor control obtainable from MFD. This again puts an effective limit on how far hyperfractionation can be taken because delivery of low fraction sizes requires MFD to prevent unreasonable extension of the overall treatment time. All of this has stimulated interest in using hypofractionation rather than MFD as a means of accelerating radiotherapy, although such schedules must still be considered experimental.
2 The time factor (i.e., the dose recovered per day) is greater for normal mucosa than for unselected HNSCC. 62 This effectively limits how large a dose per week can be delivered without excessive mucositis and therefore how strongly radiotherapy can be accelerated.
3 The dose recovered per day after 5 or 6 weeks of fractionated radiotherapy cannot be back-extrapolated all the way down to very short schedules, again effectively limiting the utility of such schedules for definitive radiotherapy. No further gain in tumor control is expected by shortening a schedule with an overall treatment time equal to the kick-off time for accelerated proliferation.
Two major clinical developments have limited the further rational development of dose-fractionation schedules based on the experience from the randomized trials. The first is that improvements in radiotherapy planning and delivery technology have led to a new class of deliverable dose distributions in head and neck radiotherapy in which three-dimensional (3-D) conformal radiotherapy or IMRT may allow organ- and function-sparing techniques. This has created a window of opportunity for escalating the dose per fraction to the gross tumor volume. The second development is the wider use of drugs combined with radiation therapy also in HNSCC. This limits the tolerance to the radiation therapy component of the treatment, and it may, for example, not be feasible to use a very strongly accelerated radiation schedule. Furthermore, it has raised the question as to the extent that radiation-alone radiobiologic parameters can be extrapolated to concurrent or sequential chemoradiation therapy.

Breast Cancer
In the 1970s, many centers introduced hypofractionated radiotherapy for postoperative treatment of breast cancer. The use of the Ellis nominal standard dose formula for adjusting the total dose in these schedules combined with dosimetric problems resulting in substantial overdosage of some tissue structures led to an unacceptably high incidence of late side effects after these schedules. It is a further limitation of this historical experience that most published institutional series did not have sufficient statistical power to estimate the corresponding locoregional tumor control rate with any clinically relevant precision. It was therefore not clear if these schedules were hot on the tumors as well as on the late endpoints. Because of the favorable prognosis in early stage cases, many long-term survivors suffered from a reduced health-related quality of life as a result of these dose-fractionation schedules. Understandably, this created a strong reluctance to use hypofractionation in this indication.
Data are now available from four large randomized controlled trials 63 - 67 including a total of more than 7000 patients receiving standard 50 Gy in 25 F whole-breast radiotherapy versus hypofractionated radiotherapy. The dose per fraction varied from 2.65 to 3.3 Gy among the tested hypofractionation schedules. These large trials have demonstrated that the α/β ratio for subclinical breast cancer is in the same range as those for the relevant late side effects 65 : α/β for tumor control was estimated at 4.6 Gy (95% confidence interval [CI], 1.1-8.1 Gy) and for late change in breast appearance at 3.4 Gy (95% CI, 2.3-4.5 Gy). Further attempts at reducing the number of fractions in this indication are in progress in the United Kingdom with John Yarnold as the principal investigator.
Improved precision in the α/β estimate for tumor control would be very helpful in terms of extrapolating the trial experience to new dose-fractionation combinations. Also, as the tumor data mature it should be possible to derive a time factor for subclinical breast cancer that again would allow rational consideration of the effect of short, intensive schedules. On the other hand, what is already now clear from the phase III trials is the near-equivalence in efficacy between the conventional and hypofractionated regimens for very similar levels of side effects. This has renewed interest for hypofractionation for postoperative radiation therapy for breast cancer in many centers.

Prostate Cancer
The seminal analysis by Brenner and Hall 68 published 10 years ago, stimulated a flurry of theoretical studies on the α/β ratio for prostate cancer. Brenner and Hall’s analysis was based on comparing the biochemical failure-free rate after 125 I permanent prostate implants (PPIs) and after external-beam radiotherapy (EBRT). In the case of 125 I, the Lea-Catcheside factor, g in Equation 5 , is essentially zero. 68 Now assume that we can estimate two doses, D EBRT and D PPI , that are isoeffective (i.e., that produce the same tumor control after EBRT and brachytherapy, respectively). Then, by rearrangement of Equation 5 , we find a simple expression for α/β:
(Eq. 7)
If, for example, 140 Gy from PPI is isoeffective with 60 Gy from EBRT, then α/β becomes 1.5 Gy. This whole argument hinges on the recurrences seen after PPI being dose-limited rather than geographic misses. If the tumor control curve at 140 Gy has maxed out, then it is possible that a lower dose than 140 Gy would produce the same tumor control probability (i.e., that the “true” value of D PPI would be lower than 140 Gy in Equation 7 ). This would lead to a higher estimate of α/β; in the hypothetical limit in which D PPI approaches D EBRT , the α/β estimate would tend to infinity.
The stark difference in the dose distributions and the possibility that some (or most?) recurrences after PPI are geographical misses (rather than dose-limited ) weakened the original estimation of α/β. A subsequent study by Brenner et al. 51 got around the first of these concerns by analyzing data from patients treated with EBRT plus two or three HDR brachytherapy implants, still arriving at a low estimate of α/β = 1.2 Gy (95% CI: 0.03, 4.1 Gy). Further support for a low α/β for prostate cancer comes from large randomized studies of conventional versus hypofractionated EBRT. 69 , 70 A definitive analysis of α/β from the EBRT trials has not yet been published; an analysis 71 of a preliminary report from the National Cancer Institute of Canada trial 72 estimated α/β at 1.1 Gy. The upper bound on the 95% confidence interval for this estimate was 5.6 Gy, but the method of estimation requires knowledge of the steepness of the dose-response curve for prostate cancer, and the confidence interval should be taken with a pinch of salt. There is one further caveat—all analyses published to date have assumed a zero time factor for prostate cancer—an assumption that is not supported by strong clinical data.
Several prostate cancer fractionation trials are in progress and it is beyond the scope of the present chapter to review these. One trial that does deserve mention because of its design is the large multicenter phase I/II trial conducted by Ritter et al. 73 The design is a three-level dose-per-fraction escalation phase I/II trial: 2.94 Gy × 22 (= 64.68 Gy); 3.63 Gy × 16 (= 58.08 Gy); 4.3 Gy × 12 (= 51.6 Gy), for level I, II, and III. The dose-fractionations were selected 74 to yield near-isoeffective schedules with respect to both rectal toxicity (assuming α/β = 3 Gy) and tumor control (assuming α/β = 1.5 Gy). Levels I and II are closed, having accrued 103 and 109 patients, respectively. The median follow-up for level I patients was 47 months and for level II patients 24 months (analyzed March 2009).
A number of large randomized phase III trials are in progress. As an example, the RTOG 0415 compares 70 Gy in 28 F versus 73.8 Gy in 41 F delivered is 3-D—conformal radiation therapy (CRT) or IMRT in patients with favorable-risk prostate cancer. The target sample size is 1067 patients. Another large phase III trial, CHHiP (conventional or hypofractionated high-dose IMRT for prostate cancer) is in progress in the United Kingdom, in which Dearnaley and colleagues are randomizing between 74 Gy in 37 F over 7.5 weeks versus 60 Gy in 20 F over 4 weeks versus 57 Gy in 19 F over 3.8 weeks. Eligible patients have stage T 1B to T 3A N 0 M 0 disease with an estimated risk of seminal vesicle involvement of 30% or less and prostate-specific antigen of 30 ng/ml or less. The target sample size has recently beer incrrased to 3163. The statistical power of these large trials could effectively establish a new standard of fractionation for prostate cancer and will allow estimation of α/β with much improved accuracy. This in turn could provide a rational basis for going one step further in exploring even lower fraction numbers.

Non–Small Cell Lung Cancer
The largest phase III trial of altered fractionation in patients with non–small cell lung cancer (NSCLC) is the UK Medical Research Council CHART Lung trial. 75 The CHART schedule used in NSCLC was identical to that used in HNSCC delivering 54 Gy in 36 F (1.5 Gy per fraction) with 3 F per day in only 12 consecutive days: patients would start radiotherapy on a Monday morning and would finish their treatment course on Friday afternoon of week 2. Between April 1990 and March 1995, 563 patients with disease confined to the thorax were accrued. A 9% absolute improvement in 2-year survival, from 20% to 29% ( P = 0.004), was seen in the CHART arm relative to the conventional arm in which patients got 60 Gy in 30 F over 6 weeks. 75
Despite the significant advantage of CHART over conventional fractionation—and the proof of principle demonstration of improved survival from intensified locoregional therapy—wider uptake of the CHART schedule has been slow, 76 most likely because of the logistical difficulties in treating three times a day as well as on Saturday and Sunday. This led to attempts to use the radiobiologic model parameters derived from the CHART trial to develop a weekend-less, dose-escalated schedule (CHARTWEL) 77 and a 2-F-per-day, weekend-less, dose-escalated regimen (Hi-CHART). The latter has been piloted in a large phase I/II trial by the University Medical Center in Maastricht, the Netherlands. 78
A CHARTWEL variant, hyperfractionated, accelerated radiotherapy (HART)—dropping “continuous”—was tested in the randomized phase III Eastern Cooperative Oncology Group 2597 trial 79 but preceded by induction chemotherapy consisting of two cycles of carboplatin (area under time concentration curve 6 mg/ml/min) on days 1 and 22 plus paclitaxel (225 mg/m 2 ) on day 1. HART delivered 57.6 Gy in 36 F, 3 F per day, 5 days a week. The first and third fraction in each day delivered 1.5 Gy on parallel-opposing anterior-posterior fields, and the mid fraction delivered 1.8 Gy on smaller-sized oblique or lateral fields excluding the spinal cord and striving to reduce the esophageal volume irradiated. The inter-fraction interval was only 4 hours. Conventional fractionation consisted of 64 Gy in 2-Gy fractions, 5 F per week. The trial was stopped prematurely after reaching approximately one-third of the planned accrual: 60 patients were randomized to HART and 59 patients to conventional radiotherapy. The early stopping of the trial reduced its power and the differences between arms were not statistically significant. However, from data in the published report it is possible to derive point estimates of the HR for survival between the two trial arms: HART reduced the HR to 0.73 when estimated from the median survival and to HR = 0.55 when estimated from the 3-year survival figures. These estimates are remarkably similar to the HR = 0.76 estimated from the CHART trial, 80 supporting the role of strongly accelerated fractionation in NSCLC.
Although patients in the CHART trial were typically treated using parallel-opposing fields, treating a relatively large volume in the first phase of the treatment course (to 44 Gy in the conventional arm, 37.5 Gy in the CHART arm), the introduction of 3-D CRT and IMRT techniques—combined with advances in anatomic and molecular imaging for better target selection and delineation—may allow intensification of therapy, especially when combined with personalized dose-fractionation schedules. One such strategy was tested in a phase I/II trial at the University of Wisconsin–Madison 81 and included acceleration by dose-per-fraction escalation with patients stratified according to the risk of developing radiation toxicity estimated from NTCP modeling.

Stereotactic Body Radiotherapy in Lung Cancer
An unexpected region of dose-per-fraction and time space has opened up for exploration through the use of SBRT, mainly in early stage NSCLC but it is also of interest in an increasing number of other tumor types. 82 Intracranial stereotactic radiosurgery was originally developed by Leksell 83 in 1951 and later for extracranial sites (SBRT) by Lax et al. at the Karolinska Hospital in Stockholm. 84 , 85 SBRT is often referred to as ablative radiotherapy 86 and is typically delivered using fraction sizes larger than 8.0 Gy. Application of the simple LQ model at these large fraction sizes gives rise to ludicrous EQD 2 s, especially for endpoints with a low α/β ratio. As an example, 22 Gy × 3 has been used in stage I NSCLC 87 and for an endpoint with α/β = 3 Gy, the EQD 2 is estimated at 330 Gy, a dose that is difficult to relate to experience with larger-volume radiation therapy. Fowler et al. 88 have called SBRT “a challenge to traditional radiation oncology”—which, in a way, it is not. Rather, the apparent clinical feasibility of such regimens shows the limitations of our current radiobiologic models when extrapolated far outside the range of data in which they have been validated. The feasibility of SBRT fractionation regimens is mainly driven by the volume effect in parallel tissues with a physiologic reserve capacity. In addition, the simple LQ model overestimates the biologic effect of large-dose fractions, as mentioned previously.
A flurry of modifications of the LQ model have been proposed (e.g., see Park et al. 89 and related correspondence) to achieve a mathematic expression that smoothly transitions from the LQ behavior at dose per fraction of less than some 5 Gy to the log-linear behavior seen in vitro at higher doses per fraction. Most of these are purely mathematic manipulations without any underlying mechanistic background. At the time of writing, none of these have come into wider use, and the real question is whether it is possible, or even useful, to extrapolate the clinical outcome after SBRT-type fractionation schedules all the way back to 2-Gy fraction sizes.
Several of the other four Rs of radiotherapy could be influenced by these very short, intensive schedules: reoxygenation, redistribution, and proliferation (for early normal tissue endpoints and for tumors) could affect toxicity as well as efficacy. However, very little data are available on these effects, and the standard position among SBRT proponents seem to be that the very large fraction sizes will dominate efficacy considerations and that the volume effect will take care of any normal tissue concerns. Data are emerging, however, suggesting that toxicity may become an issue, also in the beam transit zone away from the target volume. 90 , 91
SBRT has a lot to offer in terms of both efficacy and patient convenience, but should be used with great caution outside centers with expertise in the planning and delivery of this kind of treatment. The ongoing clinical studies will undoubtedly add much to our understanding of the radiobiologic and clinical aspects of SBRT.

Other Tumor Histologies
There is every reason to believe that the biology of fractionation effects is similar in other tumor histologies than those discussed previously—the problem is that radiobiologic parameters allowing a quantitative discussion of fractionation effects are sparse in all of these cases. Estimates of α/β are available from single studies of malignant melanoma 92 and liposarcoma 93 ; both turn out to be low: 0.6 Gy and 0.4 Gy, respectively. There are also values for skin cancer, estimated at 8.5 Gy with 95% CI (4.5 Gy, 11.3 Gy) and for esophageal cancer 4.9 Gy with 95% CI (1.5 Gy, 17 Gy), but, as can be seen, the confidence intervals for these estimates are wide. That is all that’s available; for most other tumor types there are no strong empirical human data. Most isoeffect estimates in other histologic grades are derived from assumed, “generic” values, typically α/β = 10 Gy for tumors, but as the breast and prostate examples show, this could turn out to be very misleading.
When it comes to time factors (D prolif ) and recovery kinetics (T 1/2 ) there are virtually no good human estimates for other histologies than those discussed in the previous sections.

Palliative Therapy
Palliation is an important indication for radiation therapy in a large number of patients. Hypofractionated schedules are attractive because of their convenience to the patient, especially when the treatment course spans a sizable proportion of the patient’s life expectancy. Decision making in prescription of palliative radiation therapy is complex and involves weighing a number of factors against each other, including the patient’s age, performance status, disease burden, and prognosis. It cannot be assumed that hypofractionation always offers the best balance between symptom relief and patient convenience, although in many cases it probably does. 94 - 96
From the perspective of clinical radiobiology, the largest body of evidence on fractionation in palliative radiotherapy stems from several randomized, controlled trials comparing single-radiation-dose fractions versus multifraction (typically also hypofractionated) schedules. Wu et al. 97 performed a meta-analysis of trials published before 2001 and concluded that there was no significant difference in complete and overall pain relief between single and multifraction palliative radiotherapy for bone metastases. This conclusion was further strengthened in 2005 when the results of the RTOG 9714 trial were published. 98 This trial randomized 898 patients to 8 Gy × 1 versus 3 Gy × 10. Both regimens were equivalent in terms of pain and narcotic relief at 3 months and were well tolerated. The 8-Gy arm had a higher rate of retreatment, but had less acute toxicity than the 30-Gy arm. Further analyses of the Dutch bone pain trial have shown equal levels of palliation also in patients with a relatively long survival time 99 and have shown a favorable cost-utility of the single fraction schedule. 100 Nevertheless, single-fraction schedules for metastatic bone pain have obtained wider acceptance in Europe and Canada than in the United States. 101 Speculating on the implications for evidence-based radiation oncology or on the possible influence of remuneration in various health care systems on the choice of fractionation 102 is not the topic of the present chapter.
With respect to radiobiology, the experience summarized in this section suggests that standard isoeffect calculations probably are of little value in comparing palliative fractionation schedules, pointing to the mechanism of palliation being different from that of curative treatment. Studies on urinary or serum markers of bone resorption suggest that pain relief is more closely related to an effect on normal bone than on the tumor itself. 103 , 104

Where Next in Fractionated Radiotherapy?
Fractionation biology pops up everywhere when considering current strategies for improving the outcome of radiation oncology. The fact that the physical dose of ionizing radiation can be precisely titrated and modulated in space and time is what makes radiation unique among the anticancer agents. The field is much too rich to be reviewed here, but three main areas of research should be highlighted.

Dose Distribution and Fractionation
Advances in radiation therapy planning and delivery technology have improved the conformality between the high-dose volume and the target volume. From a tumor perspective, this has stimulated exploration of hypofractionated radiation therapy, taking advantage of the improved exclusion of critical structures from the high-dose volume, and also techniques like simultaneous integrated boosts, in which specific target subvolumes are treated with different doses per fraction and, as a consequence, with different rates of dose accumulation per week. Isoeffect calculations for such dose plans require detailed knowledge of the relevant radiobiologic parameters as well as a consideration of the tumor volume effect. It also complicates the assignment of a definitive equivalent dose that can be related to tumor control or local failure. This is a ripe area for clinical radiobiologic research.
At the same time, the typical dose distribution in surrounding organs at risk (OAR) have changed from partial organ irradiation, in which a fraction of the organ receives a dose equal to the prescription dose and very limited dose (except for a relative narrow penumbra) elsewhere, to a broad spectrum of absorbed doses throughout the OAR. The varying dose to voxels in the OAR also means that these are irradiated with a varying dose per fraction ( Fig. 3-7 ). How this local fractionation effect influences local damage, and how this in turn determines organ-level functional endpoints, is not yet clear in most tissues and organs. What is clear is that fractionation effects and dose distribution cannot be considered separately. An American Society for Therapeutic Radiology and Oncology and American Association of Physicists in Medicine task force, Quantitative Analysis of Normal Tissue Effects in the Clinic, has undertaken a systematic review of our current knowledge of dose-volume effects in approximately 20 OARs. These organ-related reviews together with a number of methodologic reviews and scientific vision papers are planned to be published at the end of 2009. Much has been learned during the preceding 15 years, but there are still large white areas on the map, and further research is urgently needed.

FIGURE 3-7 • Differential dose-volume histograms for the urinary bladder in a patient with prostate cancer receiving hypofractionated radiotherapy (5 Gy per fraction) using intensity-modulated radiation therapy on a 6-mV linear accelerator. The two histograms represent the physical dose and the equivalent dose from each fraction corrected using α/β = 2 Gy, assumed to be representative for bladder wall fibrosis. Correcting for local fraction size will push the equivalent dose up for voxels receiving more than 2 Gy per fraction and push the dose down for voxels receiving less than 2 Gy per fraction. A logarithmic y-axis has been used for graphic effect.

Combined Modality Therapy and Fractionation
Indications for multimodality therapy, with radiation therapy as one component, are widening. Cytotoxic and molecular targeted agents are being tested in combination with fractionated radiotherapy in clinical trials. Among the emerging rationales for combining drugs and radiation are the possibilities of hitting distinct cellular targets as well as modulation of radiation fractionation effects. 20 , 105 The improved locoregional relapse-free survival seen in a large HNSCC trial 106 randomizing between radiation therapy with and without cetuximab, an antibody against the epidermal growth factor receptor (EGFR), has created much excitement among radiobiologists. Although several biologic mechanisms may be targeted by cetuximab, it is very likely that partly abrogating the accelerated proliferation response during fractionated radiotherapy is a main contributor. This would open exciting possibilities for reconsidering hyperfractionated regimens if the tumor time factor really is reduced by administering cetuximab.
Few studies have directly addressed the possible interaction between fractionation and chemotherapy. One example is the elegant randomized study with a factorial design, conducted by Anne Lee and her colleagues, 107 testing conventional (66 Gy in 33 F, 5 F/week) versus accelerated (same total dose and fraction size but 6 F/week) radiation therapy with or without chemotherapy in patients with T 3-4 N 0-1 M 0 nasopharyngeal carcinoma. Chemotherapy was given as concurrent cisplatin plus adjuvant cisplatin and 5-FU. Preliminary results suggested a significant interaction between chemotherapy and accelerated fractionation, with the combination of the two being the superior arm of the four. A full analysis of the outcome of this trial awaits final analysis of the mature data from the study.
Optimizing fractionation in the context of combined surgery and radiotherapy is also an under-studied field. In postoperative radiotherapy for HNSCC it is conceivable that the accelerated proliferation is triggered at the time of surgery (i.e., the 3- to 4-week delay before the onset of accelerated proliferation will already have passed at the time of commencement of radiation therapy). This would revive the rationale for very short, intensive schedules like CHART. Support for this hypothesis comes from the randomized phase III trial from the National Cancer Institute in Cairo, Egypt. 108 An independent trial addressing this issue was activated by the UK Medical Research Council, but was closed prematurely because of slow accrual.
The take-home message in combined modality therapy is that one needs to consider the whole package. Optimal fractionation for radiation therapy alone may no longer be optimal in a combination with other modalities.

Personalized Medicine and Fractionation
High-throughput assays and a large number of immunohistochemical biomarkers have opened new research avenues in predictive oncology. Although many prognostic markers are of relevance for patients receiving radiation therapy as well as other therapies, relatively few studies have looked specifically for biomarkers that would select for a specific dose-fractionation. An example of a study in which a molecular biomarker did prove to be a predictive factor for a benefit from a specific dose-fractionation schedule has come from the CHART HNSCC trial. 109 Although overall there was no statistically significant benefit from being randomized to CHART rather than to conventional fractionation, patients harboring tumors with an EGFR index assessed by immunohistochemistry above the population median value did have a significant benefit from CHART. The interaction between the randomization arm and high or low EGFR expression was statistically significant with respect to locoregional control. This is the first among a large number of clinic-pathologic and immunohistochemical markers that has shown a significant association with a benefit from strongly accelerated radiotherapy. In the future, it may be possible to use an array of markers for personalized prescription of dose fractionation.
Because radiation therapy is a locoregional modality, predictive competing risks models 110 , 111 that allow stratification of patients according to their relative risks of local, regional, and distant failure would be of great potential value in stratifying patients for differently weighted combinations of locoregional and systemic therapy. Radiation dose fractionation and scheduling of the combined modalities would need to be optimized in various subgroups of patients. Again, more research is needed in this area.
Finally, recent advances in functional and molecular imaging are hugely interesting in the context of dose fractionation. Novel imaging targets may allow noninvasive mapping of inter- and intralesion variations in surrogates for the four Rs of radiotherapy. Imaging surrogates for hypoxia and cellular proliferation are the most intensely investigated at the moment. Ultimately, this may pave the way for theragnostic radiation oncology 112 : voxel-based dose-fraction prescriptions, so-called dose painting by numbers, according to maps of cellular or microenvironmental variation throughout a tumor volume. Although this research field is still in its infancy, the potential is huge. Theragnostic radiation oncology is at the crossroad between technology and biology and is close to the heart of what makes radiation unique in cancer management.

References

1 Bentzen SM. Design of clinical trials in radiation oncology: saving lives, not Grays. In: Dale RG, Jones B, editors. Modelling in radiation oncology . British Institute of Radiology, 2007.
2 Sjögren TAU. Fall av epitheliom behandlat med röntgenstrålar. Forh Svenska Läkare Sällskapets Sammankomster . 1899:208.
3 Stenbeck T. Fall av hudkräfta, läkt genom behandling med röntgenstrålar. Hygiea . 1900;68:18.
4 Teleky L. Discussion in the K.K. Gesellschaft der Ärzte in Wien, 1902. Fortschritte auf dem Gebiete der Röntgenstrahlen . 1902;5:362.
5 Regaud C, Ferroux R. Discordance des effects des rayons X, d’une part dans la peau, d’autre part dans le testicule, par le fractionement de la dose: Diminution de l’afficacite dans la peau, maintien de l’efficacite dans le testicule. Comptes Rendus Societé Biologique . 1927;97:431.
6 Juul J, Experimental studies on roentgen treatment of malignant tumors, Acta Radiol; Suppl. VIII; 1929:1.
7 Bentzen SM, Thames HD. A 100-year Nordic perspective on the dose-time problem in radiobiology. Acta Oncol . 1995;34:1031.
7a Paterson R, Tod M, Russell M. The results of radium and X-ray therapy in malignant disease, being the third statistical report from the Christie Hospital and Holt Radium Institute . Manchester, Edinburg: E.S. Livingstone Ltd; 1950.
8 Withers HR. The four R’s of radiotherapy. Adv Radiat Biol . 1975;5:241.
9 Steel GG, McMillan TJ, Peacock JH. The 5Rs of radiobiology. Int J Radiat Biol . 1989;56:1045.
10 Bentzen SM, Christensen JJ, Overgaard J, Overgaard M. Some methodological problems in estimating radiobiological parameters from clinical data. Alpha/beta ratios and electron RBE for cutaneous reactions in patients treated with postmastectomy radiotherapy. Acta Oncol . 1988;27:105.
11 Montague ED. Experience with altered fractionation in radiation therapy of breast cancer. Radiology . 1968;90:962.
12 Bates TD, Peters LJ. Dangers of the clinical use of the NSD formula for small fraction numbers. Br J Radiol . 1975;48:773.
13 Overgaard M, Bentzen SM, Christensen JJ, Hjøllund Madsen E. The value of the NSD formula in equation of acute and late radiation complications in normal tissue following 2 and 5 fractions per week in breast cancer patients treated with postmastectomy radiotherapy. Radiother Oncol . 1987;9:1.
14 Ellis F. Dose, time and fractionation: a clinical hypothesis. Clin Radiol . 1969;20:1.
15 Nguyen TD, Panis X, Froissart D, et al. Analysis of late complications after rapid hyperfractionated radiotherapy in advanced head and neck cancers. Int J Radiat Oncol Biol Phys . 1988;14:23.
16 Fowler JF, Lindstrom M. Loss of local control with prolongation in radiotherapy. Int J Radiat Oncol Biol Phys . 1992;23:457.
17 Bentzen SM. Time-dose relationships for human tumors: estimation from non-randomized studies. In: Beck-Bornholt HP, editor. Current topics in clinical radiobiology of tumours. Medical radiology . Berlin: Springer-Verlag; 1993:11.
18 Horiot JC, Le Fur R, N’Guyen T, et al. Hyperfractionated compared with conventional radiotherapy in oropharyngeal carcinoma: an EORTC randomized trial. Eur J Cancer . 1990;26:779.
19 Withers HR. The EORTC hyperfractionation trial. Radiother Oncol . 1992;25:229.
20 Wilson GD, Bentzen SM, Harari PM. Biologic basis for combining drugs with radiation. Semin Radiat Oncol . 2006;16:2.
21 Koh WJ, Bergman KS, Rasey JS, et al. Evaluation of oxygenation status during fractionated radiotherapy in human nonsmall cell lung cancers using [F-18]fluoromisonidazole positron emission tomography. Int J Radiat Oncol Biol Phys . 9-30-1995;33:391.
22 Harris AL. Hypoxia—a key regulatory factor in tumour growth. Nat Rev Cancer . 2002;2:38.
23 Dewhirst MW, Cao Y, Moeller B. Cycling hypoxia and free radicals regulate angiogenesis and radiotherapy response. Nat Rev Cancer . 2008;8:425.
24 Chan N, Milosevic M, Bristow RG. Tumor hypoxia, DNA repair and prostate cancer progression: new targets and new therapies. Future Oncol . 2007;3:329.
25 Williams KJ, Telfer BA, Xenaki D, et al. Enhanced response to radiotherapy in tumours deficient in the function of hypoxia-inducible factor-1. Radiother Oncol . 2005;75:89.
26 Adams GE, Dische S, Fowler JF, et al. Hypoxic cell sensitisers in radiotherapy. Lancet . 1-24-1976;1:186.
27 Brown JM. Tumor hypoxia in cancer therapy. Methods Enzymol . 2007;435:297-321. 297
28 Overgaard J. Hypoxic radiosensitization: adored and ignored. J Clin Oncol . 2007;25:4066.
28a Lea DE, Catcheside DG. The mechanism of the induction by radiation of chromosome aberrations in Tradescantia . J Genet . 1942;44:216-245.
28b Lea DE. A theory of the actions of radiation on biological materials capable of recovery. Part I: The time-intensity factor. Brit J Radiol . 1938;11:489-497.
28c Catcheside DG, Lea DE, Thoday JM. The production of chromosome structural changes in TRADESCANTIA microspores in relation to dosage, intensity and temperature. J Genet . 1945;47:137-149.
29 Kellerer AM, Rossi HH. The theory of dual radiation action. Curr Top Radiat Res . 1972;8:85.
30 Douglas BG, Fowler JF. The effect of multiple small doses of x-rays on skin reactions in the mouse and a basic interpretation. Rad Res . 1976;66:401.
31 Thames HD, Withers HR, Peters LJ, et al. Changes in early and late radiation responses with altered dose fractionation: Implications for dose-survival relationships. Int J Radiat Oncol Biol Phys . 1982;8:219.
32 Bentzen SM. Preventing or reducing late side effects of radiation therapy: radiobiology meets molecular pathology. Nat Rev Cancer . 2006;6:702.
33 Dorr W. Three A’s of repopulation during fractionated irradiation of squamous epithelia: asymmetry loss, acceleration of stem-cell divisions and abortive divisions. Int J Radiat Biol . 1997;72:635.
34 Withers HR, Thames HD, Peters LJ. A new isoeffect curve for change in dose per fraction. Radiother Oncol . 1983;1:187.
35 Bentzen SM, Thames HD, Travis EL, et al. Direct estimation of latent time for radiation injury in late-responding normal tissues: gut, lung and spinal cord. Int J Radiat Biol . 1989;55:27.
36 Taylor JMG, Withers HR, Vegesna V, et al. Fitting the linear-quadratic model using time of occurrence as the endpoint for quantal response multifraction experiments. Int J Radiat Biol . 1987;52:459.
37 Joiner MC, Bentzen SM. Fractionation: the linear-quadratic approach. In: Joiner MC, van der Kogel AJ, editors. Basic clinical radiobiology , vol 4. London, UK: Edward Arnold; 2009:102.
38 Bentzen SM, Joiner MC. The linear-quadratic approach in clinical practice. In: Joiner MC, van der Kogel AJ, editors. Basic clinical radiobiology , vol 4. London, UK: Edward Arnold; 2009:120.
39 Elkind MM, Sutton H. Radiation response of mammalian cells grown in culture. 1. Repair of x-ray damage in surviving Chinese hamster cells. Radiat Res . 1960;13:556.
40 Overgaard J, Hjelm-Hansen M, Johansen LV, et al. Comparison of conventional and split-course radiotherapy as primary treatment in carcinoma of the larynx. Acta Oncol . 1988;27:147.
41 Holthusen H. Erfahrungen über die Verträglichkeitsgrenze für Röntgenstrahlen und deren Nutzanwendung zur Verhütung von Schäden. Strahlentherapie . 1936;57:254.
42 Bentzen SM, Dorr W, Anscher MS, et al. Normal tissue effects: reporting and analysis. Semin Radiat Oncol . 2003;13:189.
43 Kim JJ, Tannock IF. Repopulation of cancer cells during therapy: an important cause of treatment failure. Nat Rev Cancer . 2005;5:516.
44 Bentzen SM. Repopulation in radiation oncology: perspectives of clinical research. Int J Radiat Biol . 2003;79:581.
45 Withers HR, Taylor JMG, Maciejewski B. The hazard of accelerated tumor clonogen repopulation during radiotherapy. Acta Oncol . 1988;27:131.
46 Bentzen SM, Thames HD. Clinical evidence for tumor clonogen regeneration: interpretations of the data. Radiother Oncol . 1991;22:161.
47 Fowler JF. What next in fractionated radiotherapy? Br J Cancer . 1984;49(Suppl. VI):285.
48 Marples B, Collis SJ. Low-dose hyper-radiosensitivity: past, present, and future. Int J Radiat Oncol Biol Phys . 2008;70:1310.
49 Honore HB, Bentzen SM. A modelling study of the potential influence of low dose hypersensitivity on radiation treatment planning. Radiother Oncol . 2006;79:115.
50 Guerrero M, Li XA. Extending the linear-quadratic model for large fraction doses pertinent to stereotactic radiotherapy. Phys Med Biol . 2004;49:4825.
51 Brenner DJ, Martinez AA, Edmundson GK, et al. Direct evidence that prostate tumors show high sensitivity to fractionation (low alpha/beta ratio), similar to late-responding normal tissue. Int J Radiat Oncol Biol Phys . 2002;52:6.
52 Thames HD, Peters LJ, Withers HR, et al. Accelerated fractionation vs. hyperfractionation: Rationales for several treatments per day. Int J Radiat Oncol Biol Phys . 1983;9:127.
53 Bentzen SM. Quantitative clinical radiobiology. Acta Oncol . 1993;32:259.
54 Horiot JC, Le Fur R, N’Guyen T, et al. Hyperfractionation versus conventional fractionation in oropharyngeal carcinoma: Final analysis of a randomized trial of the EORTC cooperative group of radiotherapy. Radiother Oncol . 1992;25:231.
55 Bentzen SM. Dose-response relationships in radiotherapy. In: Joiner MC, van der Kogel AJ, editors. Basic clinical radiobiology , vol 4. London, UK: Edward Arnold; 2009:56.
56 Bentzen SM, Saunders MI, Dische S. Repair halftimes estimated from observations of treatment-related morbidity after CHART or conventional radiotherapy in head and neck cancer. Radiother Oncol . 1999;53:219.
57 Parsons JT, Bova FJ, Million RR. A re-evaluation of split-course technique for squamous cell carcinoma of the head and neck. Int J Radiat Oncol Biol Phys . 1980;6:1645.
58 Bentzen SM, Overgaard J. Clinical normal-tissue radiobiology. In: Tobias JS, Thomas PRM, editors. Current radiation oncology . London: Arnold; 1996:37.
59 Overgaard J, Hansen HS, Specht L, et al. Five compared with six fractions per week of conventional radiotherapy of squamous-cell carcinoma of head and neck: DAHANCA 6 and 7 randomised controlled trial. Lancet . 2003;362:933.
60 Bourhis J, Overgaard J, Audry H, et al. Hyperfractionated or accelerated radiotherapy in head and neck cancer: A meta-analysis. Lancet . 2006;368:843.
61 Fu KK, Pajak TF, Trotti A, et al. a Radiation Therapy Oncology Group (RTOG) phase III randomized study to compare hyperfractionation and two variants of accelerated fractionation to standard fractionation radiotherapy for head and neck squamous cell carcinomas: first report of RTOG 9003. Int J Radiat Oncol Biol Phys . 2000;48:7.
62 Bentzen SM, Saunders MI, Dische S, et al. Radiotherapy-related early morbidity in head and neck cancer: quantitative clinical radiobiology as deduced from the CHART trial. Radiother Oncol . 2001;60:123.
63 Whelan T, MacKenzie R, Julian J, et al. Randomized trial of breast irradiation schedules after lumpectomy for women with lymph node-negative breast cancer. J Natl Cancer Inst . 2002;94:1143.
64 Bentzen SM, Agrawal RK, Aird EG, et al. The UK Standardisation of Breast Radiotherapy (START) Trial A of radiotherapy hypofractionation for treatment of early breast cancer: a randomised trial. Lancet Oncol . 2008;9:331.
65 Bentzen SM, Agrawal RK, Aird EG, et al. The UK Standardisation of Breast Radiotherapy (START) Trial B of radiotherapy hypofractionation for treatment of early breast cancer: a randomised trial. Lancet . 2008;371:1098.
66 Owen J, Ashton A, Regan J, et al. Fractionation sensitivity of breast cancer. Results of a randomised trial. Eur J Cancer Suppl . 2003;1:S9.
67 Yarnold J, Ashton A, Bliss J, et al. Fractionation sensitivity and dose response of late adverse effects in the breast after radiotherapy for early breast cancer: long-term results of a randomised trial. Radiother Oncol . 2005;75:9.
68 Brenner DJ, Hall EJ. Fractionation and protraction for radiotherapy of prostate carcinoma. Int J Radiat Oncol Biol Phys . 1999;43:1095.
69 Lukka H, Hayter C, Julian JA, et al. Randomized trial comparing two fractionation schedules for patients with localized prostate cancer. J Clin Oncol . 2005;23:6132.
70 Yeoh EE, Holloway RH, Fraser RJ, et al. Hypofractionated versus conventionally fractionated radiation therapy for prostate carcinoma: updated results of a phase III randomized trial. Int J Radiat Oncol Biol Phys . 2006;66:1072.
71 Bentzen SM, Ritter MA. The alpha/beta ratio for prostate cancer: what is it, really? Radiother Oncol . 2005;76:1.
72 Lukka H, Hayter C, Warde P, et al. A randomized trial comparing two fractionation schedules for patients with localized prostate cancer. Radiother Oncol . 2003;69(Suppl. 1):S7.
73 Ritter MA, Forman JD, Kupelian PA, et al. A phase I/II trial of dose-per-fraction escalation for prostate cancer. Int J Radiat Oncol Biol Phys . 2007;69:S174.
74 Ritter M. Rationale, conduct, and outcome using hypofractionated radiotherapy in prostate cancer. Semin Radiat Oncol . 2008;18:249.
75 Saunders MI, Dische S, Barrett A, et al. Continuous hyperfractionated accelerated radiotherapy (CHART) versus conventional radiotherapy in non-small-cell lung cancer: a randomised multicentre trial. CHART Steering Committee. Lancet . 1997;350:161.
76 Macbeth F. An uncharted country. Clin Oncol (R Coll Radiol) . 1999;11:71.
77 Bentzen SM, Saunders MI, Dische S. From CHART to CHARTWEL in non-small cell lung cancer: clinical radiobiological modelling of the expected change in outcome. Clin Oncol (R Coll Radiol) . 2002;14:372.
78 De Ruysscher D, Wanders R, van Haren E, et al. HI-CHART: a phase I/II study on the feasibility of high-dose continuous hyperfractionated accelerated radiotherapy in patients with inoperable non-small-cell lung cancer. Int J Radiat Oncol Biol Phys . 2008;71:132.
79 Belani CP, Wang W, Johnson DH, et al. Phase III study of the Eastern Cooperative Oncology Group (ECOG 2597): induction chemotherapy followed by either standard thoracic radiotherapy or hyperfractionated accelerated radiotherapy for patients with unresectable stage IIIA and B non-small-cell lung cancer. J Clin Oncol . 6-1-2005;23:3760.
80 Saunders M, Dische S, Barrett A, et al. Continuous hyperfractionated accelerated radiotherapy (CHART) versus conventional radiotherapy in non-small-cell lung cancer: a randomised multicentre trial. CHART Steering Committee. Lancet . 1997;350:161.
81 Adkison JB, Khuntia D, Bentzen SM, et al. Dose escalated, hypofractionated radiotherapy using helical tomotherapy for inoperable non-small cell lung cancer: preliminary results of a risk-stratified phase I dose escalation study. Technol Cancer Res Treat . 2008;7:441.
82 Lo SS, Cardenes HR, Teh BS, et al. Stereotactic body radiation therapy for nonpulmonary primary tumors. Expert Rev Anticancer Ther . 2008;8:1939.
83 Leksell L. The stereotaxic method and radiosurgery of the brain. Acta Chir Scand . 1951;102:316.
84 Lax I, Blomgren H, Naslund I, et al. Stereotactic radiotherapy of malignancies in the abdomen. Methodological aspects. Acta Oncol . 1994;33:677.
85 Blomgren H, Lax I, Naslund I, et al. Stereotactic high dose fraction radiation therapy of extracranial tumors using an accelerator. Clinical experience of the first thirty-one patients. Acta Oncol . 1995;34:861.
86 Timmerman RD. An overview of hypofractionation and introduction to this issue of seminars in radiation oncology. Semin Radiat Oncol . 2008;18:215.
87 Fakiris AJ, McGarry RC, Yiannoutsos CT, et al. Stereotactic body radiation therapy for early-stage non-small-cell lung carcinoma: four-year results of a prospective phase II study. Int J Radiat Oncol Biol Phys . 2009;75:677-682.
88 Fowler JF, Tome WA, Fenwick JD, et al. A challenge to traditional radiation oncology. Int J Radiat Oncol Biol Phys . 2004;60:1241.
89 Park C, Papiez L, Zhang S, et al. Universal survival curve and single fraction equivalent dose: Useful tools in understanding potency of ablative radiotherapy. Int J Radiat Oncol Biol Phys . 2008;70:847.
90 Pettersson N, Nyman J, Johansson KA. Radiation-induced rib fractures after hypofractionated stereotactic body radiation therapy of non-small cell lung cancer: a dose- and volume-response analysis. Radiother Oncol . 2009;91:360.
91 Dunlap NE, Cai J, Biedermann GB, et al. Chest wall volume receiving >30 Gy predicts risk of severe pain and/or rib fracture after lung stereotactic body radiotherapy. Int J Radiat Oncol Biol Phys . 2009;76:796-801.
92 Bentzen SM, Overgaard J, Thames HD, et al. Clinical radiobiology of malignant melanoma. Radiother Oncol . 1989;16:169.
93 Thames HD, Suit HD. Tumor radioresponsiveness versus fractionation sensitivity. Int J Radiat Oncol Biol Phys . 1986;12:687.
94 Timothy AR, Girling DJ, Saunders MI, et al. Radiotherapy for inoperable lung cancer. Clin Oncol (R Coll Radiol) . 2001;13:86.
95 Hoskin PJ, Yarnold JR, Roos DR, et al. Radiotherapy for bone metastases. Clin Oncol (R Coll Radiol) . 2001;13:88.
96 Hoskin PJ, Brada M. Radiotherapy for brain metastases. Clin Oncol (R Coll Radiol) . 2001;13:91.
97 Wu JS, Wong R, Johnston M, et al. Meta-analysis of dose-fractionation radiotherapy trials for the palliation of painful bone metastases. Int J Radiat Oncol Biol Phys . 2003;55:594.
98 Hartsell WF, Scott CB, Bruner DW, et al. Randomized trial of short- versus long-course radiotherapy for palliation of painful bone metastases. J Natl Cancer Inst . 2005;97:798.
99 Van der Linden YM, Steenland E, van Houwelingen HC, et al. Patients with a favourable prognosis are equally palliated with single and multiple fraction radiotherapy: Results on survival in the Dutch Bone Metastasis Study. Radiother Oncol . 2006;78:245.
100 Van den Hout WB, van der Linden YM, Steenland E, et al. Single- versus multiple-fraction radiotherapy in patients with painful bone metastases: cost-utility analysis based on a randomized trial. J Natl Cancer Inst . 2003;95:222.
101 Kachnic L, Berk L. Palliative single-fraction radiation therapy: how much more evidence is needed? J Natl Cancer Inst . 2005;97:786.
102 Lievens Y, van den Bogaert W, Rijnders A, et al. Palliative radiotherapy practice within Western European countries: impact of the radiotherapy financing system? Radiother Oncol . 2000;56:289.
103 Papatheofanis FJ. Serum PICP as a bone formation marker in 89Sr and external beam radiotherapy of prostatic bony metastases. Br J Radiol . 1997;70:594.
104 Hoskin PJ, Stratford MR, Folkes LK, et al. Effect of local radiotherapy for bone pain on urinary markers of osteoclast activity. Lancet . 2000;355:1428.
105 Bentzen SM, Harari PM, Bernier J. Exploitable mechanisms for combining drugs with radiation: concepts, achievements and future directions. Nat Clin Pract Oncol . 2007;4:172.
106 Bonner JA, Harari PM, Giralt J, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med . 2006;354:567.
107 Lee AW, Tung SY, Chan AT, et al. Preliminary results of a randomized study (NPC-9902 Trial) on therapeutic gain by concurrent chemotherapy and/or accelerated fractionation for locally advanced nasopharyngeal carcinoma. Int J Radiat Oncol Biol Phys . 2006;66:142.
108 Awwad HK, Lotayef M, Shouman T, et al. Accelerated hyperfractionation (AHF) compared to conventional fractionation (CF) in the postoperative radiotherapy of locally advanced head and neck cancer: influence of proliferation. Br J Cancer . 2002;86:517.
109 Bentzen SM, Atasoy BM, Daley FM, et al. Epidermal growth factor receptor expression in pretreatment biopsies from head and neck squamous cell carcinoma as a predictive factor for a benefit from accelerated radiation therapy in a randomized controlled trial. J Clin Oncol . 2005;23:5560.
110 Ataman OU, Bentzen SM, Saunders MI, et al. Failure-specific prognostic factors after continuous hyperfractionated accelerated radiotherapy (CHART) or conventional radiotherapy in locally advanced non-small-cell lung cancer: a competing risks analysis. Br J Cancer . 2001;85:1113.
111 Ataman OU, Bentzen SM, Wilson GD, et al. Molecular biomarkers and site of first recurrence after radiotherapy for head and neck cancer. Eur J Cancer . 2004;40:2734.
112 Bentzen SM. Theragnostic imaging for radiation oncology: dose-painting by numbers. Lancet Oncol . 2005;6:112.

* We follow the in many ways unsatisfactory convention in the literature of using therapeutic ratio in a loose sense when referring to efficacy relative to toxicity. For a discussion of attempts to treat this more quantitatively, see Bentzen. 1
* A formal analysis of this problem requires that the uncertainty in D prolif be taken into account, an analysis that is beyond the scope of this chapter.
* Values of γ 50 equivalent to 1.8 for tumor control and 3.0 for neck fibrosis are assumed. For details of this method, see Bentzen. 55
4 Chemical Modifiers of Radiation Response

Cameron J. Koch, PhD, Matthew B. Parliament, MD, J. Martin Brown, DPhil, Raul C. Urtasun, MD, FASTRO
This chapter deals with chemical agents that have been used solely as modifiers of radiation response and with methods to detect hypoxic cells in tumors. For a discussion of the interaction of cytotoxic cancer chemotherapeutic agents with radiation (actinomycin D, 5-fluorouracil, doxorubicin [Adriamycin], hydroxyurea, and paclitaxel [Taxol]), the reader is referred to Chapter 6 .

Rationale for the Use of Radiosensitizers and Radioprotectors
There is a need in clinical practice to enhance the differential effect of radiation in tumor and normal tissues. This can be achieved by the use of chemical agents that either increase the damage to the tumor or protect normal tissues that are included in the radiation volume. However, to effect an increase in the therapeutic ratio of radiotherapy, any radiosensitizing, or radioprotective agent has to be specific for normal or malignant tissues. For example, an agent blocking the activity of any of the genes controlling double-strand break repair would be a radiation sensitizer, but to be effective, it would have to work only on the tumor cells. Even though radiotherapy focuses the radiation on the tumor mass, the dose that can be given is still limited by the cells of the surrounding normal tissue. Thus, any radiation sensitizer has to be more effective on the tumor cells, and any radiation protector has to be more specific for normal cells. Unless this is achieved, there will be no therapeutic gain. Unfortunately, this requirement is difficult to achieve. Nonetheless, there has been considerable activity in this area in the past few years and several promising agents are in advanced clinical testing.

Radiosensitizers
Radiosensitizers are compounds that, when combined with radiation, achieve greater tumor inactivation than would have been expected from the additive effect of each modality. The application of chemical agents that simply have an additive effect in normal tissues is equivalent to the administration of an increment of radiation dose with no differential benefit. The toxicities of the chemical agent and the radiation overlap with no major gain. 1 The addition of a chemical modifier to a course of radiation to improve treatment results should be considered only in those tumor sites where there is already evidence that an increase in dose intensity by 20% to 30% will translate into an increase in tumor control, because most of the sensitizer enhancement ratios (SERs) are in the range of 1.2 to 1.3 (a 20% to 30% increase in the effective dose to the tumor). Examples of such tumors are head and neck tumors and carcinoma of the cervix, in which it has been demonstrated that there is a high fraction of hypoxic tumor cells and also a rapid cell turnover. 2 , 3

Radioprotectors
Radioprotectors are compounds that protect against radiation damage to targeted normal cells, but do not provide similar protection to tumor cells.

Chemical Radiosensitizers of Hypoxic Cells and the Tumor Microenvironment
The role of the oxygen effect in promoting tumor cell inactivation by ionizing radiation has been well demonstrated in vitro and in animal tumor models for several decades. Mounting evidence of this influence in tumor control and patient survival has become available. 2 - 7 Clearly, the microenvironment, the nutritional state of the tumor, and the presence of hypoxia are only some of the many factors that contribute to tumor radioresistance, and it is likely that this resistance is multifactorial, that is, caused by tumor hypoxia, proliferation rates, and inherent cell radioresistance, as well as other biologic microenvironmental factors such as the presence of cytokines. Nonetheless, the clinical data that hypoxia, by conferring radiation resistance, is a prognostic indicator of poor response to standard radiotherapy, at least for some tumors, is reasonably clear. Hypoxia, however, has other consequences beyond conferring radiation resistance. First, hypoxia causes cells to slow their rate of proliferation and to come out of cycle. Because most anticancer drugs are more effective against rapidly proliferating than slowly or nonproliferating cells, this slowing of cell proliferation leads to decreased cell killing in the hypoxic cells. In addition, because the concentration of anticancer drugs is higher closer to blood vessels than further away, both as a consequence of geometry and the reactivity of the drugs, there is less killing of the hypoxic cells, which are invariably the farthest from the blood vessels.
In summary, there are a number of well-established phenomena that cause a gradient of reduced cell killing by most anticancer agents as a function of distance from the vasculature ( Fig. 4-1 ). Such a gradient has been shown in experimental tumors and in spheroid systems. 8 , 9

FIGURE 4-1 • Gradient of reduced cell killing as a function of distance from the vasculature as seen in experimental tumors and spheroids.
Recent studies have also shown that hypoxia in solid tumors has an important consequence in addition to conferring a direct resistance to radiation and chemotherapy. Graeber and colleagues 10 have demonstrated that low oxygen levels cause apoptosis in minimally transformed mouse embryo fibroblasts, and by selecting for mutant p53, might predispose tumors to a more malignant phenotype. Clinical data support this conclusion: Studies both with soft tissue sarcomas 5 and with carcinoma of the cervix 11 , 12 have shown that hypoxia is an independent and highly significant prognostic factor predisposing tumors to metastatic spread. Thus a more hypoxic tumor, in addition to being more difficult to control locally, is also more likely to have spread to distant sites and hence be more difficult to cure. In addition, hypoxia stabilizes the transcription factor HIF-1α, which increases levels of various survival factors such as vascular endothelial growth factor, which can protect against radiation damage to the tumor vasculature and hence, at least theoretically, protect the tumor from radiation damage. 13 , 14
The rationale for developing hypoxic cell sensitizers has been based on the assumption that sensitizing hypoxic cells to radiation killing would improve the outcome of radiotherapy. The possibility of doing this was based on pioneering studies by Adams and Chapman and colleagues on the use of electron-affinic drugs to sensitize hypoxic bacteria and mammalian cells in vitro. 15 - 17 The first drug of this class to show significant activity in sensitizing mouse tumors was the 5-nitroimidazole metronidazole, a drug that was already in clinical use. 18 , 19 Data on this and other such compounds are discussed in the following text.

Nitroimidazole Compounds
Under hypoxic tissue conditions, electron-affinic nitroimidazoles oxidize the radiation-induced free radicals on deoxyribonucleic acid (DNA), thereby mimicking oxygen for the fixation of DNA damage. However, unlike oxygen, nitroimidazoles are not rapidly metabolized by the cells through which they penetrate and are thus able to reach areas beyond the oxygen-diffusion distance. They were shown by various groups to be effective in preclinical studies with transplanted mouse tumors in radiosensitizing the tumors to large single radiation doses without sensitizing normal tissues. 20 , 21
The first compound to be investigated clinically in terms of oral and intravenous pharmacokinetics, toxicity, and efficacy in patients with solid tumors was metronidazole (a 5-nitroimidazole). The plasma β-half-life of the drug was 9.8 hours and the absolute oral availability was estimated to approximate 100%. 19 The dose-limiting toxicity was manifested in gastrointestinal and peripheral neuropathic effects; therefore, this compound reached an estimated SER of only 1.2. Given this limitation, the first randomized control clinical trial of a hypoxic radiosensitizer showing efficacy was performed in patients with glioblastoma multiforme. This study demonstrated the relevance of tumor hypoxia in terms of patient survival, showing that the results with a less-than-optimal radiation fractionation regimen approached the level of the results obtained with conventional fractionation. 22
Of interest, a major spin-off of these early investigations with the pharmacokinetics of metronidazole, particularly the use of a high-dose intravenous route, was not in the field of oncology but in the practice of abdominal surgery and infectious diseases, in which the compound was used as a parenteral agent for anaerobic bacteria. These initial investigations prompted a level of high activity in the investigation of nitroimidazole compounds in human solid tumors and the search for new and better nitroimidazole compounds with less toxicity and higher SER. The first clinical studies on the second generation of these drugs, misonidazole, a 2-nitroimidazole, were initiated in both Europe and North America during the early 1980s ( Table 4-1 ). The structures of the hypoxic sensitizers discussed are shown in Fig. 4-2 .

Table 4-1 The Evaluation from First- to Third-Generation Nitroimidazole Compounds During the Past 25 Years

FIGURE 4-2 • The structures of the hypoxic sensitizers tested in clinical studies.

Misonidazole and Nimorazole
Misonidazole, a 2-nitroimidazole, was developed as a more efficient radiosensitizer because of its known increased electron affinity. In the oral form, the dose-limiting toxicity was again manifested in gastrointestinal effects (nausea, vomiting) and peripheral neuropathy, which limits the effective SER. 23 , 24 Not surprisingly, almost all of the clinical trials of radiotherapy combined with misonidazole turned out to be negative, 25 an outcome consistent with the small degree of radiosensitization expected with the clinically used low doses. 26 Once more the inability to deliver a sufficient dose may have been one of the reasons that a significant proportion of large international clinical trials showed no benefit to misonidazole ( Table 4-2 ). However, it has been shown that selected populations of patients with specific tumors did benefit by using this compound. 27 This observation was made in patients with head and neck cancers. Overgaard and colleagues 28 reported that not only with misonidazole but also with nimorazole (a 5-nitroimidazole), there was a significant benefit in a cohort of patients with stage T 1 and T 4 pharynx carcinoma in terms of local regional control ( Fig. 4-3 ). Patients with hemoglobin levels lower than 9 mmol/L showed particular improvement. These two studies also showed that the compounds had a similar benefit, despite the fact that fewer fractions of radiation were “sensitized” in the misonidazole groups than in the nimorazole groups, in which the drug was administrated with every fraction of radiation (first 30 fractions). The significant improvement of the effect of the radiotherapeutic management of supraglottic and pharynx tumors was again demonstrated in a randomized double-blind phase III study of 414 patients receiving nimorazole or placebo in association with conventional primary radiotherapy (62-68 Gy, 2 Gy per fraction, 5 fractions per week). 28 Of interest is that in this study the nimorazole could be given without major side effects. Nimorazole is currently standard treatment for patients in Denmark receiving radiation therapy for head and neck cancer. 29 The benefit of misonidazole was not observed in another large clinical trial conducted in North America under the Radiation Therapy Oncology Group (RTOG) in patients with stage III and IV head and neck tumors, in which misonidazole was used with two of the five radiation fractions per week. In this study, the administration of the radiosensitizer was limited by neurologic complications, and the use of efficient doses was prevented. 30 A prospective randomized trial was initiated afterward in the same cooperative group with a newer third-generation compound, etanidazole. A significantly greater dose of etanidazole than misonidazole could be administered for a sensitizer effect with acceptable gastrointestinal and neurologic toxicity. 31

Table 4-2 Summary of Efficacy of Clinical Trials With Nitroimidazoles 1974-2008

FIGURE 4-3 • Results from the Danish Head and Neck Cancer Study Group 5 study. Patients with carcinoma of the pharynx and supraglottic larynx were randomly assigned to receive nimorazole or placebo in conjunction with radiotherapy.
(From Overgaard J, Hansen SH, Overgaard M, et al: The Danish Head and Neck Cancer Study Group [DAHANCA] randomized trials with hypoxic radiosensitizers in carcinoma of the larynx and pharynx. In Dewey WC, Edington M, Fry MRG, et al [eds]: Radiation Research: A 20th century perspective, vol 2, Toronto, 1992, Academic Press, p 576.)

Etanidazole
Etanidazole (originally known as SR2508) was developed by a team led by Brown and Lee with the aim of reducing the neurotoxicity seen with misonidazole. 31 It was postulated that because etanidazole is less lipophilic, it would be associated with a lower incidence of neuropathies. This was confirmed in a phase I pharmacokinetic and toxicity study in which doses three times higher than with misonidazole were delivered and fewer peripheral neuropathies were observed. 31
Based on the encouraging results of the Danish Group in pharynx carcinoma with misonidazole and nimorazole, the RTOG initiated a two-part study in advanced head and neck cancer. The first part, a toxicity and logistic study in which etanidazole was used three times a week for 17 doses in combination with standard radiation, was completed with acceptable toxicity (although the 22% incidence of peripheral neuropathies seen in that study still presented a problem). Subsequently, a phase III study in a similar patient population and with the same frequency of sensitizer administration was completed in 1992. The results showed that adding etanidazole to conventional radiotherapy produced no benefit for patients with advanced head and neck carcinomas, except for a suggestion of benefit in a subset of patients with early stages (N 0 -N 1 disease). 32 A randomized study of 374 patients from 27 European centers, conducted between 1987 and 1990, adding etanidazole to conventional radiotherapy did not afford any benefit for patients with head and neck carcinoma. Furthermore the study failed to confirm the hypothesis of benefit for patients with early disease. 33
A similar study in patients with locally advanced cancer of the prostate—T 2b , T 3 , and T 4 —was also initiated in North America (RTOG). This study was designed to deliver the sensitizer with as many fractions of conventional radiation as possible. Nineteen doses of 1.8 g/m 2 were delivered three times a week during the course of radiation, with tolerable toxicity. The results of this trial with regard to prostatic-specific antigen response and clinical disappearance of tumor are similar to those of historical control subjects, and are not considered to represent an improvement. 34
A single large dose of etanidazole (12 g/m 2 ) administered with intraoperative radiation was considered an ideal setting to assess hypoxic cell sensitizers in a phase I study, which was also completed under the RTOG. The serum and tissue concentrations of etanidazole observed in the trial were 5 to 10 times higher than the levels seen when this compound was given with fractionated radiation. The estimated SER was 2.5 to 3. In 1993, a phase III trial involving intraoperative radiation and single-dose etanidazole was initiated in patients with locally recurrent rectosigmoid carcinoma. In addition, an RTOG study was initiated to test the use of etanidazole in combination with stereostatic radiosurgery in recurrent malignant gliomas or central nervous system metastases. None of these studies demonstrated an improvement in patient outcome, but were limited in power by small numbers of patients.
Evidence exists that prolonged exposure to severe hypoxia can lead to increased sensitization beyond the oxygen effect, owing to the formation of reactive reduced metabolic species. Based on this evidence, an evaluation was made of etanidazole administered in 48-hour and 96-hour continuous intravenous infusions to patients undergoing brachytherapy. 35 The use of etanidazole under these particular conditions is considered worth pursing.
Hypoxia marker studies (see the following text) that used 3 H-misonidazole or 123 I-iodoazomycin arabinoside showed evidence of tumor hypoxia in more than 50% of patients with small cell carcinoma (SCC) of the lung and indicated that tumor hypoxia could be one of the causes of chemoresistance and radioresistance in these patients. Therefore, a phase I and II clinical prospective study was initiated in patients with limited stage of small cell lung cancer, in which etanidazole was given in doses of 1.7 g/m 2 three times a week with concomitant chemotherapy and thoracic irradiation. In patients with limited-stage disease, the median and the crude rates of survival at 5 years with no evidence of disease were superior to the best results reported in the literature from similar radiotherapy and chemotherapy regimens in which etanidazole was not used. 36
Pimonidazole (a 2-nitroimidazole) was developed in Europe at the same time that etanidazole was developed as a third-generation sensitizer, and was considered to be more potent than misonidazole because of its potential to be concentrated in tumors as a result of its pH-dependent sidechain charge. The maximum tolerated dose, when administered with a daily 20-fraction course of radiotherapy, was established at 750 mg/m 2 . The dose-limiting toxicity has been in the central nervous system, manifesting as disorientation and malaise. A randomized clinical trial in advanced carcinoma of the cervix was conducted by 16 centers in Western Europe under the guidance of the Medical Research Council of the UK. Patient accrual was completed in May 1989. Overall and disease-free survival rates were found to be poor among the patients who received pimonidazole in combination with external radiation. 37

Conclusions
The current status after decades of clinical investigations with hypoxic cell sensitizers can be summarized by stating that these compounds have not become part of the standard practice of radiotherapy, at least in North America. In Denmark, however all head and neck cancer patients routinely are given nimorazole based on the positive phase III randomized trial of this drug with conventional radiotherapy. 28 The only patients who appear to have benefited significantly from this approach are those with advanced pharyngeal and supraglottic carcinomas treated with radiation and either misonidazole or nimorazole. In the immediate future and with the advent of noninvasive markers of tumor hypoxia, it should be possible to select patients for the use of these agents. In addition, the increased use of stereotactic body radiotherapy is likely to make these compounds more attractive in the future because hypoxia is a bigger problem for large single or a limited number of large radiation doses and the individual doses of the particular sensitizers that can be delivered will be higher.

Possible Explanation for the Inconclusive Therapeutic Benefits Seen With Nitroimidazoles in Most Solid Tumors
The failure to demonstrate a major improvement of the therapeutic ratio with nitroimidazoles could be related to the following:
1 It has been postulated that the response of tumors to multiple doses of radiation similar to those used in therapy is governed not by the most hypoxic cells in the tumor, but by the more abundant cells at intermediate oxygenation. 38 Such cells require much higher concentrations of nitroimidazoles to give adequate radiosensitization, levels that are higher than clinically tolerated doses. 39
2 Some of the tumor sites chosen did not have a steep radiation-dose response when a 10% dose increment could have made a difference in the results.
3 Patients with proven tumor hypoxic fractions were not preselected with hypoxia markers, and therefore most trials were not large enough to detect significant statistical difference at the 10% level. 40
4 Resistance contributed by high tumor thiol concentrations was not accounted for.
Of interest is an updated review of all randomized clinical trials on approximately 10,108 patients combining tumor hypoxia modifiers (hyperbaric and normobaric oxygen plus carbogen and hypoxic radiosensitizers) with curative radiation, showing an improved effect of radiotherapy on local-regional control, without evidence of increase of radiation side effects in normal tissues. 40

What Has Been Learned?
It has been learned to predict (1) which patients could be at risk for drug-related neurotoxicity according to the initial pharmacokinetics of the drug in the individual patient, (2) that intravenous administration is the preferable route, and (3) that it is necessary to assess the characteristics of each tumor prior to the course of radiation by developing practical techniques to measure tumor hypoxia. It has also been recognized that sensitizers might be best tested in those tumors in which the dose response is sharp. Thus, to see a 20% to 30% improvement in local tumor control, there is only a need to increase the dose by 5%.
The resistance of tumor hypoxic cells to conventional fractionated radiation in humans is a more complex problem than it appeared to be 25 years ago, when the first clinical investigations were initiated with these agents. We now know that in addition to the classically described state of chronic tumor hypoxia, there is intermediate acute hypoxia, and that both states could be influenced by manipulation of the tumor microenvironment. We also know that drugs that kill, rather than sensitize, hypoxic cells, might be a preferable way of dealing with, and indeed of exploiting tumor hypoxia, than the classic nitroimidazole radiosensitizers.

Current Status and Future Directions in Tumor Hypoxia

Microinvasive Methods of Measuring Tumor Hypoxia
One possible interpretation of the clinical sensitizer trials is that hypoxia is less important for the therapy of human tumors than for murine tumors and other model systems. Arguing against this is a series of studies using a newly developed semiautomatic needle electrode (Eppendorf Histograph). 41 - 44 These studies not only confirm the radiation resistance of hypoxic human tumors, but demonstrate their more “aggressive” phenotype for other types of treatment failure (chemotherapy–resistance, metastasis, surgical). The overall “aggressiveness” of hypoxic tumors determined by this pioneering work is now much better understood since the discovery and subsequent elucidation of the central role played by hypoxia-inducible factor-1 (HIF-1) in many aspects of tumor behavior. 45 The resolution of the Eppendorf system is roughly 0.7 mm and several assays capable of much finer resolution have been developed for immunohistochemical analysis.
Some of these microassays involve endogenous molecular changes (often HIF -mediated 46 ) or inherent radiation resistance. 47 The most extensively developed microscopic assay of hypoxia, also central to the development of noninvasive assays (see the following text), was based on the finding that 2-nitroimidazole metabolism led to the hypoxia-dependent formation of adducts between the metabolized drug (activated by nitro-reduction) and cellular macromolecules, later shown to be primarily thiol-containing proteins. 48 , 49 Chapman first proposed that this metabolism could be used for the practical measurement of tissue hypoxia, and one resulting technique (autoradiography after excision of the labeled tumor) progressed to a clinical trial. 50 The measurement of tritiated misonidazole metabolism by autoradiography was problematic for a general-use assay, but this problem was solved through the development of antibody-based assays for the detection of 2-nitroimidazole adducts, particularly using pimonidazole or EF5 as the hypoxia-detecting agent. 51 , 52
As described previously for the sensitizer trials, most former drug development has emphasized the use of polar drugs to reduce access to the central nervous system (thus avoiding neurotoxicity) while promoting rapid excretion. In contrast, EF5 is highly lipophilic, with the recognition that neurotoxicity is unlikely at the low drug concentrations used for hypoxia detection.
One endogenous hypoxia marker currently capable of predicting outcome is carbonic anhydrase IX (CA-IX). CA-IX is one of several carbonic anhydrase enzymes, and is strongly upregulated under hypoxic conditions through the HIF-1α mechanism. CA-IX is expressed on the surface of cells and therefore can be detected by antibodies. However, its correlation with gold-standard measures of hypoxia is suboptimal. 53

Noninvasive Methods of Measuring Tumor Hypoxia
Determination of therapy-relevant tumor hypoxia using nuclear medicine techniques holds many potential benefits, including timely stratification of treatment, prediction of outcome, and even the spatial optimization of radiation therapies using image-guided radiation therapy (IGRT), intensity-modulated radiation therapy (IMRT), and protons. 54 It has become clear that the first of these is much more important than previously considered—that is, heterogeneity of the extent and degree of hypoxia between tumors or patients with otherwise similar characteristics can ultimately stifle the development and interpretation of hypoxia-specific therapies, resulting in the waste of clinical trial resources on patient cohorts for which the hypoxia-specific therapy is inappropriate. With the exception of Copper(II)-diacetyl-bis(N 4 -methylthiosemicarbazone) (Cu-ATSM) and the antibody developed against CA-IX, most agents being considered for the noninvasive detection of hypoxia are also 2-nitroimidazoles. Thus, it is fitting that the first agent developed for noninvasive imaging of hypoxia [ 18 F]fluoromisonidazole ( 18 F-FMISO) was first used to demonstrate the predictive value of the first clinically developed hypoxic cell cytotoxin (tirapazamine [TPZ]). 55 , 56 Nevertheless, this delay in the successful application of 18 F-FMISO imaging has resulted in the extensive development of other possible noninvasive hypoxia markers.
Several different principles have formed the basis for potential improvements (compared with FMISO) in marker properties.

Half-Life of Isotope versus Drug
Chapman and colleagues 17 suggested that the relatively short isotope half-life of 18 F (≈110 minutes) was suboptimal in detecting tumor hypoxia because, first, this does not allow adequate clearance (pharmacologic half-life of drug) of nonmetabolized drug (which forms the image background), and second, binding to hypoxic cells should increase with time, so that inherent limitations exist in generating optimal tumor versus normal tissue contrast during the relatively short imaging times suitable for 18 F. 57 Use of iodine as the detecting isotope was considered superior because of its variety of half-lives and decay types. However, directly iodinating the imidazole ring of misonidazole did not lead to suitably stable compounds, so a variety of nucleoside derivatives were devised, with the iodine conjugated to the sugar moiety rather than the 2-nitroimidazole. These compounds (exemplified by iodoazomycin arabinoside [IAZA] and iodoazomycin galactopyranose [IAZGP]) were also more polar than FMISO (see next paragraph). Both single photon emission computed tomography (SPECT) ( 138 I) and positron emission tomography (PET) ( 139 I) isotopes have been used to evaluate the utility of these drugs. Clinical trials of IAZA successfully imaged hypoxia but because of deiodination this agent was not considered optimal. 57 IAZGP is under current investigation clinically at the Memorial Sloan Kettering Institute. Although the positive aspects of longer-lived isotopes seem clear, there are also negative aspects that must be mentioned. First, to provide a suitable image quality, the number of counts and imaging time must be specified. For these to be equivalent for long- versus short-lived isotopes, the former must necessarily cause a substantially higher total radiation dose burden to the patient. This is exaggerated for many PET isotopes (e.g., Cu and I), because the fraction of positron emissions per decay can be much less than one. This problem cannot be resolved by use of SPECT isotopes, because, other factors being equal, SPECT imaging has an inherently lower resolution than does PET imaging. Ultimate resolution of PET depends on the energy spectrum of the positron, and 18 F is optimal in this respect.

Drug Polarity (Octanol-to-Water Partition Coefficient)
Because of the potential high resolution and low patient radiation dose from 18 F, several other fluorinated drugs have been developed. Some of these compounds have been significantly more polar than FMISO. The goal of minimizing neurotoxicity seems “off-target” for the use of noninvasive imaging agents, because their concentration is orders of magnitude lower than was used in the sensitizer trials. Additionally, there are data suggesting that highly polar nitroimidazoles do not achieve suitable access to brain and possibly other tissues. 58 Thus, it is possible that polar imaging agents will have to be evaluated much more thoroughly with respect to their biodistribution and ability to diffuse into actively metabolizing tissue.

Drug Biodistribution and Stability
EF5’s design was based on specific properties of oxygen dependence of bioreduction, drug stability, and biodistribution determined in former pharmacologic and biochemical studies. It was hypothesized that emphasis on the uniform biodistribution allowed by a lipophilic molecule, in effect minimizing excretion, might allow relatively low hypoxia-dependent bioreduction to be detected above the uniform background of the parent drug. 59 These properties were validated at the relatively high concentration used for immunohistochemistry (≈100 µm—using monoclonal antibodies against EF5 adducts) and appear to be maintained at approximately 1000-fold lower drug concentrations used for PET. 60 Recently, two other 18F-containing hypoxia markers have been tested in humans: [ 18 F]fluoroazomycin arabinoside, 61 the fluorinated equivalent of IAZA; and EF3, 62 a tri-fluoro analog of EF5. These drugs are somewhat more lipophilic than FMISO, with octanol-to-water partition coefficients close to 1.
Several imaging agents have been found to concentrate in tumors, but their precise mechanism binding is not fully understood. The best characterized of these is Cu-ATSM. ATSM is a copper chelator related to the perfusion marker copper pyruvaldehyde bis (N 4 -methylthiosemicarbazone). 63 ATSM is very lipophilic but unexpectedly shows rapid biodistribution and binding. Like iodine, copper has a number of radioactive isotopes with selectable properties. Thus this hypoxia marker has many appealing properties.

What Is the “Optimal” Hypoxia Marker?
Because of the large number of methods and compounds under current study, the answer to this difficult question may finally be answered based on current and planned clinical trials. As indicated previously, the question is very complex, and, in fact, the range of oxygen levels important for tumor aggressiveness or therapeutic outcome is only minimally understood. Like many other aspects of tumor “heterogeneity” there may be no single answer. With the current ability to partition radiotherapy to specific volumes of larger tumors or likely metastatic sites (IGRT; protons), it may soon be possible to deliver more radiation dose to the more hypoxic tumor volumes (“dose-painting”).

Manipulation of the Tumor Microenvironment

Increasing the Oxygen-Carrying Capacity of Blood: Hyperbaric Oxygen and Fluosol
Clinical studies have been completed in which hyperbaric oxygen, 64 carbogen, 65 packed red cell transfusions, 66 , 67 and oxygen-carrier substances such as perfluorocarbons (Fluosol-DA) have being used. 68 , 69 Although the use of the hyperbaric oxygen chamber at three atmospheres presented technical difficulties, such as barotrauma and the limitations of its use to only a few high-dose fractions of radiation, 3 out of 10 clinical trials showed significant positive results. The beneficial effect was seen particularly in patients with advanced cancer of the head and neck and of the cervix. 70
A phase II study of Fluosol-DA and 100% oxygen in combination with radiotherapy in advanced head and neck tumors has shown promising results. 68 , 69 Investigators have also been assessing the compound 2-(4-[{3,5-dimethylanilino}carbonyl]methyl]phenoxy)-2-methylproprionic acid derivative, an allosteric hemoglobin modifier, and preliminary reports are encouraging. 71
An alternative approach is to increase the level of hypoxia in tumor cells and to treat them with hypoxic cytotoxic agents. Attempts have been made in the past to reduce tumor perfusion by the use of agents like hydralazine. This has not proved to be of value because of the potential systemic side effects. Another approach to increase the level of tumor hypoxia by modifying the oxyhemoglobin disassociation curve with a specific chemical agent such as 5-(2-formyl-3-hydroxyphenoxy) pentanoic acid, combining this approach with the hypoxic cytotoxic agent, mitomycin C. This approach was investigated in patients with advanced gastrointestinal cancer. 72

Increasing Tumor Blood Flow: Nicotinamide and Carbogen/Arcon
Improvement in tumor P O 2 following carbogen breathing (95% O 2 , 5% CO 2 ) has been shown in both animal and human tumors. Tumor tissue oxygenation was measured in humans with the polarographic electrode system (Eppendorf) P O 2 histograph. In one study, in 12 out of 17 patients with solid tumors there was a significant increase in median tumor P O 2 during the first 10 minutes of carbogen breathing. Measurements were taken in accessible superficial tumors; 15 were epithelial tumors (most of them breast and lung carcinomas) and 2 were soft tissue sarcomas. 73
It is hypothesized that nicotinamide decreases the presence of acute intermittent hypoxia and that carbogen breathing reoxygenates the chronic hypoxic cells. 74 , 75 The benefit of this combination could be further enhanced in tumors with rapidly proliferating stem cells if accelerated radiotherapy schedules are used. This approach, accelerated radiotherapy with carbogen, nicotinamide (ARCON), was initiated originally in a pilot study in the United Kingdom. A multiple-institution ARCON phase I trial was conducted under the European Organization for Research and Treatment of Cancer. In this three-step study, 115 patients with glioblastoma multiforme were registered. The overall survival was not different when compared with results of other series using radiotherapy alone. 76 Nicotinamide produced gastrointestinal toxicity, necessitating dose reduction. Two other phase I and II clinical trials using ARCON in non-small cell lung cancer (NSCLC) 77 and in 215 patients with advanced head and neck squamous cell carcinoma 78 , 79 were conducted recently with encouraging results in regard to tumor responses, but continued to require a reduction in the dose of nicotinamide because of the incidence of gastrointestinal acute toxicity. The use of ARCON in advanced bladder cancer has been studied to assess its potential gain, with encouraging results and no overt increase in normal tissue radiosensitivity. 80
The use of carbogen (without nicotinamide) has recently been explored in a randomized study comparing definitive hyperfractionated radiation therapy to the same radiation plus carbogen in T 2 -T 4 head and neck tumors. This study did not appear to improve the local tumor control. 81 However, another trial using carbogen breathing combined with radical radiotherapy also in advanced head and neck cancer patients suggested that carbogen breathing may be an alternative form for patients who are unfit to receive concomitant chemotherapy with the radiation treatments. 82

Hypoxic Cytotoxins

Tirapazamine
The development of hypoxic radiosensitizers led to that of hypoxic cytotoxins, also known as bioreductive drugs. These are agents that can sensitize a solid tumor to radiotherapy or to chemotherapy by killing, rather than sensitizing, the resistant hypoxic cells. Hypoxic cells in tumors are not only resistant to radiation, they are also resistant to most anticancer drugs. This is because hypoxic cells, by definition, must be those farthest from functioning blood vessels, and also because cells at low oxygen levels divide much less rapidly than when fully oxygenated. These two factors lead to resistance to anticancer drugs, first because the majority of anticancer drugs are only effective against rapidly proliferating cells, and second because drugs have to reach all tumor cells regardless of their distance from blood vessels. Thus hypoxic cytotoxins are fundamentally different from conventional agents in that they target a different subpopulation of cells within the tumor. Typically, hypoxic cytotoxins have maximum cytotoxicity to the cells at maximum distance from tumor blood vessels, thereby complementing the pattern of cytotoxicity for both radiation and anticancer drugs, which is maximum for the cells immediately adjacent to the blood vessels (see Fig. 4-1 ). Thus these agents have the potential of overcoming a major cause of resistance of solid tumors to conventional therapies, namely that resulting from the inadequate oxygenation and drug delivery to tumor cells distant to blood vessels. The structures of the hypoxic cytotoxins discussed in this Chapter are shown in Fig. 4-4 .

FIGURE 4-4 • Structures of the hypoxic cytotoxins that have been tested in the clinic.

Mitomycin C
Mitomycin C, a quinone antibiotic that requires reductive metabolism for activity, is the prototype bioreductive agent. Introduced into clinical use in 1958, mitomycin C has demonstrated activity toward a number of different tumors in combination with other chemotherapeutic drugs and radiation. Sartorelli and colleagues suggested that the lower oxidation reduction (redox) potential of tumor relative to normal tissue might be exploited to obtain greater activation of this compound to its cytotoxic species. 83 Although tumor redox potential did not turn out to be key for the activity of mitomycin C, Sartorelli and Rockwell were able to show that this drug preferentially kills hypoxic rather than aerobic cells in vitro. 84 However, the differential toxicity is modest: The ratio of drug concentrations under aerobic to hypoxic conditions for the same level of cell kill (hypoxic cytotoxicity ratio [HCR]) is in the range of 1 (no differential) to 5. 85 Nonetheless, this can be sufficient to overcome the resistance of hypoxic cells in animal tumors, and clinical trials have reported higher cure rates for head and neck cancers by adding mitomycin C to radiotherapy compared with radiotherapy alone, 86 although because mitomycin C is a chemotherapy drug with toxicity toward all cells, it is not clear whether the improved cure rates over radiotherapy alone were the result of selective killing of hypoxic cells.

RB 6145
A second class of hypoxic cytotoxins was developed by Adams and colleagues, who showed that nitroheterocyclic structures containing a bifunctional side chain with alkylating properties were not only more active as radiosensitizers of hypoxic cells, they were also potent and selective killers of hypoxic cells, both in vitro and in vivo. The lead compound of this group, RB 6145, showed considerable activity in vitro and in animal tumors, but proved too toxic to warrant clinical development.

Tirapazamine
A group led by Brown and Lee introduced a third class of bioreductive drugs in 1975. The compound introduced, SR 4233, now known as tirapazamine (TPZ), a benzotriazene di-N-oxide, had an HCR of 50 to 300 for different cell lines 87 ( Fig. 4-5 ), and (unlike the classic hypoxic radiosensitizers) is active when combined with fractionated radiation at doses comparable with those used clinically. 88 The mechanism for the selective toxicity of TPZ (and other members of this class) toward hypoxic cells is that the drug is reduced (an electron is added) by intracellular reductases to form a highly reactive radical that produces both single- and double-strand breaks in DNA that result in cell death, although the exact mechanism of this is quite complex. 89 , 90 However, under aerobic conditions, oxygen removes the electron from the TPZ radical, thereby back-oxidizing it to the nontoxic parent with a concomitant production of superoxide radical. Thus the differential hypoxic cytotoxicity results from the fact that the TPZ radical is much more cytotoxic than the superoxide radical. In addition to its toxicity to hypoxic cells, TPZ was shown to be remarkably efficient at enhancing the cytotoxicity of some chemotherapeutic agents, notably cisplatin (CIS), in experimental animal tumors. 91 Following favorable results in phase I and II studies with the combination of CIS and TPZ, a phase III, multicenter, randomized clinical trial with TPZ combined with CIS in patients with advanced NSCLC showed a doubling of the overall response when TPZ was combined with CIS compared with CIS only and a significant increase in the median survival time of the patients. 92 This increase of antitumor activity occurred without any evidence of increased systemic toxicity of the anticancer drug CIS, as was also seen in experimental animal systems. Promising results of phase I and II trials of TPZ combined with both CIS and fractionated irradiation have been reported for cervix and ovarian cancer 93 - 95 and for head and neck cancer. 96 Currently, there are phase III trials underway with TPZ combined either with chemotherapy in NSCLC or with radiotherapy and CIS in head and neck cancer, and the results are awaited. 96 However, the results of the large (861-patient) multicenter phase III trial of SCC were recently reported at the 2008 American Society of Clinical Oncology (ASCO) meeting. 97 Patients with previously untreated stage III or IV SCC of the oral cavity, oropharynx, hypopharynx, or larynx were randomized to receive definitive radiotherapy (70 Gy in 7 weeks) concurrently with either CIS (100 mg/m 2 ) on day 1 of weeks 1, 4, and 7; or CIS (75 mg/m 2 ) plus TPZ (290 mg/m 2 /day) on day 1 of weeks 1, 4, and 7, and TPZ alone (160 mg/m 2 /day) on days 1, 3, and 5 of weeks 2 and 3 (CIS/TPZ). There were no significant differences in failure-free survival or time to locoregional failure (LRF) between the two groups. Interestingly, however, 20% of patients were found to have major deviations in the radiotherapy plan, which was associated with an increased risk of death (hazard ratio [HR] = 1.56; p = <0.0001), and LRF (HR = 1.82; p = 0.0002), and there was a trend of a benefit with the addition of TPZ in patients without major radiotherapy deviations, HR for risk of LRF (CIS/TPZ:CIS) 0.74, 95% CI: 0.53 to 1.04. As well, there is an ongoing phase III study under the Gynecological Oncology Group (GOG) studying the role of TPZ in combination with CIS and radiation in the management of patients with advanced cervical cancer. One of the drawbacks to the use of TPZ is muscle and gastrointestinal toxicities, 95 , 96 although with the chemoradiotherapy studies, myelotoxicity is the dose-limiting toxicity. TPZ remains the most widely studied hypoxic cytotoxin at this time. One of the major conclusions from the TPZ trials is the importance of selection of those patients with hypoxic tumors for trials with hypoxic cytotoxins. This was not done and there is evidence that in patients with hypoxic tumors there is a substantial benefit of the addition of the drug. 98

FIGURE 4-5 • Effect of tirapazamine on tumor cells surviving fraction under air and hypoxic conditions.

AQ4N (Banoxantrone)
The anthraquinone AQ4N was designed specifically as a hypoxia selective cytotoxin. It resembles TPZ in being a di- N- oxide, but has a distinct mechanism of activation and cytotoxicity. AQ4N is a prodrug of a potent DNA intercalator/topoisomerase poison, AQ4, which is formed by reduction of the two tertiary amine N -oxide groups that mask DNA binding in the prodrug form. 99 AQ4N has substantial activity against hypoxic cells in a variety of transplanted tumors, 100 and has recently completed phase I and II clinical trials with lymphomas and leukemias and a phase I and II trial in combination with radiotherapy and temozolomide, with glioblastoma multiforme in progress.

PR-104
PR-104 is a dinitrobenzamide mustard, developed by Wilson and colleagues at the University of Auckland, with considerable advantages over TPZ or AQ4N. 101 The prototype for this class is the prodrug CB 1954, which first came to attention because of its dramatic curative activity against the Walker rat tumor. 102 It was subsequently shown to be a bioreductive prodrug, activated within the tumors by rat DT-diaphorase, which reduces its 4-nitro group to the corresponding hydroxylamine, a potent DNA crosslinking agent. 103 , 104
PR-104 is a phosphate ester that is in effect a “pre-prodrug”; systemic or tumor phosphatases generate the corresponding alcohol (prodrugs), which is subsequently activated by reduction of one or more of the nitro groups by nitroreductases, including one-electron reductases, to produce hypoxia-selective cytotoxicity by virtue of its ability to form DNA interstrand crosslinks. 105
PR-104 has an interesting advantage over TPZ in that it is highly effective not only in killing the hypoxic cells in the tumor, but also better oxygenated cells, by virtue of the fact that the toxic metabolite formed in hypoxic cells can diffuse to kill nearby aerobic cells. PR-104 is therefore active against tumors alone, not requiring the addition of a second agent (such as radiation) to kill the aerobic cells of the tumor. The drug has completed a multicenter phase I trial and is progressing to phase II trials.

Modifiers of Hemoglobin Levels

Erythropoietin
Erythropoietin is a growth factor that has been synthesized in the laboratory. It has shown efficacy in the treatment of anemia related to systemic chemotherapy, 106 as well as in combined chemoradiation. A significant increase in hemoglobin levels compared with controls has been shown in patients receiving radiotherapy. 107 , 108 These studies did not address the question of whether the increase in hemoglobin levels seen when administering erythropoietin results also in improvement in local tumor control. A randomized phase III trial to assess the effect of erythropoietin on local-regional control in anemic patients treated with radiotherapy for advanced carcinoma of the head and neck was initiated by the RTOG. This study was terminated before completion of patient accrual because of negative results. Further, a review of all phase III studies have shown mixed results, with some studies reporting a decrease in patient survival despite an improvement in hemoglobin levels. 109 - 111 One phase III study evaluating erythropoietin in cervical cancer closed prematurely because of potential concerns with thromboembolic events with the use of this compound. The tumor recurrence status between treatment regimens were not statistically significant (GOG 191). 112 Also, similar results, with no improvement in tumor control, have been reported on head and neck cancer. 113

Glutathione Depletion
Sulfhydryls are scavengers of free radicals, protecting chemical damage induced by either ionizing radiation or alkylating agents. It has been postulated and demonstrated in the laboratory that one approach to increasing the efficacy of the nitroimidazoles as sensitizers is to decrease the levels of the competing endogenous sulfhydryls. Glutathione is one of the major endogenous sulfhydryls. Buthionine sulfoximine was developed as a specific inhibitor of glutathione synthesis. It has been shown to deplete glutathione levels in both in vitro and in vivo systems, therefore making misonidazole a more effective sensitizer. Earlier studies in laboratory experiments showed little or no increase in the toxicity of misonidazole in normal tissue, but showed an increased sensitizing efficacy in the tumor tissue. 114
The use of buthionine sulfoximine as a modulator for hypoxic cell sensitizers in combination with radiation and as a modulator of chemotherapeutic drugs such as alkylating agents and bioreductive hypoxic cytotoxics has been of interest in the laboratory. 115 - 118

An Alternative to Nitroimidazole Hypoxic Cell Radiosensitizers

Nitric Oxide
The hypoxic cell radiosensitization properties and vasodilator effects on tumor vasculature of nitric oxide gas have been described, and there is continued laboratory interest on the possible practical therapeutic use of nitric oxide–releasing compounds (NONOates) under specific physiologic conditions. Studies done in the laboratory under in vitro conditions has shown a marked radiosensitizer effect under hypoxic conditions. 119 Further studies are being conducted in in vivo models. For the time being, this approach is still limited to the laboratory level.

Nonhypoxic Cell Sensitizers
The cancer chemotherapy agents hydroxyurea and 5-fluorouracil are not discussed in this chapter. The reader is referred to Chapter 6 for description of these agents as possible nonhypoxia-specific cell radiosensitizers.

Halogenated Pyrimidine Analogues

5-Bromodeoxyuridine and 5-Iodoeoxyuridine
The pyrimidine analogues 5-bromodeoxyuridine (BUDR) and 5-iodoeoxyuridine (IUDR) are considered cell-cycle–specific radiosensitizers and act independently of the oxygen effect. As previously discussed, the radioresistance of human solid tumors could be multifactorial, in which, in addition to the microenvironment (nutrition oxygenation and presence of endogenous sulfhydryl compounds), tumor cell kinetics play an important role. The presence of rapidly proliferating clonogens may substantially influence the control of tumors by irradiation. Because BUDR and IUDR sensitize only rapidly proliferating cells, in such tissues either normal or tumor cells could be effectively sensitized. Rapidly proliferating tumor cells surrounded by slowly proliferating and supporting normal tissue present the ideal scenario for an improved therapeutic ratio.
Unlike the hypoxic cell sensitizers, these agents require extended exposure of the cells for the necessary incorporation into DNA, while the cells are undergoing DNA synthesis.

Importance of Cell Labeling and DNA Incorporation

Clinical Investigations
The degree of incorporation and thymidine replacement and the SER are intimately related. Therefore, measurements of thymidine replacement in individual human tumors by flow cytometry to establish the potential doubling time, and assessment of thymidine replacement after short and long infusions are needed as part of the design of future clinical trials with these cell-cycle drugs.
The means of achieving an optimal incorporation of these compounds in the cell in the clinical situation has been extensively explored over the years, particularly the route of administration and length of drug exposure. Early on, BUDR was used intra-arterially both to avoid dehalogenation by the liver and to increase the drug tumor concentration. 120 However, the necessary prolonged use of this route in patients over several weeks was laborious and had a high incidence of complications. Although there have been reports of rapid debromination of halopyrimidines occurring after intravenous therapy, Goffinet and Brown 121 showed that following intravenous infusion, enough halopyrimidine apparently passes through the hepatic vessels to permit tumor radiosensitization, despite dilution of the drug by the systemic circulation. This has also been shown in human studies. There was a renewed interest in the 1980s and 1990s in the use of continuous intravenous infusion of halopyrimidines. It was observed that adequate steady-state arterial plasma levels could be maintained with this route of administration with acceptable systemic toxicities. 122 A large study was reported by the Northern California Oncology Group in 160 patients with glioblastoma treated with 96-hour infusion of BrUdR at 800 mg/m 2 a day for a total of 6 weeks, in combination with 60 Gy irradiation directed to tumor plus a margin. The patients in this series received chemotherapy with procarbazine, lomustine (CCNU), and vincristine (PCV) for 1 year following radiotherapy. The median survival time was 12.8 months. Patients with anaplastic astrocytoma had a median survival time of almost 5 years, and the observation was made that the use of pyrimidine analogues in combination with radiation may be of greater benefit in this group of patients. 123 However a randomized study in anaplastic astrocytoma conducted by the RTOG using radiation and PCV chemotherapy compared with radiation and PCV plus BrUdR was terminated earlier because of the inferior time to tumor recurrence and survival observed in the arm using BrUdR. 124
In addition, because of the toxicities and limited tumor efficacy, the use of this drug with radiation was not warranted in patients with glioblastoma. 125

Conclusions on Radiosensitizers
After more than two decades of clinical investigations, the goal of using hypoxic cell or cell-cycle specific radiosensitizers in everyday standard radiotherapy remains elusive. There is, however, a better understanding of the multifactorial cause of radiation resistance and current efforts are concentrated on the selection of a specific tumor site in which one or two factors of resistance have been uncovered. Numerous studies indicate tumor hypoxia as one of the important factors in clinical radiotherapy. The application of radiosensitizers will not be universal in all solid tumors, but could be specifically directed to tumor sites according to their characteristics, including cell kinetics and presence or absence of hypoxia using noninvasive markers of hypoxia.

Chemical Radioprotectors
The first chemical radioprotector compounds intended for use on humans were developed for the purpose of protecting individuals from whole-body irradiation, such as in the event of nuclear warfare. The sulfhydryl compounds, including β-mercaptoethylamine and thiophosphates, were considered. An extensive drug developmental program was initiated by the U.S. Department of Defense (Walter Reed Army Research Institute [WR]). Out of 4000 screened compounds, the thiophosphate WR-2721 was the most promising. Clearly, these compounds were designed to protect all tissues, a very different requirement from their possible use in the field of oncology, in which protection of normal tissues to the exclusion of tumor tissues is essential to improve the therapeutic ratio. Initially, there were serious questions about whether these agents could also protect the tumor from the effects of radiation. 126 , 127
The assumption that the tumor tissues are not protected to the same degree as normal tissues is based on the probability that there is poor drug penetration in tumors because of their poor blood perfusion, the partition coefficient of the drug, and the probability that there is a higher concentration of the drug in normal tissues because of their higher pH. In addition, for WR-2721 to be active, the phosphate group must be cleaved by the enzyme alkaline phosphatase to form the dephosphorylated free thiol WR-1065. This enzyme is not as abundant in tumor tissues as in normal tissues; therefore, the levels of alkaline phosphatase and pH in tissues determine the uptake of WR-2721. There is also a final assumption that thiol compounds could have less protective effect on hypoxic cells. The mechanisms of radioprotection fit the competition-model, dual-action theory. Once inside the cell, the active free thiol WR-1065 can chemically reduce free radicals. However because protectors will have maximum effect at intermediate oxygen levels, the possibility of tumor protection can occur presumably at levels of intermediate hypoxia. It also takes part in the repair reaction of DNA damage. Of interest in the field of cancer chemotherapy is the fact the dephosphorylated WR-2721 can bind to the active species of alkylating agents, as well as prevent the formation of CIS-DNA adducts. 128
Biologic agents such as interleukin-1 have been shown to protect normal tissues in animal systems. 129

Amifostine WR-2721 (Ethyol)
The dose-modifying factor (DMF) of amifostine WR-2127 for both normal and tumor tissues was studied in animals carrying solid tumors. 130 , 131 For normal tissues, the greatest protection is found in bone marrow, with a DMF of 2.7 to 3, and in the gastrointestinal tract, with a DMF of 1.6. The lowest DMF, at 1.2, is in lung tissue. However, this drug also protects tumor tissues, with DMFs ranging from 1.3 for cure of EMT-6 carcinoma to 2.2 for mean survival time of P-388 leukemia. Once more, the degree of protection to tumors appears to be related to the tumor blood perfusion, degree of hypoxia, the tissue pH, and the levels of alkaline phosphatase. A cautionary note is that most of these experiments were performed with single-radiation doses. It is possible that the differential protective effect between normal and tumor tissues would be less if multiple, daily, small doses in combination with radiation were used.
Experimental work has also been done in which amifostine is used as a chemoprotector of normal tissues. Three studies were performed with different animal tumor models and all three showed protection of bone marrow and intestine with no protection of the tumor when amifostine was used with melphalan 132 or in combined chemotherapy regimes with nitrogen mustard, cyclophosphamide, carmustine, CIS, and 5-fluorouracil. 133 , 134
It should be noted that amifostine does not protect the central nervous system tissues from radiation effects because of the blood-brain barrier. 135

Clinical Experience
Amifostine has been approved for clinical use and is available for intravenous route in a sterile lyophilized powder mixture with mannitol, requiring reconstitution for intravenous administration. It is administered over a 15-minute period prior to radiation or chemotherapy. Initial single-dose toxicity and pharmacokinetic studies were performed in 1983. The plasma initial-half-life is 9 minutes, and it is assumed that the protective concentrations are maintained in normal tissues for approximately 2 hours. 136 , 137
The maximum tolerated dose for multiple doses is 340 mg/m 2 given 4 days a week for 5 weeks, 15 minutes prior to external radiation. Toxicities at this dose level are manifested as nausea, vomiting, anorexia, malaise, transient moderate hypotension, and occasional hypocalcemia. It is recommended that the amifostine infusion be interrupted if there is a 25% decrease in systolic blood pressure. The maximum tolerated dose with single doses is 740 mg/m 2 , although recently the dose was increased to 910 mg/m 2 given twice a week for at least five treatments, and this dose has been considered acceptable. 138
Since 2000, this compound has led most of the reported activity on clinical trials with chemical modifiers of radiation response. This interest has been triggered by the increasing use of chemoradiotherapy in solid tumors and in an effort to avoid the incidence of toxicities with this therapeutic combination on major sites such as NSCLC (protection for esophagitis and pneumonitis), head and neck tumors (protection from xerostomia and mucositis), as well as pelvic tumors (protection of rectal mucosa and the small bowel).
Amifostine in combination with chemoradiation therapy for small cell lung cancer and NSCLC has been studied in several randomized phase III clinical trials, which have shown a statistically significant decrease in esophagitis and pneumonitis with no observed tumor protection. 139 - 142 A meta-analysis of all published clinical trials (seven randomized involving 601 patients, with locally advanced NSCLC treated with radiotherapy with or without chemotherapy) revealed that amifostine has not protected the tumor from the therapeutic effect of either radiation or chemoradiation. 143 However, amifostine toxicity consisting of hypotension, nausea, and vomiting with the use of the intravenous route prevented a rather large proportion of patients from receiving the full dose according to protocol. 142 To avoid the amifostine toxicity seen with intravenous use, studies have been initiated to explore the subcutaneous administration based on previous phase I and II trials demonstrating a reduction of amifostine toxicities when using this route. 144 - 147
Head and neck tumors treated with chemoradiotherapy is another area in which a large number of randomized phase III clinical trials were initiated over the past 7 years using amifostine as a radioprotector. Most of the reported clinical trials on this disease site have demonstrated a decreased incidence of xerostomia and mucositis, with no clear evidence of tumor protection as measured by local regional tumor control. 148 A significant reduction in the incidence of radiation-induced xerostomia at 12 months posttreatment was reported with the use of amifostine in a multinational phase III trial of 315 patients with head and neck cancer. 149 An update of this study at 18 and 24 months after initial treatment indicated that amifostine reduces the severity and duration of xerostomia 2 years after treatment and does not seem to compromise local-regional tumor control rates, progression-free survival, or overall survival. 150 Amifostine-related nausea, vomiting, and hypotension at a dose of 200 mg IV daily with each radiation fraction led to discontinuation of the drug in approximately 20% of patients with, in some cases, a delay of radiotherapy. 151
Early phase II efficacy trials have been initiated using amifostine during chemoradiotherapy of pelvic tumors in an attempt to protect rectal mucosa, indicating significant protective effect in patients with cancers of the prostate, cervix, and rectum using either the intravenous or subcutaneous routes as well as with the intrarectal aqueous solution in a 48-ml enema. 152 , 153
Concerns about amifostine toxicity as well as the question of tumor tissue protection have been raised by some authors. 154 However, contradicting this view, a review of all clinical studies done up to 2003 have clearly indicated no evidence of amifostine tumor protection. 155
The practical application of amifostine in the standard practice of radiation oncology continues to be a controversial one. Up to 2008, the use of amifostine in the chemoradiotherapy of head and neck tumors to protect from xerostomia has been recommended by the ASCO and is approved by the U.S. Food and Drug Administration for (1) reduction of the incidence of moderate to severe xerostomia in patients undergoing postoperative radiation treatment for head and neck cancer, and (2) reduction in the cumulative renal toxicity associated with CIS in patients with advanced ovarian cancer. 156 , 157 Recent observations of the efficacy of IMRT in reducing xerostomia have raised questions about the expense of using amifostine for this purpose.

Chemoprotection
The clinical effectiveness of amifostine as a chemoprotector was first assessed with the use of bone marrow as the normal tissue endpoint and cyclophosphamide as the chemotherapeutic agent. Definitive evidence of bone marrow protection was observed. Tumor protection was not assessed. 158 In another study, renal damage and peripheral neuropathy were assessed as endpoints and CIS was used; normal tissue protection with no tumor protection was observed. 159 Another clinical study demonstrated the protective effect of amifostine in bone marrow, kidney, and peripheral nerves when cyclophosphamide is used in combination with CIS. 160 Protection of carboplatin myelotoxicity was observed in one study, 161 but findings were inconclusive in another preliminary study. 162 The use of amifostine in combination with chemotherapy and radiation is currently being used in clinical research protocols for both adult and pediatric patient populations.

Tempol
Nitroxide superoxide dismutase mimic (Tempol), a nonthio nitroxide, has been shown both in vitro and in vivo to be an effective radioprotector with the absences of tumor radioprotection. In addition, nitroxide compounds are known for their modest hypoxic cell radiosensitization. The potential of Tempol as an aerobic cell radioprotector has been described by Mitchell and colleagues. 163

Conclusions on Radioprotectors and Chemoprotectors
A spin-off of the work on radioprotectors is manifested in the increasing interest in the use of these compounds as chemoprotectors in the field of medical oncology. 164 The protective effects of amifostine have been seen in bone marrow during the use of alkylating and platinum compound agents. Phase III large clinical trials are ongoing to demonstrate conclusively normal tissue protection without tumor protection in esophageal mucosa, salivary glands, and bone marrow when these protectors are combined with radiation, and in kidney, peripheral nerves, and bone marrow when they are used with alkylating agents and platinum compounds.
An exciting evolving potential is in the combination of chemical protectors (amifostine or Tempol) with biologic stimulators (cytokines). Furthermore, the effect of thiol compounds might be even greater in the laboratory, based on their promising use as a probe in examining the mechanisms of radiation cell damage. It is likely that vital new information will be available in the near future on the molecular mechanisms of radiation protection. 164

References

1 Steel GG. Combined radiotherapy-chemotherapy: principles. In: Steel GG, Adams GE, Horwich E, editors. The biological basis of radiotherapy . ed 2. Amsterdam: Elsevier; 1989:267.
2 Gatenby RA, Kessler HB, Rosenblum JS, et al. Oxygen distribution of squamous cell carcinoma metastases and its relationship to outcome of radiation therapy. Int J Radiat Oncol Biol Phys . 1988;14(5):831.
3 Hockel M, Knoop C, Schlenger K, et al. Intratumoral Po 2 predicts the survival in advanced cancer of the uterine cervix: updated analysis of an open prospective clinical study. Radiother Oncol . 1992;26:45.
4 Bush RS, Jenkin RDT, Eltt WEC, et al. Definitive evidence for hypoxic cells influencing cure in cancer therapy. Br J Cancer . 1978;37(3):302.
5 Brizel DM, Scully SP, Harrelson JM, et al. Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma. Cancer Res . 1996;56:941.
6 Nordsmark M, Overgaard M, Overgaard J. Pretreatment oxygenation predicts radiation response in advanced squamous cell carcinoma of the head and neck. Radiother Oncol . 1996;41:31.
7 Nordsmark M, Bentzen SM, Rudat V, et al. prognostic value of tumor oxygenation in 396 head and neck tumors after primary radiation therapy. An international multi-center study. Radiother Oncol . 2005;77(1):18-24.
8 Durand RE. Distribution and activity of antineoplastic drugs in a tumor model. J Natl Cancer Inst . 1989;81:146-152.
9 Sutherland RM. Cell and environment interaction in tumor microregions. The multicell spheroid model. Science . 1988;24:177.
10 Graeber TG, Osmanian C, Jacks T, et al. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature . 1996;379:88.
11 Hockel M, Schlenger K, Aral B, et al. Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res . 1996;56:4509.
12 Sundfor K, Lyng H, Rofstad EK. Tumour hypoxia and vascular density as predictors of metastasis in squamous cell carcinoma of the uterine cervix. Br J Cancer . 1998;78:822-827.
13 Wachsberger P, Burd R, Dicker AP. Tumor response to ionizing radiation combined with antiangiogenesis or vascular targeting agents: exploring mechanisms of interaction. Clin Cancer Res . 2003;9(6):1957.
14 Moeller BJ, Cao Y, Li CY, Dewhirst MW. Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules. Cancer Cell . 2004;5(5):429.
15 Adams GE, Cooke MS. Electron-affinic sensitization. I. A structural basis for chemical radiosensitizers in bacteria. Int J Radiat Biol . 1969;15:457.
16 Adams GE, Asquith JC, Watts ME, et al. Radiosensitization of hypoxic cells in vitro: a water-soluble derivative of paranitroacetophenone. Nature [New Biol] . 1972;239:23.
17 Chapman JD, Reuvers AP, Borsa J, et al. Chemical radioprotection and radiosensitization of mammalian cells growing in vitro. Radiat Res . 1973;56(2):291.
18 Foster JL, Willson RL. Radiosensitization of anoxic cells by metronidazole. Br J Radiol . 1973;46:234.
19 Rabin HR, Urtasun RC, Partington J, et al. Pharmacokinetics and bioavailability of metronidazole using an IV preparation and application of its use as a radiosensitizer. Cancer Treatment Reports . 1980;64:1087.
20 Brown JM. Selective radiosensitization of the hypoxic cells of mouse tumors with the nitroimidazoles metronidazole and Ro 7-0582. Radiat Res . 1975;64(3):633.
21 Denekamp J, Harris SR. Tests of two electron-affinic radiosensitizers in vivo using regrowth of an experimental carcinoma. Radiat Res . 1975;61:191.
22 Urtasun RC, Band P, Chapman JD, et al. Radiation and high dose metronidazole in supratentorial glioblastomas. N Engl J Med . 1976;294:1364.
23 Urtasun RC, Chapman JD, Feldstein L, et al. Peripheral neuropathy related to misonidazole: Incidence and pathology. Br J Cancer . 1978;3:271.
24 Dische S, Saunders MI, Anderson P, et al. Neurotoxicity of misonidazole, pooling of data from five centers. Brit J Radiol. . 1978;51:1023.
25 Dische S. Chemical sensitizers for hypoxic cells: A decade of experience in clinical radiotherapy. Radiother Oncol . 1985;3:97.
26 Brown JM. Clinical trials of radiosensitizers: What should we expect? Int J Radiat Oncol Biol Phys . 1984;10:425.
27 Overgaard J. Clinical evaluation of nitroimidazoles as modifiers of hypoxia in solid tumors. Oncol Res . 1994;6:509.
28 Overgaard J, Sand Hansen H, Overgaard M, et al. A randomized double-blind phase III study on nimorazole as a hypoxic radiosensitizer of primary radiotherapy in supraglottic larynx and pharynx. Results of the Danish Head and Neck Cancer Study (Dahanca). Radiother Oncol . 1998;46(2):135.
29 Horsman MR, Bohm L, Margison GP, et al. Tumor radiosensitizers—current status of development of various approaches: Report on an International Atomic Energy Agency Meeting. Int J Radiat Oncol Biol Phys . 2006;64(2):551.
30 Fazekas J, Pagak TF, Wasserman TH, et al. Failure of misonidazole-sensitized radiotherapy to impact upon outcome among stage III/IV squamous cancers of the head and neck. Int J Radiat Oncol Biol Phys . 1987;13(8):1155.
31 Wasserman TH, Lee DJ, Cosmatos D, et al. Clinical trials with etanidazole (SR-2508) by the Radiation Oncology Group (RTOG). Radiother Oncol . 1991;20:129.
32 Lee DJ, Cosmatos D, Marcial VA, et al. Results of an RTOG phase III trial (RTOG 85-27) comparing radiotherapy plus etanidazole with radiotherapy alone for locally advanced head and neck carcinomas. Int J Radiat Oncol Biol Phys . 1995;32(3):567.
33 Eschwege R, Sancho-Garnier H, Chassagne D, et al. Results of a European randomized trial of etanidazole combined with radiotherapy in head and neck carcinomas. Int J Radiat Oncol Biol Phys . 1997;39(2):275.
34 Lawton CA, Coleman CN, Buzydlowsky JW, et al. Results of a phase II trial of external beam radiation with etanidazole for the treatment of locally advanced prostatic cancer (RTOG protocol 90-20). Int J Radiat Oncol Biol Phys . 1996;36(3):673.
35 Coleman CN, Noll L, Howes AE, et al. Initial results of the phase I trial of the continuous infusion of SR-2508 (etanidazole). Int J Radiat Oncol Biol Phys . 1989;16:1085.
36 Urtasun RC, Palmer M, Kinney B, et al. Intervention with the hypoxic tumor cell sensitizer etanidazole in the combined modality treatment of limited stage small-cell lung cancer. A one-institution study. Int J Rad Oncol Biol Phys . 1998;40:337.
37 Dische S. Radiotherapy, carcinoma of cervix and the radiosensitizer Ro 03-8799 (pimonidazole). In: Dewey WC, Edington M, Fry MRG, et al, editors. Radiation Research: A 20th Century Perspective , vol 2. Toronto: Academic Press; 1992.
38 Wouters BG, Brown JM. Cells at intermediate oxygen levels can be more important than the “hypoxic fraction” in determining tumor response to fractionated radiotherapy. Radiat Res . 1997;147:541.
39 Koch CJ, Howell RL. Combined radiation-protective and radiation-sensitizing agents: II Radiosensitivity of hypoxic or aerobic Chinese hamster fibroblasts in the presence of cysteamine and misonidazole: implications for the “oxygen effect” (with appendix on calculation of dose-modifying factors). Radiat Res . 1981;87(2):265.
40 Overgaard J. Hypoxic radiosensitization: Adored and Ignored. J Clin Oncol . 2007;25(26):4066.
41 Höckel M, Knoop C, Schlenger K, et al. Intratumoral P O 2 predicts survival in advanced cancer of the uterine cervix. Radiother Oncol . 1993;26(1):45.
42 Brizel DM, Rosner GL, Harrelson J, et al. Pretreatment oxygenation profiles of human soft tissue sarcomas. Int J Radiat Oncol Biol Phys . 1994;30(3):635.
43 Fyles AW, Milosevic M, Wong R, et al. Oxygenation predicts radiation response and survival in patients with cervix cancer. Radiother Oncol . 1998;48(2):149.
44 Nordsmark M, Overgaard J. Tumor hypoxia is independent of hemoglobin and prognostic for loco-regional tumor control after primary radiotherapy in advanced head and neck cancer. Acta Oncol . 2004;43(4):396.
45 Wang GL, Semenza GL. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J Biol Chem . 1993;268(29):21513.
46 Olive PL, Aquino-Parsons C, MacPhail SH, et al. Carbonic anhydrase 9 as an endogenous marker for hypoxic cells in cervical cancer. Cancer Res . 2001;61(24):8924.
47 Olive PL, Johnston PJ, Banáth JP, et al. The comet assay: a new method to examine heterogeneity associated with solid tumors. Nat Med . 1998;4(1):103.
48 Varghese AJ, Whitmore GF. Binding of nitroreduction products of misonidazole to nucleic acids and protein. Cancer Clin Trials . 1980;3(1):43.
49 Raleigh JA, Koch CJ. Importance of thiols in the reductive binding of 2-nitroimidazoles to macromolecules. Biochem Pharmacol . 1990;40(11):2457.
50 Urtasun RC, Chapman JD, Raleigh JA, et al. Binding of 3H-misonidazole to solid human tumors as a measure of tumor hypoxia. Int J Radiat Oncol Biol Phys . 1986;12(7):1263.
51 Kennedy AS, Raleigh JA, Perez GM, et al. Proliferation and hypoxia in human squamous cell carcinoma of the cervix: first report of combined immunohistochemical assays. Int J Radiat Oncol Biol Phys . 1997;37(4):897.
52 Evans SM, Hahn S, Pook DR, et al. Detection of hypoxia in human squamous cell carcinoma by EF5 binding. Cancer Res . 2000;60(7):2018.
53 Mayer A, Höckel M, Vaupel P. Endogenous hypoxia markers: case not proven!. Adv Exp Med Biol . 2008;614:127.
54 Tatum JL, Kelloff GJ, Gillies RJ, et al. Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy. Int J Radiat Biol . 2006;82(10):699.
55 Koh WJ, Rasey JS, Evans ML, et al. Imaging of hypoxia in human tumors with [F-18]fluoromisonidazole. Int J Radiat Oncol Biol Phys . 1992;22(1):199.
56 Rischin D, Hicks RJ, Fisher R, et al. Prognostic significance of [18F]-misonidazole positron emission tomography-detected tumor hypoxia in patients with advanced head and neck cancer randomly assigned to chemoradiation with or without tirapazamine: a substudy of Trans-Tasman Radiation Oncology Group Study 98.02. J Clin Oncol . 2006;24(13):2098.
57 Parliament MB, Chapman JD, Urtasun RC, et al. Non-invasive assessment of human tumour hypoxia with 123I-iodoazomycin arabinoside: Preliminary report of a clinical study. Br J Cancer . 1992;65(1):90.
58 Brown JM, Workman P. Partition coefficient as a guide to the development of radiosensitizers which are less toxic than misonidazole. Radiat Res . 1980;82(1):171.
59 Laughlin KM, Evans SM, Jenkins WT, et al. Biodistribution of the nitroimidazole EF5 (2-[2-nitro-1H-imidazol-1-yl]-N-[2,2,3,3,3-pentafluoropropyl] acetamide) in mice bearing subcutaneous EMT6 tumors. J Pharmacol Exp Ther . 1996;277(2):1049.
60 Ziemer LS, Evans SM, Kachur AV, et al. Noninvasive imaging of tumor hypoxia in rats using the 2-nitroimidazole 18F-EF5. Eur J Nucl Med Mol Imaging . 2003;30(2):259.
61 Piert M, Machulla HJ, Picchio M, et al. Hypoxia-specific tumor imaging with 18F-fluoroazomycin arabinoside. J Nucl Med . 2005;46(1):106.
62 Christian N, Bol A, De Bast M, et al. Determination of tumour hypoxia with the PET tracer [18F]EF3: improvement of the tumour-to-background ratio in a mouse tumour model. Eur J Nucl Med Mol Imaging . 2007;34(9):1348.
63 Matsumoto K, Szajek L, Krishna MC, et al. The influence of tumor oxygenation on hypoxia imaging in murine squamous cell carcinoma using [64Cu]Cu-ATSM or [18F] Fluoromisonidazole positron emission tomography. Int J Oncol . 2007;30(4):873.
64 Hank JM. Does hyperbaric oxygen have a future in radiation therapy? Int J Rad Oncol Biol Phys . 1981;7:1125.
65 Rubin P, Hanley J, Keys HM, et al. Carbogen breathing during radiation therapy. Radiation Oncology Group study. Int J Radiat Oncol Biol Phys . 1979;5:1963.
66 Bush RS. The significance of anemia in clinical radiation therapy. Int J Radiat Oncol Biol Phys . 1986;12:2047.
67 Dische S, Saunders MI, Warvurton MF. Hemoglobin, radiation, morbidity and survival. Int J Radiat Oncol Biol Phys . 1986;12:1335.
68 Rose C, Lustig R, McIntosh LN, et al. A clinical trial of fluosol-DA 20% in advanced squamous cell carcinoma of the head and neck. Int J Radiat Oncol Biol Phys . 1988;12:1325.
69 Lustig R, McIntosh LN, Rose C, et al. Phase I/II, study of fluosol-DA and 100% oxygen as adjuvant to radiation in the treatment of advanced squamous cell tumors of the head and neck. Int J Radiat Oncol Biol Phys . 1989;16(6):1587.
70 Dische S. Hypoxia in local tumor control: Part 2. Radiother Oncol . 1991;20:9.
71 Amorino GP, Lee H, Holburn GE, et al. Enhancement of tumor oxygenation and radiation response by the allosteric effector of hemoglobin, RSR13. Radiat Res . 2001;156:294.
72 Ramsay JRS, Bleehan NM, Dennis I, et al. Phase I: A study of BW12C in combination with mitomycin C in patients with advanced gastrointestinal cancer. Int J Rad Oncol Biol Phys . 1992;22:21.
73 Falk SJ, Ward R, Bleehan NM. The influence of carbogen breathing on tumor tissue oxygenation in man evaluated by computerized P O 2 histography. Br J Cancer . 1992;66:919.
74 Rojas A. Radiosensitization with normobaric oxygen and carbogen. Radiother Oncol . 1991;20:65.
75 Horsman MR, Kristjansen PEG, Mizuno M, et al. Biochemical and physiological changes induced by nicotinamide in a C3H mouse mammary carcinoma and CDF1 mice. Int J Radiat Oncol Biol Phys . 1992;22:451.
76 Miralbell R, Mornex F, Greiner R, et al. Accelerated radiotherapy, carbogen and nicotinamide in glioblastoma. J Clin Oncol . 1999;17:3143.
77 Bernier J, Denekamp J, Rojas A, et al. ARCON: Accelerated radiotherapy with carbogen and nicotinamide in non-small cell lung cancer: A phase I/II study by the EORTC. Radiotherapy and Oncology . 1999;52(2):149-156.
78 Hoogsteen IJ, Pot LAM, Marres HAH. Oxygen modifying treatment with ARCON reduces the prognostic significance of hemoglobin in squamous cell carcinoma of the head and neck. Int J Radiat Oncol Biol Phys . 2006;61(1):83.
79 Kaanders JH, Pop LA, Marres HA, et al. ARCON experience into 115 patients with advanced head and neck cancer. Int J Radiat Oncol Biol Phys . 2002;52(3):769.
80 Hoskin PJ, Rojas AM, Phillips H, et al. Acute and late morbidity in the treatment of advanced bladder carcinoma with accelerated radiotherapy, carbogen, and nicotinamide. Cancer . 2005;103(11):2287.
81 Mendenhall WM, Morris CG, Amdur RS, et al. Radiotherapy alone or combined with carbogen breathing for squamous cell carcinoma of the head and neck: A prospective randomized trial. Cancer . 2005;104(2):332.
82 Vees H, Allal AS. Carbogen breathing combined with radical radiotherapy in advanced head and neck cancer patients, Clinical Oncology. Royal College of Radiologists . 2006;18(6):493-496.
83 Lin AJ, Cosby LA, Shansky CW, et al. Potential bioreductive alkylating agents. 1. Benzoquinone derivatives. J Med Chem . 1972;15:1247.
84 Rockwell S, Kennedy KA, Sartorelli AC. Mitomycin-C as a prototype bioreductive alkylating agent: in vitro studies of metabolism and cytotoxicity. Int J Radiat Oncol Biol Phys . 1982;8:753.
85 Brown JM, Siim BG. Hypoxia-specific cytotoxins in cancer therapy. Seminars in Rad Onc . 1996;6:22.
86 Haffty BG, Son YH, Sasaki CT, et al, Mitomycin C as an adjunct to postoperative radiation therapy in squamous cell carcinoma of the head and neck: results from two randomized clinical trials, Int J Radiat Oncol Biol Phys; 27; 1993:241.
87 Zeman EM, Brown JM, Lemmon MJ, et al. SR-4233: a new bioreductive agent with high selective toxicity for hypoxic mammalian cells. Int J Radiat Oncol Biol Phys . 1986;12:1239.
88 Brown JM, Lemmon MJ. SR-4233: a tumor specific radiosensitizer active in fractionated radiation regimes. Radiother Oncol . 1991;1:151.
89 Evans JW, Chernikova SB, Kachnic LA, et al. Homologous recombination is the principal pathway for the repair of DNA damage induced by tirapazamine in mammalian cells. Cancer Res . 2008;68(1):257.
90 Peters KB, Brown JM. Tirapazamine: a hypoxia-activated topoisomerase II poison. Cancer Res . 2002;62(18):5248.
91 Dorie MJ, Brown JM. Tumor-specific, schedule-dependent interaction between tirapazamine (SR 4233) and cisplatin. Cancer Research . 1993;53:4644.
92 von Pawel J, von Roemeling R, Gatzemeier U, et al, Tirapazamine plus cisplatin versus cisplatin in advanced non-small-cell lung cancer: A report of the international CATAPULT I study group, [In Process Citation]; J Clin Oncol; 48; 2000:791.
93 Craighead PS, Pearcey R, Stuart G. A phase I/II evaluation of tirapazamine administered intravenously concurrent with cisplatin and radiotherapy in woman with locally advanced cervical cancer. Int J Radiat Oncol Biol Phys . 2000;48:791.
94 Maluf FC, Leiser AL, Aghajanian, et al. Phase II study of Tirapazamine plus cisplatin in patients with advanced or recurrent cervical cancer. Int J Gynecological Cancer . 2006;16(3):1165.
95 Covens A, Blessing J, Bender D, et al. A phase II evaluation of Tirapazamine plus cisplatin-sensitive ovarian or primary peritoneal cancer: A Gynecological Oncology Group Study. Gynecologic Oncology . 2006;100(3):585.
96 Rischin D, Peters L, Fisher R, et al. Tirapazamine, Cisplatin, and Radiation versus Fluorouracil, Cisplatin and Radiation in patients with locally advanced head and neck cancer: A randomized phase II trial of the Trans-Tasman Radiation Oncology Group (TROG 98-02). J Clin Oncol . 2005;23:79.
97 Rischin D, Peters L, O’Sullivan B, et al, Phase III study of tirapazamine, cisplatin and radiation versus cisplatin and radiation for advanced squamous cell carcinoma of the head and neck, J Clin Oncol; 26; 2008, (May 20 suppl; abstr LBA6008)
98 Peters LJ, Rischin D, Fisher R, et al: Identification and therapeutic targeting of hypoxia in H&N cancer. Abstract PS1042 International Congress of Radiation Research, July 2007.
99 Patterson LH. Rationale for the use of aliphatic N-oxides of cytotoxic anthraquinones as prodrug DNA binding agents: a new class of bioreductive agent. Cancer Metastasis Rev . 1993;12(2):119.
100 Patterson LH, McKeown SR, Ruparelia K, et al. Enhancement of chemotherapy and radiotherapy of murine tumours by AQ4N, a bioreductively activated anti-tumour agent. Br J Cancer . 2000;82(12):1984.
101 Wilson WR, Edmunds SJ, Valentine S, et al: Mechanism of action and antitumour activity of PR-104, A dinitrobenzamide mustard pre-prodrug that is activated selectively under hypoxia. Poster at National Cancer Research Institute Conference, Birmingham, Oct 3, 2005.
102 Cobb LM, Connors TA, Elson LA, et al. 2,4-Dinitro-5-ethyleneiminobenzamide (CB 1954): A potent and selective inhibitor of the growth of the Walker carcinoma 256. Biochem Pharmacol . 1969;18:1519.
103 Knox RJ, Friedlos F, Jarman M, Roberts JJ. A new cytotoxic, DNA interstrand crosslinking agent, 5-(Aziridin-1-YL)-4-hydroxylamino-2-nitrobenzamide, is formed from 5-(Aziridin-1-YL)-2,4-dinitrobenzamide (CB 1954) by a nitroreductase enzyme in Walker carcinoma cells. Biochem Pharmacol . 1988;37(24):4661.
104 Knox RJ, Boland MP, Friedlos F, et al. The nitroreductase enzyme in Walker cells that activates 5-(Aziridin-1-YL)-2,4-dinitrobenzamide (CB 1954) to 5-(Aziridin-1-YL)-4-hydroxylamino-2-nitrobenzamide is a form of NAD(P)H dehydrogenase (quinone) (EC 1.6.99.2). Biochem Pharmacol . 1988;37(24):4671.
105 Patterson AV, Ferry DM, Edmunds SJ, et al. Mechanism of action and preclinical antitumor activity of the novel hypoxia-activated DNA cross-linking agent PR-104. Clin Cancer Res . 2007;13(13):3922.
106 Bunn H. Recombinant erythropoietin therapy in cancer patients. J Clin Oncol . 1990;8:949.
107 Vijayakumar S, Roach N, Wara W, et al. Effects of subcutaneous recombinant human erythropoietin in cancer patients receiving radiotherapy: Preliminary results of a randomized, open-label phase II trial. Int J Radiat Oncol Biol Phys . 1993;26:721.
108 Lavey RS, Dempsey WH. Erythropoietin increases hemoglobin in cancer patients during radiation therapy. Int J Radiat Oncol Biol Phys . 1993;27:1147.
109 Lavoy RS, Liu PY, Greer BE, et al. Recombinant human erythropoietin as a adjunct to radiation therapy and cisplatin for stage IIB-IVA carcinoma of the cervix. A Southwest Oncology Group Study. Gynecol Oncol . 2004;95(1):145.
110 Varlotto J, Stevenson MA. Anemia, tumor hypoxia and the cancer patient (Review). Int J Radiat Oncol Biol Phys . 2005;63(1):25.
111 Thomas G, Ali S, Hoevers FJ, et al. Phase III trial to evaluate the efficacy of maintaining hemoglobin levels above 12.0 g/dl with erythropoietin above 10.0 g/dl without erythropoietin in anemic patients receiving concurrent radiation and cisplatin for cervical cancer. Gynecol Oncol . 2008;108(2):317.
112 Jacobson GM, Kamth RS, Smith BJ, et al. Thromboembolic events in patients treated with definitive chemotherapy and radiation therapy for invasive cervical cancer. Gynecol Oncol . 2005;6(2):470.
113 Nordsmark M, Overgaard J. Tumor hypoxia is independent of hemoglobin and prognostic for loco-regional tumor control after primary radiotherapy in advanced head and neck cancer. Acta Oncol . 2004;43(4):396.
114 Yu NY, Brown MJ. Depletion of glutathione in vivo as a method of improving the therapeutic ratio of misonidazole and SR 2508. Int J Radiat Oncol Biol Phys . 1984;10(8):1265.
115 Mitchell JB, Cook JA, DeGraff W, et al. Glutathione modulation in cancer treatment: Will it work? Int J Radiat Oncol Biol Phys . 1989;16(5):1289.
116 Kramer RA, Soble M, Howes AE. The effect of glutathione (GSH) depletion in vivo by buthionine sulfoximine (BSO) in the radiosensitization SR2508. Int J Radiat Oncol Biol Phys . 1989;16(5):1325.
117 Allalunis-Turner MJ, Barrone GM, Day RS, et al. Heterogeneity in response to treatment with buthionine or sulfoximine or interferon in human malignant glioma cells. Int J Radiat Oncol Biol Phys . 1992;22:765.
118 Britten RA, Warenius HM, White R. BSO-induced reduction of glutathione levels increases the cellular radiosensitivity of drug-resistant human tumor cells. Int J Radiat Oncol Biol Phys . 1992;22:769.
119 Mitchell JB, Wink DA, DeGraff W, et al. Hypoxic mammalian cell radiosensitization by nitric oxide. Cancer Res . 1993;53:5845.
120 Bagsaw MA, Doggett RSL, Smith KC, et al. Intra-arterial 5-bromodeoxyuridine and x-ray therapy. Radiology . 1967;99:886.
121 Goffinet DR, Brown JM. Comparison of intravenous and intra-arterial pyrimidine infusion as a means of radiosensitizing tumors. Radiology . 1977;124:819.
122 Kinsella TJ, Collins J, Rowald J, et al. Pharmacology in phase I/II study of continuous infusions of iododeoxyuridine and hyperfractionated radiotherapy in patients with glioblastoma multiforme. J Clin Oncol . 1988;6:871.
123 Phillips TL, Levin VA, Ahn DK. Evaluation of bromodeoxyuridine in glioblastoma multiforme, a NCOG phase II study. Int J Radiat Oncol Biol Phys . 1991;21:709.
124 Prados M, Scott C, Sander H, et al. A phase III randomized study of radiotherapy with or without BUDR plus procarbazine, CCNU and Vincristine (PCV) for the treatment of anaplastic astrocytoma: A preliminary report of RTOG 94-04 radiation and BUDR in malignant gliomas. Int J Radiat Oncol Biol Phys . 1999;45(5):1109-1115.
125 Urtasun RC, Cosmatos D, DelRowe J, et al. Iododeoxyuridine (IUdR) combined with radiation in the treatment of malignant glioma: A comparison of short vs long intravenous dose schedules (RTOG86-12). Int J Radiat Oncol Biol Phys . 1993;27:207.
126 Yuhas MJ, Storer JB. Differential chemo protection of normal and malignant tissues. J Nat Cancer Inst . 1969;32:331.
127 Denekamp J, Rojas A, Stewart FA. Is radioprotection by WR-2731 restricted to normal tissues? In: Nygaard OF, Simic MG, editors. Radioprotectors and Anticarcinogens . New York: Academic Press; 1983:655.
128 Tresher M, Nitjmans LG, Shepman A, et al. Effects of modulating agent WR-2721 and its main metabolites on the formation and stability of cisplatin/DNA adducts in vitro. Bio Chem Pharmacol . 1992;33:1013.
129 Neta R, Oppenheim JJ, Douche S. Interdependence of the radioprotective effects of human recombinant interleukin 1 alpha, tumor necrosis factor alpha, granulocyte colony-stimulating factor, and murine recombinant granulocyte-macrophage colony-stimulating factor. J Immunol . 1988;140:108.
130 Phillips TL, Kane L, Utley JF. Radioprotection of tumor and normal tissues by thiol phosphate compounds. Cancer . 1973;32:528.
131 Milas L. Improving radiotherapy by reducing normal tissue damage with radioprotectors. Cancer Bull . 1986;38:223.
132 Millar JL, McElwain TJ, Clutterbuck RD, et al. Demodification of melphalan toxicity as single agent in tumor bearing mice by WR 2721. Am J Clin Oncol (CCT) . 1992;5:321.
133 Wasserman TH, Phillips TL, Ross G, et al. Differential protection against cytotoxic chemotherapeutic effects on bone marrow CFU’s by WR-2721. Cancer Clin Trials . 1981;4:3.
134 Peters GJ, Vanderwilt CL, Gyergyay F, et al. Protection by WR-2721 of the toxicity induced by the combination of cisplatin and 5-fluorouracil. Int J Radiat Oncol Biol Phys . 1992;22:785.
135 Washburn LC, Rafter JJ, Hayes RL:. Prediction of the effective radioprotective dose of WR-2721 in humans through an interspecies tissue distribution study. Radiat Res . 1976;66:100-101.
136 Kligerman MN, Glover DJ, Turrisi AT, et al. Toxicity of WR-2721 administered in single and multiple doses. Int J Radiat Oncol Biol Phys . 1984;10:1773.
137 Turrisi AT, Glover DJ, Hurwitz, et al. Final report of the phase I trial of single dose WR-2721. Can Treat RTTS . 1986;70:1389.
138 Coia L, Crigel R, Hanks G, et al. Phase I study WR-2721 in combination with total body radiation in patients with refractory lymphoid malignancies. Int J Rad Oncol Biol Phys . 1992;22:791.
139 Garces YI, Okuno SH, Schild SE, et al. Phase I North Central Cancer Treatment Group Trial—N9923 of escalating dose of twice daily thoracic radiation therapy with amifostine with alternating chemotherapy in limited stage small cell lung cancer. Int J Radiat Oncol Biol Phys . 2007;67(4):995.
140 Antonadu D, Coliarakis N, Synodinou M, et al. Randomized Phase III trial of radiation treatment +/− Amifostine in patients with advanced-stage lung cancer. Int J Radiat Oncol Biol Phys . 2001;51:915.
141 Komaki R, Lee JS, Kaplan B, et al. Randomized Phase III study of chemoradiation with or without Amifostine for patients with favorable performance status inoperable Stage II-III non-small cell lung cancer: Preliminary results. Semin Rad Oncol . 2002;12:S46.
142 Movsas B, Scott C, Langer C, et al. Phase III study of Amifostine in patients with locally advanced NSCLC receiving chemotherapy and hyperfractionated radiation. RTOG 98-01. Proc Am Soc Clin Oncol . 2003;22:636. (Abstract 2559)
143 Mell LK, Malik R, Komaki R, et al. Effect of Amifostine on response rates in locally advanced non-small cell lung cancer patients treated with unrandomized control trials: A meta-analysis. Int J Radiat Oncol Biol Phys . 2007;68(1):111.
144 Werner-Wasik M, Langer C, Movsas B. Amifostine in chemoradiation therapy for non-small cell lung cancer: Review of experience and design of a phase II trial assessing subcutaneous and intravenous bolus administration. Semin Oncol . 2005;32(Suppl 3):S105.
145 Law A, Kennedy T, Pellitteri P, et al. Efficacy and safety of subcutaneous Amifostine in minimizing radiation-induced toxicity in patients receiving combined-modality treatment for a squamous cell carcinoma of the head and neck. Int J Radiat Oncol Biol Phys . 2007;69(5):1361.
146 Koukourakis MI, Kyrias G, Kakolyris S, et al. Subcutaneous administration of Amifostine during fractionated radiotherapy: A randomized Phase II study. J Clin Oncol . 2000;18:2226.
147 Anne PR, Machtay M, Rosenthal DI, et al. A Phase II trial of subcutaneous amifostine and radiation therapy in patients with head and neck cancer. Int J Radiat Oncol Biol Phys . 2007;67(2):445.
148 Jellema AP, Slotmann BJ, Muller MJ, et al. Radiotherapy alone, versus radiotherapy with amifostine 3 times weekly, versus radiotherapy with amifostine 5 times weekly: A prospective randomized study in squamous cell head and neck cancer. Cancer . 2006;107(3):544.
149 Brizel DM, Wasserman TH, Henke M, et al. Phase III randomized trial of Amifostine as a radioprotector in head and neck cancer. J Clin Oncol . 2000;18:3339.
150 Wasserman TH, Brizel DM, Henke M, et al. Influence of IV Amifostine on xerostomia, tumor control and survival after radiotherapy for head and neck cancer: Two year follow-up of a prospective randomized phase III trial. Int J Radiat Oncol Biol Phys . 2005;63(4):985.
151 Rades D, Fehlauer F, Bajrovic A, et al. Serious adverse effects of amifostine during radiotherapy in head and neck cancer patients. Radiother Oncol . 2004;70(3):261.
152 Kouvaris JR, Kouloulias V, Malaise E, et al. Amifostine as radioprotective agent for the rectal mucosa during radiation of pelvic tumors. A Phase II randomized study using various toxicities scales and rectal sigmoidoscopy. Sonderb Z Strahlenther Onkol . 2003;179(3):167.
153 Small WJr. Cytoprotection/radioprotection with amifostine: Potential role in cervical cancer and early findings in the Radiation Therapy Oncology Group C-0116 Trial. Semin Oncol . 2003;30((6), Suppl 18):68.
154 Overgaard J. Does Amifostine have a role in chemoradiation treatment? Lancet . 2003;4:380.
155 Koukourakis MI. Amifostine: Is there evidence of tumor protection? Semin Oncol . 2003;30((6)Suppl 18):18.
156 Schuchter LM, Hensley ML, Meropol NJ, et al. 2002 Update of recommendations for the use of chemotherapy and radiotherapy protectants: Clinical practice guidelines of the American Society of Clinical Oncology. J Clin Oncol . 2002;20:2895.
157 Kouvaris JR, Kouloulias VE, Vlahos LJ. Amifostine: The first selective-target and broad-spectrum radioprotector. Oncologist . 2007;12:738.
158 Glick JH, Glover D, Weiler C, et al. Phase I controlled trials of WR-2721 and cyclophosphamide. Int J Radiat Oncol Biol Phys . 1984;10:1777.
159 Glover D, Glick JH, Weiler C, et al. Phase I/II trials of WR-2721 and cis-platinum. Int J Radiat Oncol Biol Phys . 1986;12:1509.
160 Glick J, Kemp J, Rose P, et al. A randomized trial of cyclophosphamide and cisplatin plus or minus WR-2721 in the treatment of advanced epithelial ovarian cancer. ASCO . 1992;11:122.
161 Muggia F, Parker R, Reed E, et al. WR-2721 pretreatment protects against the bone marrow toxicity of carboplatin and cisplatin. ASCO . 1992;11:132.
162 Luginbuhl W, Tester W, Shaw L, et al. One or two doses of WR-2721: Does it protect patients receiving carboplatin? ASCO . 1992;11:312.
163 Mitchell JB, DeGraff W, Kaufman D, et al. Inhibition of oxygen-dependent radiation-induced damage by nitroxide superoxide dismutase mimic, Tempol. Arch-Biochem Biophys . 1991;289(1):62.
164 Coleman CN, Mitchell JB. Radiation modifiers. In: Chabner BA, Longo DL, editors. Cancer chemotherapy and biotherapy: Principles and practice . 3. Lippincott Williams and Wilkins; 2001:707-751.
5 Prediction of Radiation Response

Adrian C. Begg, PhD
The prescription for clinical radiotherapy is usually based on such factors as tumor site, stage, and grade. All patients with tumors falling in the same category with regard to these clinical parameters receive the same radiation schedule, conforming to the institute’s current policy. An increasing body of evidence now shows that even for similar histologic type and extent of tumor, wide variations exist in the response to irradiation. For example, the fractionation scheme (e.g., conventional, accelerated, hyper- or hypo-fractionation) has been shown to influence outcome in clinical trials. 1 - 5 Total dose also influences outcome. It is therefore likely that a single radiotherapy prescription is not optimum for all patients, even those within the same clinical category. Some patients, but not all, may benefit from accelerated fractionation (depending on proliferation rate of the tumor), some but not all may benefit from hyperfractionation (depending on the shape of the survival curve of the tumor cells), some may require higher doses (radioresistant tumors), others may be overtreated with conventional doses (radiosensitive tumors), while for others, conventional radiotherapy of 1.8 to 2 Gy per day for 6 to 7 weeks will be the best choice.
In addition, many patients will also receive chemotherapy, before, during, or after radiotherapy. Some drugs are known to interact with radiation to produce synergistic cell kill, and some aren’t. Some tumors will be sensitive to a particular drug, either from cell killing or radiosensitization, and some won’t. And so, in addition to dose and fractionation, treatment choice also includes chemotherapy or not, as well as which drug. In making the correct choice, being able to predict accurately how the tumor and relevant normal tissues will respond to each treatment would be of great benefit. The goal of prediction is, therefore, to give each patient a tailored treatment so that improved local control and survival rates with reduced morbidity can be achieved for the patient population as a whole.
There has been rapid progress in the last decade in knowledge of the molecular pathways that are deregulated in tumors and that can affect response to treatment. This has been facilitated by a revolution in molecular biologic techniques. Genome-wide methods in particular have increased enormously in power and reliability, allowing DNA copy number, single nucleotide polymorphisms, gene expression, epigenetic changes, and others to be measured rapidly in individual tumor or normal tissue samples. These are increasingly the methods of choice for developing predictive assays for outcome after radiation therapy, and will be a major focus of this chapter. However, cell based and functional assays have provided us with important insights into factors affecting outcome, and the major findings of these assays are also summarized.
Genome-wide screening methods facilitate the search for “signatures” (genes, genetic loci, mutations) with the ability to separate patients with good and poor outcome independent of any underlying hypothesis. These are data-driven approaches. In addition, three main tumor parameters are known to influence outcome after radiation therapy: intrinsic radiosensitivity, the degree of tumor hypoxia, and the rate of repopulation of tumor cells. A fourth important factor is the radiosensitivity of normal tissues, which determines the dose that can safely be delivered. Testing specific signatures for these factors falls under the hypothesis-driven approach. Current studies on predictive assay development have focused on both data-driven and hypothesis-driven approaches. Any promising biologic predictor will ultimately need to be tested in a multivariate analysis with current known clinical predictors to show independence from them and the added value of the biologic assay.

Genetic Assays: Data-Driven Approach
With this approach, the aim is to find sets of genes or other genetic parameters that give the best discrimination between good and poor outcome. For gene expression, all known genes can be measured simultaneously. Statistical analyses are then employed to look for discriminating gene sets, taking no account of the function of the genes or their interrelationships. Analogous methods can be used for sets or patterns of genomic loci that are amplified or deleted, or for loci that are methylated or not, or for other genetic measurements. The rationale here is that we know that the genetic and epigenetic makeup of a tumor or normal tissue determines its behavior, but in most cases we don’t know all the genes involved or how they interact to produce the observed response to treatment. By restricting analyses to a few well-studied genes, important factors may be missed. The genome-wide approach should provide greater predictive discriminatory power than possible when using just one or a few genes, and in principle could lead to elucidation of response genes and pathways. The disadvantage is the high chance of false positives, and thus the need for large studies to both find and validate potential genetic signatures.

Tumors
The clinical value of microarrays was shown by studies in breast cancer, 6 , 7 lymphoma, 8 lung adenocarcinoma, 9 glioma, 10 and others that showed tumors could be subdivided into groups based on their gene expression profiles—and that these subdivisions have clinical relevance, since the different groups have different prognoses ( Fig. 5-1 ). In addition, the prognostic potential of microarrays was shown by van’t Veer and colleagues, 11 who defined a 70-gene signature that predicted the chance of distant metastases in young women with breast cancer. Several other signatures with the same utility have been found in subsequent studies, often containing different genes. 12 , 13 Of note is that these different signatures usually select the same patients as being at high or low risk, suggesting that many different gene sets may have similar prognostic potential for the same clinical situation and may represent different genes on common deregulated pathways. 14 - 16 In addition to distant metastases, it also appears to be possible to predict the chance of developing regional lymph node metastases in sites such as head and neck, breast, and others. 17 - 19

FIGURE 5-1 • Examples of the clinical value of gene expression profiling by microarrays. (A) Study by Sorlie and colleagues 7 on breast tumors showing that clusters defined by differences in expression patterns have significantly different outcomes. (B) Study by Garber et al. 9 showing a similar result for lung tumors.
(Redrawn from Sorlie T, Tibshirani R, Parker J, et al: Repeated observation of breast tumor subtypes in independent gene expression data sets, Proc Natl Acad Sci USA 100:8418–8423, 2003; Garber ME, Troyanskaya OG, Schluens K, et al: Diversity of gene expression in adenocarcinoma of the lung, Proc Natl Acad Sci USA 98:13784–13789, 2001.)
It is useful at this stage to distinguish between the terms prognostic and predictive. Several gene signatures have been described that appear to discriminate between good and poor outcome, but in a variety of disease sites and in patients given a variety of different treatments. Such signatures can be described as prognostic and probably reflect degree of malignancy. Examples are the “wound” signature, 20 hypoxic signatures, 21 genetic instability signatures, 22 and stem cell signatures. 23 By contrast, a signature that discriminates between good and bad responders to a specific treatment can be described as predictive. Such signatures can help in treatment selection, whereas prognostic signatures are inherently less useful. A predictive signature can also, in principle, help unravel causes of resistance, leading to potential new intervention strategies.
To date, very few predictive signatures have been described. Chung and colleagues 24 defined a 75-gene high risk signature for patients with head and neck cancer treated with primary surgery followed by radiation and/or chemotherapy. Pramana and colleagues 25 subsequently showed that this signature also predicted locoregional control in an independent series of head and neck cancer patients treated with radiotherapy and concomitant cisplatin ( Fig. 5-2 ). However, the specificity of this signature for radiotherapy, with or without chemotherapy, remains to be determined. Nuyten and colleagues 26 described a signature for predicting local recurrence after breast conserving therapy (local excision plus radiotherapy). Signatures have also been described that predict tamoxifen resistance in breast cancer. 27 , 28 Validated signatures predicting response to radiotherapy alone in cancer patients have not yet been reported.

FIGURE 5-2 • Chung et al. 24 defined an expression signature comprising 75 genes associated with high risk of relapse in head and neck cancer treated with primary surgery with or without subsequent treatment with radiation and/or chemotherapy. The signature significantly distinguished good and poor prognosis groups in an initial group of 28 patients (A) and in a subsequent group of 60 patients (B) . Pramana et al. 25 found this signature to predict locoregional control after concurrent radiotherapy and cisplatin in an independent group of 70 head and neck tumors (C) .
(Redrawn from Chung CH, Parker JS, Ely K, et al: Gene expression profiles identify epithelial-to-mesenchymal transition and activation of nuclear factor–kappaB signaling as characteristics of a high-risk head and neck squamous cell carcinoma, Cancer Res 66:8210–8218, 2006; Pramana J, Van den Brekel MW, van Velthuysen ML, et al: Gene expression profiling to predict outcome after chemoradiation in head and neck cancer, Int J Radiat Oncol Biol Phys 69:1544–1552, 2007.)
MicroRNAs (miRNA) also have been shown to have predictive or prognostic potential. These are small 18- to 22-nucleotide single-stranded RNAs. They are transcribed from genomic DNA like messenger RNA (mRNA), but do not code for proteins. Instead, they bind to partially complementary sequences on target messenger RNAs, thereby inhibiting translation and sometimes causing mRNA degradation. It is estimated that the expression of up to half of all genes are regulated by miRNAs. It is therefore not surprising that they have been shown to be involved in carcinogenesis and treatment response. Reports are now appearing of the predictive potential of miRNAs, 29 , 30 which may ultimately prove to be at least as powerful as mRNA profiling for prediction ( Fig. 5-3 ). To date, no study testing miRNA as a predictor of outcome after radiotherapy has been reported, although specific miRNAs have been shown to be induced by hypoxia and to correlate with outcome in breast cancer patients treated by surgery and adjuvant therapies. 29

FIGURE 5-3 • The prognostic potential of microRNAs (miRNA). Yu et al. 30 showed that a 5-gene miRNA signature distinguished good and poor outcome groups in non–small cell lung cancer in a training group of 56 patients (A) and in a test group of 56 patients (B). Camps et al. 29 showed that a single miRNA, hsa-miR-210, which is induced under hypoxia, could distinguish good and poor outcome in 219 breast cancer patients. Camps et al. showed that a single miRNA, hsa-miR-210, which is induced under hypoxia, could distinguish good and poor outcome in 219 breast cancer patients (C).
(Redrawn from Yu SL, Chen HY, Chang GC, et al: MicroRNA signature predicts survival and relapse in lung cancer, Cancer Cell13:48-57, 2008, and Camps C, Buffa FM, Colella S, et al: hsa-miR-210 Is induced by hypoxia and is an independent prognostic factor in breast cancer, Clin Cancer Res 14:1340-1348, 2008.)
Methylation of DNA in the so-called CpG islands in gene promoter regions can silence expression of those genes. Conversely, demethylation can activate transcription. Deregulation of methylation can thus lead to silencing of tumor suppressors or activation of oncogenes. Deregulation of genes involved in drug metabolism, the DNA damage response, and others can affect response to chemotherapy and radiotherapy. The most consistent result concerning prediction is in glioblastoma, where methylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter is associated with improved survival in patients treated with radiotherapy plus alkylating agents, particularly temozolamide. 31 , 32 The MGMT enzyme can reverse drug-induced DNA alkylation, thus reversing its cytotoxic effects. Gene methylation will reduce expression of this DNA repair gene, leading to increased tumor responses. Studies in other tumors have also shown correlations between methylation status of specific genes and outcome in colorectal cancer, 33 neuroblastoma, 34 and others. No clinical studies on radiotherapy alone have reported on methylation status. Several methods have been developed to measure DNA methylation, some of them approaching a genome-wide scale, and are now being increasingly applied to test their predictive potential.
Comparative genomic hybridization (CGH) measures copy number variations (CNV; amplifications and deletions) in regions of genomic DNA, currently at a 5 to 10 kilobase resolution. This method allows characterization of recurrent chromosome changes in tumors, which can pinpoint relevant known or novel oncogenes and suppressor genes. In addition, CNVs can be correlated with outcome to define CNV predictors, analogous to gene expression predictors. The resolution of current arrays is such that candidate genes in affected loci can be rapidly traced and tested further for their involvement if desired or necessary. Reports have appeared showing the prognostic potential of CNVs in several cancer types, including breast, 35 gliomas, 36 colorectal cancers, 37 Wilms’ tumor 38 and others ( Fig. 5-4 ). Few studies have been done on radiotherapy patients, although van den Broek and colleagues 39 reported finding specific gains and losses in head and neck cancers that correlated with outcome after treatment with combined chemoradiotherapy. It is likely that CGH will complement gene expression and other genome wide assays in defining robust predictors.

FIGURE 5-4 • The prognostic potential of comparative genomic hybridization (CGH). Idbaih et al. 36 defined three groups of gliomas based on their patterns of genomic amplifications and deletions that showed prognostic significance for overall survival (A) and progression free survival (B).
(Redrawn from Chin SF, Teschendorff AE, Marioni JC, et al: High-resolution aCGH and expression profiling identifies a novel genomic subtype of ER negative breast cancer, Genome Biol 8:R215, 2007.)

Normal Tissues
Predicting the chance of adverse normal tissue reactions after radiation would also be a valuable aid, allowing dose adjustments and/or protective measures (radioprotective or ameliorating drugs) to be prescribed to patients at risk. It further opens the possibility of increasing tumor radiation doses for the remainder (the majority) of patients, leading to potential increases in cure rates for the patient population as a whole. Several factors are known to affect the response of normal tissues to irradiation. The extent of cell kill of parenchymal cells in the organ at risk is clearly important but is not the only factor. Cytokines are known to play an important role, 40 , 41 TGF-β being a key cytokine in fibrosis development, influencing fibroblast proliferation and differentiation.
Attempts have been made to predict the chance of adverse reactions from the transcriptional response of patient’s lymphocytes irradiated ex vivo. 42 The underlying assumption is that the genetic profile of the patient will affect radiation response in all tissues and can therefore be monitored in easily accessible peripheral blood lymphocytes. The further assumption is that sensitivity is best monitored not necessarily by the basal level of gene expression but by transcriptional response to radiation. In the Svensson study, 42 an expression signature in ex vivo irradiated lymphocytes was identified that discriminated prostate cancer patients with severe late complications following radiotherapy (over-responders) from patients without such complications (nonresponders). Lymphocytes have also been used with some success for predicting radiation induced complications using colony survival 43 and apoptosis assays, 44 supporting the use of these cells for predictive purposes.
Gene polymorphisms have also been studied in relation to adverse radiotherapy induced reactions. Single-nucleotide polymorphisms (SNP) in several genes have been found to correlate with radiation induced fibrosis, 45 particularly in the TGF-β gene. Lymphocytes represent ideal test material, since they are easy to obtain and SNPs will be the same in all cells (in contrast to gene expression). SNP predictors of adverse reactions are not yet robust, because the studies carried out to date have been small and only a few candidate genes have been investigated. The method appears promising, but evaluation will have to await the results of several ongoing larger studies looking at genome-wide SNPs.

Genetic Assays: Hypothesis-Driven Approach
For radiotherapy, three main factors are known to influence outcome: intrinsic radiosensitivity of the tumor cells, the degree of tumor hypoxia, and the rate of tumor cell repopulation during treatment. Radioresistant cells, high hypoxia, and high repopulation capacity have all been associated with poor outcome. Attempts have therefore been made to create signatures characterizing each of these factors; genetic profiles of individual tumors could then be compared against each signature to estimate treatment sensitivity. For example, expression signatures have been defined for genes that are most up-regulated under hypoxia. Low or high expression of such genes in a particular tumor would indicate whether that tumor had low or high degree of hypoxia, respectively. Similar approaches can be used for radiosensitivity and repopulation capacity. The status of these signatures is described below.
Two approaches have been used. The first is a hybrid approach, using a data-driven strategy to define signatures for a particular biologic process (e.g., radiosensitivity) in the laboratory (e.g., on cell lines with varying radiosensitivities), followed by application of these signatures to human tumors. This second step is hypothesis-driven, the hypothesis being that the factor (intrinsic radiosensitivity) determines clinical outcome after radiotherapy and that the in vitro-derived signature is relevant to estimate the magnitude of the factor in a tumor.

Radiosensitivity
Human tumor cells in tissue culture exhibit wide variation in radiosensitivity despite being irradiated under standard conditions, indicating the presence of inherent genetic factors influencing the radiation response of mammalian cells. It is expected that tumors comprising cells inherently resistant to radiation will be more difficult to cure with radiotherapy than those comprising radiosensitive cells. It is also likely that patients with radiosensitive tumors may be overtreated by “conventional” radiotherapy, undergoing the unnecessary risk of excessive complications to normal tissue, while some radioresistant tumors are undertreated, and would benefit either from a higher dose, an added therapy, or an alternative therapy. The goal of predicting inherent sensitivity is thus to select out tumors at the extremes of the radiosensitivity spectrum for adjusted or alternative therapies, with the aim of improving cure rates of the population as a whole.
Many radiosensitivity genes have been discovered using knockout, knock-down or chemical inhibitor strategies. Many of these genes are involved in DNA repair. However, in recent years it has become clear that the DNA damage response is highly complex, so that predicting the extent of radiation induced killing for any random cell line or tumor has proven to be difficult. One approach has therefore been to use a genome-wide strategy, not dependent on known data or pathways, in which genetic signatures are sought that correlate with intrinsic radiosensitivity in a series of cell lines or tumors. Torres-Roca et al. 46 described such an approach on selected cell lines from the National Cancer Institute (NCI) panel and found a small set of genes correlating with radiosensitivity. More recently, Amundson and colleagues 47 extended the studies to the complete NCI panel of cell lines and found a larger set of genes correlating with intrinsic radiosensitivity, with some overlap with the Torres-Roca set. Khodarev and colleagues 48 used a different approach combining animal tumor and cell line studies, creating a radioresistant and radiosensitive pair of cell lines, to define a radiosensitivity signature. Little has so far been reported on testing radiosensitivity signatures. Pramana et al. 25 tested one of these signatures (Torres-Roca) on a group of head and neck cancer patients treated with radiation plus cisplatin, but no significant correlation was found. Recently, this signature has been independently refined, and the updated signature showed a strong trend ( P = .06) that patients with tumors predicted to be radiosensitive had a better outcome after chemoradiotherapy (Torres-Roca J, et al., presented at ASTRO, Boston, 2008).

Hypoxia
It has been known for more than half a century that hypoxic cells are up to three times more radioresistant than normoxic cells. In addition, hypoxic cells in tumors are often more slowly proliferating and harder to reach with drugs, because they often are at a distance from blood vessels, making them more chemoresistant. Studies using glass electrodes to measure oxygen tension in human tumors have shown, firstly, how ubiquitous hypoxia is, and secondly, that it is a negative prognostic factor for all the three current major treatment modalities of surgery, radiotherapy, and chemotherapy. 49 - 54
Cells react to hypoxia by reducing the expression of many genes. However, in contrast, there are a smaller number of genes that are up-regulated. Many of these are dependent on HIF-1α (hypoxia inducible factor), a protein that is stabilized under hypoxic conditions, leading to accumulation in the cell. HIF-1α, a transcription factor, then switches on transcription of a plethora of other genes that have an HRE (HIF responsive element, a specific short DNA sequence) in their promoter region. This allows the cell to adapt to hypoxic stress by, among others, increasing glucose uptake and stimulating angiogenesis. Such up-regulated genes represent a hypoxic signature. Several such signatures have been defined by growing cells under hypoxic conditions and subsequently measuring their expression profiles. Chi et al. 21 showed that such in vitro–defined signatures have prognostic significance and are more predictive of outcome (overall and relapse-free survival) in breast and ovarian cancer than present clinical parameters. As expected, patients with tumors showing high expression of hypoxia genes, indicating high tumor hypoxia, did worse.
The cell’s response to hypoxia also depends on the degree and duration of hypoxia. More severe hypoxia and longer times under hypoxia are reflected by altered gene expression patterns. Signatures have therefore been described for both acute and chronic hypoxia, and for different degrees of hypoxia. 21 , 55 These are potentially important distinctions, since there is accumulating evidence that acute hypoxia is more dangerous than chronic hypoxia (equally radioresistant, more viable, more DNA repair–proficient). 56 , 57 Indeed, Seigneuric and colleagues 55 showed that in vitro signatures derived from short exposures to hypoxia (acute) predicted outcome in breast cancers whereas those derived from longer exposures (chronic) did not ( Fig. 5-5 ).

FIGURE 5-5 • The prognostic potential of gene expression signatures for hypoxia. Seigneuric et al. 55 analyzed cell line data to define signatures for genes up-regulated at early times during hypoxic incubation (“early”; acute hypoxia) or after long times (“late long”) chronic hypoxia. Acute signatures (A) had greater potential to distinguish good and poor prognosis in breast cancer patients than chronic signatures (B).
(From Seigneuric R, Starmans MH, Fung G, et al: Impact of supervised gene signatures of early hypoxia on patient survival, Radiother Oncol 83:374-382, 2007.)
An alternative method of deriving hypoxic signatures was described by Winter et al. 58 They first chose 10 genes known to be HIF-1α (and thus hypoxia)–dependent. They then looked for other genes whose expression correlated with expression of these 10 “seed” genes across a series of human head and neck carcinomas. In this way they defined a hypoxic metagene of 99 genes. This metagene had prognostic value in both head and neck and breast cancer, supporting the notion that hypoxia is an important negative prognostic factor and that gene signatures can be used to monitor it.

Repopulation
During the course of fractionated radiotherapy, tumor cells that have survived (remained clonogenic) up to that point can begin to divide and will increase the number of cells to be killed with the remaining dose fractions. Such tumor cell repopulation is a particular risk during weekends and other gaps in therapy. Cell kill by radiotherapy is thus counteracted by cell production (repopulation) in the treatment gaps. The greater the capacity of a tumor for repopulation, the greater the effective resistance of that tumor will be. Further, the more gaps in treatment there are, the greater the opportunity will be for repopulation. Evidence for this is strongest in head and neck cancer where a number of studies on the influence of treatment time, including split dose and accelerated fractionation, have consistently shown either worse local control if the treatment time is longer, or that higher radiation doses are needed for a given level of local control. 1 - 4 This is most likely to be due to repopulation, which can therefore limit cure in some cancers. The influence of repopulation will vary between cancers of the same type, and between different types of cancer. Cancers that are in general more slowly growing, (e.g., breast and prostate), may be at less risk from repopulation, although some tumors within these types may be capable of rapid repopulation. It is therefore important to be able to predict repopulation in individual tumors.
Great strides have been made in understanding the molecular events driving and accompanying cell proliferation. Cell culture studies have employed cell populations synchronized in different cell cycle phases, as well as resting cells stimulated to proliferate by serum addition. These have defined a host of cycle phase–specific and proliferation-specific genes. Such proliferation-associated gene signatures include genes that regulate cell cycle progression, such as the cyclin-dependent kinases, and the many external and signal transduction pathways that control entry into and exit from the cell cycle, including cytokines, hormones, growth and antigrowth factors, checkpoints, cell adhesion, and others. Some of these signatures have been shown to have prognostic potential for outcome of breast cancer. 58a In addition, using data-driven approaches, several expression-profiling studies on clinical material have found gene signatures correlating with outcome that contain a preponderance of proliferation-associated genes. Such studies include those on lymphoma, 59 , 60 breast cancer, 61 hepatocellular carcinoma, 62 and others. 63 These signatures, and those derived from cell culture studies, have thus been shown to have prognostic potential. These have not yet been tested on patients receiving curative radiotherapy as the primary treatment.

Validation of Genetic Signatures
Genetic signatures must be validated before clinical application. This is especially so for genome-wide measurements, where the possibility of false positives is high. During development of a signature on a clinical series (the training set), internal validation is usually performed (e.g., leave-one-out cross-validation). This is the first of several steps. In the case of the van’t Veer et al. 70-gene signature for predicting metastasis in women with breast cancer, 11 this was defined on a test series of 78 patients, was subsequently validated on a larger series of patients, 64 and is currently being tested in a large randomized trial. 65 - 68 The progression from definition in a training set to validation in a separate clinical series followed by testing in a randomized trial is a route now being followed for several promising gene signatures in different diseases and therapies. 69 These are necessary steps before routine application of such signatures in the clinic.

Genetic Assay Considerations

Multiple Causes of Resistance
The previous discussion assumes that one gene signature will be sufficient for predicting outcome after a specific treatment such as radiotherapy. This may be too simplistic. Radiotherapy may fail for several reasons: for example, because the tumor is too hypoxia, too radioresistant, or repopulates too rapidly. Furthermore, if it is too intrinsically radioresistant, this may result from increased double-strand break (DSB) repair via homologous recombination, from nonhomologous endjoining, or through down-regulation of apoptosis pathways. Given these multiple reasons for failure, one genetic signature may not be predictive for all tumors. Future developments may therefore include testing each tumor with several signatures, each with proven efficacy and specificity for a biologic process associated with resistance. In this way, the cause of potential resistance in an individual patient can be estimated, leading to an optimum treatment choice.

Combining Assays
No single genome wide assay is likely to be perfect for predictive purposes. For mRNA expression profiling, the most widely used to date, a potential deficiency is that mRNA expression often does not correlate with protein expression, that one would ultimately like to monitor. Nor does it measure posttranslational modifications of proteins, which are central to transmission of signals within the cell, and therefore important for all aspects of cell behavior, including response to damage. Despite this, expression signatures are clearly successful at discriminating cell types, tissue types, tumor subtypes and show promise as response predictors.
At the DNA level, amplifications and deletions detected by CGH do not indicate which genes on these genomic loci are the most important, although the increased resolution of current CGH arrays coupled with the availability of complete DNA sequencing of the genome have facilitated the tracing of relevant genes. A second potential confounding factor is that amplifications do not always correlate with increased gene expression. However, several CGH signatures have been shown to have prognostic potential in a variety of cancers, although all need further validation. Methylation profiles indicate which genes are deregulated in cancer, affecting gene expression. Methylation assays are now becoming genome wide, but whether they will provide additional information to gene expression assays remains to be tested. This may depend on the extent of methylation at a given locus.
Given the potential deficiencies of individual assays, a combination of assays may prove superior. Indeed, combining CGH data with expression profiling allowed Adler and colleagues to separate relevant from irrelevant genetic changes and discover the most important genes driving the “wound” signature, a set of genes with prognostic significance in breast cancer. 70 Other studies have also shown the added value of combining expression profiling with CGH 35 , 71 for defining better predictors and better subclassifications of tumors. Combining three approaches (CGH, methylation, and mRNA expression) is now possible, as shown by Sadikovic and colleagues, 72 providing a more complete picture of genetic and epigenetic changes.

Stem Cells
Another important consideration for predictive assays concerns stem cells. There is good evidence that a small proportion of tumor cells are stem cells and are the only important cells that need to be eradicated for tumor control. In addition, there is evidence that they may be more resistant to treatment by both radiation and drugs than bulk tumor cells. Furthermore, it is also possible, if not likely, that their genetic profiles differ, particularly their gene expression patterns. The relevance of measuring expression in bulk tumor cells can therefore be called into question. This problem is likely to be more important for predicting response where the minority stem cell fraction is crucial than for tumor subtyping, where bulk tumor also defines the subclass. The extent of this problem is as yet unknown, since some prediction success has already been obtained by measuring genetic parameters on bulk tumor. However, it is possible that better predictors can be found by isolating and measuring only cancer stem cells. This awaits better technical procedures and understanding of stem cell characteristics.

Cell Based and Functional Assays
Ideally, functional assays are the preferred way of measuring factors affecting response, since they can provide the most direct and relevant estimates. Such assays have been developed for the three main factors determining outcome after fractionated radiotherapy. Disadvantages of such assays include their technical difficulty, the time required to obtain a result, and the difficulty of combining assays for more than one factor, making many of them difficult to apply in a routine clinical setting. However, studies using these assays have provided valuable information on the importance of each factor.

Radiosensitivity
The most relevant measure of radiosensitivity is based on the fraction of cells surviving a particular radiation dose, defined as the ability of a cell to undergo at least six doublings, thus forming a clone of at least 50 cells. This is termed the colony-forming, or clonogenic, assay. A summary of predictive assays studies for radiosensitivity is shown in Table 5-1 . The most convincing study is that of West and colleagues 73 , 74 on cervix carcinomas treated by radiotherapy alone. Explanted tumor cells irradiated in vitro had SF 2 values that correlated with outcome. Patients with tumors exhibiting SF 2 values higher than the median value (radioresistant) had significantly worse local control and significantly worse survival rates than did those with tumors with SF 2 values below the median. This trend was the same for all tumor stages. Two of the larger studies on head and neck tumors also showed a positive correlation of in vitro radiosensitivity with local control. 75 , 76 These clinical studies support the notion that in vitro measurements of radiosensitivity, with all their potential limitations, have relevance to the response of tumors in situ.

Table 5-1 Clinical Studies Correlating Outcome After Radiotherapy with Intrinsic Radiosensitivity Measured on Cells Taken from Primary Tumor Material Before Treatment and Irradiated and Assayed in Vitro
Although these results are encouraging, it is unlikely that either the colony assay or similar assays could be used as predictors for routine clinical application, because they take several weeks to complete—unacceptably long for many radiotherapy departments. They also require a highly skilled laboratory team with extensive experience. Other assays have therefore been sought that are more rapid and more suitable for a routine clinical laboratory. These alternative assays are indirect, measuring parameters that have shown correlations with cell kill. DNA DSBs are thought to be the most important and toxic DNA lesion after irradiation. Techniques for their measurement can be completed within a few days rather than the few weeks necessary for colony assays. These include gel electrophoresis and, more recently, antibody detection of a nuclear histone protein that becomes phosphorylated at DSB sites, called γH2AX. The latter provides a method of measuring breaks in tumors irradiated in situ. However, results of studies correlating DSB induction or repair with cell kill have been variable, suggesting that DSBs will not be a reliable predictor of radiation-induced cell kill.
Ionizing radiation also induces chromosome aberrations, including fragments, translocations (dicentrics or reciprocal), rings, chromatid exchanges, gaps, complex types, and micronuclei, all being dose related. Many studies have shown a good correlation between chromosome damage and cell kill. 77 - 79 Chromosome aberrations can also be measured in a matter of days and can be detected in cells after repair with doses less than 1 Gy, making it as sensitive as γH2AX detection of DSBs. However, although chromosome damage assays have a lot of attractive features as radiosensitivity predictors, ex vivo culturing and irradiations would be required, making it unlikely to prove robust enough for routine clinical use.

Repopulation
Here the goal is to predict, before treatment begins, which tumors are capable of rapid proliferation during treatment. These could be then selected for adjusted radiotherapy schedules or alternative or extra-treatment modalities. Several methods have been tried, including simply counting the frequency of mitoses. Flow cytometry has advantages over counting cells under a microscope, allowing the quantitative measurements of many thousands of cells per minute. By using fluorescent dyes that bind to DNA, DNA histograms can be generated and analyzed for the fraction of cells in each cycle phase (G 1 , S, and G 2 /M). A more functional assessment of proliferation can be obtained using analogs of thymidine, bromodeoxyuridine (BrdU), and iododeoxyuridine (IdU), which are incorporated into DNA during the S phase. Fluorescent conjugated antibodies allow the degree of analog incorporation per cell to be rapidly measured by flow cytometry. Cell kinetics can be measured in patients with this method by injection or infusion of thymidine analogs at nontoxic tracer doses. The combination of thymidine analogs and flow cytometry allows rapid measurement of the proportion of labeled cells (labeling index [LI]). In addition, by taking samples a few hours after analog administration, one can determine both the LI and the rate of movement through the S phase ( T S ). 80 The ratio of the two ( T S /LI) approximates the potential doubling time, T pot , a parameter describing the cell number doubling time of a tumor population in the absence of cell loss. Staining and measuring can be accomplished in 1 day. Disadvantages include the necessity of administering a drug (the thymidine analog) and the inability to reliably distinguish malignant from non-malignant cells in a biopsy.
A multicenter study from 11 different centers was carried out for head and neck tumor patients receiving radiotherapy alone given in an overall time of at least 6 weeks, with a total of 476 patients. 81 All patients received the thymidine analog prior to treatment. A univariate analysis showed that only LI was significantly associated with local control ( P < .03), higher values correlating with worse outcome. T pot showed no trend. In a multivariate analysis of local control, LI lost its significance ( P < .16). Two potential confounding factors in this study were that each center carried out its own flow cytometry and analysis (rather than a standard reference center), and in none of these analyses was an adequate distinction made between normal and malignant cells. This study suggests that LI but not T pot may predict repopulation during radiotherapy, but not strongly.
Finding a proliferation marker that does not require administration of a potentially toxic substance remains a worthwhile goal. Such markers include antibodies to Ki67 (cycle-specific), PCNA (S phase–specific), 82 cyclin A (S/G2 phase–specific) 83 and DNA polymerase alpha (cycle-specific). 84 These all provide static parameters and can be measured by either immunohistochemistry or flow cytometry. The rapidly increasing knowledge of cell cycle control gives hope that expression profiles will be found that can predict repopulation capacity.
Ideally, measurements of proliferation during, and not before, treatment are desired, since this is when the dangerous repopulation takes place. Labeling measurements can be made during treatment, but the data will be strongly dominated by doomed and dying cells that constitute the vast majority of cells after the first few 2 Gy fractions. Such measurements are therefore likely to be misleading, as shown by animal studies. 85 Until ways can be found to distinguish doomed but intact cells from surviving cells, measurements during treatment will remain unreliable at best, and often come too late to change treatment.
In summary, many of the studies mentioned above have indicated the relevance and importance of predicting tumor proliferation for radiotherapy schedules 6 weeks or longer. Better methods and better knowledge of the biology (e.g., role of cytokines and receptors in irradiated tissue) are now needed. Genome-wide assays (see above) are proving promising for achieving these goals.

Hypoxia
The most direct method to date for measuring tumor hypoxia is the use of glass oxygen electrodes inserted into the tumor. Multiple measurements can then be taken along several tracks, allowing the distribution of oxygen tension to be assessed. The mean or median oxygen tension can be calculated, as well as the fraction of values below a cut-off, usually 5 or 10 mm Hg, giving an estimate of the hypoxic fraction. Several studies have correlated such measurements with outcome after radiotherapy. 49 - 53 These studies have shown remarkable uniformity in that most, but not all, found that pretreatment oxygen tension was a significant prognostic indicator. These included different tumor sites and all three major treatment modalities, although no sufficiently large series have been published for surgery alone or chemotherapy alone. Hypoxia could affect chemotherapy outcome through lower drug concentrations at hypoxic sites, and the fact that hypoxic cells tend to proliferate slower, reducing the effectiveness of many drugs. Exposure to hypoxia can also lead to selection of apoptosis-resistant cells, 86 and consequently to malignant progression and an increase in metastatic capacity. 87 , 88 These may be contributing reasons why hypoxia is also a bad prognostic indicator for surgery. A major disadvantage of electrode methods is its invasive nature and its restriction to accessible tumors.
One of the current most widely used alternative methods to electrodes is the administration of a bioreductive drug, in particular, the nitroimidazoles. Such drugs have been shown to be selectively reduced in and bind to hypoxic cells and can be detected with a labeled drug or by antibodies developed against bound products. 89 , 90 Two of these nitroimadazoles, pimonidazole and EF5, are approved for human use as hypoxic markers. 90 , 91 Of interest is that the pimonidazole staining fraction in head and neck tumors does not appear to correlate with electrode oxygen measurements in the same tumors. 92 Possible reasons include the influence of stroma and necrosis on the polarographic measurements, or that one method may be more influenced than the other by acute (fluctuating) hypoxia. Of further interest is that the only study measuring both pimonidazole staining fraction and oxygen tension with Eppendorf electrodes found that neither parameter correlated with outcome in cervix cancer patients treated with radiotherapy alone. 93
Several other methods, both direct and indirect, have also been applied in the clinic for measuring tumor hypoxia. 54 These include noninvasive assessment of hypoxia using the imaging techniques of PET or SPECT or with MRI, and measuring expression of endogenous markers associated with hypoxia, such as HIF-1α and CA9 (see discussion of genetic assays above). Few studies with sufficient statistical power have yet been carried out to test the predictive potential of these techniques for radiotherapy patients. Finally, it should be noted that none of the methods can distinguish between clonogenic and nonclonogenic cells. Extrapolation from changes in the measured hypoxia parameter occurring during treatment to reoxygenation patterns of the hypoxic, clonogenic cells therefore cannot be made with any degree of certainty.
These data collectively imply that hypoxia can limit cure of cancers by radiotherapy and other modalities in at least three cancer sites. Pretreatment hypoxia measurements with oxygen electrodes have shown the best, although not universal, prognostic significance. Results with exogenous markers (pimonidazole, EF5, and others) and endogenous markers (HIF1α, CA9, and others) have shown mixed results as predictors and do not yet appear to be robust. The ability to predict outcome based on hypoxia measurements is therefore, as yet, suboptimal, and improvements will require better knowledge of which type of hypoxia is important (e.g., acute or chronic) and what each technique is measuring. It remains to be seen whether hypoxia signatures derived from genome wide studies (see above) prove to be more reliable predictors and better indicators of how to treat.

Normal Tissues
Several studies have tested the relationship between the in vitro radiosensitivity of either fibroblasts or lymphocytes and the severity of normal tissue reactions. Geara et al. 94 and Johansen and colleagues 95 found a significant correlation between fibroblast radiosensitivity and late reactions. These and other studies indicated that colony survival of fibroblasts after in vitro irradiation may predict for late normal tissue damage, primarily fibrosis. However, two subsequent larger studies could not confirm these results. 96 , 97 In vitro radiosensitivity of lymphocytes, measured either by colony, cytogenetic or apoptosis assays, have been reported to predict normal tissue morbidity in some studies. 43 , 44 , 98
Problems with cell-based assays include their technical difficulty and the long assay times. Clinical confounding factors include an often inaccurate estimate of dose in the target tissue. 99 While cell based assays have been useful in showing that intrinsic radiosensitivity of somatic cells is probably a contributing cause to differences observed between patients in their reactions to radiotherapy, it is unlikely that they will be routinely useful as predictors for reasons stated above. In addition, intrinsic radiosensitivity is not the only determinant of radiation morbidity. There are a number of biologic factors that influence treatment response. For several tissues (e.g., lung, skin, and intestinal mucosa), the involvement of cytokine-mediated multicellular interactions are implicated, including those mediated by interleukins 2 and 6 (IL-2, IL-6), and interferon alpha (IFN-α). 100 Transforming growth factor beta (TGF-β) clearly also plays an important role in generating and modulating tissue fibrosis in many tissues and organs. 40 , 41 , 101 Understanding the mechanisms of normal tissue radiation response other than the conventional radiobiologic paradigm of target cell death will ultimately lead to better prediction.
Analogous to tumors, response of normal tissues to radiation will be determined by multiple factors. With enough knowledge of the relevant genes and pathways, looking at expression or polymorphisms in a far wider range of genes than is now being done may provide a viable approach to predicting normal tissue morbidity (see above). Large microarray studies, analogous to those in tumors, have not yet been reported.

Action Based on Assay Results
The obvious question concerning the use of predictive assays is what action should be taken based on the assay result? It should be emphasized that prospective trials should be done only after an assay or assays have been sufficiently validated in retrospective trials, and shown to provide additional and better information than that provided by present clinical predictors. This has so far been done with very few of the assays described. However, if assays for intrinsic radiosensitivity, proliferation and hypoxia were validated and made sufficiently reliable and simple to use routinely, how should they influence the choice of treatment?
For rapidly repopulating tumors, accelerated radiotherapy (shorter overall treatment time) is the obvious choice to minimize the number of possible cell divisions. Treatments shorter than 4 to 5 weeks have to be accompanied by a dose reduction to reduce the chance of unacceptable early reactions in proliferating normal tissues such as buccal mucosa. Slowly proliferating tumors would be disadvantaged by any dose reduction accompanying acceleration and could therefore be treated with conventional schedules, with or without concomitant chemotherapy (depending on institute policy), or with hyperfractionation to effectively increase the tumor dose. In addition, if the molecular cause of the rapid proliferation is indicated by the predictor (for example, overexpression of a growth factor receptor), drugs specifically targeting that receptor—of which there are now an increasing number—could be used in combination with radiation.
Several options are available for high hypoxic fraction tumors. The main current ones are the use of a chemical hypoxic cell radiosensitizer such as nimorazole, 102 or applying carbogen (increases blood oxygen) with or without nicotinamide (counteracts acute hypoxia), which is also undergoing clinical testing. 103 An alternative approach would be to selectively kill the hypoxic cells using a bioreductive agent such as tirapazamine. This promising approach is also undergoing clinical testing. 104 Future possibilities include the delivery of gene-encoded toxins coupled to hypoxia-specific promoters and the use of anaerobic bacteria as tumor (hypoxia)-specific delivery vectors. 105 - 107
The question is more complicated for intrinsic radiosensitivity. If the tumor is radiosensitive, it is likely that conventional radiotherapy (e.g., 1.8 to 2.0 Gy per fraction, 60 to 70 Gy total) will be successful. If the tumor is resistant, adjuvant treatments could be considered, or highly conformal radiotherapy (allowing an increased dose to the tumor). Changing the fractionation scheme is also a possibility, although information on survival curve shape on which to base such a change is usually not available. If the tumor is extremely radioresistant, radiotherapy may not be the best treatment choice and an alternative modality should be considered. If gene signatures are used for prediction, these may give a clue to the cause of resistance, such as a particular DNA repair pathway, signal transduction pathway, or cell death pathway being deregulated. An increasing number of drugs have been developed or are being developed with specificity for many such pathways, and it is the hope for the future that knowledge of causes of resistance will allow the best drug to be chosen in combination with radiation in an individual patient to achieve the optimal chance of cure.
For normal tissues, lower total doses could be considered for highly radiosensitive patients and somewhat increased doses for highly resistant patients. It should be emphasized that the assays must be proven to be reliable if treatment choices are to be based on them. There are also methods being developed for treating late reactions, such as fibrosis, and patients predicted to be radiosensitive could be monitored more closely and offered these treatments, as they become available, as soon as adverse side effects become apparent.

The Future
Many functional assays tested to date have shown some significant correlations with outcome. Examples include SF 2 for radiosensitivity and oxygen electrode determinations for hypoxia. However, most of these assays have not proved robust, practical, or successful enough for use as routine assays on a wide scale. This is particularly true for cell-based assays (colony assays of tumor cells, fibroblasts or lymphocytes, chromosome damage assays). In addition, a major disadvantage is that most of the assays are limited to measuring one factor. This means that other biologic factors known to affect outcome remain unexplored. Applying multiple different assays to one patient is often impractical or impossible because of burden to the patient and the limited amount of tumor material available. Genome-wide assays can overcome these problems.
An important consideration, in addition to whether an assay is a reliable predictor, is whether it is biologically informative. For example, if a tumor were found to have a high SF 2 , this does not indicate why that tumor is radioresistant. The choice of therapy therefore remains somewhat arbitrary (although the patient could be considered for more aggressive or alternative treatments). The information gained in studies applying such a predictor is therefore limited and will not ultimately provide a greater understanding of the response of tumors to therapy. What is needed for better prediction and ultimately improving therapy is a greater knowledge of the biology governing treatment response. These should be coupled with methods to measure rapidly and accurately what the dominant deregulated pathways are in any given tumor giving rise to resistance.
Genome-wide assays will play an increasingly major role, having the dual advantages of measuring multiple biologic characteristics, as well as providing direct or indirect clues as to what genes are important in determining response. This will in turn provide potential leads for drug development and thus eventual therapy improvements. Increasing numbers of signatures will be found showing predictive potential, at the DNA level (SNP, CGH, methylation, mutations) and the RNA level (messenger and micro RNA expression). Combining information from more one more genome-wide assays will help discriminate relevant from irrelevant changes, with a consequent increase in predictive power and knowledge of the important response pathways. Proteomic methods are becoming more high-throughput and powerful, although the study of proteins, with their inherently variability, is an order of magnitude more complex and difficult than for DNA or RNA. It is the proteome, however, that ultimately defines the cell’s behavior. Therefore, despite the complexity, it is expected that proteomic methods such as mass spectrometry, antibody arrays, and others will soon complement or even replace other assays. Lastly, DNA sequencing is now becoming extremely powerful and rapid, such that it could be envisioned that each tumor could undergo full sequencing to characterize its genome, including mutations and SNPs. This would in principle be feasible in some large centers. It is also probable that signatures will be refined and reduced to just the essential most predictive genes or loci. This will open up the possibility of replacing genome wide methods for routine use with more practical, cheap and widely applicable assays such as immunohistochemistry, and perhaps more quantitative assays such as polymerase chain reaction (PCR)-based methods.
For normal tissue response prediction, as with tumor response prediction, cell-based assays hold little promise for routine use, despite having provided useful information in the past. Future efforts will depend on progress in understanding the fundamental biology of radiation pathogenesis. For example, radiation can stimulate cytokine release from a variety of cells, including endothelial cells, leading to increased vascular permeability, increased platelet adhesion, increased leukocyte adhesion, and invasion. These can in turn lead to short- and long-term disturbances in normal tissue function resulting from vascular damage. Prediction of specific types of normal tissue damage will therefore require more sophisticated tests in the future than colony-forming ability. High throughput array technology will certainly help here (SNPs, CNVs, methylation, mRNA expression). The question remains whether this can be done in a surrogate tissue such as peripheral blood lymphocytes or whether tissues at risk need to be tested.
Finally, accurate prediction will only be really useful when it not only indicates whether the tumor in an individual patient will be resistant to radiotherapy or other therapies, but when it can also guide the physician in choosing the best therapy. This will depend on the availability of pathway-specific drugs. An optimum predictor should then indicate which radioresistance pathway is activated or deregulated so that the appropriate drug inhibiting that pathway can be chosen for combining with radiotherapy. Indications from studies on hypoxia, DNA repair, growth receptor signaling, proliferation, and others suggest that this will be possible in the medium-term future. At the time of writing, there are as yet no validated markers or signatures for predicting response to available pathway specific agents in combination with radiotherapy, 108 and this must come through integrating marker assays into clinical trials, preferably those where the effectiveness of such agents are specifically being tested within the trial.

Summary
The four main factors likely to be relevant for predicting outcome after radiation therapy are intrinsic tumor cell radiosensitivity, normal tissue radiosensitivity, tumor hypoxia, and tumor cell proliferation. Positive correlations with outcome have been reported for all four parameters, measured by clonogenic assays, oxygen electrodes, and BrdU/IdU-flow cytometry, although variability in results have been seen between studies for all parameters. Problems with these functional assays include reproducibility difficulties, long assays times, the necessity for ex vivo cell cultures, the need for invasive procedures, addition of tracer drugs, and the difficulty of carrying out more than one assay per patient. These have led to the use of genome-wide assays, in principle allowing multiple factors to be assessed simultaneously, as well as indicating which genes and pathways cause resistance. Gene expression, SNP, CGH, microRNA, methylation, and others have all shown promise as predictors, as well as some assay combinations. None have yet been validated fully in randomized trials, although several are in this phase of testing. Many prognostic signatures have been described, but few predictive signatures have been reported that are specific for a particular therapy, such as radiotherapy. Such therapy-specific predictors will be the most useful for choosing optimum treatments for individuals and are achieving increasingly more attention for both chemotherapy and radiotherapy. On a final note, it should not be forgotten that one of the most robust predictors of outcome of radiotherapy is the size of the tumor ( Fig. 5-6 ). Measuring tumor volume dose not indicate the way ahead for biologists or clinicians in the future, but it should always be taken into account, as with other clinical factors of proven importance, when assessing potential predictive assays.

FIGURE 5-6 • Examples of studies showing that one of the most important clinical predictors is tumor volume. (A) Study of Kim et al. 109 on 106 cervix cancer patients treated with concurrent chemotherapy and radiotherapy. (B) Study of Begg et al. 80 on head and neck cancer patients treated with radiotherapy alone (numbers against curves are tumor diameters).
( A, Redrawn from Coco Martin JM, Mooren E, Ottenheim C, et al: Potential of radiation-induced chromosome aberrations to predict radiosensitivity in human tumour cells, Int J Radiat Biol 75:1161–1168, 1999; Liu SC, Minton NP, Giaccia AJ, et al: Anticancer efficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis, Gene Ther 9:291–296, 2002. B, Reprinted by permission from Macmillan Publishers Ltd, Gene Ther 9:291–296, 2002.)

References

1 Overgaard J, Hjelm-Hansen M, Johansen LV, et al. Comparison of conventional and split-course radiotherapy as primary treatment in carcinoma of the larynx. Acta Oncol . 1988;27:147-152.
2 Overgaard J, Hansen HS, Specht L, et al. Five compared with six fractions per week of conventional radiotherapy of squamous-cell carcinoma of head and neck: DAHANCA 6 and 7 randomised controlled trial. Lancet . 2003;362:933-940.
3 Fu KK, Pajak TF, Trotti A, et al. A Radiation Therapy Oncology Group (RTOG) phase III randomized study to compare hyperfractionation and two variants of accelerated fractionation to standard fractionation radiotherapy for head and neck squamous cell carcinomas: first report of RTOG 9003. Int J Radiat Oncol Biol Phys . 2000;48:7-16.
4 Bourhis J, Overgaard J, Audry H, et al. Hyperfractionated or accelerated radiotherapy in head and neck cancer: a meta-analysis. Lancet . 2006;368:843-854.
5 Skladowski K, Maciejewski B, Golen M, et al. Continuous accelerated 7-days-a-week radiotherapy for head-and-neck cancer: long-term results of phase III clinical trial. Int J Radiat Oncol Biol Phys . 2006;66:706-713.
6 Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature . 2000;406:747-752.
7 Sorlie T, Tibshirani R, Parker J, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA . 2003;100:8418-8423.
8 Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature . 2000;403:503-511.
9 Garber ME, Troyanskaya OG, Schluens K, et al. Diversity of gene expression in adenocarcinoma of the lung. Proc Natl Acad Sci USA . 2001;98:13784-13789.
10 Phillips HS, Kharbanda S, Chen R, et al. Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell . 2006;9:157-173.
11 van’t Veer LJ, Dai H, van de Vijver MJ, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature . 2002;415:530-536.
12 Wang HP, Long XH, Sun ZZ, et al. Identification of differentially transcribed genes in human lymphoblastoid cells irradiated with 0.5 Gy of gamma-ray and the involvement of low dose radiation inducible CHD6 gene in cell proliferation and radiosensitivity. Int J Radiat Biol . 2006;82:181-190.
13 Foekens JA, Atkins D, Zhang Y, et al. Multicenter validation of a gene expression-based prognostic signature in lymph node-negative primary breast cancer. J Clin Oncol . 2006;24:1665-1671.
14 Fan C, Oh DS, Wessels L, et al. Concordance among gene-expression-based predictors for breast cancer. N Engl J Med . 2006;355:560-569.
15 Yu JX, Sieuwerts AM, Zhang Y, et al. Pathway analysis of gene signatures predicting metastasis of node-negative primary breast cancer. BMC Cancer . 2007;7:182.
16 Roepman P, Kemmeren P, Wessels LF, et al. Multiple robust signatures for detecting lymph node metastasis in head and neck cancer. Cancer Res . 2006;66:2361-2366.
17 Roepman P, Wessels LF, Kettelarij N, et al. An expression profile for diagnosis of lymph node metastases from primary head and neck squamous cell carcinomas. Nat Genet . 2005;37:182-186.
18 Chung CH, Levy S, Yarbrough WG. Clinical applications of genomics in head and neck cancer. Head Neck . 2006;28:360-368.
19 O’Donnell RK, Kupferman M, Wei SJ, et al. Gene expression signature predicts lymphatic metastasis in squamous cell carcinoma of the oral cavity. Oncogene . 2005;24:1244-1251.
20 Chang HY, Nuyten DS, Sneddon JB, et al. Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival. Proc Natl Acad Sci USA . 2005;102:3738-3743.
21 Chi JT, Wang Z, Nuyten DS, et al. Gene expression programs in response to hypoxia: cell type specificity and prognostic significance in human cancers. PLoS Med . 2006;3:e47.
22 Carter SL, Eklund AC, Kohane IS, et al. A signature of chromosomal instability inferred from gene expression profiles predicts clinical outcome in multiple human cancers. Nat Genet . 2006;38:1043-1048.
23 Glinsky GV, Berezovska O, Glinskii AB. Microarray analysis identifies a death-from-cancer signature predicting therapy failure in patients with multiple types of cancer. J Clin Invest . 2005;115:1503-1521.
24 Chung CH, Parker JS, Ely K, et al. Gene expression profiles identify epithelial-to-mesenchymal transition and activation of nuclear factor-kappaB signaling as characteristics of a high-risk head and neck squamous cell carcinoma. Cancer Res . 2006;66:8210-8218.
25 Pramana J, Van den Brekel MW, van Velthuysen ML, et al. Gene expression profiling to predict outcome after chemoradiation in head and neck cancer. Int J Radiat Oncol Biol Phys . 2007;69:1544-1552.
26 Nuyten DS, Kreike B, Hart AA, et al. Predicting a local recurrence after breast-conserving therapy by gene expression profiling. Breast Cancer Res . 2006;8:R62.
27 Loi S, Haibe-Kains B, Desmedt C, et al. Predicting prognosis using molecular profiling in estrogen receptor-positive breast cancer treated with tamoxifen. BMC Genomics . 2008;9:239.
28 Chanrion M, Negre V, Fontaine H, et al. A gene expression signature that can predict the recurrence of tamoxifen-treated primary breast cancer. Clin Cancer Res . 2008;14:1744-1752.
29 Camps C, Buffa FM, Colella S, et al. hsa-miR-210 Is induced by hypoxia and is an independent prognostic factor in breast cancer. Clin Cancer Res . 2008;14:1340-1348.
30 Yu SL, Chen HY, Chang GC, et al. MicroRNA signature predicts survival and relapse in lung cancer. Cancer Cell . 2008;13:48-57.
31 Brandes AA, Franceschi E, Tosoni A, et al. MGMT promoter methylation status can predict the incidence and outcome of pseudoprogression after concomitant radiochemotherapy in newly diagnosed glioblastoma patients. J Clin Oncol . 2008;26:2192-2197.
32 Eoli M, Menghi F, Bruzzone MG, et al. Methylation of O6-methylguanine DNA methyltransferase and loss of heterozygosity on 19q and/or 17p are overlapping features of secondary glioblastomas with prolonged survival. Clin Cancer Res . 2007;13:2606-2613.
33 Shen L, Catalano PJ, Benson ABIII, et al. Association between DNA methylation and shortened survival in patients with advanced colorectal cancer treated with 5-fluorouracil based chemotherapy. Clin Cancer Res . 2007;13:6093-6098.
34 Yang Q, Kiernan CM, Tian Y, et al. Methylation of CASP8, DCR2, and HIN-1 in neuroblastoma is associated with poor outcome. Clin Cancer Res . 2007;13:3191-3197.
35 Chin SF, Teschendorff AE, Marioni JC, et al. High-resolution aCGH and expression profiling identifies a novel genomic subtype of ER negative breast cancer. Genome Biol . 2007;8:R215.
36 Idbaih A, Marie Y, Lucchesi C, et al. BAC array CGH distinguishes mutually exclusive alterations that define clinicogenetic subtypes of gliomas. Int J Cancer . 2008;122:1778-1786.
37 Kim MY, Yim SH, Kwon MS, et al. Recurrent genomic alterations with impact on survival in colorectal cancer identified by genome-wide array comparative genomic hybridization. Gastroenterology . 2006;131:1913-1924.
38 Natrajan R, Little SE, Sodha N, et al. Analysis by array CGH of genomic changes associated with the progression or relapse of Wilms’ tumour. J Pathol . 2007;211:52-59.
39 van den Broek GB, Wreesmann VB, Van den Brekel MW, et al. Genetic abnormalities associated with chemoradiation resistance of head and neck squamous cell carcinoma. Clin Cancer Res . 2007;13:4386-4391.
40 Rodemann HP, Blaese MA. Responses of normal cells to ionizing radiation. Semin Radiat Oncol . 2007;17:81-88.
41 Bentzen SM. Preventing or reducing late side effects of radiation therapy: radiobiology meets molecular pathology. Nat Rev Cancer . 2006;6:702-713.
42 Svensson JP, Stalpers LJ, Esveldt-van Lange RE, et al. Analysis of gene expression using gene sets discriminates cancer patients with and without late radiation toxicity. PLoS Med . 2006;3:e422.
43 West CM, Davidson SE, Elyan SA, et al. Lymphocyte radiosensitivity is a significant prognostic factor for morbidity in carcinoma of the cervix. Int J Radiat Oncol Biol Phys . 2001;51:10-15.
44 Ozsahin M, Crompton NE, Gourgou S, et al. CD4 and CD8 T-lymphocyte apoptosis can predict radiation-induced late toxicity: a prospective study in 399 patients. Clin Cancer Res . 2005;11:7426-7433.
45 Alsner J, Andreassen CN, Overgaard J. Genetic markers for prediction of normal tissue toxicity after radiotherapy. Semin Radiat Oncol . 2008;18:126-135.
46 Torres-Roca JF, Eschrich S, Zhao H, et al. Prediction of radiation sensitivity using a gene expression classifier. Cancer Res . 2005;65:7169-7176.
47 Amundson SA, Do KT, Vinikoor LC, et al. Integrating global gene expression and radiation survival parameters across the 60 cell lines of the National Cancer Institute Anticancer Drug Screen. Cancer Res . 2008;68:415-424.
48 Khodarev NN, Beckett M, Labay E, et al. STAT1 is overexpressed in tumors selected for radioresistance and confers protection from radiation in transduced sensitive cells. Proc Natl Acad Sci USA . 2004;101:1714-1719.
49 Hockel M, Knoop C, Schlenger K, et al. Intratumoral pO2 histography as predictive assay in advanced cancer of the uterine cervix. Adv Exp Med Biol . 1994;345:445-450.
50 Nordsmark M, Overgaard J. A confirmatory prognostic study on oxygenation status and loco-regional control in advanced head and neck squamous cell carcinoma treated by radiation therapy. Radiother Oncol . 2000;57:39-43.
51 Brizel DM, Dodge RK, Clough RW, et al. Oxygenation of head and neck cancer: changes during radiotherapy and impact on treatment outcome. Radiother Oncol . 1999;53:113-117.
52 Fyles A, Milosevic M, Hedley D, et al. Tumor hypoxia has independent predictor impact only in patients with node-negative cervix cancer. J Clin Oncol . 2002;20:680-687.
53 Janssen HL, Haustermans KM, Balm AJ, et al. Hypoxia in head and neck cancer: how much, how important? Head Neck . 2005;27:622-638.
54 Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev . 2007;26:225-239.
55 Seigneuric R, Starmans MH, Fung G, et al. Impact of supervised gene signatures of early hypoxia on patient survival. Radiother Oncol . 2007;83:374-382.
56 Bristow RG, Hill RP. Hypoxia and metabolism. Hypoxia, DNA repair and genetic instability. Nat Rev Cancer . 2008;8:180-192.
57 Martinive P, Defresne F, Bouzin C, et al. Preconditioning of the tumor vasculature and tumor cells by intermittent hypoxia: implications for anticancer therapies. Cancer Res . 2006;66:11736-11744.
58 Winter SC, Buffa FM, Silva P, et al. Relation of a hypoxia metagene derived from head and neck cancer to prognosis of multiple cancers. Cancer Res . 2007;67:3441-3449.
58a Starmans MH, Krishnapuram B, Steck H, et al. Robust prognostic value of a knowledge-based proliferation signature across large patient microarray studies spanning different cancer types. Br J Cancer . 2008;99:1884-1890.
59 Cuadros M, Dave SS, Jaffe ES, et al. Identification of a proliferation signature related to survival in nodal peripheral T-cell lymphomas. J Clin Oncol . 2007;25:3321-3329.
60 Rosenwald A, Wright G, Wiestner A, et al. The proliferation gene expression signature is a quantitative integrator of oncogenic events that predicts survival in mantle cell lymphoma. Cancer Cell . 2003;3:185-197.
61 Dai H, Van’T Veer L, Lamb J, et al. A cell proliferation signature is a marker of extremely poor outcome in a subpopulation of breast cancer patients. Cancer Res . 2005;65:4059-4066.
62 Villanueva A, Toffanin S, Llovet JM. Linking molecular classification of hepatocellular carcinoma and personalized medicine: preliminary steps. Curr Opin Oncol . 2008;20:444-453.
63 Whitfield ML, George LK, Grant GD, et al. Common markers of proliferation. Nat Rev Cancer . 2006;6:99-106.
64 van de Vijver MJ, He YD, van’t Veer LJ, et al. A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med . 2002;347:1999-2009. 19
65 Mook S, Schmidt MK, Viale G, et al. The 70-gene prognosis-signature predicts disease outcome in breast cancer patients with 1–3 positive lymph nodes in an independent validation study. Breast Cancer Res Treat . 2009;116:295-302.
66 Desmedt C, Piette F, Loi S, et al. Strong time dependence of the 76-gene prognostic signature for node-negative breast cancer patients in the TRANSBIG multicenter independent validation series. Clin Cancer Res . 2007;13:3207-3214.
67 Bogaerts J, Cardoso F, Buyse M, et al. Gene signature evaluation as a prognostic tool: challenges in the design of the MINDACT trial. Nat Clin Pract Oncol . 2006;3:540-551.
68 Buyse M, Loi S, Van’T Veer L, et al. Validation and clinical utility of a 70-gene prognostic signature for women with node-negative breast cancer. J Natl Cancer Inst . 2006;98:1183-1192.
69 Bonnefoi H, Potti A, Delorenzi M, et al. Validation of gene signatures that predict the response of breast cancer to neoadjuvant chemotherapy: a substudy of the EORTC 10994/BIG 00-01 clinical trial. Lancet Oncol . 2007;8:1071-1078.
70 Adler AS, Lin M, Horlings H, et al. Genetic regulators of large-scale transcriptional signatures in cancer. Nat Genet . 2006;38:421-430.
71 Blenk S, Engelmann JC, Pinkert S, et al. Explorative data analysis of MCL reveals gene expression networks implicated in survival and prognosis supported by explorative CGH analysis. BMC Cancer . 2008;8:106.
72 Sadikovic B, Yoshimoto M, Al Romaih K, et al. In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma. PLoS ONE . 2008;3:e2834.
73 West CM, Davidson SE, Roberts SA, et al. Intrinsic radiosensitivity and prediction of patient response to radiotherapy for carcinoma of the cervix. Br J Cancer . 1993;68:819-823.
74 West CM, Davidson SE, Roberts SA, et al. The independence of intrinsic radiosensitivity as a prognostic factor for patient response to radiotherapy of carcinoma of the cervix. Br J Cancer . 1997;76:1184-1190.
75 Bjork-Eriksson T, West C, Karlsson E, et al. Tumor radiosensitivity (SF2) is a prognostic factor for local control in head and neck cancers. Int J Radiat Oncol Biol Phys . 2000;46:13-19.
76 Stausbol-Gron B, Overgaard J. Relationship between tumour cell in vitro radiosensitivity and clinical outcome after curative radiotherapy for squamous cell carcinoma of the head and neck. Radiother Oncol . 1999;50:47-55.
77 Carrano AV. Chromosome aberrations and radiation-induced cell death. II. Predicted and observed cell survival. Mutat Res . 1973;17:355-366.
78 Cornforth MN, Bedford JS. A quantitative comparison of potentially lethal damage repair and the rejoining of interphase chromosome breaks in low passage normal human fibroblasts. Radiat Res . 1987;111:385-405.
79 Coco Martin JM, Mooren E, Ottenheim C, et al. Potential of radiation-induced chromosome aberrations to predict radiosensitivity in human tumour cells. Int J Radiat Biol . 1999;75:1161-1168.
80 Begg AC, McNally NJ, Shrieve DC, et al. A method to measure the duration of DNA synthesis and the potential doubling time from a single sample. Cytometry . 1985;6:620-626.
81 Begg AC, Haustermans K, Hart AA, et al. The value of pretreatment cell kinetic parameters as predictors for radiotherapy outcome in head and neck cancer: a multicenter analysis. Radiother Oncol . 1999;50:13-23.
82 Celis JE, Bravo R, Larsen PM, et al. Cyclin: a nuclear protein whose level correlates directly with the proliferative state of normal as well as transformed cells. Leuk Res . 1984;8:143-157.
83 Juan G, Li X, Darzynkiewicz Z. Correlation between DNA replication and expression of cyclins A and B1 in individual MOLT-4 cells. Cancer Res . 1997;57:803-807.
84 Wahl AF, Geis AM, Spain BH, et al. Gene expression of human DNA polymerase alpha during cell proliferation and the cell cycle. Mol Cell Biol . 1988;8:5016-5025.
85 Begg AC, Hofland I, Kummermehr J. Tumour cell repopulation during fractionated radiotherapy: correlation between flow cytometric and radiobiological data in three murine tumours. Eur J Cancer . 1991;27:537-543.
86 Graeber TG, Osmanian C, Jacks T, et al. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature . 1996;379:88-91.
87 Cairns RA, Kalliomaki T, Hill RP. Acute (cyclic) hypoxia enhances spontaneous metastasis of KHT murine tumors. Cancer Res . 2001;61:8903-8908.
88 Hasan NM, Adams GE, Joiner MC, et al. Hypoxia facilitates tumour cell detachment by reducing expression of surface adhesion molecules and adhesion to extracellular matrices without loss of cell viability. Br J Cancer . 1998;77:1799-1805.
89 Hodgkiss RJ, Jones G, Long A, et al. Flow cytometric evaluation of hypoxic cells in solid experimental tumours using fluorescence immunodetection. Br J Cancer . 1991;63:119-125.
90 Evans SM, Hahn S, Pook DR, et al. Detection of hypoxia in human squamous cell carcinoma by EF5 binding. Cancer Res . 2000;60:2018-2024.
91 Raleigh JA, Chou SC, Calkins-Adams DP, et al. A clinical study of hypoxia and metallothionein protein expression in squamous cell carcinomas. Clin Cancer Res . 2000;6:855-862.
92 Nordsmark M, Loncaster J, Chou SC, et al. Invasive oxygen measurements and pimonidazole labeling in human cervix carcinoma. Int J Radiat Oncol Biol Phys . 2001;49:581-586.
93 Nordsmark M, Loncaster J, Aquino-Parsons C, et al. The prognostic value of pimonidazole and tumour pO2 in human cervix carcinomas after radiation therapy: a prospective international multi-center study. Radiother Oncol . 2006;80:123-131.
94 Geara FB, Peters LJ, Ang KK, et al. Prospective comparison of in vitro normal cell radiosensitivity and normal tissue reactions in radiotherapy patients. Int J Radiat Oncol Biol Phys . 1993;27:1173-1179.
95 Johansen J, Bentzen SM, Overgaard J, et al. Evidence for a positive correlation between in vitro radiosensitivity of normal human skin fibroblasts and the occurrence of subcutaneous fibrosis after radiotherapy. Int J Radiat Biol . 1994;66:407-412.
96 Russell NS, Grummels A, Hart AA, et al. Low predictive value of intrinsic fibroblast radiosensitivity for fibrosis development following radiotherapy for breast cancer. Int J Radiat Biol . 1998;73:661-670.
97 Peacock J, Ashton A, Bliss J, et al. Cellular radiosensitivity and complication risk after curative radiotherapy. Radiother Oncol . 2000;55:173-178.
98 Barber JB, Burrill W, Spreadborough AR, et al. Relationship between in vitro chromosomal radiosensitivity of peripheral blood lymphocytes and the expression of normal tissue damage following radiotherapy for breast cancer. Radiother Oncol . 2000;55:179-186.
99 Russell NS, Begg AC. Editorial radiotherapy and oncology 2002: predictive assays for normal tissue damage. Radiother Oncol . 2002;64:125-129.
100 Okunieff P, Cornelison T, Mester M, et al. Mechanism and modification of gastrointestinal soft tissue response to radiation: role of growth factors. Int J Radiat Oncol Biol Phys . 2005;62:273-278.
101 Martin M, Lefaix J, Delanian S. TGF-beta1 and radiation fibrosis: a master switch and a specific therapeutic target? Int J Radiat Oncol Biol Phys . 2000;47:277-290.
102 Overgaard J, Hansen HS, Overgaard M, et al. A randomized double-blind phase III study of nimorazole as a hypoxic radiosensitizer of primary radiotherapy in supraglottic larynx and pharynx carcinoma. Results of the Danish Head and Neck Cancer Study (DAHANCA) Protocol 5-85. Radiother Oncol . 1998;46:135-146.
103 Kaanders JH, Pop LA, Marres HA, et al. ARCON: experience in 215 patients with advanced head-and-neck cancer. Int J Radiat Oncol Biol Phys . 2002;52:769-778.
104 Rischin D, Peters L, Fisher R, et al. Tirapazamine, Cisplatin, and Radiation versus Fluorouracil, Cisplatin, and Radiation in patients with locally advanced head and neck cancer: a randomized phase II trial of the Trans-Tasman Radiation Oncology Group (TROG 98.02). J Clin Oncol . 2005;23:79-87.
105 Brown JM, Wilson WR. Exploiting tumour hypoxia in cancer treatment. Nat Rev Cancer . 2004;4:437-447.
106 Nuyts S, Van Mellaert L, Theys J, et al. Clostridium spores for tumor-specific drug delivery. Anticancer Drugs . 2002;13:115-125.
107 Liu SC, Minton NP, Giaccia AJ, et al. Anticancer efficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis. Gene Ther . 2002;9:291-296.
108 Riesterer O, Milas L, Ang KK. Use of molecular biomarkers for predicting the response to radiotherapy with or without chemotherapy. J Clin Oncol . 2007;25:4075-4083.
109 Kim H, Kim W, Lee M, et al. Tumor volume and uterine body invasion assessed by MRI for prediction of outcome in cervical carcinoma treated with concurrent chemotherapy and radiotherapy. Jpn J Clin Oncol . 2007;37:858-866.
6 Radiotherapy and Chemotherapy

Prakash Chinnaiyan, MD, George David Wilson, PhD, Paul M. Harari, MD
The logical integration of distinct modalities of cancer therapy, including surgery, radiation, and chemotherapy, has been a major focus of clinical investigation for several decades. As a result of these studies, combining chemotherapy with radiation has contributed to improved clinical outcomes and now represents the standard management approach for a majority of cancer patients with solid tumors. In this chapter, fundamental processes providing the rationale for integrating chemotherapeutic agents with radiation therapy are discussed. A firm mechanistic understanding of the antitumor activity of chemotherapeutic agents is of critical importance for optimal integration with radiation regimens. Therefore, commonly applied agents used in chemoradiation strategies are reviewed and preclinical models used to define interactions between specific agents and radiation response discussed. Lastly, a brief review of chemoradiation regimens that have become standard management approaches for individual tumor types is provided.

Exploitable Strategies in Combining Chemotherapy With Radiation
The primary objective of combining chemotherapy with radiation is to achieve an improved therapeutic result, which can be evaluated as a function of enhanced tumor response or reduced normal tissue toxicity. In the late 1970s, Steel and Peckham developed a conceptual framework for analyzing drug-radiation interactions. 1 In this seminal work, four mechanisms were described in which combined modality therapy could improve therapeutic outcome: spatial cooperation, toxicity independence, protection of normal tissue, and enhancement of tumor response. For more than 20 years, these mechanisms provided the backbone for evaluating chemoradiation combinations clinically. Based on lessons learned from these clinical investigations, coupled with the rapid emergence of molecularly targeted agents, Bentzen and colleagues proposed an updated conceptual framework to evaluate drug-radiation combinations, which are summarized in the following text and in Fig. 6-1 . 2

FIGURE 6-1 • Schematic diagram representing the interplay between spatial cooperation, cytotoxic enhancement, biologic cooperation, temporal modulation, and normal tissue protection. RT, Radiotherapy; SF, surviving fraction; X, drug.
(From Bentzen SM, Harari PM, Bernier J: Exploitable mechanisms for combining drugs with radiation: concepts, achievements and future directions. Nat Clin Pract Oncol 4(3):172–180, 2007.)
Spatial cooperation refers to combining a drug that is efficacious against systemic disease with radiation, which is effective against locoregional disease. Because a full dose of radiotherapy and chemotherapy is required, and spatial cooperation does not require an interaction at the cellular level, these modalities are typically administered sequentially in an effort to reduce toxicity. Examples of spatial cooperation include adjuvant chemotherapy and radiation therapy in breast cancer and the combination of hormonal therapy and radiation in patients with high-risk prostate cancer. Although this approach is advantageous in terms of toxicity, the issue of overall treatment must also be considered in the design of these schedules. By integrating a chemotherapeutic agent either before or after definitive radiation therapy, the overall treatment time would consequently increase, potentially contributing toward accelerated cellular repopulation and diminished response. 3 , 4 For example, a recent overview of randomized controlled trials of combined chemoradiation for small cell lung cancer concluded that shorter time from the start of any cytotoxic chemotherapy to the end of radiotherapy was significantly correlated with improved treatment outcome. 5 Similar findings have been identified for head and neck cancer.
Cytotoxic enhancement refers to the capacity of chemotherapy to interact with radiation and produce a greater effect on the local tumor than would be expected from simple additivity of cell killing. A drug with additive cell killing properties might be seen as a borderline case of cytotoxic enhancement, but unless it possesses active biologic targeting, it will not provide an advantage over a simple radiation dose escalation. With the rapid development of molecular oncology, which has identified numerous molecularly targeted agents that target tumor-specific biologic targets, the identification of tumor-specific agents is being actively investigated. Interestingly, preclinical models suggest that many of these agents also appear to enhance radiation response. Numerous trials are currently underway in multiple tumor types to determine the capacity of these agents to serve as radiation sensitizers. The mechanism that underlies this favorable interaction likely involves induction or repair of radiation-induced cellular deoxyribonucleic acid (DNA) damage. For example, incorporation of halogenated pyrimidines such as 5-fluorouracil into DNA seems to enhance the initial induction of DNA damage by radiation. 6 The diverse and complex biologic processes that may be targeted by chemotherapy occurring during the interval between fractionated radiotherapy, including tumor-cell repopulation, reoxygenation, and cellular redistribution, have been collectively termed temporal modulation. For example, combining epidermal growth factor receptor (EGFR) blockade with fractionated radiation may reduce cellular proliferation during therapy, thereby attenuating radiation-induced accelerated cellular proliferation. 7
Both toxicity independence and normal tissue protection were initially described by Steel and Peckham as exploitable strategies of chemoradiation; however, lessons learned from clinical investigations over the years suggest these mechanisms may no longer have direct clinical relevance. Toxicity independence refers to the concept of combining a drug that caused systemic toxicity with radiation, in which toxicity is expressed locally. It was suggested that this combination could allow for treatment intensification without unacceptable toxicity, even if the anticancer effects of the two modalities were simply additive. Although this approach was eagerly pursued, a number of studies have shown that effective radiochemotherapy combinations did, in fact, increase radiation-related side effects. 8 - 10 Normal tissue protection was initially based on the potential of some agents, such as cyclophosphamide and methotrexate, given before radiation, to reduce the effect of a subsequent irradiation in some normal tissues, including bone marrow and intestinal epithelium. This effect of dosing chemotherapy before radiotherapy is now understood to be a result of induced cellular repopulation after the first cytotoxic insult. Unfortunately, attempts to exploit this mechanism clinically have been unsuccessful. However, normal tissue protection is continuing to be actively investigated using agents specifically designed to provide cytoprotection or modulate the cytotoxic response of normal tissue. A number of strategies in preclinical or early clinical testing fall under this category, such as the stimulation of stem-cell proliferation in early responding normal tissues, 11 , 12 for example the use of keratinocyte growth factors to ameliorate mucositis, 13 or cytoprotection using the thiol-based free radical scavenger amifostine. 14 , 15
Advances in molecular oncology have fostered the development of the mechanism termed biologic cooperation, which has been proposed by Bentzen and colleagues to refer to strategies that target distinct cell populations, or employ different mechanisms for cell killing or delaying tumor regrowth. 2 An example could be a drug that targets hypoxic tumor cells, thereby complementing the effect of radiation, which has greater response in well-oxygenated cells. This approach has been applied with such agents as misonidazole, nimorazole, tirapazamine, and mitomycin C, which target hypoxic tumor cells either as radiation sensitizers or bioreductive cytotoxins. 16 More recently, clinical interest has focused on agents that affect hypoxia indirectly by targeting the tumor microenvironment. For example, vascular-targeted antiangiogenic agents such as combretastatin cause a shutdown of tumor vasculature, leading to tumor cell death via hemorrhagic necrosis. Agents such as combretastatin are mainly effective in the central regions of the tumor where hypoxic radioresistance may be a therapeutic challenge. Biologic cooperation arises because radiation is particularly effective against the well-oxygenated cells in the periphery of the tumor where vascular shut-down is less effective. Biologic cooperation can even be applied when combining angiogenic agents with radiation. For example, Jain recently presented the case of antiangiogenic agents inducing a normalization of tumor blood vessels, which in turn reduced tumor hypoxia, creating potential biologic cooperation between this class of drugs and radiation. 17

Methods for Assessing and Defining Drug–Radiation Interactions
Preclinical experimentation is the backbone for initial evaluation of the clinical potential of investigational agents to be used in concert with radiation. The overarching goals of these studies are to identify agents that may provide therapeutic benefit when combined with radiation in a specific tumor type, and to define mechanisms contributing to response enhancement. The mechanistic understanding of drug–radiation interactions is of critical importance, because this may help contribute to a personalized approach to cancer therapy. For example, clinical trials could be rationally designed and specifically enriched with patients who would be most likely to respond to a proposed therapy, rather than testing a specific therapy in an otherwise biologically heterogeneous population.
Preclinical studies are typically initiated in vitro, using cell-culture systems. With respect to assays that may be used, the clonogenic assay, which detects all forms of radiation-induced cell death, is considered the “gold standard” for radiosensitivity analysis. 18 The underlying biologic end-point evaluated in this assay involves reproductive integrity. Following irradiation, a cell may be physically present and apparently intact, and may even progress through one to two cycles of mitosis; however, if it has lost the capacity to divide indefinitely and produce a large number of progeny, it loses its reproductive integrity. In the context of radiation treatment, losing reproductive integrity prevents continued growth and metastases; therefore, from a practical perspective, the tumor is eradicated. A surviving cell, however, retains its reproductive integrity and is able to proliferate indefinitely to produce a large clone or colony, referred to as clonogenic. In the clonogenic assay, cell survival is determined as a function of radiation dose, with the surviving fraction of cells (colonies) plotted on a logarithmic scale and the dose of radiation plotted linearly. To assess the effect of a drug on cell radiosensitivity, the combined drug–radiation curve is commonly plotted after the cytotoxicity produced by the drug alone is excluded, referred to as normalization. The radiation cell-survival curve is not changed if the drug does not influence cell radiosensitivity, regardless of whether the drug is cytotoxic on it own. In this case, the cytotoxicity of the drug contributes only to the overall cell killing by the combined treatment of both agents, referred to as an additive effect. Investigational agents may interact with radiation by altering cell radiosensitivity such that the combination results in a supra-additive or subadditive effect, depending on whether the cell killing is greater or smaller than the sum of cell killings produced by individual agents. This type of interaction often changes the shape of the cell-survival curve. For instance, a modification of the shoulder region indicates an interaction affecting the repair of radiation-induced DNA damage. An example of a clonogenic survival experiment demonstrating the capacity of an investigational agent to enhance radiation response is depicted in Fig. 6-2 A .

FIGURE 6-2 • Preclinical models used to define drug radiation interactions. In the clonogenic assay (A), cell survival is defined as a function of radiation dose, with the surviving fraction of cells (colonies) plotted on a semi-logarithmic scale. The dose enhancement factor (DEF), which is used to quantify radiation sensitization, is calculated as the dose of radiotherapy (RT) alone (4 Gy) divided by the dose of drug + RT (3 Gy) to elicit equivalent cell kill (surviving fraction of 0.1). In the presented example, the investigational agent appears to enhance radiation response, with a DEF at a surviving fraction of 0.1 = 1.33. B, Delay in tumor growth (in vivo) after drug, radiation, or a combination of both. Points at B, C, and D represent absolute growth delays after treatment with drug, RT, or drug + RT, respectively. Normalized growth delay is defined as B, C, or D minus A .
Favorable findings in vitro are often followed by in vivo exploration of drug–radiation interactions. A commonly applied technique involves a mouse xenograft model, in which human tumor cells are grown in culture and inoculated in the flank region of immunodeficient mice. The efficacy of treatment is then determined by the extent of tumor growth delay 19 or the rate of tumor cure. 20 When the treatment endpoint is delay in tumor growth in drug and radiation combinations, typically four treatment arms are required (control, radiation alone, drug alone, and drug and radiation combined), and tumors are followed until they meet predetermined size criteria. As in the clonogenic assay, growth delays are normalized by subtracting delays of independent treatments (radiation alone and drug alone) from the difference between the drug–radiation combination and the untreated control arm. An example of a growth delay experiment is depicted in Figure 6-2 B . The 50% tumor control dose (TCD 50 ; i.e., the dose of radiation that achieves 50% tumor control) assay is used when the treatment endpoint is rate of tumor cure. In this experiment, tumor cure is measured as a function of radiation dose, and the dose that is required to cure or control 50% of the tumors are compared between mice treated with radiation alone and those treated with combined treatment. If combined treatment displaces the curve to the left (i.e., a lower TCD 50 ), then addition of the drug improves tumor curability 21 ; however, this assay does not discriminate between independent drug activity and a direct influence on radiation response. The TCD 50 assay has been suggested to be a more relevant model to assess the curative potential of radiation when combined with an investigational agent, as its primary endpoint involves clonogenic cell death. 22
Although these in vivo methodologies certainly have their limitations, their potential advantage over traditional in vitro studies is that they also provide insight into the exploitable strategy of biologic cooperation, because both fractionation and tumor microenvironment are evaluated. In an effort to better replicate tumor environment for a specific tumor type, this assay may be of further value if performed orthotopically (e.g., brain tumor grown intracranially, breast tumor grown in mammary fat pads, etc.). 23 - 25 It has been historically suggested that this model may be applied to address normal tissue toxicity; however, its overall clinical application has been limited thus far. Therefore, it is of great importance to identify novel model systems that may be used in this context, because this would provide insight into an improved therapeutic ratio, rather than solely cytotoxic enhancement, which would be the necessary endpoint to further clinical gains.
Although the specific proteins and cell-signaling networks contributing to radiosensitization of a chemotherapeutic or molecularly targeted agent are very complex and multifactorial in nature, the fundamental biologic processes that eventually lead to enhanced radiation response fall under five broad categories. These are the initial induction of radiation-induced DNA damage, DNA damage repair, modulation of cell-cycle kinetics, tumor microenvironment, and cell repopulation. These general categories are summarized in the following text.

Initial Radiation Damage
The critical mechanism of radiation injury is damage to DNA. Radiation induces several types of lesions, including single-strand breaks (SSBs), double-strand breaks (DSBs), base damage, and DNA–DNA or DNA–protein crosslinks. The most common types of lesions caused by radiation are SSBs; however, these are readily repaired and rarely lead to cytotoxicity. DSBs, on the other hand, are the primary contributor to cell death if left unrepaired. One example of a chemotherapeutic agent that influences initial radiation-induced damage is the halogenated pyrimidines. Bromodeoxyuridine (BrdU) and iododeoxyuridine (IUdR) are thymidine analogs that become incorporated into DNA during S phase. The mechanism for strand-break formation involves electron attachment to BrdU or IUdR, followed by the departure of a bromide (or iodide) anion and the generation of a uracil-5-yl radical that further reacts to create strand breaks. Consequently, the cells are more susceptible to radiation-induced damage. The rationale for using these agents was that tumor cells may be cycling more rapidly than normal cells in surrounding tissues so that more drug could be incorporated into tumor DNA, resulting in “selective” radiosensitization. Head and neck tumors were among the first treated, because of the need for intra-arterial injection (liver dehalogenates the drugs rapidly). Tumor responses were good, but normal tissue toxicity was unacceptable because of rapid mucosal proliferation. The most appropriate tumors are those with a high growth fraction or labeling index, and a tumor site in which normal tissues are not proliferating rapidly (e.g., high-grade gliomas or large, unresectable sarcomas).
The primary methodology employed to assess radiation damage preclinically is the neutral comet or single-cell gel electrophoresis assay, which is a rapid and sensitive method for detecting DNA damage at the level of individual cells. 26 It is based on the ability of negatively charged loops or fragments of DNA to be drawn through an agarose gel in response to an electric field. The extent of DNA migration is directly influenced by the amount of DNA damage present in the cell. In this assay, a suspension of cells are mixed with agarose and spread onto a microscope glass slide. DNA unwinding and electrophoresis is carried out at a specific pH. Unwinding of the DNA and electrophoresis at neutral pH (termed neutral comet ) predominantly facilitates the detection of DSBs and crosslinks, whereas, when performed at pH levels greater than 12 (termed alkaline comet ), the test facilitates the detection of SSBs and DSBs, incomplete excision repair sites, and crosslinks. When subjected to an electric field, the DNA migrates out of the cell in the direction of the anode, appearing like a “comet” with a distinct head composed of intact DNA, and a tail consisting of damaged or broken pieces of DNA. The size and shape of the comet and the distribution of DNA within the comet correlate with the extent of DNA damage. 27

DNA Damage Repair
Immediately following radiation-induced DNA damage, a dynamic and well-orchestrated repair process is initiated, which includes DNA damage recognition, chromatin relaxation, formation of multiunit repair protein complexes at sites of DNA damage, DNA DSB repair, and finally repair protein dissolution and chromatin restoration. Despite its complexity, the entire repair process of DNA DSBs is rapid; a majority of base pairs are repaired within 6 hours. Several modalities have been used preclinically to evaluate DNA damage repair, including constant or pulsed-field gel electrophoresis, but these have been criticized because of the need for large radiation doses. The neutral comet assay, which, as described previously, determines initial damage repair, can also be applied to measure DNA DSB repair when evaluated in a time course manner. A methodology that has gained significant popularity in recent years to assess DNA damage repair involves immunofluorescent cytochemistry to assess phosphorylation of H2AX. At DSB sites, the histone H2AX becomes rapidly phosphorylated (γH2AX), forming readily visible foci. The dephosphorylation and dispersal of γH2AX in irradiated cells correlates with repair of the DNA DSBs. Therefore, prolonged expression of γH2AX, for example, when an investigational agent is combined with radiation, suggests abrogation of DNA repair processes. In addition, the γH2AX assay is sensitive enough to detect damage and repair at very low doses in the clinical and subclinical range. 28 - 31 Common methods used to assess DNA damage repair and initial DNA damage are depicted in Fig. 6-3 .

FIGURE 6-3 • Preclinical models used to evaluate radiotherapy (RT)-induced DNA damage and repair. Following RT, H2AX is rapidly phosphorylated (γH2AX), forming readily visible foci with a cell (A). γH2AX foci kinetics can be evaluated as a function of time, with foci dispersion correlating to repaired DNA. In the presented example (B), the investigational agent prolongs expression of γH2AX foci at 12 and 24 hours, suggesting it has a role in abrogating DNA repair. In the neutral comet assay (C), an individual cell is depicted with a distinct head, comprising intact DNA and a radiation-induced tail, consisting of damaged or broken fragments of DNA.
Many chemotherapeutic agents used in combination with radiation interact with and abrogate cellular repair mechanisms, leading to radiosensitization. One example is halogenated pyrimidines. In addition to the previously described mechanism of increased initial radiation damage, these agents also appear to influence the DNA repair. Fludarabine represents one example, which is a potent inhibitor of DNA primase, DNA polymerase-α, and ribonucleotide reductase. Nucleoside analogs, such as gemcitabine also appear to modulate the repair of radiation-induced DNA and chromosome damage through inhibition of ribonucleotide reductase. Preclinical studies combining gemcitabine with radiation are promising, and this combination is currently under clinical evaluation.

Cell Cycle Kinetics
The mechanistic interplay between radiation and chemotherapy through cell cycle kinetics may be appreciated at multiple levels. One potential interaction involves cell cycle redistribution. Focused radiobiologic studies have determined differential radiosensitivity based on cell cycle phase. In general, cells in the G 2 or M phases are the most sensitive, and cells in the S phase are the most resistant cells to radiation. This variation in radiosensitivity during the cell cycle can be potentially exploited when designing effective chemoradiation therapy strategies. One example involves the mitotic-spindle poison taxane. These agents act to stabilize microtubules, and thereby prevent chromosome separation at M phase, leading to cell cycle arrest in the radiosensitive G 2 and M phases. Another approach by which cell cycle kinetics may be exploited in radiation and chemotherapy interactions involves differential cytotoxicity. Although the S phase of the cell cycle appears to render cells more resistant to the cytotoxic effects radiation, this phase is particularly sensitive to nucleoside analogs that become incorporated in cellular DNA, such as fludarabine or gemcitabine. Therefore, the preferential targeting of cells in radioresistant phases of the cell cycle represents a form of biologic cooperation underlying the observed radioenhancement. An important point to remember is that although chemotherapeutic agents may have a specific cell cycle phase during which they inhibit cell cycle progression, it is often cells in another phase that might be more sensitive. Classic examples are the vinca-alkaloids, which arrest cells in mitosis but are most toxic when cells are exposed in S phase. Fig. 6-4 summarizes these relationships for a series of chemotherapeutic agents.

FIGURE 6-4 • A summary of the influence of chemotherapeutic agents on the cell cycle.
In addition to cell cycle redistribution, the modulation of checkpoint response represents another approach by which chemotherapeutic agents may influence radiosensitization. The activation of the G 2 checkpoint allows for DNA repair before progression into mitosis and is considered to protect against radiation-induced cell death. 32 Cell cycle kinetics is typically assessed in vitro by way of flow cytometry. By using a DNA-specific stain (typically propidium iodide or Hoechst) the DNA profile can be determined based on the relative amount of DNA. When relative fluorescence (i.e., the amount of DNA per cell) is plotted as a function of cell number, cells in G 1 have the least amount of DNA per cell (2n) and therefore represent the first spike on this plot. Cells accumulate DNA during the S phase, representing the intermediate region, and finally, cells in G 2 and M have the most quantitative DNA per cell (4n), and therefore represent the final spike on this plot. However, this approach, which assesses DNA quantity per cell, is unable to differentiate cells in G 2 and M phases. Therefore, to distinguish between G 2 and mitotic cells, in addition to labeling cellular DNA, cells are also labeled with an antibody specific to phosphorylated histone H3, which is specifically expressed in mitotic cells. Done as a function of time after irradiation, this analysis provides a measure of the progression of G 2 cells into M phase and thus the activation of the G 2 checkpoint. 33

Tumor Microenvironment/Hypoxia
In addition to the aberrant genetic alterations driving uncontrolled tumor growth, the surrounding microenvironment of a tumor plays a critical role in its continued growth and may also influence therapeutic response. An important way in which the microenvironment influences tumor growth is by way of its supporting vasculature. The continued growth of a tumor requires the formation of new blood vessels to facilitate the delivery of nutrients and oxygen, a process called angiogenesis. A critical mediator of tumor angiogenesis is the vascular endothelial growth factor, and targeting this protein using a variety of molecular agents represents a dominant theme in anticancer therapy in nearly all solid tumor types. Despite a robust angiogenic response, the oxygenation concentrations are quite heterogeneous within a tumor, with a gradient of intratumoral oxygenation governed by the diffusion capacity of host vessels. Therefore, regions within a tumor distant from the supporting vasculature develop subpopulations of hypoxic cells. In addition to distance from vasculature, hypoxic cells are also induced from defective vascularization within a tumor, both in the number of blood vessels and vessel function. For example, tumor blood vessels are commonly irregular and tortuous, and have blind ends, arteriovenous shunts, incomplete endothelial linings, and basement membranes, leading to areas of poor oxygenation within a tumor. It has long been suggested that hypoxia contributes to radiation resistance, 34 a phenomenon that was initially interpreted as reflecting the requirement for oxygen as a source of radiation-induced free radicals that mediate tumor cell killing. Although mechanisms still remain unclear, a more generally accepted principle is that hypoxia influences radiation response at a molecular level, modulating tumor phenotype and angiogenesis through upregulation of key mediators, including hypoxia-inducible factor (HIF-1). 35
Targeting these characteristics of tumor microenvironment represents potential for mechanistic interplay between drugs and radiation. For example, several chemotherapeutic agents, including tirapazamine and mitomycin, selectively target hypoxic cells, likely because of their reductive activation under hypoxic conditions. Hypoxic radiosensitizers represent another approach in which chemotherapy may be applied to enhance response in this resistant subpopulation of cells. These drugs selectively target hypoxic cells by mimicking the effect of oxygen in increasing radiation damage. Misonidazole and other hypoxic cell radiosensitizers are highly effective in enhancing radioresponse in preclinical models; however, their clinical application has been limited, primarily by excessive neurotoxicity.
Although the evaluation of angiogenesis inhibitors as anticancer agents is apparent, their application when combined with radiation is not as intuitive. For example, based on these proposed mechanisms, although angiogenesis inhibitors purportedly prevent continued growth of the tumor, it may also contribute to inducing hypoxia and theoretically minimizing therapeutic efficacy when combined with radiation. However, tumor vasculature is often functionally and structurally abnormal and contributes to hypoxia through spatial and temporal heterogeneity in tumor blood flow. Recent findings suggest that certain angiogenic agents may normalize the abnormal structure and function of tumor vasculature to abrogate hypoxia and potentially even increase the efficacy of conventional therapies. 17

Cell Repopulation
Both radiation therapy and chemotherapy are typically administered in multiple, temporally spaced, doses. With respect to radiation treatments, this is to allow for recovery from sublethal radiation-induced damage and allow repopulation of normal tissues between treatments. However, repopulation of surviving tumor cells can also occur during this interval, increasing tumor burden. In addition, the rate of repopulation may increase during a protracted course of therapy, a phenomenon termed accelerated repopulation, which may further limit the effectiveness of therapy. 4 Although studies aimed to quantify accelerated repopulation are often confounded by multiple factors, often being derived from retrospective data with a heterogeneous dose of radiation per fraction, their findings are striking. In an important paper, Withers and colleagues 36 analyzed pooled clinical data for TCD 50 in squamous cell carcinoma of the head and neck. A marked increase in TCD 50 was demonstrated if the treatment lasted more than 4 weeks, which was attributed to accelerated repopulation. Further, the added radiation dose required to overcome repopulation has been estimated to range from 0.5 to 1 Gy per day of prolonged treatment. 4 The critical importance of minimizing treatment time in cancer therapy has been corroborated by a large prospective randomized trial that demonstrated clinical gains in head and neck cancer with accelerated fractionated radiotherapy (delivered for 6 weeks) when compared with a conventional fractionated radiotherapy (delivered for 7 weeks). 37
As described previously, one potential way to overcome accelerated tumor repopulation is to decrease overall treatment time through altered radiation fractionation. Another approach that reduces or eliminates accelerated repopulation is to integrate chemotherapeutic agents into the course of radiation treatment. These agents may reduce the rate of proliferation when given concurrently with radiation therapy, and hence mitigate cellular repopulation. This may be a particularly important application of molecularly targeted agents, which are typically both tumor-selective and cytostatic, rather than cytotoxic; therefore, these agents may have a similar effect on repopulation with an improved toxicity profile.

Commonly Applied Chemotherapies and Mechanisms
A fundamental issue in the application of conventional chemotherapeutic agents with radiation involves determining the molecular mechanism contributing to the improved therapeutic index. However, to appreciate this level of interaction, it is important to consider how these chemotherapy agents function, so as to best exploit their clinical development potential as radiation sensitizers. Although complex, a simplified overview of biologic processes underlying the most common chemotherapeutic agents used in concert with radiation are described in the following text, and potential levels of interaction that these agents may have with radiation response are discussed.

Cisplatin and Analogs
Cisplatin is probably the most widely used anticancer agent in combination with radiation. Cisplatin is a water-soluble, coplanar complex that is converted to its active form by replacing its chloride ions with hydroxyl groups. 38 The active metabolite reacts with cellular DNA to form interstrand and intrastrand crosslinks that impair DNA replication and ribonucleic acid (RNA) transcription. This leads to DNA breaks and miscoding that may be repaired, mutagenic, or lethal, causing activation of an irreversible apoptotic program.
The cytotoxicity of cisplatin is primarily ascribed to its interaction with nucleophilic N 7 -sites of purine bases in DNA 39 to form both DNA–protein and DNA–DNA interstrand and intrastrand crosslinks. Although there is controversy involving which lesion is dominant in cisplatin toxicity, 40 evidence favors the intrastrand adducts. The intrastrand cis -Pt(NH3)2-d(GpG) and cis -Pt(NH3)2-d(ApG) crosslinks represent approximately 65% and 25%, respectively, of the total lesions present in DNA. Binding of the drug causes physical distortions in DNA that attract the attention of a myriad of proteins. More than 20 candidate proteins have been suggested, including components of the mismatch repair complex and the nonhistone chromosomal high-mobility group 1 and 2 proteins. 41 It has been suggested that each of the recognition proteins may initiate one or more specific events resulting in several seemingly unrelated biologic effects. This concept is consistent with the cisplatin-induced disruption of replication and transcription, simultaneously initiating responses that may be both survival and death signals. 41 , 42
The consequence of cisplatin adduct formation is a cascade of cellular events involving signaling pathways, checkpoint activation, DNA repair activity, and apoptosis. Previously, the ataxia-telangiectasia RAD3 -related protein has been implicated as the main kinase activated by cisplatin, 43 , 44 resulting in phosphorylation of CHK1 kinase, although recent evidence suggests that cisplatin may activate CHK2 in an ataxia-telangiectasia mutated–dependent manner. 45 CHK1 and CHK2 orchestrate a complex pathway of cell cycle checkpoint activation involved in G 1 , S, and G 2 arrest. 46 In particular, activation of p53 and its downstream mediators 47 and the mitogen-activated protein kinase cascade 48 are considered key events determining the ultimate fate of the cell.

Cisplatin and Radiation
The basis for interaction between these two modalities may be appreciated at multiple levels, including (1) enhanced formation of toxic platinum intermediates in the presence of radiation-induced free radicals, (2) the capacity of cisplatin to scavenge free electrons formed by the interaction between radiation and DNA that may fixate otherwise reparable damage to DNA, (3) a radiation-induced increase in cellular cisplatin uptake, (4) a synergistic effect because of cell cycle disruption, and (5) the inhibition of repair of radiation-induced DNA lesions.
In vitro studies have shown that the most effective combinations between the two agents tend to be achieved with lower doses of the two modalities. Myint and colleagues 49 showed increased radiosensitivity of murine embryonic fibroblast cells at low levels of cisplatin, but when concentrations were increased, instead of observing an increase in radiosensitivity, the data revealed an increasing radioresistance. Gorodetsky and colleagues 50 showed that when human ovarian carcinoma (OV-1063) and murine mammary adenocarcinoma (EMT-6) cell lines were pre-irradiated at a low dose of 2 Gy, a clear additional effect of the drug was observed, but this was almost totally eliminated when cells were irradiated with a higher dose (6 Gy). This dose sensitivity argues against a simple model of physical interaction because of proximity between the lesions caused by the two modalities.
The most plausible explanation, supported by the timing of optimal exposure to the agents, is that synergy is a consequence of cisplatin inhibition of repair of radiation-induced DNA damage. 50 - 52 In two cell lines, the EMT-6 and OV-1063 cells, a 2-hour postradiation drug exposure resulted in a supra-additive combined effect, whereas a 24-hour pre-irradiation exposure or protracted postirradiation exposure yielded an additive or slightly subadditive response. 50 In experimental tumors, the greatest dose-enhancement factors were observed when cisplatin was administered immediately before a daily fraction of radiation. 52
The concept of sublethal damage repair was developed to explain the recovery of cells to full mitotic potential after exposure to a radiation dose insufficient to cause cell death. Early studies suggested that the addition of cisplatin to radiation inhibited this process through modification of two critical pathways involved in DNA DSB repair, homologous recombination 53 and nonhomologous end-joining (NHEJ). 49 , 54 The first support for the involvement of NHEJ in the mechanism of cisplatin and radiation interaction came from Frit and colleagues, 55 who showed that cross-resistance to ionizing radiation and cisplatin was associated with increased KU80 activity. It was later found that cisplatin-induced DNA damage reduced the ability of the DNA-dependent protein kinase to interact with duplex DNA molecules in vitro 56 and that cisplatin radiosensitization was not evident in KU80 -deficient cells compared with their wild-type counterparts. 49
The involvement of homologous recombination in the sensitization mechanism has been suggested from studies in repair-proficient wild-type and recombinational repair-deficient ( RAD52 ) strains of the yeast Saccharomyces cerevisiae. 53 Cisplatin exposure sensitized wild-type yeast cells with a competent recombinational repair mechanism, but could not sensitize cells defective in recombinational repair, indicating that the radiosensitizing effect of cisplatin involved inhibition of RAD52 -dependent recombinational repair of DNA. This study also suggested that excision repair was not involved in the sensitization mechanism because strains homozygous for RAD3-2 showed synergy between radiation and cisplatin.

5-Fluoruracil and Analogs
5-Fluoruracil (5-FU) is used widely in the treatment of solid tumors and belongs to a class of anticancer agents termed antimetabolites . It is an analogue of uracil with a fluorine atom at the C 5 position in place of hydrogen. The mechanism of cytotoxicity of 5-FU has been ascribed to the misincorporation of fluoronucleotides into RNA and DNA and to the inhibition of the nucleotide synthetic enzyme thymidylate synthase (TS). 5-FU rapidly enters the cell using the same facilitated transport mechanism as uracil, and is converted intracellularly to several active metabolites that disrupt RNA synthesis and the action of TS. 57 , 58
TS represents a critical enzyme for the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), a precursor of thymidylate, which is necessary for DNA replication and repair. The 5-FU metabolite fluorodeoxyuridine monophosphate competitively inhibits dUMP from binding to TS, thereby abrogating dTMP synthesis. Interestingly, thymidylate can be salvaged from thymidine through the action of thymidine kinase, thereby alleviating the effects of TS deficiency. Therefore, this salvage pathway may represent a potential mechanism of resistance to 5-FU.
Another metabolite of 5-FU, fluoridine triphosphate (FUTP), is extensively incorporated into RNA, disrupting normal RNA processing and function. The toxicity of 5-FU–induced toxicity to RNA is present at several levels. 57 This includes inhibiting the processing of pre-rRNA into mature rRNA, 59 , 60 disrupting posttranslational modifications of tRNAs, 61 , 62 and the assembly and activity of snRNA/protein complexes, thereby inhibiting splicing of pre-mRNA. 63 , 64 In addition to their effect on cellular RNA, 5-FU may also be misincorporated into cellular DNA. This is accomplished through another one of its active metabolites, fluorodeoxyuridine triphosphate (FdUTP). Based on this mechanism, 5-FU has particular activity in cells in S phase through incorporation into DNA, leading to both SSBs and DSBs.

5-FU and Radiation
There are a number of mechanisms by which 5-FU could increase radiation sensitivity at the cellular level. As described previously, the toxicity of 5-FU appears to be S phase–specific, a phase in the cell cycle that is particularly resistant to radiation. However, this mechanism is not sufficient to account for all of the increase in radiation sensitivity produced by the drug because noncytotoxic concentrations of 5-FU have also been demonstrated to increase radiation sensitivity. 65 , 67 Recent data suggest that increased radiation sensitivity occurs in cells that have inappropriate progression though S phase in the presence of drug, suggesting a disordered S-phase checkpoint response. 67
Preclinical studies suggest that protracted infusions of 5-FU during a course of fractionated radiotherapy would be required to achieve optimal radiosensitization. This is logical if one considers the mechanism of 5-FU inhibition of TS and the short half-life of 5-FU and its metabolites. 67 Indeed, protracted venous infusion of 5-FU has become a standard therapy in several tumor types. The introduction of oral forms of 5-FU, such as the prodrug capecitabine, allow for added convenience for protracted treatment. In addition to ease of use, this agent may also offer further advantages when combined with radiation. Capecitabine is converted to its active form in a multistep process that relies on the enzyme thymidine phosphorylase (TP). TP has higher concentrations in many tumor types compared with matched normal tissue, and therefore may serve as a potent prodrug. 68 , 69 However, over and above this favorable activation profile, interest in integrating capecitabine in combination with radiation has been heightened by the observation that radiation itself can induce TP activity. 70 , 71 Local irradiation with a single dose of 5 Gy increased TP levels by up to 13-fold at 9 days after irradiation. Therefore, there seems to be great potential for synergy between radiation and capecitabine through its capacity to act as a prodrug and enhanced drug activation during irradiation, in addition to the established interaction between the two modalities through cell-cycle effects.

Gemcitabine
Gemcitabine is another nucleoside analogue that acts as a very potent radiation sensitizer. 72 , 73 Early studies in leukemic cells noted a dramatic effect of gemcitabine on DNA metabolism, with notable decreases in cellular deoxynucleotide triphosphates, which are required for DNA synthesis and repair. 74 It was later shown than once inside the cell, gemcitabine is rapidly phosphorylated by deoxycytidine kinase, the rate-limiting enzyme for the formation of the active metabolites gemcitabine diphosphate (dFdCDP) and gemcitabine triphosphate (dFdCTP). Ribonucleotide reductase is inhibited by dFdCDP, which is responsible for producing the deoxynucleotides. In addition, the subsequent decrease in cellular deoxynucleotides (particularly dCTP) favors dFdCTP in its competition with dCTP for incorporation into DNA. Therefore, this reduction in cellular dCTP represents an important self-potentiating mechanism resulting in increased gemcitabine nucleotide incorporation into DNA. Once the gemcitabine nucleotide is incorporated on the end of the elongating DNA strand, one more deoxynucleotide is added, following which the DNA polymerases are unable to proceed. This action, termed masked chain termination, appears to lock the drug into DNA, and is strongly correlated with the inhibition of further DNA synthesis, which is likely attributed to the inability of proof-reading exonucleases to remove the gemcitabine nucleotide. 75

Gemcitabine and Radiation
Gemcitabine demonstrates a significant enhancement of radiation-induced cell killing at both nontoxic and cytotoxic concentrations. 76 There appears to be no radiosensitization when cells are irradiated before gemcitabine, whereas the greatest enhancement of radiation was observed when cells were incubated for 24 hours before irradiation. Subsequent studies explained these results by revealing that maximum sensitization was produced under conditions in which cells were depleted of phosphorylated deoxynucleotides, which, as previously described, is one of their primary mechanisms of action through inhibition of ribonucleotide reductase. 72 This mechanism is further supported by the finding that ribonucleotide reductase overexpressing cells were resistant to gemcitabine-mediated radiosensitization. 77 Another key mechanism that likely underlies gemcitabine-mediated radiosensitization involves cell cycle distribution. Similar to 5-FU, incorporation of gemcitabine’s active metabolites into dividing DNA renders cells more susceptible in the radiation resistant S phase. It has therefore been suggested that maximum sensitization requires simultaneous redistribution into S phase along with deoxyadenosine triphosphate pool depletion. 78 Early clinical trials combining gemcitabine with radiation has indeed demonstrated potent radiosensitization; however, its continued development has been hindered by normal tissue toxicity. 67

Temozolomide
Temozolomide is an imidazotetrazine derivative of the alkylating agent dacarbazine. It undergoes rapid chemical conversion in the systemic circulation at physiologic pH to the active compound monomethyl triazeno imidazole carboxamide (MTIC). In contrast, MTIC is formed from dacarbazine only after metabolism by the liver. Because hepatic metabolism can be influenced by agents commonly taken by brain-tumor patients such as anticonvulsant drugs and corticosteroids, it is thought that bioavailability of MTIC may be more consistent with temozolomide than with dacarbazine. In addition, temozolomide is administered orally and has strong capacity to enter the cerebrospinal fluid without accumulation with repeat dosing, further contributing to its rapidly developing clinical interest and applications. 79 - 81
The antitumor activity of temozolomide has largely been attributed to methylation of DNA, specifically at the O 6 position of guanine, which has been found to be especially mutagenic and cytotoxic. Methyl adducts at the O 6 guanine in DNA are repaired by the cytoprotective DNA repair protein methyl guanine methyl transferase (MGMT; also referred to as arginine glycine amidinotransferase ), which transfers the methyl group to an internal cysteine acceptor residue. This reaction results in an irreversible inactivation of MGMT, requiring increased de novo protein synthesis to restore repair activity. Although methylation at the O 6 position of guanine is attributed to temozolomide toxicity, it makes up only a small proportion (≈5%) of the DNA that is methylated. For example, nearly 70% of total DNA methylation by temozolomide occurs at the N 7 position of guanine and approximately 9% of adducts formed at the N 3 position at adenine. 80 , 81 However, these sites are typically repaired rapidly, using the highly conserved and efficient base excision repair pathway. Although methylation at the O 6 position of guanine is also readily repaired by MGMT, a subset of tumors has been identified with epigenetic silencing of this repair protein through promoter methylation. In a seminal work reported by Hegi and colleagues, it was demonstrated that promoter methylation of MGMT in glioblastoma could serve as a biomarker for predicting response to temozolomide. 82 Although these findings are currently being validated prospectively, this is perhaps the first study that has identified a predictive marker for a cytotoxic agent, and therefore serves as an important step in the direction of individualized therapy based on tumor biology. However, despite representing progress, MGMT methylation is clearly not an exclusive factor, as nearly half of these patients still do not benefit from therapy, and a subset of patients who do not carry this methylation demonstrate long-term survival; therefore, more specific biomarkers are still required.

Temozolomide and Radiation
The addition of temozolomide with definitive radiation has doubled 2-year survival among patients with glioblastoma, an achievement that had not been accomplished in the prior 30 years of clinical investigations of this tumor. 83 Clearly, temozolomide has independent activity in glioblastoma, although preclinical investigations suggest it may also enhance radiation response. Kil and colleagues demonstrated modest in vitro enhancement of radiation response in a glioma cell line and a breast cancer cell line with a proclivity to form brain metastases, although in vivo sensitization was more striking, with a dose enhancement factor of 2.8 in tumor growth delay. 84 Of the numerous mechanisms studied, it appeared that mitotic catastrophe played a dominant role in the observed sensitization. Chakravarti and colleagues demonstrated similar findings and went on to suggest that sensitization was most effective in MGMT-negative tumors. 85

Timing of Drug Administration
Depending on the principal aim, chemotherapeutic agents may be administered before (neoadjuvant), during (concurrent), or following (adjuvant) the course of radiation therapy. In general, when a chemotherapeutic agent is delivered alone, using either a neoadjuvant or adjuvant approach, the primary goal is for the treatment of disseminated micrometastatic disease. However, activity at the primary tumor site is also important. For example, a potential application of neoadjuvant chemotherapy is for tumor down-sizing, which would attempt to convert patients with locally advanced tumors, previously unresectable, into surgical candidates. By tumor down-sizing, neoadjuvant chemotherapy can also potentially be used to decrease radiation field sizes, thereby reducing toxicity. Although both of these approaches clearly have their respective roles, there is likely little interaction between the chemotherapeutic agent and the biologic processes underlying radiation response. In addition, one must judiciously choose such an approach, as it may have negative effects on clinical outcome. For example, neoadjuvant chemotherapy would actually increase overall treatment time, which has been shown to negatively correlate with prognosis and contribute to accelerated cellular repopulation. 4
When delivering a chemotherapeutic agent concurrently with radiation, in addition to the treatment of disseminated micrometastases, a primary objective is to influence molecular processes involved in radiation response, in an effort to enhance overall outcomes. As described previously, potential levels of interaction between a specific agent and radiation may include initial DNA damage and its subsequent repair, cell cycle phase distribution, and their influence on tumor microenvironment and repopulation. An inherent advantage to this approach, when compared with a neoadjuvant treatment, is that overall treatment time is not prolonged. However, acute toxicities are typically heightened using this approach; therefore, drug doses and scheduling should be considered when designing clinical trials integrating these agents concurrently with radiation.
When delivering a chemotherapeutic agent concurrently with radiation, if the primary rationale is to influence radiation response, it seems logical to deliver the specific agent daily to coincide with the typical radiation regimen. This approach has been previously hampered by intravenous formulations and dosing regimens that have been previously applied in the adjuvant setting (for example, intravenous bolus delivered every 3 to 4 weeks). However, such approaches as protracted venous infusion, which has been applied to 5-FU in rectal cancer, and oral formulations of agents, including temozolomide in brain tumors and capecitabine, have allowed for more practical integration of chemotherapeutic agents into established radiation regimens.

Normal Tissue Effects
Although maximizing tumor control is the underlying rationale for integrating chemotherapeutic agents with radiation, the potential for enhancing cytotoxic effects in early responding normal tissues, including mucosa and epithelium, poses a clear challenge. For example, in head and neck cancer, chemoradiation regimens nearly doubled the incidence of grade 3 and 4 mucositis and increase the necessity for feeding tubes to maintain nutritional support during therapy. Similar increases in acute toxicities were demonstrated in lung cancer and esophageal cancer. However, recent advances in methods of radiation delivery and diagnostic imaging will likely assist in tempering such toxicities. For example, the radiation oncology community has rapidly embraced such technologies as intensity-modulated radiation therapy (IMRT), which will assist in minimizing normal tissue toxicity through conformal avoidance. In conjunction with improved treatment delivery, progress in diagnostic imaging will also play an important role in minimizing toxicities. For example, in non–small cell lung cancer (NSCLC), the integration of positron emission tomography–computed tomography (PET-CT) imaging into treatment planning systems has allowed physicians to tailor fields to target PET-positive regions, rather than comprehensive coverage of elective lymph nodes. Thus, radiation field size and subsequent treatment-related toxicities would be reduced.
Integrating agents that may serve as normal tissue radioprotectors also represents an active area of investigation. 86 A majority of work has focused on the thiol-containing prodrug amifostine, the primary mechanism of which involves free-radical scavenging. A phase III randomized trial was conducted from 1995 to 1997 to assess the ability of this drug to reduce the incidence of grade ≥ 2 acute and late xerostomia and grade ≥ 3 acute mucositis in locally advanced head and neck cancer. Amifostine did not reduce the incidence of mucositis, although it did appear to reduce both acute and late xerostomia. 14 Although amifostine has an approved xerostomia indication in the setting of radiation alone, its activity when combined with chemoradiation or modern radiation techniques that significantly minimizes dose to salivary glands, including IMRT, has yet to be defined. A similar study has been performed in NSCLC to determine the capacity of amifostine to mitigate radiation-induced esophagitis. 15 Although no reduction in esophagitis was observed, less swallowing dysfunction was demonstrated in patients who received amifostine. Although it has been a challenge to identify compounds that could selectively protect normal tissue (and not protect tumor) and integrate them in the clinic, success in such an approach would demonstrate a clear method of improving the therapeutic ratio.

Strategies for Improvement
Despite clear clinical gains demonstrated with the integration of chemotherapeutic agents into definitive radiation treatment regimens, investigators are actively seeking novel approaches to continue these improvements. One sought-after strategy for improvement in current treatment regimens has been to profile tumors using gene microarrays in an effort to identify patients most likely to respond to a particular chemotherapeutic agent, and tailor treatment accordingly. However, because of complex mechanisms of action and the multitiered biologic processes driving tumorigenesis and response and resistance, modest progress has been made to date using this approach. One promising example has involved predicting response to temozolomide in glioblastoma patients. Although findings are currently being prospectively validated, the promoter-methylation status of repair protein MGMT, which is involved in the repair of temozolomide-induced DNA damage, may serve as an important biomarker for temozolomide response. 82
Another strategy that has generated enthusiasm in furthering clinical gains involves integrating molecularly targeted agents into the chemoradiation platform. Such investigations are supported by preclinical investigations demonstrating the capacity of many of these agents to enhance radiation response. When developing these early phase clinical trials, it is important to recognize that these targeted agents are not benign and clearly have their own toxicity profile. However, as their toxicities are typically distinct from those commonly attributed to radiation and standard chemotherapeutics, this may allow for an improved therapeutic ratio. Targeting the tumor microenvironment with angiogenesis inhibitors represents another actively investigated approach to improve current chemoradiation regimens. In addition to having independent activity, these agents may have added benefits by inducing vascular normalization to alleviate hypoxia and increase intratumoral drug concentrations. 17

Clinical Results
One of the first modern chemoradiation platforms involved the use of the antimetabolite 5-FU in the late 1950s, 87 which interestingly still plays a significant role in the treatment of gastrointestinal malignancies. This was followed by one of the Radiation Therapy Oncology Group’s (RTOG’s) first studies, which was a prospective randomized trial evaluating methotrexate combined with radiation in head and neck cancer. 88 Since these initial investigations, combining chemotherapy with radiation has been an active area of investigation, contributing to improved clinical outcomes in a majority of solid tumors. Key clinical investigations leading to current chemoradiation platforms are summarized in the following text.

Lung
The Cancer and Leukemia Group B was one of the earliest groups evaluating the role of combining chemotherapy with radiation in locally advanced NSCLC. 89 This study used induction chemotherapy consisting of 5 weeks of cisplatin and vinblastine, which was subsequently followed by radiotherapy. Results demonstrated a statistically significant improvement in median survival of 4.1 months in patients receiving induction chemotherapy, which was also demonstrated by a similar trial performed by the RTOG and the Eastern Cooperative Oncology Group (ECOG). 90 Following these studies, the next critical question was to determine if chemotherapy delivered concurrently with radiation could further clinical gains. Both the RTOG and the West Japan Lung Cancer Group 91 identified concurrent chemoradiation to be superior, using cisplatin/vinblastine and mitomycin/vindesine/cisplatin platforms, respectively. Another regimen that is commonly used is concurrent paclitaxel and carboplatin, which has shown promising results in phase II studies. 92
As the natural history of small cell carcinoma (SCC) involves early seeding of distant metastases, chemotherapy has been the mainstay of therapy. However, in limited-stage small cell, radiation has demonstrated both an improvement in local control and survival. The use of concurrent cisplatin and etoposide with radiation has been generally accepted, largely based on results presented by ECOG (in collaboration with RTOG and the Southwest Oncology Group [SWOG]), using this platform with a hyperfractionated radiation regimen. 93

Head and Neck
In a report from the journal Lancet in 2000, the Meta-Analysis of Chemotherapy in Head and Neck Cancer Group identified that the addition of chemotherapy to locoregional treatment provides a modest overall improvement in survival (4% at 5 years) for patients with locoregionally advanced head and neck cancer. 94 The most promising benefit (8%) was evident with the use of concomitant chemoradiation with no significant benefit for the use of induction or adjuvant chemotherapy. These results were recently updated by incorporating data from randomized trials performed between 1994 and 2000. 95 Twenty-four new trials, most of them involving concomitant chemotherapy, were included, totaling 87 trials with more than 17,000 total patients. This updated analysis confirmed the small overall survival benefit of chemotherapy of 4% at 5 years. Similarly, analysis of the 50 concomitant chemoradiation trials confirmed the previous results, demonstrating an absolute survival benefit of 8% at 5 years. As with the prior meta-analysis, there was no significant survival benefit identified for the use of induction or adjuvant chemotherapy for patients with head and neck cancer, only in the concurrent chemoradiation setting.
Key clinical trials demonstrating benefit of concurrent chemotherapy in head and neck cancer include results presented by the Groupe d’Oncology Radiothérapie Téte et Cou, which used a carboplatin and 5-FU platform in SCC of the oropharynx. 96 Improved local control and survival came at a cost of increased acute mucositis and bone marrow toxicity, although it did not lead to an increase in late toxicities. In nasopharyngeal cancer, cisplatin delivered with radiation, followed by adjuvant cisplatin and 5-FU, was also shown to improve local control and survival. 97 In addition to the potential of improved survival, another key rationale for concurrent chemoradiation is organ preservation. Improved local control offered by concurrent chemoradiation in locally advanced laryngeal cancer, when compared with both radiation alone and induction chemotherapy, led to an improvement in larynx preservation. 98
Molecularly targeted agents have also been actively investigated in head and neck cancer. Results from an international, randomized phase III clinical trial of 424 locoregionally advanced head and neck cancer patients treated with curative intent using either high dose radiotherapy alone or high-dose radiotherapy plus the anti-EGFR monoclonal antibody cetuximab demonstrated a near doubling of the median survival for patients treated with radiotherapy plus cetuximab over radiotherapy alone, 49 months versus 29 months. 99 The percentage of patients who achieved locoregional control at 1 year and at 2 years following treatment was 69% and 56% in the cetuximab-treated patients, compared with 59% and 48% for those treated with radiotherapy alone. There was a statistically significant improvement (p = 0.02) in locoregional disease control (9% at 2 years) and overall survival (10% at 3 years) favoring the cetuximab arm. Because there has been a shift in the management of locally advanced head and neck cancer since the initiation of this study, with concurrent cisplatin now being accepted as a standard regimen with radiation, current trials are examining whether cetuximab combined with concurrent cisplatin and radiation may lead to further clinical gains.

Cervical Cancer
In 1999 the National Cancer Institute advised that cisplatin-based chemotherapy administered concurrently with radiotherapy exhibited a marked superiority over standard radiotherapy alone in cervical cancer and therefore was the new standard of care for this disease. 100 This was largely based on five of six randomized prospective studies that demonstrated a survival advantage of concurrent chemotherapy in the definitive treatment of cervical cancer. 101 In addition, concurrent chemotherapy also demonstrated benefit in the postoperative setting for patients exhibiting high-risk features, including positive margins, parametrial invasion, or positive lymph node. 102 The RTOG is now evaluating the benefit of integrating the angiogenesis inhibitor bevacizumab to this platform.

Gastrointestinal Disease
Nearly all subtypes of tumor in the gastrointestinal track have demonstrated clear benefit when chemotherapy is administered concurrently with radiation. Many of these regimens are based on a 5-FU platform, which was one of the first chemotherapeutic agents combined with radiation nearly 50 years ago. 87 In esophageal cancer, the definitive trial was performed by the RTOG, demonstrating a 5-year survival rate of 26% when cisplatin and 5-FU was combined with radiation, compared with 0% in the radiation-alone arm in unresectable disease. 103 In gastric cancer, an Intergroup study using a 5-FU and leucovorin platform demonstrated an improved local control and overall survival in patients with T 3 , T 4 , or N+ disease when delivered in the postoperative setting. 104
In rectal cancer, the Gastrointestinal Tumor Study Group performed one of the initial studies demonstrating benefit with combined modality therapy. 105 In this study, following surgery, patients were randomized to fours arms: no further therapy, radiation alone, chemotherapy alone (consisting of 5-FU and methyl-CCNU), and combined 5-FU and radiotherapy followed by adjuvant chemotherapy. Results demonstrated an improved local control with combined modality therapy. The North Central Cancer Treatment Group went on to demonstrate that protracted venous infusion of 5-FU improved recurrence rate and overall survival when compared with bolus 5-FU when delivered concurrently with radiation, suggesting its direct influence on radiation response. 106 With the established role of chemoradiation in rectal cancer, another important question that needed to be answered was the most effective time of delivery, either pre- or postoperative. This was addressed by the German Rectal Cancer Study group, which showed that preoperative concurrent 5-FU and radiation led to improvements in local control, colostomy-free survival, and an improved toxicity profile. 107 This regimen has now been established as standard therapy for locally advanced rectal cancer.
The initial rationale for combining chemotherapy with radiation in anal cancer was to increase resectability. However, in 1974, Nigro and coworkers observed complete tumor regression in its initial patients treated with a 5-FU and mitomycin C preoperative regimen, suggesting the potential for cure without surgery and colostomy. 108 A subsequent report described the outcome for 45 patients treated with the same regimen with a complete response rate of 84% on biopsy 6 weeks after completion of treatment. 109 The benefit of a concurrent chemoradiation regimen over radiation alone was demonstrated in two randomized phase III trials by the European Organization for Research and Treatment of Cancer (EORTC) and the United Kingdom Coordinating Committee on Cancer Research, both demonstrating increased local control and colostomy-free survival. 110 , 111 The potential of replacing mitomycin C with cisplatin was investigated in the randomized Intergroup RTOG trial that showed no difference between the two regimens in disease-free survival, its primary endpoint; however, the colostomy-free survival rate was significantly higher in patients receiving mitomycin C. 112 Therefore, the 5-FU and mitomycin regimen remains standard concurrent chemotherapy in anal cancer.

Glioblastoma
For nearly 30 years, surgical resection to the extent that is safely feasible, followed by radiotherapy has been the standard of care in glioblastoma. Because an overwhelming majority of patients experience local recurrence and eventually succumb to uncontrolled disease progression despite therapy, numerous attempts had been made at integrating chemotherapy to this regimen. Although individual trials did not show benefit, a meta-analysis demonstrated approximately a 5% improvement in overall survival with the addition of chemotherapy. 113 Despite the discouraging historical context involving glioblastoma management, novel therapeutic strategies offering clinical gains have emerged. A survival benefit in glioblastoma has recently been published by the EORTC. 83 This regimen, which is now the current standard of care in newly diagnosed glioblastoma patients, consists of concomitant low-dose temozolomide with radiation, followed by high-dose adjuvant temozolomide. Determining ways to integrate novel agents upon this platform to further clinical gains in this disease remains an active area of investigation.

Conclusions
The delivery of chemotherapy concurrently with radiation has become a common therapeutic strategy in locally advanced tumors. The concurrent chemoradiation regimen has contributed significantly to improved clinical outcomes, measured by superior local control, overall survival, and organ preservation. Continued progress in our understanding of the underlying biology contributing to unregulated cellular growth and therapeutic resistance of tumors has fostered the development of an array of novel, molecularly targeted agents, which, in addition to demonstrating independent antitumor activity, have also demonstrated the capacity to enhance both chemotherapeutic and radiation-induced cell death. A major objective of current investigations is to integrate these molecularly targeted agents into established chemoradiation platforms in an effort to further improve clinical outcomes. Translational research designed to identify and apply more relevant tumor model systems and identify specific patient populations that will likely respond to a particularly regimen is critical for continued progress in cancer therapy.

References

1 Steel GG, Peckham MJ. Exploitable mechanisms in combined radiotherapy-chemotherapy: the concept of additivity. Int J Radiat Oncol Biol Phys . 1979;5(1):85-91.
2 Bentzen SM, Harari PM, Bernier J. Exploitable mechanisms for combining drugs with radiation: concepts, achievements and future directions. Nat Clin Pract Oncol . 2007;4(3):172-180.
3 Bentzen SM. Repopulation in radiation oncology: perspectives of clinical research. Int J Radiat Biol . 2003;79(7):581-585.
4 Kim JJ, Tannock IF. Repopulation of cancer cells during therapy: an important cause of treatment failure. Nat Rev Cancer . 2005;5(7):516-525.
5 De Ruysscher D, Pijls-Johannesma M, Bentzen SM, et al. Time between the first day of chemotherapy and the last day of chest radiation is the most important predictor of survival in limited-disease small-cell lung cancer. J Clin Oncol . 2006;24(7):1057-1063.
6 Nishimura Y. Rationale for chemoradiotherapy. Int J Clin Oncol . 2004;9(6):414-420.
7 Harari PM, Huang SM. Head and neck cancer as a clinical model for molecular targeting of therapy: combining EGFR blockade with radiation. Int J Radiat Oncol Biol Phys . 2001;49(2):427-433.
8 Bentzen SM, Overgaard M, Thames HD, et al. Early and late normal-tissue injury after postmastectomy radiotherapy alone or combined with chemotherapy. Int J Radiat Biol . 1989;56(5):711-715.
9 Denis F, Garaud P, Bardet E, et al. Late toxicity results of the GORTEC 94-01 randomized trial comparing radiotherapy with concomitant radiochemotherapy for advanced-stage oropharynx carcinoma: comparison of LENT/SOMA, RTOG/EORTC, and NCI-CTC scoring systems. Int J Radiat Oncol Biol Phys . 2003;55(1):93-98.
10 Trotti A, Bentzen SM. The need for adverse effects reporting standards in oncology clinical trials. J Clin Oncol . 2004;22(1):19-22.
11 Bentzen SM. Preventing or reducing late side effects of radiation therapy: radiobiology meets molecular pathology. Nat Rev Cancer . 2006;6(9):702-713.
12 Trotti A. Toxicity antagonists in head and neck cancer. Semin Radiat Oncol . 1998;8(4):282-291.
13 Spielberger R, Stiff P, Bensinger W, et al. Palifermin for oral mucositis after intensive therapy for hematologic cancers. N Engl J Med . 2004;351(25):2590-2598.
14 Brizel DM, Wasserman TH, Henke M, et al. Phase III randomized trial of amifostine as a radioprotector in head and neck cancer. J Clin Oncol . 2000;18(19):3339-3345.
15 Movsas B, Scott C, Langer C, et al. Randomized trial of amifostine in locally advanced non-small-cell lung cancer patients receiving chemotherapy and hyperfractionated radiation: radiation therapy oncology group trial 98-01. J Clin Oncol . 2005;23(10):2145-2154.
16 Peters KB, Brown JM. Tirapazamine: a hypoxia-activated topoisomerase II poison. Cancer Res . 2002;62(18):5248-5253.
17 Jain RK. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science . 2005;307(5706):58-62.
18 Hall EJ. Radiobiology for the radiologist . Philadelphia: Lippincott Williams & Wilkins; 1993.
19 Begg AC. Rodent Tumor Models in Experimental Cancer Therapy. Principles and Practices of the Tumor Growth Delay Assay . 1987:114-121.
20 Suit HD, Sedlacek R, Thames HDJr. Radiation dose-response assays of tumor control. principles and practices of the tumor growth delay assay . 1987:138-148.
21 Mason KA, Hunter NR, Milas M, et al. Docetaxel enhances tumor radioresponse in vivo. Clin Cancer Res . 1997;3(12 Pt 1):2431-2438.
22 Krause M, Zips D, Thames HD, et al. Preclinical evaluation of molecular-targeted anticancer agents for radiotherapy. Radiother Oncol . 2006;80(2):112-122.
23 Morikawa K, Walker SM, Nakajima M, et al. Influence of organ environment on the growth, selection, and metastasis of human colon carcinoma cells in nude mice. Cancer Res . 1988;48(23):6863-6871.
24 Nakajima M, Morikawa K, Fabra A, et al. Influence of organ environment on extracellular matrix degradative activity and metastasis of human colon carcinoma cells. J Nat Cancer Inst . 1990;82(24):1890-1898.
25 Yokoi K, Sasaki T, Bucana CD, et al. Simultaneous inhibition of EGFR, VEGFR, and platelet-derived growth factor receptor signaling combined with gemcitabine produces therapy of human pancreatic carcinoma and prolongs survival in an orthotopic nude mouse model. Cancer Res . 2005;65(22):10371-10380.
26 Singh NP, McCoy MT, Tice RR, et al. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res . 1988;175(1):184-191.
27 Olive PL. DNA damage and repair in individual cells: applications of the comet assay in radiobiology. Int J Radiat Biol . 1999;75(4):395-405.
28 Banath JP, Macphail SH, Olive PL. Radiation sensitivity, H2AX phosphorylation, and kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lines. Cancer Res . 2004;64(19):7144-7149.
29 Olive PL, Banath JP. Phosphorylation of histone H2AX as a measure of radiosensitivity. Int J Radiat Oncol Biol Phys . 2004;58(2):331-335.
30 Taneja N, Davis M, Choy JS, et al. Histone H2AX phosphorylation as a predictor of radiosensitivity and target for radiotherapy. J Biol Chem . 2004;279(3):2273-2280.
31 Rothkamm K, Lobrich M. Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses. Proc Natl Acad Sci U S A . 2003;100(9):5057-5062.
32 Carson CT, Schwartz RA, Stracker TH, et al. The Mre11 complex is required for ATM activation and the G2/M checkpoint. EMBO J . 2003;22(24):6610-6620.
33 Xu B, Kim ST, Lim DS, Kastan MB. Two molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation. Mol Cell Biol . 2002;22(4):1049-1059.
34 Gray LH, Conger AD, Ebert M, et al. The concentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherapy. Br J Radiol . 1953;26(312):638-648.
35 Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer . 2003;3(10):721-732.
36 Withers HR, Taylor JM, Maciejewski B. The hazard of accelerated tumor clonogen repopulation during radiotherapy. Acta Oncol . 1988;27(2):131-146.
37 Fu KK, Pajak TF, Trotti A, et al. A Radiation Therapy Oncology Group (RTOG) phase III randomized study to compare hyperfractionation and two variants of accelerated fractionation to standard fractionation radiotherapy for head and neck squamous cell carcinomas: first report of RTOG 9003. Int J Radiat Oncol Biol Phys . 2000;48(1):7-16.
38 Rosenberg B, VanCamp L, Trosko JE, et al. Platinum compounds: a new class of potent antitumour agents. Nature . 1969;222(5191):385-386.
39 Eastman A, Barry MA. Interaction of trans-diamminedichloroplatinum(II) with DNA: formation of monofunctional adducts and their reaction with glutathione. Biochemistry . 1987;26(12):3303-3307.
40 Jones JC, Zhen WP, Reed E, et al. Gene-specific formation and repair of cisplatin intrastrand adducts and interstrand cross-links in Chinese hamster ovary cells. J Biol Chem . 1991;266(11):7101-7107.
41 Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene . 2003;22(47):7265-7279.
42 Jordan P, Carmo-Fonseca M. Molecular mechanisms involved in cisplatin cytotoxicity. Cell Mol Life Sci . 2000;57(8–9):1229-1235.
43 Damia G, Filiberti L, Vikhanskaya F, et al. Cisplatinum and Taxol induce different patterns of p53 phosphorylation. Neoplasia . 2001;3(1):10-16.
44 Zhao H, Piwnica-Worms H. ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol Cell Biol . 2001;21(13):4129-4139.
45 Agner J, Falck J, Lukas J, et al. Differential impact of diverse anticancer chemotherapeutics on the Cdc25A-degradation checkpoint pathway. Exp Cell Res . 2005;302(2):162-169.
46 Wilson GD. Radiation and the cell cycle, revisited. Cancer Metastasis Rev . 2004;23(3–4):209-225.
47 Appella E, Anderson CW. Post-translational modifications and activation of p53 by genotoxic stresses. Eur J Biochem . 2001;268(10):2764-2772.
48 Losa JH, Parada Cobo C, Viniegra JG, et al. Role of the p38 MAPK pathway in cisplatin-based therapy. Oncogene . 2003;22(26):3998-4006.
49 Myint WK, Ng C, Raaphorst GP. Examining the non-homologous repair process following cisplatin and radiation treatments. Int J Radiat Biol . 2002;78(5):417-424.
50 Gorodetsky R, Levy-Agababa F, Mou X, et al. Combination of cisplatin and radiation in cell culture: effect of duration of exposure to drug and timing of irradiation. Int J Cancer . 1998;75(4):635-642.
51 Bartelink H, Kallman RF, Rapacchietta D, et al. Therapeutic enhancement in mice by clinically relevant dose and fractionation schedules of cis-diamminedichloroplatinum (II) and irradiation. Radiother Oncol . 1986;6(1):61-74.
52 Kanazawa H, Rapacchietta D, Kallman RF. Schedule-dependent therapeutic gain from the combination of fractionated irradiation and cis-diamminedichloroplatinum (II) in C3H/Km mouse model systems. Cancer Res . 1988;48(11):3158-3164.
53 Dolling JA, Boreham DR, Brown DL, et al. Cisplatin-modification of DNA repair and ionizing radiation lethality in yeast, Saccharomyces cerevisiae. Mutat Res . 1999;433(2):127-136.
54 Haveman J, Castro Kreder N, Rodermond HM, et al. Cellular response of x-ray sensitive hamster mutant cell lines to gemcitabine, cisplatin and 5-fluorouracil. Oncol Rep . 2004;12(1):187-192.
55 Frit P, Canitrot Y, Muller C, et al. Cross-resistance to ionizing radiation in a murine leukemic cell line resistant to cis-dichlorodiammineplatinum(II): role of Ku autoantigen. Mol Pharmacol . 1999;56(1):141-146.
56 Turchi JJ, Henkels KM, Zhou Y. Cisplatin-DNA adducts inhibit translocation of the Ku subunits of DNA-PK. Nucleic Acids Res . 2000;28(23):4634-4641.
57 Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer . 2003;3(5):330-338.
58 Shewach DS, Lawrence TS. Antimetabolite radiosensitizers. J Clin Oncol . 2007;25(26):4043-4050.
59 Ghoshal K, Jacob ST. Specific inhibition of pre-ribosomal RNA processing in extracts from the lymphosarcoma cells treated with 5-fluorouracil. Cancer Res . 1994;54(3):632-636.
60 Kanamaru R, Kakuta H, Sato T, et al. The inhibitory effects of 5-fluorouracil on the metabolism of preribosomal and ribosomal RNA in L-1210 cells in vitro. Cancer Chemother Pharmacol . 1986;17(1):43-46.
61 Randerath K, Tseng WC, Harris JS, et al. Specific effects of 5-fluoropyrimidines and 5-azapyrimidines on modification of the 5 position of pyrimidines, in particular the synthesis of 5-methyluracil and 5-methylcytosine in nucleic acids. Recent Results Cancer Res . 1983;84:283-297.
62 Santi DV, Hardy LW. Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase: evidence for covalent catalysis. Biochemistry . 1987;26(26):8599-8606.
63 Doong SL, Dolnick BJ. 5-Fluorouracil substitution alters pre-mRNA splicing in vitro. J Biol Chem . 1988;263(9):4467-4473.
64 Patton JR. Ribonucleoprotein particle assembly and modification of U2 small nuclear RNA containing 5-fluorouridine. Biochemistry . 1993;32(34):8939-8944.
65 Byfield JE. 5-Fluorouracil radiation sensitization—a brief review. InvestNew Drugs . 1989;7(1):111-116.
66 Wilson GD, Bentzen SM, Harari PM. Biologic basis for combining drugs with radiation. Semin Radiat Oncol . 2006;16(1):2-9.
67 Lawrence TS, Blackstock AW, McGinn C. The mechanism of action of radiosensitization of conventional chemotherapeutic agents. Semin Radiat Oncol . 2003;13(1):13-21.
68 Hotta T, Taniguchi K, Kobayashi Y, et al. Increased expression of thymidine phosphorylase in tumor tissue in proportion to TP-expression in primary normal tissue. Oncol Rep . 2004;12(3):539-541.
69 Nishimura G, Terada I, Kobayashi T, et al. Thymidine phosphorylase and dihydropyrimidine dehydrogenase levels in primary colorectal cancer show a relationship to clinical effects of 5′-deoxy-5-fluorouridine as adjuvant chemotherapy. Oncol Rep . 2002;9(3):479-482.
70 Barton-Burke M. Gemcitabine: a pharmacologic and clinical overview. Cancer Nurs . 1999;22(2):176-183.
71 Sawada N, Ishikawa T, Sekiguchi F, et al. X-ray irradiation induces thymidine phosphorylase and enhances the efficacy of capecitabine (Xeloda) in human cancer xenografts. Clin Cancer Res . 1999;5(10):2948-2953.
72 Lawrence TS, Chang EY, Hahn TM, et al. Delayed radiosensitization of human colon carcinoma cells after a brief exposure to 2′,2′-difluoro-2′-deoxycytidine (Gemcitabine). Clin Cancer Res . 1997;3(5):777-782.
73 Shewach DS, Lawrence TS. Radiosensitization of human tumor cells by gemcitabine in vitro. Semin Oncol . 1995;22(4 Suppl 11):68-71.
74 Huang P, Chubb S, Hertel LW, et al. Action of 2′,2′-difluorodeoxycytidine on DNA synthesis. Cancer Res . 1991;51(22):6110-6117.
75 Plunkett W, Huang P, Xu YZ, et al. Gemcitabine: metabolism, mechanisms of action, and self-potentiation. Semin Oncol . 1995;22(4 Suppl 11):3-10.
76 Shewach DS, Hahn TM, Chang E, et al. Metabolism of 2′,2′-difluoro-2′-deoxycytidine and radiation sensitization of human colon carcinoma cells. Cancer Res . 1994;54(12):3218-3223.
77 Zhou BS, Hsu NY, Pan BC, et al. Overexpression of ribonucleotide reductase in transfected human KB cells increases their resistance to hydroxyurea: M2 but not M1 is sufficient to increase resistance to hydroxyurea in transfected cells. Cancer Res . 1995;55(6):1328-1333.
78 Ostruszka LJ, Shewach DS. The role of cell cycle progression in radiosensitization by 2′,2′-difluoro-2′-deoxycytidine. Cancer Res . 2000;60(21):6080-6088.
79 Denny BJ, Wheelhouse RT, Stevens MF, et al. NMR and molecular modeling investigation of the mechanism of activation of the antitumor drug temozolomide and its interaction with DNA. Biochemistry . 1994;33(31):9045-9051.
80 Danson SJ, Middleton MR. Temozolomide: a novel oral alkylating agent. Expert Rev Anticancer Ther . 2001;1(1):13-19.
81 Ostermann S, Csajka C, Buclin T, et al. Plasma and cerebrospinal fluid population pharmacokinetics of temozolomide in malignant glioma patients. Clin Cancer Res . 2004;10(11):3728-3736.
82 Hegi ME, Diserens AC, Gorlia T, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med . 2005;352(10):997-1003.
83 Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med . 2005;352(10):987-996.
84 Kil WJ, Cerna D, Burgan WE, et al. In vitro and in vivo radiosensitization induced by the DNA methylating agent temozolomide. Clin Cancer Res . 2008;14(3):931-938.
85 Chakravarti A, Erkkinen MG, Nestler U, et al. Temozolomide-mediated radiation enhancement in glioblastoma: a report on underlying mechanisms. Clin Cancer Res . 2006;12(15):4738-4746.
86 Brizel DM. Pharmacologic approaches to radiation protection. J Clin Oncol . 2007;25(26):4084-4089.
87 Heidelberger C, Chaudhuri NK, Danneberg P, et al. Fluorinated pyrimidines, a new class of tumour-inhibitory compounds. Nature . 1957;179(4561):663-666.
88 Kramer S. Use of methotrexate and radiation therapy for advanced cancer of the head and neck. Front Radiat Ther Oncol . 4(116), 1969.
89 Dillman RO, Herndon J, Seagren SL, et al. Improved survival in stage III non-small-cell lung cancer: seven-year follow-up of cancer and leukemia group B (CALGB) 8433 trial. J Natl Cancer Inst . 1996;88(17):1210-1215.
90 Sause WT, Scott C, Taylor S, et al. Radiation Therapy Oncology Group (RTOG) 88-08 and Eastern Cooperative Oncology Group (ECOG) 4588: preliminary results of a phase III trial in regionally advanced, unresectable non-small-cell lung cancer. J Natl Cancer Inst . 1995;87(3):198-205.
91 Furuse K, Fukuoka M, Kawahara M, et al. Phase III study of concurrent versus sequential thoracic radiotherapy in combination with mitomycin, vindesine, and cisplatin in unresectable stage III non-small-cell lung cancer. J Clin Oncol . 1999;17(9):2692-2699.
92 Choy H, Akerley W, Safran H, et al. Multiinstitutional phase II trial of paclitaxel, carboplatin, and concurrent radiation therapy for locally advanced non-small-cell lung cancer. J Clin Oncol . 1998;16(10):3316-3322.
93 Turrisi AT, Kim K, Blum R, et al. Twice-daily compared with once-daily thoracic radiotherapy in limited small-cell lung cancer treated concurrently with cisplatin and etoposide. N Engl J Med . 1999;340(4):265-271.
94 Pignon JP, Bourhis J, Domenge C, et al. Chemotherapy added to locoregional treatment for head and neck squamous-cell carcinoma: three meta-analyses of updated individual data. MACH-NC Collaborative Group. Meta-Analysis of Chemotherapy on Head and Neck Cancer. Lancet . 2000;355(9208):949-955.
95 Pignon JP, le Maitre A, Bourhis J. Meta-Analyses of Chemotherapy in Head and Neck Cancer (MACH-NC): an update. Int J Radiat Oncol Biol Phys . 2007;69(2 Suppl):S112-114.
96 Denis F, Garaud P, Bardet E, et al. Final results of the 94-01 French Head and Neck Oncology and Radiotherapy Group randomized trial comparing radiotherapy alone with concomitant radiochemotherapy in advanced-stage oropharynx carcinoma. J Clin Oncol . 2004;22(1):69-76.
97 Al-Sarraf M, LeBlanc M, Giri PG, et al. Chemoradiotherapy versus radiotherapy in patients with advanced nasopharyngeal cancer: phase III randomized Intergroup study 0099. J Clin Oncol . 1998;16(4):1310-1317.
98 Forastiere AA, Goepfert H, Maor M, et al. Concurrent chemotherapy and radiotherapy for organ preservation in advanced laryngeal cancer. N Engl J Med . 2003;349(22):2091-2098.
99 Bonner JA, Harari PM, Giralt J, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med . 2006;354(6):567-578.
100 Moore DH. Chemotherapy for advanced, recurrent, and metastatic cervical cancer. J Natl Compr Canc Netw . 2008;6(1):53-57.
101 Monk BJ, Tewari KS, Koh WJ. Multimodality therapy for locally advanced cervical carcinoma: state of the art and future directions. J Clin Oncol . 2007;25(20):2952-2965.
102 Peters WA, Liu PY, Barrett RJ, et al. Concurrent chemotherapy and pelvic radiation therapy compared with pelvic radiation therapy alone as adjuvant therapy after radical surgery in high-risk early-stage cancer of the cervix. J Clin Oncol . 2000;18(8):1606-1613.
103 Cooper JS, Guo MD, Herskovic A, et al. Chemoradiotherapy of locally advanced esophageal cancer: long-term follow-up of a prospective randomized trial (RTOG 85-01). Radiation Therapy Oncology Group. JAMA . 1999;281(17):1623-1627.
104 Macdonald JS, Smalley SR, Benedetti J, et al. Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction. N Engl J Med . 2001;345(10):725-730.
105 Gastrointestinal Tumor Study Group. Prolongation of the disease-free interval in surgically treated rectal carcinoma. Gastrointestinal Tumor Study Group. N Engl J Med . 1985;312(23):1465-1472.
106 O’Connell MJ, Martenson JA, Wieand HS, et al. Improving adjuvant therapy for rectal cancer by combining protracted-infusion fluorouracil with radiation therapy after curative surgery. N Engl J Med . 1994;331(8):502-507.
107 Sauer R, Becker H, Hohenberger W, et al. Preoperative versus postoperative chemoradiotherapy for rectal cancer. N Engl J Med . 2004;351(17):1731-1740.
108 Nigro ND, Vaitkevicius VK, Considine BJr. Combined therapy for cancer of the anal canal: a preliminary report. Dis Colon Rectum . 1974;17(3):354-356.
109 Leichman L, Nigro N, Vaitkevicius VK, et al. Cancer of the anal canal. Model for preoperative adjuvant combined modality therapy. Am J Med . 1985;78(2):211-215.
110 Epidermoid anal cancer: results from the UKCCCR randomised trial of radiotherapy alone versus radiotherapy, 5-fluorouracil, and mitomycin. UKCCCR Anal Cancer Trial Working Party. UK Co-ordinating Committee on Cancer Research. Lancet . 1996;348(9034):1049-1054.
111 Bartelink H, Roelofsen F, Eschwege F, et al. Concomitant radiotherapy and chemotherapy is superior to radiotherapy alone in the treatment of locally advanced anal cancer: results of a phase III randomized trial of the European Organization for Research and Treatment of Cancer Radiotherapy and Gastrointestinal Cooperative Groups. J Clin Oncol . 1997;15(5):2040-2049.
112 Ajani JA, Winter KA, Gunderson LL, et al. Fluorouracil, mitomycin, and radiotherapy vs fluorouracil, cisplatin, and radiotherapy for carcinoma of the anal canal: a randomized controlled trial. JAMA . 2008;299(16):1914-1921.
113 Stewart LA. Chemotherapy in adult high-grade glioma: a systematic review and meta-analysis of individual patient data from 12 randomised trials. Lancet . 2002;359(9311):1011-1018.
7 Principles of Radiation Physics

Lynn J. Verhey, PhD, Paula L. Petti, PhD
In this chapter, we present the basic radiation physics needed for the practice of radiation oncology. Many of the specific applications of these basic physics principles, such as brachytherapy and treatment planning, are presented elsewhere in this textbook. Since the primary audience for this textbook is assumed to be practicing radiation oncologists and resident clinicians, many topics are not treated in the detail that would be required by radiation physicists. Excellent physics textbooks that present the topics in more depth are available. 1 - 4

Basic Concepts

Atomic and Nuclear Structure
Matter is composed of individual elements distinguishable from each other according to their physical and chemical properties. The fundamental building block for these elements is the individual atom, which consists of a small central core, called the nucleus , surrounded by diffuse clouds of electrons. The nucleus, containing almost all the mass in the atom, consists of uncharged neutrons (mass of 1.675 × 10 −27  kg) and positively charged protons (mass of 1.673 ×10 −27  kg), bound together by the strongly attractive nuclear force. The electrons (mass of 9.11 × 10 −31  kg) are negatively charged particles with the same absolute value of charge as the protons. Since the atoms have the same number of electrons and protons, they are electrically neutral. The radius of the atom is about 10 −10  m, whereas the radius of the nucleus is about 10 −14  m.
The atom is completely specified by the atomic number Z, which is the number of protons in the nucleus (or electrons in the atom), and the mass number A, which is the number of neutrons plus protons in the nucleus. Obviously, the number of neutrons in the nucleus, N, is just A – Z. Element X is specified as . Isotopes of an element have the same Z but different A and, therefore, have the same chemical properties but can have different physical properties. Isobars are atoms with the same A but different Z, and isotones are atoms with different A and Z but the same N.
In the planetary model of the atom, first described by Niels Bohr, the electron orbits make up discrete concentric shells, with a different binding energy associated with each shell. The shells are labeled K, L, M, and so on from the innermost to the outermost shell. The maximum number of electrons allowed in each shell is 2n 2 , where n = 1 for the K shell, n = 2 for the L shell, and so on. Therefore, there are up to 2 electrons in the K shell, 8 in the L shell, 18 in the M shell, and so on. A simple schematic drawing of the Bohr model is shown in Fig. 7-1 .

FIGURE 7-1 • Schematic drawing of the Bohr model of the atom. The nucleus contains neutrons and protons, bound together by the attractive nuclear force. The nucleus is surrounded by electrons moving in specific orbits with discrete energy levels. By convention, the orbits are labeled K, L, M, and so on, from inside out.
(From Perez CA, Brady LW: Principles and practice of radiation oncology, ed 2, Philadelphia, 1992, JB Lippincott, p 184.)
The electrons are bound to the protons in the nucleus by the attractive electromagnetic force; therefore, the inner K orbit electrons are bound most tightly because they are closest. The electrons in the outer orbit are called valence electrons and they are typically bound very loosely. The process of excitation is the movement of an electron from an inner orbit to an outer orbit. This requires the addition of energy to the atom. If an electron is completely removed from an atom, the process is called ionization. The energy required to just barely remove an electron from an atom is called the binding energy. In a heavy atom such as tungsten, the binding energy of K shell electrons is about 70,000 electron volts (eV), whereas the valence electrons have binding energies of a few eV. Note that 1 eV is defined as the energy acquired by an electron accelerated through a potential difference of 1 volt, and 1 eV = 1.602 × 10 −19 Joules.

Electromagnetic Radiation
Electromagnetic radiation is energy transmitted at a fixed velocity through sinusoidally varying electric and magnetic fields. The frequency of variation of this energy, represented by the Greek letter ν, is the number of oscillations per second, measured in hertz (Hz). The wavelength, λ, is the distance in meters between two crests of the sine wave. The velocity of propagation of the radiation is the product of the wavelength and the frequency, which, in a vacuum, is equal to the speed of light, c = 3 × 10 8  m/sec.
The range of wavelengths encountered in conventional physics is from 10 5  m for AM radio waves, to 10 −7  m for visible light, to 10 −12  m for x-rays and cosmic rays. Although electromagnetic radiation is conventionally described as waves of energy, quantum physics tells us that it is equally valid to describe the radiation as particle-like packets of energy called photons. Experiments that scatter x-rays off particles have been used to validate this concept. In general, the shorter wavelength radiation is more “particle-like” than the longer wavelength radiation. The energy of a photon is directly proportional to the frequency of the radiation, with a constant of proportionality called Planck’s constant. That is, E = hν, where h = 6.626 × 10 −34  J/s and the energy is in Joules.

Nuclear Transformations
Henri Becquerel discovered natural radioactivity in 1896 when he observed the blackening of wrapped photographic plates when they were placed in contact with certain elements. This event was preceded by the discovery of an invisible form of energy, dubbed x-rays, by Röntgen, who observed the glowing of fluorescent material placed near a gas discharge tube.
We now understand that Becquerel observed the natural decay of radioactive nuclei into one of three types of radiation: positively charged alpha particles (α), which we now know are helium nuclei; negatively charged beta particles (β), which are electrons; and uncharged gamma-rays (γ), which are a type of electromagnetic radiation emitted from nuclei. It is entirely possible that Becquerel also observed other types of elementary particles that can exist in the nucleus, or can be produced in nuclear decay processes.
The explanation of nuclear decay depends on understanding the interplay between the very strong, attractive nuclear force that binds the neutrons and protons together in the nucleus, and the moderately strong repulsive electromagnetic force between the protons. As the protons and neutrons move about within the nucleus, there is some probability that one of them will acquire enough kinetic energy to escape from the nuclear potential energy “well.” Large nuclei with many neutrons and protons tend to be less stable because of the increasing repulsion of the protons. For large nuclei to be stable, an excess of neutrons is required to provide the nuclear attraction to keep the protons from escaping. Fig. 7-2 shows the line of stable nuclei for the range of known elements.

FIGURE 7-2 • Plot of numbers of neutrons and protons in stable nuclei.
(From Khan FM: The physics of radiation therapy, ed 3, Philadelphia, 2003, Lippincott Williams & Wilkins, p 4.)

Decay Constant
The decay of a large collection of nuclei can be described as a statistical phenomenon. At any moment in time, the number of nuclei disintegrating per unit of time is proportional to the number of nuclei available. Mathematically,

where N is the number of radioactive nuclei and λ is the decay constant. The minus sign indicates that the number of nuclei is decreasing with time. Integration of this differential equation leads to the following equation:

where N 0 is the number of radioactive nuclei initially present, N(t) represents the number remaining at time t, and e is the base of the natural logarithm (2.71828).
The rate of decay, previously referred to as dN/dt, is also called the activity of a radioactive substance, referred to by the symbol A. Then A = − λN and

where A 0 is the initial activity and A(t) is the activity at time t. The unit of activity is the Curie (Ci) or the Becquerel (Bq), which is 1 disintegration per second:

The Ci was originally defined to be the activity associated with 1 g of radium, although the number of disintegrations per second from 1 g of radium is now known to be somewhat less than 3.7 × 10 10 .

Half-Life and Mean Life
The half-life (T ½ ) of a radioactive substance is the time required for a population of atoms to decay to half the original number. That is,

Solving this equation,

the mean or average life (T λ ) is the time for the number of atoms to decay to 1/e of the initial number, so

The mean life can also be thought of as the time required for all the atoms to decay if the decay rate could be maintained at its initial value.

Modes of Radioactive Decay
Just as electrons have energy levels related to the orbit or shell they occupy, nucleons (protons and neutrons) are thought to occupy shells that represent discrete energy levels within the nucleus. When nucleons are excited into higher levels, they can subsequently decay back to their initial energy level, emitting a photon in the process. This process is called γ decay. Because of the very strong nature of nuclear forces, these excited states are very short-lived, with half-lives on the order of 10 −15 seconds. If, in the process of leaving the nucleus, the γ-decay photon transfers enough energy to an inner shell electron to eject it from the atom, the process is called internal conversion. The subsequent cascade of an outer shell electron into the vacated hole in the inner shell releases a characteristic x-ray photon with energy equal to the difference between the energy levels of the inner and outer shells. In some situations, the characteristic x-ray is absorbed and re-emitted by the atom in the form of an ejected orbital electron. Such electrons, emitted in lieu of x-rays, are called Auger electrons.
The decay of a nucleus accompanied by the ejection of an electron (e − ) or positron (e + ) is called β decay. In β (−) decay, a nucleus that has an excess of neutrons can reduce its energy by converting a neutron into a proton, an electron, and an antineutrino (which has energy but no mass). In β (+) decay, a neutron-deficient nucleus can become more stable by converting a proton into a neutron, a positron, and a neutrino. The typical disintegration energy of 1 to 3 MeV is shared between the emitted particles, the neutrino (antineutrino), and positron (electron). For heavy nuclei, the average binding energy of the nucleons inside the nucleus may be less than 10 MeV. Half-lives for β decay can range from a few seconds to many years.
A second way in which neutron-deficient nuclei can gain stability is by the process of electron capture. In this process, an electron in one of the inner shells is captured by the nucleus, thus, transforming a proton into a neutron. That is, a proton plus the captured electron is converted to a neutron plus a neutrino. In general, the inner electron orbital vacancy is quickly filled with an electron from an outer shell, resulting in the emission of characteristic x-rays or Auger electrons. Very heavy nuclei with Z > 82 frequently decay with the emission of an α particle, which is identical to the helium nucleus and is composed of two protons and two neutrons tightly bound together. The binding energy of this configuration is particularly high, making this decay mode a very efficient way to decrease the energy and increase the stability of a heavy nucleus. From a specific nuclide, the α decay is monoenergetic, that is, the α particle is emitted with a single, well-defined energy. Typically, α particles are emitted with kinetic energies of between 5 and 10 MeV.

Radioactive Decay Series and Equilibrium
A total of 106 elements are known. Of these, the first 92 occur naturally; that is, they are either stable or radioactive with half-lives long enough that they can still be found naturally in trace amounts on the earth. Elements 93 to 106 are very unstable and can only be produced artificially in accelerators with heavy ion bombardment of heavy nuclei. Lead, with Z = 82 is the heaviest known element that has completely stable isotopes. All naturally occurring isotopes of elements with Z between 83 and 92 are members of one of three radioactive series referred to as the uranium (Z = 92) series, the actinium (Z = 89) series, and the thorium (Z = 90) series. The name of each series refers to the parent, a long-lived nuclide that decays into a daughter nuclide, that then decays into another daughter, followed by successive transformations that continue until a stable isotope of lead is reached.
In each series, the half-life of the parent is longer than any of the half-lives of any of the daughter nuclides. This phenomenon leads to a state of transient or secular equilibrium. Since each daughter is less stable than the original parent, the activity of the daughter will increase with time, until it approaches the activity of the long-lived parent. If the half-lives are very different, the activities will become essentially identical after a number of daughter half-lives, and this is called secular equilibrium. An example is shown in Fig. 7-3 . If the half-lives of the parent and daughter are very similar, transient equilibrium is reached in a few half-lives, after which the daughter has an activity in excess of the parent activity but decays with the same rate as the limiting parent decay rate. An example of this phenomenon is shown in Fig. 7-4 . For either case, after a time that is long compared to the half-life of the shorter-lived nuclide,

FIGURE 7-3 • Example of secular equilibrium in the decay of 226 R a to 222 R n .
(From Khan FM: The physics of radiation therapy, ed 3, Philadelphia, 2003, Lippincott Williams & Wilkins, p 19.)

FIGURE 7-4 • Example of transient equilibrium in the decay of 99 Mo to 99m Tc. This figure is drawn as if 100% of the 99 Mo decays to 99m Tc. In fact, only about 88% of the 99 Mo atoms decay in this fashion, and in actuality the 99m Tc activity curve lies slightly below the 99 Mo curve.
(Redrawn from Khan FM: The physics of radiation therapy, ed 3, Philadelphia, Lippincott Williams & Wilkins, p 18.)

where A i and T i refer to the activity and half-life of the daughter (d) or parent (p).

X-Ray Production
Most radiotherapy is delivered with beams of x-rays that are produced as a result of the interactions of accelerated electrons with matter. This can be either through excitation or ionization of the atom via interactions of the accelerated electrons with target electrons leading to the emission of characteristic x-rays, mentioned earlier, or through direct interaction between the electrons and the electromagnetic field of the target nuclei, leading to bremsstrahlung x-rays.

Characteristic X-Rays
When electrons are incident on target atoms, they can ionize those atoms by depositing sufficient energy to eject an inner shell electron. The inner shell vacancy is subsequently filled by an outer shell electron, causing the emission of a characteristic x-ray with energy equal to the difference between the binding energies of the inner and outer shells. This process is diagrammed in Fig. 7-5 . Although this process can take place in both low-Z and high-Z atoms, only for high-Z atoms are the binding energies sufficient to produce radiation in the x-ray portion of the electromagnetic spectrum. For example, the binding energy of K-shell electrons in tungsten, a common target for x-ray tubes and accelerators, is about 70 keV, whereas for aluminum, it is only about 1.5 keV. As mentioned earlier, the characteristic x-ray energy is occasionally transferred directly to an orbital electron, leading to the production of an Auger electron.

FIGURE 7-5 • Illustration of the production of characteristic radiation.
(From Khan FM: The physics of radiation therapy, ed 3, Philadelphia, Lippincott Williams & Wilkins, p 35.)

Bremsstrahlung X-Rays
When high-energy electrons interact directly with the electromagnetic field of a target nucleus, they are deflected and lose a portion of their energy due to deceleration. The energy lost is then emitted in the form of radiation called bremsstrahlung (literally, braking radiation ).
Since the incident electron can lose any portion of its energy in this process, the energy of these bremsstrahlung x-rays can vary continuously from nearly zero to the full energy of the incident electron. The angle of the x-rays, relative to the beam direction, depends on the energy of the electron. At low energies typical of those used for diagnostic radiology, the x-ray has equal probability of being emitted in any direction, whereas at high energies typically used for radiotherapy, the x-rays are emitted preferentially in the forward direction. These facts explain the use of thick targets and 90-degree angles between electrons and x-rays for diagnostic applications and thin transmission targets for radiotherapy.
The probability of bremsstrahlung production varies with Z 2 of the target material, whereas the efficiency of x-ray production depends on the product of Z and E, the energy of the electrons. At 100 keV in a tungsten target, only about 1% of the incident energy of the beam is converted into x-rays, the remainder ending up in heat.
In general, both bremsstrahlung and characteristic x-rays are present when electrons strike a target. Fig. 7-6 shows x-ray spectra for a thick tungsten target for electron energies of from 65 keV to 200 keV. Note the appearance of the characteristic x-rays on top of the bremsstrahlung spectrum once the electron energy is in excess of the K-shell binding energy of 70 keV. Any material in the x-ray beam, including the target itself, will absorb some of the energy of the beam. The effect of such absorption is to preferentially filter out the low energy portion of the bremsstrahlung spectrum. The process of preferentially attenuating the low-energy component of the beam is referred to as “beam hardening.” When the beam is hardened, the average energy of the x-rays increases at the expense of reduced intensity.

FIGURE 7-6 • Spectral distribution of x-rays calculated for a thick tungsten target. Dotted curves are for no filtration and solid curves are for a filtration of 1 mm aluminum. Note the characteristic radiation peaks on top of the continuous bremsstrahlung spectra.
(From Khan FM: The physics of radiation therapy, ed 3, Philadelphia, Lippincott Williams & Wilkins, p 36.)

Interaction of X-Rays and Particles With Matter

X-Ray Interactions
As described earlier, x-rays can be considered as packets of energy, called photons, for purposes of considering their interactions with matter. There are a number of different interaction processes that the photons can experience, and each of these has a probability described by an attenuation coefficient. Mathematically, this can be written as

where dN is the reduction in the number of photons due to interactions in a thickness dx of an absorber, N is the number of incident photons, and µ i is the attenuation coefficient for the ith interaction process. After integration over the thickness, this equation becomes

where N(x) is the number of photons left in the beam (without interaction) as a function of the thickness x of material traversed, and µ tot is the sum of the individual attenuation coefficients. (µ i /ρ) is called the mass-attenuation coefficient for process i, where ρ is the density of the material. Dividing the conventional attenuation coefficients by density removes the dependence on the physical density of materials and the resulting mass attenuation coefficients are approximately equal from material to material. The corresponding thickness, ρx, is in units of grams per square centimeter (g/cm 2 ). The total mass attenuation coefficients for water and lead are shown as functions of energy in Fig. 7-7 . There are five different photon interaction processes, including coherent scattering, photoelectric effect, Compton scattering, pair production, and photodisintegration. These will be discussed in turn.

FIGURE 7-7 • Mass attenuation coefficients for lead and water. The sharp discontinuities in the lead curve are due to absorption edges.
(From Johns HE, Cunningham JR: The physics of radiology, ed 4, Springfield, IL, 1983, Charles C. Thomas.)
The thickness of material that reduces the number of transmitted photons to one half the initial value is referred to as the half-value layer (HVL) and depends on the nature of the material and the energy of the photons. This occurs when

implying that

Although not constant with depth, HVL is often used to describe the quality of the beam or its ability to penetrate material.

Coherent Scattering
Coherent scattering, also called classical scattering, is a low-energy phenomenon in which the electromagnetic field of the incident photon interacts with the field of a bound electron in the atom without any transfer of energy. The bound electron is set into oscillation by the passing photon, but then reirradiates the energy of oscillation back to the outgoing photon. The net effect is only a change in direction for the photon. Since there is no transfer of energy, this process is of little importance to radiotherapy.

Photoelectric Effect
In the photoelectric effect, the energy of the photon is totally absorbed by the atom and subsequently transferred to an orbital electron, which is then ejected from the atom with an energy equal to the original energy of the photon minus the binding energy of the electron. The vacancy created by the ejection of the photoelectron from a shell gets filled by an electron from a shell with lower binding energy, followed by the emission of a characteristic x-ray with energy equal to the difference in binding energies of the two shells. Again, an Auger electron can be emitted in lieu of the x-ray in some cases.
The most likely angle of ejection of the photoelectron relative to the incident photon direction is 90 degrees for low-energy photons (50 keV or less), becoming smaller (more forward) as the photon energy increases. The mass attenuation coefficient for the photoelectric effect is proportional to Z 3 /E 3 , where Z is the atomic number of the target atom and E is the energy of the photon.
The photoelectric effect is important at diagnostic x-ray energies of 100 keV and is the basis for the radiographic contrast of bone versus soft tissue. However, because of the 1/E 3 energy dependence, the photoelectric effect is relatively unimportant at typical radiotherapy energies of several MeV.

Compton Scattering
Compton scattering is a process in which the incident photon interacts with an orbital electron as if it were a free particle, since the binding energy is small compared to the photon energy. The dynamics of the interaction can be described as a typical particle–particle scattering interaction whereby the photon transfers some of its energy to the electron and is scattered at an angle φ relative to the incident direction. The electron is ejected at an angle θ relative to the forward direction. The energy of the outgoing Compton-scattered photon is equal to the difference between the incident photon energy and the energy transferred to the electron. A diagram of the Compton process is shown in Fig. 7-8 .

FIGURE 7-8 • Illustration of the Compton effect.
(From Khan FM: The physics of radiation therapy, ed 3, Philadelphia, Lippincott Williams & Wilkins, p 67.)
As with any particle–particle scattering process, energy and momentum conservation can be applied to obtain relationships between energy and angle.
From conservation of energy:

From conservation of momentum:

and

where v e is the velocity of the electron, m e is the rest mass of the electron, c is the speed of light, and

Solving these three simultaneous equations, we obtain the following relationships:



where α = hν o / m e c 2 and f = (1 − cos φ).
From these equations, we can examine several limiting cases of the Compton effect. A grazing hit corresponds to θ = 90 degrees, in which case the photon is undeflected, and continues in the forward direction. A direct hit corresponds to θ = 0 degrees, meaning that φ = 180 degrees, or in other words, the photon scatters backwards. For intermediate situations, for example, when the photon is deflected by 90 degrees, the angle and energy of the electron depend on the energy of the photon. When α is much greater than 1 (high energy), θ is small and E is essentially equal to hν o . When α is much less than 1 (low energy), the photon energy remains nearly unchanged and the Compton process approaches that of coherent scattering.
The mass attenuation coefficient for Compton scattering is independent of Z, decreases slowly with photon energy, and is directly proportional to the number of electrons per gram, which varies by only 20% from the lightest to the heaviest elements (with the exception of hydrogen). Therefore, in the energy region where Compton processes dominate, the attenuation of the beam will vary according to the integrated density of the material traversed. This fact is responsible for the relatively poor contrast observed in portal verification films exposed with megavoltage x-rays exiting from the irradiated patient.

Pair Production
Pair production is an interaction of a photon with the electromagnetic field of a nucleus in which the energy of the photon is converted into an electron (e − ) and a positron (e + ). Since the rest mass of each of these particles is 0.511 MeV, the threshold energy for pair production is 1.02 MeV. The total shared kinetic energy of this pair of particles is just the photon energy minus 1.02 MeV. The mass attenuation coefficient for pair production increases logarithmically with energy above the threshold, and is proportional to Z 2 . Subsequent to the production of the electron-positron pair, the positron has a high probability of combining with a free electron during the process of energy loss in the medium and converting the combined e + -e − mass into a pair of annihilation photons, each of energy 0.511 MeV, that will leave the annihilation region in opposite directions.

Photodisintegration
At very high energies, the photon can deposit so much energy into the nucleus that partial or complete disintegration of the nucleus takes place. Since the binding energies of nucleons within the nucleus tend to be typically 7 MeV or higher, this process is of little importance at therapy energies. However, photodisintegration is a source of low-level neutron production that must be considered when designing radiation shielding around high-energy linear accelerators.

Relative Importance of Interaction Types
The total mass absorption coefficient is the sum of the coefficients for coherent, photoelectric, Compton, and pair production. For low-Z targets such as tissue (average Z = 7), Compton processes are overwhelmingly dominant throughout the range of energies used in therapy. Fig. 7-9 shows the relative importance of photoelectric, Compton, and pair production as a function of photon energy and the atomic number of the target material. 4 For tissue, Compton dominates between approximately 30 keV and 30 MeV, whereas for lead, photoelectric dominates up to approximately 800 keV and pair production above about 5 MeV. Because of its calcium content, bone has a higher absorption by photoelectric and pair production. This leads to high bone doses for orthovoltage and kilovoltage and for higher energy accelerator beams.

FIGURE 7-9 • Relative importance of the three main modes of energy loss for photons as a function of energy and atomic number of the medium.
(From Hendee WR: Medical radiation physics: roentgenology, nuclear medicine and ultrasound, Chicago, 1979, Year Book Medical Publishers, p 115.)

Charged Particles

Electrons
Electrons lose energy to the medium by two processes, ionization and radiation. Ionization processes are interactions with the atomic electrons, whereas radiation losses are interactions with the field of the nucleus, leading to bremsstrahlung x-ray production. Whereas the microscopic interactions are well understood, the macroscopic description of the energy and range of the particles at any point in the medium is not simple. This is because the electrons are very much lighter than the atomic nuclei. Therefore, the electron can lose a very large fraction of its energy in a single process and can be deflected by very large angles. This means that even if the electron beam is monoenergetic when entering a medium, there will be a large amount of range-straggling, or a large variation from electron to electron as to where, in the phantom, the electron will stop. Fig. 7-10 shows a plot of absorbed dose as a function of depth for monoenergetic electrons incident on water. There is a depth beyond which the dose is almost zero, where all the incident electrons have been stopped and where the remaining dose is due to bremsstrahlung x-rays produced by the electrons in the medium. This is in sharp contrast to the exponential falloff of dose for x-rays. This ranging out of electrons in matter is responsible for the popularity of electrons for treatment of superficial disease.

FIGURE 7-10 • Depth-dose curve for monoenergetic electrons incident on water. R 50 is the mean range and R p is the extrapolated or practical range.
(From Johns HE, Cunningham JR: The physics of radiology, ed 4, Springfield, IL, 1983, Charles C. Thomas.)

Protons and Heavier Ions
The use of protons and heavy ions for radiotherapy was pioneered in the 1970s and 1980s at the Massachusetts General Hospital in collaboration with the now decommissioned Harvard Cyclotron Laboratory and at the Lawrence Berkeley National Laboratory in Berkeley, California. 5 - 15 Since then, several proton and charged-particle facilities have been built around the world, a list of which can be found on the website for the Particle Therapy Co-Operative Group (PTCOG; http://ptcog.web.psi.ch/ ). Similar to electrons, protons lose energy primarily by electromagnetic interactions with the atomic electrons. A major difference between protons and electrons is that protons are much heavier than electrons and therefore lose only a very small fraction of their energy in an individual interaction, scattering minimally in the process. Protons lose energy at an increasing rate as they slow down, yielding an enhanced region of energy deposition called the Bragg Peak just before they stop. One can predict very accurately the depth at which the protons will come to rest if the initial energy of the protons and the electron density of the material traversed are known. The absence of exit dose beyond the intended target makes protons nearly ideally suited for optimal physical dose delivery. A typical depth dose distribution for a clinical, energy-modulated proton beam, compared to that of a 10-MV bremsstrahlung x-ray beam, is shown in Fig. 7-11 . A small percentage of proton interactions is via nuclear interactions with the nucleus. These rare interactions are qualitatively different than electromagnetic interactions and are thought to be responsible for enhancing the cell kill per unit dose by about 10% to 20% over that of x-rays. 16

FIGURE 7-11 • Depth-dose curve for a modulated proton beam of 160 MeV compared to that for a 10-MV bremsstrahlung x-ray beam. The region of the flat dose is referred to as the spread-out bragg peak (SOBP).
Heavy ions such as helium, carbon, neon, and argon nuclei have also been used in radiotherapy. 9 - 12 ,14 ,15 ,17 ,18 Similar to protons, they lose energy by interacting with the atomic electrons. Being even heavier than protons, they scatter even less and have even more rapid dose falloff outside the central beam and even faster falloff of dose beyond the end of range. In addition to these electromagnetic interactions, heavy ions have a probability of interacting with the nucleus via nuclear interactions that increases with the mass of the incident ion. In particular, as the heavy ions begin to slow down, they tend to suffer nuclear interactions in which they lose a very large amount of their energy in a single event. This high density of deposited energy kills cells in a far more efficient way per unit dose than x-rays. These particles are said to have a high radiobiologic efficiency (RBE). For more information on these particles, the reader is referred to the literature. 13

Neutrons
Neutrons have also been used in clinical treatments. 19 - 24 These particles are neutral, so they cannot lose energy by any means other than the nuclear interaction. All of their interactions are “catastrophic,” because a significant portion of their energy is deposited in an individual event. Their primary interactions are with protons within the nucleus. These nuclear events result in recoil protons and charged nuclear fragments that have rather low energy and deposit large amounts of energy very close to the site of the original interaction. The falloff of neutron dose with depth is exponential and similar to that of low-energy x-rays, with the depth of the 50% dose being around 10 cm for typical treatment energies. Neutrons are very efficient in producing cell kill per unit dose (high RBE) relative to x-rays for both tumor tissue and normal tissues. Clinical trials have been conducted to investigate the areas where neutrons might have an advantage over x-rays. A summary of the physical characteristics and results of early clinical results is available. 13
In addition, clinical trials are under way to investigate the use of slow neutrons, either thermal or epithermal in energy, to treat boronated tumor cells in patients. 25 - 32 The clinical efficacy of these neutrons for cancer therapy depends on the very high cross section for neutron capture by boron and on a high ratio of boron in the tumor cells to that in the surrounding normal cells. To date, clinical results of these trials have been disappointing.

Radiation Therapy Treatment Machines
Before about 1950, external beam radiation therapy was primarily carried out with x-ray beams produced in evacuated x-ray tubes by electrons accelerated with an electric field, impinging on a target and interacting with the nuclei of target atoms. The details of the interactions and the properties of the resulting x-ray beams are described elsewhere in this chapter. The maximum energy of these electrons and the photon beams that they produced was about 400 kV. Therefore, this period of radiation therapy history is called the kilovoltage era. Although most of these machines have been replaced in the intervening years with 60 Co teletherapy machines and electron linear accelerators, low-energy x-ray generators still play a limited role in the treatment of superficial disease.

Kilovoltage Units
A schematic diagram of an x-ray tube suitable for radiation therapy is shown in Fig. 7-12 . Since a high-voltage supply is used to generate the accelerating potential for the electron beams, a maximum energy of approximately 300 kV is achievable with this design. The resulting x-ray beams have a spectrum of photon energies with a maximum equal to the energy of the electron beam.

FIGURE 7-12 • Schematic diagram of an x-ray tube that could be used for radiation therapy.
(From Khan FM. The physics of radiation therapy, ed 3, Philadelphia, Lippincott Williams & Wilkins, p 29.)

Contact Therapy Machines
X-ray machines that operate at potentials of 40 to 50 kV are referred to as contact units. They typically operate at tube currents of 2 mA and the beams are usually filtered with 0.5 to 1.0 mm aluminum in order to remove the very low energy x-rays in the beam. Treating at a typical source-to-skin (SSD) distance of 2 cm, the dose in this beam drops off to 50% of its surface value in less than 5 mm of water or soft tissue. Such a beam would be useful only for the most superficial of targets.

Superficial Therapy Machines
Units with x-ray beams produced by electrons with energies between 50 and 150 kV are usually referred to as superficial therapy units. These units normally are filtered with 1 to 4 mm aluminum and treated at distances of 20 cm SSD. The 50% depth in water or soft tissue in this energy range would be typically 1 to 2 cm. By using thicker filters, it is possible to further harden the beam and move the 50% dose somewhat deeper with a reduction in dose rate. Superficial units can be very useful for treatment of skin lesions, using either regular fields defined by interchangeable cones or irregular fields defined with custom lead cutouts.

Orthovoltage Therapy Machines
Orthovoltage x-ray units are defined as those that operate in the 150 to 300 kV range. Typical SSDs are 50 cm, field sizes up to 20 × 20 cm and filters of 1 to 4 mm of copper. Depths of 50% are usually between 5 and 7 cm depending on filter thickness and field size. Regular fields are defined with detachable cones or adjustable collimators and irregular fields with lead cutouts or special hand blocking. Before 1950, these units were the workhorses of radiation therapy. Although useful for treating disease in thin sections of the body such as the neck, in thicker areas of the body, where tumors may lie 10 to 15 cm below the surface, the dose to normal tissue can be quite high when using beams in this energy range. Very few of these machines remain in current use in radiation therapy centers.

Megavoltage Units
X-ray and γ-ray beams of energy greater than 1 MV are classified as megavoltage beams. In the 1930s a number of transformer-based and van de Graaff generator–based units working in the 800 KeV to 2 MeV range were installed around the world. However, it was not until the introduction of the 60 Co teletherapy machine and the linear accelerator that the routine use of megavoltage x-ray and γ-ray beams occurred, significantly changing the practice of radiotherapy and leading to substantial improvements in clinical results. 33

Teletherapy Machines
The megavoltage era began with the introduction of the 60 Co teletherapy machine into radiotherapy clinics beginning in 1951. 34 , 35 The development of nuclear reactors in the late 1940s made possible the production of small 60 Co sources with specific activities (in Curies per gram) high enough to produce clinically acceptable dose rates of more than 1 Gray (Gy) per minute at a typical treatment distance of 80 cm from the source. These machines quickly became the standard of radiotherapy because of their simplicity of design and operation, low cost, and availability. The two photons produced by the decay of 60 Co have energies of 1.17 and 1.33 MeV, yielding a depth-dose curve that falls to 50% of maximum in about 10 cm of water or tissue. In addition, the relatively high energy of the photons leads to “skin sparing” due to a reduced entrance dose that builds up for a distance of 5 mm (the maximum range of the secondary electrons from 60 Co photons) to a maximum value. The half-life of 60 Co is 5.27 years, making the source change a relatively infrequent requirement. With the highest specific activities currently achievable, dose rates of at least 1.5 Gy per minute for a field size of 40 cm × 40 cm can be achieved at 80 cm source-to-treatment distance.
Aside from the requirement to replace the sources every few years, additional disadvantages to 60 Co isotope machines include the need to shield against continuous leakage radiation and the lack of field flatness for the largest field sizes. In addition, the physical size of the source capsule, typically 1.5 cm to 2.0 cm in diameter, is much larger than the effective source size available on any machine that uses an electron beam (typically 4 mm or less for linear accelerators). This large source size contributes to a geometric penumbra (the penumbra is the area at the edge of the beam where the dose falls from high dose to low dose) that can be significantly wider than with accelerators.
Before the development of 60 Co isotope machines, both radium and cesium sources were used for teletherapy treatments. However, the limitations of low specific activity for radium and low energy and limited specific activity for cesium limited their usefulness for general radiotherapy.

Betatrons
The next chronological development in treatment machines is the betatron, first developed by D. W. Kerst at the University of Illinois 36 primarily for physics experimentation. Electrons with energies of up to 45 MeV have been produced in betatrons for radiotherapy and used either directly or to produce bremsstrahlung x-ray beams. The massive size of these machines, their high cost, and relatively low dose rate combined to limit their usefulness in radiation therapy.

Linear Accelerators
The use of microwaves to accelerate electrons to high energies for radiotherapy was first demonstrated in Great Britain in 1953. 37 This became possible primarily because of the development of high-power microwave generators for military radar use during World War II. The major components of a typical medical linear accelerator (linac) are shown in a block diagram in Fig. 7-13 . The acceleration of the electron beam takes place in the accelerator wave guide, consisting of a stack of cylindrical cavities with a hole through the center, into which resonant electromagnetic waves of frequency in the microwave range (∼3000 MHz) have been coupled. The electron beam is created and preaccelerated to approximately 50 keV in a conventional electrostatic electron gun, injected into the resonating waveguide, spatially bunched and accelerated through interactions with the electromagnetic field in the individual cavities, and then emerges from the accelerating structure as a narrow pencil beam that can be magnetically bent or focused onto a scattering foil (for electron treatments) or a bremsstrahlung target (for x-ray treatments) in the treatment head of the linac. For low energies of 6 MeV or less, the beam can be magnetically focused in the forward (vertical) direction onto the axis of the treatment head, whereas at higher energies, the beam is usually bent through a 90-degree or 270-degree angle before entering the treatment head since the accelerating structure is much longer for high energies and is frequently oriented horizontally to save space. For more details about the acceleration process, the reader is referred to several excellent review articles in the literature. 38 - 40

FIGURE 7-13 • A block diagram of a typical medical linear accelerator.
(From Khan FM: The physics of radiation therapy, ed 3, Philadelphia, 2003, Lippincott Williams & Wilkins, p 43.)
Although the details of the acceleration process can be quite technical, the shaping of the beam in the treatment head is both important and conceptually easy to understand. Fig. 7-14 shows a schematic diagram of the head of a typical linear accelerator in x-ray and electron treatment mode. In the x-ray mode, the beam first strikes an x-ray target made of high-Z material such as tungsten and produces a bremsstrahlung beam that is forward peaked—the higher the electron energy, the more forward the angular distribution of the resulting x-rays. After primary collimation, the beam strikes a flattening filter made of high-Z material that is thick in the center and thin toward the outside, thereby flattening the angular distribution of the x-ray beam. Although the beam can be made precisely flat only for one particular field size and at one particular depth in a patient, the filters are selected to produce beams that have acceptable flatness over the entire range of field sizes and depths normally used in therapy. If the accelerator is designed to deliver x-ray bremsstrahlung beams produced by electrons of more than one energy, a second flattening filter, optimized for that energy, will be rotated into position on the carousel. Following the flattening filter, the beam encounters the transmission ion chamber used to monitor the beam intensity (and, if segmented, the beam position), and then a secondary collimator that, along with the primary collimator, defines the rectangular field size. Patient-specific collimators and compensators, as well as wedges, if required, are placed beyond the secondary collimator as shown. In all contemporary machines dual ion chambers are used.

FIGURE 7-14 • Schematic diagram showing the basic components of the treatment head of a modern linear accelerator. A , Components in place for x-ray therapy. B , Components in place for electron therapy.
(From Khan FM: The physics of radiation therapy, ed 3, Philadelphia, 2003, Lippincott Williams & Wilkins, p 46.)
In the electron mode the x-ray target is out of position and the beam first strikes a thin scatterer that is rotated into position on the multielement carousel. This scatterer is designed to produce flat electron beams when used in combination with a field size–specific combination of secondary collimator settings and electron applicators as shown in Fig. 7-14 . The scatterer is commonly a dual system of lead foils, with the second foil thicker in the center to remove the forward peaking prevalent at high energies. Bremsstrahlung x-rays will inevitably be produced in the scattering foils, but since the foils are thin, they will typically account for less than 5% of the dose received by the patient. The transmission monitor ion chamber remains in the beam as for x-ray therapy.
In the 1990s a new X-band linear accelerator was developed for medical applications. This accelerator operates at ∼9000 MHz frequency, resulting in a smaller diameter wave guide. X-band linacs capable of accelerating electrons to 6 or 12 MeV are relatively compact and are now being used with a robotic delivery system 41 ; a portable IORT device; and a tomotherapy device, which mounts it on a computed tomography gantry. 42

Microtrons
The microtron is an electron accelerator that combines the linear acceleration principle of the linac with a fixed magnetic field to confine the electrons, similar to a cyclotron (see later). The electrons move in circular orbits of increasing radii as they repeatedly recirculate through a resonant accelerating cavity. A moveable deflection tube can be placed in any location inside the magnet to select the desired electron energy for extraction. The advantages of the microtron over a linear accelerator are its simplicity, compact size, and ease of energy selection. In addition, compared to a linac, the energy spread, beam divergence, and beam size are all small, simplifying subsequent beam transport. The first example of a microtron in clinical use was a 10-MeV unit described in 1972. 43 The first commercial unit, manufactured by AB Scanditronix in Sweden, was a 22 MeV unit installed at the University of Umeå, Sweden. 44 Currently, microtron units of up to 50 MeV are installed at various facilities in Europe and the United States.

Cyclotrons
Cyclotrons are used to accelerate heavy charged particles such as protons, deuterons, and heavier ions. The cyclotron was invented by Ernest Lawrence in the 1930s as a tool for physics research and isotope production. It has been used to produce protons for radiotherapy since the 1960s at several physics laboratories around the world including locations in the United States, Russia, Europe, and Japan. Cyclotrons have also been used to produce therapeutic neutron beams through the interaction of accelerated proton or deuteron beams with beryllium or other light targets.
In its simplest form, the cyclotron is composed of two conducting D-shaped half-cylinders that are evacuated and placed between the poles of a direct current magnet. An alternating potential difference is applied between the two Ds such that when protons are injected into the center of the Ds, they are accelerated toward the negative potential. Under the constant magnetic field, they travel in circular orbits, experiencing acceleration each time they reach the gap. Energies of 200 MeV or more are needed for proton therapy if all areas of the body are to be reached. Deuteron energies of approximately 50 MeV are adequate for the production of neutron beams for therapy. Proton and deuteron beams of these energies can be produced in appropriately designed cyclotrons. Most of the accelerators in use today for proton therapy are cyclotrons. 45 , 46

Synchrotrons
Whereas cyclotrons use a fixed magnetic field and variable radius orbits as the particles increase energy, synchrotrons use a variable magnetic field and a fixed radius orbit to confine the protons or other heavy charged particles. The synchrotron consists of a ring of magnets with interspersed resonating structures that together can confine and accelerate the charged particles to high energy. Loma Linda University Medical Center has the world’s first dedicated proton facility that uses a synchrotron to produce proton beams that are used in any of several treatment rooms. 8 Synchrotrons have also been used to create heavy ion charged particle beams for radiotherapy at Lawrence Berkeley Laboratory in California and at the National Institute for Radiological Sciences in Chiba, Japan. 17

Radiation Dosimetry
Biologic damage depends on how much energy is absorbed from the radiation beam and deposited in the tissue. The absorbed dose is defined as the energy absorbed per unit mass of material and can be used to describe the interactions of all types of ionizing radiation with matter—both directly ionizing (charged particles such as electrons and protons) and indirectly ionizing (neutral particles such as photons and neutrons). Since it is difficult to measure absorbed dose in tissue-like material directly, radiation dosimetry is typically performed by measuring ionization in air and then converting this measurement into absorbed dose.

Radiation Exposure
When ionizing radiation passes through a volume of air, some of the atoms of the air are ionized by interactions of the beam with the atoms. Exposure is defined as the ratio of the number of ions of either sign created by the passage of a beam through a sample of air, divided by the mass of that volume of air from which the ions are produced, assuming all of the ions are completely stopped in the air volume. The unit of exposure is the Roentgen (R) defined as

Although this definition is difficult to use operationally, the free-air chamber that can directly measure exposure for photons of energy less than approximately 3 MeV has been constructed and used in standards laboratories for absolute measurements. 1 The difficulty with satisfying the definition for higher energies is that the mean free path length in air for electrons produced by high energy photons is several meters, and therefore the free air chamber, which must be large enough to stop these electrons, is not practical above a few MeV. As a result, exposure is not a valid concept at energies above 3 MeV.
In practice, thimble ionization chambers are used to measure exposure. These chambers, either cylindrical or spherical in shape, typically contain less than 1 ml of air and, in combination with an electrometer, can be used conveniently and reproducibly to measure the quantity of charge produced when ionizing radiation passes through those volumes. National standards laboratories, using either free-air chambers or other methods, can provide calibrations of these chambers in units of R/C (i.e., exposure per unit of collected charge in a 60 Co beam that can then be converted to dose per unit collected charge in beams of other energies and in phantoms of various materials). A schematic diagram of a typical cylindrical thimble ionization chamber is shown in Fig. 7-15 .

FIGURE 7-15 • Diagram of Farmer thimble ionization chamber.
(From Khan FM: The physics of radiation therapy, ed 3, Philadelphia, 2003, Lippincott Williams & Wilkins, p 88.)

Absorbed Dose
Whereas exposure, as defined earlier, is a property of the beam, absorbed dose is a measure of the energy deposited by the beam and absorbed by the target and is assumed to be closely related to the observed biologic effects. The units of absorbed dose are: 1 Gy = 1 J/kg, that is, 1 Gray, (Gy) is the dose associated with the absorption of 1 Joule of energy per kg of the medium of interest. Clinical doses are often described in units of centigray (cGy), which is equal to the historical unit rad. That is,

Another useful concept is kerma , an acronym for kinetic energy released in the medium. Although it has the same units as dose, the distinction is that kerma refers to energy transferred to the medium, whereas dose refers to energy absorbed by the medium. As a beam of photons enters a medium, there is an initial distance in which secondary electrons are being produced more rapidly than they are being stopped (the “buildup region”) due to the interactions of the photons with the material. In this region, the absorbed dose is increasing with depth, whereas the kerma is decreasing with depth because of the exponential attenuation of the beam in the medium. After a distance that corresponds approximately to the path length of the highest energy secondary electron that can be produced by a photon in the beam, the number of electrons produced per unit pathlength becomes approximately equal to the number that come to the end of their range. From this depth on, the photons are said to be in electronic equilibrium with the secondary electrons and kerma is approximately equal to dose. Fig. 7-16 shows the relationship between kerma and dose as a function of depth.

FIGURE 7-16 • Schematic plot of absorbed dose and kerma as functions of depth.
(From Khan FM: The physics of radiation therapy, ed 3, Philadelphia, 2003, Lippincott Williams & Wilkins, p 164.)

Dose Determination
Absolute dose determinations are difficult. They require a detector that responds linearly to deposited energy and that has an absolute calibration factor in units of response per unit dose that is known or that can be determined independently. Most practical dosimetry for clinical linear accelerator beams is performed with ionization chambers that are calibrated by accredited radiation laboratories whose measurements can be traced to the National Institute of Standards and Technology (NIST). These laboratories calibrate ionization chambers in one of two ways, either in terms of the absorbed-dose-to-water or in terms of the exposure in air. Before 1999 the standard practice for clinical dosimetry was to use in-air calibration factors for ionization chambers, and to apply a number of beam-specific and chamber-specific factors to convert the exposure rate in air to absorbed dose in water. One protocol used by medical physicists for doing this is called TG-21. 47 More recently, a new dosimetry protocol has been introduced by the American Association of Physicists in Medicine (AAPM), which relies on the absorbed-dose-to-water as opposed to the exposure rate in air. This protocol is called TG-51, 48 and in many ways is simpler to implement than TG-21.

Absolute Dose Measurements

Calorimeters
The most widely used direct dose measuring device is the calorimeter. The calorimeter can be used to determine absorbed dose by measuring a change in temperature in an irradiated material since, for most materials, energy deposited eventually appears as heat within the material, although for some materials a small portion of the absorbed energy (called the thermal defect) may be lost because of lattice deformations or chemical changes. The primary detector in the calorimeter is a thermistor embedded in the absorbing phantom. A thermistor is a solid state device that has a rapidly varying resistance with temperature. In carefully controlled situations, a thermistor can measure a change in temperature of as little as 10 −5 °C. This corresponds to a deposited dose of approximately 4 cGy in water. To accurately measure clinically relevant doses delivered at achievable dose rates, the thermistor needs to be very well isolated from the outside environment so that temperature changes due to atmospheric conditions are much smaller than those produced by the energy absorbed from the beam during the time required to make a single measurement. Successful calorimeters have been constructed out of carbon, 49 water, 50 - 52 and tissue-equivalent conducting plastic. 53 An excellent review of the field of calorimeters can be found in Attix’s textbook on radiation dosimetry. 54 Although calorimeters are the most direct method of measuring dose, they are generally expensive, bulky, and difficult to use. Therefore, they are rarely used for routine clinical dosimetry, although water calorimetry is becoming close to a standard for calibrations laboratories worldwide. 55 For example, the NIST uses water calorimetry as its standard.

Fricke Dosimetry
The Fricke dosimeter 56 is based on the conversion of ferrous sulfate ions in a solution to ferric sulfate ions due to the deposition of energy by ionizing radiation. It is not truly a direct dosimeter because there is no a priori way of knowing the calibration constant in units of molecules of ferric ion produced per unit of absorbed dose. This calibration factor, called the G value, is dependent on both energy and modality. However, since the dosimeter response is proportional to absorbed dose, it can be a useful device, particularly since the solution is tissue-equivalent and the dosimeter is very small.

Exposure-Based Dosimetry
Much of the practical dosimetry of clinical x-ray beams is based on ionization measurements made with thimble ionization chambers. The number of ion pairs produced by interactions of the beam in the gas of the chamber is first converted to exposure using an exposure calibration factor, N x , that is obtained from a standards laboratory and is appropriate for a particular energy x-ray beam (typically 60 Co) and a particular set of measurement conditions. In general,

where
X=exposure in roentgens,
M=meter reading in coulombs,
N x =calibration factor for the energy of interest in roentgens/coulombs, and
C=correction factor to account for any differences in measurement technique between the calibration condition and the experimental condition.
The corrections that must be made to obtain exposure include, most importantly, a correction for differences in temperature and pressure between the calibration condition (normally N x is valid for a temperature of 22° C and atmospheric pressure of 760 mm Hg) and the experimental condition. 1 If the walls of the chamber are assumed to be made of condensed air-equivalent material and are thick enough to produce electronic equilibrium, then the dose to the air of the chamber is obtained by multiplying the exposure by W/e, the average energy required to create an ion pair for electrons in air per unit charge collected, where W/e = 33.97 J/C:

To obtain this equation we have also used the definitions 1R = 2.58 × 10 −4  C/(kg air) and 1 cGy = 10 −2  J/kg. The term A eq is a small correction that accounts for the attenuation of the photon beam in the condensed air walls needed to produce the electronic equilibrium. If we wish to know the dose at the point of measurement to a small mass of some material other than air, this is:
(1)
where f med , the term in brackets in this equation, was referred to historically as the roentgen-to-rad conversion factor. 2 It must be realized that since the roentgen is only defined for energies 3 MeV or less, this exposure-based dosimetry method is also valid only in this low-energy region. The variation of f med with energy and material is shown in Fig. 7-17 . Further corrections are necessary if an exposure determination in free space, such as just described, is to be used to predict the dose to a finite phantom rather than a small mass, due to the effects of scatter. 2

FIGURE 7-17 • The ratio of exposure to dose for bone, muscle, and water as a function of photon energy.
(From Johns HE, Cunningham JR: The physics of radiology, ed 4, Springfield, IL, 1983, Charles C. Thomas.)

Dose Determination Using Bragg-Gray Cavity Theory
As mentioned, the exposure-based calibration method cannot be used above 3 MeV photon energies, so another technique must be used to evaluate dose at higher energies. The Bragg-Gray cavity theory is designed to directly relate the charge measured in a small air cavity in a phantom to the dose that would be delivered to the same point in the phantom in the absence of the air cavity. Fig. 7-18 shows a schematic drawing of the geometry of such a measurement. The Bragg-Gray theory 2 states that if the air cavity is so small that it does not interfere with either the photons or their associated secondary electrons (an assumption that cannot be exactly true unless no ionizations are produced in the cavity) and if the cavity is located in a place where electronic equilibrium has been established, then

FIGURE 7-18 • Schematic drawing of a Bragg-Gray cavity in a medium exposed to an x-ray beam.
(From Johns HE, Cunningham JR: The physics of radiology, ed 4, Springfield, IL, 1983, Charles C. Thomas.)
(2)
where
D med is the dose to the medium in the absence of the cavity (in Gy),
(s/ρ) x is the mass stopping power for the secondary electrons in substance x in units of MeV cm 2 /g, 57
Q is the quantity of charge of either sign released by ionizations in the gas in units of coulombs (C),
m air is the mass of air in the cavity in kg, and
W/e is the average energy required to create an ion pair in air (= 33.97 J/C for dry air). 58
It should also be noted that

that is, the product of the last two bracketed factors in Equation 2 is just the dose absorbed by the air in the cavity due to the interactions of the secondary electrons with the air. Note that because the cavity is very small, it is assumed that there are no direct interactions of the passing photons with the air molecules in the cavity, only the secondary electrons.
If we now replace the theoretical small air cavity with a realistic thimble ionization chamber as shown in Fig. 7-19 , then we must assume that the chamber has a finite wall thickness of a material that may be different than the medium of the phantom. In this case, we have to modify the previous formula to account for electrons that are produced in the wall as well as in the phantom material and for a possible difference in the attenuation of the photons in the wall material versus the phantom. The Bragg-Gray theory, modified to account for this situation, leads to the following expression:

FIGURE 7-19 • Determination of absorbed dose in a medium with a practical ion chamber. A , Homogeneous phantom showing the point P at which the absorbed dose is desired. B , Practical ion chamber with outer radius c and inner radius a shown centered at position P′, which is identical in position to P.
(From Johns HE, Cunningham JR: The physics of radiology, ed 4, Springfield, IL, 1983, Charles C. Thomas.)
(3)
where
α is the fraction of electrons crossing the air cavity that come from the wall,
(µ/ρ) x is the mass attenuation coefficient for photons of this energy in material x, and
A accounts for the perturbation of the beam by the chamber wall (probably very close to 1.00 for thin, low-Z walls).
The first term in Equation 3 corresponds to electrons produced in the wall that interact in the air of the chamber and the second term to electrons that are produced in the medium. Obviously, if the wall is very thin, α is very close to zero and A is very close to 1.0, in which case Equation 3 becomes nearly identical to Equation 2 .

Practical Dosimetry With Thimble Ionization Chambers
In 1983, the AAPM published a formalism for photon and electron dosimetry based on existing exposure or dose calibrations at 60 Co energies. 47 This formalism, called TG21, defines a parameter called N gas = D air /M c (dose to the air of the chamber per unit meter reading in the calibration beam) that contains all the chamber-specific and calibration beam-specific parameters, including the 60 Co exposure constant (N x ) or dose calibration constants. Knowledge of N gas is equivalent to knowledge of the effective mass of air in the chamber. The quantity N gas /N x is a function of only the geometry of the chamber and the well-known properties of 60 Co photons and is tabulated for specific thimble ionization chambers in the literature. 47 This ratio can also be calculated from a knowledge of the materials and dimensions of the chamber. Once the N gas for a chamber is known, the above formalism of Equation 3 , based on Bragg-Gray cavity theory, can be used to obtain the dose to a medium for photons or electrons of arbitrary energy. That is, for photons of energy λ,
(4)
where
M λ is the meter reading in this photon beam,
P λ is a factor to account for the perturbation of the beam by the presence of the chamber, and
L/ρ is the restricted mass stopping power, only accounting for those electrons with energy large enough to cross the air cavity.
Similarly, for electrons of energy E
(5)
since one normally assumes that the electron beam will not be modified by the wall material, so the equation is simplified.

Dosimetry Based on Absorbed-Dose-to-Water Calibration
In 1999 the AAPM published the TG-51 dosimetry protocol for high-energy photons and electrons, 48 which replaces TG-21 in most cases. In this protocol an absorbed-dose-to-water calibration factor for a 60 Co beam, , is determined for ionization chambers by a NIST-traceable calibration laboratory. Using the AAPM TG-51 protocol, the absorbed dose in the medium, , is related to the charge collected in the ionization chamber according to the formula

where M is the electrometer reading (i.e., the charge collected by the electrodes in the ion chamber) corrected for environmental temperature and pressure, ion recombination and polarity effects. is the NIST-traceable absorbed-dose-to-water calibration factor for the ionization chamber in a 60 Co beam. The factor, k Q , is a beam-quality conversion factor that is specified for different types of ionization chambers and adjusts for the quality, Q, of the beam in question. Quality conversion factors, k Q , have been tabulated for different types of ionization chambers and different photon beam energies (see Almond et al. 48 : Table I, p 1857). The value of k Q ranges from about 0.944 for a 24-MV photon beam to 1.0 for 60 Co.
TG-51 is fundamentally different from previous dosimetry protocols, because the ionization chamber is calibrated in terms of the dose to water as opposed to exposure in air. A requirement of the protocol is that dosimetry measurements must be performed in water at a calibration depth of 10 cm. The protocol does not rely on Bragg-Gray or Spencer-Attix cavity theory to convert exposure in air to dose in water or tissue. No stopping power ratios or mass-energy absorption coefficients are required. The relative simplicity of the TG-51 protocol makes it less likely that errors will be made in the calibration of clinical radiation beams, and this is one of the primary reasons why the protocol was adopted.

Thermoluminescent Dosimetry
Certain crystalline materials display a property called thermoluminescence (TL) that can be exploited for dosimetry. When such a crystal is irradiated, a portion of the absorbed energy can be stored in the lattice and recovered later in the form of visible light emission if the material is heated. If the emission of light after radiation is spontaneous, the phenomenon is called fluorescence.
In a crystal lattice, discrete electron energy levels are perturbed by the interactions between atoms, forming both allowed and forbidden energy bands. In the presence of certain impurities, energy traps can be formed in the forbidden region, and when irradiated, electrons can be excited out of their ground states into one of these forbidden energy traps. Since they are in the forbidden region, spontaneous decays back to the ground state are rare and the imprint of the absorbed dose remains until extra energy is provided to force the transition. This extra energy can be externally applied heat. By observing the heated TL material with a light-sensitive phototube, an electronic signal can be created that is proportional to the number of electrons in forbidden traps, which is, in turn, related to the energy absorbed by the TL material due to irradiation. Over a restricted range of absorbed dose, the response can be linear. 59
The TL materials most commonly used for dosimetry are lithium fluoride, lithium borate, and calcium fluoride. They can be prepared in the form of powders, solid chips, and solid rods. By careful handling procedures, 3% to 5% reproducibility can be achieved after individual calibration of dosimeters with a photon beam. 1 , 59 Because of their small size (typically <1 mm 3 in volume) they are very conveniently used to attach to patient surfaces or place in patient cavities as in vivo dosimeters to verify dose calculations.

Solid State Detectors
Silicon diodes and other semiconductors can also be used to produce signals that are proportional to radiation dose. In a typical diode, there is a “p” region having an excess of positively charged “holes” and a depletion layer, or “n” region that has an excess of electrons. Radiation to the depletion layer produces electron-hole pairs that can cause current to flow across the junction between the layers if the diode has a reverse bias across it. The amount of current flow is proportional to the energy deposited by charged particles passing through the depletion layer. 54 Silicon diodes have the great advantage of small size and a low-energy threshold for producing an ion pair, resulting in a high sensitivity. The disadvantage of the diode is that radiation damage causes a decrease in sensitivity with radiation dose.
Another solid state detector is the so-called “MOSFET,” an acronym for metal oxide semiconductor field effect transistor. A MOSFET detector for radiation dosimetry consists of two MOSFETs operating at different voltages. The difference in the threshold voltage shifts of the two MOSFETs is proportional to absorbed dose. Like diodes, these detectors are very small and sensitive and are now being used clinically at some institutions to evaluate dose in a phantom for highly complex dose distributions. 60 They have been shown to be very linear over the range of doses of interest for radiotherapy verification. However, they have an angular dependence to radiation sensitivity that can limit their accuracy in clinical situations.

Special Purpose Dosimeters
Arrays of diodes have been constructed on a grid of 10 cm × 10 cm using fixed spacing of 5 mm. These arrays are extremely useful for efficient characterization of a beam for dosimetric purposes. Implantable dosimeters have also been developed that are small enough to be inserted into tissue with conventional needles. These dosimeters register cumulative dose during a course of radiotherapy and the dose can be externally readout using a special RF receiver.

Film Dosimetry

Radiographic Film
Radiographic film consists of an emulsion of fine silver bromide crystals on a transparent film base. When the film is exposed to ionizing radiation, a chemical reaction takes place within the exposed crystals, forming what is called a latent image. The development process reduces the exposed crystals to small grains of metallic silver that remain attached to the base during the fixing process, which removes all of the unexposed crystals of silver bromide. The degree of blackening of an area of the film depends on the amount of free silver deposited; therefore, it is related to the radiation energy absorbed by the film. A densitometer can be used to measure the optical density of the film, defined as

where I o is the amount of light transmitted through an unexposed portion of the film and I t is the amount of light transmitted through the exposed portion of interest. By carefully exposing and developing film to a series of known doses using the same photon energies as will be used for the test exposure, it is possible to obtain curves of optical density versus dose for specific emulsions from which absolute dose information can be subsequently extracted with some substantial uncertainty. This uncertainty is due to the fact that the silver in the emulsions strongly absorbs radiation below about 150 keV by the photoelectric process. For electrons, the information is much more trustworthy.
In spite of the dose uncertainties in photon beams, films are very useful for measuring relative dose distributions in phantoms and can be used with high-energy photon beams to obtain absolute information with only about 3% to 5% uncertainty. Computer-controlled densitometers can be used to quickly scan complex films and convert the information into dose contours.

Radiochromic Film
Radiochromic film consists of thin layers (of the order of several to about 20 microns) of colorless radiosensitive dye sandwiched between a clear plastic material (e.g., Mylar or polyester). Unexposed film is a light blue-gray in color. After the film is exposed it darkens to a deep blue as a result of a polymerization process induced by ionizing radiation. The amount of darkening is related to the dose exposure of the film. Earlier forms of radiochromic film required high doses of radiation to achieve measurable film darkening. For example, GAFCHROMIC HD-810 film (International Specialty Products [ISP], Wayne, NJ) has been used in the dose range of 50 to 2500 Gy, and GAFCHROMIC MD-55 film (ISP, Wayne, NJ or Nuclear Associates, Carle Place, NY) is useful in the range of about 3 to 100 Gy. In 2004, a new form of radiochromic film, GAFCHROMIC EBT dosimetry film (ISP, Wayne, NJ) was introduced specifically for radiotherapy quality assurance (QA), in particular for intensity-modulated-radiotherapy (IMRT) fields. Several scientific papers have been published on the use of EBT film, 61 , 62 and a white paper describing the product in detail can be found on the ISP website ( http://www.ispcorp.com ). GAFCHROMIC EBT film is ten times more sensitive than previous generation radiochromic films, with a dose range sensitivity of about 1 to 800 cGy. This implies that QA films can be irradiated in significantly less time, rendering QA procedures more efficient. Furthermore, all forms of radiochromic film exhibit significantly less energy dependence than radiographic film, primarily because radiochromic film is made with essentially water-equivalent materials (i.e., these films contain no high-Z elements such as silver).
All types of radiochromic film have the advantage that they do not require chemical processing, and in addition, they are not very sensitive to ambient room lighting, and hence, film handling is significantly simpler than with radiographic film. The film darkening process (called film density growth) is complete within 1 to 2 days with HS or MD-55 film and within less than 2 hours with EBT film. This further facilitates QA dosimetry with EBT film, because the films can be analyzed shortly after they are irradiated.
Film read-out is accomplished with transmission densitomiters, film scanners or spectrophotometers, depending on which type of film is employed. The response of the film is enhanced if the spectral response of the scanner is matched to the absorbance of the film.

Dose Distribution in Media
The clinical use of radiation in patients requires the specification of dose at any point within the irradiation field. In general, if the dose at a reference point is known, the dose at any other point can be calculated if the field size, distance to source, field shape, depth of point of interest, and energy of the beam are known, as well as the constituents of the irradiated medium. In this section, we consider methods of determining the dose at any point of interest, relative to a reference point dose.

Phantoms
Since it is seldom possible to measure dose distributions directly in patients, phantoms have been developed. These phantoms have absorption and scattering properties that mimic those in human patients. The phantoms can be constructed to have the same geometry as humans and are then said to be anthropomorphic. An example of such a phantom is the Alderson Rando Phantom (Alderson Research Laboratories, Inc, Stamford, CT.), which is sectioned transversely for dosimetric studies and incorporates materials to simulate specific body tissues including muscle, bone, lung, and air. To be equivalent to a given tissue in terms of the interactions of photons and electrons, the phantom material must have the same electron density (in electrons per gram or electrons per gram per square centimeter) as the tissue to be simulated. Basic dose distribution data are frequently measured in a water phantom that closely approximates the radiation absorption and scattering properties of soft tissue.

Percentage Depth Dose
The percentage depth dose is a way of characterizing the change in dose with depth along the central axis of the beam. The definition for any beam is

where D d and D ref are dose rates at depth d below the surface and at the reference depth. This definition is described schematically in Fig. 7-20 . In general, P depends on the depths d and ref, on the distance (f) from the source to the surface (often referred to as SSD), the field size on the surface (r), and the beam energy (E). The definition assumes that the field is square, so corrections must be made if rectangular or irregular field shapes are used.

FIGURE 7-20 • Schematic drawing illustrating the definition of percentage depth-dose. D d , dose at the depth d; D ref , dose at the point on the central axis which is at depth ref; S, field size at the surface of the phantom; SSD, source-to-surface distance.
(From Perez CA, Brady LW: Principles and practice of radiation oncology, ed 2, Philadelphia, 1992, JB Lippincott, p 198.)
Percentage depth doses are generally tabulated for a specific SSD (usually 100 cm) as a function of depth and field size. Given some dose D 1 at depth d 1 along the central axis, one can determine the dose D 2 at any other depth d 2 from the relation:

In particular, given the prescription dose at a specified depth, one can calculate the dose any other point on the central axis.
In the absence of scattering effects, the dose to any point in space varies as the inverse of the square of the distance from the source to the point (the so-called inverse square law). Using this assumption, if we compare the percentage depth dose for different SSDs, one would expect that

The term on the right-hand side of the equation is called the Mayneord F factor. This factor is greater than 1 for f 1 > f 2 . Therefore, the percent depth dose is seen to increase with increasing SSD.
Percentage depth dose also increases with increasing field size due to the increasing contribution, with field size, of scatter to the dose on the central axis. As the field size is increased from zero, where we have only primary radiation, to a finite field size of radius r, the dose at all depths will increase due to the contribution of scatter. At deeper depths, where the field size is larger, the increase in dose will be larger than for shallower depths. For low energies and for small field sizes, the rate of change of the percent depth dose with field size will be fairly rapid. This variation of percent depth dose with field size for various energies is displayed in Fig. 7-21 .

FIGURE 7-21 • Plot showing variation of percentage depth-dose with field size for three photon beams, at a depth of 10 cm. HVL, Half-value layer; SSD, source-to-skin distance.
(From Johns HE, Cunningham JR: The physics of radiology, ed 4, Springfield, IL, 1983, Charles C. Thomas.)
Compilations of central axis depth dose for various energies have been published 63 , 64 and are summarized, for a few energies, in Fig. 7-22 for a fixed-field size of 10 cm × 10 cm. Of interest in this figure is the buildup of dose from the surface to a maximum value at a depth of d max for high energy photons, and an increasing percentage depth dose with energy, for a fixed depth. The so-called buildup region near the surface for high-energy photon beams is due to the increase of secondary electron fluence with depth from the surface to a depth that corresponds approximately to the maximum range of the secondary electrons produced by the photons in the material of the phantom. The photon fluence, on the other hand, is exponentially decreasing with depth starting from the surface itself. The combination of these two effects yields a dose that is low at the surface, builds up to a maximum value at a depth that increases with the energy of the beam (and the maximum range of the secondary electrons), and then falls exponentially with depth beyond d max . The rate of the exponential falloff in dose with depth beyond d max is determined by the mean attenuation coefficient µ such that

FIGURE 7-22 • Plot showing variation of dose with depth for 6 different energy photon beams.
(From Johns HE, Cunningham JR: The physics of radiology, ed 4, Springfield, IL, 1983, Charles C. Thomas.)

where K s is the contribution of scatter to the dose on the central axis at depth d. It should be noted that the exact value of the dose in the buildup region and the depth of d max are critically dependent on the design of the treatment head of the machine and the field size. The addition of any material near the surface of the patient or phantom, such as special blocks, produces electrons and thereby increases the surface dose 65 and potentially changes the depth of maximal dose. For low-energy photons the buildup region is vanishingly small because of the combined effects of backscatter in the phantom and the short ranges of the secondary electrons produced in the phantom.
Correction of the PDD for rectangular fields (or irregular fields) requires that the integrated scatter from all points along the periphery of the field be calculated. The “equivalent square” field is that field, of radius r, that has the same integrated scatter as the field of interest. For rectangular fields, the equivalent square field must be found using published tables 63 or by using the approximation

where S is the side of the equivalent square and L and W are the sides of the rectangle.

Tissue-Air Ratio
The use of PDD depends on SSD, as discussed earlier. This makes its use rather cumbersome, since in practical circumstances, the SSD may vary substantially across the field and from field to field. The tissue-air ratio (TAR) was introduced to allow dosimetry for rotational therapy, where the gantry moves around a fixed point in the patient. 66 The TAR is defined as

where D d is the dose rate at depth d in a phantom for field size r d defined at that depth, and D fs is the dose rate in free space at the same point. The TAR concept is schematically represented in Fig. 7-23 . The D fs is measured with a small equilibrium mass around the point of interest to provide dose buildup. Since the point in the phantom and in free space are at the same distance from the source, TAR depends only on scatter and attenuation in the phantom. To the extent that phantom scatter is nearly independent of divergence of the beam, TAR is usually considered to be independent of SSD, an assumption that has been shown correct to within about 2% over the range of SSDs used clinically. 67

FIGURE 7-23 • Illustration demonstrating the definition of tissue-air ratio (TAR). d, Depth; D d , dose at depth d; D fs , dose in free space; r d , field size depth d; S, source.
(From Khan FM: The physics of radiation therapy, ed 3, Philadelphia, 2003, Lippincott Williams & Wilkins, p 168.)
The TAR at d max is given the special name backscatter factor (BSF), since the variation of this TAR from unity is a measure of the importance of scatter dose at d max . As might be expected, BSF increases with increasing field size and decreases with increasing energy. The variation of BSF with HVL and field size is shown in Fig. 7-24 . Above about 8 MV, the scatter contribution to the dose at d max for all field sizes becomes negligibly small and BSF approaches unity.

FIGURE 7-24 • Variation of backscatter factor with beam quality for circular fields of different sizes.
(From Khan FM: The physics of radiation therapy, ed 3, Philadelphia, 2003, Lippincott Williams & Wilkins, p 170.)

Scatter-Air Ratio
The scatter-air ratio (SAR) is that portion of the TAR that is due to scatter. That is,

where TAR(d, 0, E) is the TAR for zero field size and represents the primary component of the TAR. The SAR concept is useful in calculating the net scatter dose to a point inside an irregular field. 68 In this method, referred to as Clarkson’s method, 69 radii are drawn at regular angular intervals from a point of interest inside an irregular field to the periphery of the field. The (SAR) avg is then calculated as the average SAR for all the intersections of these radii with the field edge, using the appropriate circular field SARs for each radial distance. Finally, the average TAR for the point of interest can be calculated as

More information on this technique can be found in textbooks on medical physics. 1 , 2

Tissue-Phantom Ratio and Tissue-Maximum Ratio
The concept of TAR was developed partially to overcome the difficulty created by the dependence of PDD on SSD. As we go to higher photon energies, we encounter a difficulty with TAR due to the requirement of measuring a dose in free space. At low energies, the free space dose can be measured with a small buildup cap placed around the ion chamber to provide electron equilibrium. At high energies, however, this buildup cap becomes very thick and the attenuation of the beam in the cap as well as the effect of different materials in the cap and the wall must be considered. To avoid these difficulties, the concepts of tissue-phantom ratio (TPR) 70 and tissue-maximum ratio (TMR) 71 were developed. For both concepts, the reference dose and the dose of interest are measured in the phantom, thus, overcoming the difficulty of measuring a dose in free space. Like TAR, however, TMR and TPR are considered to be independent of SSD. The definitions are given by

and

where D d is the dose rate on the central axis of the beam at the depth d for field size f d and D ref is the dose rate at the reference depth for the same field size. For the case of TMR this depth is d max , whereas for TPR, the depth is some standard depth greater than d max . One difficulty with TMR is created by the fact that the depth of d max is a function of field size and SSD, thereby presenting a measurement problem.
For an isocentric patient setup, given the dose D(d iso ) at isocenter, the dose D(d) at any other depth d on the central axis can be calculated as follows:

where SAD is the source-to-isocenter distance, and [SAD + (d – d iso )] is the source-to-calculation-point distance.
In a manner analogous to SAR, a scatter-maximum ratio (SMR) or scatter-phantom ratio (SPR) can be defined by

where is the ratio of the phantom scatter at the reference depth for the field size f d to that for zero field size and where the reference depth is either d max or d ref for SMR or SPR. The phantom scatter factor ratio can be considered the same as the ratio of backscatter factors.

Dose Distributions
To this point, we have considered methods for determining the absolute dose at a point in free space and in a phantom, and then methods for determining the ratio of doses on the central axis to doses at a reference point. We now consider the distribution of dose outside the central axis as a way of evaluating the dose at any point of interest in an irradiated phantom.
An isodose curve connects points of equal dose in a single plane. Frequently, one of the axes of the isodose curve display is the central axis of the beam, in which case the curves represent the variation in dose as a function of depth and transverse distance from the central axis. Fig. 7-25 shows two such displays for a 4-MV x-ray beam, one normalized to the maximum dose, and the other to the depth of the isocenter, that is the axis of rotation for an isocentric therapy unit. The shape of the isodose curves is affected by the beam parameters such as SSD, field size, and beam filter characteristics, as well as the shape of the entrance surface. Such curves are normally reconstructed after measuring at a large number of fixed points in a water phantom with an ionization chamber or diode. Computer-driven scanning probes are commercially available to help with this task.

FIGURE 7-25 • Examples of isodoses for a 4-MV x-ray beam. A , 80 cm SSD with field size 10 cm × 10 cm at the surface. B , 80 cm SAD with field size 10 cm × 10 cm at isocenter. SSD, Source-to-skin distance.
(From Perez CA, Brady LW: Principles and practice of radiation oncology, ed 2, Philadelphia, 1992, JB Lippincott.)
A dose profile is a display of dose at a fixed depth along a single axis transverse to the central axis. These displays are useful as a way to characterize beam flatness and beam penumbra (the rate of lateral dose falloff) as a function of field size and depth.

Compensators and Wedge Filters
Most linear accelerators and 60 Co machines contain flattening filters designed to produce beam profiles that are flat at a typical depth for a typical field size. These filter designs assume that the patient entrance is flat and that the patient tissues have uniform electron density. Special compensating filters can be used when this is not the case or when nonflat isodose contours are desired, to shape the isodose curves at depth.
The most common form of compensator is the wedge filter. These wedges are normally constructed of brass, steel, or lead. When placed in the beam, they cause the isodose curves to be angled relative to the central axis. The wedge angle is defined as the angle between the isodose curve and a perpendicular to the central axis at some reference depth, often 10 cm. Wedges are normally furnished with commercial linear accelerators to produce wedge angles of 15, 30, 45, and 60 degrees. Examples of wedge isodose curves are shown in Fig. 7-26 . When a wedge is used, a wedge factor must be defined, that is, the ratio of the dose rate at d max on the central axis with the wedge in place to that with the wedge removed.

FIGURE 7-26 • Wedged isodose curves for 30-, 45-, and 60-degree wedges for a 10 cm × 10 cm field size.
(From Abrath FG, Purdy JA: Wedge design and dosimetry for 25-MV x-rays, Radiology 136:757, 1980.)
Patient-specific compensators are sometimes made to compensate for rapid variations in the patient surface over the field, 72 although wedge filters are more commonly used for this purpose. Patient-specific compensators can be made of cells of high density metal distributed so as to attenuate the beam where the patient surface is far from the source, relative to those areas where the patient surface is closer.
The presence of any high-density compensator or wedge filter can lead to beam hardening, which can result in significant changes in the percent depth dose, particularly for low-energy beams.

Output Factor
The final dosimetric factor that needs to be defined is output factor. This factor is defined as the ratio of the dose rate in air at a reference source-to-calibration (SCD) distance for a given field size to that for the reference field size (usually 10 cm × 10 cm). For field sizes greater than the reference field size, the output factor will be greater than 1.0 because of the increased scatter from the collimator for the larger fields. This factor is sometimes called the collimator scatter factor.

Dose Calculations
Before the late 1980s, photon dose calculations were based primarily on interpolations of measured depth-dose data. Since these data are measured in homogeneous water phantoms for standard field sizes, corrections have to be made to account for tissue heterogeneities, sloping patient surfaces, and irregular field shapes. Mackie et al. 73 has characterized this type of dose calculation as “correction-based.” Cunningham 74 has described several commonly used tissue heterogeneity correction algorithms.
Advances in imaging and computing technologies have made it possible to incorporate sophisticated model-based dose calculation algorithms in clinical treatment planning systems. In model-based dose calculations, the treatment parameters, including the setup and patient geometry, are simulated from first principles by taking into account the physics of radiation transport. Measured relative dose quantities (e.g., percent depth dose and tissue phantom ratios) are used to verify the results of the model-based dose calculations, but they are not used directly to perform the calculation. Salient aspects of model-based photon dose calculation algorithms can be found in the review article of Ahnesjö and Aspradakis. 75

Dose Calculations Based on Correction Methods
Traditionally, dose calculations for irregularly shaped fields and for points not on the central axis generally require that the effects of scatter and primary dose are calculated separately. In this case, the TAR and SAR formalisms are used in conjunction with the Clarkson calculation method described earlier. The dose at an arbitrary point in an irregular field can be calculated in the following way:

where
D p is the dose to the arbitrary point p at depth d in the phantom,
D fs is the dose in free space, as described in the TAR discussion earlier,
OF is the output factor for the collimator opening compared to 10 cm ×10 cm,

(the inverse square factor),
SSD c and SSD p are the source-to-skin distances along the central axis and along the ray that connects the source to point p,
OAF is the off-axis factor measured with dose profiles in air,
TAR(d, 0) is the TAR for zero field size (primary dose fraction), and
SAR avg (d) is the average SAR to point p from the periphery of the irregular field (scatter dose fraction).
Further information on this can be found in the standard textbooks on medical physics. 1 , 2

Correction for Non-Normal Beam Incidence
In the earlier dose calculation discussions, we assumed that the beam was normally incident on a uniform medium of unit density. For real patients, these assumptions are never exactly correct and we must therefore deal with corrections to our dose calculation formalism to account for this fact.

Effective Source-to-Skin Method
Fig. 7-27 illustrates the effective SSD method of correcting for surface obliquity. In this figure, S represents the patient surface, which is at a distance F from the source on the central axis, at a distance F + h′ above point p′ and a distance F − h′′ above point p′′. In this case, the dose on the central axis, D c , gets modified at p′ and p′′ due to the decrease or increase in the overlying tissue relative to the central axis. However, an inverse square correction must be made because the points of interest are still at a distance of F + d from the source. The complete correction is

FIGURE 7-27 • Demonstration of the missing tissue method of calculating the effect of surface obliquity.
(From Williams JR, Thwaites DI: Radiotherapy physics in practice, Oxford, 1993, Oxford University Press.)

and

This correction is appropriate for SSD treatment techniques, and is simple enough to be done either manually or by computer treatment-planning programs. Note that in general, there is an additional modification to the dose at points such as p′ because of changes in scatter, but this technique adequately corrects the primary dose component and is a good approximation to the total dose at such points.

Tissue-Air Ratio Correction Method
For isocentric treatments, a better approach involves the use of TARs, which are independent of SSD. For this reason, we can simply calculate

Again, this corrects only the primary component of the dose, but for most situations is sufficiently accurate.

Corrections for Tissue Heterogeneities
The presence of tissues with electron densities different than water leads to a further modification of dose to points in the irradiated medium. The primary component of dose to any point is modified by any change in attenuation properties of overlying tissues. In addition, the scatter component of the dose will be influenced by the presence of neighboring inhomogeneities. This latter effect will be reduced for greater distances from the heterogeneities and for higher energies. There are a number of ways of correcting for the presence of tissue heterogeneities from very simple and fast approximations to more complex three-dimensional methods that are more accurate but much more computation-intensive. We discuss a few of these methods and refer the interested reader to other sources for more detailed information. 1, 3, 76

Effective Depth Method
The simplest method of correcting for tissue heterogeneities involves simply calculating an effective depth, d eff , for the point of interest, which is defined as the depth in unit density material that would result in the same net attenuation as the depth d in the phantom. Referring to Fig. 7-28 , the effective depth of point p would be

FIGURE 7-28 • Illustration of the power law tissue-air ratio method of correcting for inhomogeneity beneath a slab of material of density 0.3.
(From Williams JR, Thwaites DI: Radiotherapy physics in practice, Oxford, 1993, Oxford University Press.)

Since the distance of point p from the source has not changed, the dose at p would also have to be corrected by an inverse square factor, so

where PDD is the percent depth dose, and F is the source-to-surface distance. This method does not take account of the proximity of the inhomogeneity to the point of interest but is sufficiently accurate for many applications and simple enough to be calculated manually.

Tissue-Air Method
For isocentric techniques, a correction to the dose at point p can be calculated with TARs using d eff as defined earlier:

where D p,uniform is the dose that would be measured at a true depth of d in a uniform phantom of water-equivalent material. This method considers both the beam size and the depth of the point of calculation and is probably more accurate than the effective depth method.

Equivalent Tissue-Air (ETAR) Method
A further improvement to the tissue-air method can be accomplished by using an effective field size as well as an effective depth. 77 In this case,

where r eff is the radius of a circular field that, when incident on unit density material, produces the same scatter dose to the point of interest as is observed for the inhomogeneous medium. For a uniform phantom of non–unit density material, the value of r eff has been shown to scale directly with the density of the medium. 78

Power Law Tissue-Air Ratio Method
In this method, the thickness and composition of overlying inhomogeneities are accounted for as well as the position of the calculation point relative to the inhomogeneity, 79 , 80 although the lateral extent and shape of the inhomogeneity are not. Under the assumption that the calculation point is not very close to a boundary between two tissues of varying composition, the dose at point P in Fig. 7-28 can be written as

for the general case where the point of interest is imbedded in the nth layer of tissues,

where ρ i is the density of the ith layer.

Other Methods of Correcting for Tissue Heterogeneities
The specific methods discussed to this point have been relatively simple approximations of the effect of tissue heterogeneities on the dose at an arbitrary point in a phantom. The most exact method would need to account for all scatter contributions to the dose at the point of interest. In principle, this can most be accomplished most accurately with a Monte Carlo calculation. This method uses the known cross sections for electron and photon interactions in matter and follows individual photons and their associated electrons through the entire phantom. By calculating the trajectories and interactions of a very large number of photon and electrons, one can accurately model the dose to all points within the heterogeneous phantom or patient. Recently, several Monte Carlo codes have been developed for radiotherapy treatment planning. These are discussed by a number of authors. 81 - 90 Even with today’s fast computers such calculations are time consuming and not practical to use routinely. Monte Carlo is, however, a useful tool in testing the accuracy of simpler dose calculation methods as well as in providing input for the convolution/superposition methods discussed below.
Computed tomography is now routinely used as a basis for treatment planning. It provides the basis for target and normal tissue identification and location as well as a three-dimensional array of computed tomography numbers that are directly related to the electron density of the tissues. This array of electron densities can be used to calculate, the dose at each point, corrected for scatter from all other points in the phantom. It has been shown that such an approach yields only a marginal improvement in accuracy relative to the simpler tissue heterogeneity correction methods, described earlier. 91

Model-Based Dose Calculations
At present, model-based dose calculation methods can be divided into two categories, those that use convolution/superposition principles to calculate dose 73, 92, 93 and those that rely purely on Monte Carlo methods. Principles of the Monte Carlo method have been already been briefly described. Convolution/superposition calculations are defined by the basic equation

where
D represents the dose at some point ,
T E represents the total energy released by primary photon interactions per unit mass (or terma ), and
is the point-spread function (also called the energy deposition kernel).
The point-spread function represents the fraction of the energy deposited (per unit volume) at some point ( ) in the calculation volume that is subsequently transported to the calculation point ( ). The dose at any given point is, thus, computed by adding together (i.e., integrating over all space) the contributions from photons and electrons produced at all other points in the phantom or patient. Ahnesjö et al. 94 found that the point-spread function changes only slightly as a function of energy (due to beam hardening), and that in the convolution integral from earlier, can be replaced by , the average dose-spread function weighted by the spectral components of the beam. Dose-spread functions for mono-energetic photons are generally precomputed using Monte Carlo methods. 94 The energy dependence of the terma, T E , can be expressed by applying the inverse-square law and exponential attenuation to the photon fluence at the surface of the phantom or patient.
Once the energy dependence of the terma in the convolution equation has been simplified, the four-dimensional integral in this equation reduces to a three-dimensional integral over all space. This evaluation is usually divided into two parts. First, it is necessary to compute the energy fluence at the phantom or patient surface. This requires that the treatment planning program models specific aspects of the linear accelerator including the finite source size, the primary collimator, the flattening filter, dynamic or physical wedges, multileaf collimators or cerrobend blocks, and compensators. Secondly, one must apply the inverse square law and exponential attenuation to this incident fluence to determine the terma, , at each point within the phantom or patient and convolve the result with the point-spread function, . Essentially, the first part of the calculation takes into account the properties of the accelerator (including any beam-modifying devices used for the treatment), and the second part of the calculation takes into account the patient anatomy.
The convolution equation given earlier is strictly valid only for homogeneous media (i.e., the point-spread function must be spatially invariant). One way to model the effects of tissue heterogeneities is to replace all physical distances in the convolution integral with effective pathlengths. Many investigators have used this approximation. 93 - 95 Details of this and other approximations that allow the convolution/superposition integral to be evaluated efficiently under many different conditions are not discussed here, but an excellent review is found in Webb’s book. 96
The convolution/superposition algorithm can account for the effects of non–unit density material anywhere in the vicinity of the calculation point because it requires a three-dimensional integral over the entire radiation field. By contrast, most correction-based dose-calculation techniques require only a simple one-dimensional evaluation of effective pathlength, and can thus account for the effects of only those tissue heterogeneities that lie along a ray connecting the radiation source to the calculation point. Ahnesjö 97 has tested the convolution method against Monte Carlo–generated data for simple layered as well as more complicated phantom geometries and found that the convolution model predicted the buildup dose and penumbra in the low-density regions fairly accurately. Lydon 98 has tested the convolution model for clinical beams in homogeneous media and also obtained generally good results.

Monitor Unit Calculations

Monitor Unit Calculations for Correction-Based Dose Calculations
We have now defined all the parameters needed to calculate the number of MUs (or timer setting) as well as the dose to any point of interest in the irradiated field. The dose normalization for each field is usually done with one of two techniques, depending on whether the patient is set up to a fixed SSD or is set up isocentrically. For a fixed-SSD setup, the dose from each field is normalized to 100% on the central axis at a depth of d max . For an isocentric setup, the dose for each beam is normalized to 100% at the isocenter, that is, at the point corresponding to the intersection between the central axis and the axis of gantry rotation. We now consider how to calculate the number of Motor Units required to be set to give a desired dose at the depth of interest, for these two techniques.

Fixed Source-to-Skin Distance Technique
Since by definition there is a fixed SSD distance for this technique, PDD is the appropriate choice of parameterizations for the dose variation along the central axis. PDD is normalized to be 100% at the depth of the maximum dose, d max . The number of MUs is then calculated as follows, where r is the equivalent square field size defined at the surface:

where
MU=monitor unit setting on console,
D=dose prescribed at depth d (cGy),
CF=calibration factor for machine in cGy/MU at d max in phantom,
OF C =output factor for collimator field size r compared to 10 cm × 10 cm,
PDD=percentage depth dose for equivalent square field size r at surface, for SSD and depth d of treatment,
S p (r)=BSF(r)/BSF(10 × 10), that is, the phantom scatter factor,

(the inverse square factor), and
f mod = product of transmission factors of beam modifiers not in the calibration (e.g., wedge factor, blocking tray).
The reader should note that the product CF × OF C in the above equation is simply the effective calibration factor for the treatment at d max for the actual field size used in units of cGy/MU. Also, the product PDD × S p (r) is the PDD at depth d corrected for the actual field size r at the surface. The product of all four terms CF × OF C × (PDD/100) × S p (r) is the dose per MU at depth d for the equivalent square field size r (defined at the surface) in units of cGy/MU.

Isocentric (Source-to-Isocenter) Technique
For the SAD technique, the SSD might be changing across the field, so an appropriate choice of variables to describe the change in dose with depth on the central axis is one that is independent of SSD. The best choice seems to be TPR, since it can be specified at a fixed depth for all field sizes. For this technique, the MUs can be calculated as follows:

where
D=dose prescribed at depth d iso (cGy),
CF=calibration factor for machine in cGy/MU at d ref in phantom,
OF C =output factor for collimator field size r compared to 10 cm × 10 cm,
TPR=tissue-phantom ratio evaluated at d iso where the field size is of an equivalent square of side r (defined at d iso ),

which is the phantom scatter factor and where r is defined at d iso ,

(SCD is the source-to-calibration distance and SAD is the source-to-isocenter distance).
Note that CF × OF C is the dose/MU at d ref corrected for the actual collimator field size; TPR × S p (r) is the TPR (corrected for phantom scatter) at depth d iso for equivalent square field size r defined at d iso ; and CF × OF C × TPR × S p (r) is the dose/MU at depth d iso for field size r at d iso .

Monitor Unit Calculations for Convolution/Superposition Dose Calculations
If the convolution/superposition model is used for the dose calculations, then the MUs for a given beam may be calculated from the following equation (Pinnacle treatment planning system, 1999, Philips Medical Systems, Andover, MA):

where D Q is the dose at the dose-specification point Q (usually isocenter). The calibration factor, CF, is the measured dose per MU at the reference point for the calibration field. For convolution/superposition calculations, this reference point is typically selected to be sufficiently large (e.g., 10 cm) such that the effects of electron and photon contamination from the treatment head are negligible. ND is the normalized dose, defined as the dose per unit energy fluence at point Q relative to the dose per unit energy fluence at the reference point for the calibration field (i.e., the point at which CF is measured). f mod is the tray transmission factor, and OF C is the collimator output factor. OAR is the off-axis factor, which is required if the calculation point is not on the central axis.
OF C is determined from the relationship between phantom scatter and collimator scatter proposed by Khan 1 ; that is, OF C = S c,p /S p , where S c,p is the total scatter factor, and S p is the phantom scatter factor. The total scatter factor is easily measured and is the ratio of the dose per MU in a phantom at the reference point for field size r to that measured at the reference depth for the standard field size (typically 10 cm × 10 cm). The phantom scatter factor, S p , represents the ratio of the dose contributed from scattered radiation for a given field size at the reference point to that for a standard field size. This quantity is calculated directly for each clinical treatment field using the convolution/superposition model. The factor OF C is then obtained by dividing the measured S c,p value by the calculated S p value.
Comparing the MU equation for the convolution/superposition model to that used for standard correction-based dose calculations for points along the central axis, we see that, for a water phantom with normally incident beam in an isocentric treatment setup, the term ND corresponds to

Thus, ND may be thought of as a patient-specific, treatment-field-specific TPR. All of the correction terms that must be included in the standard MU equation (e.g, S p , and f SAD ) are implicitly included in the value of ND, which is calculated by the convolution/superposition method for each treatment field.

References

1 Khan FM. The physics of radiation therapy , ed 3. Philadelphia: Lippincott Williams and Wilkins; 2003.
2 Johns HE, Cunningham JR. The physics of radiology , ed 4. Springfield, Ill: Charles C Thomas; 1983. p 796
3 Williams JR, Thwaites DI. Radiotherapy physics in practice . Oxford: Oxford University Press; 1993. p 280
4 Hendee WR. Radiation therapy physics , ed 2. Chicago: Yearbook Medical Publishers; 1979. p 517
5 Amaldi U. Cancer therapy with particle accelerators. Nuclear Physics . 1999;A:375c.
6 Austin-Seymour M, Munzehrider JE, Goitein M, et al. Progress in low-LET heavy particle therapy: Intracranial and paracranial tumors and uveal melanomas. Radiat Res . 1985;104:S219.
7 Miller D. A review of proton beam radiation therapy. Med Phys . 22(Part 2), 1995.
8 Slater JM, Miller DW, Archambeau JO. Development of a hospital-based proton beam treatment center. Int J Radiat Oncol Biol Phys . 1988;14:761.
9 Castro JR, Chen GTY, Blakeley EA. Current considerations in heavy charged-particle radiotherapy: A clinical research trial of the University of California Lawrence Berkeley Laboratory, Northern California Oncology Group and Radiation Therapy Oncology Group. Radiat Res . 1985;104:S263.
10 Castro J, Phillips T, Prados M, et al. Neon heavy charged particle radiotherapy of glioblastoma of the brain. Int J Radiat Oncol Biol Phys . 1997;38:257.
11 Futami Y, Kanai T, Fujita M, et al. Broad-beam three-dimensional irradiation system for heavy-ion radiotherapy at HIMAC. Nuclear Instr Meth Physics Res . 1999;A:143.
12 Kraft G, Arndt U, Becher W, et al. Heavy ion therapy at GSI. Nuclear Instr Meth Physics Res . 1995;A:66.
13 Raju MR. Heavy particle radiotherapy . New York: Academic Press; 1980. p 500
14 Suzuki M, Kase Y, Yamaguchi H, Kanai T, Ando K. Relative bological effectiveness for cell-killing effect on various human cell lines irradiated with heavy-ion medical accelerator in Chiba (HIMAC) carbon-ion beams. Int J Radiat Oncol Biol Phys . 2000;48:241.
15 Orecchia R, Zurlo A, Loasses M, et al. Particle beam therapy (hadrontherapy): Basis for interest and clinical experience. Eur J Cancer . 1998;34:459.
16 Verhey L, Koehler A, McDonald J, et al. The determination of absorbed dose in a proton beam for purposes of charged-paraticle radiation therapy. Radiat Res . 1979;79:34.
17 Kanai T, et al. Biophysical characteristics of himac clinical irradiation system for heavy-ion radiation therapy. Int J Radiat Oncol Biol Phys . 1999;44:201.
18 Kraft G. Tumortherapy with ion beams. Nuclear Instr Meth Physics Res . 2000;A:1.
19 Douglas J, Laramore G, Austin-Seymour M, et al. Neutron radiotherapy for adenoid cystic carcinoma of minor salivary glands. Int J Radiat Oncol Biol Phys . 1996;36:87.
20 Douglas J, Laramore G, Autin-Seymour M, et al. Treatment of locally advanced adenoid cystic carcinoma of the head and neck with neutron radiotherapy. Int J Radiat Oncol Biol Phys . 2000;46:551.
21 Huber P, Debus J, Latz D, et al. Radiotherapy for advanced adenoid cystic carcinoma: Neutrons, photons or mixed beam? Radiother Oncol . 2001;59:161.
22 Raymond J, Vuong M, Russell K. Neutron beam radiotherapy for recurrent prostate cancer following radical prostatectomy. Int J Radiat Oncol Biol Phys . 1998;41:93.
23 Schwartz D, Einck J, Bellon J, et al. Fast neutron radiotherapy for soft tissue and cartilaginous sarcomas at high risk for local recurrence. Int J Radiat Oncol Biol Phys . 2001;50:449.
24 Bittner N, Koh WJ, Laramore G, et al. Treatment of locally advanced adenoid cystic carcinoma of the trachea with neutron radiotherapy. Int J Radiat Oncol Biol Phys . 2008.
25 Sauerwein W, Zurlo A. The EORTC Boron Neutron Capture Therapy (BNCT) Group: Achievements and future projects. Eur J Cancer . 2002;38:S31.
26 Yamamoto T, Matsumura A, Nakai K, et al. Current Clinical Results of the Tsukuba BNCT Trial. Appl Radiat Isot . 2004;61:1089.
27 Harling O, Riley K, Binns P, et al. The MIT user center for neutron capture therapy research. Radiat Res . 2005;165:221.
28 Henriksson R, Capala J, Michanek A, et al. Boron neutron capture therapy (BNCT) for globlastoma multiforme: A phase II study evaluating a prolonged high dose of boronophenylalanine (BPA). Radiother Oncol . 2008;88:183.
29 Yamamoto T, Nakai K, Matsumura A. Boron neutron capture therapy for glioblastoma. Cancer Letters . 2008;262:143.
30 Kankaanranta L, Seppälä T, Koivunoro H, et al. Boron Neutron Capture Therapy in the treatment of locally recurred head and neck cancer. Int J Radiat Onc Biol Phys . 2007;69:475.
31 Scalliet P, Remouchamps F, Lhoas M, et al. A retrospective analysis of the results of p(65) + Be neutrontherapy for the treatment of prostate adenocarcinoma at the Cyclotron of Louvain-la-Neuve. Part 1: Survival and progression-free survival. Cancer Radiother . 2001;5:262.
32 Palmer M, Goorley T, Kiger W, et al. Treatment planning and dosimetry for the Harvard-Mit phase I clinical trial of cranial neutron capture therapy. Int J Radiat Oncol Biol Phys . 2002;53:1361.
33 Allt WE. Supervoltage radiation treatment in advanced cancer of the uterine cervix: A preliminary report. J Can Med Assoc . 1969;100:792.
34 Johns HE, Bates LM, Watson TA. 1,000 Curie cobalt units for radiation therapy. 1. The Saskatchewan cobalt 60 unit. Br J Radiol . 1952;25:296.
35 Green DT, Errington RF. Design of a cobalt 60 beam therapy unit. Brit J Radiol . 1952;25:309.
36 Kerst DW. The betatron. Radiology . 1943;40:115.
37 Miller CW. Travelling-wave linear accelerator for x-ray therapy. Nature . 1953;171:297.
38 Purdy JA, Goer DA. Dual energy x-ray beam accelerators in radiation therapy: An overview. Nucl Inst Methods Phys Res . 1985;10/11:1090.
39 Karzmark CJ, Pering NC. Electron linear accelerators for radiation therapy: History, principles and contemporary developments. Phys Med Biol . 1973;18:321.
40 Karzmark CJ, Morton RJ. A Primer on Theory and Operation of Linear Accelerators in Radiation Therapy . Rockville, Md: US Bureau of Radiological Health; 1981.
41 Adler J, Murphy MJ, Chang SD, et al. Image-guided robotic radiosurgery. Neurosurg . 1999;44:1299.
42 Mackie T, Balog J, Ruchalak K, et al. Tomotherapy. Semin Radiat Oncol . 1999;9:108.
43 Reistad D, Brahme A. The microtron, a new accelerator for radiation therapy. In: 3rd International Conference on Medical Physics . Gøtenberg, Sweden: Chalmers University of Technology; 1972.
44 Svensson H, Jonsson L, Larsson LG, et al. A 22 MeV mictrotron for radiation therapy. Acta Radiol Ther Phys Biol . 1977;16:145.
45 Jongen Y, Beeckman W, Cohilis P. The proton therapy system for MGH’s NPTC: Equipment description and progress report. Bull Cancer Radiother . 1996;82(Suppl):219s.
46 Pedroni E, Bohringer T, Coray A, et al. Initial experience of using an active beam delivery technique at PSI. Strahlenther Onkol . 1999;175(suppl 2):18.
47 AAPM. American Association of Physicists in Medicine, A protocol for the determination of absorbed dose from high-energy photon and electron beams. Med Phys . 1983;10:741.
48 Almond PR, Biggs PJ, Coursey BM, Saiful Huq M, Nath R, Rogers DWO. AAPM’s TG-51 protocol for clinical reference dosimetry of high energy photon and electron beams. Med Phys . 1999;26:1847.
49 Domen SR, Lamperti PJ. A heat-loss compensated calorimeter: Theory, design and performance. J Res NBS . 1974;78A:595.
50 Schulz RJ, Wuu CS, Weinhous MS. The direct determination of dose-to-water using a water calorimeter. Med Phys . 1987;14:790.
51 Domen SR. A sealed water calorimeter for measuring absorbed dose. NIST J Res . 1994;99:121.
52 Domen SR. Absorbed dose water calorimeter. Med Phys . 1980;7:157.
53 McDonald JC, Domen SR. A-150 plastic radiometric calorimetry for charged particles and other radiations. Nucl Instr Methods Phys Res . 1986;752:35.
54 Attix F. Introduction to radiological physics and radiation dosimetry . New York: John Wiley and Sons; 1986. p 607
55 Seuntjens J, Palmans H. Correction factors and performance of a 4° C sealed water calorimeter. Phys Med Biol . 1999;44:627.
56 Fricke H, Hart EJ. Chemical dosimetry. In: Attix FH, Roesch WC, editors. Radiation dosimetry . New York: Academic Press, 1966.
57 ICRU: Stopping Powers for Electron Beams with Energies Between 1 and 50 MeV. International Commission on Radiation Units and Measurements, 1984.
58 CRU: Average Energy Required to Produce an Ion Pair. International Commission on Radiation Units and Measurements, 1979.
59 Cameron JR, Suntharalingam N, Kenney GN. Thermoluminescent dosimetry . Madison, Wisc: University of Wisconsin Press; 1968.
60 Chuang C, Verhey L, Xia P. Investigation of the use of MOSFET for clinical IMRT dosimetric verification. Med Phys . 2002;29:1109.
61 van Battum LJ, Hoffmans D, Piersma H, Heukelom S. Accurate dosimetry with GafChromic™ EBT film of a 6 MV photon beam in water: What level is achievable? Med Phys . 2008;35:704.
62 Wilcox E, Daskalov G, Nedialkova L. Comparison of the Epson Expression 1680 flatbed and the Vidar VXR-16 Dosikmetry PRO™ film scanners for use in IMRT dosimetry using Gafchromic and radiographic film. Med Phys . 2007;34:41.
63 Cohen M, Jones DEA, Greene D. Central axis depth dose data for use in radiotherapy. Br J Radiol . 1972;11(suppl):1.
64 Health Physics Association. Depth dose tables for use in radiotherapy. Br J Radiol . 1961;10(suppl):1.
65 Velkley DE, Manson DJ, Purdy JA. Build-up region of megavoltage photon radiation sources. Med Phys . 1975;2:14.
66 Johns HE, Whitmore G, Watson T, et al. A system of dosimetry for rotation therapy with typical rotation distributions. J Can Assn Radiol . 1953;4:1.
67 Johns HE, Bruce WR, Reid WB. The dependence of depth dose on focal skin distance. Br J Radiol . 1958;31:254.
68 Cunningham JR. Scatter-air ratios. Phys Med Biol . 1972;17:42.
69 Clarkson JR. A note on depth doses in fields of irregular shape. Br J Radiol . 1941;14:265.
70 Karzmark CJ, Deubert A, Loevinger R. Tissue-phantom ratios—An aid to treatment planning. Br J Radiol . 1965;38:158.
71 Holt JG, Laughlin JS, Moroney JP. The extension of the concept of tissue-air ratios (TAR) to high energy x-ray beams. Radiology . 1970;96:437.
72 Ellis F, Hall EJ, Oliver R. A compensator for variations in tissue thickness for high energy beams. Br J Radiol . 1959;32:421.
73 Mackie T, Scrimger J, Battista J. A convolution method for calculating dose for 15-MV x rays. Med Phys . 1985;12:188.
74 Cunningham J, Tissue inhomogeneity corrections in photon-beam treatment planning, ed; Orton C, editor; Progress in medical radiation physics; 1982; Plenum, New York:45.
75 Ahnesjö A, Aspradakis M. Dose calculations for external photon beams in radiotherapy. Phys Med Biol . 1999;44:99.
76 Coffey CW, Hines HC. Inhomogeneity corrections in treatment planning of the thorax. Am Assoc Med Dosimetrists J . 1983;8:6.
77 Sontag MR, Cunningham JR. The equivalent tissue-air ratio method for making absorbed dose calculations in a heterogeneous medium. Radiology . 1978;129:787.
78 O’Connor JE. The variation of scattered x-rays with density in an irradiated body. Phys Med Biol . 1957;1:352.
79 Young MEJ, Gaylord JD. Experimental tests of corrections for tissue inhomogeneities in radiotherapy. Br J Radiol . 1970;43:349.
80 Batho HF. Lung corrections in cobalt 60 beam therapy. J Can Assoc Radiol . 1964;15:79.
81 Ma C-M, Faddegon B, Rogers DWO, et al. Accurate characterization of the Monte Carlo beams for radiotherapy. Med Phys . 1997;24:401.
82 Ma C-M, Mok E, Kapur A, et al. Clinical implementation of a Monte Carlo treatment planning system. Med Phys . 1999;26:2133.
83 Ma C-M, Li J, Pawlicki T, et al. A Monte Carlo dose calculation tool for radiotherapy treatment planning. Phys Med Biol . 2002;47:1671.
84 Hartmann-Siantar C, Walling R, Daly T, et al. Description and dosimetric verification of the PEREGRINE Monte Carlo dose calculation system for photon beams incident on a water phantom. Med Phys . 2001;28:1322.
85 Faddegon B, Blogh J, Mackerzie R, et al. Clinical considerations of Monte Carlo for electron radiotherapy treatment planning. Radiat Phys Chem . 1998;35:217.
86 DeMarco J, Solberg T, Smathers J. A CT-based Monte Carlo simulation tool for dosimetry planning and analysis. Med Phys . 1998;25:1.
87 Wang L, Chui C, Lovelock M. A patient-specific Monte Carlo dose-calculation method for photon and electron radiotherapy treatment planning dose calculations. Med Phys . 1998;25:867.
88 Sempau J, Wilderman S, Bielajew A. DPM, a fast, accurate Monte Carlo code optimized for photon and electron radiotherapy treatment planning dose calculations. Phys Med Biol . 2000;45:2263.
89 Kawrakow I, Fippel M, Friedrich K. 3D electron dose calculation using voxel based Monte Carlo algorithm. Med Phys . 1996;23:479.
90 Chetty I, Moran J, Nurusher T, et al. Experimental validation of the DPM Monte Carlo code using minimally scattered electron beams in heterogeneous media. Phys Med Biol . 2002;47:1837.
91 Parker RP, Hobday PA, Cassell KJ. The direct use of CT numbers in radiotherapy dosage calculations for inhomogeneous media. Phys Med Biol . 1979;24:802.
92 Boyer A, Mok E. A photon dose distribution model employing convolution calculations. Med Phys . 1985;12:169.
93 Mohan R, Chui C, Lidofsky L. Differential pencil beam dose computation model for photons. Med Phys . 1986;13:64.
94 Ahnesjö A, Andreo P, Brahme A. Calculation and application of point spread functions for treatment planning with high energy photon beams. Acta Oncol . 1987;26:49.
95 Nilsson M, Knöös T. Application of the Fano theorem in inhomogeneous media using a convolution algorithm. In: 1st Biennial ESTRO Meeting on Physics in Clinical Radiotherapy . Budapest: European Society for Therapeutic Radiology and Oncology; 1991.
96 Webb S. The physics of three-dimensional radiation therapy. Medical Science Series . Bristol and Philadelphia: IOP Publishing, Ltd; 1993. p 373
97 Ahnesjö A. Collapsed cone convolution of radiant energy for photon dose calculation in heterogeneous media. Med Phys . 1989;16:577.
98 Lydon J. Photon dose calculations in homogeneous media for a treatment planning system using a collapsed cone superposition convolution algorithm. Phys Med Biol . 1998;43:1813.
8 Imaging in Radiation Oncology *

Luc M. Bidaut, PhD, John L. Humm, PhD, Gikas S. Mageras, PhD, Lawrence N. Rothenberg, PhD
Accurate, patient-specific anatomic information is a prerequisite for planning and implementing the delivery of radiation to the entire extent of the malignancy while minimizing exposure to critical structures. For this reason, anatomic images are of utmost importance in radiotherapy. In fact, most of the significant recent advances in radiation oncology have resulted from developments in imaging modalities such as computed tomography (CT), magnetic resonance imaging (MRI), magnetic resonance spectroscopy imaging (MRSI), positron emission tomography (PET), and digital planar image receptors. With information from the new imaging modalities, it is now possible to define treatment volumes and critical structures with great precision, thus reducing marginal misses and irradiation of normal tissues. Such capabilities may permit higher tumor doses, potentially leading to improved local control, while maintaining the same level of normal tissue morbidity or even reducing it. 1 - 4
Images of various types are employed at virtually every step of the radiation treatment process, including diagnosis, assessment of the extent of disease, treatment planning, treatment delivery, follow-up, and outcome evaluation. These images may be classified in several ways. Images may be cross-sectional or projectional. The former category includes CT, MRI, MRSI, PET, and single-photon emission computed tomography (SPECT); examples of the latter are simulation and portal films and CT scout views. In some cases, useful projectional images (e.g., digitally reconstructed radiographs [DRRs]) can be reconstructed from the cross-sectional data. The acquired image data may be in either analog or digital format. Conventional images captured on photographic film are analog, whereas most tomographic images like CT and MRI are acquired as digital data. In general, analog images have better spatial resolution but a smaller dynamic range. Digital images can be mathematically processed (e.g., filtered and enhanced). Analog images can be similarly manipulated, provided t