Lung Cancer:: Prevention, Management, and Emerging Therapies (Current Clinical Oncology)

Lung Cancer

Current Clinical Oncology Maurie Markman, MD, Series Editor

For other titles published in this series, go to www.springer.com/series/7631

Lung Cancer Prevention, Management, and Emerging Therapies

Edited by

David J. Stewart, md, frcpc Professor and Deputy Chair, Department of Thoracic Head and Neck Medical Oncology, Division of Cancer Medicine, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Editor David J. Stewart, MD, FRCPC Professor and Deputy Chair Department of Thoracic Head and Neck Medical Oncology Division of Cancer Medicine The University of Texas M.D. Anderson Cancer Center Houston, TX USA [email protected]

ISBN 978-1-60761-523-1 e-ISBN 978-1-60761-524-8● DOI 10.1007/978-1-60761-524-8 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010921199 © Humana Press, a part of Springer Science+Business Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press, a part of Springer Science+Business Media (www.springer.com)

This book is dedicated to Lesley, Megan, Adam, Andrew, Jenika, Grayson and Cameron whose love gives purpose to my life and work, and whose support, understanding and patience make all things possible.

Preface

Defining the Lung Cancer Problem Lung cancer is the leading cause of cancer death in the world.1 It kills almost as many Americans as cancers of the breast, prostate, colon, rectum, pancreas, and kidney combined, and accounts for 28.6% of all US cancer deaths.2 With an increase in the 5-year relative survival rate from 13% to only 16% in the more than 30 years from 1974 to the present,2 it will take us another 840 years to eradicate lung cancer deaths if we do not improve the current rate of progress. As discussed in this text, lung cancer prevention has received substantial attention. The decrease in smoking in recent decades has helped, but smoking is not the only problem. Lung cancer in people who have never smoked is currently the 5th leading cause of cancer death in the United States.3 Several factors contribute to the lethality of lung cancer, including the rapidity of tumor growth, advanced stage at diagnosis (due to nonspecificity of early symptoms and the uncertain efficacy of screening), early development of metastases, and resistance to therapy. Several chapters in this book discuss new molecular targets that may be potentially exploitable in the future, as well as discussing our track record to date in exploiting them. Over the last few decades, we have made several errors that have slowed our pace in the war against lung cancer. For example, until recently, most nonsmall cell lung cancers were treated more or less as if they were the same disease. It has been postulated that common cancers are both common and resistant to therapy since many different mutations may give rise to them, and that each underlying mutation may require a different treatment approach.4 Hence, there may never be one silver bullet for lung cancer. We may need instead 20 or 30 different agents, each targeting a molecularly distinct subpopulation of patients. Large randomized trials ignore this possibility and try to overpower biological realities by using the statistical power of large patient numbers to achieve a significant p value. Hence, we have ended up with a variety of therapies that achieve statistical significance, but with survival gains of mere weeks.5 There are two major problems with this. The first is that if the p value is not significant, a drug may be abandoned despite being of marked benefit in a small

vii

viii

Preface

subpopulation of patients, as happened with gefitinib. The other side of the problem is that with p<0.05, the drug may be accepted as being “effective”, and the drug may be applied widely at high cost and potential toxicity, despite being of value in only a small subpopulation of patients. We feel that progress against lung cancer and other malignancies has been slowed by our placing the efficacy bar too low, using large randomized trials to eke out small gains.5 We would argue that we need small trials looking for large gains, not large trials looking for small gains. We need to molecularly characterize the tumors of all patients from the earliest phase I trials onward and use the results of this molecular profiling to identify those most and least likely to benefit from the therapy.5 If we do randomized trials without fully characterizing patients, we may well be misled. For example, simulations suggest to us that if a new therapy quintuples survival in a 10% subpopulation of patients whose tumors express a particular target, this will be missed unless around 2,000 patients are included in the trial. If the new agent only doubles survival in this 10% subpopulation, then more than 5,000 patients may be needed to detect the benefit. As discussed earlier, at the end of the study, we will either conclude that the therapeutic approach is “effective” and inappropriately apply it widely, or we will conclude that it is “ineffective” and inappropriately discard it. If, however, one correctly identifies the required target, then one may get the correct answer (that the agent is effective in the subpopulation with the target) with fewer than 20 patients if survival is quintupled and with fewer than 100 patients if survival is only doubled. At $26,000 per patient (the amount that it currently costs per patient on a phase III trial6), the money saved by reducing phase III trial sizes would pay for very extensive molecular profiling of every patient to ever participate in phase I and II trials of the agent, and correlating these profiles with % tumor shrinkage or with some other measure of tumor cell kill would go a long way toward defining the population that should subsequently be targeted in phase III trials. There are also other problems with randomized trials in unselected patients. The study may indicate that the agent was not helpful when in fact benefit in one subpopulation was balanced by harm in another, as may be the case with EGFR inhibitors in patients with EGFR vs. Kras mutations.7 Furthermore, if an agent hitting a target present in 5% of the population is compared to another hitting a target present in 40% of the population, the agent hitting the more common target will win and will be the new “standard of care”. It will be incorrectly concluded that this agent is the “better” agent when in fact it is not: it just hits a more common target. If there is no exploitable target that is present in >40% of patients, progress will plateau and we would make no further gains. In addition, an agent which increases the survival of all patients by 30% (equivalent to increasing median survival from 6 months to 7.8 months) may consistently beat an agent which increases survival 5-fold in a 10% subpopulation, but we would argue that the latter agent is the more important one. Contrary to this, some newer statistical approaches such as randomized discontinuation strategies8 are specifically designed to try to identify small advances, and in our opinion contribute to the problem. Overall, as stated above, we feel that it is of paramount importance to molecularly characterize all

Preface

ix

patients on study, and then to aim for large gains in appropriate subpopulations rather than using unselected patients to aim for small gains in large studies.5 In addition to the efficacy bar being set too low, we feel that the safety bar has been set too high for fatal, incurable diseases like cancer.5 We recently calculated that increasingly stringent research regulations might have decreased toxic death rates by 0.3% for patients on study. However, with the cost of complying with these regulations running at an estimated $8,000 per patient studied and an estimated life expectancy for patients on study of 1 year, this translates into $2.7 million per year of life gained - an amount far higher than either other preventive measures or the figure of $50,000–$100,000 per year of life gained that is regarded as being acceptable for therapies.9 In addition, if 5,000–10,000 patients need to be treated on studies to make a small advance (eg., a new therapy that increases cure rate of lung cancer by 1% through improved adjuvant therapy and that increases survival of incurable patients by a median of 3 months), the regulations would have led to a savings of 15–30 life-years (5,000–10,000 × 0.3% × 1 year), but if the regulations slow the advance by a conservatively estimated 5 years, the regulation-induced delays will have cost 285,000 life-years in the United States and almost 2 million life-years worldwide, seriously challenging equipoise. We feel that the regulations governing cancer research need to change.5 Overall, lung cancer remains a formidable foe. While we have made some progress, much remains to be done. In this book, we give a brief description of where we stand today, as well as offering a glimpse of the path forward. Houston, TX

David J. Stewart, MD, FRCPC

References 1. Lopes Pegna A, Picozzi G (2009) Lung cancer screening update. Curr Opin Pulm Med 15:327–33 2. Jemal A, Siegel R, Ward E et al (2008) Cancer statistics. CA Cancer J Clin 58:71–96 3. Gazdar AF, Minna JD Molecular techniques for early detection of lung cancer and for studying preneoplasia. In: Pass HI, Carbone DP, Johnson DH, Minna JD, Turrisi AT (eds) Lung cancerprinciples and practice. 3rd edn. New York: Lippincott, Williams & Wilkins; 2004:201 4. Braiteh F, Kurzrock R (2007) Uncommon tumors and exceptional therapies: paradox or paradigm? Mol Cancer Ther 6:1175–1179 5. Stewart DJ, Kurzrock R (2009) Cancer: the road to Amiens. J Clin Oncol 27:328–333 6. Phase 3 clinical trial costs exceed $26,000 per patient. Life sciences world 2006. http://www. lifesciencesworld.com/news/view/11080 7. Eberhard DA, Johnson BE, Amler LC et al (2005) Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol 23:5900–5909 8. Rosner GL, Stadler W, Ratain MJ (2002) Randomized discontinuation design: application to cytostatic antineoplastic agents. J Clin Oncol 20:4478–4484 9. Berry SR, Neumann PJ, Bell C et al (2009) What price for a year of life? A survey of US and Canadian oncologists. Proc ASCO 27:Abstract 6565

Contents

Molecular Pathology of Lung Cancer............................................................ Alejandro Corvalan and Ignacio I. Wistuba

1

Tumor Microenvironment............................................................................... Tonya C. Walser, Jane Yanagawa, Edward Garon, Jay M. Lee, and Steven M. Dubinett

27

Racial and Ethnic Diversity in Lung Cancer................................................ Carol J. Etzel and Sumesh Kachroo

71

Pharmacogenetics of Lung Cancer................................................................. Xifeng Wu and Jian Gu

87

Lung Cancer Prevention................................................................................. 107 Nir Peled, Robert L. Keith, and Fred R. Hirsch Adjuvant and Neoadjuvant Therapy of NSCLC.......................................... 139 Katherine Pisters Chemoradiotherapy for Inoperable Non-small Cell Lung Cancer............. 161 James D. Cox and David J. Stewart Management of Advanced Non-small Cell Lung Cancer: Front Line Treatment...................................................................................... 177 William N. William Jr. and David J. Stewart Chemotherapy in Previously Treated Patients with Non-small Cell Lung Cancer.................................................................. 195 Frank V. Fossella Epidermal Growth Factor Receptor Inhibitors in the Treatment of Non-small Cell Lung Cancer...................................................................... 205 Paul Wheatley-Price and Frances A. Shepherd xi

xii

Contents

Angiogenesis Inhibitors in Lung Cancer....................................................... 227 Leora Horn and Alan Sandler Other Molecular Targeted Agents in Non-small Cell Lung Cancer............ 253 Benjamin Besse and Jean-Charles Soria Vaccine Therapy for Lung Cancer................................................................. 279 John Nemunaitis and Jack Roth Gene-Based Therapies for Lung Cancer....................................................... 305 John Nemunaitis and Jack Roth Lung Cancer Resistance to Chemotherapy................................................... 331 David J. Stewart Small Cell Carcinoma of the Lung................................................................. 395 Emer O. Hanrahan and Bonnie Glisson Mesothelioma.................................................................................................... 435 Mary Frances McAleer, Reza J. Mehran, and Anne Tsao Advances in Oncology Clinical Research: Statistical and Study Design Methodologies.................................................................... 467 B. Nebiyou Bekele Palliative Care for Patients with Lung Cancer............................................. 483 David Hui and Eduardo Bruera The Future of Lung Cancer............................................................................ 503 Sophie Sun, Joan H. Schiller, Monica Spinola, and John D. Minna Index.................................................................................................................. 515

Contributors

B. Nebiyou Bekele, Ph.D. Associate Professor, Department of Biostatistics, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Benjamin Besse, M.D. Assistant Professor, Head of Thoracic Oncology Group, Institut de Cancerologie, Gustave Roussy, Villejuif, France Eduardo Bruera, M.D. Professor and Chair, Department of Palliative Care and Rehabilitation Medicine, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Alejandro Corvalan, M.D. Assistant Professor, Departments of Pathology and Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA James D. Cox, M.D. Professor and Head, Division of Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Steven M. Dubinett, M.D. Professor and Division Chief, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA; Chief, Division of Pulmonary and Critical Care Medicine, Department of Medicine; Director, UCLA Lung Cancer Research Program, Jonsson Comprehensive Cancer Center, Los Angeles, CA, USA Carol J. Etzel, Ph.D. Assistant Professor, Department of Epidemiology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Frank V. Fossella, M.D. Professor, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

xiii

xiv

Contributors

Edward Garon, M.D. Assistant Professor, Division of Hematology and Oncology, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Bonnie Glisson, M.D. Professor, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Jian Gu, Ph.D. Assistant Professor, Department of Epidemiology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Emer O. Hanrahan, M.D. Assistant Professor, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Fred R. Hirsch, M.D., Ph.D. Professor, Division of Medical Oncology, University of Colorado, Denver, CO, USA Leora Horn, M.D. Thoracic Oncology Fellow, Vanderbilt Ingram Cancer Center, Nashville, TN, USA David Hui, M.D. Fellow, Department of Symptomatic Center and Palliative Care, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Sumesh Kachroo, M.S. Department of Epidemiology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Robert L. Keith, M.D. Assistant Professor, Pulmonary Sciences and Critical Care Medicine, University of Colorado, Denver, CO, USA Jay M. Lee, M.D. Assistant Professor-in-Residence, Division of Pulmonary and Critical Care Medicine, Department of Medicine; Division of Cardiothoracic Surgery, Department of Surgery, UCLA Lung Cancer Research Program, Jonsson Comprehensive Cancer Center, Los Angeles, CA, USA Mary Frances McAleer, M.D., Ph.D. Assistant Professor, Department of Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Reza J. Mehran, M.D. Professor, Department of Thoracic and Cardiovascular Surgery, The University of Texas M.D. Anderson Cancer Center, TX, USA

Contributors

xv

John D. Minna, M.D. Professor, Division of Hematology and Oncology and Director, the Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, USA John Nemunaitis, M.D. Executive Director, Mary Crowley Cancer Research Centers, Texas Oncology PA, Dallas, TX, USA; Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, USA Nir Peled, M.D., Ph.D. Visiting Professor, Pulmonary Medicine and Medical Oncology, University of Colorado, Denver, CO, USA Katherine Pisters, M.D. Professor, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Jack Roth, M.D. Professor, Department of Thoracic and Cardiovascular Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Alan Sandler, M.D. Professor and Chief, Division of Hematology and Medical Oncology, Oregon Health and Science University, Portland, OR, USA Joan H. Schiller, M.D. Professor, Division of Hematology and Oncology and the Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, USA Frances A. Shepherd, M.D., FRCPC Professor of Medicine, University Health Network, Princess Margaret Hospital Division and the University of Toronto, Toronto, ON, Canada Jean-Charles Soria, M.D., Ph.D. Professor, Chief of Service, Institut Gustave Roussy, Villejuif, France Monica Spinola, Ph.D. Postdoctoral Researcher, Division of Hematology and Oncology and the Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, USA David J. Stewart, M.D., FRCPC Professor and Deputy Chair, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

xvi

Contributors

Sophie Sun, M.D. Assistant Professor, Division of Medical Oncology, British Columbia Cancer Agency, Vancouver, BC, Canada Anne Tsao, M.D. Assistant Professor, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Tonya C. Walser, Ph.D. Postdoctoral Scholar, Division of Pulmonary and Critical Care Medicine, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Paul Wheatley-Price, MBChB, MRCP Specialist Registrar in Medical Oncology, Guy’s Hospital, London, UK William N. William, Jr., M.D. Assistant Professor, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Ignacio I. Wistuba, M.D. Professor, Departments of Pathology and Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Xifeng Wu, M.D., Ph.D. Professor, Department of Epidemiology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Jane Yanagawa, M.D. Postdoctoral Scholar, Division of Pulmonary and Critical Care Medicine, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

Molecular Pathology of Lung Cancer Alejandro Corvalan and Ignacio I. Wistuba

Abstract  In contrast to most other organs, the lungs demonstrate a very wide range of epithelial tumors that vary in their location and histology. These tumors show varying degrees of relationship to smoke exposure, with the central carcinomas showing the greatest relationship. The molecular lesions found in the tumors share certain common elements and have characteristic changes. Their precursor lesions also differ, with some being well defined, whereas others are poorly understood because of the difficulty in identifying them before surgical resection of an existing tumor. Thus, their natural history is also poorly understood. The advent of newer molecular genetic methods to examine lung tumor and preneoplastic lesion tissue specimens will help delineate all the significant molecular abnormalities responsible for lung cancer development and progression. Gene-specific and copynumber alteration approaches have identified mutations that have proven to be unique in lung cancer. Simultaneously, molecular profiling studies at DNA, RNA, and protein levels have provided a molecular classification of lung cancer while also improving the ability to predict prognosis and response to treatment. The integration of these different platforms might overcome the overtraining and instability of the identified signatures. Combining clinical covariates with molecular profiling approaches may be the optimal approach for building new models for lung cancer. The ultimate goal is to be able to identify all molecular changes present in any one patient’s tumor and to use this information for early molecular detection, prediction of biological/clinical behavior and prognosis, and selection or rational development of therapeutics. Keywords  Molecular pathology • Lung cancer • Oncogenes • Tumor suppressor genes • Preneoplasia • Pathogenesis

A. Corvalan (*) Department of Pathology, Unit 85, MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030-4009, USA e-mail: [email protected] D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_1, © Humana Press, a part of Springer Science+Business Media, LLC 2010

1

2

A. Corvalan and I.I. Wistuba

Introduction Accurate pathological classification of lung cancer is essential for patients to receive appropriate therapy. From a histopathological and biological perspective, lung cancer is a highly complex neoplasm (1, 2). There are several histological types, with the most frequent being small cell lung carcinoma (SCLC, 15%) and the non-small cell lung carcinoma (NSCLC) variants such as squamous cell carcinoma (30%), adenocarcinoma (45%), and large cell carcinoma (9%) (3). Advances in molecular technologies have provided insight into the biological processes involved in the pathogenesis of lung cancer. Recent findings have indicated that clinically evident lung cancers are the result of the accumulation of numerous genetic and epigenetic changes, including abnormalities in the inactivation of tumor suppressor genes and the activation of oncogenes (1, 2). All of these molecular abnormalities involve the “hallmarks of cancer,” including abnormalities in the self-sufficiency of growth signals, insensitivity to antigrowth signals, sustained angiogenesis, evasion of apoptosis, limitless replicative potential, and tissue invasion and metastasis (4, 5). Recent molecular advances have provided unique opportunities to develop rational targeted therapies for lung cancer. These advances have led to an emerging and exciting new area of therapy that takes advantage of cancer-specific molecular defects which render the cancer cells more likely to respond to specific agents (6, 7). In this setting, the analysis of molecular abnormalities of lung cancers is becoming increasingly important, and the adequate integration of routine pathological and molecular examinations into the diagnosis, classification, and choice of therapy options presents an interesting challenge. Although many molecular abnormalities have been described in clinically evident lung cancers, relatively little is known about the molecular events preceding the development of lung carcinomas and about the underlying genetic basis of lung carcinogenesis (2, 8, 9). In the past decade, several studies have provided information regarding the molecular characterization of the preneoplastic changes involved in the pathogenesis of lung cancer, especially squamous cell carcinoma and adenocarcinoma (8, 10). In this chapter, we will describe the most relevant molecular abnormalities observed in lung cancer with regard to their pathological and clinical characteristics. In addition, we will review the current understanding of this cancer’s early pathogenesis and progression.

Molecular Pathology of Lung Cancer It has been shown that multiple genetic changes are found in clinically evident lung cancers and involve several dominant oncogenes as well as known and putative recessive oncogenes (tumor suppressor genes) (1, 2). Many growth factors or regulatory peptides and their receptors are overexpressed by cancer cells and adjacent normal-appearing cells in the lung and thus provide a series of autocrine and paracrine

Molecular Pathology of Lung Cancer

3

growth stimulatory loops in this neoplasm (11). The list of recessive oncogenes that are involved in lung cancer is likely to include as many as 10–15 known and putative genes (1, 2). Recessive oncogenes are believed to be inactivated via a two-step process involving both alleles. Knudson (12) proposed that the first “hit” frequently is a point mutation, whereas the second allele is subsequently inactivated via a chromosomal deletion, translocation, or other event, such as methylation of gene promoter regions. Until recently, chromosomal rearrangements have mainly been linked to bloodrelated cancers and seldom to solid tumors. Recently, it has been shown that a small inversion within chromosome 2p results in the formation of a fusion gene composed of portions of the echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene in NSCLC cells (EML-ALK fusion) (13, 14). This discovery indicates that activated fusion genes associated with chromosomal rearrangements are probably both common and important in lung cancer. Studies of large numbers of lung cancers have demonstrated different patterns of molecular alterations between the two major groups of lung carcinomas (SCLC and NSCLC) (Table  1) (1) and among the two major histologic types of NSCLC (squamous cell carcinomas and adenocarcinomas; Table 2) (15–19).

Non-small Cell Lung Carcinoma Pathology NSCLC comprises a heterogeneous group of histology types, with the most frequent types being adenocarcinoma, squamous cell carcinoma, large cell carcinoma, adenosquamous carcinoma, and sarcomatoid carcinoma (3). Adenocarcinoma accounts for nearly 40% of all lung cancers. According to the 2004 World Health Organization classification, adenocarcinoma can be subclassified into five major subtypes: acinar, papillary, solid with mucin production, bronchioloalveolar (BAC), and mixed adenocarcinomas (3). Most adenocarcinomas (~90%) are heterogeneous, consisting of two or more of the histological subtypes and are thus categorized as mixed subtype (20). When tumor cells grow in a purely lepidic fashion without the evidence of invasion, they are regarded as BAC (21). Unfortunately, this strict definition of BAC as a true noninvasive tumor is not uniformly applied, and pathologists frequently label mixed tumors with varying degrees of lepidic growth as either BAC tumors or adenocarcinomas with BAC features. This inconsistency of terminology has led to considerable confusion. Squamous cell carcinoma accounts for approximately 30% of all lung cancers. Most squamous cell carcinomas of the lung (~70%) present as central lung tumors (22). The tumor may grow to a large mass and then cavitate; most cavitating lung cancers are squamous cell carcinomas (23). Large cell carcinoma accounts for approximately 9% of all lung cancers, they have a spectrum of morphologies, and most of them consist of large cells with

4

A. Corvalan and I.I. Wistuba

Table 1  Molecular differences between non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC) Molecular abnormalities SCLC NSCLC Oncogenes EGFR TK domain mutations No 10–40% EGFR gain copy number No 25–50% HER2 mutations Not studied 4% HER2 gain copy number No 10% RAS mutations <1% 15–20% MYC amplification 18–31% 8–20% NKX2-1 (TITF-1) amplification Not studied 14% BCL-2 IHC 75–95% 10–35% Tumor suppressor genes TP53 abnormalities LOH 90% 65% Mutation 75% ~50% p53 IHC 40–70% 40–60% RB abnormalities LOH 67% 31% rb abnormalities (IHC) 90% 15–30% p16Ink4 abnormalities LOH 53% 66% Mutation <1% 10–40% p16 IHC 0–10% 30–70% LKB1 Mutation or deletion Not studied 26% PTEN/MMAC1 loci LOH 91% 41% TSG101 abnormal transcripts ~100% 0% DMBT1 abnormal expression 100% 43% 3p LOH various regions >90% >80% 8p21–23 LOH 80–90% 80–100% Other specific LOH regions 1q23, 9q22–32, 10p15, 13q34 13q11, Xq22.1 Promoter hypermethylation RASSF1 gene >90% ~40% 72% 41% RARb gene LOH loss of heterozygosity; IHC immunohistochemistry; % percent of tumors that have the abnormality

abundant cytoplasm and large nuclei with prominent nucleoli (23). These carcinomas also include some specific variants, including the large cell neuroendocrine carcinomas (LCNEC) (24).

Genetic Abnormalities Several studies have shown that the major types of NSCLCs harbor multiple molecular similarities and differences (Table 2). The genetic abnormalities of lung adenocarcinomas include point mutations of dominant oncogenes, such as KRAS,

Molecular Pathology of Lung Cancer Table  2  Summary of characteristics of adenocarcinoma and lung Abnormality Squamous cell carcinoma Precursor lesions Histopathology Known: Squamous dysplasia and carcinoma in situ Gene abnormalities TP53 LOH and mutation LOH Chromosomal regions 8p21–23, 9p21, 17p/TP53 Invasive tumors KRAS mutation Very rare BRAF mutation 3% EGFR mutation Very rare EGFR copy gain 30% EGFR IHC expression ~80% HER2 mutation Very rare HER2 amplification 2% LKB1 inactivation 19% MET mutation 12% MET amplification 21% Very rare EML-ALK fusion

5 squamous cell carcinoma of the Adenocarcinoma Probable: AAH

KRAS mutationEGFR mutation 9p21, 17p/TP53 10–20%a 2% 10–30%a 15% ~50% 4% 6% 34% 14% 20% 7%

IHC immunohistochemistry; LOH loss of heterozygosity a With variations based in patients’ smoking history and ethnicity

BRAF, and EGFR, and tumor suppressor genes such as TP53 and p16Ink4 (1, 25–28). In lung cancer, activating KRAS mutations preferentially cause adenocarcinomas (20–30%) (1). Most KRAS mutations in lung cancer are G→T or G→C transversions, and they affect exons 12 (~90% of the mutations), 13, and 61 (29). These types of KRAS mutations have been associated with tobacco-related carcinogens (30). However, recently it has been shown that KRAS mutations are found in 15% of adenocarcinomas from never smokers, and these patients were significantly more likely than former or current smokers to have a transition mutation (G→A) rather than the transversion mutations known to be smoking-related (29). Activating mutations of BRAF, a Raf serine–threonine kinase pathway component, have been also detected in lung adenocarcinoma cell lines (11%) (27) and primary tumors (3%) (28). Recent studies indicate that EGFR mutations affecting the tyrosine kinase domain of the gene (exons 18–21) are present in approximately 10–55% of adenocarcinomas, but are almost entirely absent in other types of lung carcinomas (25). EGFR mutations are somatic in origin, and they occur significantly more frequently in adenocarcinomas of patients who have never smoked (51–68%), women (42–62%), and patients from countries in East Asia (30–50%) when compared with patients from Western countries (~10%) (25, 31–36). These EGFR mutations are clinically relevant because most of them have been associated with sensitivity of lung adenocarcinoma to small molecule tyrosine kinase inhibitors (TKIs; gefitinib and erlotinib) (31–33, 37). More than 80% of the mutations detected in EGFR are

6

A. Corvalan and I.I. Wistuba

in-frame deletions in exon 19 or a single missense mutation in exon 21 (L858R) (25, 31–36). It has been proposed that lung cancer cells with mutant EGFR might become physiologically dependent on the continued activity of the gene for the maintenance of their malignant phenotype, leading to an accelerated development of lung adenocarcinoma (38). An increase in the number of EGFR copies, including high polysomy and gene amplification, has been detected by fluorescent in situ hybridization (FISH) in 22% of patients with surgically resected (stages I–IIIA) NSCLC, and the increase in EGFR copy number correlated with EGFR protein overexpression (39). Higher frequencies (40–50%) of a high number of EGFR copies have been reported in patients with advanced NSCLC (40–45). Recent studies have demonstrated that tumor cell high EGFR copy number, identified using FISH, may also be a predictor for response to EGFR TK inhibitors (40–46). Recent studies have identified a new EGFR mutation (T790M, exon 20) in patients who had a relapse after initial response to therapy with EGFR tyrosine kinase inhibitors. This mutation confers resistance to treatment with EGFR tyrosine kinase small molecules (47, 48). However, this mutation has also been detected in tumors from patients not exposed to EGFR inhibitors (47, 48). Amplification of the MET oncogene seems to be another major mechanism of acquired resistance to EGFR tyrosine kinase inhibitors (49). Other proposed resistance mechanisms include activation of other receptor tyrosine kinases, such as insulin-like growth factor 1 receptor (which can bypass EGFR to activate critical downstream signaling pathways) (50), KRAS mutations (44), and the epithelial-to-mesenchymal transition (EMT) (51). In addition, HER2 gene mutations, although infrequent (3%), have been detected in lung cancer, predominantly in lung adenocarcinomas and in patients with an East Asian ethnic background (26). There are remarkable similarities between EGFR and HER2 gene mutations in lung cancer, including predilection for adenocarcinoma histological type, mutation type, gene location (tyrosine kinase domain), and specific patient subpopulations targeted. These similarities are unprecedented and suggest similar etiological factors. Of great interest are EGFR, HER2, and KRAS mutations which are mutually exclusive, suggesting different pathways to lung cancer in smokers and never smokers. Recently, using high-resolution gene copy analysis of a large number of lung adenocarcinomas, it was shown that the most common focal event in this tumor type was amplification of the NKX2-1 gene (also known as TITF1) located at the 14.q13.3 region (52). NKX2-1 is a transcription factor that plays an essential role in the formation of type II pneumocytes, the cell type that lines lung alveoli (53). The protein coded by NKX2-1 has been called thyroid transcription factor-1 (TTF-1) and is considered to be a reliable marker for primary adenocarcinoma of the lung. On the basis of findings of higher levels of protein expression of nuclear TTF-1 in EGFR-mutant lung adenocarcinomas than in wild-type tumors, it has been suggested that EGFR-mutant lung adenocarcinoma originates from the terminal respiratory unit (54). Recently, amplification of NKX2-1 has been also detected in squamous cell carcinoma histology (55).

Molecular Pathology of Lung Cancer

7

Recently, Ding et al. (56) reported the results of a collaborative study to discover new somatic mutations for lung adenocarcinomas (Table 3). By DNA sequencing of 623 specific genes with known or potential relationships to cancer, these authors found more than 1,000 somatic mutations across approximately 400 samples. Detailed analysis identified 26 genes mutated at significantly high frequencies. These genes include, among others, tyrosine kinases (ERBB4), ephrin receptor genes (EPH3, EPH10), vascular endothelial growth factor receptor genes (VEGFR2), basic fibroblast growth factor receptor-4 (FGFR4), and tumor suppressor genes (NF1, APC, RB1 and ATM). Remarkably, the frequency of genetic abnormalities in most genes in lung adenocarcinoma is not greater than 20% (Table 3). TP53 mutations are frequent in lung adenocarcinomas, with different patterns detected by sex and smoking status (57). p16Ink4 inactivation by multiple mechanisms occurs frequently in adenocarcinomas and may be related to smoking (1). In addition, gene methylation studies have shown that methylation rates of APC, CDH13, and RARb genes are significantly higher in adenocarcinomas than in

Table  3  Summary of the frequency of genes with mutation and copy number abnormalities detected by profiling DNA analysis in adenocarcinoma of the lung from US patients (52, 56) Gene Copy number change Frequency changes (%) Mutation + amplification 38 RAS TP53 Mutation + deletion 36 EGFR Mutation + amplification 27 STK11 Mutation + deletion 23 FGFR Mutation + amplification 19 CDKN2A (p16) Mutation + deletion 15 MAPK Mutation 13 NKX2-1 (TITF-1) Amplification 12 NTRK Mutation 10 EPHA/B Mutation + amplification 10 VEGFR Mutation + amplification  9 MDM2 Mutation + amplification  9 PI3K Mutation + amplification  8 ATM Mutation + deletion  8 NF1 Mutation + deletion  7 APC Mutation + deletion  7 INSR Mutation  5 CDK Mutation + amplification  5 PDGFR Mutation  4 RB1 Mutation  4 PTPRD Deletion  4 TSC1/2 Mutation  2 PTEN Mutation + deletion  2 RAF Mutation  2 GSK3 Mutation  2 AKT Mutation  2 Mutation  1 CTNNB1

8

A. Corvalan and I.I. Wistuba

squamous cell carcinomas (58, 59). Among other chromosomal abnormalities, localized chromosome 3p deletions are also frequently detected in lung adenocarcinomas (15). Squamous cell carcinomas demonstrate most of the genetic abnormalities commonly present in lung NSCLCs, except for KRAS and EGFR gene mutations, which are more frequent in adenocarcinomas (Table 2) (1, 25). However, squamous cell carcinomas are characterized by a very high frequency (84%) of EGFR expression as determined by immunohistochemical methods (39). Disruption of the TP53 and RB gene pathways is frequent in squamous cell carcinomas (1). Most tumors demonstrate large segments of chromosome 3p deletions (15). Recently, it is has been shown that the inactivation of the tumor suppressor gene LKB1 by mutation and deletion is a relatively frequent event in both squamous cell carcinomas (19%) and adenocarcinomas (34%) of the lung (60). Angiogenesis Nutrients and oxygen supplied by the vasculature are crucial for cell survival; therefore, angiogenesis is critical for tumor growth. Angiogenesis involves interactions between tumor cells, endothelial cells, and stromal cells (61). A number of angiogenic factors, including inducers and inhibitors regulating endothelial cell proliferation and migration, have been identified in lung cancer. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a key regulator of angiogenesis (62). High VEGF expression, reported in approximately 60% of stage I NSCLCs, is closely associated with high intratumoral angiogenesis and poor prognosis (63). In NSCLC, the majority of studies support a correlation between high VEGF expression, high microvessel density (MVD), and poor prognosis (61). Other angiogenic factors recently studied in lung cancer include platelet-derived growth factor and its receptor (64), hypoxia inducible factor-1a (65), and basic fibroblast growth factor and its receptors 1 and 2 (66). Fibroblast growth factor 2 (FGF2), or basic FGF (bFGF), and its transmembrane tyrosine kinase receptors (the FGFRs) make up a large, complex family of signaling molecules involved in several physiologic processes, and the dysregulation of these molecules has been associated with cancer development (67, 68). bFGF belongs to a family of ubiquitously expressed ligands that bind to the extracellular domain of FGFRs, initiating a signal transduction cascade that promotes cell proliferation, motility, and angiogenesis (67–69). As with some other angiogenesis pathways, the bFGF pathway has been shown to be activated in lung cancer (66, 70–76). Elevated levels of bFGF, FGFR1, and FGFR2 proteins have been detected in NSCLC cell lines (66, 77). bFGF, FGFR1, and FGFR2 are frequently overexpressed in squamous cell carcinoma and adenocarcinoma of the lung. bFGF signaling pathway activation may be an early phenomenon in the pathogenesis of squamous cell carcinoma and is thus an attractive novel target for lung cancer chemopreventive and therapeutic strategies (78).

Molecular Pathology of Lung Cancer

9

Epithelial-to-Mesenchymal Transition This is a process in which cells undergo a developmental switch from an epithelial to a motile mesenchymal phenotype (79). This process has been related to embryologic morphogenesis, fibrosis, and lately, to the progression and metastasis of epithelial tumors (80). Epithelial to mesenchymal transition has been described among many types of cancer, including lung cancer (51, 81, 82). In lung cancer, EMT has been studied in  vitro; the expression of individual markers in EMT has been described, and these markers are associated with prognosis (51, 83). We have demonstrated that EMT phenotype (loss of E-cadherin and gain of N-cadherin, integrinavb6, vimentin, and matrix metalloproteinase-9) is commonly expressed in primary squamous cell carcinoma and adenocarcinoma of the lung (Prudkin et al., unpublished). These findings have led to the hypothesis that EMT is a target for lung cancer therapy. The EMT phenomenon has also been associated with resistance to therapy with EGFR inhibitors (51). Markers Associated with Response to Chemotherapy The current standard of treatment for patients with advanced NSCLC is a doublet chemotherapy regimen, which commonly includes, among others, a platinumbased drug and gemcitabine (84). Platinum compounds are heavy metal complexes that form adducts with and cross-link between DNA molecules, blocking DNA replication and transcription. Repair of these adducts and cross-links is dependent on the excision repair cross-complementation group 1 (ERCC1) (85). In NSCLC, high level of mRNA ERCC1 expression correlated with better survival in surgically resected tumors (86, 87), and low level of mRNA correlated with longer overall survival in patients with advanced tumors treated with platinum-based therapy. (88, 89) Patients with completely resected NSCLC and negative tumors for ERCC1 protein expression by immunohistochemistry appear to benefit from adjuvant cisplatin-based chemotherapy, whereas patients with ERCC1 positive tumors do not (90). It has been postulated that an intact DNA repair mechanism may reduce the accumulation of genetic aberrations that are thought to contribute to a tumor’s malignant potential and therefore the risk of relapse after definitive treatment. Conversely, a defective DNA repair mechanism reflected by low ERCC1 expression may be responsible for better response to platinum-based chemotherapy and longer survival in treated patients. Several studies have demonstrated that the ribonucleotide reductase M1 polypeptide (RRM1) is a molecular target of gemcitabine and, thus, a key cellular determinant of its therapeutic efficacy (87). RRM1 is located on chromosome segment 11p15.5, a region with a frequent deletion in NSCLC (91). Low levels of expression of the gene are associated with poor survival among patients with NSCLC (92). RRM1 is also the predominant cellular determinant of the efficacy of the nucleoside analogue gemcitabine (2¢,2¢-difluorodeoxycytidine) (93). In surgically resected NSCLC, a high level of RRM1 protein expression by immunofluorescence correlated with better survival (87).

10

A. Corvalan and I.I. Wistuba

In addition, elevated levels of mRNA RRM1 have been shown to be predictive of a lack of gemcitabine efficacy in advanced NSCLC patients (93, 94).

Small Cell Lung Carcinoma Pathology SCLCs account for approximately 15% of all lung cancers (95). They are characterized by small epithelial tumor cells with finely granular chromatin and absent or inconspicuous nucleoli (95). Necrosis is frequent and extensive, and the mitotic count is high. Fewer than 10% of SCLCs also demonstrate a mixture of NSCLC histological types—usually adenocarcinoma, squamous cell carcinoma, and large cell carcinoma and these are termed “combined SCLCs” (95). Molecular Abnormalities The etiology of SCLC is strongly tied to cigarette smoking, and now there is considerable information concerning molecular abnormalities involved in its pathogenesis (1, 17, 96). Autocrine growth factors such as neuroendocrine regulatory peptides (e.g., bombesin/gastrin-releasing peptide) are prominent in SCLC (17). Dominant oncogenes of the MYC family are frequently overexpressed (and may be amplified) in both SCLC and NSCLC, whereas the KRAS oncogene is never mutated in SCLC. TP53 is mutated in more than 90% of SCLCs, and the RB gene is inactivated in more than 90% of SCLCs. In contrast to NSCLC, p16INK4a, the other component of the retinoblastoma/p16 pathway, is almost never abnormal in SCLC. A genome-wide allelotyping study of approximately 400 polymorphic markers distributed at around 10 cm resolution across the human genome found that on average, 17 loci showed loss of heterozygosity (LOH) in individual SCLCs and 22 for NSCLC, with an average size of loss of 50–60  cm and an average frequency of microsatellite abnormalities of five per tumor (97). There were 22 different “hot spots” for loss of heterozygosity, 13 with a preference for SCLC, 7 with a preference for NSCLC, and 2 affecting both. This provides clear evidence on a genome-wide scale that SCLC and NSCLC differ significantly in the tumor suppressor genes that are inactivated during their pathogenesis. In addition, differences in gene methylation profiles have been detected between SCLC and NSCLC tumors (16).

Molecular Profiling Studies in Lung Cancer Molecular profiling studies that began with single or relatively small groups of genes or proteins have now progressed to large-scale and high-throughput methods using DNA, RNA and protein-based approaches. These large-scale methods analyze

Molecular Pathology of Lung Cancer

11

thousands of genes at one time and have led to a better understanding of the complexity of gene abnormality patterns of lung cancer.

RNA Signatures Among RNA-based methods, cDNA microarray is a powerful technique for the global analysis of gene expression that has become a standard tool in molecular biology and has succeeded in identifying multiple crucial genes that are up- or down-regulated in a variety of tumors, including lung (18, 19, 98–101). Several groups have reported cDNA microarray-based profiles that are potentially useful in assessing molecular classification (18, 19, 98), prognosis, (99–101) and response to treatment (102) of lung cancer. Most studies focusing on molecular classification have shown that cDNA microarray profiles recapitulate the morphological classification of lung cancer (18, 19). Potti et  al. (100) developed a genomic strategy to determine prognosis in early-stage NSCLC by identifying a gene expression profile that predicted the risk of recurrence in a cohort of 89 patients with stage I and II tumors. Then, they evaluated the predictor profile in two independent cohorts to find an overall predictive accuracy of 72 and 79%, respectively. In addition, a subgroup of patients with stage IA disease was identified who were at high risk for recurrence and who might be best treated by adjuvant chemotherapy. Using cDNA microarray strategy coupled with quantitative polymerase chain reaction (PCR) analysis, Chen et  al. (101) developed a five-gene signature (DUSP6, MMD, STAT1, ERBB3, and LCK) that correlated with clinical outcome in stages I and II NSCLC. This five-gene signature model was validated in two independent cohorts, and was closely associated with relapse-free and overall survival. With respect to response to treatment, the study of Oshita et  al. (102) showed that gene expression profiles in peripheral blood obtained from 31 patients before chemotherapy (paclitaxel and irinotecan) correlated with the outcome in patients with advanced NSCLC. Multivariate analysis revealed that genes encoding protein phosphatase, interleukin-1alfa and IgA were independent predictive factors for chemosensitivity (102). Using in  vitro drug sensitivity data coupled with cDNA microarray data, Potti et al. (103) developed gene expression signatures that predicted sensitivity to individual chemotherapeutic drugs for several human solid tumors, including lung tumors. Each signature was validated with response data from an independent set of cell line studies. Of interest, signatures developed to predict response to individual agents, when combined, could also predict response to multidrug regimens. In relation to molecular predictors of metastatic pattern in lung cancer, Zohrabian et  al. (104) and Kikuchi et  al. (105) studied gene expression profiling of brain metastases from primary lung adenocarcinoma and found 1,561 genes consistently altered. Further functional classification placed the genes into seven categories: cell cycle and DNA damage repair, apoptosis, signal transduction molecules, transcription

12

A. Corvalan and I.I. Wistuba

factors, invasion and metastasis, adhesion, and angiogenesis. Interestingly, genes involved in adhesion, motility, and angiogenesis were consistently up-regulated in metastatic brain tumors, while genes involved in apoptosis, neuroprotection, and suppression of angiogenesis were markedly down-regulated, collectively making these cancer cells prone to metastasis (104, 105). Although prognostic gene expression signatures for survival in early-stage lung cancer have been proposed, for clinical application, it is critical to establish their performance across different subject populations and in different laboratories. In this scenario, Shedden et al. (106) studied clinical and histopathological prognostic factors and their relationship to molecular prognostic factors. These authors reported a large, training-testing, multisite, blinded validation study to characterize the performance of several prognostic models based on gene expression for 442 lung adenocarcinomas. They examined whether gene expression profile either alone or combined with clinical covariates such as gender, age or stage could be used to predict the overall survival in lung cancer patients. After building several models of risk scores that correlated with patient outcome they found that most of these models performed better when gene expression profiles were combined with clinical covariates. These results support the idea that the combined use of gene expression profiles and clinical covariates will be necessary when building prognostic models for early-stage lung cancer.

MicroRNA Profiles Another RNA-based approach involves the assessment microRNAs (miRNAs). miRNAs are a recently discovered class of small (~18–24 nt) nucleic acids that negatively regulate gene expression (107). This novel class of molecules modulates a wide array of growth and differentiation processes in human cancers (107). An emerging number of studies have shown that miRNAs can act as oncogenes, as tumor suppressor genes, or sometimes as both (108). High-throughput analyses have demonstrated that miRNA expression is commonly dysregulated in human cancer (107), including lung cancer (109–111). However, considerable disagreement remains with respect to the optimal miRNA signature for specific cancer cell types, which appears to depend largely on the analytical platform (107). In lung cancer, miRNA profiles have been shown to correlate with disease outcome (109, 111). Using real-time reverse transcription PCR, Yu et al. (111) identified a five-miRNA signature in NSCLC that predicts treatment outcome. In that study, patients with high risk scores in their miRNA signatures showed poor overall and disease-free survival when compared with patients with low risk scores (111). In addition, it has been shown that miRNAs regulate several important pathways in lung cancer. Weiss et al. (112) showed that loss of miRNA-128b, located on chromosome 3p and a putative regulator of EGFR, correlated with response to targeted EGFR inhibition. miRNA is an area of very active research that will have an impact on lung cancer pathogenesis and therapy.

Molecular Pathology of Lung Cancer

13

DNA Copy Number Profiles Chromosomal regions harboring tumor suppressor genes and oncogenes are often deleted or amplified. Deletions have been analyzed mostly by LOH studies using microsatellites (15, 97, 113). Amplifications have been investigated by comparative genomic hybridization (CGH) and single-nucleotide polymorphism (SNP) arrays (52, 114, 115). Although lung cancers have been profiled using CGH (115), few high-throughput and comprehensive whole genome efforts examining lung cancer tissue specimens are available (52, 114). Weir et al. reported a large-scale characterization of copy number alterations in a large set of lung adenocarcinomas using dense SNP arrays (52). They discovered the amplification of the 14.q13.3 region spanning the NKX2-1 (TITF-1) gene. In addition, other genes were shown to have an increased number of copies in adenocarcinoma, including MDM2, MYC, CDK4, KRAS, TERT, and VGFA. However, the results of the study indicated that many of the genes that are involved in lung adenocarcinoma remain to be discovered.

Epigenetic Methylation Profiling The term “epigenetic” refers to a heritable change in the pattern of gene expression that is mediated by mechanisms other than alterations in the primary nucleotide sequence of a gene (116). Normal epigenetic modifications of DNA encompass three types of changes: chromatin modifications, DNA methylation, and genomic imprinting, each of which is altered in cancer cells. These processes control the packaging and function of the human genome and play an important role in normal development and in diseases such as cancer (117). Epigenetic modifications are frequent among human tumors and epigenetic alterations often serve as potent surrogates for genetic mutations. The study of epigenetic modifications in cancer has ranged from early assessments of global DNA methylation content to the recently proposed epigenetic progenitor model. These observations provide a common unifying mechanism for cancer development (118). Multiple known and putative tumor suppressor genes have been reported to be inactivated by hypermethylation in lung cancer. Shames et  al. (119) performed a genome-wide screen using a global expression profiling approach in NSCLC cell lines and identified 132 genes for which expression was induced by the DNA demethylating agent 5¢-aza-2¢-deoxycytidine. Methylation analysis of the promoter region of a subset of these genes in primary lung tumors and adjacent nonmalignant tissues showed that 31 genes were methylated in tumors but not in normal lung tissue or peripheral blood cells. Tsou et al. (120) identified 13 loci showing significant differential DNA methylation levels between tumoral and nontumoral lung tissue, and eight of these showed highly significant hypermethylation in lung adenocarcinomas. Different patterns of gene methylation have been found in the major histological types of NSCLCs, with the methylation of APC, CDH13, and RAR-b being significantly higher in adenocarcinoma than in squamous cell carcinoma (58).

14

A. Corvalan and I.I. Wistuba

It has been shown that methylation of three genes—RASSF1A, RUNX3, and CDH13—correlated with worse prognosis in patients with surgically resected NSCLC (121), and interestingly, RUNX3 methylation correlated with a worse prognosis in adenocarcinoma, whereas methylation of RASSF1A was associated with a worse prognosis in squamous histology. In multivariate analysis, both genes have been found to be independent prognostic factors of worse outcome (121). Methylation of p16Ink4, CDH13, RASSF1A, and APC in NSCLC stage I tumors and in histologically tumor-negative lymph nodes was associated with disease recurrence, independently of other clinical and pathological factors (122). These findings suggest that methylation of four genes in patients with stage I NSCLC resected with curative intent is associated with early recurrence.

Proteomic Signatures It has been suggested that proteomics-based approaches complement the genomic initiatives and represent the next step in attempting to understand the biology of cancer. Because mRNA expression does not always correlate with levels of protein expression, cDNA-based gene expression analysis cannot always indicate which proteins are expressed or how their activity might be modulated after translation (123). Accordingly, a comprehensive analysis of protein expression patterns in tissues might improve our ability to understand the molecular complexities of tumor cells. Among others, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can profile proteins in tissues (124). This technology can not only address peptides and proteins in sections of tumor tissues, but can also be used for high-resolution imaging of individual biomolecules present in tissue sections (124). Recently, proteomic pattern analysis using MALDI-TOF MS directly on small amounts of frozen lung tumor tissues was used to accurately classify and predict histological groups as well as nodal involvement and patient survival in resected NSCLCs (125). If these data are confirmed in larger series, the resulting analysis will have great prognostic and therapeutic implications for patients with NSCLC. Recently, a MALDI-TOF MS algorithm developed from serum specimens was able to classify NSCLC patients with respect to good vs. poor outcomes after treatment with EGFR TKIs (126). Thus, these algorithms have a potential role in assisting in the pretreatment selection of appropriate subgroups of NSCLC patients for treatment with targeted therapy.

Integrative Approaches to Profiling Data Direct profiling of human cancers using an unbiased approach presents intrinsic problems connected with the high genetic noise embedded in the system. This leads to overtraining the data with consequent instability of the signatures identified.

Molecular Pathology of Lung Cancer

15

To circumvent this problem, Bianchi et al. (127) has proposed a biased approach which exploits the molecular knowledge of cancer obtained in model systems. However, biased approaches failed to capture the complex repertoire of alterations of human cancers. Using a cDNA microarray strategy coupled with miRNA profiling, we are currently developing combined signatures (mRNA & miRNA) that might overcome some of these problems. Integrated strategies, which combine different platforms, could potentially lead to the identification of stable and reliable predictive signatures in lung cancer.

Pathogenesis of Lung Cancer Lung cancers are believed to arise after a series of progressive pathological changes that generate preneoplastic or precursor lesions in the respiratory mucosa (Fig. 1). The recent 2004 World Health Organization International Association for the Study of Lung Cancer histological classification of preinvasive lesions of the lung lists three main morphologic forms of preneoplastic lesions in the lung: (3) (a) squamous dysplasia and carcinoma in situ (CIS), (b) atypical adenomatous hyperplasia (AAH), and (c) diffuse idiopathic pulmonary neuroendocrine cell hyperplasia. Although the sequential preneoplastic changes have been defined for centrally arising squamous carcinomas, they have been poorly documented for large cell carcinomas, adenocarcinomas, and SCLCs (Table 3) (128, 129).

Squamous Cell Carcinoma Preneoplastic Lesions Mucosal changes in the large airways that may precede or accompany invasive squamous cell carcinoma include hyperplasia, squamous metaplasia, squamous dysplasia, and CIS (128, 129). Dysplastic squamous lesions may be of different intensities (i.e., mild, moderate, and severe); however, these lesions represent a continuum of cytological and histological atypical changes that can show some overlap between categories. Little is known about the rate and risks of progression of squamous dysplasia to CIS and, ultimately, to invasive squamous cell carcinoma. The current working model of the sequential molecular abnormalities in the pathogenesis of squamous cell lung carcinoma (Fig. 1) indicates that genetic abnormalities commence in histologically normal epithelium and increase with increasing severity of histologic change (130). Mutations follow a sequence, with progressive allelic losses at multiple 3p chromosome sites (3p21, 3p14, 3p22–24, and 3p12) and at 9p21 (p16INK4a) as the earliest detected changes. Later changes include 8p21–23, 13q14 (RB), and 17p13 (TP53) (15, 113, 130). p16INK4a methylation has also been detected in early stage squamous preinvasive lesions with a

16

A. Corvalan and I.I. Wistuba

Normal Alveoli

Normal Epithelium 3p LOH 9p LOH

Smoking

Telomerase Activation

Non-Smoking Hyperplasia

Methylation of TSG

?

EGFR Mutation

Squamous Metaplasia 8p LOH

KRAS Mutation

FHIT-TP53 Genes Inactivation

AAH

Dysplasia

5q LOH BAC

Adenocarcinoma

EGFR Amplification

Carcinoma In Situ

Squamous Cell Ca

Fig. 1  Major molecular pathways involved in the pathogenesis of NSCLC. While the sequence of preneoplastic and molecular events involved in the pathogenesis of squamous cell carcinoma has been partially elucidated, there is a lack of knowledge in the development of adenocarcinomas of the lung. However, there is evidence suggesting that at least two molecular pathways are involved in adenocarcinoma early pathogenesis: smoking and KRAS-related and nonsmoking and EGFR-related. AAH atypical adenomatous hyperplasia; BAC bronchioloalveolar carcinoma; LOH loss of heterozygosity; TSG tumor suppressor gene

frequency that increases during histopathological progression of disease (24% in squamous metaplasia and 50% in CIS) (131). Molecular changes in the respiratory epithelium are extensive and multifocal throughout the bronchial tree of smokers and lung cancer patients, indicating a field effect (“field cancerization”), resulting in widespread mutagenesis of the respiratory epithelium, presumably caused by exposure to tobacco-related carcinogens (15, 130, 132, 133). Using fluorescent bronchoscopy, one can find multiple clonal and subclonal patches with molecular abnormalities that include allelic losses and genomic instability. These patches (which typically consist of an estimated ~40,000–360,000 cells) can be detected in the normal and slightly abnormal bronchial epithelium of patients with lung cancer (134). Despite this progress, neither histological features nor molecular changes of squamous cell carcinoma precursor lesions have been shown to be useful to predict their progression to invasive carcinoma (9, 135).

Molecular Pathology of Lung Cancer

17

Adenocarcinoma Precursor Lesions It has been suggested that adenocarcinomas may be preceded by AAH in peripheral airway cells; (10, 128) however, the respiratory structures and the specific epithelia cell types involved in the origin of most lung adenocarcinomas are unknown (Fig. 1). AAH is considered to be a putative precursor of adenocarcinoma (10, 128). AAH is a discrete parenchymal lesion arising in the alveoli close to terminal and respiratory bronchioles. Because of their size, AAH cells are usually incidental histological findings, but they may be detected grossly, especially if they are 0.5 cm or larger. The increasing use of high-resolution computed tomography scans for screening purposes has led to an increasing awareness of AAH, which remains one of the most important differential diagnoses of air-filled peripheral lesions (called “ground glass opacities”). AHH maintains an alveolar structure lined by rounded, cuboidal, or low columnar cells. The postulated progression of AAH to adenocarcinoma with BAC features, apparent from the increasingly atypical morphology, is supported by the results of morphometric, cytofluorometric, and molecular studies (10, 129). The origin of AAH is still unknown, but the differentiation phenotype derived from immunohistochemical and ultrastructural features suggests an origin from the progenitor cells of the peripheral airways, such as Clara cells and type II pneumocytes (136, 137). There is an increasing body of evidence to support the concept of AAH as a precursor of at least a subset of adenocarcinomas. Several molecular changes frequently present in lung adenocarcinomas are also present in AAH lesions, and they provide further evidence that AAH might represent true preneoplastic lesions (136). The most important finding is the presence of KRAS (codon 12) mutations in up to 39% of AAHs, and these mutations are also relatively frequent alterations in lung adenocarcinomas (10, 138). Other molecular alterations detected in AAH are overexpression of the cyclin D1 (~70%), p53 (10–58%), survivin (48%), and HER2/neu (7%) proteins (10, 139, 140). Of great interest, EGFR mutations have been detected in some cases of atypical AAH accompanying resected peripheral adenocarcinomas, providing further evidence that they represent precursor lesions of peripheral adenocarcinomas (54).

Precursors of Nonsmoking-Related Adenocarcinoma Although most lung cancers are smoking-related tumors, a subset of adenocarcinomas arises in patients who have never smoked. As stated above, somatic mutations in the EGFR and HER2 tyrosine kinase members of the ErbB family have been reported, and these are most likely to occur in a subset of female patients with lung adenocarcinoma who were never smokers or light smokers and of East Asian ethnicity (25, 31–36).

18

A. Corvalan and I.I. Wistuba

To better understand the pathogenesis of EGFR mutant lung adenocarcinomas, our group has investigated the presence of EGFR mutations in the normal bronchial and bronchiolar epithelium adjacent to mutant tumors. As reported by Tang et al. (141), we detected EGFR mutations in normal appearing peripheral respiratory epithelium in 9 (44%) of 21 adenocarcinoma patients but not in patients without mutations in the tumors (141). The finding of more frequent EGFR mutations in normal epithelium within the tumor (43%) than in adjacent sites (24%) suggests a localized field effect phenomenon for this abnormality in the respiratory epithelium of the lung. Although the cell type having those mutations is unknown, we hypothesize that stem or progenitor cells of the bronchial and bronchiolar epithelium are the cell type bearing such mutations. The finding of relatively infrequent EGFR mutations in AAH lesions (three of 40 examined) (54, 142) and the finding of no mutation (25) or relatively low frequency of mutation in true BACs of the lung support the concept that genetic abnormalities of EGFR are not relevant in the pathogenesis of alveolar-type lung neoplasia. The most recent data by Tang et al. (143) suggest that the EGFR mutation precedes an increase in the number of copies of the gene in the pathogenesis of lung adenocarcinoma (143), and that the increase in the number of copies of EGFR is a phenomenon associated with tumor progression and metastasis (Fig. 1). In summary, two different molecular pathways have been identified in the pathogenesis of lung adenocarcinoma (Fig.  1), a smoking-associated activation of KRAS-signaling, and nonsmoking-associated activation of EGFR signaling, the latter of which is detected in histologically normal bronchial and bronchiolar epithelium (144).

Conclusion In contrast to most other organs, the lungs demonstrate a very wide range of epithelial tumors that vary in their location and histology. These tumors show varying degrees of relationship to smoke exposure, with the central carcinomas showing the greatest relationship. The molecular lesions found in the tumors share certain common elements and have characteristic changes. Their precursor lesions also differ, with some being well defined, whereas others are poorly understood because of the difficulty of identifying them before surgical resection of an existing tumor. Thus, their natural history is also poorly understood. The advent of newer molecular genetic methods to examine lung tumor and preneoplastic lesion tissue specimens will help delineate all the significant molecular abnormalities responsible for lung cancer development and progression. Gene-specific and copy-number alteration approaches have identified mutations that have proven to be unique in lung cancer. Simultaneously, molecular profiling studies at DNA, RNA, and protein levels have provided a molecular classification of lung cancer while also improving the ability to predict prognosis and response to treatment. The integration of these different platforms might overcome the overtraining and instability of the identified signatures.

Molecular Pathology of Lung Cancer

19

Combining clinical covariates with molecular profiling approaches may be the optimal approach for building new models for lung cancer. The ultimate goal is to be able to identify all molecular changes present in any one patient’s tumor and to use this information for early molecular detection, prediction of biological/clinical behavior and prognosis, and selection or rational development of therapeutics.

References 1. Minna JD, Gazdar A (2002) Focus on lung cancer. Cancer Cell 1:49–52 2. Herbst RS, Heymach JV, Lippman SM (2008) Lung cancer. N Engl J Med 359:1367–1380 3. Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC (2004) Tumours of the lung. In: Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC (eds) Pathology and genetics: tumours of the lung, pleura, thymus and heart. International Agency for Research on Cancer (IARC), Lyon, pp 9–124 4. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70 5. Fong KM, Sekido Y, Gazdar AF, Minna JD (2003) Lung cancer. 9: molecular biology of lung cancer: clinical implications. Thorax 58:892–900 6. Herbst RS, Sandler AB (2004) Overview of the current status of human epidermal growth factor receptor inhibitors in lung cancer. Clin Lung Cancer 6(Suppl 1):S7–S19 7. Herbst RS, Onn A, Sandler A (2005) Angiogenesis and lung cancer: prognostic and therapeutic implications. J Clin Oncol 23:3243–3256 8. Wistuba II, Mao L, Gazdar AF (2002) Smoking molecular damage in bronchial epithelium. Oncogene 21:7298–7306 9. Wistuba II (2007) Genetics of preneoplasia: lessons from lung cancer. Curr Mol Med 7:3–14 10. Westra WH (2000) Early glandular neoplasia of the lung. Respir Med 1:163–169 11. Fong KM, Minna JD (2002) Molecular biology of lung cancer: clinical implications. Clin Chest Med 23:83–101 12. Knudson AG (1989) Hereditary cancers: clues to mechanisms of carcinogenesis. Br J Cancer 59:661–666 13. Soda M, Choi YL, Enomoto M et al (2007) Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 448:561–566 14. Inamura K, Takeuchi K, Togashi Y et al (2008) EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers. J Thorac Oncol 3:13–17 15. Wistuba II, Behrens C, Virmani AK et al (2000) High resolution chromosome 3p allelotyping of human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple, discontinuous sites of 3p allele loss and three regions of frequent breakpoints. Cancer Res 60:1949–1960 16. Toyooka S, Toyooka KO, Maruyama R et al (2001) DNA methylation profiles of lung tumors. Mol Cancer Ther 1:61–67 17. Wistuba II, Gazdar AF, Minna JD (2001) Molecular genetics of small cell lung carcinoma. Semin Oncol 28:3–13 18. Bhattacharjee A, Richards WG, Staunton J et al (2001) Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. Proc Natl Acad Sci USA 98:13790–13795 19. Garber ME, Troyanskaya OG, Schluens K et al (2001) Diversity of gene expression in adenocarcinoma of the lung. Proc Natl Acad Sci USA 98:13784–13789 20. Brambilla E, Travis WD, Colby TV, Corrin B, Shimosato Y (2001) The new World Health Organization classification of lung tumours. Eur Respir J 18:1059–1068

20

A. Corvalan and I.I. Wistuba

21. Travis WD, Garg K, Franklin WA et  al (2005) Evolving concepts in the pathology and computed tomography imaging of lung adenocarcinoma and bronchioloalveolar carcinoma. J Clin Oncol 23:3279–3287 22. Hammar SP, Brambilla C, Pugatch B et al (2004) Tumours of the lung. Squamous cell carcinoma. In: Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC (eds) Pathology and genetics: tumours of the lung, pleura, thymus and heart. IARC, Lyon, pp 26–34 23. Colby TV, Koss MN, Travis WD (1995) Tumors of the lower respiratory tract, 3rd. series, Fascicle 13. Armed Forces Institute of Pathology, Washington, DC 24. Brambilla C, Pugatch B, Geisinger K et al (2004) Tumours of the lung. Large cell carcinoma. In: Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC (eds) Pathology and genetics: tumours of the lung, pleura, thymus and heart. IARC, Lyon, pp 45–50 25. Shigematsu H, Lin L, Takahashi T et al (2005) Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 97:339–346 26. Shigematsu H, Takahashi T, Nomura M et al (2005) Somatic mutations of the HER2 kinase domain in lung adenocarcinomas. Cancer Res 65:1642–1646 27. Davies H, Bignell GR, Cox C et  al (2002) Mutations of the BRAF gene in human cancer. Nature 417:949–954 28. Brose MS, Volpe P, Feldman M et al (2002) BRAF and RAS mutations in human lung cancer and melanoma. Cancer Res 62:6997–7000 29. Riely GJ, Kris MG, Rosenbaum D et al (2008) Frequency and distinctive spectrum of KRAS mutations in never smokers with lung adenocarcinoma. Clin Cancer Res 14:5731–5734 30. Slebos RJ, Rodenhuis S (1992) The ras gene family in human non-small-cell lung cancer. Monogr Natl Cancer Inst 13:23–29 31. Paez JG, Janne PA, Lee JC et al (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304:1497–1500 32. Lynch TJ, Bell DW, Sordella R et  al (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129–2139 33. Pao W, Miller V, Zakowski M et al (2004) EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA 101:13306–13311 34. Huang SF, Liu HP, Li LH et al (2004) High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan. Clin Cancer Res 10:8195–8203 35. Kosaka T, Yatabe Y, Endoh H, Kuwano H, Takahashi T, Mitsudomi T (2004) Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications. Cancer Res 64:8919–8923 36. Tokumo M, Toyooka S, Kiura K et al (2005) The relationship between epidermal growth factor receptor mutations and clinicopathologic features in non-small cell lung cancers. Clin Cancer Res 11:1167–1173 37. Amann J, Kalyankrishna S, Massion PP et al (2005) Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancer. Cancer Res 65:226–235 38. Gazdar AF, Shigematsu H, Herz J, Minna JD (2004) Mutations and addiction to EGFR: the Achilles ‘heal’ of lung cancers? Trends Mol Med 10:481–486 39. Hirsch FR, Varella-Garcia M, Bunn PA Jr et al (2003) Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 21:3798–3807 40. Cappuzzo F, Hirsch FR, Rossi E et al (2005) Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst 97:643–655 41. Tsao MS, Sakurada A, Cutz JC et al (2005) Erlotinib in lung cancer – molecular and clinical predictors of outcome. N Engl J Med 353:133–144

Molecular Pathology of Lung Cancer

21

42. Hirsch FR, Varella-Garcia M, McCoy J et al (2005) Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes: a Southwest Oncology Group Study. J Clin Oncol 23(28):6838–6845 43. Jackman DM, Holmes AJ, Lindeman N et al (2006) Response and resistance in a non-smallcell lung cancer patient with an epidermal growth factor receptor mutation and leptomeningeal metastases treated with high-dose gefitinib. J Clin Oncol 24:4517–4520 44. Massarelli E, Varella-Garcia M, Tang X et al (2007) KRAS mutation is an important predictor of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer. Clin Cancer Res 13:2890–2896 45. Bunn PA Jr, Dziadziuszko R, Varella-Garcia M et al (2006) Biological markers for non-small cell lung cancer patient selection for epidermal growth factor receptor tyrosine kinase inhibitor therapy. Clin Cancer Res 12:3652–3656 46. Zhu CQ, da Cunha Santos G, Ding K et al (2008) Role of KRAS and EGFR as biomarkers of response to erlotinib in National Cancer Institute of Canada Clinical Trials Group Study BR.21. J Clin Oncol 26:4268–4275 47. Pao W, Miller VA, Politi KA et  al (2005) Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2:1–11 48. Kobayashi S, Boggon TJ, Dayaram T et  al (2005) EGFR mutation and resistance of nonsmall-cell lung cancer to gefitinib. N Engl J Med 352:786–792 49. Engelman JA, Zejnullahu K, Mitsudomi T et al (2007) MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316:1039–1043 50. Morgillo F, Kim WY, Kim ES, Ciardiello F, Hong WK, Lee HY (2007) Implication of the insulin-like growth factor-IR pathway in the resistance of non-small cell lung cancer cells to treatment with gefitinib. Clin Cancer Res 13:2795–2803 51. Yauch RL, Januario T, Eberhard DA et al (2005) Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res 11:8686–8698 52. Weir BA, Woo MS, Getz G et al (2007) Characterizing the cancer genome in lung adenocarcinoma. Nature 450:893–898 53. Ikeda K, Clark JC, Shaw-White JR, Stahlman MT, Boutell CJ, Whitsett JA (1995) Gene structure and expression of human thyroid transcription factor-1 in respiratory epithelial cells. J Biol Chem 270:8108–8114 54. Yatabe Y, Kosaka T, Takahashi T, Mitsudomi T (2005) EGFR mutation is specific for terminal respiratory unit type adenocarcinoma. Am J Surg Pathol 29:633–639 55. Perner S, Wagner P, Soltermann A et al (2009) TTF1 expression in non-small cell lung carcinoma: association with TTF1 gene amplification and improved survival. J Pathol 217:65–72 56. Ding L, Getz G, Wheeler DA et  al (2008) Somatic mutations affect key pathways in lung adenocarcinoma. Nature 455:1069–1075 57. Toyooka S, Tsuda T, Gazdar AF (2003) The TP53 gene, tobacco exposure, and lung cancer. Hum Mutat 21:229–239 58. Toyooka S, Maruyama R, Toyooka KO et  al (2003) Smoke exposure, histologic type and geography-related differences in the methylation profiles of non-small cell lung cancer. Int J Cancer 103:153–160 59. Toyooka S, Suzuki M, Maruyama R et al (2004) The relationship between aberrant methylation and survival in non-small-cell lung cancers. Br J Cancer 91:771–774 60. Ji H, Ramsey MR, Hayes DN et al (2007) LKB1 modulates lung cancer differentiation and metastasis. Nature 448:807–810 61. Sandler AB, Johnson DH, Herbst RS (2004) Anti-vascular endothelial growth factor monoclonals in non-small cell lung cancer. Clin Cancer Res 10:4258s–4262s 62. Asahara T, Takahashi T, Masuda H et al (1999) VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J 18:3964–3972

22

A. Corvalan and I.I. Wistuba

63. Han H, Silverman JF, Santucci TS et al (2001) Vascular endothelial growth factor expression in stage I non-small cell lung cancer correlates with neoangiogenesis and a poor prognosis. Ann Surg Oncol 8:72–79 64. Shikada Y, Yonemitsu Y, Koga T et al (2005) Platelet-derived growth factor-AA is an essential and autocrine regulator of vascular endothelial growth factor expression in non-small cell lung carcinomas. Cancer Res 65:7241–7248 65. Kim SJ, Rabbani ZN, Dewhirst MW et al (2005) Expression of HIF-1alpha, CA IX, VEGF, and MMP-9 in surgically resected non-small cell lung cancer. Lung Cancer 49:325–335 66. Kuhn H, Kopff C, Konrad J, Riedel A, Gessner C, Wirtz H (2004) Influence of basic fibroblast growth factor on the proliferation of non-small cell lung cancer cell lines. Lung Cancer 44:167–174 67. Dailey L, Ambrosetti D, Mansukhani A, Basilico C (2005) Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev 16:233–247 68. Ribatti D, Vacca A, Rusnati M, Presta M (2007) The discovery of basic fibroblast growth factor/fibroblast growth factor-2 and its role in haematological malignancies. Cytokine Growth Factor Rev 18(3–4):327–334 69. Mohammadi M, Olsen SK, Ibrahimi OA (2005) Structural basis for fibroblast growth factor receptor activation. Cytokine Growth Factor Rev 16:107–137 70. Takanami I, Tanaka F, Hashizume T et al (1996) The basic fibroblast growth factor and its receptor in pulmonary adenocarcinomas: an investigation of their expression as prognostic markers. Eur J Cancer 32A:1504–1509 71. Takanami I, Tanaka F, Hashizume T, Kodaira S (1997) Tumor angiogenesis in pulmonary adenocarcinomas: relationship with basic fibroblast growth factor, its receptor, and survival. Neoplasma 44:295–298 72. Volm M, Koomagi R, Mattern J, Stammler G (1997) Prognostic value of basic fibroblast growth factor and its receptor (FGFR-1) in patients with non-small cell lung carcinomas. Eur J Cancer 33:691–693 73. Guddo F, Fontanini G, Reina C, Vignola AM, Angeletti A, Bonsignore G (1999) The expression of basic fibroblast growth factor (bFGF) in tumor-associated stromal cells and vessels is inversely correlated with non-small cell lung cancer progression. Hum Pathol 30:788–794 74. Shou Y, Hirano T, Gong Y et al (2001) Influence of angiogenetic factors and matrix metalloproteinases upon tumour progression in non-small-cell lung cancer. Br J Cancer 85:1706–1712 75. Iwasaki A, Kuwahara M, Yoshinaga Y, Shirakusa T (2004) Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) levels, as prognostic indicators in NSCLC. Eur J Cardiothorac Surg 25:443–448 76. Bremnes RM, Camps C, Sirera R (2006) Angiogenesis in non-small cell lung cancer: the prognostic impact of neoangiogenesis and the cytokines VEGF and bFGF in tumours and blood. Lung Cancer 51:143–158 77. Berger W, Setinek U, Mohr T et al (1999) Evidence for a role of FGF-2 and FGF receptors in the proliferation of non-small cell lung cancer cells. Int J Cancer 83:415–423 78. Behrens C, Lin HY, Lee JJ et al (2008) Immunohistochemical expression of basic fibroblast growth factor and fibroblast growth factor receptors 1 and 2 in the pathogenesis of lung cancer. Clin Cancer Res 14:6014–6022 79. Thiery JP (2002) Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2:442–454 80. Thiery JP, Sleeman JP (2006) Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7:131–142 81. Thomson S, Buck E, Petti F et al (2005) Epithelial to mesenchymal transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. Cancer Res 65:9455–9462 82. Elayadi AN, Samli KN, Prudkin L et al (2007) A Peptide Selected by Biopanning Identifies the Integrin {alpha}v{beta}6 as a Prognostic Biomarker for Nonsmall Cell Lung Cancer. Cancer Res 67:5889–5895

Molecular Pathology of Lung Cancer

23

83. Bremnes RM, Veve R, Hirsch FR, Franklin WA (2002) The E-cadherin cell-cell adhesion complex and lung cancer invasion, metastasis, and prognosis. Lung Cancer 36:115–124 84. Schiller JH, Harrington D, Belani CP et al (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 346:92–98 85. Altaha R, Liang X, Yu JJ, Reed E (2004) Excision repair cross complementing-group 1: gene expression and platinum resistance. Int J Mol Med 14:959–970 86. Simon GR, Sharma S, Cantor A, Smith P, Bepler G (2005) ERCC1 expression is a predictor of survival in resected patients with non-small cell lung cancer. Chest 127:978–983 87. Zheng Z, Chen T, Li X, Haura E, Sharma A, Bepler G (2007) DNA synthesis and repair genes RRM1 and ERCC1 in lung cancer. N Engl J Med 356:800–808 88. Lord RV, Brabender J, Gandara D et al (2002) Low ERCC1 expression correlates with prolonged survival after cisplatin plus gemcitabine chemotherapy in non-small cell lung cancer. Clin Cancer Res 8:2286–2291 89. Cobo M, Isla D, Massuti B et al (2007) Customizing cisplatin based on quantitative excision repair cross-complementing 1 mRNA expression: a phase III trial in non-small-cell lung cancer. J Clin Oncol 25:2747–2754 90. Olaussen KA, Mountzios G, Soria JC (2007) ERCC1 as a risk stratifier in platinum-based chemotherapy for nonsmall-cell lung cancer. Curr Opin Pulm Med 13:284–289 91. Bepler G, Garcia-Blanco MA (1994) Three tumor-suppressor regions on chromosome 11p identified by high-resolution deletion mapping in human non-small-cell lung cancer. Proc Natl Acad Sci USA 91:5513–5517 92. Bepler G, Sharma S, Cantor A et al (2004) RRM1 and PTEN as prognostic parameters for overall and disease-free survival in patients with non-small-cell lung cancer. J Clin Oncol 22:1878–1885 93. Bepler G, Kusmartseva I, Sharma S et al (2006) RRM1 modulated in vitro and in vivo efficacy of gemcitabine and platinum in non-small-cell lung cancer. J Clin Oncol 24:4731–4737 94. Bepler G (2007) Phase II pharmacogenomics-based adjuvant therapy trial in patients with non-small-cell lung cancer: Southwest Oncology Group Trial 0720. Clin Lung Cancer 8:509–511 95. Travis WD, Nicholson S, Hirsch F et al (2004) Tumours of the lung. Small cell carcinoma. In: Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC (eds) Pathology and genetics: tumours of the lung, pleura, thymus and heart. IARC, Lyon, pp 31–34 96. Wistuba II, Gazdar AF (2000) Molecular pathology of lung cancer. Verh Dtsch Ges Pathol 84:96–105 97. Girard L, Zochbauer-Muller S, Virmani AK, Gazdar AF, Minna JD (2000) Genome-wide allelotyping of lung cancer identifies new regions of allelic loss, differences between small cell lung cancer and non-small cell lung cancer, and loci clustering. Cancer Res 60:4894–4906 98. Beer DG, Kardia SL, Huang CC et al (2002) Gene-expression profiles predict survival of patients with lung adenocarcinoma. Nat Med 8:816–824 99. Larsen JE, Pavey SJ, Passmore LH, Bowman RV, Hayward NK, Fong KM (2007) Gene expression signature predicts recurrence in lung adenocarcinoma. Clin Cancer Res 13:2946–2954 100. Potti A, Mukherjee S, Petersen R et  al (2006) A genomic strategy to refine prognosis in early-stage non-small-cell lung cancer. N Engl J Med 355:570–580 101. Chen HY, Yu SL, Chen CH et al (2007) A five-gene signature and clinical outcome in nonsmall-cell lung cancer. N Engl J Med 356:11–20 102. Oshita F, Sekiyama A, Saito H, Yamada K, Noda K, Miyagi Y (2006) Genome-wide cDNA microarray screening of genes related to the benefits of paclitaxel and irinotecan chemotherapy in patients with advanced non-small cell lung cancer. J Exp Ther Oncol 6:49–53 103. Potti A, Dressman HK, Bild A et al (2006) Genomic signatures to guide the use of chemotherapeutics. Nat Med 12:1294–1300 104. Zohrabian VM, Nandu H, Gulati N et al (2007) Gene expression profiling of metastatic brain cancer. Oncol Rep 18:321–328

24

A. Corvalan and I.I. Wistuba

105. Kikuchi T, Daigo Y, Ishikawa N et al (2006) Expression profiles of metastatic brain tumor from lung adenocarcinomas on cDNA microarray. Int J Oncol 28:799–805 106. Shedden K, Taylor JM, Enkemann SA et al (2008) Gene expression-based survival prediction in lung adenocarcinoma: a multi-site, blinded validation study. Nat Med 14:822–827 107. Jay C, Nemunaitis J, Chen P, Fulgham P, Tong AW (2007) miRNA profiling for diagnosis and prognosis of human cancer. DNA Cell Biol 26:293–300 108. Fabbri M, Croce CM, Calin GA (2008) MicroRNAs. Cancer J 14:1–6 109. Yanaihara N, Caplen N, Bowman E et  al (2006) Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 9:189–198 110. Volinia S, Calin GA, Liu CG et al (2006) A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA 103:2257–2261 111. Yu SL, Chen HY, Chang GC et al (2008) MicroRNA signature predicts survival and relapse in lung cancer. Cancer Cell 13:48–57 112. Weiss GJ, Bemis LT, Nakajima E et al (2008) EGFR regulation by microRNA in lung cancer: correlation with clinical response and survival to gefitinib and EGFR expression in cell lines. Ann Oncol 19:1053–1059 113. Wistuba II, Behrens C, Virmani AK et al (1999) Allelic losses at chromosome 8p21–23 are early and frequent events in the pathogenesis of lung cancer. Cancer Res 59:1973–1979 114. Jiang F, Yin Z, Caraway NP, Li R, Katz RL (2004) Genomic profiles in stage I primary non small cell lung cancer using comparative genomic hybridization analysis of cDNA microarrays. Neoplasia 6:623–635 115. Garnis C, Lockwood WW, Vucic E et al (2006) High resolution analysis of non-small cell lung cancer cell lines by whole genome tiling path array CGH. Int J Cancer 118:1556–1564 116. Callinan PA, Feinberg AP (2006) The emerging science of epigenomics. Hum Mol Genet 15 Spec No 1:R95–R101 117. Herman JG, Baylin SB (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349:2042–2054 118. Iacobuzio-Donahue CA (2009) Epigenetic changes in cancer. Annu Rev Pathol 4:229–249 119. Shames DS, Girard L, Gao B et al (2006) A genome-wide screen for promoter methylation in lung cancer identifies novel methylation markers for multiple malignancies. PLoS Med 3:e486 120. Tsou JA, Galler JS, Siegmund KD et  al (2007) Identification of a panel of sensitive and specific DNA methylation markers for lung adenocarcinoma. Mol Cancer 6:70 121. Yanagawa N, Tamura G, Oizumi H et al (2007) Promoter hypermethylation of RASSF1A and RUNX3 genes as an independent prognostic prediction marker in surgically resected non-small cell lung cancers. Lung Cancer 58:131–138 122. Brock MV, Hooker CM, Ota-Machida E et al (2008) DNA methylation markers and early recurrence in stage I lung cancer. N Engl J Med 358:1118–1128 123. Wilkins-Haug L (1993) The emerging genetic theories of unstable DNA, uniparental disomy, and imprinting. Curr Opin Obstet Gynecol 5:179–185 124. Caprioli RM, Farmer TB, Gile J (1997) Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS. Anal Chem 69:4751–4760 125. Yanagisawa K, Shyr Y, Xu BJ et  al (2003) Proteomic patterns of tumour subsets in nonsmall-cell lung cancer. Lancet 362:433–439 126. Taguchi F, Solomon B, Gregorc V et al (2007) Mass spectrometry to classify non-small-cell lung cancer patients for clinical outcome after treatment with epidermal growth factor receptor tyrosine kinase inhibitors: a multicohort cross-institutional study. J Natl Cancer Inst 99:838–846 127. Bianchi F, Nicassio F, Di Fiore PP (2008) Unbiased vs. biased approaches to the identification of cancer signatures: the case of lung cancer. Cell Cycle 7:729–734 128. Colby TV, Wistuba II, Gazdar A (1998) Precursors to pulmonary neoplasia. Adv Anat Pathol 5:205–215 129. Kerr KM (2001) Pulmonary preinvasive neoplasia. J Clin Pathol 54:257–271

Molecular Pathology of Lung Cancer

25

130. Wistuba II, Behrens C, Milchgrub S et  al (1999) Sequential molecular abnormalities are involved in the multistage development of squamous cell lung carcinoma. Oncogene 18:643–650 131. Belinsky SA, Nikula KJ, Palmisano WA et al (1998) Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis. Proc Natl Acad Sci USA 95:11891–11896 132. Wistuba II, Lam S, Behrens C et al (1997) Molecular damage in the bronchial epithelium of current and former smokers. J Natl Cancer Inst 89:1366–1373 133. Mao L, Lee JS, Kurie JM et al (1997) Clonal genetic alterations in the lungs of current and former smokers. J Natl Cancer Inst 89:857–862 134. Park IW, Wistuba II, Maitra A et al (1999) Multiple clonal abnormalities in the bronchial epithelium of patients with lung cancer. J Natl Cancer Inst 91:1863–1868 135. Wistuba II (2005) Histologic evaluation of bronchial squamous lesions: any role in lung cancer risk assessment? Clin Cancer Res 11:1358–1360 136. Kitamura H, Kameda Y, Ito T, Hayashi H (1999) Atypical adenomatous hyperplasia of the lung. Implications for the pathogenesis of peripheral lung adenocarcinoma [see comments]. Am J Clin Pathol 111:610–622 137. Osanai M, Igarashi T, Yoshida Y (2001) Unique cellular features in atypical adenomatous hyperplasia of the lung: ultrastructural evidence of its cytodifferentiation. Ultrastruct Pathol 25:367–373 138. Westra WH, Baas IO, Hruban RH et al (1996) K-ras oncogene activation in atypical alveolar hyperplasias of the human lung. Cancer Res 56:2224–2228 139. Tominaga M, Sueoka N, Irie K et al (2003) Detection and discrimination of preneoplastic and early stages of lung adenocarcinoma using hmRNP B1, combined with the cell cyclerelated markers p16, cyclin D1, and Ki-67. Lung Cancer 40:45–53 140. Nakanishi K, Kawai T, Kumaki F, Hiroi S, Mukai M, Ikeda E (2003) Survivin expression in atypical adenomatous hyperplasia of the lung. Am J Clin Pathol 120:712–719 141. Tang X, Shigematsu H, Bekele BN et al (2005) EGFR tyrosine kinase domain mutations are detected in histologically normal respiratory epithelium in lung cancer patients. Cancer Res 65:7568–7572 142. Yoshida Y, Shibata T, Kokubu A et al (2005) Mutations of the epidermal growth factor receptor gene in atypical adenomatous hyperplasia and bronchioloalveolar carcinoma of the lung. Lung Cancer 50(1):1–8 143. Tang X, Varella-Garcia M, Xavier AC et al (2008) EGFR abnormalities in the pathogenesis and progression of lung adenocarcinomas. Cancer Prev Res 1:404–408 144. Wistuba I, Gazdar A (2006) Lung cancer prenoplasia. Annu Rev Pathol Mech Dis 1:331–348

Tumor Microenvironment Tonya C. Walser, Jane Yanagawa, Edward Garon, Jay M. Lee, and Steven M. Dubinett

Abstract  While genetic changes are critical for the malignant transformation of epithelial cells, the microenvironment in which the cells reside also governs carcinogenesis. Most tumors arise within a cellular microenvironment characterized by suppressed host immunity, dysregulated inflammation, and increased production of cellular growth and survival factors that induce angiogenesis and inhibit apoptosis. The studies highlighted in this chapter indicate that the lung tumor and its microenvironment interact, together informing the process of carcinogenesis. Understanding the molecular mechanisms driving the contributions of the tumor microenvironment to lung carcinogenesis may afford us the opportunity to develop new drugs that target these reversible nonmutational events in the prevention and treatment of lung cancer. Findings from recent microenvironment-related clinical studies have implications for understanding the immunopathobiology of lung cancer, for targeting surgery and adjuvant therapy, and for designing future trials of adjuvant therapy. If the field is to progress and promising leads in the laboratory are to translate into anticancer therapeutics, future trials targeting the tumor microenvironment must incorporate improved patient risk assessment and selection, in addition to the continued evaluation of combination therapies using the optimal biological dose of each compound being tested. Appropriately targeting the tumor microenvironment in a highly selected patient population is a newly emerging strategy that holds unique potential for advancing the current state of lung cancer prevention and treatment. Keywords  Tumor microenvironment • NSCLC prognosis • Mast cells • Macrophage • Dendritic cells • Ectopic lymph nodes • T regulatory cells • MMP • COX-2 • PGE2 • PPARg • 15-PGDH • Inflammation • EMT • NF-kB • HGF • c-MET • Angiogenesis • Molecular signatures

S.M. Dubinett (*) Division of Pulmonary & Critical Care Medicine, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA e-mail: [email protected]

D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_2, © Humana Press, a part of Springer Science+Business Media, LLC 2010

27

28

T.C. Walser et al.

Introduction While genetic changes are critical for the malignant transformation of epithelial cells, the microenvironment in which the cells reside also governs carcinogenesis. Most tumors arise within a cellular microenvironment characterized by suppressed host immunity, dysregulated inflammation, and increased production of cellular growth and survival factors that induce angiogenesis and inhibit apoptosis. The pulmonary microenvironment, in particular, represents a unique milieu in which lung carcinogenesis proceeds in complicity with the structural (extracellular matrix or ECM), soluble (cytokines, proteases, hormones, etc.), and cellular (fibroblasts, inflammatory cells, endothelial cells, etc.) components of the microenvironment. Understanding the molecular mechanisms driving the contributions of the tumor microenvironment to lung carcinogenesis may afford us the opportunity to develop new drugs that target these reversible nonmutational events in the prevention and treatment of lung cancer. In recent years, gene expression profiling studies of several tumor types have described molecular signatures associated with progression. Identification of robust biomarkers predictive of cancer progression and prognosis could have a clinically significant impact on non-small cell lung cancer (NSCLC) management, as these biomarkers would aid in the appropriate selection of patients who would benefit from therapy beyond surgery. The molecular signatures that have emerged from these progression-associated gene sets are composed mainly of cytokine genes involved in inflammatory and immune responses. In one such study by Bhattacharjee and colleagues in 2001, microarray-based expression profiling of 139 resected adenocarcinoma specimens allowed the investigators to discriminate between biologically distinct subclasses of adenocarcinomas, as well as primary lung adenocarcinomas versus metastases of nonlung origin (1). A gene expression profiling study that closely followed came from Beer et al. (2). This group used expression profiling to predict survival among patients with early stage lung adenocarcinomas. Using the top 50 differentially expressed genes, the investigators developed a survival-based risk index, whereby patients were determined to have high-risk or low-risk stage I adenocarcinomas and poor or favorable predicted survival, respectively, based on their molecular signature. Novel survival-associated genes were identified, but, more importantly, the molecular profile that emerged predicted survival of the patient population. Likewise, the high hsa-mir-155 and low hsa-let7a-2 miRNA expression signature described by Yanaihara and colleagues correctly predicted the poor survival of patients with stage I adenocarcinomas (3). And finally, an mRNA expression profile described by Potti and colleagues identified a subset of stage IA NSCLC patients at high risk of recurrence (4). Together, these studies provided an early indication of the diagnostic potential of expression profiling and clear evidence that molecular signatures composed mainly of inflammation- and immune-related cytokines correlate with important clinical parameters. A recent investigation of the role of the lung tumor microenvironment in promoting carcinogenesis was conducted by Seike et al. (5). To inquire whether gene

Tumor Microenvironment

29

expression changes in the noncancerous tissue surrounding tumors could be used as a biomarker to predict cancer progression and prognosis, this group conducted a molecular profiling study of paired noncancerous and tumor tissues from 80 patients with adenocarcinoma. Many of the genes identified were part of a unique inflammatory and immune response signature that this group previously observed in noncancerous hepatic tissue from HCC patients (6). Ultimately, however, they identified an 11-gene signature, called Cytokine Lung Adenocarcinoma Survival Signature of 11 genes (CLASS-11), which predicted lymph node status and disease prognosis. The results of this well designed trial demonstrate that molecular signatures associated with the tumor microenvironment can serve as robust biomarkers predictive of cancer progression and prognosis. Though not in lung cancer, a recent publication by Farmer and colleagues was the first to report a major contribution of stromal genes to drug sensitivity in the context of a randomized clinical trial (7). Using 63 tumor biopsies from individuals in the EORTC 10994/BIG 00-01 trail with estrogen receptor-negative breast cancer treated with 5-fluorouracil, epirubicin, and cyclophosphamide (FET), the Farmer group described a stromal gene signature that predicts resistance to preoperative chemotherapy. This study expands the clinical significance of the identification of tumor microenvironment-associated gene signatures, and it encourages the development of antistromal agents as a new method by which to overcome resistance to chemotherapy. A translational study by the Kurie Laboratory in 2008 also defined tumor cell and stromal cell interactions that inform the course of NSCLC progression (8). By coculturing a K-ras mutant lung adenocarcinoma cell line with one of three lung stromal cells lines (macrophage, endothelial cell, or fibroblast) and subsequently profiling the secreted proteins, they developed an in vitro model for evaluating the mechanisms by which stromal cells regulate the biological properties of lung adenocarcinoma cells. The group confirmed that the in vitro model robustly recapitulates many of the features of their K-ras mutant murine model and, most importantly, NSCLC, suggesting that it can serve as a useful model of the NSCLC tumor microenvironment. By two different proteomic approaches, the investigators profiled the secretome of the tumor cells and evaluated its regulation by the stromal cells. They concluded that stromal cells in the tumor microenvironment do alter the tumor cell secretome, including proteins required for tumor growth and dissemination. Specifically, enhanced stromal cell migration, induced endothelial tube formation, increased tumor cell proliferation, and differentially expressed of proteins involved in angiogenesis, inflammation, cell proliferation, and epithelial–mesenchymal transition (EMT) were all observed when tumor cells were cocultured with stromal cells. These findings suggest that stromal cells drive the aggressiveness of tumor cells via their effect on the tumor cell secretome. By extension, inhibition of specific interactions between tumor cells and the tumor-adjacent stroma holds significant potential in the search for novel cancer therapeutics. Cancer progression depends on both genetic and epigenetic changes that affect gene expression by the tumor and surrounding stroma, and it depends on the immunologic status of the host. The studies highlighted in this chapter indicate that the

30

T.C. Walser et al.

lung tumor and its microenvironment interact, together informing the process of carcinogenesis. Appropriately targeting the tumor microenvironment in a highly selected patient population is a newly emerging strategy that holds unique potential for advancing the current state of lung cancer prevention and treatment.

Macrophages and Mast Cells Macrophages and mast cells are components of the innate immune cell infiltrate present in nearly every malignancy. In basic, translational, and clinical research investigations, these particular immune effector cells have been found to both thwart and support tumor growth depending upon their microenvironmental context (9–12). While studies correlating macrophage and mast cell infiltrates with NSCLC prognosis are relatively few, the data that exist are sharply divided between support for a correlation to favorable prognosis and support for a correlation to poor prognosis (13–23). Several recent publications suggest that these discrepancies may reflect differences in the number, grade, stage, and size of tumors included in each study, all of which varied considerably across the studies. The lack of consensus both within and between tumor types may also be related to the diverse approaches used to assess the infiltrates. For example, in two publications by Chen and colleagues, tumor-associated macrophage density correlated with poor prognosis in NSCLC, but macrophages within the tumor and those in the adjacent stroma were counted together (13, 14). Toomey and colleagues found no association between macrophage counts and NSCLC outcome, but macrophages within the tumor and those in the adjacent stroma were again counted together 23, as in Chen et al. (13,14). Johnson and colleagues found no correlation between tumor- or stroma-associated macrophages and NSCLC prognosis, however, their assessment was semiquantitative, and the number of cases evaluated was relatively small (16). The original study to demonstrate the importance of the microanatomical location of macrophages as related to prognosis was in gastric cancer rather than lung cancer. Ohno and colleagues specifically counted macrophages within gastric carcinoma tumor cell islets/nests and the adjacent stroma and found that tumor-infiltrating macrophages were associated with increased survival (24). The 5-year disease-free survival rate was significantly increased in patients with a high number of macrophages in the tumor islets when compared to those with a low number of macrophages in the tumor islets (87% versus 44%, respectively; p = 0.0002). In fact, the density of tumor-infiltrating macrophages was an independent predictor of patient survival by Cox’s multivariate analysis (p = 0.016). When combined with additional immunohistochemical staining data, these results led the investigators to conclude that aggregation of macrophages within gastric tumors has a beneficial effect on host survival via augmented cytotoxicity and antigen presentation. One of the more recent investigations of the prognostic significance of macrophage and mast cell infiltration in NSCLC was launched by Welsh et  al. (25). Like the Ohno group, the authors of this study suggest that the microanatomical

Tumor Microenvironment

31

location of macrophages and mast cells associated with a tumor must be taken into account when considering their correlation to prognosis. Because the microenvironment is a key determinant of immune cell phenotype and function, the authors suspected that it might also influence the nature of the immunocyte–tumor interaction. Using immunohistochemistry to identify CD68+ macrophages and tryptase+ mast cells in the tumor islets and adjacent stroma of 175 patients with surgically resected NSCLC, the authors identified tumor islet CD68+ macrophage density as a powerful independent predictor of survival. Specifically, increasing tumor islet macrophage density (p < 0.001) and increasing tumor islet/stromal macrophage ratio (p < 0.001) were favorable prognostic indicators, while increasing stromal macrophage density was an independent predictor of reduced survival (p = 0.001). The presence of tumor islet mast cells (p = 0.018) and increasing tumor islet/stromal mast cell ratio (p = 0.032) were also favorable independent prognostic indicators. When the data were divided into two groups above and below the median cell count value, additional associations with survival became clear. Tumor islet macrophage density showed the strongest effect: 5-year survival was 52.9% in patients with a tumor islet macrophage density greater than the median versus 7.7% when less than the median (p < 0.0001). In the same groups, respectively, median survival was 2,244 days versus 334 days (p < 0.0001). Thus, with a tumor islet macrophage density above the median, 5-year survival was approximately seven times that of the group below the median. In contrast, with a stromal macrophage density above the median, survival was reduced by half when compared with the group below the median. Tumor islet mast cell density and the tumor islet/stromal mast cell ratio had a similar but less marked association with survival: 5-year survival was 40% in patients with a tumor islet mast cell density greater than the median versus 22% when less than the median, and median survival increased threefold. Similar differences in survival with respect to macrophage and mast cell counts were also evident within each tumor stage. In fact, survival of patients with stage IIIa disease and greater than the median tumor islet macrophage density was increased when compared with survival of patients with stage I or stage II disease and less than the median tumor islet macrophage density. Further, where surgical resection data were available, patients with a high tumor islet macrophage density had a significantly better 5-year survival rate (62.5%) than those with a low tumor islet macrophage density (13.2%), even when the former had incomplete resection and the latter had complete resection (p < 0.0001). This supports the notion that a high tumor islet macrophage count limits disease progression. These findings also indicate that the location of immune cells within the tumor microenvironment is critical in determining their relationship to prognosis. The importance is demonstrated most clearly by the strong direct relationship between survival and tumor islet macrophage density and the strong inverse relationship between survival and stromal macrophage density. In a study of 144 patients with resected NSCLC, Kim and colleagues also investigated the prognostic value of tumor-associated macrophages with a focus on their location within the tumor microenvironment (26). Patients with a high tumor islet macrophage density survived longer than patients with a low density (5-year overall

32

T.C. Walser et al.

survival 63.9% versus 38.9%, respectively; p = 0.0002). By multivariate analysis, tumor islet macrophage density was an independent prognostic factor for survival (p = 0.001). The total macrophage (tumor islet plus stromal) density was not significantly correlated with survival, nor was the stromal macrophage density when gauged independently. However, high stromal macrophage counts were associated with poor survival when measured within the context of both high and low tumor islet macrophage groups (p = 0.0011). More recently, Kawai and colleagues evaluated the number of macrophages and mast cells in tumor islets and the adjacent stroma in pretreatment biopsy specimens obtained from 199 patients with stage IV NSCLC treated by platinum-based combination chemotherapy (27). Patients with more macrophages in the tumor islets than in the adjacent stroma had significantly better median survival than patients with the macrophage numbers reversed (440 days versus 199 days; p < 0.0001). The associated 1-year survival rates were 60.8 and 21.4%, respectively. A high proportion of tumor-infiltrating macrophages when compared with stromal macrophages emerged as an independent prognostic factor by multivariate analysis (p < 0.001). Mast cell density in the tumor islets and in the adjacent stroma was also evaluated, but neither correlated with the clinical outcome of this patient population. Additionally, there was no correlation between chemotherapy response and macrophage or mast cell density within the tumor islets or adjacent stroma. These findings suggest that the number of macrophages in tumor islets and the adjacent stroma is a useful biomarker for predicting prognosis of stage IV NSCLC patients treated with chemotherapy, without being a predictor of chemotherapy response specifically. The authors suggest that the host immune system in the stage IV NSCLC tumor microenvironment has the ability to control malignant disease beyond its response to chemotherapy. They further suggest that the transfer of macrophages to tumor islets or elimination of macrophages from tumor-adjacent stroma may prolong the survival of stage IV NSCLC patients with an unfavorable prognosis. Considering preclinical evidence for a dual function of macrophages, there are likely two types of macrophages within the tumor microenvironment, now referred to as the “macrophage balance theory” (9, 10, 12). A simple explanation is that stromal macrophages contribute to tumor stroma formation and angiogenesis, thus supporting tumor growth, while tumor islet macrophages are cytotoxic, thus limiting tumor growth. Macrophages in tumor-adjacent stroma secrete several growth factors and proteases involved in angiogenesis. A recent study demonstrated that interaction between lung cancer cells and these particular macrophages promotes the invasiveness and matrix-degrading activity of the tumor cells (13). Likewise, mast cells produce histamine, basic fibroblast growth factor, heparin, chymase, and tryptase, all of which have been shown to promote cancer progression (11, 12). Conversely, tumor islet macrophages, but not stromal macrophages, express nitric oxide synthase and tumor necrosis factor alpha, both factors involved in tumor-killing mechanisms (9, 10). Tumor-infiltrating macrophages can also act as antigen-presenting cells (APC) to activate cytotoxic T cells, suggesting that they take part in the activation of T cells and subsequent tumor cell destruction (9, 10).

Tumor Microenvironment

33

Likewise, previous studies reveal that mast cells have antitumor functions, including serving as natural cytotoxic effectors and producers of antitumor compounds (11, 12). Taken together, the highlighted clinical studies also suggest that tumor-associated macrophages and mast cells are multifaceted and functionally versatile, and they emphasize the importance of microenvironmental stimuli in the regulation of immunocyte function. Discrepancies between studies still exist, but a much clearer picture of the immunocyte–tumor interaction and its relation to NSCLC prognosis has emerged since investigators began incorporating an evaluation of the microanatomical location of immunocytes into their study design. Findings from these recent clinical studies have implications for understanding the immunopathobiology of NSCLC, for targeting surgery and adjuvant therapy, and for designing future trials of adjuvant therapy. For example, determining tumor islet macrophage density may be particularly useful when considering which late stage NSCLC patients to select for surgery since those with high tumor islet macrophage density often do better than some stage I patients with low tumor islet macrophage density. Similarly, when considering adjuvant therapy, it would be useful to target patients most likely to benefit. At present, adjuvant chemotherapy for NSCLC improves survival only minimally overall, so the majority of patients do not benefit from the potentially toxic treatment. Tumor islet macrophage density may help identify those for whom treatment would be beneficial. These findings may also be useful in the design of future clinical trials, as those who are already predicted to do well based on their tumor islet macrophage density should be excluded to avoid confounding the interpretation of the experimental treatment being tested. However, before being truly useful clinically, the microanatomical location of these cell types must be considered routinely, counting methods must be standardized, and a clinically practical cutoff value other than the median number must be established. In addition to quantifying macrophage and mast cell numbers, use of activation markers would likely add to our current understanding of the role of these immune cells in NSCLC tumor development and as predictors of prognosis.

Dendritic Cells and Ectopic Lymph Nodes The potential for the immune system to induce tumor regression has stimulated much research into development of vaccines that unmask tumor antigens and lead to a specific host immune response against the tumor (28). However, the poor immunogenicity of human lung cancer due to low expression of MHC antigens, a deficit of transporter-associated antigen processing, and lack of costimulatory molecules have rendered most such immunotherapeutic efforts ineffective (29). In addition, tumor cell-derived inhibitory factors and immune suppressive cells, such as T regulatory (T reg) cells, are common components of the tumor microenvironment that impede the immune response to NSCLC (30–36). Dendritic cells (DC) are the most potent APC capable of inducing primary immune responses (37). DCs express high levels of MHC and costimulatory molecules,

34

T.C. Walser et al.

such as CD40, CD80, and CD86. DCs also release high levels of cytokines and chemokines into the tumor microenvironment that attract antigen-specific T cells in vivo. These properties, combined with efficient capture of antigens by immature DCs, allow them to efficiently present antigenic peptides and costimulate antigenspecific naïve T cells (37). Presentation of tumor-associated antigens by DCs and their recognition by cytotoxic T lymphocytes (CTL) play an important role in the eradication of tumor cells (38). Based upon the importance of DCs in tumor immunity, a variety of strategies have been used to exploit this cell type in cancer immunotherapy (39–41). Advances in the isolation and in  vitro propagation of DCs combined with the identification of specific tumor antigens have allowed initiation of clinical trials testing DC-based vaccines (39–41). Additionally, DC-transfer has been demonstrated to be a safe approach in clinical studies (42–47). Strategies employing DCs in immunotherapy have included pulsing isolated DCs with tumor antigen peptides, apoptotic tumor cells, or tumor lysates ex vivo (48–50). DCs have also been genetically modified with genes encoding tumor antigens or immunomodulatory proteins (51–53). There is evidence that DCs transduced with adenoviral vectors (AdV) have a prolonged survival and resistance to spontaneous and Fas-mediated cell death, suggesting their utility in delivering immunotherapy more efficiently and robustly (54). AdV transduction itself can also augment the capacity of DCs to induce protective antitumor immunity (55). In addition, enhanced local and systemic antitumor effects have been demonstrated when AdV-transduced DCs expressing cytokine genes have been injected intratumorally (56). AdVs have been utilized to transduce DCs because they efficiently induce strong heterologous gene expression in these cells (55, 56). Prototypical vectors have now been extensively used in a variety of contexts (55, 56). CCL21 is a CC chemokine that belongs to a family of proteins involved in leukocyte chemotaxis and activation. Expressed in high endothelial venules and T cell zones of spleen and lymph nodes, CCL21 exerts potent attraction of naïve T cells and mature DCs promoting their colocalization in secondary lymphoid organs and promoting cognate T cell activation (57). Potent antitumor properties of CCL21 in murine cancer models were previously reported (58–60). CCL21 has also shown antiangiogenic activities in mice, thus strengthening its immunotherapeutic potential in cancer (61, 62). Intratumoral administration of clinical grade CCL21transduced DCs in a phase I clinical trial for late stage NSCLC is currently being evaluated. Previously noted in nonmalignant contexts and in the setting of autoimmune diseases and chronic inflammation, the concept of ectopic lymph nodes or tertiary lymphoid structures has recently made a resurgence. As described in a review by Carragher and colleagues, tertiary lymphoid structures are similar to conventional secondary lymphoid organs, with separated B cell and T cell areas, specialized DC populations, and well differentiated stromal cells and high endothelial venules (63). In a recent investigation, Singh and colleagues identified immune clusters in the livers of geriatric mice, subsequently characterizing them as ectopic lymphoid structures (64). As described by Timmer and colleagues, ectopic lymphoid structures are also present in the synovial tissues of rheumatoid arthritis patients,

Tumor Microenvironment

35

perhaps activated by the IL-7 pathway (65). While ectopic lymph nodes are often identified in association with local pathology resulting from chronic infection or inflammation, there is limited evidence supporting the existence of ectopic lymph nodes in solid tumors as well. Their role in exacerbating or attenuating all of these disease states is still the subject of debate, however. Accumulating evidence that adaptive immunity can be initiated independent of secondary lymphoid organs, likely via induction of these tertiary lymphoid structures, suggests that ectopic lymph nodes may contribute to local protective immune responses in certain microenvironmental contexts (66, 67). Data from both basic and clinical research investigations support the existence of ectopic lymph nodes within the tumor microenvironment and support their role in protecting the host against the tumor. In a murine melanoma model, for example, targeting lymphotoxin alpha to the tumor enables priming of T cells and an accelerated immune response independent of secondary lymphoid tissue (67). The authors observed tertiary lymphoid tissues composed of compartmentalized T cell and B cells zones and elevated numbers of tumor-specific T cells to which they ascribed the immune-mediated tumor destruction. Likewise, DCs genetically modified to secrete CCL21 induce these lymphoid cell aggregates that prime naïve T cells within the tumor mass, resulting in tumor-specific T cells and subsequent tumor regression (68). In clinical samples of infiltrating ductal carcinoma of the breast, Coronella and colleagues identified ectopic follicles characterized by B cell aggregates and T cell zones with interdigitating CD21+ follicular DCs (69). Bell and colleagues described similar structures resembling DC/T cell clusters of secondary lymphoid organs in 32 breast cancer tissues evaluated by immunohistochemistry (70). The immature DCs were located within the tumor bed, while the mature DCs were confined to peritumoral areas. In a retrospective study of 18 ovarian cancer cases, low numbers of these DC aggregates were linked to recurrence, suggesting that ectopic lymph node status may be a favorable prognostic indicator in ovarian carcinoma (71). In their evaluation of 69 NSCLC specimens, Kurabayashi and colleagues described S100+ DC aggregates that associated with apoptotic tumor cells though no correlations with prognosis were explored (72). While not evaluating ectopic lymph nodes per se, a study by Zeid and colleagues also examined S100+ DCs in normal trachea, lung, bronchial lymph nodes, lung tumors (n = 130), and lymph nodes (n = 100) regional to the tumors (73). Patients whose tumors contained a high density of S100+ DCs had a more favorable outcome. For the first time in human NSCLC, Dieu–Nosjean and colleagues retrospectively identified ectopic lymph nodes or tertiary lymphoid structures and demonstrated that there is a correlation between their cellular content and clinical outcome (74, 75). The structures, called tumor-induced bronchus-associated lymphoid tissue (Ti-BALT), were characterized as follicle-like, containing germinal centers similar to those in secondary lymphoid follicles of the lymph nodes. The density of DC-Lamp+ mature DCs within the Ti-BALT structures was found to be a predictor of long-term survival within the NSCLC patient population surveyed, suggesting that Ti-BALTs have clinical relevance and participate in the host antitumor immune response. Furthermore, tumors poorly infiltrated by DC-Lamp+ mature DCs were

36

T.C. Walser et al.

also poorly infiltrated by immune-reactive T cell subpopulations, again suggesting the potential role of Ti-BALTs in mediating an adaptive immune reaction in the setting of NSCLC. More specifically, 74 early stage NSCLC and five nonpathologic lung biopsies were examined. To evaluate the prognostic value of Ti-BALTs, the authors attempted to quantify the structures. Because the Ti-BALTs were variably sized and often aggregated, their cellular composition was instead characterized. By immunohistochemical staining, CD3+ T cells, CD20+ B cells, and DC Lamp+ mature DCs within Ti-BALT structures were quantified. The identity of mature DCs was also confirmed by CD83 expression. Densities of DCs, T cells, and B cells were strongly correlated to each other within the tumor. Additionally, mature DC/T cell clusters were often surrounded by CD20+ B cell follicles characterized by the presence of both a CD21+ follicular DC network and Ki67+ proliferating germinal center B cells. While the TiBALT structures were heterogeneous between tumors, the data suggest that they are composed of mature DCs, T cells, and B cells organized in a fashion reminiscent of secondary lymphoid organs to which mature DCs exclusively home. Because DC-Lamp+ mature DCs were selectively detected in Ti-BALTs, DC-Lamp was chosen as a specific marker of Ti-BALTs, and the authors concluded that DC-Lamp+ mature DCs are a predictive marker of survival for patients with early stage NSCLC. The density of T cells and B cells also closely correlated with the density of mature DCs (p = 0.0018 and <0.0001, respectively). The ectopic lymphoid structures described in the preceding study were not observed in sites distant from the tumor, suggesting that they were induced in response to the tumor microenvironment. The B cell areas included proliferating germinal center B cells and a follicular DC network, both features of an ongoing immune response. Nonproliferating germinal center B cells have been described in ectopic lymphoid structures of idiopathic lung fibrosis, indicating that Ti-BALTs might not have the same maturation status in different lung diseases and that the local microenvironment influences Ti-BALT development and function. The Dieu–Nosjean group demonstrated that the density of mature DCs, which home exclusively to Ti-BALT structures, is highly predictive of survival in early-stage NSCLC. The authors suggest that with increasing infiltration of tumors by mature DCs, an increasing number of T cells and B cells are organized and proliferate within the Ti-BALT structures. Conversely, the absence of mature DCs and associated Ti-BALT structures leads to poor T cell priming with improper T cell localization and/or activity, thus resulting in inefficient antitumor immunity. While the highlighted studies suggest that ectopic lymph nodes composed primarily of DC cells are associated with favorable outcomes, few other studies examining clinical outcome are available. Why these structures arise, how they are generated, and how they impact the host are questions still being evaluated. The answers to these questions may be very different depending upon the cellular microenvironment in which the ectopic lymph nodes are being investigated. For example, impaired homing of T reg cells to lymph nodes is partially responsible for the formation and maintenance of ectopic lymphoid tissues, termed iBALTs, in

Tumor Microenvironment

37

CCR7-deficient mice (76). These mice have few T reg cells in their lymph nodes and develop iBALTs shortly after birth. Additionally, iBALT formation is prevented by adoptive transfer of CCR7+/+ T regs to CCR7-deficient recipients. These data suggest that poor T reg cell activity or low T reg cell numbers result in the formation of ectopic lymphoid tissues. Yet where there should be supportive evidence in autoimmune disease models, the data are mixed. Therefore, CCR7 and T reg cells appear to play multiple and contradictory roles in the formation and function of ectopic lymph nodes. As previously noted, tertiary lymphoid structures have now been identified in nonmalignant, autoimmune, chronic inflammatory, and malignant microenvironmental contexts. In an editorial associated with the Dieu–Nosjean article, Coppola and Mule suggest that an examination of the relationship between smoking history and the formation of nonmalignant- versus malignant-associated lung-related lymphoid structures is warranted (74). Additionally, they suggest that routine lung sections from nonmalignant areas of patients with cancer and from patients with nonmalignant conditions should be assessed to gauge the true nature of the ectopic lymph nodes. The functional activity of lymphocytes residing within ectopic lymph nodes in human solid tumors, their tumor specificity, and their ability to predict clinical outcome are also heralded as the most pressing questions yet to be addressed.

T Regulatory Cells Active immune suppression induced by tumors has been well documented in lung cancer and other malignancies. Often, tumor-reactive T cells accumulate in lung cancer tissues, but fail to respond to the tumor (36). A high proportion of these NSCLC tumor-infiltrating lymphocytes (TIL) are CD4+CD25high T reg cells (34, 35). Tumor cells may promote immune suppression by directing surrounding inflammatory cells to release suppressive cytokines into the tumor microenvironment, augmenting the trafficking of T reg cells to the tumor site and/or promoting differentiation of effector lymphocytes into T reg cells (32). One major impediment to effective NSCLC therapy is our inadequate understanding of how lung cancer cells escape immune surveillance and inhibit antitumor immunity. Thus, identification of T regs in cancer patients was a finding of great clinical importance. The June laboratory was the first to document the increase in the CD4+CD25+ T reg cell population at the tumor site in NSCLC patients (34, 35). These studies clearly demonstrated that greater than one third of NSCLC CD4+ TIL were CD4+CD25+ T reg cells capable of inhibiting autologous T cell proliferation (35). A more recent examination of normal and tumor tissue from 46 patients with NSCLC also indicated that tumor tissues have significantly higher expression of FOXP3 mRNA than normal tissues (77). FOXP3 is one of the most specific markers of functional T reg cells currently available. Expression of FOXP3 mRNA and NSCLC tumor diameter were inversely proportional in this

38

T.C. Walser et al.

study. A subsequent evaluation of T reg cells in the peripheral blood of patients with NSCLC (n = 17) and breast cancer (n = 22) indicated that T reg cells are significantly increased in the blood of NSCLC patients when compared with healthy volunteers (n = 10), but not in the blood of breast cancer patients (78). In their flow cytometry evaluations, patients with recurrent NSCLC when compared with healthy volunteers had significantly higher percentages of T reg cells in their CD3+ (47.6% versus 33.7%; p = 0.02) and CD4+ (71.0% versus 52.2%; p < 0.03) T cell subsets. Zhang and colleagues also analyzed peripheral blood samples from 55 NSCLC patients who underwent paclitaxel-based chemotherapy (79). Paclitaxel is a mitotic inhibitor that is considered a promising agent for the treatment of advanced NSCLC. The authors discovered that among lymphocyte subsets, paclitaxel selectively decreased the size of the T reg cell population and not the effector T cell subsets. Subsequent evaluation indicated that the mechanism was up-regulation of the cell death receptor Fas (CD95) which selectively induced apoptosis of the T reg cell population. While T reg cell function was significantly impaired, the authors reported that production of the Th1 cytokines, interferon-gamma and interleukin 2, and expression of the activation marker CD44 was intact and even elevated within the CD4+ and CD8+ T cell subsets after paclitaxel treatment. In addition to these studies in lung cancer, several other research groups have reported increased CD4+CD25+ T reg cells in peripheral blood lymphocytes (PBL) and TILs in several different malignancies (80–83). These findings are consistent with studies in murine models demonstrating that the depletion of CD4+CD25+ T reg cells can significantly augment the efficacy of cancer vaccination (84). Together, these data suggest that T reg cells are selectively recruited to NSCLC tumors, where they contribute to the immune-suppressive microenvironment characteristic of NSCLC progression. Though not in lung cancer, one of the first studies to link T reg cell recruitment and prognosis came from Curiel and colleagues and their investigation of CD4+CD25+FOXP3+ T reg cells in 104 patients with ovarian carcinoma (85). Where data were available, tumor T reg cell content was associated with reduced survival in the group as a whole (n = 70; p < 0.001) and within stage II (p = 0.0362), stage III (p = 0.0003), and stage IV (p = 0.0001) groups. By Cox’s proportional hazards model, tumor T reg cells were a significant predictor of death hazard after controlling for stage, surgical debulking, and other factors affecting survival (p < 0.0001). Taken together, an increase in the number of tumor T regs was a significant predictor of increased risk for death and reduced survival in ovarian cancer. This group also discovered that in contrast to homeostatic CD4+CD25+ T cells, which preferentially home to lymph nodes, T reg cells from patients with late stage ovarian cancer preferentially homed to tumors and ascites and only rarely to draining lymph nodes. Additionally, tumor cells and tumor-adjacent macrophages were contributors of the CCL22 chemokine that mediated trafficking of the T reg cells to the tumor. This was the first report of functional tumor microenvironmental CCL22 and the earliest indication that blocking CCL22 in vivo reduces human T reg cell tumor trafficking. While these data provide the basis for developing novel immuneboosting strategies based on eradication of the T reg cell population in cancer

Tumor Microenvironment

39

patients, they also suggest that novel pathways regulating T reg cell biology and trafficking are still being discovered and elucidated. Studies demonstrate that cyclooxygenase-2 (COX-2) and its metabolite, prostaglandin E2 (PGE2), inhibit immune responses in lung cancer by promoting T reg cell activity. Studies have now demonstrated that PGE2 enhances the in vitro inhibitory function of human purified CD4+CD25+ T reg cells and induces a regulatory phenotype in CD4+CD25− T cells (86, 87). PGE2-treated T reg cells inhibited responder anti-CD3-stimulated lymphocytes following coculture and when the cells were separated by a transwell (86). PGE2 exposure induced the T reg cell specific transcription factor FOXP3 in CD4+CD25− T cells and significantly up-regulated its expression in CD4+CD25+ T reg cells. Supernatants from COX-2 over-expressing lung cancer cells that secrete high levels of PGE2 significantly induced FOXP3 in CD4+CD25− T cells. PGE2 up-regulated FOXP3 at both mRNA and protein levels and enhanced FOXP3 promoter activity in Jurkat T cells. Based upon in vitro results using human T reg cells, the effect of tumor COX-2 expression on the frequency and activity of CD4+CD25+ T reg cells in murine lung cancer models was determined. Tumor-derived COX-2/PGE2 induced expression of the T reg cell-specific transcription factor FOXP3 and increased T reg cell activity (87). Utilization of lymphocytes from EP2 and EP4 receptor knockout mice showed that the PGE2-mediated increase in FOXP3 gene expression in T reg cells was EP2 receptor-dependent. In vivo, COX-2 inhibition reduced T reg cell frequency and activity, attenuated FOXP3 expression in TIL, and decreased tumor burden. Transfer of T reg cells or administration of PGE2 to mice receiving COX-2 inhibitors reversed these effects. These and other basic and translational research investigations have informed our understanding of the role of CD4+CD25+ T reg cells in the lung cancer microenvironment, collectively suggesting that the development of clinical strategies to reduce the suppressive effects of these T reg cells in lung cancer is warranted. Efforts directed at ablating the suppressive activities of CD4+CD25+ T reg cells include clinical trials that utilize total lymphodepletion (88–90). Other trials are evaluating immunotoxins, such as denileukin diftitox, a fusion protein combining truncated diphtheria toxin and human IL-2 (Ontak), to specifically ablate the CD4+CD25+ T reg cell population (91). Ongoing clinical investigations are also assessing the role of celecoxib in controlling T reg cell number, activity, and differentiation in human NSCLC. While lymphodepletion or therapy with denileukin diftitox may prove beneficial, COX-2/PGE2 inhibition has additional potential benefits in the setting of NSCLC. In addition to the potential capacity to clinically decrease T reg cell function, COX-2 inhibition has been found to limit angiogenesis, decrease tumor invasiveness, and decrease tumor resistance to apoptosis in NSCLC (92–94). These pathways and malignant phenotypes may be inhibited by several different agents in the non-steroidal anti-inflammatory drug (NSAID) class (95). Therefore, trials are evaluating COX inhibition in combination with other therapies (94). Such studies will help further define the required interventions in this pathway and lead to more specifically targeted agents to diminish T reg cell activities in cancer. These agents could then be combined with other immune-based clinical therapies in an informed manner.

40

T.C. Walser et al.

Matrix Metalloproteinases There are more than 25 structurally- and functionally-related matrix metalloproteinases (MMPs) capable of degrading various components of the ECM, making MMPs critical for physiologic and pathologic processes characterized by ECM remodeling. MMP substrates are numerous, including both ECM and non-ECM molecules, like growth factors and their receptors, cell adhesion molecules, cytokines, chemokines, apoptotic ligands, and angiogenic factors. Over the past 25 years, a role for MMPs in carcinogenesis has been well established and well reviewed (96–102). MMPs increase the invasive behavior of tumor cells, and increase the ability of those tumor cells to metastasize in animal models. Knock-out mice deficient in specific MMPs exhibit decreased tumor growth, invasion, and metastasis when compared to their normal counterparts. In clinical specimens, MMPs are up-regulated in virtually every human malignancy evaluated, and elevated MMP expression is associated with increased invasion, advanced stage, and poor prognosis. Conversely, overexpression of tissue inhibitors of MMPs (TIMPs), the endogenous inhibitors of MMPs, decreases the ability of tumors to grow and metastasize both in vitro and in vivo, while antisense depletion of TIMPs results in cells that are more tumorigenic, invasive, and metastatic. In clinical specimens, elevated TIMP expression is associated with less aggressive tumor behavior and favorable prognosis. In addition to their degradation of ECM, MMPs have also been shown to contribute to carcinogenesis by promoting angiogenesis, stimulating tumor growth, regulating innate immunity, and exhibiting antiapoptotic properties. The MMP-related proteases, a disintegrin and metalloproteinase (ADAM) and ADAM with thrombospondin repeats (ADAMTS), have also recently come to the attention of investigators for their role in promoting cancer progression (99). Despite promising results from basic research investigations and preclinical animal models, large scale phase III trials of MMP inhibitors (MMPIs) failed to demonstrate a statistically significant survival advantage in most of the advanced stage malignancies surveyed. In fact, in some trials where MMPI therapy was compared to placebo, the MMPI arm was associated with worse outcomes. Root causes for the collective failure of these large and costly MMPI clinical trials of the 1990s are numerous, and they have been well reviewed as recently as 2008 (96–102). Briefly, mouse models cannot model human disease entirely, and dose-limiting toxicities are hard to evaluate in animals. However, the promising activity in animal models was clearly associated with early stage disease, yet most clinical trials were initiated in patients with late stage disease. Appropriate MMPI dosing was also never achieved, as the optimal biological dose (OBD) was never clearly established prior to initiation of the clinical trials. Further, investigators started patients on a fraction of the dose proven effective in mice, and that dose was reduced, or patients were given drug holidays, when musculoskeletal toxicity developed. Interestingly, the toxicity was likely an indicator of good response. The broad spectrum nature of the MMPIs used in the trials was also inappropriate since MMPs have diverse and sometime opposite effects depending upon their microenvironmental context. We now know that not all MMPs should be eliminated to achieve an antitumor response.

Tumor Microenvironment

41

Since the unsuccessful conclusion of the MMPI clinical trials, there has been a slow return to execution of studies evaluating the association of MMPs and their inhibitors with cancer prognosis. There has been a similar slow return to optimism that MMPs represent a component of the tumor microenvironment that can be successfully targeted therapeutically. Before that time, however, investigators must identify which MMPs are “good” and which are “bad” within each histologic subtype of each malignancy during each stage of that particular malignancy, and clinical trials must be tailored to fit the knowledge gathered from these inquiries. Detailed knowledge about the targeted MMP, its substrate, and its mechanism of action in vivo is also essential if the most appropriate and most effective antitumor drugs are to be designed. MMP-related research investigations launched within the last 3–5 years have been informed by the research communities’ past failures. Most investigators attempting to link MMP expression to prognosis are now more cognizant that the expression and ultimate function of MMP proteases during carcinogenesis is frequently modulated by the microenvironment, yielding MMPs with diverse and sometimes opposite effects depending upon their source, their tissue location, and the disease stage during which they are expressed. The following are recent studies relating MMP expression to clinical outcome in a manner clearly informed by the past failures of MMP-related cancer therapeutics. Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147) is a transmembrane protein present on the surface of numerous tumor cells. By stimulating the production of MMPs by tumor-adjacent stromal cells, EMMPRIN contributes to the accumulation of select MMPs in the tumor microenvironment and is, therefore, associated with cancer invasion and metastasis in certain settings. Leinonen and colleagues analyzed the expression of MMP-2 and its inducer, EMMPRIN, in 212 surgically resected NSCLC specimens (103). Immunohistochemical staining of MMP-2 and EMMPRIN was evaluated in both the tumor cells and the tumoradjacent stroma. High expression of MMP-2 was observed in the tumor cells in 44% of the cases, with adenocarcinomas showing more expression than other NSCLC tumor types. High expression of MMP-2 by the tumor cells was associated with increased tumor recurrence (p = 0.001). The tumor stroma was positive for MMP-2 expression in 98% of the cases, with 72% of those cases scoring the highest possible staining intensity. High expression of MMP-2 in the tumor-adjacent stroma was more often associated with large cell carcinomas when compared with other NSCLC tumor types. High expression of EMMPRIN by tumor cells was found in 61% of the cases and correlated with high MMP-2 expression by the tumor cells (p = 0.006). High tumor cell MMP-2 expression correlated with reduced overall survival and reduced disease-free survival (p = 0.018 and 0.001, respectively), as did high stromal MMP-2 expression (p = 0.010 and 0.045, respectively). By multivariate analysis, however, only elevated stromal expression of MMP-2 was an independent prognostic indicator of poor overall survival and disease-free survival (p = 0.028 and 0.039, respectively). These data indicate that when MMP-2 expression by tumor cells and the tumor-adjacent stroma is considered separately, MMP-2 has significant prognostic value in the setting of NSCLC and could be useful in determining which patients have the most aggressive disease.

42

T.C. Walser et al.

Using 150 resected NSCLC tumors, Sienel and colleagues assessed expression of EMMPRIN in association with MMP-2 expression, MMP-9 expression, and overall survival (104). Like the Leinonen group, this group assessed the intensity, extent, and cellular localization of EMMPRIN staining. Of the 145 tumors evaluated, 61 of the tumors displayed intense EMMPRIN staining, and the staining was localized to the tumor cell membrane in 102 of the tumors. EMMPRIN expression was not associated with MMP-2 or MMP-9 expression in this study. By multivariate analysis, membrane localization of EMMPRIN was associated with shortened survival in patients with adenocarcinoma (p = 0.03) but not squamous cell carcinoma. In fact, by multivariate analysis, membrane expression of EMMPRIN was an independent predictor of patient survival for the adenocarcinoma group (p = 0.04). The authors suggest that, in some settings, EMMPRIN may have a role in the progression of NSCLC that is independent of its function as an inducer of MMPs. Taken together, the Leinonen and Sienel studies suggest that even within NSCLC, MMP expression patterns and associations with prognosis vary across the histologic subtypes. Hakuma and colleagues conducted a similar immunohistochemical evaluation of 208 surgically resected NSCLC specimens (105). While EMMPRIN was expressed by 92% of the NSCLC samples, this group found no significant differences in overall survival between patients with high EMMPRIN tumor expression and those with low expression. It should be noted, however, that this study was semiquantitative, and levels of EEMPRIN in the tumor-adjacent stroma were not evaluated as in the previous studies. Kim and colleagues examined MMP enzyme activity in normal and tumor tissue from 34 patients with surgically resected stage I NSCLC (106). MMP-2 enzyme activity in nontumor tissue was significantly different in patients later found with recurrence when compared to those without recurrence, and it was associated with the 5-year survival rate. The authors suggest that MMP-2 enzyme activity in nontumor tissue can be used as a prognostic biomarker that predicts postoperative tumor recurrence and survival for early stage NSCLC patients. Further, the authors suggest that it could be used to assist in deciding which patients are most in need of postoperative adjunct chemotherapy. In their examination of bone marrow microinvolvement and MMP expression in the bone aspirates of 57 NSCLC patients, Hsu and colleagues found that MMP-13 expression by the tumor cells is an independent prognostic factor by Cox’s multivariate analysis (107). The overall 5-year survival rate was 36.8%. While the existence of bone marrow microinvolvement did not influence prognosis, patients whose bone marrow aspirates were positive for MMP-13 expression (n = 34) had a reduced 5-year survival rate of 26.5% (p = 0.025). The authors suggest that NSCLC cells with high MMP-13 expression shed and aggregate in the bone marrow leading to worse survival. By examining MMP-7 expression in 147 NSCLC specimens, Liu and colleagues demonstrated that MMP-7 expression is associated with tumor proliferation and poor prognosis in NSCLC, likely via Wnt1 regulation of tumor cell expression of MMP-7 (108). Overall survival was significantly lower in patients with MMP-7positive tumor cells when compared with those with MMP-7-negative tumor cells

Tumor Microenvironment

43

(p = 0.0018). By multivariate analysis, MMP-7 status was a significant prognostic factor (hazard radio 2.187; p = 0.0023). Mino and colleagues examined 143 resected NSCLC specimens, evaluating TIMP-3 expression in association with a large number of clinical parameters, including prognosis (109). TIMP-3 is an endogenous inhibitor of select MMPs, including MMP-2. TIMP-3 expression was higher in squamous cell carcinoma than in adenocarcinoma, and low level TIMP-3 expression was significantly associated with nodal involvement (p = 0.016) and advanced stage (p = 0.036). MMP-2 expression was also reduced in association with TIMP-3 expression (p = 0.010). By multivariate analysis, high TIMP-3 expression wan an independent indicator of favorable prognosis (p = 0.037). Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is an endogenous MMP inhibitor shown to inhibit angiogenesis, invasion, and metastasis in association with its inhibition of MMP-2, MMP-9, and MTP-1/MMP-14 (110). Takemoto and colleagues analyzed RECK expression, along with MMP-2, MMP-9, and MMP-14 expression, in 83 surgically resected NSCLC specimens and 20 matched normal lung tissue specimens (111). Expression of RECK in the tumor specimens was significantly lower than in the control tissues. Additionally, RECK was higher in stage IA adenocarcinomas than in stage IB-IIIA adenocarcinomas. By Cox’s multivariate analysis, low RECK expression (p = 0.036) was a significant negative prognostic predictors of relapse-free survival. No associations between RECK expression and survival were observed in squamous cell carcinoma. This suggests that suppression of RECK expression contributes to the progression of the adenocarcinoma histologic subtype of NSCLC and that expression of RECK is a favorable predictor of prognosis. These highlighted studies suggest that within NSCLC the expression patterns of MMPs and MMP inhibitors vary across histologic subtype and across disease stage. On whole, these studies also suggest that the compartment (tumor versus stroma) and even the cell type expressing the MMP or MMP inhibitor must be considered if strong correlations are to be made to clinically relevant parameters, like survival. Overexpression or treatment of normal tissues with RECK still needs to be evaluated preclinically, and the effect of RECK on developing versus established vasculature must still be considered. Interestingly, HDAC inhibitors (trichostatin A) increase RECK levels by minimizing promoter inhibition of RECK (112). Also, NSAIDs increase RECK expression, perhaps by inhibition of the ras/ERK/Sp1 pathway. This particular action appears to be via RECK up-regulation that is independent of NSAID action on cyclooxygenase since concurrent administration of PGE2 or overexpression of COX-2 in lung cancer cells does not affect the levels of RECK (112). This makes HDAC inhibitors a means by which to increase levels of RECK present in the tumor microenvironment. While future MMP-related cancer therapeutics could take the form of antisense oligonucleotides, siRNA, shRNA, or recombinant antibodies, small molecular weight inhibitors will likely continue to be the cheapest and the easiest to engineer, produce, and deliver. As reviewed by Konstantinopoulos et al. (98), broad spectrum MMPIs will likely give way to more specific MMPIs that target one or a few MMPs

44

T.C. Walser et al.

expressed in specific tumor types during specific disease stages, which may eliminate some of the musculoskeletal toxicity associated with the use of broad spectrum MMPIs. Next generation MMPIs will also likely be evaluated initially in acute settings, such as stokes, before being used clinically in the setting of chronic conditions, like cancer. As suggested in a review by Overall and Kleifeld (102), validating MMPs as therapeutic targets and understanding the role of the targets and nontargets requires the use of surrogate markers that can guide dose-escalation. Identifying molecular markers that will predict response would also be useful. The reviewers suggest that generation of additional MMP-knockout mice crossed with models of spontaneous cancers is essential if we are to study MMP-related redundancies and drug-resistance preclinically. Additionally, combinations of MMPIs with other chemotherapeutic or molecularly-targeted agents must be evaluated preclinically. If these suggestions are implemented, future clinical trials in carefully selected patient populations with very specific malignancies at predetermined stages are warranted despite the disappointing past of MMPIs in cancer therapy.

Cyclooxygenase-2 and Prostaglandin E2 Cyclooxygenases (COX) are the rate-limiting enzymes in prostanoid synthesis that convert arachidonic acid into prostaglandin H2, a substrate for specific prostaglandin synthases. Two isoforms of COX, ubiquitously expressed COX-1 and inducible COX-2, have been isolated and characterized. Numerous studies have now demonstrated high level constitutive COX-2 expression in human NSCLC. In the initial report in NSCLC, Huang and colleagues assessed COX-2 expression in resected NSCLC specimens and normal adjacent lung tissue by immunohistochemistry (33). All of the 15 tumor specimens evaluated displayed cytoplasmic staining for COX-2 in tumor cells. In contrast, adjacent normal lung showed no COX-2 staining in the alveolar lining epithelium, but often demonstrated positive cytoplasmic staining in alveolar macrophages and occasionally in the bronchiolar epithelium. Other studies have since corroborated and expanded this initial finding, further documenting the importance of COX-2 in lung cancer, as recently reviewed (113). Mounting evidence indicates that tumor COX-2 activity has a multifaceted role in conferring the malignant and metastatic phenotype of lung cancer. Although multiple genetic alterations are necessary for lung carcinogenesis, COX-2 appears to be a central element in orchestrating this process. For example, COX-2 has been implicated in resistance to apoptosis (114–116), enhanced angiogenesis (117, 118), decreased host immunity (30, 119, 120), and enhanced invasion and metastasis (121, 122). These newly discovered molecular mechanisms in the pathogenesis of lung cancer provide novel opportunities for targeted therapies. COX-2 is one of the targets under investigation for both lung cancer chemoprevention and therapy (92, 113, 123). Prostaglandin E2 (PGE2) is a COX-2 metabolite present in the lung cancer microenvironment that is both an autocrine and paracrine mediator of immune

Tumor Microenvironment

45

regulation (93), epithelial cell growth and invasion (124), and epithelial survival (116). PGE2 exerts its multiple effects through four G protein-coupled receptors (GPCR). Blocking COX-2-dependent PGE2 production or activity by targeting the signaling pathways downstream of COX-2, such as the EP receptors, may produce more profound antitumor effects than COX-2 inhibition alone. Many of the most recent approaches to lung cancer chemoprevention and therapy have been based on this rationale. There is also an established role for GPCRs in the transactivation of the epidermal growth factor receptor (EGFR) pathway which leads to increased cancer cell growth and motility. Krysan and colleagues were the first to demonstrate PGE2-mediated EGFR-independent activation of the MAPK/Erk pathway, which suggests that COX-2 overexpression contributes to EGFR inhibitor resistance in NSCLC (124). These findings provide the rationale for the pharmacologic inhibition of PGE2 synthesis in combination with EGFR inhibitors in NSCLC therapy. In one of the initial reports linking COX-2 expression to NSCLC prognosis, Khuri and colleagues evaluated COX-2 expression in 160 stage I NSCLC specimens by in situ hybridization and reported that COX-2 overexpression appears to predict shorter survival among patients with early stage disease (125). The strength of COX-2 expression was associated with both decreased overall survival (p = 0.001) and decreased disease-free survival (p = 0.022). Further, the correlation between COX-2 overexpression and poor prognosis was independent of disease stage in this surgically resected NSCLC patient population. Tsubochi and colleagues described a similar relationship between COX-2 expression and poor prognosis in stage I adenocarcinomas (126). These reports, together with other studies documenting an increase in COX-2 expression in precursor lesions (127, 128), a common polymorphism in the COX-2 gene associated with increased risk of lung cancer (129), and epidemiological studies that indicate a decreased incidence of lung cancer in patients who regularly take aspirin (130), all support the involvement of COX-2 in the pathogenesis of lung cancer. High constitutive expression of COX-2 documented in both precursor lesions and established lung cancers has lead to use of COX-2 inhibitors in chemoprevention trials. Mao and colleagues reported on the feasibility of celecoxib as a chemopreventative agent for lung cancer by administering heavy current smokers with a 6 month course of oral celecoxib and performing serial bronchoscopies with bronchoalveolar lavage and biopsy (131). Treatment with celecoxib significantly reduced the Ki-67 labeling index in smokers by 35% (p = 0.016) and increased the expression of nuclear survivin by 23% (p = 0.036) without significantly changing that of cytoplasmic survivin. These findings support the hypothesis that oral administration of celecoxib is capable of modulating Ki-67 labeling index in the bronchial tissue of active smokers at high risk for developing lung cancers. Larger randomized placebo-controlled clinical trials are underway to determine the efficacy of COX-2 inhibitors in preventing the development of bronchogenic carcinoma (132, 133). Several recent studies have also evaluated the combined inhibition of the EGFR and COX-2 pathways in patients with established NSCLC. Gadgeel and colleagues

46

T.C. Walser et al.

reported a phase II study of gefitinib and celecoxib in patients with platinum refractory NSCLC (134, 135). Patients received gefitinib 250 mg daily and celecoxib 400 mg twice daily. The response rate to the combination of celecoxib and gefitinib was similar to that observed with gefitinib alone. Agarwala and colleagues conducted a similar study in an unselected population of chemotherapy naïve patients with advanced NSCLC (136). Again, the combination of gefitinib 250  mg daily and celecoxib 400 mg twice daily did not yield a greater response rate or better efficacy than control therapy. O’Byrne and colleagues reported a phase I/II trial of combination therapy with gefitinib 250 mg daily and rofecoxib 50 mg daily in patients with platinum-pretreated relapsed NSCLC (137). Gefitinib combined with rofecoxib was found to provide disease control rates equivalent to that expected with singleagent gefitinib. Finally, Fidler and colleagues recently evaluated the safety and efficacy of erlotinib 150 mg daily plus celecoxib 400 mg twice daily in the setting of advanced NSCLC (138). Combination therapy was not superior to erlotinib alone in the unselected patients. However, longer progression-free survival was observed in patients with high tumor COX-2, suggesting that the combination trial would be more appropriate in a patient population selected for high tumor COX-2 expression. The lack of benefit from combining EGFR tyrosine kinase inhibitor (TKI) and COX-2 inhibitor therapy in these studies also raises the question of whether the OBD to inhibit COX-2 was achieved and whether a higher dosage may have a critical effect on efficacy. Reckamp and colleagues conducted a phase I trial evaluating escalating doses of celecoxib (200–800 mg twice daily) in combination with a fixed dose of erlotinib (150 mg/day) in late stage NSCLC patients, establishing an OBD of 600 mg twice daily, as defined by the maximal decrease in urinary prostaglandin E-M (PGE-M) (139). This study revealed an acceptable toxicity profile with combination therapy and demonstrated a disease control rate above that expected for erlotinib alone. Based on these results, a phase II trial is currently in progress to assess combination therapy with celecoxib at 600  mg twice daily and erlotinib 150  mg daily versus single agent erlotinib. The use of COX-2 inhibitors at the OBD may improve efficacy of combination therapy and may explain the lack of benefit in some trials in which lower doses of COX-2 inhibitors were used. Although the use of COX-2 inhibitors at the OBD may promote responses to combination therapy, there may be associated toxicities with the use of COX-2 inhibitors. Gridelli and colleagues evaluated the addition of rofecoxib (50 mg/day) to cisplatin and gemcitabine in stage IV or IIIB NSCLC subjects (140, 141). The groups receiving rofecoxib were closed early because of safety issues surrounding the higher frequency of cardiac ischemia in subjects that received rofecoxib at 50 mg/day. In a cumulative meta-analysis of 18 randomized controlled trials and 11 observational studies, Juni and colleagues reported on the increased risk of myocardial infarction in subjects who received rofecoxib (142). Other reports have shown that rofecoxib exhibits a greater risk of cardiovascular toxicity to compared celecoxib, and the risk may be dose-dependent (143). Solomon and colleagues found that rofecoxib was associated with a greater incidence of cardiovascular toxicity compared to celecoxib and NSAIDS, and patients taking rofecoxib at >25 mg doses

Tumor Microenvironment

47

were associated with higher risk than lower doses (143). These studies suggest that COX-2 inhibitors may have differing cardiovascular risks, and dose may also determine safety profile. It is unclear if cardiac ischemia will occur at a higher rate with short-term usage of COX-2 inhibitors alone or in combination with targeted therapies or conventional chemotherapy. Critical to the interpretation of all of these studies and to the design of future studies is the consideration of patient selection. Chan and colleagues compared the impact of use of aspirin on the relative risk of colorectal cancer in relation to the expression of COX-2 in the tumor (144). The authors found that regular use of aspirin reduces the risk of colorectal cancer development when COX-2 is overexpressed, but not when COX-2 expression is either weak or absent. Similarly, a randomized phase II trial to assess whether there was benefit with dual eicosanoid inhibition or with either agent (celecoxib or zileuton) alone in addition to chemotherapy found an advantage for celecoxib and chemotherapy only in advanced lung cancer patients with moderate to high tumor expression of COX-2 (145). These studies illustrate the importance of a more individualized approach to therapy that ideally minimizes the risk-benefit ratio and improves efficacy in future clinical trials.

Peroxisome Proliferator-Activated Receptor-Gamma and 15-Prostaglandin Dehydrogenase The presence of PGE2 in the tumor microenvironment fuels lung carcinogenesis, making PGE2 the target of both chemopreventative and chemotherapeutic strategies for lung cancer management, as described earlier. Increasing PGE2 metabolism and clearance represents an alternative therapeutic approach to interfering with COX-2 activity. The catabolic enzyme and newly identified tumor suppressor, 15-prostaglandin dehydrogenase (15-PGDH), converts PGE2 to biologically inactive 15-keto derivatives. Therefore, augmenting 15-PGDH production in the tumor microenvironment has the potential to inhibit tumor growth by lowering the concentration of PGE2 available to the tumor. Hazra and colleagues recently reported that the thiazolidinediones (TZDs), rosiglitazone and pioglitazone, diminish PGE2 production in lung tumor cell lines, and siRNA targeting 15-PGDH completely thwarted the effect of the TZDs (146). TZDs are a class of anti-diabetic and insulinsensitizing drugs that includes troglitazone, ciglitazone, rosiglitazone, and pioglitazone. Biologically, TZDs are agonists of the peroxisome proliferator-activated receptor gamma (PPARg), a ligand-activated nuclear receptor. While the role of PPARg in regulation of metabolism and inflammation is well established, in recent years, a role for PPARg and the TZDs in carcinogenesis has been defined and has been the subject of extensive review (147–149). TZDs alter expression of COX-2 and consequent production of PGE2 and the PGE2 catabolic enzyme, 15-prostaglandin dehydrogenase (15-PGDH), through both PPARg-dependent and -independent mechanisms, resulting in reduced expression of COX-2, inhibition of NSCLC cell growth in  vitro, and inhibition of tumor progression in xenograft models.

48

T.C. Walser et al.

The interrelatedness of PPARg, TZDs, and the COX-2/PGE2 pathways in lung cancer is complex, but understanding the mechanisms underlying this relationship may be helpful in designing anticancer therapies. Lee and colleagues investigated the effect of the PPARg agonist rosiglitazone on growth of NSCLC cells in  vitro (150). Specifically, they evaluated expression of PTEN and the antitumor activity of the EGFR TKI, gefitinib. Rosiglitazone treatment reduced the growth of A549 cells in a dose-dependent manner and facilitated the anti-proliferative effects of gefitinib. PPARg and PTEN expression were increased in gefitinib- and rosiglitazone-treated cells. These data indicate that the PPARg agonist rosiglitazone potentiates gefitinib’s anti-proliferative effects via increased PTEN expression, suggesting that PPARg ligands may serve as therapeutic targets for NSCLC. Reddy and colleagues investigated the potential use of the PPARg ligand, troglitazone, in combination with cisplatin or paclitaxel, in the setting of NSCLC (151). These in vitro studies demonstrated a synergistic interaction between troglitazone and cisplatin or paclitaxel that mediated inhibition of NSCLC cell growth in a sequence-specific manner; growth inhibition occurred only if troglitazone treatment followed chemotherapy, not vice versa. Both cisplatin and paclitaxel upregulated PPARg protein expression, which likely accounts for the sequence-specific nature of the benefit with combination therapy. Similar results were obtained in an NSCLC xenograft mouse model. These data demonstrate a novel sequence-specific synergy between PPARg ligands and chemotherapeutic agents in the treatment of NSCLC. Girnun and colleagues determined the efficacy of combining rosiglitazone and carboplatin therapy in a preclinical model of drug-resistant NSCLC (152). Mice bearing K-ras or EGFR mutant tumors were given either rosiglitazone or carboplatin monotherapy or a combination of both drugs. Tumor burden and pathology, along with cell proliferation and apoptosis, were evaluated. Tumor burden was unchanged or increased in mice after monotherapy. In contrast, significant tumor inhibition occurred in response to combination therapy. Immunohistochemical analysis revealed that therapy was mediated by increased apoptosis and decreased tumor cell proliferation. Importantly, the synergy between carboplatin and rosiglitazone did not increase systemic toxicity. These results suggest that platinum-based drugs and rosiglitazone interact synergistically to reduce NSCLC tumor burden preclinically. They further suggest that the PPARg ligand and carboplatin combination is worthy of further investigation clinically, particularly in those cancers that show primary resistance to platinum therapy or acquired resistance to targeted therapy. Kim and colleagues examined the expression and function of a truncated splice variant of human PPARg in primary human lung cancer specimens (153). While PPARg expression was mostly restricted to the nucleus of nontumor tissues, it was present in the nucleus and cytoplasm of SCC tumors. In vitro studies using Chinese hamster ovary cells indicated that overexpression of the PPARg splice variant dampens their sensitivity to cell death induced by oxidative stress or by the chemotherapeutic agent, cisplatin; conversely, down-regulation of the splice variant renders the tumor cells sensitive to cisplatin. Overexpression of this PPARg splice

Tumor Microenvironment

49

variant may be one mechanism by which tumor cells become resistant to drug- and chemical-induced cell death, suggesting that the splice variant may be linked to tumor progression and poor clinical outcome. In a recent retrospective study, Govindarajan and colleagues observed a significant reduction in lung cancer risk in a Veterans’ Administration population of diabetics aged >40 years who were using the TZD rosiglitazone for at least 1 year (154). Recently, several chemoprevention trials were initiated using TZDs (155). However, several clinical studies in diabetes patients have demonstrated an increased risk of cardiovascular events associated with chronic rosiglitazone or pioglitazone treatment (156–158). Prospective clinical studies specifically designed to address the effects of TZDs on cancer and cardiac outcomes are now required. If the anti-inflammatory and antitumor effects of TZDs are derived through pathways distinct from those leading to cardiovascular toxicity, more selective candidate drug molecules may be therapeutically effective, without leading to adverse cardiac events. As is the case with COX-2 inhibitors, the use of TZDs for the chemoprevention of lung cancer will require careful patient risk assessment and selection that ideally minimizes the risk-benefit ratio and improves efficacy in future clinical trials. Both elevated COX-2 and reduced PPARg expression are associated with poor prognosis in lung cancer patients (125, 159), and recent work has revealed multiple interactions between PPARg signaling and the COX-2 pathway. The targets downstream of COX-2 may be useful in light of recent evidence that interfering with COX-2 enzymatic activity may increase risk of cardiovascular events (160). The discovery that certain PPARg agonists can specifically reduce PGE2 concentration or expression of EP receptors may aid in the design of strategies to reduce the effects of harmful prostaglandins without impacting production of critical cardioprotective eicosanoids. More research is required to define these and other opportunities to specifically interfere with PGE2 production, metabolism, and its downstream effects.

Inflammation and Epithelial–Mesenchymal Transition Within the tumor microenvironment, inflammatory cells and their secretomes influence nearly every aspect of cancer progression (123). The role of chronic inflammation in carcinogenesis is well documented (161–166), such that Montavani recently suggested that inflammation represents the seventh hallmark of cancer (167, 168). The tobacco-induced pulmonary microenvironment, in particular, represents a unique milieu in which carcinogenesis proceeds in complicity with surrounding lung inflammatory, structural, and stromal cells. Pulmonary diseases associated with the greatest risk for lung cancer, COPD and IPF, are characterized by abundant and deregulated inflammation (169–171). Among the cytokines, growth factors, and mediators released in these lung diseases and the developing tumor microenvironment, interleukin-1 beta (IL-1b), PGE2, and transforming growth factor beta

50

T.C. Walser et al.

(TGF-b) have deleterious properties that simultaneously inhibit cell-mediated immune responses against tumor antigens and promote EMT (30, 172–175). Advances in risk assessment play a crucial role in the development of preventive measures. For example, COPD has long been associated with lung cancer risk (176–178), and recent studies emphasize the integral role of inflammation as a potential central shared pathway in the pathogenesis of COPD and lung cancer (123, 179, 180).This raises the intriguing possibility that agents used to limit inflammation in patients with COPD may also serve to prevent lung cancer. For example, a cohort study has already suggested that inhaled corticosteroids may have a role in lung cancer prevention in patients who have COPD (181). By studying another high risk group for lung cancer – current and ex-smokers – radiographic assessments of emphysema and spirometric evaluation of airflow obstruction have been correlated to lung cancer risk in this population, providing potential clinical and imaging parameters for lung cancer risk assessment (182). Such assessments direct the appropriate attention and potential chemopreventive measures to the people who need it most. While the mechanisms linking inflammation, COPD, and lung cancer are not yet defined, a recent editorial by Houghton et al. discusses the role of bronchoalveolar stem cells (BASC) (179, 183). Chronic inflammation may drive otherwise quiescent BASCs to proliferate in an uncontrolled manner, potentially leading to cancer initiation given the carcinogen-rich inflammatory milieu characteristic of COPD. In fact, BASC cells do give rise to lung adenocarcinoma in a mouse model of K-ras-induced malignancy (183). Adair–Kirk et al. suggest other mechanisms, including activation of a proinflammatory cytokine cascade initiated by the “inflammasome” (180, 184) and expression of proinflammatory genes induced by an endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) (180, 185). Increased expression of proteins characteristic of ER stress and of UPR is a mechanism not previously described for the initiation of the inflammatory response to cigarette smoke by lung epithelial cells. The discovery that the lung epithelium responds to cigarette smoke with UPR followed by antioxidant and cytokine gene expression suggests that efforts should be made to identify clinically useful makers of lung epithelial stress. Sin and colleagues recently reported that surfactant-associated protein-D (SP-D) is a biomarker of COPD activity that is relatively lung specific (180, 186). In a retrospective analysis of serum samples from a COPD treatment trial, SP-D abundance decreased during treatment with inhaled corticosteroids and increased during treatment withdrawal, making SP-D a potential marker of cigarette smoke-induced lung epithelial cell stress. Additionally, Bulk and colleagues recently discovered that the S100 family of inflammation-related proteins predicted survival in a large cohort (n = 196) of NSCLC patients. By microarray analysis, high mRNA expression of several S100 proteins, especially S100A2, associated with poor survival in surgically resected NSCLC patients, suggesting that these inflammationrelated proteins also represent potential biomarkers and novel therapeutic targets in NSCLC.

Tumor Microenvironment

51

Recent studies highlight the connection between inflammation and EMT in the development of lung cancer and resistance to therapy (113, 172, 187). For example, IL-1b and PGE2 have the capacity to decrease E-cadherin expression and promote EMT. These inflammatory mediators up-regulate the zinc-finger E-box-binding transcriptional repressors of E-cadherin, including Zeb1, Snail, and Slug, thus leading to EMT progression (113, 172, 175). Elevated levels of Snail exist in both human lung cancers as well as in premalignant lesions, and Snail overexpression enhances diverse malignant phenotypes in NSCLC cell lines as well as in immortalized human bronchial epithelial cell lines (188, 189). Factors that play a pathologic role across the spectrum of carcinogenesis – from premalignancy to advanced disease – hold unique potential as targets for therapy. For example, 30% of lung cancer patients after resection of early disease develop recurrence. In this population, targeting these factors could potentially treat patients for any remnant of the cancer they already have while simultaneously preventing the cancer they are at risk of developing. EMT requires alterations in cell morphology, adhesion, and migration (190). These cellular changes result in variable expression of proteins which serve as EMT markers. Decreased E-cadherin, which permits reduced cell-to-cell adhesion and enhanced cell migration, is the hallmark feature of EMT. Dohadwala and colleagues have previously shown a COX-2-dependent transcriptional regulation of E-cadherin expression and cellular aggregation in NSCLC and a reciprocal relationship between COX-2 and E-cadherin, as well as ZEB1 and E-cadherin (172). COX-2 and PGE2 expression resulted in significant reduction in E-cadherin via the transcriptional repressors ZEB1 and Snail. Inhibition of COX-2 resulted in rescue of E-cadherin expression. Thus, therapies targeting the COX pathway may diminish the propensity for tumor metastasis in NSCLC by blocking the PGE2-mediated induction of E-cadherin transcriptional repressors. This newly defined pathway for transcriptional regulation of E-cadherin in NSCLC has important implications for chemoprevention and treatment of NSCLC using COX-2 inhibitors in combination with other agents. For example, E-cadherin expression in NSCLC has recently been implicated as a marker of sensitivity to EGFR TKIs (191–193). Concordantly, low serum E-cadherin levels correlate with response to combination therapy with erlotinib and celecoxib in patients with NSCLC (194). By enhancing E-cadherin expression, COX-2 inhibitors may augment sensitivity to EGFR TKI therapy (139). Recent work from the Weinberg Laboratory suggests a direct link between EMT and gain of epithelial stem cell properties (195). Thus, in the pathogenesis of lung cancer, inflammation may promote stem cell-like properties via EMT-dependent events. While EMT-induced alterations are implicated in the metastatic process, the work of Mani and colleagues suggests that the EMT genetic program may also regulate early events in carcinogenesis, therefore implicating the inflammatory pulmonary environment in both lung cancer initiation and metastatic progression, as recently summarized by Sanchez– Garcia (195, 196). Development of mouse models of EMT, identification of endogenous inhibitors of EMT, and development of synthetic inhibitors is now the focus of intense investigation given that E-cadherin transcriptional repressors may lie at the intersection of inflammation, EMT, and lung cancer progression.

52

T.C. Walser et al.

NF-kB The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway is considered the central mediator of immune responses (197). The pathway is induced by many different stimuli in the developing tumor microenvironment, and there are hundreds of downstream effector molecules (198). Five NF-kB proteins all bind DNA and regulate transcription, and the action of NF-kB is inhibited by IkB. When specific serine residues on IkB are phosphorylated, IkB is targeted for degradation by the proteasome (199). The IkB kinase (IKK) family has five members (200). In response to stimulation, one action of this family is to phosphorylate IkB, leading to its degradation. A variety of agents targeting IKK family members are in different stages of development. CHS828 and SU6668 are two molecules that are currently being evaluated in early phase clinical studies. The proteasome is a multisubunit protein, the role of which is destruction of proteins (201). The proteasome degrades many different ubiquinated proteins. The antineoplastic activity of proteasome inhibition is hypothesized to be impaired destruction of IkB, as elevated levels of IkB lead to greater inhibition of NF-kB. There are many clinically evaluated agents for which inhibition of NF-kB is considered one of the mechanisms of action. Bortezomib is the only agent under extensive evaluation in lung cancer for which inhibition of NF-kB is considered its primary mechanism of action. Bortezomib is the first FDA approved proteasome inhibitor. It is a modified dipeptidyl boronic acid that reversibly inhibits the chymotrypsin-like activity of the 26S proteasome. The FDA approved bortezomib on May 13, 2003 for patients with multiple myeloma (202). In December 2006, the approval of bortezomib was extended to patients with mantle cell lymphoma (203). Bortezomib has been extensively evaluated in solid malignancies. After the single agent dose of bortezomib was established, the agent was evaluated in combination with frontline chemotherapy for NSCLC, second line therapy for NSCLC, and second line therapy for small cell lung cancer. Phase I studies have evaluated bortezomib in combination with gemcitabine and carboplatin (204) and in combination with gemcitabine and cisplatin (205). Bortezomib 1.0 mg/m2 on days 1, 4, 8 and 11 plus gemcitabine 1,000 mg/m2 on days 1 and 8 and carboplatin AUC 5.0 on day 1 of a 21 day cycle was found to be safe. Bortezomib 1.0 mg/m2 on days 1 and 8 plus gemcitabine 1,000 mg/m2 on days 1 and 8 and cisplatin 70 mg/m2 on day 1 of a 21 day cycle was also considered safe. Because of gastrointestinal toxicity and myelosuppression, the frequency of bortezomib was less in the cisplatin containing study. A phase II study evaluated bortezomib in combination with gemcitabine and carboplatin at the doses stated above in previously untreated patients with NSCLC. Median overall survival was 11 months, and progression-free survival was 5 months. Side effects were mainly hematologic. The regimen was reasonably well tolerated, but the overall survival and progression-free survival were similar to historical data (206). There are three approved second line therapies in NSCLC, docetaxel (207, 208), pemetrexed (209), and erlotinib (210). All three have been evaluated in combination

Tumor Microenvironment

53

with bortezomib. Bortezomib and docetaxel have been evaluated in multiple studies (211, 212). A phase I study evaluated 36 patients with mostly NSCLC. Two patients achieved a partial response, and seven achieved stable disease. The recommended phase II dose was bortezomib 1.0  mg/m2 on days 1, 4, 8, and 11 plus docetaxel 75 mg/m2 on day 1 every 21 days. In a randomized phase II study of 155 patients, bortezomib 1.5  mg/m2 was compared to bortezomib 1.3  mg/m2 plus docetaxel 75 mg/m2 on day 1. Treatment cycles were 21 days and bortezomib was given on days 1, 4, 8, and 11. The response rate was 8% with disease control rates of 29% for bortezomib alone, and the response rate was 9% with disease control rate of 54% for the combined treatment arm. Bortezomib and pemetrexed were evaluated in a phase I trial (213). Responses were seen, and the recommended phase II dose of pemetrexed was 500 mg/m2 day 1 and bortezomib 1.3 mg/m2 days 1, 4, 8 and 11 of a 21-day cycle. Bortezomib 1.6 mg/m2 on days 1 and 8 of a 21-day cycle and erlotinib 150 mg orally were compared to erlotinib alone in a randomized phase II study (214). Although the combined therapy was well tolerated, the study was halted after 50 patients were treated as a result of a planned interim analysis that demonstrated insufficient clinical activity in the combination arm. Small cell lung cancer after recurrence is a difficult clinical scenario. Bortezomib has been evaluated as a single agent for the treatment of small cell lung cancer (215). Thirty-six patients were enrolled on a phase II trial of bortezomib as a single agent in previously treated patients at 1.3 mg/m2 intravenously on days 1, 4, 8, and 11 every 21 days. Median progression-free survival and overall survival were 1 and 3 months, respectively, and there was a single responder. These results were felt to be insufficient to proceed with the development of bortezomib for this clinical situation. Although a great deal of clinical data has been generated evaluating bortezomib in lung cancer, at this time, there are no data that indicate a role of the agent in the treatment of lung cancer. Responses have been seen, even in the single agent setting, but these responses have been rare. There is also no convincing data that the addition of bortezomib to standard regimens improves the outcome. It is still possible that such differences could be detected in larger studies, or by identifying a subgroup of patients with an increased likelihood of response to bortezomib. It is also possible that other proteasome inhibitors in development will demonstrate greater efficacy.

HGF and c-Met c-Met is a receptor tyrosine kinase that is currently receiving considerable attention from the lung cancer research community. c-Met was originally identified in 1984 (216). The ligand for c-Met is hepatocyte growth factor (HGF), which is abundantly expressed in the developing tumor microenvironment. In response to activation of c-Met by HGF, homodimerization of c-Met occurs, leading to downstream signaling, including increased motility, invasion, proliferation, and angiogenesis (217).

54

T.C. Walser et al.

c-Met is overexpressed in a variety of tumor types, including both small cell lung cancer (218) and NSCLC (219). Increased signaling via the c-Met axis has been seen as a result of multiple mechanisms. Increased expression of the ligand or receptor, amplification, and mutation of c-Met have all been observed (220). In the setting of lung cancer, c-Met amplification has recently received increased attention for its role in resistance to inhibitors of EGFR in NSCLC (221). Although most of the work to establish such a role for c-Met has been in vitro, there is newly available clinical data in which biopsies prior to therapy with EGFR-targeted therapies did not have c-Met amplification, while biopsies at the time of resistance did show evidence of amplification. Although c-Met signaling is only partially responsible for resistance to EGFR inhibitors, these data have spurred clinical studies to evaluate the role of c-Met inhibition along with EGFR inhibition. Clinical trials with these inhibitors in the setting of lung cancer have not yet been completed. Based on the preclinical data described above, several studies are currently being planned in which inhibitors of both EGFR and c-MET are used. Many agents are being evaluated in phase I testing as well. AMG102 is a fully humanized monoclonal antibody against HGF that has been the subject of clinical investigation (222). In addition to single agent studies, this agent has been studied with other antiangiogenic agents, including bevacizumab, and the investigational small molecule tyrosine kinase inhibitor, motesanib diphosphate (223). These combinations were well tolerated and will be investigated further in additional studies. AMG102 is being evaluated alone or along with cytotoxic chemotherapy or targeted agents in studies of nonthoracic malignancies. In one study currently recruiting patients, AMG102 is used in combination with etoposide and a platinum agent (carboplatin or cisplatin) in extensive stage small cell lung cancer. Tyrosine kinase inhibition is another attractive approach to inhibiting c-Met signaling. PF-02341066 is one such tyrosine kinase inhibitor (224), and phase I studies of this agent are currently recruiting patients. ARQ197 is another agent directed against MET for which data from phase I studies are already available (225). This agent is being extensively evaluated in several different studies. In addition to single agent studies, ARQ197 is being evaluated as part of a study in which all patients receive erlotinib, but are also randomly assigned to ARQ197 or placebo. The MET inhibitor XL 880 has completed phase I study (226). It is currently being evaluated in phase II studies in several nonthoracic malignancies. In summary, HGF and c-Met are attractive targets for therapy in both NSCLC and small cell lung cancer. Agents targeting this pathway are currently under evaluation, but it is still too early to know whether this will be a successful strategy in clinical situations.

Angiogenesis The induction of tumor vasculature, termed the angiogenic switch, is a required discrete step in tumor progression and an example of how tumors inherently depend on their microenvironment. Interaction between tumor and stroma leads to the

Tumor Microenvironment

55

elevation of proangiogenic factors and the inhibition of antiangiogenic factors, creating an imbalance that promotes the blood vessel formation necessary for exponential growth and metastases (227). Tumor angiogenesis has been shown to play an important role in the progression of a number of solid tumors, including lung cancer, and potential therapies targeted to antiangiogenic pathways have been a focus of extensive study (141). However, the recent results of phase III clinical trials of an antiangiogenic drug provide a preview of the complexities of optimizing such therapies. In particular, they suggest a need for further preclinical studies that elucidate interactions between cancer cells and the whole organism in the context of other therapies. VEGF is a proangiogenic factor that has been associated with tumor progression and poor prognosis in NSCLC, and has been the center of attention for antiangiogenic investigations. Only because of the modest benefits seen with the use of antiangiogenic therapies, studies have begun to focus on their combination with other agents. It is believed that the early antiangiogenic effect is, in fact, a morphologic normalization of poorly organized and inefficient tumor vasculature. The result is transiently improved tumor blood flow, oxygenation, and decreased interstitial fluid pressure before continued suppression ultimately leads to decreased perfusion (228). This creates an opportunity for the optimal delivery of cytotoxic agents, and a strong argument for the use of antiangiogenics in combination with standard chemotherapy. Although multiple clinical trials at all phases are currently evaluating targeted therapies to different factors of the VEGF pathway, the most advanced work has focused on the addition of bevacizumab to standard chemotherapy. Bevacizumab is a monoclonal anti-VEGF antibody that has been approved by FDA for use as firstline therapy for advanced nonsquamous cell NSCLC in conjunction with carboplatin and paclitaxel (229). Leading up to this decision was the phase III trial conducted by the Eastern Cooperative Oncology Group (ECOG), which randomized 878 advanced nonsquamous cell NSCLC patients to standard carboplatin and paclitaxel doublet chemotherapy with or without bevacizumab (94). Because of a prior phase II trial that suggested patients with squamous cell lung cancer had an increased incidence of severe or fatal hemoptysis, patients with this histology were excluded from the phase III trial. Concern over hemorrhage also led to the exclusion of patients with brain metastases, a history of gross hemoptysis, or those requiring therapeutic anticoagulation. The results of the trial revealed superior response rate and progression-free survival in the bevacizumab group. Importantly, the investigators found that the addition of bevacizumab conferred an advantage in overall survival by approximately 2 months. Upon subset analysis, however, it was found that no significant benefit was achieved in overall survival for women (94) or the elderly (>70 years of age) (230) included in the study. A second large multicenter phase III trial, Avastin in Lung Cancer (AVAiL), was conducted in Europe that corroborated the ECOG trial by demonstrating increased progression-free survival in patients with the addition of bevacizumab to their chemotherapy regimen (although only by 18 days). However, unlike the ECOG study, a recent announcement revealed that there was no overall survival advantage associated with the addition of bevacizumab in this study (231).

56

T.C. Walser et al.

The details of these clinical trials contribute important insights for the development of future antiangiogenic therapies and clinical trials. For example, one potential explanation for the disparate results between the two phase III trials is the fact that the AVAiL patients were treated with a different chemotherapy regimen – gemcitabine and cisplatin, as opposed to the carboplatin and paclitaxel that was used in the ECOG study. A recent study by Shaked and colleagues suggests that the beneficial clinical effect of VEGF antagonism may not simply involve potentiating cytotoxic drug delivery, but also result from the manipulation of angiogenic effects specifically initiated by only certain chemotherapies (232). The authors demonstrate how paclitaxel, but not gemcitabine, initiates the mobilization of circulating endothelial progenitors, which home to viable tumor tissue and contribute to tumor recovery after the administration of chemotherapy. They also provide evidence that this effect can at least in part be inhibited by anti-VEGF receptor therapy, thereby enhancing the overall antitumor effect of chemotherapy. The fact that an improvement in overall survival was only achieved in the ECOG trial but not in the AVAiL study invites more focused preclinical data to elucidate the mechanisms of action for disease prog­ ression, interaction between drug therapies, and physiological treatment responses, possibly addressing the questions raised by the results of these clinical trials. An appreciation for both the existence of compensatory mechanisms that take effect in response to treatment as well as the fact that the same pathologic outcomes can be reached by different avenues further supports the argument for using combined therapies. In addition to the VEGF pathway, a number of angiogenic mechanisms have been demonstrated to play an important role in NSCLC. One such pathway is the CXCR2 axis. CXC chemokines are a family of pro-angiogenic and angiostatic peptides. Angiogenic peptides possess a glutamic acid–leucine– arginine (ELR) motif and are all bound by a single receptor, CXCR2. Murine models of lung tumorigenesis have found that the expression of CXCR2 ligands contribute to inflammation, neovascularization, and neoplasia. Importantly, CXCR2 blockade in these models is capable of reducing tumor burden (233). In K-ras mice, administration of CXCR2 neutralizing antibody was also able to inhibit premalignant alveolar lesions, in addition to inducing apoptosis of vascular endothelial cells in these same lesions (234). These effects are dependent on interactions with the tumor microenvironment and have been compared with VEGF activity in key studies. For example, Mizukami and colleagues demonstrated that knockdown of the expression of tumor cell-derived HIF-1a in a preclinical xenograft mouse model markedly inhibited the expression of VEGF in tumors, without eradicating tumor-associated angiogenesis. The study further suggested that this was a result of reactive oxygen species and subsequent NF-kB activation with the induction of angiogenic ELR+ CXC chemokine CXCL8 (235). In another study, Sun and colleagues illustrated how IL-1b, which is produced by macrophages and monocytes in the tumor microenvironment, can induce a variety of angiogenic ELR+ CXC chemokines and consequent angiogenesis in NSCLC. They also showed that the angiogenic effect produced by IL-1b was reversible with blockade of CXCR2, but not with anti-VEGF treatment. These studies and others support the critical concept that studies for targeted therapies must evaluate their role in

Tumor Microenvironment

57

the context of the tumor microenvironment, and suggest that targeting multiple antiangiogenic factors or alternative angiogenic pathways may provide a more dramatic clinical benefit. Ultimately, the optimization of antiangiogenic therapies will require the ability to identify patients for whom the therapeutic index is greatest. To address this, another goal of the ECOG study was to identify predictors of response. Although the ECOG study did not find a correlation between pre- and posttreatment serum levels of VEGF, bFGF, or E-selectin with response to therapy, the benefit for those receiving bevacizumab appeared to be greatest in those patients with low baseline intracellular adhesion molecule (ICAM-1) levels (236). In addition to specifying the populations that benefit with currently tested therapy, efforts have already branched out to identify multimodality strategies to expand treatment to subpopulations otherwise excluded for their bleeding risk (237). A phase II trial, known as PASSPORT, is evaluating the use of bevacizumab with first- or second-line therapy in patients whose brain metastases from nonsquamous NSCLC have already been treated with radiation. Other trials are investigating whether bevacizumab can be used safely in patients with squamous cell histology if they have been pretreated with either chemotherapy or thoracic radiation therapy. The results of the advanced trials involving the combination of bevacizumab and chemotherapy have already had an important clinical impact on how patients with lung cancer are treated today. They also inspire further investigations regarding mechanisms of disease and drug therapy in the context of the tumor microenvironment. They have also contributed to defining appropriate patient selection, which will continue to be a crucial subject of study. In addition to the ground-breaking studies described here, a multitude of new studies (at both the clinical and preclinical levels) are ongoing that include targeting other factors in the VEGF signaling pathway, combining anti-VEGF activity with EGFR-TKI therapy, testing antiangiogenic therapy in earlier stages of lung cancer, and defining alternative angiogenic pathways. In summary, the development of antiangiogenic therapies in the treatment of NSCLC is a burgeoning field that revolves around the critical interaction between cancer and its microenvironment.

Conclusion While genetic changes are essential for the malignant transformation of epithelial cells, the tumor microenvironment plays an equally significant role in cancer progression and metastasis. Molecular signatures that are most often composed of cytokines involved in inflammatory and immune responses correlate with important clinical parameters. There is mounting evidence that these signatures serve as robust biomarkers predictive of cancer progression, prognosis, and therapeutic resistance. Molecular profiling studies that have assessed the tumor-adjacent stroma and in vitro models of the tumor microenvironment have been particularly

58

T.C. Walser et al.

suggestive that the secretome of the tumor microenvironment drives the aggressiveness of tumors. In the clinical studies highlighted in this chapter, macrophage, mast cell, and DC recruitment to the tumor bed are all associated with improved clinical parameters, while ablation of immune-suppressive T reg cells is associated with a favorable prognosis. Redistribution of these immune effectors in favor of a host antitumor immune response and improved clinical outcome is the focus of numerous ongoing clinical investigations. While past trials targeting MMPs, NF-kB, and HGF have met with limited success, there is a renewed optimism that improved inhibitors and better informed patient selection will yield more favorable outcomes in the future. Inflammation and angiogenesis are cancer-associated phenotypes clearly involved in lung carcinogenesis. Clinical studies that successfully target these aspects of the tumor microenvironment will also likely require patient selection and combination therapies that utilize the OBD of each agent being evaluated. Likewise, targeting EMT will require careful trial design appropriately informed by all available preclinical data. Taken together, further elucidation of the microenvironmental and molecular mechanisms involved in lung carcinogenesis is required. Targeting the reversible events in the microenvironment that contribute to tumor progression continues to hold great clinical promise.

References 1. Bhattacharjee A, Richards WG, Staunton J et al (2001) Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. Proc Natl Acad Sci USA 98:13790–13795 2. Beer DG, Kardia SL, Huang CC et  al (2002) Gene-expression profiles predict survival of patients with lung adenocarcinoma. Nat Med 8:816–824 3. Yanaihara N, Caplen N, Bowman E et al (2006) Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 9:189–198 4. Potti A, Mukherjee S, Petersen R et al (2006) A genomic strategy to refine prognosis in earlystage non-small-cell lung cancer. N Engl J Med 355:570–580 5. Seike M, Yanaihara N, Bowman ED et al (2007) Use of a cytokine gene expression signature in lung adenocarcinoma and the surrounding tissue as a prognostic classifier. J Natl Cancer Inst 99:1257–1269 6. Budhu A, Forgues M, Ye QH et al (2006) Prediction of venous metastases, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment. Cancer Cell 10:99–111 7. Farmer P, Bonnefoi H, Anderle P et al (2009) A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer. Nat Med 15:68–74 8. Zhong L, Roybal J, Chaerkady R et al (2008) Identification of secreted proteins that mediate cell-cell interactions in an in vitro model of the lung cancer microenvironment. Cancer Res 68:7237–7245 9. Allavena P, Sica A, Garlanda C, Mantovani A (2008) The Yin-Yang of tumor-associated macrophages in neoplastic progression and immune surveillance. Immunol Rev 222: 155–161

Tumor Microenvironment

59

10. Bingle L, Brown NJ, Lewis CE (2002) The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. J Pathol 196:254–265 11. Dimitriadou V, Koutsilieris M (1997) Mast cell-tumor cell interactions: for or against tumour growth and metastasis? Anticancer Res 17:1541–1549 12. Murdoch C, Muthana M, Coffelt SB, Lewis CE (2008) The role of myeloid cells in the promotion of tumour angiogenesis. Nat Rev Cancer 8:618–631 13. Chen JJ, Lin YC, Yao PL et  al (2005) Tumor-associated macrophages: the double-edged sword in cancer progression. J Clin Oncol 23:953–964 14. Chen JJ, Yao PL, Yuan A et  al (2003) Up-regulation of tumor interleukin-8 expression by infiltrating macrophages: its correlation with tumor angiogenesis and patient survival in nonsmall cell lung cancer. Clin Cancer Res 9:729–737 15. Imada A, Shijubo N, Kojima H, Abe S (2000) Mast cells correlate with angiogenesis and poor outcome in stage I lung adenocarcinoma. Eur Respir J 15:1087–1093 16. Johnson SK, Kerr KM, Chapman AD et al (2000) Immune cell infiltrates and prognosis in primary carcinoma of the lung. Lung Cancer 27:27–35 17. Kerr KM, Johnson SK, King G, Kennedy MM, Weir J, Jeffrey R (1998) Partial regression in primary carcinoma of the lung: does it occur? Histopathology 33:55–63 18. Koukourakis MI, Giatromanolaki A, Kakolyris S et al (1998) Different patterns of stromal and cancer cell thymidine phosphorylase reactivity in non-small-cell lung cancer: impact on tumour neoangiogenesis and survival. Br J Cancer 77:1696–1703 19. Nagata M, Shijubo N, Walls AF, Ichimiya S, Abe S, Sato N (2003) Chymase-positive mast cells in small sized adenocarcinoma of the lung. Virchows Arch 443:565–573 20. Takanami I, Takeuchi K, Kodaira S (1999) Tumor-associated macrophage infiltration in pulmonary adenocarcinoma: association with angiogenesis and poor prognosis. Oncology 57:138–142 21. Takanami I, Takeuchi K, Naruke M (2000) Mast cell density is associated with angiogenesis and poor prognosis in pulmonary adenocarcinoma. Cancer 88:2686–2692 22. Tomita M, Matsuzaki Y, Onitsuka T (1999) Correlation between mast cells and survival rates in patients with pulmonary adenocarcinoma. Lung Cancer 26:103–108 23. Toomey D, Smyth G, Condron C et al (2003) Infiltrating immune cells, but not tumour cells, express FasL in non-small cell lung cancer: no association with prognosis identified in 3-year follow-up. Int J Cancer 103:408–412 24. Ohno S, Inagawa H, Dhar DK et  al (2003) The degree of macrophage infiltration into the cancer cell nest is a significant predictor of survival in gastric cancer patients. Anticancer Res 23:5015–5022 25. Welsh TJ, Green RH, Richardson D, Waller DA, O’Byrne KJ, Bradding P (2005) Macrophage and mast-cell invasion of tumor cell islets confers a marked survival advantage in non-smallcell lung cancer. J Clin Oncol 23:8959–8967 26. Kim DW, Min HS, Lee KH et al (2008) High tumour islet macrophage infiltration correlates with improved patient survival but not with EGFR mutations, gene copy number or protein expression in resected non-small cell lung cancer. Br J Cancer 98:1118–1124 27. Kawai O, Ishii G, Kubota K et al (2008) Predominant infiltration of macrophages and CD8(+) T Cells in cancer nests is a significant predictor of survival in stage IV nonsmall cell lung cancer. Cancer 113:1387–1395 28. Rosenberg SA, Yang JC, Restifo NP (2004) Cancer immunotherapy: moving beyond current vaccines. Nat Med 10:909–915 29. Auberger J, Loeffler-Ragg J, Wurzer W, Hilbe W (2006) Targeted therapies in non-small cell lung cancer: proven concepts and unfulfilled promises. Curr Cancer Drug Targets 6:271–294 30. Baratelli F, Lin Y, Zhu L et al (2005) Prostaglandin E2 induces FOXP3 gene expression and T regulatory cell function in human CD4+ T cells. J Immunol 175:1483–1490 31. Batra RK, Lin Y, Sharma S et al (2003) Non-small cell lung cancer-derived soluble mediators enhance apoptosis in activated T lymphocytes through an I kappa B kinase-dependent mechanism. Cancer Res 63:642–646

60

T.C. Walser et al.

32. Huang M, Sharma S, Mao JT, Dubinett SM (1996) Non-small cell lung cancer-derived soluble mediators and prostaglandin E2 enhance peripheral blood lymphocyte IL-10 transcription and protein production. J Immunol 157:5512–5520 33. Huang M, Stolina M, Sharma S et al (1998) Non-small cell lung cancer cyclooxygenase2-dependent regulation of cytokine balance in lymphocytes and macrophages: up-regulation of interleukin 10 and down-regulation of interleukin 12 production. Cancer Res 58:1208–1216 34. Woo EY, Chu CS, Goletz TJ et al (2001) Regulatory CD4(+)CD25(+) T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer. Cancer Res 61:4766–4772 35. Woo EY, Yeh H, Chu CS et  al (2002) Cutting edge: regulatory T cells from lung cancer patients directly inhibit autologous T cell proliferation. J Immunol 168:4272–4276 36. Yoshino I, Yano T, Murata M et al (1992) Tumor-reactive T-cells accumulate in lung cancer tissues but fail to respond due to tumor cell-derived factor. Cancer Res 52:775–781 37. Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392:245–252 38. Steinman RM, Hemmi H (2006) Dendritic cells: translating innate to adaptive immunity. Curr Top Microbiol Immunol 311:17–58 39. Cerundolo V, Hermans IF, Salio M (2004) Dendritic cells: a journey from laboratory to clinic. Nat Immunol 5:7–10 40. Nestle FO, Farkas A, Conrad C (2005) Dendritic-cell-based therapeutic vaccination against cancer. Curr Opin Immunol 17:163–169 41. Svane IM, Soot ML, Buus S, Johnsen HE (2003) Clinical application of dendritic cells in cancer vaccination therapy. APMIS 111:818–834 42. Holtl L, Ramoner R, Zelle-Rieser C et al (2005) Allogeneic dendritic cell vaccination against metastatic renal cell carcinoma with or without cyclophosphamide. Cancer Immunol Immunother 54:663–670 43. Nestle FO, Alijagic S, Gilliet M et al (1998) Vaccination of melanoma patients with peptideor tumor lysate-pulsed dendritic cells. Nat Med 4:328–332 44. Redfern CH, Guthrie TH, Bessudo A et  al (2006) Phase II trial of idiotype vaccination in previously treated patients with indolent non-Hodgkin’s lymphoma resulting in durable clinical responses. J Clin Oncol 24:3107–3112 45. Timmerman JM, Czerwinski DK, Davis TA et al (2002) Idiotype-pulsed dendritic cell vaccination for B-cell lymphoma: clinical and immune responses in 35 patients. Blood 99:1517–1526 46. Morse MA, Clay TM, Hobeika AC et al (2005) Phase I study of immunization with dendritic cells modified with fowlpox encoding carcinoembryonic antigen and costimulatory molecules. Clin Cancer Res 11:3017–3024 47. Kimura H, Iizasa T, Ishikawa A et al (2008) Prospective phase II study of post-surgical adjuvant chemo-immunotherapy using autologous dendritic cells and activated killer cells from tissue culture of tumor-draining lymph nodes in primary lung cancer patients. Anticancer Res 28:1229–1238 48. Albert ML, Pearce SF, Francisco LM et al (1998) Immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J Exp Med 188:1359–1368 49. Fields RC, Shimizu K, Mule JJ (1998) Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo. Proc Natl Acad Sci USA 95:9482–9487 50. Hsu FJ, Benike C, Fagnoni F et al (1996) Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells. Nat Med 2:52–58 51. Boczkowski D, Nair SK, Nam JH, Lyerly HK, Gilboa E (2000) Induction of tumor immunity and cytotoxic T lymphocyte responses using dendritic cells transfected with messenger RNA amplified from tumor cells. Cancer Res 60:1028–1034 52. Miller PW, Sharma S, Stolina M et al (2000) Intratumoral administration of adenoviral interleukin 7 gene-modified dendritic cells augments specific antitumor immunity and achieves tumor eradication. Hum Gene Ther 11:53–65

Tumor Microenvironment

61

53. Wan Y, Bramson J, Carter R, Graham F, Gauldie J (1997) Dendritic cells transduced with an adenoviral vector encoding a model tumor-associated antigen for tumor vaccination. Hum Gene Ther 8:1355–1363 54. Lundqvist A, Choudhury A, Nagata T et al (2002) Recombinant adenovirus vector activates and protects human monocyte-derived dendritic cells from apoptosis. Hum Gene Ther 13:1541–1549 55. Song W, Tong Y, Carpenter H, Kong HL, Crystal RG (2000) Persistent, antigen-specific, therapeutic antitumor immunity by dendritic cells genetically modified with an adenoviral vector to express a model tumor antigen. Gene Ther 7:2080–2086 56. Basak SK, Kiertscher SM, Harui A, Roth MD (2004) Modifying adenoviral vectors for use as gene-based cancer vaccines. Viral Immunol 17:182–196 57. Hromas R, Kim CH, Klemsz M et al (1997) Isolation and characterization of Exodus-2, a novel C-C chemokine with a unique 37-amino acid carboxyl-terminal extension. J Immunol 159:2554–2558 58. Riedl K, Baratelli F, Batra RK et al (2003) Overexpression of CCL-21/secondary lymphoid tissue chemokine in human dendritic cells augments chemotactic activities for lymphocytes and antigen presenting cells. Mol Cancer 2:35 59. Yang SC, Batra RK, Hillinger S et al (2006) Intrapulmonary administration of CCL21 genemodified dendritic cells reduces tumor burden in spontaneous murine bronchoalveolar cell carcinoma. Cancer Res 66:3205–3213 60. Yang SC, Hillinger S, Riedl K et  al (2004) Intratumoral administration of dendritic cells overexpressing CCL21 generates systemic antitumor responses and confers tumor immunity. Clin Cancer Res 10:2891–2901 61. Arenberg DA, Zlotnick A, Strom SR, Burdick MD, Strieter RM (2001) The murine CC chemokine, 6C-kine, inhibits tumor growth and angiogenesis in a human lung cancer SCID mouse model. Cancer Immunol Immunother 49:587–592 62. Vicari AP, Ait-Yahia S, Chemin K, Mueller A, Zlotnik A, Caux C (2000) Antitumor effects of the mouse chemokine 6Ckine/SLC through angiostatic and immunological mechanisms. J Immunol 165:1992–2000 63. Carragher DM, Rangel-Moreno J, Randall TD (2008) Ectopic lymphoid tissues and local immunity. Semin Immunol 20:26–42 64. Singh P, Coskun ZZ, Goode C, Dean A, Thompson-Snipes L, Darlington G (2008) Lymphoid neogenesis and immune infiltration in aged liver. Hepatology 47:1680–1690 65. Timmer TC, Baltus B, Vondenhoff M et al (2007) Inflammation and ectopic lymphoid structures in rheumatoid arthritis synovial tissues dissected by genomics technology: identification of the interleukin-7 signaling pathway in tissues with lymphoid neogenesis. Arthritis Rheum 56:2492–2502 66. Moyron-Quiroz JE, Rangel-Moreno J, Kusser K et  al (2004) Role of inducible bronchus associated lymphoid tissue (iBALT) in respiratory immunity. Nat Med 10:927–934 67. Schrama D, Voigt H, Eggert AO et al (2008) Immunological tumor destruction in a murine melanoma model by targeted LTalpha independent of secondary lymphoid tissue. Cancer Immunol Immunother 57:85–95 68. Kirk CJ, Hartigan-O’Connor D, Mule JJ (2001) The dynamics of the T-cell antitumor response: chemokine-secreting dendritic cells can prime tumor-reactive T cells extranodally. Cancer Res 61:8794–8802 69. Coronella JA, Spier C, Welch M et al (2002) Antigen-driven oligoclonal expansion of tumorinfiltrating B cells in infiltrating ductal carcinoma of the breast. J Immunol 169:1829–1836 70. Bell D, Chomarat P, Broyles D et al (1999) In breast carcinoma tissue, immature dendritic cells reside within the tumor, whereas mature dendritic cells are located in peritumoral areas. J Exp Med 190:1417–1426 71. Eisenthal A, Polyvkin N, Bramante-Schreiber L, Misonznik F, Hassner A, Lifschitz-Mercer B (2001) Expression of dendritic cells in ovarian tumors correlates with clinical outcome in patients with ovarian cancer. Hum Pathol 32:803–807 72. Kurabayashi A, Furihata M, Matsumoto M, Hayashi H, Ohtsuki Y (2004) Distribution of tumor-infiltrating dendritic cells in human non-small cell lung carcinoma in relation to apoptosis. Pathol Int 54:302–310 73. Zeid NA, Muller HK (1993) S100 positive dendritic cells in human lung tumors associated with cell differentiation and enhanced survival. Pathology 25:338–343

62

T.C. Walser et al.

74. Coppola D, Mule JJ (2008) Ectopic lymph nodes within human solid tumors. J Clin Oncol 26:4369–4370 75. Dieu-Nosjean MC, Antoine M, Danel C et  al (2008) Long-term survival for patients with non-small-cell lung cancer with intratumoral lymphoid structures. J Clin Oncol 26:4410–4417 76. Kocks JR, Davalos-Misslitz AC, Hintzen G, Ohl L, Forster R (2007) Regulatory T cells interfere with the development of bronchus-associated lymphoid tissue. J Exp Med 204:723–734 77. Ishibashi Y, Tanaka S, Tajima K, Yoshida T, Kuwano H (2006) Expression of Foxp3 in nonsmall cell lung cancer patients is significantly higher in tumor tissues than in normal tissues, especially in tumors smaller than 30 mm. Oncol Rep 15:1315–1319 78. Okita R, Saeki T, Takashima S, Yamaguchi Y, Toge T (2005) CD4+CD25+ regulatory T cells in the peripheral blood of patients with breast cancer and non-small cell lung cancer. Oncol Rep 14:1269–1273 79. Zhang L, Dermawan K, Jin M et al (2008) Differential impairment of regulatory T cells rather than effector T cells by paclitaxel-based chemotherapy. Clin Immunol 129:219–229 80. Ichihara F, Kono K, Takahashi A, Kawaida H, Sugai H, Fujii H (2003) Increased populations of regulatory T cells in peripheral blood and tumor-infiltrating lymphocytes in patients with gastric and esophageal cancers. Clin Cancer Res 9:4404–4408 81. Liyanage UK, Moore TT, Joo HG et al (2002) Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol 169:2756–2761 82. Sasada T, Kimura M, Yoshida Y, Kanai M, Takabayashi A (2003) CD4+CD25+ regulatory T cells in patients with gastrointestinal malignancies: possible involvement of regulatory T cells in disease progression. Cancer 98:1089–1099 83. Wolf AM, Wolf D, Steurer M, Gastl G, Gunsilius E, Grubeck-Loebenstein B (2003) Increase of regulatory T cells in the peripheral blood of cancer patients. Clin Cancer Res 9:606–612 84. Sutmuller RP, van Duivenvoorde LM, van Elsas A et  al (2001) Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25(+) regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses. J Exp Med 194:823–832 85. Curiel TJ, Coukos G, Zou L et al (2004) Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10:942–949 86. Baratelli F, Takedatsu H, Hazra S et  al (2008) Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in non-small cell lung cancer. J Transl Med 6:38 87. Sharma S, Yang SC, Zhu L et al (2005) Tumor cyclooxygenase-2/prostaglandin E2-dependent promotion of FOXP3 expression and CD4+ CD25+ T regulatory cell activities in lung cancer. Cancer Res 65:5211–5220 88. Dudley ME, Rosenberg SA (2003) Adoptive-cell-transfer therapy for the treatment of patients with cancer. Nat Rev Cancer 3:666–675 89. Klebanoff CA, Khong HT, Antony PA, Palmer DC, Restifo NP (2005) Sinks, suppressors and antigen presenters: how lymphodepletion enhances T cell-mediated tumor immunotherapy. Trends Immunol 26:111–117 90. Rosenberg SA, Dudley ME (2004) Cancer regression in patients with metastatic melanoma after the transfer of autologous antitumor lymphocytes. Proc Natl Acad Sci USA 101(Suppl 2):14639–14645 91. Kreitman RJ, Pastan I (2003) Immunobiological treatments of hairy-cell leukaemia. Best Pract Res Clin Haematol 16:117–133 92. Dubinett SM, Sharma S, Huang M, Dohadwala M, Pold M, Mao JT (2003) Cyclooxygenase-2 in lung cancer. Prog Exp Tumor Res 37:138–162 93. Riedl K, Krysan K, Pold M et  al (2004) Multifaceted roles of cyclooxygenase-2 in lung cancer. Drug Resist Updat 7:169–184 94. Sandler A, Gray R, Perry MC et al (2006) Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 355:2542–2550

Tumor Microenvironment

63

  95. Dannenberg AJ, Subbaramaiah K (2003) Targeting cyclooxygenase-2 in human neoplasia: rationale and promise. Cancer Cell 4:431–436   96. Coussens LM, Fingleton B, Matrisian LM (2002) Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science 295:2387–2392   97. Fingleton B (2008) MMPs as therapeutic targets – still a viable option? Semin Cell Dev Biol 19:61–68   98. Konstantinopoulos PA, Karamouzis MV, Papatsoris AG, Papavassiliou AG (2008) Matrix metalloproteinase inhibitors as anticancer agents. Int J Biochem Cell Biol 40:1156–1168   99. Murphy G (2008) The ADAMs: signalling scissors in the tumour microenvironment. Nat Rev Cancer 8:929–941 100. Noel A, Jost M, Maquoi E (2008) Matrix metalloproteinases at cancer tumor-host interface. Semin Cell Dev Biol 19:52–60 101. Overall CM, Kleifeld O (2006) Towards third generation matrix metalloproteinase inhibitors for cancer therapy. Br J Cancer 94:941–946 102. Overall CM, Kleifeld O (2006) Tumour microenvironment – opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. Nat Rev Cancer 6:227–239 103. Leinonen T, Pirinen R, Bohm J, Johansson R, Kosma VM (2008) Increased expression of matrix metalloproteinase-2 (MMP-2) predicts tumour recurrence and unfavourable outcome in non-small cell lung cancer. Histol Histopathol 23:693–700 104. Sienel W, Polzer B, Elshawi K et  al (2008) Cellular localization of EMMPRIN predicts prognosis of patients with operable lung adenocarcinoma independent from MMP-2 and MMP-9. Mod Pathol 21:1130–1138 105. Hakuma N, Betsuyaku T, Kinoshita I et  al (2007) High incidence of extracellular matrix metalloproteinase inducer expression in non-small cell lung cancers. Association with clinicopathological parameters. Oncology 72:197–204 106. Kim SH, Choi HY, Lee J et al (2007) Elevated activities of MMP-2 in the non-tumorous lung tissues of curatively resected stage I NSCLC patients are associated with tumor recurrence and a poor survival. J Surg Oncol 95:337–346 107. Hsu CP, Shen GH, Ko JL (2006) Matrix metalloproteinase-13 expression is associated with bone marrow microinvolvement and prognosis in non-small cell lung cancer. Lung Cancer 52:349–357 108. Liu D, Nakano J, Ishikawa S et  al (2007) Overexpression of matrix metalloproteinase-7 (MMP-7) correlates with tumor proliferation, and a poor prognosis in non-small cell lung cancer. Lung Cancer 58:384–391 109. Mino N, Takenaka K, Sonobe M et al (2007) Expression of tissue inhibitor of metalloproteinase-3 (TIMP-3) and its prognostic significance in resected non-small cell lung cancer. J Surg Oncol 95:250–257 110. Clark JC, Thomas DM, Choong PF, Dass CR (2007) RECK – a newly discovered inhibitor of metastasis with prognostic significance in multiple forms of cancer. Cancer Metastasis Rev 26:675–683 111. Takemoto N, Tada M, Hida Y et al (2007) Low expression of reversion-inducing cysteinerich protein with Kazal motifs (RECK) indicates a shorter survival after resection in patients with adenocarcinoma of the lung. Lung Cancer 58:376–383 112. Liu LT, Chang HC, Chiang LC, Hung WC (2003) Histone deacetylase inhibitor up-regulates RECK to inhibit MMP-2 activation and cancer cell invasion. Cancer Res 63:3069–3072 113. Peebles KA, Lee JM, Mao JT et al (2007) Inflammation and lung carcinogenesis: applying findings in prevention and treatment. Expert Rev Anticancer Ther 7:1405–1421 114. Krysan K, Dalwadi H, Sharma S, Pold M, Dubinett S (2004) Cyclooxygenase 2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer. Cancer Res 64:6359–6362 115. Krysan K, Merchant FH, Zhu L et al (2004) COX-2-dependent stabilization of survivin in non-small cell lung cancer. FASEB J 18:206–208 116. Tsujii M, DuBois RN (1995) Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 83:493–501

64

T.C. Walser et al.

117. Gately S, Li WW (2004) Multiple roles of COX-2 in tumor angiogenesis: a target for antiangiogenic therapy. Semin Oncol 31:2–11 118. Leahy KM, Koki AT, Masferrer JL (2000) Role of cyclooxygenases in angiogenesis. Curr Med Chem 7:1163–1170 119. Liu VC, Wong LY, Jang T et al (2007) Tumor evasion of the immune system by converting CD4+CD25- T cells into CD4+CD25+ T regulatory cells: role of tumor-derived TGF-beta. J Immunol 178:2883–2892 120. Stolina M, Sharma S, Lin Y et al (2000) Specific inhibition of cyclooxygenase 2 restores antitumor reactivity by altering the balance of IL-10 and IL-12 synthesis. J Immunol 164:361–370 121. Dohadwala M, Batra RK, Luo J et al (2002) Autocrine/paracrine prostaglandin E2 production by non-small cell lung cancer cells regulates matrix metalloproteinase-2 and CD44 in cyclooxygenase-2-dependent invasion. J Biol Chem 277:50828–50833 122. Dohadwala M, Luo J, Zhu L et al (2001) Non-small cell lung cancer cyclooxygenase-2-dependent invasion is mediated by CD44. J Biol Chem 276:20809–20812 123. Lee JM, Yanagawa J, Peebles KA, Sharma S, Mao JT, Dubinett SM (2008) Inflammation in lung carcinogenesis: new targets for lung cancer chemoprevention and treatment. Crit Rev Oncol Hematol 66:208–217 124. Krysan K, Reckamp KL, Dalwadi H et al (2005) Prostaglandin E2 activates mitogen-activated protein kinase/Erk pathway signaling and cell proliferation in non-small cell lung cancer cells in an epidermal growth factor receptor-independent manner. Cancer Res 65:6275–6281 125. Khuri FR, Wu H, Lee JJ et al (2001) Cyclooxygenase-2 overexpression is a marker of poor prognosis in stage I non-small cell lung cancer. Clin Cancer Res 7:861–867 126. Tsubochi H, Sato N, Hiyama M et al (2006) Combined analysis of cyclooxygenase-2 expression with p53 and Ki-67 in nonsmall cell lung cancer. Ann Thorac Surg 82:1198–1204 127. Hosomi Y, Yokose T, Hirose Y et al (2000) Increased cyclooxygenase 2 (COX-2) expression occurs frequently in precursor lesions of human adenocarcinoma of the lung. Lung Cancer 30:73–81 128. Wolff H, Saukkonen K, Anttila S, Karjalainen A, Vainio H, Ristimaki A (1998) Expression of cyclooxygenase-2 in human lung carcinoma. Cancer Res 58:4997–5001 129. Campa D, Zienolddiny S, Maggini V, Skaug V, Haugen A, Canzian F (2004) Association of a common polymorphism in the cyclooxygenase 2 gene with risk of non-small cell lung cancer. Carcinogenesis 25:229–235 130. Schreinemachers DM, Everson RB (1994) Aspirin use and lung, colon, and breast cancer incidence in a prospective study. Epidemiology 5:138–146 131. Mao JT, Fishbein MC, Adams B et al (2006) Celecoxib decreases Ki-67 proliferative index in active smokers. Clin Cancer Res 12:314–320 132. Lee JM, Mao JT, Krysan K, Dubinett SM (2007) Significance of cyclooxygenase-2 in prognosis, targeted therapy and chemoprevention of NSCLC. Future Oncol 3:149–153 133. Mao JT, Cui X, Reckamp K et al (2005) Chemoprevention strategies with cyclooxygenase-2 inhibitors for lung cancer. Clin Lung Cancer 7:30–39 134. Gadgeel SM, Ali S, Philip PA, Ahmed F, Wozniak A, Sarkar FH (2007) Response to dual blockade of epidermal growth factor receptor (EGFR) and cycloxygenase-2 in nonsmall cell lung cancer may be dependent on the EGFR mutational status of the tumor. Cancer 110:2775–2784 135. Gadgeel SM, Ruckdeschel JC, Heath EI, Heilbrun LK, Venkatramanamoorthy R, Wozniak A (2007) Phase II study of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), and celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, in patients with platinum refractory non-small cell lung cancer (NSCLC). J Thorac Oncol 2:299–305 136. Agarwala A, Fisher W, Bruetman D et al (2008) Gefitinib plus celecoxib in chemotherapynaive patients with stage IIIB/IV non-small cell lung cancer: a phase II study from the Hoosier Oncology Group. J Thorac Oncol 3:374–379 137. O’Byrne KJ, Danson S, Dunlop D et al (2007) Combination therapy with gefitinib and rofecoxib in patients with platinum-pretreated relapsed non small-cell lung cancer. J Clin Oncol 25:3266–3273

Tumor Microenvironment

65

138. Fidler MJ, Argiris A, Patel JD et al (2008) The potential predictive value of cyclooxygenase-2 expression and increased risk of gastrointestinal hemorrhage in advanced non-small cell lung cancer patients treated with erlotinib and celecoxib. Clin Cancer Res 14:2088–2094 139. Reckamp KL, Krysan K, Morrow JD et al (2006) A phase I trial to determine the optimal biological dose of celecoxib when combined with erlotinib in advanced non-small cell lung cancer. Clin Cancer Res 12:3381–3388 140. Gridelli C, Gallo C, Ceribelli A et al (2007) Factorial phase III randomised trial of rofecoxib and prolonged constant infusion of gemcitabine in advanced non-small-cell lung cancer: the GEmcitabine-COxib in NSCLC (GECO) study. Lancet Oncol 8:500–512 141. Gridelli C, Maione P, Rossi A, De Marinis F (2007) The role of bevacizumab in the treatment of non-small cell lung cancer: current indications and future developments. Oncologist 12:1183–1193 142. Juni P, Nartey L, Reichenbach S, Sterchi R, Dieppe PA, Egger M (2004) Risk of cardiovascular events and rofecoxib: cumulative meta-analysis. Lancet 364:2021–2029 143. Solomon DH, Schneeweiss S, Levin R, Avorn J (2004) Relationship between COX-2 specific inhibitors and hypertension. Hypertension 44:140–145 144. Chan AT, Ogino S, Fuchs CS (2007) Aspirin and the risk of colorectal cancer in relation to the expression of COX-2. N Engl J Med 356:2131–2142 145. Edelman MJ, Watson D, Wang X et al (2008) Eicosanoid modulation in advanced lung cancer: cyclooxygenase-2 expression is a positive predictive factor for celecoxib + chemotherapy – Cancer and Leukemia Group B Trial 30203. J Clin Oncol 26:848–855 146. Hazra S, Batra RK, Tai HH, Sharma S, Cui X, Dubinett SM (2007) Pioglitazone and rosiglitazone decrease prostaglandin E2 in non-small-cell lung cancer cells by up-regulating 15-hydroxyprostaglandin dehydrogenase. Mol Pharmacol 71:1715–1720 147. Hazra S, Peebles KA, Sharma S, Mao JT, Dubinett SM (2008) The role of PPARgamma in the cyclooxygenase pathway in lung cancer. PPAR Res 2008:790568 148. Ondrey F (2009) Peroxisome proliferator-activated receptor gamma pathway targeting in carcinogenesis: implications for chemoprevention. Clin Cancer Res 15:2–8 149. Nemenoff RA (2007) Peroxisome proliferator-activated receptor-gamma in lung cancer: defining specific versus “off-target” effectors. J Thorac Oncol 2:989–992 150. Lee SY, Hur GY, Jung KH et al (2006) PPAR-gamma agonist increase gefitinib’s antitumor activity through PTEN expression. Lung Cancer 51:297–301 151. Reddy RC, Srirangam A, Reddy K et  al (2008) Chemotherapeutic drugs induce PPARgamma expression and show sequence-specific synergy with PPAR-gamma ligands in inhibition of non-small cell lung cancer. Neoplasia 10:597–603 152. Girnun GD, Chen L, Silvaggi J et al (2008) Regression of drug-resistant lung cancer by the combination of rosiglitazone and carboplatin. Clin Cancer Res 14:6478–6486 153. Kim HJ, Hwang JY, Choi WS et al (2007) Expression of a peroxisome proliferator-activated receptor gamma 1 splice variant that was identified in human lung cancers suppresses cell death induced by cisplatin and oxidative stress. Clin Cancer Res 13:2577–2583 154. Govindarajan R, Ratnasinghe L, Simmons DL et al (2007) Thiazolidinediones and the risk of lung, prostate, and colon cancer in patients with diabetes. J Clin Oncol 25:1476–1481 155. Nemenoff RA, Weiser-Evans M, Winn RA (2008) Activation and molecular targets of peroxisome proliferator-activated receptor-gamma ligands in lung cancer. PPAR Res 2008:156875 156. (2007) Thiazolidinediones and cardiovascular disease. Med Lett Drugs Ther 49:57–58 157. Nissen SE, Wolski K (2007) Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes. N Engl J Med 356:2457–2471 158. Rosen CJ (2007) The rosiglitazone story – lessons from an FDA Advisory Committee meeting. N Engl J Med 357:844–846 159. Sasaki H, Tanahashi M, Yukiue H et al (2002) Decreased perioxisome proliferator-activated receptor gamma gene expression was correlated with poor prognosis in patients with lung cancer. Lung Cancer 36:71–76 160. Finckh A, Aronson MD (2005) Cardiovascular risks of cyclooxygenase-2 inhibitors: where we stand now. Ann Intern Med 142:212–214

66

T.C. Walser et al.

161. Balkwill F, Charles KA, Mantovani A (2005) Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell 7:211–217 162. de Visser KE, Eichten A, Coussens LM (2006) Paradoxical roles of the immune system during cancer development. Nat Rev Cancer 6:24–37 163. DeNardo DG, Johansson M, Coussens LM (2008) Immune cells as mediators of solid tumor metastasis. Cancer Metastasis Rev 27:11–18 164. Walser T, Cui X, Yanagawa J et al (2008) Smoking and lung cancer: the role of inflammation. Proc Am Thorac Soc 5:811–815 165. Coussens LM, Werb Z (2002) Inflammation and cancer. Nature 420:860–867 166. Mantovani A, Allavena P, Sica A, Balkwill F (2008) Cancer-related inflammation. Nature 454:436–444 167. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70 168. Mantovani A (2009) Cancer: inflaming metastasis. Nature 457:36–37 169. O’Donnell R, Breen D, Wilson S, Djukanovic R (2006) Inflammatory cells in the airways in COPD. Thorax 61:448–454 170. Sevenoaks MJ, Stockley RA (2006) Chronic obstructive pulmonary disease, inflammation and co-morbidity – a common inflammatory phenotype? Respir Res 7:70 171. Taraseviciene-Stewart L, Voelkel NF (2008) Molecular pathogenesis of emphysema. J Clin Invest 118:394–402 172. Dohadwala M, Yang SC, Luo J et  al (2006) Cyclooxygenase-2-dependent regulation of E-cadherin: prostaglandin E(2) induces transcriptional repressors ZEB1 and snail in nonsmall cell lung cancer. Cancer Res 66:5338–5345 173. Keshamouni VG, Michailidis G, Grasso CS et al (2006) Differential protein expression profiling by iTRAQ-2DLC-MS/MS of lung cancer cells undergoing epithelial-mesenchymal transition reveals a migratory/invasive phenotype. J Proteome Res 5:1143–1154 174. Leng Q, Bentwich Z, Borkow G (2006) Increased TGF-beta, Cbl-b and CTLA-4 levels and immunosuppression in association with chronic immune activation. Int Immunol 18:637–644 175. Heinrich EL, Charuworn B, Dohadwala M, Dubinett SM. IL-1B dependent epithelialmesenchymal transition in non-small cell lung cancer. Proceedings of the frontiers in cancer prevention research conference, Abstract vol A26, p 76 176. Skillrud DM (1986) COPD: causes, treatment, and risk for lung cancer. Compr Ther 12:13–16 177. Skillrud DM, Offord KP, Miller RD (1986) Higher risk of lung cancer in chronic obstructive pulmonary disease. A prospective, matched, controlled study. Ann Intern Med 105:503–507 178. de Torres JP, Bastarrika G, Wisnivesky JP et al (2007) Assessing the relationship between lung cancer risk and emphysema detected on low-dose CT of the chest. Chest 132:1932–1938 179. Houghton AM, Mouded M, Shapiro SD (2008) Common origins of lung cancer and COPD. Nat Med 14:1023–1024 180. Adair-Kirk TL, Atkinson JJ, Senior RM (2008) Smoke particulates stress lung cells. Nat Med 14:1024–1025 181. Parimon T, Chien JW, Bryson CL, McDonell MB, Udris EM, Au DH (2007) Inhaled corticosteroids and risk of lung cancer among patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 175:712–719 182. Wilson R, McConnell EE, Ross M, Axten CW, Nolan RP (2008) Risk assessment due to environmental exposures to fibrous particulates associated with taconite ore. Regul Toxicol Pharmacol 52:S232–S245 183. Kim CF, Jackson EL, Woolfenden AE et al (2005) Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121:823–835 184. Dostert C, Petrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J (2008) Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science 320:674–677

Tumor Microenvironment

67

185. Kelsen SG, Duan X, Ji R, Perez O, Liu C, Merali S (2008) Cigarette smoke induces an unfolded protein response in the human lung: a proteomic approach. Am J Respir Cell Mol Biol 38:541–550 186. Sin DD, Man SF, Marciniuk DD et al (2008) The effects of fluticasone with or without salmeterol on systemic biomarkers of inflammation in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 177:1207–1214 187. Krysan K, Lee JM, Dohadwala M et  al (2008) Inflammation, epithelial to mesenchymal transition, and epidermal growth factor receptor tyrosine kinase inhibitor resistance. J Thorac Oncol 3:107–110 188. Walser TC, Yanagawa J, Luo J et al (2008) Snail-induced and EMT-mediated early lung cancer development: promotion of invasion and expansion of stem cell populations. Proceedings of the frontiers in cancer prevention research conference, Abstract vol PR-11, p 67 189. Yanagawa J, Walser TC, Zhu LX, et al (2008) The zinc-finger E-box-binding transcriptional repressor Snail promotes tumor progression and angiogenesis in non-small cell lung cancer. Proceedings of the frontiers in cancer prevention research conference, Abstract vol A1, p 69 190. Lee JM, Dedhar S, Kalluri R, Thompson EW (2006) The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol 172:973–981 191. Buck E, Eyzaguirre A, Barr S et al (2007) Loss of homotypic cell adhesion by epithelialmesenchymal transition or mutation limits sensitivity to epidermal growth factor receptor inhibition. Mol Cancer Ther 6:532–541 192. Witta SE, Gemmill RM, Hirsch FR et al (2006) Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines. Cancer Res 66:944–950 193. Yauch RL, Januario T, Eberhard DA et al (2005) Epithelial versus mesenchymal phenotype determines in  vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res 11:8686–8698 194. Reckamp KL, Gardner BK, Figlin RA et al (2008) Tumor response to combination celecoxib and erlotinib therapy in non-small cell lung cancer is associated with a low baseline matrix metalloproteinase-9 and a decline in serum-soluble E-cadherin. J Thorac Oncol 3:117–124 195. Mani SA, Guo W, Liao MJ et  al (2008) The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133:704–715 196. Sanchez-Garcia I (2009) The crossroads of oncogenesis and metastasis. N Engl J Med 360:297–299 197. Wang XW, Tan NS, Ho B, Ding JL (2006) Evidence for the ancient origin of the NF-kappaB/ IkappaB cascade: its archaic role in pathogen infection and immunity. Proc Natl Acad Sci USA 103:4204–4209 198. Xiao W (2004) Advances in NF-kappaB signaling transduction and transcription. Cell Mol Immunol 1:425–435 199. Dey A, Tergaonkar V, Lane DP (2008) Double-edged swords as cancer therapeutics: simultaneously targeting p53 and NF-kappaB pathways. Nat Rev Drug Discov 7:1031–1040 200. Lee DF, Hung MC (2008) Advances in targeting IKK and IKK-related kinases for cancer therapy. Clin Cancer Res 14:5656–5662 201. Mitchell BS (2003) The proteasome – an emerging therapeutic target in cancer. N Engl J Med 348:2597–2598 202. Richardson PG, Barlogie B, Berenson J et  al (2003) A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med 348:2609–2617 203. Fisher RI, Bernstein SH, Kahl BS et al (2006) Multicenter phase II study of bortezomib in patients with relapsed or refractory mantle cell lymphoma. J Clin Oncol 24:4867–4874 204. Davies AM, Ruel C, Lara PN et al (2008) The proteasome inhibitor bortezomib in combination with gemcitabine and carboplatin in advanced non-small cell lung cancer: a California Cancer Consortium Phase I study. J Thorac Oncol 3:68–74 205. Voortman J, Smit EF, Honeywell R et al (2007) A parallel dose-escalation study of weekly and twice-weekly bortezomib in combination with gemcitabine and cisplatin in the first-line treatment of patients with advanced solid tumors. Clin Cancer Res 13:3642–3651

68

T.C. Walser et al.

206. Davies AM, Chansky K, Lara PN Jr et al (2009) Bortezomib plus gemcitabine/carboplatin as first-line treatment of advanced non-small cell lung cancer: a phase II Southwest Oncology Group Study (S0339). J Thorac Oncol 4:87–92 207. Fossella FV, DeVore R, Kerr RN et al (2000) Randomized phase III trial of docetaxel versus vinorelbine or ifosfamide in patients with advanced non-small-cell lung cancer previously treated with platinum-containing chemotherapy regimens. The TAX 320 Non-Small Cell Lung Cancer Study Group. J Clin Oncol 18:2354–2362 208. Shepherd FA, Dancey J, Ramlau R et al (2000) Prospective randomized trial of docetaxel versus best supportive care in patients with non-small-cell lung cancer previously treated with platinum-based chemotherapy. J Clin Oncol 18:2095–2103 209. Hanna N, Shepherd FA, Fossella FV et al (2004) Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy. J Clin Oncol 22:1589–1597 210. Shepherd FA, Rodrigues Pereira J, Ciuleanu T et al (2005) Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 353:123–132 211. Fanucchi MP, Fossella FV, Belt R et  al (2006) Randomized phase II study of bortezomib alone and bortezomib in combination with docetaxel in previously treated advanced nonsmall-cell lung cancer. J Clin Oncol 24:5025–5033 212. Lara PN Jr, Koczywas M, Quinn DI et  al (2006) Bortezomib plus docetaxel in advanced non-small cell lung cancer and other solid tumors: a phase I California Cancer Consortium trial. J Thorac Oncol 1:126–134 213. Davies AM, Ho C, Metzger AS et  al (2007) Phase I study of two different schedules of bortezomib and pemetrexed in advanced solid tumors with emphasis on non-small cell lung cancer. J Thorac Oncol 2:1112–1116 214. Lynch TJ, Fenton DW, Hirsh V, et al Randomized phase II study of erlotinib alone and in combination with bortezomib in previously treated advanced non-small cell lung cancer (NSCLC). J Clin Oncol 2007; ASCO annual meeting proceedings, part I, vol 25, no 18S (June 20 Supplement), p 7680 215. Lara PN Jr, Chansky K, Davies AM et al (2006) Bortezomib (PS-341) in relapsed or refractory extensive stage small cell lung cancer: a Southwest Oncology Group phase II trial (S0327). J Thorac Oncol 1:996–1001 216. Cooper CS, Park M, Blair DG et al (1984) Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature 311:29–33 217. Sattler M, Salgia R (2007) c-Met and hepatocyte growth factor: potential as novel targets in cancer therapy. Curr Oncol Rep 9:102–108 218. Ma PC, Tretiakova MS, Nallasura V, Jagadeeswaran R, Husain AN, Salgia R (2007) Downstream signalling and specific inhibition of c-MET/HGF pathway in small cell lung cancer: implications for tumour invasion. Br J Cancer 97:368–377 219. Nakamura Y, Niki T, Goto A et al (2007) c-Met activation in lung adenocarcinoma tissues: an immunohistochemical analysis. Cancer Sci 98:1006–1013 220. Toschi L, Janne PA (2008) Single-agent and combination therapeutic strategies to inhibit hepatocyte growth factor/MET signaling in cancer. Clin Cancer Res 14:5941–5946 221. Engelman JA, Zejnullahu K, Mitsudomi T et al (2007) MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316:1039–1043 222. Gordon MS, Mendelson DS, Sweeney C et al (2007) Interim results from a first-in-human study with AMG102, a fully human monoclonal antibody that neutralizes hepatocyte growth factor (HGF), the ligand to cMET receptor, in patients (pts) with advanced solid tumors. J Clin Oncol; ASCO annual meeting proceedings, part 1, vol 25, no 18S (June 20 Supplement), p 3551 223. Rosen PJ, Sweeney CJ, Park DJ et al (2008) AMG 102, an HGF/SF antagonist, in combination with anti-angiogenesis targeted therapies in adult patients with advanced solid tumors. J Clin Oncol; ASCO annual meeting proceedings, part I, vol 26 (May 20 Supplement), p 3570 224. Zou HY, Li Q, Lee JH et al (2007) An orally available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy through antiproliferative and antiangiogenic mechanisms. Cancer Res 67:4408–4417

Tumor Microenvironment

69

225. Garcia A, Rosen L, Cunningham CC et  al (2007) Phase 1 study of ARQ 197, a selective inhibitor of the c-Met RTK in patients with metastatic solid tumors reaches recommended phase 2 dose. J Clin Oncol; ASCO annual meeting proceedings, part 1, vol 25, no 18S (June 20 Supplement), p 3525 226. Eder JP, Heath E, Appleman L et al (2007) Phase I experience with c-MET inhibitor XL880 administered orally to patients (pts) with solid tumors. J Clin Oncol; ASCO annual meeting proceedings, part 1, vol 25, no 18S (June 20 Supplement), p 3526 227. Bergers G, Benjamin LE (2003) Tumorigenesis and the angiogenic switch. Nat Rev Cancer 3:401–410 228. Ma J, Waxman DJ (2008) Combination of antiangiogenesis with chemotherapy for more effective cancer treatment. Mol Cancer Ther 7:3670–3684 229. Gettinger S (2008) Targeted therapy in advanced non-small-cell lung cancer. Semin Respir Crit Care Med 29:291–301 230. Ramalingam SS, Dahlberg SE, Langer CJ et al (2008) Outcomes for elderly, advanced-stage non small-cell lung cancer patients treated with bevacizumab in combination with carboplatin and paclitaxel: analysis of Eastern Cooperative Oncology Group Trial 4599. J Clin Oncol 26:60–65 231. Manegold C, von Powel J, Zatloukal P, Ramlau R, Gorbounova V, Hirsh V, Leighl N, Mezger J, Archer V, Reck M (2008) BO17704 (AVAiL): a phase III randomised study of first-line bevacizumab combined with cisplatin/gemcitabine (CG) in patients (pts) with advanced or recurrent non-squamous, non-small cell lung cancer (NSCLC). In: European society for medical oncology; Ann Oncol 19 (Suppl 8):viii1–viii4 232. Shaked Y, Henke E, Roodhart JM et al (2008) Rapid chemotherapy-induced acute endothelial progenitor cell mobilization: implications for antiangiogenic drugs as chemosensitizing agents. Cancer Cell 14:263–273 233. Keane MP, Belperio JA, Xue YY, Burdick MD, Strieter RM (2004) Depletion of CXCR2 inhibits tumor growth and angiogenesis in a murine model of lung cancer. J Immunol 172:2853–2860 234. Wislez M, Fujimoto N, Izzo JG et al (2006) High expression of ligands for chemokine receptor CXCR2 in alveolar epithelial neoplasia induced by oncogenic kras. Cancer Res 66:4198–4207 235. Mizukami Y, Jo WS, Duerr EM et al (2005) Induction of interleukin-8 preserves the angiogenic response in HIF-1alpha-deficient colon cancer cells. Nat Med 11:992–997 236. Keedy VL, Sandler AB (2007) Inhibition of angiogenesis in the treatment of non-small cell lung cancer. Cancer Sci 98:1825–1830 237. Stinchcombe TE, Socinski MA (2007) Bevacizumab in the treatment of non-small-cell lung cancer. Oncogene 26:3691–3698

Racial and Ethnic Diversity in Lung Cancer Carol J. Etzel and Sumesh Kachroo

Abstract  Racial disparity in cancer risk and outcomes is a subject of scientific and political controversy; however, differences in lung cancer incidence and mortality as well as access to and quality of health care among differing racial populations in the United States have been well-documented. Some researchers attribute the disparities in cancer risk and outcome to interactions between genetic and environmental factors, but these theories do not hold for disparities in access to quality care. In order to fully understand these differences and to aid in deciphering the roles that genetic and environmental factors and their interactions play in lung cancer development, lung cancer research studies and trials must include participants from multiracial, multiethnic populations. Keywords  Lung cancer • Race • Ethnicity • Diet • Environmental factors

Introduction Racial disparity in cancer risk and outcomes is a subject of scientific and political controversy. Some researchers have theorized that the cause of these disparities is due to biological differences among races, whereas others have proposed inequities in socioeconomic status and access to health care (1) as the main cause of cancer disparities. In the past few decades, research aimed at understanding racial differences has seen a gradual shift with researchers looking at race as a sociologic construct rather than a biological construct (2). There is growing consensus, however, that the interaction of genetic and environmental factors including diet is at least partially responsible for the ethnic differences in cancer risk and outcome (3).

C.J. Etzel (*) and S. Kachroo Department of Epidemiology, UT MD Anderson Cancer Center, Houston, TX, USA e-mail: [email protected] D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_3, © Humana Press, a part of Springer Science+Business Media, LLC 2010

71

72

C.J. Etzel and S. Kachroo

In this chapter, we discuss differences and similarities in lung cancer incidence, risk factors, mortality, and outcomes among racial groups of the United States (US), namely Whites, African Americans, Asians/Pacific Islanders, and American Indians/Alaska Natives. The ethnic group “Hispanics” may include individuals from any of the four racial groups and is therefore not mutually exclusive from any of these four groups except for “Non-Hispanic Whites.”

Racial Differences in Lung Cancer Incidence and Mortality Incidence Although lung cancer incidence rates have gradually declined over the past few years, lung cancer is still the second most common cancer among Whites, African Americans, Asians/Pacific Islanders, and American Indians/Alaska Natives and the third most common cancer among Hispanics (4). Among men, African Americans (107.6 per 100,000) have the highest incidence rates followed by non-Hispanic Whites (66.6 per 100,000) (Table  1). Hispanic men, regardless of race, have the lowest lung cancer incidence rates (44.2 per 100,000) (5). Among women, nonHispanic Whites have the highest incidence rates (57.3 per 100,000) followed by African Americans (54.6 per 100,000). Compared to other racial and ethnic groups, Middle Eastern women who reside in the US have relatively low lung cancer incidence rates (24.1 per 100,000) (6). As a whole, American Indian/Alaskan Natives have much lower incidence rates compared to non-Hispanic Whites and African Americans, but some subgroups had higher rates compared to Whites. Kelly et al. (7) compared incidence rates for the three major ethnic groups that comprise Alaskan Natives (Eskimo, Indian, and Aleut people) to an American Indian population living in New Mexico and US Whites. They reported that lung cancer incidence rates in Alaska Indian men and women were 1.5–1.7 times greater than White men and women. New Mexico Indian men also had higher incidence rates compared to White men although New Mexico Indian women had lower rates compared to their White counterparts. The US racial group of Asian/Pacific Islanders is a broad category that can further be classified into many ethnically diverse sub groups: Japanese, Chinese, Vietnamese, Korean, Filipino, Asian Indians/Pakistanis, Native Hawaiians, Japanese, Kampucheans, Laotians, Samoans, and Tongans. Each of these subgroups is culturally diverse from one another and incidence rates vary greatly amongst these subgroups. According to SEER, incidence rates are 53.9 per 100,000 for Asian/Pacific Islander men and 28.0 per 100,000 for women (Table 1). Recent studies (8, 9) show that SEER incidence rates can be higher than 100 per 100,000 for Native Hawaiian, Samoan, and Tongan men and as low as 41 per 100,000 for Japanese men. Rates among Asian/Pacific Islander women can range as high as 69.7 for Native Hawaiian women to as low as 13.1 for Asian Indian/Pakistani women.

Racial and Ethnic Diversity in Lung Cancer

73

Table 1  Incidence and mortality rates for the four major US racial groups, Hispanics and Middle Easterners Incidence Mortality Overall Males Females Overall Males Females Racial group Non-Hispanic Whites 66.6   79.5 57.3 56.9 74.2 44.3 African Americans 76.0 107.6 54.6 60.9 93.1 39.9 Asian/Pacific Islanders 39.1   53.9 28.0 26.6 37.5 18.5 American Indian/Alaskan Natives 46.0   54.3 39.7 40.9 50.2 33.8 Ethnic group Hispanics 33.3   44.2 25.4 23.2 35.1 14.6 Middle Eastern   54.3 24.1 – – – Incidence source: All incidence rates are obtained from SEER 17 areas (San Francisco, Connecticut, Detroit, Hawaii, Iowa, New Mexico, Seattle, Utah, Atlanta, San Jose-Monterey, Los Angeles, Alaska Native Registry, Rural Georgia, California excluding SF/SJM/LA, Kentucky, Louisiana and New Jersey) (5), except for “Middle Eastern” which are compiled from Nasseri et al. (6). Seer rates are per 100,000 and are age-adjusted to the 2000 US Std Population (19 age groups – Census P25-1130). The modeled rates are the point estimates for the regression lines calculated by the Joinpoint Regression Program (Version 3.3, April 2008, National Cancer Institute). Rates for American Indian/Alaska Native are based on the CHSDA (Contract Health Service Delivery Area) counties. Hispanics and Non-Hispanics are not mutually exclusive from whites, blacks, Asian/Pacific Islanders, and American Indians/Alaska Natives. Incidence data for Hispanics and Non-Hispanics are based on NHIA and exclude cases from the Alaska Native Registry and Kentucky Mortality source: US Mortality Files, National Center for Health Statistics, CDC (4). Rates are per 100,000 and are age-adjusted to the 2000 US Std Population (19 age groups – Census P251130). The modeled rates are the point estimates for the regression lines calculated by the Joinpoint Regression Program (Version 3.3, April 2008, National Cancer Institute). Rates for American Indian/Alaska Native are based on the CHSDA (Contract Health Service Delivery Area) counties. Hispanics and Non-Hispanics are not mutually exclusive from whites, blacks, Asian/ Pacific Islanders, and American Indians/Alaska Natives. Mortality data for Hispanics and NonHispanics do not include cases from Minnesota, New Hampshire, and North Dakota

As noted by Miller et al. (9) with the exception of Japanese Americans and Native Hawaiians, the majority of the other Asian/Pacific Islander ethnic groups were born outside of the U.S.

Mortality Recent trends indicate that the lung cancer mortality rates have also shown a gradual decline over the past few years (4). African Americans consistently have the highest mortality rates (Table  1) (4). Hispanic women have the lowest lung cancer-related mortality rates. As a whole, American Indian/Alaskan Natives have lower lung cancer-specific mortality rates compared to Whites; however, Lanier et al. (10) reported that Alaska Natives, alone, have a 1.2 increased risk of mortality compared to Whites.

74

C.J. Etzel and S. Kachroo

Within the culturally diverse Asian/Pacific Islander group, Native Hawaiian men had the highest mortality rate (87.7 per 100,000) while Asian Indian women had the lowest (6.4 per 100,000) (9).

Racial and Ethnic Differences in Risk Factors for Lung Cancer Despite the differences in lung cancer incidence and mortality among racial and ethnic groups, there exist only a few studies that investigated as to why these differences exist. One of the major factors that might have contributed toward lack of such studies could be low awareness and lower screening rates among minorities resulting in underreporting and therefore lack of availability of good, analyzable data for different racial groups. Ethical dilemmas, especially in studies investigating genetic differences, and high costs associated with developing population-based and minorityspecific lung cancer registries could also have acted as obstacles. These studies have highlighted the fact that there are some risk factors that are specific to different ethnic groups and that may be directly related to their risks and outcomes. In addition, although these groups share some of the risks factors (including epidemiologic, occupational, environmental, dietary, familial, socioeconomic, and/or genetic factors), the level of risk is different, and thus differences in outcomes. Although most of these studies have essentially focused on understanding the differences between Whites and African Americans, there are some that have also looked at understanding these differences among other racial/ethnic groups.

Smoking and Tobacco Consumption Tobacco consumption is a well-established risk factor of lung cancer. Pinsky (11) used tobacco consumption data from the US Census to determine if smoking patterns predicted lung cancer rates among the US racial/ethnic groups. Pinsky found that non-Hispanic Whites were more likely to have smoked (56%) compared to Asian/Pacific Islanders (34%) and Hispanics (38%). Likewise, Non-Hispanic Whites were more likely to smoke more cigarettes on average (19 per day) for longer durations of time (30 pack-years) compared to Asian/Pacific Islanders (15 per day for 22 pack-years) and Hispanics (13 per day for 19 pack-years). These results are aligned with the increased incidence of lung cancer among Non-Hispanic Whites compared to Asian/Pacific Islanders and Hispanics. In contrast, Pinsky observed that although American Indians have lower incidence rates compared to Non-Hispanic Whites, a higher proportion (58%) of American Indians smoke although at lower intensity (17 cigarettes per day) and at lower duration (28 pack-years). African Americans, who have the highest lung cancer incidence rates of all these groups, smoke less cigarettes on average (12 per day) and for shorter duration of

Racial and Ethnic Diversity in Lung Cancer

75

time (20 pack-years). However, African Americans also have been shown to have a lower cigarette smoking quit rate (32%) compared to Whites (47%), possibly indicating longer smoking duration in African Americans which could account for their increased risks for lung cancer (12). Schwartz and Swanson (13) compared lung carcinoma incidence between African Americans and Whites in the Detroit Metropolitan Cancer Surveillance System. They concluded that after accounting for smoking habit, race is not an independent predictor of lung cancer risk. However, they did find that there exist age-related racial differences in lung cancer risk such that African American males with low or no smoking exposure had three to eight times the risk of lung cancer compared to white males with comparable smoking exposures. Stellman et al. (14) reported that lung cancer risks were comparable for Whites and African Americans with similar smoking habits, except for those African Americans who were very heavy smokers (smoked more than 21 cigarettes per day or 37.5 pack-years). They acknowledge that this heavy smoking group is uncommon for the general population of African American smokers. In contrast, Haiman et al. (15) reported that the risk of lung cancer among US racial and ethnic groups is modified by level of smoking exposure using data from the MultiEthnic Cohort Study. They found that among cigarette smokers who consumed less than 30 cigarettes per day, African American and Native Hawaiians were at a higher risk for developing lung cancer as compared to the other groups. No significant differences were observed for smokers of more than 30 cigarettes per day exposure. The authors suggest that the lack of association at these higher smoking levels could be due to the fact that “metabolic or other relevant pathways (have) become saturated.” Many studies have highlighted the fact the African Americans show higher preference for mentholated cigarettes; however, the association between mentholated cigarette consumption and the risk of lung cancer is controversial with studies reporting conflicting results (14, 16–19). Most recently, Etzel et al. (20) reported no increased risks of lung cancer among former or current smokers who had reported a history of smoking mentholated cigarettes (OR range = 0.69–0.99) but found evidence of a possible protective effect among current smokers who had reported smoking mentholated cigarettes. Among American Indians and Native Alaskans, smoking rates and tobacco consumption vary with geographical region, with smoking rates being highest among Indians residing in Alaska (45.1%) followed by Northern Plains (44.2%) and Southwest (17.0%) (21). Although American Indians and Alaska Natives smoke less cigarettes per day with lower intensity as compared to Whites; higher proportion of American Indian and Alaska Native men smoked cigars (5.3%) as compared to White men (4.8%) and African American men (3.9%) (21). In addition, the report also highlights highest prevalence rates for smoking of pipes, chewing of tobacco, and using snuff in American Indians and Alaska Natives as compared to other ethnic groups. For pipe smoking, the reported prevalence rates are 6.9% for American Indians and Alaska Natives, 2.9% for Whites, 2.4% for African Americans, and 2.3% for Asian Americans/Pacific Islanders (21). The reported prevalence rates for

76

C.J. Etzel and S. Kachroo

chewing tobacco/using snuff are 4.5% for American Indians and Alaska Natives, 3.4% for Whites, 3.0% for African Americans, 0.8% for Hispanics, and 0.6% for Asian Americans/Pacific Islanders (21). Among men, the reported prevalence rates for cigar use are 5.3% for American Indian and Alaska Natives, 4.8% for Whites, and 3.9% for African Americans (21). Although this report is 10 years old, it highlights the variation in tobacco consumption among the US racial/ethnic groups.

Family History of Lung Cancer Very few studies exist that evaluate the role of family history of lung cancer among racial or ethnically diverse populations. Cote et al. (22) and Naff et al. (23) reported that first-degree relatives of African Americans patients with early-onset lung cancer have a greater risk of lung cancer than Whites. Cote et al. (22) further concluded that cigarette smoking further increased these risks. Etzel et al. (20) observed an elevated but not statistically significant association between family history and lung cancer among African Americans; however, they observed increased risks of lung cancer for White never smokers and former smokers with a family history of any cancer and among White current smokers with a family history of smoking-related cancers (24).

Race, Diet, and Lung cancer Differences in lung cancer incidence among the racial and ethnic groups could be attributed to variation in diet. Pillow et al. (25) reported that Mexican Americans tend to consume lower levels of total fat and higher levels of fiber and vegetables compared to African Americans. Other studies have also highlighted that the diet of African Americans contains higher fat content and lower consumption of fruits and vegetables, risk factors that have been linked to increased risk for lung cancer (12, 26). African American diet includes higher intake of preserved and processed meats which have higher carcinogenic potential, while the diet of Whites includes higher consumption of raw vegetables (12, 26). Dietary studies suggested that some aspects of the Asian Indian diet, such as vegetarianism and use of spices and various food additives, especially turmeric (curcumin) and garlic, may play a protective role against cancer (27–30).

Occupational and Environmental Exposures Studies looking at the effect of different occupations on lung cancer have also reported ethnic differences. Sterling and Weinkam (31) analyzed data from the National Health Interview Survey and observed a higher proportion of African Americans, as compared to Whites, in occupations that involved exposure to potential carcinogens (31).

Racial and Ethnic Diversity in Lung Cancer

77

Muscat et al. (32) also suggested that occupational exposure to lung carcinogens may be more common in African Americans as compared to Whites and observed an increased risk of lung cancer among African American men who were exposed to asbestos and coal dust. Swanson et al. (33) observed risks for lung cancer were higher among ethnically and racial diverse groups although they held the same occupations as Whites. In a large cohort study of African American and White metal industry workers, Birdsey et  al. (34) reported higher risks of lung cancer mortality among African American oven workers (mortality odds ratio (MOR) 1.38, 95% CI = 1.10– 1.73)), but no increased risk among white-collar workers.

Genetic Variations and Variations in Role of Metabolic Pathways There exist few studies that explore the genetic differences among different racial groups and how these genetic differences result in differences in lung cancer risk. These genes are important in the metabolism of tobacco smoke and environmental carcinogens. For example, Fukami et al. (35) found a unique variant of CYP2A6, a gene that is involved in nicotine metabolism, among African Americans. This novel variant resulted in increased plasma cotinine/nicotine levels in African Americans. Gadgeel and Kalemkerian (12) reviewed other metabolic polymorphisms that have been implicated in lung cancer risk among minority populations and concluded that many of these reports provide conflicting results and have a lack of in-depth information on the biological functions of the polymorphisms and thus no valid conclusions can be made. However, consistent reports have been published on nicotine intake and metabolism. Perez-Stable et al. (36) reported that higher levels of cotinine/cigarette smoked were observed in African Americans as compared to Whites and suggested that the possible reasons could be “slower clearance of cotinine” and also “higher intake of nicotine/cigarette” in African Americans. Benowitz et  al. (37) observed slower metabolism of cotinine in African Americans is due to a “slower oxidative metabolism of nicotine to cotinine (presumably via cytochrome P-450 2A6)” and also “slower N-glucuronidation.” Caraballo et al. (38) confirmed that African American smokers have higher serum cotinine levels compared to White and Mexican American smokers. Furthermore, Benowitz et al. (39) reported that Americans of Chinese descent display slower clearance of total and nonrenal nicotine compared to Latinos and Whites.

Histology and Stage at Presentation Overall, adenocarcinoma is the most common lung cancer histology. Etzel et  al. (20) observed that adenocarcinoma was the most common histology diagnosed in African American women, but African American men presented with squamous

78

C.J. Etzel and S. Kachroo

cell carcinoma as frequently as adenocarcinoma, whereas the majority of White cases presented with adenocarcinoma followed by squamous cell carcinoma (24) Gadgeel et al. (40) reported that, compared to male Whites, African American men have shown a relatively greater increase in the incidence for adenocarcinoma and a relatively greater decline in the incidence of squamous cell carcinoma (Fig.  1). Haiman et  al. (15) reported that a higher percentage of African Americans and Native Americans present with squamous cell carcinoma compared to other groups. Haiman et al. (15) also showed Latinos had a higher percentage of large-cell carcinoma cases while the rate of small-cell carcinoma among Native Americans was double the rates of other ethnic groups. Previous studies have reported racial differences in the stage of disease at initial presentation, reporting higher rates for African Americans presenting with advanced stage disease as compared to Whites (40). However, from 2000 to 2005, approximately 57% of all Asian/Pacific Islander lung cancer cases presented with advanced disease followed by 55% of all Hispanic cases, 53% of African American cases and 49% of non-Hispanic White cases (5). In an earlier report, Gadgeel et al. (40) reported that from 1973 to 1988, presentation with advanced-stage disease increased from 44 to 53% in African Americans, while among Whites it has increased from 42 to 48%. A study by Halpern et al. (41), looking at the association of insurance status and ethnicity with the stage of cancer presentation have reported that, irrespective of their insurance status, African American and Hispanic patients were at a higher risk of presenting with lung cancer at an advanced stage as compared to their White counterparts. Goggins and Wong (42) reported that Pacific Islanders as

Fig. 1  Age-adjusted incidence rates of lung cancer by histologic subtype and race in metropolitan Detroit (1973–1998) (40). Source: Gadgeel SM, Severson RK, Kau Y, Graff J, Weiss LK, Kalemkerian GP (2001) Impact of race in lung cancer – analysis of temporal trends from a surveillance, epidemiology, and end results database. Chest 120:55–63 (with permission)

Racial and Ethnic Diversity in Lung Cancer

79

a whole were more likely to be diagnosed with advanced stage (Stage III or IV) lung cancer compared to other racial/ethnic groups although Native Hawaiians tended to have more positive stage distribution compared to African and Native Americans. Out of the Pacific Islanders subgroups, Samoans had the least favorable stage distribution with more than 80% presenting with Stage III or IV lung cancer. Finlay et  al. (43) have reported that, compared to non-Asians, Asian immigrants present with a more advanced disease stage.

Racial/Ethnic Differences in Lung Cancer Treatment Racial differences have also been reported in treatment and adherence to the treatment regimen. Bach et al. (44) compared treatment among White and African American early-stage, non-small-cell lung cancer cases who were eligible for surgical resection. They observed that the rate of surgery among the African American patients was 13% less than that of White patients. Lower rates of surgery among African Americans have been confirmed in other studies as well (45–47). Wisnivesky et al. (48) and Neighbors et al. (49) reported similar findings for Hispanics. Bach et al. (44) further observed that African Americans and Whites who received surgery had comparable survival rates, but those African Americans who did not receive surgery had the lowest survival rate. Greenwald et al. (50) concluded that lack of surgical treatment accounted for differences in 5-year survival in African Americans and that survival was also associated with income level. Potosky et al. (51) showed that Whites were more likely to receive recommended therapy compared to African Americans. McCann et  al. (52), Lathan et  al. (45), and Bryant et  al. (53) also reported that, even with access to care, African Americans were more likely to decline surgery or neo-adjuvant therapy compared to Whites. Wisnivesky et al. (48) reported that fewer Hispanic patients agreed to recommended therapy including chemotherapy. Earle et  al. (54) have reported that African Americans were even less likely to see a cancer specialist for consultation as compared to Whites. A study by Lackan et al. has also reported racial differences in hospice use, with non-Hispanic Whites having significantly higher utilization as compared to other ethnicities (55). Ngo-Metzger et al. have also reported that for all subgroups, Asian American and Pacific Islander patients were less likely to enroll for hospice care as compared to Whites (56).

Survival There have been numerous reports on racial differences in lung cancer survival with most of them highlighting the fact these variations may be due to differences in the prevalence of well-known prognostic factors, such as stage at presentation or socioeconomic status for lung cancer among these ethnic groups. Gadgeel et  al. (40)

80

C.J. Etzel and S. Kachroo

Fig. 2  Relative 2- and 5-year survival rates of lung cancer patients at all stages by race in metropolitan Detroit (1973–1997) (40). Source: Gadgeel SM, Severson RK, Kau Y, Graff J, Weiss LK, Kalemkerian GP (2001) Impact of race in lung cancer – analysis of temporal trends from a surveillance, epidemiology, and end results database. Chest 120:55–63 (with permission)

demonstrated that Whites had better (p < 0.0001) 2- and 5-year survival rates compared to African Americans (Fig. 2) although as stated previously, Bach et al. (44) showed that African American and White lung cancer patients who received surgery had comparable survival rates. Wang et  al. (57) compared conditional survival (for all patients that survived at least 1 year after initial diagnosis) among the five racial/ethnic groups and reported that overall African Americans have the lowest conditional survival of the groups (Fig. 3). When stratified by stage at presentation, Wang et al. further showed that Americans/Alaskan Natives with stage I disease had lower conditional survival rates in the first few years but this difference was nominal by year five compared with the other groups, while Hispanics with stage IV disease demonstrated an increase in conditional survival rate over time. Goggins and Wong (42) focused specifically on survival among the Pacific Islanders and reported Native Hawaiians and other Pacific Islanders also had significantly worse cause-specific (death resulting from only lung cancer) survival than did non-Hispanic Whites, but generally had better survival than African Americans or Native Americans. In addition, they found that among the Pacific Islanders subgroups, Samoans had the worst cause-specific survival (42). Differences in socioeconomic status (SES, such as education level, income, and lack of insurance coverage) have been implicated as the “sole cause” of racial disparities in lung cancer (58). Lower SES has been shown to be associated with higher rates of smoking and nicotine-dependence, a greater use of “non-filter, hightar cigarettes,” and a lower rate of cessation (12). Greenwald et al. (50) demonstrated that lower income among African Americans compared to Whites led to a decreased

Racial and Ethnic Diversity in Lung Cancer

81

Fig. 3  Five-year conditional survival for all patients with non-small cell lung cancer, grouped by ethnicity (57). Source: Wang SJ, Fuller CD, Thomas CR Jr (2007) Ethnic disparities in conditional survival of patients with non-small cell lung cancer. J Thorac Oncol 2(3):180–190 (with permission)

likelihood of receiving medical care and hence poorer survival outcomes. However, Kreiger et al. (59) showed that SES affluence does not necessarily result in lower risk for lung cancer. They observed that lung cancer incidence rates were indirectly proportional to SES status, except among Hispanics, where incidence rates increased with SES status. Health care beliefs and perceptions toward cancer also differ among different racial groups (60, 61). This, together with socioeconomic and sociocultural factors, may also play an important role in choosing treatment options. Margolis et al. (62) reported that, compared to Whites, a higher percentage of African American patients believed that tumor spread was caused by air exposure at the time of surgery, and thus refused this treatment. Similar concerns can also lead to reduced participation in screening (63) and clinical (64) trials in which African Americans and Hispanics are less likely to participate. Racial differences have also been reported for patient’s level of trust in his/her physician. Gordon et  al. (65) have reported that African American patients have lower post-visit trust in their physicians as compared to Whites. In addition, African American patients reported that their communication with their physicians was

82

C.J. Etzel and S. Kachroo

“less informative, less supportive and less partnering.” Bach et al. (66) highlighted that compared to physicians who treat White patients, physicians who treat African American patients may be clinically less well-trained and, in addition, lacking access to the necessary clinical resources.

Immigration and Acculturation Cancer incidence rates in people who have moved to the US from other countries with low incidence rates tend to become similar over time to the rates seen in US (6, 67). Nasseri et al. (6) compared lung cancer incidence rates among Middle East immigrants who settled in California to those rates in the Middle East and have reported higher rates in the immigrant population. The incidence rates among immigrant men and women were 1.54–4.09 times higher than for current Middle East resident counterparts. McCracken et al. (8) observed that the lung cancer incidence rate among Chinese-American women in California were 1.5 times the rate for women in China. Similar findings have been reported for South Korean and Vietnamese immigrants and the native Koreans from South Korea (68–71) reported higher incidence rates for South Asians living in US when compared to their native countries and that variation in incidence rates among the South Asian immigrants highlighted difference patterns of the host countries. Acculturation and adoption of the US lifestyle “Westernization” have been implicated in the increase in rates among recent immigrants (6, 8, 72). However, other reasons such as better data quality in US and the concern that those who migrate are not a true representative sample of the actual population have also been suggested (6).

Conclusion The literature shows that racial and ethnic differences in lung cancer do exist. Previous studies focused mostly on improving access to care as an approach to eliminate existing racial disparities; however, access to care may be only a mediating factor and not the main cause. There is a need to identify risk factors of lung cancer in multiracial, multiethnic groups and to not just assume that risk factors that have been documented among White populations convey the same level of exposure and hence risk to other diverse populations. A case in point is tobacco exposure. We previously discussed how African Americans have the lowest exposure to tobacco smoke, but still have the highest level of lung cancer risk. These types of observations show that other factors such as genetic polymorphisms and gene–environment interactions need to be the focus of research studies to better understand these differences. Inclusion of minority populations in lung cancer screening and clinical trials could also prove to be helpful in diminishing disparities.

Racial and Ethnic Diversity in Lung Cancer

83

References 1. Brawley OW (2006) Lung cancer and race: equal treatment yields equal outcome among equal patients, but there is no equal treatment. J Clin Oncol 24:332–333 2. Kiefe CI (2002) Race/ethnicity and cancer survival: the elusive target of biological differences. JAMA 287(16):2138–2139 3. Olden K, White SL (2005) Health-related disparities: influence of environmental factors. Med Clin North Am 89:721–738 4. CDC. Comparing lung cancer by race and ethnicity (Accessed December 12, 2008 at http:// www.cdc.gov/cancer/lung/statistics/race.htm) 5. SEER. SEER stat fact sheet (Accessed December 12, 2008 at http://seer.cancer.gov/statfacts/ html/lungb.html) 6. Nasseri K, Mills PK, Allan M (2007) Cancer incidence in the Middle Eastern population of California, 1988–2004. Asian Pac J Cancer Prev 8(3):405–411 7. Kelly JJ, Lanier AP, Alberts S, Wiggins CL (2006) Differences in cancer incidence among Indians in Alaska and New Mexico and U.S. Whites, 1993-2002. Cancer Epidemiol Biomarkers Prev 15(8):1515–1519 8. McCracken M, Olsen M, Chen MS Jr et al (2007) Cancer incidence, mortality, and associated risk factors among Asian Americans of Chinese, Filipino, Vietnamese, Korean, and Japanese ethnicities. CA Cancer J Clin 57(4):190–205 9. Miller BA, Chu KC, Hankey BF, Ries LA (2008) Cancer incidence and mortality patterns among specific Asian and Pacific Islander populations in the U.S. Cancer Causes Control 19(3):227–256 10. Lanier AP, Day GE, Kelly JJ, Provost E (2008) Disparities in cancer mortality among Alaska Native people, 1994–2003. Alaska Med 49(4):120–125 11. Pinsky PF (2006) Racial and ethnic differences in lung cancer incidence: how much is explained by differences in smoking patterns? (United States). Cancer Causes Control 17(8):1017–1024 12. Gadgeel SM, Kalemkerian GP (2003) Racial differences in lung cancer. Cancer Metastasis Rev 22(1):39–46 13. Schwartz AG, Swanson GM (1997) Lung carcinoma in African Americans and whites. A population-based study in metropolitan Detroit, Michigan. Cancer 79(1):45–52 14. Stellman SD, Chen Y, Muscat JE et al (2003) Lung cancer risk in white and black Americans. Ann Epidemiol 13(4):294–302 15. Haiman CA, Stram DO, Wilkens LR et al (2006) Ethnic and racial differences in the smokingrelated risk of lung cancer. N Engl J Med 354(4):333–342 16. Carpenter CL, Jarvik ME, Morgenstern H, McCarthy WJ, London SJ (1999) Mentholated cigarette smoking and lung-cancer risk. Ann Epidemiol 9:114–120 17. Brooks DR, Palmer JR, Strom BL, Rosenberg L (2003) Menthol cigarettes and risk of lung cancer. Am J Epidemiol 158:609–616 18. Kabat GC, Hebert JR (1991) Use of mentholated cigarettes and lung cancer risk. Cancer Res 51:6510–6513 19. Sidney S, Tekawa IS, Friedman GD, Sadler MC, Tashkin DP (1995) Mentholated cigarette use and lung cancer. Arch Intern Med 155:727–732 20. Etzel CJ, Kachroo S, Liu M et al (2008) Development and validation of a lung cancer risk prediction model for African Americans. Cancer Prev Res (Phila Pa) 1(4):255–265 21. 1998 Surgeon General’s Report – Tobacco use among U.S. Racial/Ethnic Minority Groups: American Indians and Alaska Natives and Tobacco (CDC) (Accessed November 29, 2008 at http://www.cdc.gov/tobacco/data_statistics/sgr/sgr_1998/sgr-min-nat.htm) 22. Coté ML, Kardia SL, Wenzlaff AS, Ruckdeschel JC, Schwartz AG (2005) Risk of lung cancer among white and black relatives of individuals with early-onset lung cancer. JAMA 293(24): 3036–3042 23. Naff JL, Coté ML, Wenzlaff AS, Schwartz AG (2007) Racial differences in cancer risk among relatives of patients with early onset lung cancer. Chest 131(5):1289–1294 24. Spitz MR, Hong WK, Amos CI et al (2007) A risk model for prediction of lung cancer. J Natl Cancer Inst 99(9):715–726

84

C.J. Etzel and S. Kachroo

25. Pillow PC, Hursting SD, Duphorne CM et al (1997) Case-control assessment of diet and lung cancer risk in African Americans and Mexican Americans. Nutr Cancer 29(2):169–173 26. Swanson CA, Gridley G, Greenberg RS et  al (1993) A comparison of diets of blacks and whites in three areas of the United States. Nutr Cancer 20(2):153–165 27. Sinha R, Anderson DE, McDonald SS, Greenwald P (2003) Cancer risk and diet in India. J Postgrad Med 49(3):222–228 28. Sengupta A, Ghosh S, Bhattacharjee S, Das S (2004) Indian food ingredients and cancer prevention – an experimental evaluation of anticarcinogenic effects of garlic in rat colon. Asian Pac J Cancer Prev 5(2):126–132 29. Aggarwal BB, Kunnumakkara AB, Harikumar KB, Tharakan ST, Sung B, Anand P (2008) Potential of spice-derived phytochemicals for cancer prevention. Planta Med 74(13):1560–1569 30. Anand P, Sundaram C, Jhurani S, Kunnumakkara AB, Aggarwal BB (2008) Curcumin and cancer: an “old age” disease with an “age-old” solution. Cancer Lett 267(1):133–164 31. Sterling TD, Weinkam JJ (1989) Comparison of smoking-related risk factors among black and white males. Am J Ind Med 15(3):319–333 32. Muscat JE, Stellman SD, Richie JP Jr, Wynder EL (1998) Lung cancer risk and workplace exposures in black men and women. Environ Res 76(2):78–84 33. Swanson GM, Lin CS, Burns PB (1993) Diversity in the association between occupation and lung cancer among black and white men. Cancer Epidemiol Biomarkers Prev 2(4):313–320 34. Birdsey J, Alterman T, Petersen MR (2007) Race, occupation, and lung cancer: detecting disparities with death certificate data. J Occup Environ Med 49(11):1257–1263 35. Fukami T, Nakajima M, Yamanaka H, Fukushima Y, McLeod HL, Yokoi T (2007) A novel duplication type of CYP2A6 gene in African American population. Drug Metab Dispos 35(4):515–520 36. Pérez-Stable EJ, Herrera B, Jacob P 3rd, Benowitz NL (1998) Nicotine metabolism and intake in black and white smokers. JAMA 280(2):152–156 37. Benowitz NL, Perez-Stable EJ, Fong I, Modin G, Herrera B, Jacob P 3rd (1999) Ethnic differences in N-glucuronidation of nicotine and cotinine. J Pharmacol Exp Ther 291(3):1196–1203 38. Caraballo RS, Giovino GA, Pechacek TF et al (1998) Racial and ethnic differences in serum cotinine levels of cigarette smokers: Third National Health and Nutrition Examination Survey, 1988–1991. JAMA 280(2):135–139 39. Benowitz NL, Pérez-Stable EJ, Herrera B, Jacob P 3rd (2002) Slower metabolism and reduced intake of nicotine from cigarette smoking in Chinese-Americans. J Natl Cancer Inst 94(2):108–115 40. Gadgeel SM, Severson RK, Kau Y, Graff J, Weiss LK, Kalemkerian GP (2001) Impact of race in lung cancer: analysis of temporal trends from a surveillance, epidemiology, and end results database. Chest 120(1):55–63 41. Halpern MT, Ward EM, Pavluck AL, Schrag NM, Bian J, Chen AY (2008) Association of insurance status and ethnicity with cancer stage at diagnosis for 12 cancer sites: a retrospective analysis. Lancet Oncol 9(3):222–231 42. Goggins WB, Wong GK (2007) Poor survival for US Pacific Islander cancer patients: evidence from the Surveillance, Epidemiology, and End Results database: 1991 to 2004. J Clin Oncol 25(36):5738–5741 43. Finlay GA, Joseph B, Rodrigues CR, Griffith J, White AC (2002) Advanced presentation of lung cancer in Asian immigrants: a case-control study. Chest 122(6):1938–1943 44. Bach PB, Cramer LD, Warren JL, Begg CB (1999) Racial differences in the treatment of early-stage lung cancer. N Engl J Med 341(16):1198–1205 45. Lathan CS, Neville BA, Earle CC (2006) The effect of race on invasive staging and surgery in non-small-cell lung cancer. J Clin Oncol 24(3):413–418 46. Gross CP, Smith BD, Wolf E, Andersen M (2008) Racial disparities in cancer therapy: did the gap narrow between 1992 and 2002? Cancer 112(4):900–908 47. Esnaola NF, Gebregziabher M, Knott K et al (2008) Underuse of surgical resection for localized, non-small cell lung cancer among whites and African Americans in South Carolina. Ann Thorac Surg 86(1):220–226 48. Wisnivesky JP, McGinn T, Henschke C, Hebert P, Iannuzzi MC, Halm EA (2005) Ethnic disparities in the treatment of stage I non-small cell lung cancer. Am J Respir Crit Care Med 171(10):1158–1163

Racial and Ethnic Diversity in Lung Cancer

85

49. Neighbors CJ, Rogers ML, Shenassa ED, Sciamanna CN, Clark MA, Novak SP (2007) Ethnic/racial disparities in hospital procedure volume for lung resection for lung cancer. Med Care 45(7):655–663 50. Greenwald HP, Polissar NL, Borgatta EF, McCorkle R, Goodman G (1998) Social factors, treatment, and survival in early-stage non-small cell lung cancer. Am J Public Health 88(11):1681–1684 51. Potosky AL, Saxman S, Wallace RB, Lynch CF (2004) Population variations in the initial treatment of non-small-cell lung cancer. J Clin Oncol 22(16):3261–3268 52. McCann J, Artinian V, Duhaime L, Lewis JW Jr, Kvale PA, DiGiovine B (2005) Evaluation of the causes for racial disparity in surgical treatment of early stage lung cancer. Chest 128(5): 3440–3446 53. Bryant AS, Cerfolio RJ (2008) Impact of race on outcomes of patients with non-small cell lung cancer. J Thorac Oncol 3(7):711–715 54. Earle CC, Neumann PJ, Gelber RD, Weinstein MC, Weeks JC (2002) Impact of referral patterns on the use of chemotherapy for lung cancer. J Clin Oncol 20(7):1786–1792 55. Lackan NA, Ostir GV, Freeman JL, Mahnken JD, Goodwin JS (2004) Decreasing variation in the use of hospice among older adults with breast, colorectal, lung, and prostate cancer. Med Care 42(2):116–122 56. Ngo-Metzger Q, Phillips RS, McCarthy EP (2008) Ethnic disparities in hospice use among AsianAmerican and Pacific Islander patients dying with cancer. J Am Geriatr Soc 56(1):139–144 57. Wang SJ, Fuller CD, Thomas CR Jr (2007) Ethnic disparities in conditional survival of patients with non-small cell lung cancer. J Thorac Oncol 2(3):180–190 58. Flenaugh EL, Henriques-Forsythe MN (2006) Lung cancer disparities in African Americans: health versus health care. Clin Chest Med 27(3):431–439 59. Krieger N, Quesenberry C Jr, Peng T et al (1999) Social class, race/ethnicity, and incidence of breast, cervix, colon, lung, and prostate cancer among Asian, Black, Hispanic, and White residents of the San Francisco Bay Area, 1988–92 (United States). Cancer Causes Control 10(6):525–537 60. EVAXX Inc (1981) Black Americans’ attitudes toward cancer and cancer tests: highlights of a study. CA Cancer J Clin 31(4):212–218 61. Clarke-Tasker V (2003) Socioeconomic status and African Americans’ perceptions of cancer. J Natl Black Nurses Assoc 14(1):13–19 62. Margolis ML, Christie JD, Silvestri GA, Kaiser L, Santiago S, Hansen-Flaschen J (2003) Racial differences pertaining to a belief about lung cancer surgery: results of a multicenter survey. Ann Intern Med 139(7):558–563 63. Pinsky PF, Ford M, Gamito E et al (2008) Enrollment of racial and ethnic minorities in the prostate, lung, colorectal and Ovarian Cancer Screening Trial. J Natl Med Assoc 100(3):291–298 64. Trauth JM, Jernigan JC, Siminoff LA, Musa D, Neal-Ferguson D, Weissfeld J (2005) Factors affecting older African American women’s decisions to join the PLCO Cancer Screening Trial. J Clin Oncol 23(34):8730–8738 65. Gordon HS, Street RL Jr, Sharf BF, Kelly PA, Souchek J (2006) Racial differences in trust and lung cancer patients’ perceptions of physician communication. J Clin Oncol 24(6):904–909 66. Bach PB, Pham HH, Schrag D, Tate RC, Hargraves JL (2004) Primary care physicians who treat blacks and whites. N Engl J Med 351(6):575–584 67. John EM, Phipps AI, Davis A, Koo J (2005) Migration history, acculturation, and breast cancer risk in Hispanic women. Cancer Epidemiol Biomarkers Prev 14(12):2905–2913 68. Le GM, Gomez SL, Clarke CA, Glaser SL, West DW (2002) Cancer incidence patterns among Vietnamese in the United States and Ha Noi, Vietnam. Int J Cancer 102(4):412–417 69. Lee J, Demissie K, Lu SE, Rhoads GG (2007) Cancer incidence among Korean-American immigrants in the United States and native Koreans in South Korea. Cancer Control 14(1):78–85 70. Gomez SL, Le GM, Clarke CA, Glaser SL, France AM, West DW (2003) Cancer incidence patterns in Koreans in the US and in Kangwha, South Korea. Cancer Causes Control 14(2):167–174 71. Rastogi T, Devesa S, Mangtani P et al (2008) Cancer incidence rates among South Asians in four geographic regions: India, Singapore, UK and US. Int J Epidemiol 37(1):147–160 72. Wilkinson AV, Spitz MR, Strom SS et  al (2005) Effects of nativity, age at migration, and acculturation on smoking among adult Houston residents of Mexican descent. Am J Public Health 95(6):1043–1049

Pharmacogenetics of Lung Cancer Xifeng Wu and Jian Gu

Abstract  Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer-related death in the United States and around the world. Smoking is the predominant cause of lung cancer and also affects treatment response and survival of lung cancer patients. There is compelling evidence supporting a genetic susceptibility to lung cancer. A family history of lung cancer increases the relative risk of lung cancer by approximately 1.8-fold. The associations between common genetic polymorphisms and the risk of lung cancer have been firmly established through candidate gene studies and genome-wide association studies. Genetic polymorphisms may not only directly affect lung cancer susceptibility through their functional impact on genes involved in processes of tobacco-induced carcinogenesis, including carcinogen metabolism and DNA damage response, but also indirectly influence lung cancer risk through their effect on nicotine addiction and other risk behavior. The same genes that are implicated in lung cancer risk may also be involved in the modulation of clinical outcome. For example, tobacco carcinogen metabolism genes may affect cancer risk and clinical outcome due to the dual role of smoking in lung cancer etiology and outcome, and genes involved in repairing DNA–carcinogen adducts are also responsible for removing DNA–cisplatin complex and therefore can impact chemotherapy response. This chapter summarizes current knowledge of genetic susceptibility to lung cancer and genetic determinants of lung cancer clinical outcomes, focusing on pharmacogenetics of platinum-based chemotherapy and genetic predictors of radiotherapy response and toxicity. Keywords   Pharmacogenetics  •  Pharmacokinetics  •  Pharmacodynamics •  Radiogenetics  •  Single nucleotide polymorphism  •  Platinum  •  EGFR-targeted therapy

X. Wu (*) and J. Gu Department of Epidemiology, Unit 1340, The University of Texas M. D. Anderson Cancer Center, 1155 Pressler Blvd, Houston, TX, 77030, USA e-mail: [email protected] D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_4, © Humana Press, a part of Springer Science+Business Media, LLC 2010

87

88

X. Wu and J. Gu

Introduction Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer-related death in the United States and around the world. Smoking is the predominant cause of lung cancer, responsible for approximately 85% of cases in men and 75% in women. However, only a small percentage of heavy smokers develop lung cancer, suggesting that some individuals are genetically more susceptible to lung cancer given the same level of tobacco exposure. In a systematic review of 45 case–control and cohort studies, a family history of lung cancer increases the relative risk of lung cancer by approximately 1.8-fold, and the elevated familial risk appears to be greater among relatives of young age onset patients and multiple affected family members (1). Association studies of genetic polymorphisms provide further evidence for the genetic susceptibility of lung cancer. Genetic polymorphisms may not only directly affect lung cancer susceptibility through their functional impact on genes involved in processes of tobacco-induced carcinogenesis, including carcinogen metabolism (activation and detoxification), DNA damage response (repair, cell cycle control, and apoptosis), and other cellular functions (2–4), but also indirectly influence lung cancer risk through their effect on nicotine addiction and other risk behavior (5, 6). The clinical outcomes of lung cancer are determined by a variety of factors besides tumor characteristics and treatment modalities, including patient demographics, physiological condition, comorbidity, environmental exposure (particularly smoking), and host genetics. There has been ample evidence suggesting that smoking affects treatment response and survival of lung cancer patients (7–9). There is also accumulating evidence that genetic variations play an important role in modulating treatment response, toxicity, and survival in lung cancer patients. The same genes that are implicated in lung cancer risk may also be involved in the modulation of clinical outcome. For example, tobacco carcinogen metabolism genes may affect cancer risk and clinical outcome due to the dual role of smoking in lung cancer etiology and outcome, and genes involved in repairing DNA–carcinogen adducts are also responsible for removing DNA–cisplatin complex and therefore can influence chemotherapy response (10). In this chapter, we first summarize current knowledge of genetic susceptibility to lung cancer, including genes that regulate carcinogen metabolism and repair DNA–carcinogen adducts, and genes that are involved in nicotine addiction. We then discuss genetic determinants of lung cancer clinical outcomes, focusing on pharmacogenetics of platinum-based chemotherapy, genetic predictors of radiotherapy response, and pharmacogenetics of EGFR-targeted therapy.

Genetic Predisposing Factors for Lung Cancer It is recognized that, for the majority of common sporadic cancers, the genetic susceptibility is a complex polygenic trait determined by multiple genes of modest individual effects. Thousands of candidate gene association studies (i.e., selecting one or a few polymorphisms in a candidate gene and evaluating their associations

Pharmacogenetics of Lung Cancer

89

with disease risk) have been performed to identify potential cancer susceptibility loci. However, the results from such studies have been mostly inconsistent. There are multiple reasons for the inconsistency, including heterogeneity of study populations, small sample size, false positives, no consideration of gene–environment and gene–gene interaction, and publication bias. Meta-analysis (extracting data from published results) and pooled analysis (obtaining original datasets from individual studies) are increasingly used to incorporate results from multiple independent individual studies that individually may not have sufficient power to detect statistically significant associations due to small sample sizes. Dong et  al. (4) recently performed a comprehensive literature review of candidate gene polymorphisms and cancer risk, which included 161 meta-analyses and pooled analyses encompassing 99 genes and 18 cancer sites. The majority of studies evaluated genes involved in carcinogen metabolism and DNA repair and nearly one-third of gene-variant cancer associations were statistically significant. The most consistent associations were observed for a few metabolizing gene variants, including GSTM1 null variant and bladder cancer, NAT2 slow acetylator variant and bladder cancer, MTHFR C677T and gastric cancer, and GSTM1 null variant and acute leukemia (4). The role of candidate gene polymorphisms in lung cancer susceptibility has also been heavily investigated as summarized below.

Carcinogen Metabolism Genes The metabolism of a carcinogen generally consists of two phases, phase I (activation) and phase II (detoxification). Cytochrome P-450 (CYP) family enzymes are the major phase I enzymes that bioactivate carcinogens. CYP1A1 encodes a key enzyme in the bioactivation of tobacco carcinogen polycyclic aromatic hydrocarbons (PAHs). Two single-nucleotide polymorphisms (SNPs) in CYP1A1 gene, MspI polymorphism and Ile462Val, have been widely studied for their associations with lung cancer risk. A recent meta-analysis of 46 published studies suggested that the variant alleles of both MspI and Ile462Val SNPs were associated with increased lung cancer risk in the Chinese population (OR = 1.34, 95% CI 1.08–1.67 for MspI, and OR = 1.61, 95% CI 1.24–2.08 for Ile462Val) (11). Several earlier meta-analyses or pooled analyses generally found negative overall associations but some significant associations in stratified analyses (12–17). Considering the small sample sizes of individual studies included in these meta-analyses and the lack of positive reports from any large studies so far, it appears that SNPs in CYP1A1 gene do not have a significant effect on lung cancer risk. Among phase II enzymes, glutathione-S-transferase (GST) enzymes play a major role in the detoxification of environmental carcinogens, xenobiotics, therapeutic agents, and products of oxidative stress. The human GST family consists of at least eight sub-classes (18). GSTM1 gene is the most studied, and about half of Caucasians have a null genotype due to deletion of GSTM1 gene. In a recent metaanalysis of 98 published studies of the GSTM1 null variant and lung cancer risk

90

X. Wu and J. Gu

(19,638 lung cancer cases and 25,266 controls) (19), the GSTM1 null variant was associated with an increased risk of lung cancer (OR = 1.22, 95% CI 1.14, 1.30) when all the studies were considered; however, when the five largest studies (>500 cases each) were considered, there was no increase in risk (OR = 1.01, 95% CI: 0.91, 1.12). In addition to sample size, ethnic background was a significant source of heterogeneity, with a significant association in East Asians, but not in Caucasians (19). In an earlier meta-analysis of five GST gene variants in 23,452 lung cancer cases and 30,397 controls from 130 studies, Ye et al. (20) found that the associations of the GSTM1 null and GSTT1 null variants with lung cancer risk were 1.18 (95% CI 1.14–1.23) and 1.09 (95% CI 1.02–1.16), respectively, when all studies were combined, but in the larger studies, the ORs were only 1.04 (95% CI: 0.95–1.14) and 0.99 (95% CI: 0.86–1.11), respectively. The relationship between GSTM1 and GSTT1 null genotypes and lung cancer risk warrants further investigations. SNPs in other GST family members, for example, the GSTP1 I105V and A114V and GSTM3*B (a 3-bp deletion polymorphism in intron 6) variant, were not significantly associated with lung cancer risk (20). In addition to CYP1A1 and GST family genes, there are many other metabolizing enzyme genes that have been evaluated in relation to lung cancer risk, including CYP2D6, N-acetyltransferase 2 (NAT2), microsomal epoxide hydrolase (mEH), NAD(P)H quinone oxidoreductase 1 (NQO1), myeloperoxidase (MPO), and manganese superoxide dismutase (MnSOD) (2, 3). Some of the observed associations between these variants and lung cancer risk reached statistical significance in metaanalysis or pooled analysis, but none have a high likelihood of being true associations after considering false-positive report probability (FPRP), statistical power, publication bias, and heterogeneity (4).

DNA Repair Genes Because tobacco carcinogens may induce a variety of DNA lesions, an optimal DNA repair system is critical to protect cells from DNA damage and other consequences. Conversely, a suboptimal DNA repair capacity as measured by several phenotypic assays has been consistently associated with increased cancer risk probably due to increased genetic instability (21). There are four major mammalian DNA repair pathways, namely, nucleotide-excision repair (NER), base-excision repair (BER), double-strand break repair (DSBR), and mismatch repair (MMR), each of which repairs specific types of DNA damage. Many SNPs in DNA repair genes have been reported to be associated with altered lung cancer risk in individual studies (2, 3). NER is the main pathway repairing tobacco carcinogen-induced bulky DNA adducts. A number of functional SNPs in NER pathway genes, including XPA G23A, XPC Lys939Gln, and XPD Lys751Gln, have been associated with lung cancer susceptibility in meta-analysis or pooled analysis (4). In particular, carriers of the XPD 751Gln/Gln genotype exhibited a 30% increase in lung cancer risk compared to the Lys/Lys genotype (95% CI 1.14–1.49) in meta-analysis (22).

Pharmacogenetics of Lung Cancer

91

This association was deemed noteworthy (less likely to be false positives and have a high likelihood of being a true association) after considering false-positive report probability (FPRP), statistical power, publication bias, and heterogeneity (4). Similarly, the BER gene XRCC1 Arg399Gln exhibited a noteworthy significant association with lung cancer risk, but the association was limited to Asians (4, 23).

Genes Involved in Nicotine Addiction Tobacco smoking is the dominant risk factor for lung cancer. It follows that genetic factors predisposing individuals to nicotine addiction theoretically should also be associated with an increased risk of lung cancer. Indeed, recent major advances in genome-wide association studies (GWAS) provide compelling evidence for this notion. Three independent GWAS of lung cancer unequivocally identified the same set of SNPs on chromosome 15q24/25.1, which harbors the CHRNA5–CHRNA3– CHRNB4 cluster of cholinergic nicotinic receptor genes, as a lung cancer susceptibility locus, although these three studies differ on whether the association was direct or through smoking behavior and nicotine dependence (5, 6, 24). One strong candidate causal variant was a nonsynonymous SNP on CHRNA5 (rs16969968, Asp398Asn). Recent additional studies have strongly supported that at least part of the effect of this SNP on lung cancer risk was mediated via smoking behavior and nicotine dependence (25–29). This SNP (or SNPs that are in strong linkage disequilibrium with it) was significantly associated with smoking quantity and/or nicotine dependence by several independent studies (25–29). Furthermore, functional studies showed that the risk allele of rs16969968 reduced receptor response to a nicotine agonist (29). Rs16969968 was also associated with the risk of peripheral arterial disease (5). A second independent allele on CHRNA3 has also been associated with nicotine dependence and lung cancer (24, 29). These data provide compelling evidence that genetic predisposing variants of nicotine dependence are associated with an increased risk of lung cancer.

Other Pathway Genes Genetic variants in other important cellular functions, including cell cycle control, apoptosis, telomere maintenance, microenvironment, inflammation, and methylation, have been suggested to modulate lung cancer risk (2, 3). Many of the reported associations need validation. However, two recent GWAS studies have unequivocally identified a novel lung cancer susceptibility locus at 5p15.33, which harbors the essential telomere maintenance gene, human telomerase reverse transcriptase (hTERT), and CLPTM1L, which may induce apoptosis (30, 31). In addition, a region at 6p21.33 containing BAT3 and MSH5 genes was also identified and validated as a novel locus for lung cancer from GWAS (30). BAT3 is implicated in apoptosis

92

X. Wu and J. Gu

and is required for acetylation of p53 in response to DNA damage. MSH5 is involved in DNA mismatch repair and meiotic recombination.

Genetic Predictors of Clinical Outcome of Lung Cancer Lung cancer is the leading cause of cancer death in both men and women in the United States. Non-small cell lung cancer (NSCLC) comprises over 75% of all lung cancer cases. Surgery remains the frontline therapy for stage I NSCLC. For patients with operable stage II and IIIA NSCLC, adjuvant systemic chemotherapy has been shown to improve 5-year survival by approximately 5% compared with surgery alone and has recently become the standard treatment. Patients with inoperable but potentially curable stage IIIA or IIIB (without a malignant effusion) NSCLC are typically treated with concurrent platinum-based chemotherapy with definitive radiation. Stage IIIB patients with malignant effusions and stage IV patients are offered platinumbased chemotherapy and may also receive palliative radiotherapy. More than 90% of all front-line NSCLC chemotherapy treatments include the platinum derivatives cisplatin or carboplatin. A large volume of clinical experience shows that NSCLC patients vary widely with respect to benefit and toxicity with standard platinum-based chemotherapy. Currently, only clinical variables are used to guide treatment decisions with only modest ability to predict overall survival (32). Genome-wide molecular signatures of chromosome alteration, gene expression, and microRNA expression have shown promise to predict clinical outcome of lung cancer (33–35). However, molecular profiling requires tumor tissue and is limited by the difficulty in sample procurement, tissue storage, experimental procedures, and tumor heterogeneity, which often resulted in the nonreproducibility of the signatures. The use of germline genetic variants is an alternative and complementary approach to predict clinical outcome. Pharmacogenetics and radiogenetics are two relevant disciplines that focus on the effect of genetic variations on drug therapy and radiotherapy. In lung cancer, since platinum drug chemotherapy with or without thoracic radiation is the major front-line therapy, the pharmacogenetics of platinum drugs has attracted wide interest.

Pharmacogenetics of Platinum Drug Therapy There are two components of pharmacogenetics: pharmacokinetics (the absorption, distribution, metabolism, and excretion of a drug) and pharmacodynamics (the action of a drug) (Fig. 1) (36, 37). Genetic variations in genes that are involved in any functions along this path could potentially affect clinical outcomes after a drug therapy. The pharmacokinetics and pharmacodynamics of platinum drugs have been largely elucidated (Fig. 2). Platinum drugs cross cell membranes and enter cells mainly through passive diffusion, although an active import through

Pharmacogenetics of Lung Cancer

93

Fig. 1  Components of pharmacogenetics. Pharmacokinetics is the process of absorption, distribution, metabolism, and excretion of a drug that determines the level of the drug at the site of action. Pharmacodynamics deals with the biological mechanisms of the pharmacologic effect of a drug, including drug target and signaling pathways that are involved in the execution of the pharmacologic effect  

Drug Delivery

Pharmacokinetics (ADME) Absorption Distribution Metabolism Excretion Site of Action

Pharmacodynamics Drug target Signaling pathways Biologic mechanism of drug action Pharmacologic Effect Clinical Outcomes Efficacy Adverse drug reaction (ADR)

copper-transporting proteins is also involved. SLC31A1 (CTR1), a high-affinity copper transporter, is a major component in ushering cisplatin into cells through the active mechanism. For efflux, the copper-transporting P-type adenosine triphosphatases (ATP7A and ATP7B) play an important role. In addition, several ABC membrane transporter family proteins, such as ABCG2 and ABCC2 (MRP2), are also involved in platinum efflux. It appears that the most prominent member of the ABC family, ABCB1 (MDR1), does not play a prominent role in cisplatin efflux. Once entering cells, platinum drugs are susceptible to intracellular detoxification. Detoxification of platinum drugs is mainly carried out by the glutathione detoxification system. GSTs catalyze the reaction of glutathione with drugs, yielding glutathione S-conjugates that are more water-soluble and less reactive than their parent compounds. The primary antitumor mechanism of platinum drugs is through the forming of platinum–DNA adducts, which if not repaired, lead to cell death. The major cellular processes involved in the repairing of platinum–DNA adducts include damage recognition,

94

X. Wu and J. Gu

Fig. 2  Pharmacogenetic (pharmacokinetic and pharmacodynamic) pathways of platinum drugs (reproduced from PharmGKB (91), http://www.pharmgkb.org/, with permission from PharmGKB and Stanford University). Platinum drugs cross cell membranes and enter cells mainly through passive diffusion, but uptake may also involve an active pathway mediated by SLC31A1 (CTR1), a high-affinity copper transporter. The efflux of platinum drugs involves the copper-transporting P-type adenosine triphosphatases (ATP7A and ATP7B) and ABC membrane transporter family proteins, such as ABCG2 and ABCC2 (MRP2). The detoxification of platinum drugs is mainly carried out by the glutathione detoxification system and several other phase II enzymes. The primary anti-tumor mechanism of platinum drugs is through the formation of platinum–DNA adducts, which if not repaired, lead to cell death. HMGB1 is important in the cell recognition of these Pt–DNA adducts, and therefore signals cellular response to these adducts. The major cellular processes involved in the response and repairing of platinum–DNA adducts include translesional replication, NER, and mismatch repair

Pharmacogenetics of Lung Cancer

95

cell cycle arrest, translesional replication, NER, and mismatch repair. In particular, the NER pathway is the major pathway that repairs DNA–platinum adducts. Many studies have been carried out to investigate the association between interindividual variations in DNA repair ability and clinical outcome of platinum drug chemotherapy, and the data are compelling that there is an inverse relationship between DNA repair ability and platinum drug response. Kelland (38) and Cepeda et al. (39) summarized the major cellular pathways that control cisplatin action, and deficiencies in these pathways contribute to cisplatin resistance: (a) reduced drug accumulation; (b) detoxification by thiol-containing species and related enzymes (e.g., GST family proteins); (c) increased repair of platinum–DNA adducts; (d) increased adduct tolerance/replicative bypass (loss of mismatch repair); and (e) decreased apoptosis. There are numerous important genes involved in these cellular pathways and any genetic variations in these genes would affect cisplatin response. Pharmacokinetics of Platinum Drug Therapy in Lung Cancer There were only scattered reports of variations in drug absorption and disposition pathways and platinum response. The focus of the pharmacokinetic aspect (i.e., ADME) of pharmacogenetics of platinum drugs has been on the genetic variations in drug metabolism pathways, particularly the detoxification of platinum drugs by the glutathione metabolic pathway. Glutathione is a tripeptide of glutamine, cysteine, and glycine. Glutathione forms conjugates with platinum drugs, a reaction that is catalyzed by GSTs. Several GST family proteins (e.g., GSTP, GSTT, and GSTM), glutathione synthesis enzymes (e.g., glutamyl-cysteine synthetase, glutamyltransferase, glutamate cysteine ligase, glutathione peroxidase, and glutathione reductase), metallothioneins (e.g., MT1A and MT2A), and phase II enzymes (e.g., MPO, MnSOD, and NQO) have been linked to cisplatin detoxification and resistance to cisplatin (40, 41). Theoretically, variants that decrease the enzymatic activity of GSTs lead to reduced detoxification of platinum drugs and increased effective drug levels, which would be associated with better therapy response and longer survival. There were a few studies that support this hypothesis. Two studies showed that the GSTM1 null genotype was associated with significantly better survival than the wild-type genotype (10, 42). NSCLC patients carrying the variant genotypes of GSTP1 I105V and A114V had significantly longer survival than those with the wild type genotype (10). However, there were also inconsistent reports; for example, the null genotypes of GSTM1 were associated with significantly worse survival in some other studies (43, 44). One explanation is that reduced detoxification also leads to increased toxicity, which may outweigh the desired drug response. Patients with GSTM1 null genotype may have received dose reductions or delays due to toxicity and, therefore, may have received less effective treatment. This discrepancy could also be related to tumor heterogeneity, treatment heterogeneity, and small sample size. Future studies should endeavor to assess large sample size and a homogeneous patient population (in terms of tumor stage and treatment regimen) to produce consistent data.

96

X. Wu and J. Gu

Pharmacodynamics of Platinum Drug Therapy in Lung Cancer Platinum agents form intra- and inter-strand DNA adducts that result in bulky distortion of DNA. The level of platinum–DNA adducts in the circulation correlates with clinical outcome. Therefore, the major focus of platinum drug pharmacodynamics has been on DNA repair genes. Theoretically, poor DNA repair capacity (DRC) should favor cisplatin treatment since more cisplatin–DNA adducts would be formed, which causes cytotoxicity, whereas effective DRC may predispose to resistance to cisplatin. Considering the detrimental effect of poor DRC on cancer risk, DNA repair apparently is a double-edged sword: on the one hand, a poor DRC leads to increased cancer risk because of increased DNA damage and genetic instability (21), on the other hand, a poor DRC results in more platinum–DNA adducts and better chemotherapy outcome (Fig.  3). Indeed, several functional studies provided compelling evidence to support this notion. In a study investigating the role of DNA repair and resistance to NSCLC therapy, the overall DRC was estimated by the ability of cells to repair a transfected reporter plasmid damaged by cisplatin (“host cell reactivation” assay), and a positive correlation was observed between reporter gene activity and intrinsic resistance to cisplatin, suggesting that good DRC is associated with cisplatin resistance (45). In a case–control study of 375 advanced NSCLC patients, Bosken et  al. (46) measured DRC in patients’ peripheral lymphocytes using the host cell reactivation assay and reported the risk of death increased by 1.11 (95% CI = 1.02–1.21) for every percentage increase in DRC in patients treated with cisplatin-based chemotherapy. Patients in the top quartile of the DRC distribution had 2.7-fold increased death compared to those in the lowest quartile (95% CI = 1.24–5.95; P = 0.01). In contrast, good DRC was not a risk factor for death in patients who were not treated with chemotherapy (46).

DNA Repair Capacity

Removal of DNA-Carcinogen Adducts

Cancer Risk

Removal of DNA-bound Platinum

Platinum-based Therapy Response

Fig.  3  Opposing roles of interindividual variation in DNA repair capacity in cancer risk and platinum drug response. On one hand, a poor DRC leads to increased cancer risk because of increased DNA damage and genetic instability. On the other hand, a poor DRC results in more platinum–DNA adducts and better chemotherapy outcome

Pharmacogenetics of Lung Cancer

97

Gene expression data of the DNA repair gene ERCC1 (a critical gene in the NER pathway) provide the strongest epidemiologic evidence that DNA repair plays a critical role in platinum therapy response. Olaussen et al. (47) reported that cisplatinbased adjuvant chemotherapy significantly prolonged survival in NSCLC patients with ERCC1-negative tumors, but not in those with ERCC1-positive tumors. In contrast, in patients randomly assigned to the observation arm (no adjuvant treatment), patients with ERCC1-positive tumors had a better survival than those with ERCC1-negative tumors. Zheng et al. (48) confirmed that high expression of ERCC1 (hence efficient repair of cisplatin–DNA adducts) is associated with poor survival in patients with resected NSCLC who receive adjuvant platinum chemotherapy but is associated with a good prognosis in resected NSCLC patients who do not receive adjuvant therapy. More recently, Cobo et  al. (49) reported the first prospective randomized phase III clinical trial testing the concept of customized chemotherapy in stage IV NSCLC based on prebiopsy ERCC1 mRNA levels. Patients were randomly assigned in a 1:2 ratio to either the control or genotypic arm. Patients in the control arm received docetaxel plus cisplatin. In the genotypic arm, patients with low ERCC1 levels received docetaxel plus cisplatin, and those with high levels received docetaxel plus gemcitabine. An objective response was attained by 53 patients (39.3%) in the control arm and 107 patients (50.7%) in the genotypic arm (P = 0.02). There have been many studies investigating DNA repair gene polymorphisms and clinical outcomes of platinum drug chemotherapy. Wu et al. (50) showed that the variant A allele of the ERCC1 8092C>A had a significantly improved survival rate with evidence of a gene dosage effect, which is consistent with Zienolddiny et al. who reported that individuals with this variant allele might exhibit suboptimal DRC (51). This allele was previously reported to be associated with higher toxicity in NSCLC patients treated with cisplatin (52). Wu et  al. (50) also reported that patients homozygous for the variant allele of XPD Lys751Gln and the haplotypes containing both variant alleles of Asp312Asn and Lys751Gln exhibited a better overall survival, which was in line with the findings by de las Penas et al. (53) for stage IIIB and stage IV NSCLC patients treated with cisplatin and gemcitabine (HR = 0.46, P = 0.03). Functionally, the wild-type genotypes of these two SNPs had the most efficient DRC, and there was a significant trend for reduced DRC as the number of variant alleles increased (54, 55). Nevertheless, there are also inconsistent observations in literature regarding these SNPs as well as other DNA repair gene SNPs with platinum drug responses in lung cancer patients (56–58). Differences in study design, sample size, patient stage, population stratification, and treatment homogeneity may partially account for these discrepancies. Cumulative Effect of Multiple Variants Although there are numerous candidate gene studies reporting significant associations between genetic variations and platinum drug therapy outcomes in lung cancer, there has not been an unequivocal genetic marker that emerged from these studies. Association studies of clinical outcome are limited by many factors that may produce

98

X. Wu and J. Gu

false positives, including small sample size, heterogeneity in patients, tumor characteristics and treatment, and population stratification. However, from the conceptual point of view, it should not be expected that any individual genetic variation would have a dramatic effect on platinum drug response. Platinum drugs do not target a specific protein but instead cause general DNA damage. The effect of individual genetic variations on the function of the host gene is generally weak. For certain variations that have dramatic functional impact, such as the deletion of GSTM1 and GSTT1, the lost function of one enzyme may be compensated by other redundant enzymes in the same family or other pathways. Because of the expected weak associations from these candidate gene studies, they are not likely to be clinically usable as single predictors of treatment response, even if confirmed in larger well-designed studies. The inherent limitations of the candidate gene approach demonstrate the need for an improved approach for association studies. One such improvement may be the use of a polygenic analytic approach that assesses the cumulative effects of multiple variants. A large number of unfavorable alleles, each contributing to a small yet important portion of the drug response, should theoretically enhance the predictive power to a level that may be clinically relevant. In cancer association studies, a few recent reports have strongly supported this combined approach in risk assessment (59, 60). Zheng et  al. (60) recently evaluated the combined effects of five confirmed significant SNPs in five different chromosome regions and family history of prostate cancer. In men with any five or more of these known risk factors, the OR for prostate cancer was 9.46 (P = 1.29 × 10−8), as compared with men without any of these factors. The pharmacogenetics that Warfarin provides is a successful example of this polygenic approach. Warfarin targets the vitamin K epoxide reductase complex 1 (VKORC1), which is metabolized mainly by CYP2C9. VKORC1 genotypes account for about one quarter of the variance in the Warfarin maintenance dose and CYP2C9 accounts for 6–10%. However, consideration of both genes accounts for >50% of the variability in the maintenance dose of Warfarin (61, 62). Several recent studies have demonstrated the enhanced power of this polygenic approach in platinum pharmacogenetics. Quintela–Fandino et  al. (63) determined the associations between four DNA repair gene SNPs and clinical outcomes in patients with advanced head and neck cancer treated with cisplatin-based chemotherapy and found a dramatic cumulative effect of multiple variants, with each variant allele reducing the risk of death by 2.1-fold. Wu et al. (50) analyzed six polymorphisms in NER genes jointly and found a significant trend of reduced risk of death with decreasing number of unfavorable genotypes in NSCLC patients receiving firstline platinum drug therapy (P for trend <0.001 and log-rank P < 0.001). In addition to simply analyzing the cumulative effect of multiple variants, other sophisticated machine learning and analysis tools have been used to analyze the interaction of multiple polymorphisms and to provide a more powerful predictive capability. Several nonparametric statistical methods are available to model gene– gene interactions. Classification and regress tree (CART) analysis uses a binary recursive partitioning method to identify subgroups of patients with worse or better clinical outcomes. The method generates a tree-structured node with binary splits

Pharmacogenetics of Lung Cancer

99

and identifies optimal cut points at each node for the covariate. The recursive procedure is continued to yield subsequent nodes that are more homogeneous (with respect to the response variable) than the original node. Gordon et al. (64) evaluated 21 polymorphisms in 18 genes involved in the critical pathways of cancer progression and applied CART to identify a subgroup with four genes (IL-8, ICAM-1, TGF-b and FGFR4) and the TNM classification that best predict tumor recurrence in rectal cancer treated with chemoradiation. Survival tree analysis is similar to CART in that it also uses recursive partitioning procedure to categorize patients into different subgroups with different survival time based on distinct genotype profile. Wu et  al. (50) genotyped 25 functional SNPs in 16 key genes involved in cisplatin metabolism and action in 229 NSCLC patients receiving first-line cisplatin-based chemotherapy and performed an exploratory survival tree analysis. The top three splits were from NER genes (ERCC1, XPC, and XPG), confirming the importance of the NER gene pathway in the outcome of platinum drug therapy. Twelve subgroups were categorized with HR estimates ranging from 2.5 to 11.8. Kaplan–Meier survival curves of these subgroups showed median survival times of 6.7–78.5 months, demonstrating good discriminative ability of the tree analysis. The substantially increased prediction ability of multiple variants and gene–gene interactions highlights the clinical potential of taking a polygenic approach in predicting clinical outcomes.

Radiogenetics It is estimated that given the same standardized radiation dose, technical and clinical factors account for about one third of the interindividual variation in normal tissue reactions, and genetic differences between patients account for the greater proportion of interpatient radiosensitivity differences (65). Several lines of evidence support that radiotherapy response has strong genetic components. The contribution of genetic components to radiosensitivity can be best exemplified by the rare genetic syndromes such as Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and Bloom’s syndrome (BS). Patients with these syndromes have a defective DNA repair system, abnormal cell cycle control, and increased cellular and clinical radiosensitivity (66–68). Patients with immune deficiencies arising from DSB repair defects in genes, including DNA-PKcs (XRCC7), Ku70 (XRCC6), and Ku86 (XRCC5), exhibited high radiation sensitivity (69–71). It is clear that the DSB repair system plays a significant role in determining radiosensitivity. Family studies also suggest a genetic predisposition to radiosensitivity. Roberts et al. (72) studied the heritability of radiation sensitivity in peripheral blood lymphocytes in families of patients with breast cancer. They found that 62% of the first-degree relatives of radiation-sensitive patients were also sensitive compared with 7% of the firstdegree relatives of patients with normal sensitivity. More recently, there has been a rapid increase in the number of association studies that suggest germline genetic variations in a few selected cellular pathways, particularly DNA repair, cell cycle,

100

X. Wu and J. Gu

and inflammation, which may modify radiation sensitivity, treatment response, and toxicity (73–76). A broad international effort has been organized to create the GenePARE project (Genetic Predictors of Adverse Radiotherapy Effects). In a recent preliminary summary of this project of more than 2,000 radiotherapy cancer patients from five countries, a handful of selected functional polymorphisms in DSB repair, BER, and inflammation pathways were screened for their association with adverse effects of radiotherapy, and a few positive associations were identified, including polymorphisms in XRCC1, XRCC3, and TGFb1 genes (73). A recent review summarized the results of roughly 40 studies evaluating the associations between various genetic variants and different types of radiation-induced morbidity (77). No consensus can be drawn from these studies and future studies are needed to validate these findings.

Pharmacogenetics of EGFR-Target Therapy Targeted therapies are generally well tolerated with less severe adverse effects and can be highly effective against a subset of patients. Two small-molecular inhibitors of EGFR, gefitinib (Iressa) and erlotinib (Tarceva), have been approved as singledrug therapy for the treatment of advanced chemotherapy-refractory NSCLC patients. Although the use of gefitinib has been restricted in the USA due to lack of survival benefit in a large clinical trial, it is still widely used in Asia. It has long been observed that responders to these EGFR inhibitors are more likely to be female, Asian, nonsmokers, and have adenocarcinoma histology, suggesting a genetic component. Later, it was found that somatic mutation plays a determining role in tumor response to gefitinib and erlotinib (78–80). The two most common sensitizing EGFR mutations are the 15-bp nucleotide in-frame deletion in exon 19 (E746A750del) and the point mutation in exon 21 (L858R). Many retrospective and prospective studies have consistently shown a response rate of 75–80% for patients with EGFR mutation compared to a 10% response rate for patients with wild-type EGFR (81, 82). Conversely, three EGFR kinase domain mutations, exon 19 D761Y, exon 20 T790M, and exon 20 D770-N771 insertion, have been found to be associated with resistance to gefitinib and erlotinib therapy in NSCLC (83–85). In addition to these acquired somatic mutations, there are also reports that link inherited germline polymorphisms in EGFR and response to gefitinib and erlotinib, although the effects are modest compared to somatic mutations in EGFR. EGFR gene intron 1 contains a polymorphic microsatellite sequence of CA dinucleotide repeats ranging from 9 to 23 repeats. A high number of CA repeats is associated with decreased expression of EGFR. It follows that a low number of CA repeats should favor EGFR inhibitor treatment. Indeed, a number of studies have supported this hypothesis (86– 89) in showing that shorter CA repeats were associated with longer progression free survival and/or overall survival. This CA repeat polymorphism was also reported to be associated with skin toxicity related to gefitinib or erlotinib treatment (86, 90). The pharmacogenetics of gefitinib and erlotinib highlight the need for the integration of both germline and somatic variations in determining drug response.

Pharmacogenetics of Lung Cancer

101

Conclusions and Perspectives In the past decade, enormous progress has been made in identifying genetic polymorphisms as common cancer susceptibility markers. Particularly in recent years, GWAS has unequivocally demonstrated that a large number of low penetrance, high prevalent genetic polymorphisms contribute collectively to the etiology of common cancers. The validated cancer susceptibility loci identified from GWAS in very recent years have far exceeded those from candidate gene studies in the past decade. In lung cancer, although previously numerous candidate gene studies have suggested several possible susceptibility polymorphisms, only recent GWAS pinpointed three unequivocal lung cancer susceptibility loci. Similarly, in the pharmacogenetic study of lung cancer, except for the somatic mutations of EGFR in gefitinib and erlotinib therapy response, candidate gene studies have provided only suggestive evidence for an association between genetic polymorphisms and lung cancer outcome, but none of the reported associations have been validated. For polygenic traits such as platinum drug and radiation response, it is likely that the major breakthrough in identifying unequivocal genetic predictors will also come from GWAS. However, GWAS requires a very large sample size. In pharmacogenomic studies, the available sample size is much smaller than in cancer etiology studies. Nevertheless, the successful completion of multiple independent GWAS of lung cancer risk has laid a strong foundation for the application of GWAS in lung cancer pharmacogenetic studies. The primary scan data in the cases can be used to analyze for their associations with clinical outcome. The positive associations can then be validated in independent patient populations. The pooling of data from multiple GWAS is necessary to obtain a sufficient number of relatively homogeneously treated patients. Therefore, a collective effort of many investigators and institutions is essential for the success of GWAS of clinical outcomes. Most currently used cytotoxic chemotherapeutic drugs (e.g., platinum drugs) are designed to kill as many tumor cells as possible at the expense of toxicity to normal cells. The most likely application of genetic predictors in these types of therapies is to reduce toxicity by dose reduction or to avoid unnecessary toxicity to those patients who are unlikely to benefit from these therapies. Future pharmacogenomic studies should be incorporated into early-phase clinical trials during the development of drugs. Such pharmacogenomic information can be used to reduce the size and cost of phase III trials and improve the chance of developing effective novel targeted drugs.

References 1. Matakidou A, Eisen T, Houlston RS (2005) Systematic review of the relationship between family history and lung cancer risk. Br J Cancer 3:825–833 2. Wu X, Zhao H, Suk R, Christiani DC (2004) Genetic susceptibility to tobacco-related cancer. Oncogene 23:6500–6523 3. Schwartz AG, Prysak GM, Bock CH, Cote ML (2007) The molecular epidemiology of lung cancer. Carcinogenesis 28:507–518

102

X. Wu and J. Gu

4. Dong LM, Potter JD, White E, Ulrich CM, Cardon LR, Peters U (2008) Genetic susceptibility to cancer: the role of polymorphisms in candidate genes. JAMA 299:2423–2436 5. Thorgeirsson TE, Geller F, Sulem P, Rafnar T, Wiste A, Magnusson KP et al (2008) variant associated with nicotine dependence, lung cancer and peripheral arterial disease. Nature 452:638–642 6. Amos CI, Wu X, Broderick P, Gorlov IP, Gu J, Eisen T, Dong Q et al (2008) Genome-wide association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25. Nat Genet 40:616–622 7. Tsao AS, Liu D, Lee JJ, Spitz M, Hong WK (2006) Smoking affects treatment outcome in patients with advanced nonsmall cell lung cancer. Cancer 106:2428–2436 8. Tammemagi CM, Neslund-Dudas C, Simoff M, Kvale P (2004) Smoking and lung cancer survival: the role of comorbidity and treatment. Chest 125:27–37 9. Ebbert JO, Yang P, Vachon CM, Vierkant RA, Cerhan JR, Folsom AR, Sellers TA (2003) Lung cancer risk reduction after smoking cessation: observations from a prospective cohort of women. J Clin Oncol 21:921–926 10. Spitz MR, Wu X, Mills G (2005) Integrative epidemiology: from risk assessment to outcome prediction. J Clin Oncol 23:267–275 11. Shi X, Zhou S, Wang Z, Zhou Z, Wang Z (2008) CYP1A1 and GSTM1 polymorphisms and lung cancer risk in Chinese populations: a meta-analysis. Lung Cancer 59:155–163 12. Lee KM, Kang D, Clapper ML, Ingelman-Sundberg M, Ono-Kihara M, Kiyohara C et  al (2008) CYP1A1, GSTM1, and GSTT1 polymorphisms, smoking, and lung cancer risk in a pooled analysis among Asian populations. Cancer Epidemiol Biomarkers Prev 17:1120–1126 13. Houlston RS (2000) CYP1A1 polymorphisms and lung cancer risk: a meta-analysis. Pharmacogenetics 10:105–114 14. Le Marchand L, Guo C, Benhamou S, Bouchardy C, Cascorbi I, Clapper ML, Garte S, Haugen A, Ingelman-Sundberg M, Kihara M, Rannug A, Ryberg D, Stücker I, Sugimura H, Taioli E (2003) Pooled analysis of the CYP1A1 exon 7 polymorphism and lung cancer (United States). Cancer Causes Control 14:339–346 15. Vineis P, Veglia F, Benhamou S, Butkiewicz D, Cascorbi I, Clapper ML, Dolzan V, Haugen A, Hirvonen A, Ingelman-Sundberg M, Kihara M, Kiyohara C, Kremers P, Le Marchand L, Ohshima S, Pastorelli R, Rannug A, Romkes M, Schoket B, Shields P, Strange RC, Stucker I, Sugimura H, Garte S, Gaspari L, Taioli E (2003) CYP1A1 T3801 C polymorphism and lung cancer: a pooled analysis of 2451 cases and 3358 controls. Int J Cancer 104:650–657 16. Taioli E, Gaspari L, Benhamou S, Boffetta P, Brockmoller J, Butkiewicz D, Cascorbi I, Clapper ML, Dolzan V, Haugen A, Hirvonen A, Husgafvel-Pursiainen K, Kalina I, Kremers P, Le Marchand L, London S, Rannug A, Romkes M, Schoket B, Seidegard J, Strange RC, Stucker I, To-Figueras J, Garte S (2003) Polymorphisms in CYP1A1, GSTM1, GSTT1 and lung cancer below the age of 45 years. Int J Epidemiol 32:60–63 17. Hung RJ, Boffetta P, Brockmöller J, Butkiewicz D, Cascorbi I, Clapper ML, Garte S, Haugen A, Hirvonen A, Anttila S, Kalina I, Le Marchand L, London SJ, Rannug A, Romkes M, Salagovic J, Schoket B, Gaspari L, Taioli E (2003) CYP1A1 and GSTM1 genetic polymorphisms and lung cancer risk in Caucasian non-smokers: a pooled analysis. Carcinogenesis 24:875–882 18. Hayes JD, Strange RC (2000) Glutathione S-transferase polymorphisms and their biological consequences. Pharmacology 61:154–166 19. Carlsten C, Sagoo GS, Frodsham AJ, Burke W, Higgins JP (2008) Glutathione S-transferase M1 (GSTM1) polymorphisms and lung cancer: a literature-based systematic HuGE review and meta-analysis. Am J Epidemiol 167:759–774 20. Ye Z, Song H, Higgins JP, Pharoah P, Danesh J (2006) Five glutathione s-transferase gene variants in 23, 452 cases of lung cancer and 30, 397 controls: meta-analysis of 130 studies. PLoS Med 3:e91 21. Spitz MR, Wei Q, Dong Q, Amos CI, Wu X (2003) Genetic susceptibility to lung cancer: the role of DNA damage and repair. Cancer Epidemiol Biomarkers Prev 12:689–698 22. Kiyohara C, Yoshimasu K (2007) Genetic polymorphisms in the nucleotide excision repair pathway and lung cancer risk: a meta-analysis. Int J Med Sci 4:59–71

Pharmacogenetics of Lung Cancer

103

23. Kiyohara C, Takayama K, Nakanishi Y (2006) Association of genetic polymorphisms in the base excision repair pathway with lung cancer risk: a meta-analysis. Lung Cancer 54:267–283 24. Hung RJ, McKay JD, Gaborieau V, Boffetta P, Hashibe M, Zaridze D et al (2008) A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25. Nature 452:633–637 25. Saccone SF, Hinrichs AL, Saccone NL, Chase GA, Konvicka K, Madden PA et  al (2007) Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs. Hum Mol Genet 16:36–49 26. Sherva R, Wilhelmsen K, Pomerleau CS, Chasse SA, Rice JP, Snedecor SM et  al (2008) Association of a single nucleotide polymorphism in neuronal acetylcholine receptor subunit alpha 5 (CHRNA5) with smoking status and with ‘pleasurable buzz’ during early experimentation with smoking. Addiction 103:1544–1552 27. Berrettini W, Yuan X, Tozzi F, Song K, Francks C, Chilcoat H et al (2008) Alpha-5/alpha-3 nicotinic receptor subunit alleles increase risk for heavy smoking. Mol Psychiatry 13:368–373 28. Weiss RB, Baker TB, Cannon DS, von Niederhausern A, Dunn DM, Matsunami N et  al (2008) A candidate gene approach identifies the CHRNA5-A3-B4 region as a risk factor for age-dependent nicotine addiction. PLoS Genet 4:e1000125 29. Bierut LJ, Stitzel JA, Wang JC, Hinrichs AL, Grucza RA, Xuei X et al (2008) Variants in nicotinic receptors and risk for nicotine dependence. Am J Psychiatry 165:1163–1171 30. Wang Y, Broderick P, Webb E, Wu X, Vijayakrishnan J, Matakidou A et al (2008) Common 5p15.33 and 6p21.33 variants influence lung cancer risk. Nat Genet 40:1407–1409 31. McKay JD, Hung RJ, Gaborieau V, Boffetta P, Chabrier A, Byrnes G et al (2008) Lung cancer susceptibility locus at 5p15.33. Nat Genet 40:1404–1406 32. Schiller JH et al (2002) Comparison of four chemotherapy regimens for advanced non-smallcell lung cancer. N Engl J Med 346:92–98 33. Chen HY et al (2007) A five-gene signature and clinical outcome in non-small-cell lung cancer. N Engl J Med 356:11–20 34. Potti A et al (2006) A genomic strategy to refine prognosis in early-stage non-small-cell lung cancer. N Engl J Med 355:570–580 35. Thomas RK, Weir B, Meyerson M (2006) Genomic approaches to lung cancer. Clin Cancer Res 12(14 Pt 2):4384s–4391s 36. Weinshilboum RM, Wang L (2006) Pharmacogenetics and pharmacogenomics: development, science, and translation. Annu Rev Genomics Hum Genet 7:223–245 37. Eichelbaum M, Ingelman-Sundberg M, Evans WE (2006) Pharmacogenomics and individualized drug therapy. Annu Rev Med 57:119–137 38. Kelland L (2007) The resurgence of platinum-based cancer chemotherapy. Nat Rev Cancer 7:573–584 39. Cepeda V, Fuertes MA, Castilla J, Alonso C, Quevedo C, Pérez JM (2007) Biochemical mechanisms of cisplatin cytotoxicity. Anticancer Agents Med Chem 7:3–18 40. Stewart DJ (2007) Mechanisms of resistance to cisplatin and carboplatin. Crit Rev Oncol Hematol 63:12–31 41. Yang P, Ebbert JO, Sun Z, Weinshilboum RM (2006) Role of the glutathione metabolic pathway in lung cancer treatment and prognosis: a review. J Clin Oncol 24:1761–1769 42. Ge H, Lam WK, Lee J, Wong MP, Yew WW, Lung ML (1996) Analysis of L-myc and GSTM1 genotypes in Chinese non-small cell lung carcinoma patients. Lung Cancer 15:355–366 43. Sweeney C, Nazar-Stewart V, Stapleton PL, Eaton DL, Vaughan TL (2003) Glutathione S-transferase M1, T1, and P1 polymorphisms and survival among lung cancer patients. Cancer Epidemiol Biomarkers Prev 12:527–533 44. Goto I, Yoneda S, Yamamoto M, Kawajiri K (1996) Prognostic significance of germ-line polymorphisms of the CYP1A1 and glutathione S-transferase genes in patients with nonsmall cell lung cancer. Cancer Res 56:3725–3730

104

X. Wu and J. Gu

45. Zeng-Rong N, Paterson J, Alpert L, Tsao MS, Viallet J, Alaoui-Jamali MA (1995) Elevated DNA repair capacity is associated with intrinsic resistance of lung cancer to chemotherapy. Cancer Res 55:4760–4764 46. Bosken CH, Wei Q, Amos CI, Spitz MR (2002) An analysis of DNA repair as a determinant of survival in patients with non-small-cell lung cancer. J Natl Cancer Inst 94:1091–1099 47. Olaussen KA, Dunant A, Fouret P et al (2006) DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy. N Engl J Med 355:983–991 48. Zheng Z, Chen T, Li X et al (1007) DNA synthesis and repair genes RRM1 and ERCC1 in lung cancer. N Engl J Med 356:800–808 49. Cobo M, Isla D, Massuti B et al (2007) Customizing cisplatin based on quantitative excision repair cross-complementing 1 mRNA expression: a phase III trial in non-small-cell lung cancer. J Clin Oncol 25:2747–2754 50. Wu X, Lu C, Ye Y, Chang J, Yang H, Lin J, Gu J, Hong WK, Stewart D, Spitz MR (2008) Germline genetic variations in drug action pathways predict clinical outcomes in advanced lung cancer treated with platinum-based chemotherapy. Pharmacogenet Genomics 18:955–965 51. Zienolddiny S et al (2006) Polymorphisms of DNA repair genes and risk of non-small cell lung cancer. Carcinogenesis 27:560–567 52. Suk R et al (2005) Polymorphisms in ERCC1 and grade 3 or 4 toxicity in non-small cell lung cancer patients. Clin Cancer Res 11:1534–1538 53. de las Penas R et al (2006) Polymorphisms in DNA repair genes modulate survival in cisplatin/gemcitabine-treated non-small-cell lung cancer patients. Ann Oncol 17:668–675 54. Spitz MR et al (2001) Modulation of nucleotide excision repair capacity by XPD polymorphisms in lung cancer patients. Cancer Res 61:1354–1357 55. Qiao Y et al (2002) Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes. Mutat Res 509:165–174 56. Gurubhagavatula S et al (2004) XPD and XRCC1 genetic polymorphisms are prognostic factors in advanced non-small-cell lung cancer patients treated with platinum chemotherapy. J Clin Oncol 22:2594–2601 57. Zhou W et al (2004) Excision repair cross-complementation group 1 polymorphism predicts overall survival in advanced non-small cell lung cancer patients treated with platinum-based chemotherapy. Clin Cancer Res 10:4939–4943 58. Booton R et  al (2006) Xeroderma pigmentosum group D haplotype predicts for response, survival, and toxicity after platinum-based chemotherapy in advanced nonsmall cell lung cancer. Cancer 106:2421–2427 59. Wu X et al (2006) Bladder cancer predisposition: a multigenic approach to DNA-repair and cell-cycle-control genes. Am J Hum Genet 78:464–479 60. Zheng SL, Sun J, Wiklund F, Smith S, Stattin P, Li G et al (2008) Cumulative association of five genetic variants with prostate cancer. N Engl J Med 358:910–919 61. Sconce EA, Khan TI, Wynne HA, Avery P, Monkhouse L, King BP et al (2005) The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirements: proposal for a new dosing regimen. Blood 106:2329–2333 62. Wadelius M, Chen LY, Downes K, Ghori J, Hunt S, Eriksson N et al (2005) Common VKORC1 and GGCX polymorphisms associated with warfarin dose. Pharmacogenomics J 5:262–270 63. Quintela-Fandino M, Hitt R, Medina PP, Gamarra S, Manso L, Cortes-Funes H, SanchezCespedes M (2006) DNA-repair gene polymorphisms predict favorable clinical outcome among patients with advanced squamous cell carcinoma of the head and neck treated with cisplatin-based induction chemotherapy. J Clin Oncol 24:4333–4339 64. Gordon MA, Gil J, Lu B, Zhang W, Yang D, Yun J et al (2006) Genomic profiling associated with recurrence in patients with rectal cancer treated with chemoradiation. Pharmacogenomics 7:67–88 65. Awwad HK (2005) Normal tissue radiosensitivity: prediction on deterministic or stochastic basis? J Egypt Natl Canc Inst 17:221–230

Pharmacogenetics of Lung Cancer

105

66. Jeggo PA, Carr AM, Lehmann AR (1998) Splitting the ATM: distinct repair and checkpoint defects in ataxia-telangiectasia. Trends Genet 14:312–316 67. Rogers PB, Plowman PN, Harris SJ, Arlett CF (2000) Four radiation hypersensitivity cases and their implications for clinical radiotherapy. Radiother Oncol 57:143–154 68. Crompton NE, Shi YQ, Emery GC, Wisser L, Blattmann H, Maier A et al (2001) Sources of variation in patient response to radiation treatment. Int J Radiat Oncol Biol Phys 49:547–554 69. Blunt T, Finnie NJ, Tacciol GE et al (1995) Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell 80:813–823 70. Gu Y, Seidl KJ, Rathburn GA et al (1997) Growth retardation and leaky SCID phenotype of Ku70 deficient mice. Immunity 7:653–665 71. Nussenzweig A, Chen C, da Costa Soares V et al (1996) Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature 382:551–555 72. Roberts SA, Spreadborough AR, Bulman B, Barber JBP, Evans DGR, Scott D (1999) Heritability of cellular radiosensitivity: a marker of low-penetrance predisposition genes in breast cancer. Am J Hum Genet 65:784–794 73. Ho AY, Atencio DP, Peters S, Stock RG, Formenti SC, Cesaretti JA, Green S, Haffty B, Drumea K, Leitzin L, Kuten A, Azria D, Ozsahin M, Overgaard J, Andreassen CN, Trop CS, Park J, Rosenstein BS (2006) Genetic predictors of adverse radiotherapy effects: the GenePARE project. Int J Radiat Oncol Biol Phys 65:646–655 74. West CM, McKay MJ, Holscher T, Baumann M, Stratford IJ, Bristow RG et al (2005) Molecular markers predicting radiotherapy response: report and recommendations from an International Atomic Energy Agency technical meeting. Int J Radiat Oncol Biol Phys 62:1264–1273 75. De Ruyck K, Van Eijkeren M, Claes K, Morthier R, De Paepe A, Vral A, De Ridder L, Thierens H (2005) Radiation-induced damage to normal tissues after radiotherapy in patients treated for gynecologic tumors: association with single nucleotide polymorphisms in XRCC1, XRCC3, and OGG1 genes and in vitro chromosomal radiosensitivity in lymphocytes. Int J Radiat Oncol Biol Phys 62:1140–1149 76. Andreassen CN, Alsner J, Overgaard M, Sorensen FB, Overgaard J (2006) Risk of radiationinduced subcutaneous fibrosis in relation to single nucleotide polymorphisms in TGFB1, SOD2, XRCC1, XRCC3, APEX and ATM–a study based on DNA from formalin fixed paraffin embedded tissue samples. Int J Radiat Biol 82:577–586 77. Alsner J, Andreassen CN, Overgaard J (2008) Genetic markers for prediction of normal tissue toxicity after radiotherapy. Semin Radiat Oncol 18(2):126–135 78. Lynch TJ, Bell DW, Sordella R et  al (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129–2139 79. Paez JG, Janne PA, Lee JC et al (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304:1497–1500 80. Pao W, Miller V, Zakowski M et al (2004) EGF receptor gene mutations are common in lung cancers from ‘never smokers’ and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA 101:13306–13311 81. Chan SK, Gullick WJ, Hill ME (2004) Mutations of the epidermal growth factor receptor in non-small cell lung cancer-search and destroy. Eur J Cancer 42:17–23 82. Sequist LV, Bell DW, Lynch TJ, Haber DA (2007) Molecular predictors of response to epidermal growth factor receptor antagonists in non-small-cell lung cancer. J Clin Oncol 25:587–595 83. Kobayashi S, Boggon TJ, Dayaram T et  al (2005) EGFR mutation and resistance of nonsmall-cell lung cancer to gefitinib. N Engl J Med 352:786–792 84. Pao W, Miller VA, Politi KA et  al (2005) Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2:E73 85. Balak MN, Gong Y, Riely GJ et  al (2006) Novel D761Y and common secondary T790M mutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors. Clin Cancer Res 12:6494–6501

106

X. Wu and J. Gu

86. Amador ML et al (2004) An epidermal growth factor receptor intron 1 polymorphism mediates response to epidermal growth factor receptor inhibitors. Cancer Res 64:9139–9143 87. Han SW, Jeon YK, Lee KH, Keam B, Hwang PG, Oh DY, Lee SH, Kim DW, Im SA, Chung DH, Heo DS, Bang YJ, Kim TY (2007) Intron 1 CA dinucleotide repeat polymorphism and mutations of epidermal growth factor receptor and gefitinib responsiveness in non-small-cell lung cancer. Pharmacogenet Genomics 17:313–319 88. Tiseo M, Capelletti M, De Palma G, Franciosi V, Cavazzoni A, Mozzoni P et  al (2008) Epidermal growth factor receptor intron-1 polymorphism predicts gefitinib outcome in advanced non-small cell lung cancer. J Thorac Oncol 3:1104–1111 89. Liu G, Gurubhagavatula S, Zhou W, Wang Z, Yeap BY, Asomaning K et al (2008) Epidermal growth factor receptor polymorphisms and clinical outcomes in non-small-cell lung cancer patients treated with gefitinib. Pharmacogenomics J 8:129–138 90. Rudin CM, Liu W, Desai A, Karrison T, Jiang X, Janisch L et al (2008) Pharmacogenomic and pharmacokinetic determinants of erlotinib toxicity. J Clin Oncol 26:1119–1127 91. Klein TE, Chang JT, Cho MK, Easton KL, Fergerson R, Hewett M, Lin Z, Liu Y, Liu S, Oliver DE, Rubin DL, Shafa F, Stuart JM, Altman RB (2001) Integrating genotype and phenotype information: an overview of the PharmGKB project. Pharmacogenomics J 1:167–170

Lung Cancer Prevention Nir Peled, Robert L. Keith, and Fred R. Hirsch

Abstract  The poor prognosis associated with lung cancer has stimulated substantial effort directed toward lung cancer prevention. As smoking prevention and cessation have proven difficult to achieve, many chemoprevention trials have been done during the last few decades. Such trials have focused on lung cancer survivors and high-risk groups including current and former heavy smokers with or without COPD. The aims of these trials were to reduce lung cancer incidence as well as to reduce/reverse premalignant changes in the bronchial epithelium. Unfortunately, till now all the chemoprevention clinical trials have failed to show any clinical or histologic benefit with respect to lung cancer. This chapter reviews the chemoprevention trials that have been done in the last few decades, analyzes the reasons for their failure, and reviews the future directions and the targeted therapy that may be potentially relevant in the chemoprevention of lung cancer. Keywords  Lung cancer • Chemoprevention • Smoking cessation • Bronchial dysplasia • Premalignancy

Introduction Lung cancer is the leading cause of cancer death in the US and globally. In the US, 215,000 new patients are diagnosed with lung cancer every year. The prognosis is poor, with a cure rate of about 16%. The reason for the poor prognosis is the lack of early detection measures and the lack of efficient therapy for systemic disease.

N. Peled (*), R.L. Keith, and F.R. Hirsch Pulmonary Medicine and Medical Oncology, University of Colorado Denver, 12801 E 17th Ave, Aurora, CO 80045, USA e-mail: [email protected] D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_5, © Humana Press, a part of Springer Science+Business Media, LLC 2010

107

108

N. Peled et al.

Cigarette smoking is the main culprit for lung cancer, with 87% of cases occurring in subjects with a smoking history and approximately 50% of lung cancers arising in former smokers (1). Therefore, the overall “at risk” population in the United States is 90 million subjects, including 45 million current and 45 million former smokers. While never smoking has the greatest impact on decreasing the burden of this disease, it is unlikely to be accomplished. This has motivated an intense interest in the chemoprevention of this disease. Chemoprevention involves the use of natural or laboratory made substances to prevent cancer or to reduce cancer risk (2), and aims to reverse or inhibit the carcinogenic process. Therefore, identifying the high risk group is crucial for achieving this aim. This concept has been built on the progression in bronchial epithelium from normal epithelium through hyperplasia, metaplasia, dysplasia, and carcinoma in situ to invasive squamous cell cancer. Approximately 25% of lung cancer cases worldwide are not attributable to tobacco use, accounting for over 300,000 deaths each year. Striking differences in the epidemiological, clinical, and molecular characteristics of lung cancers arising in never smokers versus smokers have been identified, suggesting that they are separate entities. As discussed in more detail later in this chapter, three large randomized trials conducted several years ago evaluating vitamin A, b-carotene, and vitamin E failed to show a benefit for active treatment in current and/or former smokers (3, 4). As a matter of fact, one study suggested an increased risk of lung cancer in current smokers who received b-carotene (3). Two additional phase III studies conducted in a high-risk population of patients with resected stage I nonsmall cell lung cancer were unable to reduce the incidence of second primary cancers with vitamin A, 13-cis retinoic acid or N-acetylcysteine when compared to placebo (5, 6). Nonsteroidal anti-inflammatory drugs have been observed to reduce the relative risk of tobacco-induced lung carcinogenesis in both preclinical and clinical studies (7–9). The anti-inflammatory action of these drugs is mediated through their inhibitory effect on cyclooxygenases (COX), which are essential enzymes for the synthesis of prostaglandins generated from arachidonic acid (10). In fact, the COX-2 isoenzyme is frequently upregulated in neoplastic tissue of the lung and seems to be associated with a poor prognosis among patients with nonsmall cell lung cancer, implying a role for COX-2 in carcinogenesis (11, 12). Celecoxib, the first selective COX-2 inhibitor approved for chemoprevention of colon cancer in patients with familial adenomatous polyposis (13), has also been found to decrease the incidence of esophageal cancer in humans (14), and colon (15), gastric (16), lung (17, 18), mammary (19), oral (20), prostate (21), urinary bladder (22), and skin (23) cancer in various animal models with no associated toxicity. Moreover, a number of studies have shown that celecoxib at clinically feasible concentrations (£5.6 µ mol/L) markedly suppresses the biosynthesis of PGE2 in COX-2-expressing lung cancer cells (24, 25). Other important targets/pathways include the insulin-like growth factor axis, phosphoinositide 3-kinase pathway, cyclin D and E family members, and epigenetic events. In this chapter, we review the previous and the current randomized controlled trials (RCTs) of preventive agents and present our view for the future.

Lung Cancer Prevention

109

Principles of Chemoprevention The pathogenesis of lung cancer is the result of the stepwise accumulation of a number of genetic alterations that occur in the respiratory epithelium (26). Histologic examination of airway epithelium provides a clue for identifying epithelium that may be at risk for malignant transformation. The histology of preneoplasia in the respiratory epithelium includes the progression from squamous metaplasia through several grades of dysplasia (27, 28). However, the relationship between different steps of dysplasia and risk of invasion is not clear. Investigators have focused their attention on determining the genetic and phenotypic alterations that occur in the respiratory epithelium both in patients with lung cancer and in individuals at high risk for developing lung cancer, and correlated these alterations with the severity of the histological changes. Figure 1 shows a current understanding of the histological and molecular pathway for lung carcinogenesis (29). The term chemoprevention was coined by Sporn in 1976 to describe either pharmacologic or dietary interventions that would interfere in the carcinogenic process, resulting in a decreased cancer risk (2). Lung carcinogenesis can occur over 20–30 years, and more recent studies have appropriately chosen to evaluate the effects of treatment on premalignant lesions or inhibition of carcinogenic progression. Chemoprevention studies can further be categorized into three distinct areas (primary, secondary, and tertiary), and current investigations in each area should advance the field. Primary chemoprevention measures the development of cancer in a high-risk population (e.g., current/former smokers with/without airflow limitation

Histological/Molecular Pathway of Lung Carcinogenesis

Normal Epithelium

Hyperplasia

Dysplasia

Carcinoma in Situ

3p, 9p loss FHIT loss RAR loss EGFR overexp. P53 8p loss Fig. 1  Histological/molecular pathway of lung carcinogenesis

Cancer

HER2/NEU exp 5q loss K_RAS BC2 P16 Cyclin D1 Cyclin E

110

N. Peled et al.

on spirometry), while secondary chemopreventive studies examine the development of cancer in subjects with precursor lesions (for example, severe dysplasia or atypical adenomatous hyperplasia (AAH)). Tertiary chemoprevention studies focus on the development of lung cancer in subjects with a previous cancer. Chemoprevention has been applied with some early success to individuals at high risk for breast, prostate, and colon cancer, but there is no currently available chemoprevention for lung cancer. In fact, certain agents (b-carotene, N-acetyl cysteine) have been shown to increase cancer risk in current smokers (4, 6). Retinoids have received substantial attention in the past as potential lung cancer chemopreventive agents (30). A large body of epidemiologic, genetic, and cell biology data suggested that supplementation with b-carotene would be protective, although preclinical animal studies were not very supportive. No one would have predicted that the two large trials (the ATBC and CARET trials) conducted in the 1990s would each show a statistically significant increase in lung cancer incidence (approximately 20%) associated with b-carotene supplementation (particularly in current smokers) (3, 5). As mentioned, most of the clinical chemoprevention studies in lung cancer so far have all been focusing on lung cancer development as the primary endpoint. These studies have a long duration of many years and have included more than 100,000 individuals. In order to more rapidly develop and validate potential new agents, other approaches are being evaluated. At present, there are four major approaches to selecting promising agents for study in lung cancer chemoprevention trials: observational studies, analysis of the effects of new agents on cancer or dysplastic cell biology, preclinical animal models of lung carcinogenesis, and intermediate endpoint trials in humans. Since we currently have no validated lung cancer chemoprevention agents, none of these strategies is a reliable predictor.

Definition of High-Risk Groups Three requirements must be met for clinically effective chemoprevention. First, an adequately high-risk population must be readily identifiable (and historically, this has proven challenging for lung cancer). Second, effective agents with a tolerable side-effect profile must be available. Side effects for chemoprevention agents must be minimal, given that annual risk for cancer development is small. Subjects enrolled in tertiary chemoprevention trials have a previous history of cancer, and therefore more toxicity may be tolerated with chronically administered chemopreventive agents. This is a particularly important group of patients, as the risk of developing a second primary lung cancer after resection can be as high as 1–2% per year (31, 32), as well as 3% of local stump recurrence within 1 year of followup (32). Thirdly, endpoints to the clinical trials must be identified, defined, and validated in terms of demonstrating reduction in cancer development. For tertiary trials, this can be cancer incidence, but for secondary prevention trials, intermediate biomarkers must be present and must be associated with risk of progression.

Lung Cancer Prevention

111

For lung cancer, one could argue that there is no current gold standard biomarker, and that histologic changes are used as a similar paradigm to the development of other epithelial cancers. However, histologic changes may not prove to be the best biomarker, and advances in genomics, proteomics, and molecular imaging studies may increase our understanding and better define endpoints. One in nine smokers eventually develops lung cancer (33), and epidemiologic studies have been able to more accurately identify high-risk populations. Bach and colleagues developed and validated a model of lung cancer risk based on age, gender, tobacco smoke, and asbestos exposure history (34). Clinical experience has shown that significant variation in risk within smokers is evident, with 50% of lung cancers occurring in the highest risk quartile and only 8% in the lowest risk quartile (34). Risk of developing lung cancer in the upper quartile is up to 1.5% per year. The University of Colorado SPORE in Lung Cancer recruited and followed a cohort of high-risk current and ex-smokers with airflow obstruction (35). The overall rate of incident lung cancer in this group was 1.85 per 100 person-years, or six times that required for tamoxifen chemoprevention of breast cancer (0.3% per year) (36, 37). Therefore, high-risk groups for lung cancer can easily be identified on the basis of age, smoking history, exposure to asbestos, pulmonary function, and family history. A very recent published report also showed an association of spiral CT-detected emphysema and risk of developing lung cancer (OR 3.56, 95% CI 2.21–5.73) (38). Ongoing genetic testing should further clarify and aid in better defining lung cancer risk in the near future and improve the ability to identify even higher risk subpopulations. For example, a comprehensive study of somatic mutations in 188 human lung adenocarcinomas identified 26 genes mutated at significantly high frequencies and provided information on new signaling pathways involved in lung tumorigenesis, and may help identify new chemopreventive and chemotherapeutic targets (39). The Liverpool Lung Project Risk Model integrates several factors to quantify the risk for lung cancer within 5 years. Significant risk factors were family history of lung cancer (with particularly high risk in those with relatives diagnosed with lung cancer at age under 60), prior diagnosis of pneumonia, prior diagnosis of another type of cancer, occupational exposure to asbestos and duration of smoking (40). Such quantification may guide patient enrollment into screening programs and chemoprevention trials.

Biomarkers and High-Risk Groups High-risk groups might also be identified by biomarkers assessed using different tests, such as sputum examination (i.e., for cytology, gene methylation, FISH, etc), bronchial biopsy (i.e., histology and genetic abnormalities), plasma proteomics, urine analysis, and breath sampling for volatile organic compounds. Multiple changes are seen in the lungs of smokers, including dysplastic and angiogenic histologic abnormalities, increased proliferation, chromosomal deletions, rearrangements and gains, gene amplification, point mutations, gene silencing by

112

N. Peled et al.

promoter methylation and histone acetylation, and aberrant gene and protein expression (41). Whether these abnormalities are associated with the development of lung cancer or are epiphenomena of lifetime exposures, such as age and smoking, is not fully understood yet. However, it seems that a combined panel of such abnormalities might quantify the risk for lung cancer. The exact power of such combinations is under current evaluation in programs such as our Colorado High Risk Cohort. Biomarkers of lung cancer risk can have a variety of clinical uses, including population screening for early detection, monitoring of lung cancer therapy, and as measurements guiding clinical decision making. Several biomarkers, including sputum cytology, promoter methylation of a panel of genes, and chromosomal aneusomy in the sputum, have been prospectively assessed by our group and others (36, 42). Recently, a set of volatile organic compounds have been identified as unique for lung cancer patients (43, 44). The significance and the potential of this observation are currently under investigation by our group using novel nanotechnology (45).

Smoking Cessation Current or past tobacco smoking (the majority of which is in the form of cigarettes) is responsible for approximately 87% of lung cancers in the United States (46). Worldwide, the development of lung cancer may also be attributed to smoke exposure from the burning of wood and other solid fuels for cooking and heating. This biomass smoke exposure has been associated with asthma, COPD, and chronic bronchitis (47). A report by Delgado and colleagues characterized a Mexican cohort of 62 lung cancer patients in which 38.7% (n = 24) had an association with wood smoke exposure (48). It is abundantly clear that the absolute best method to prevent lung cancer is smoking cessation. The National Lung Health study was a randomized trial of the health benefits of smoking cessation conducted in the US and Canada. After a total of 14.5 years of follow-up, 731 of 5,887 patients had died of the following causes: lung cancer (33%); cardiovascular disease (22%); respiratory disease (7.8%); and unknown causes (2.3%). Mortality rate was significantly lower in the smoking cessation treated group, although only a minority succeed with smoking cessation (49). There was a significant mortality difference between the special intervention group and the usual care group in the youngest tertile of participants (those younger than 45 years of age). There were no significant differences in the cause for mortality between the groups except respiratory disease, although there was a trend toward higher incidence of heart disease and lung cancer in the usual care group (49). Besides complete abstinence from smoking, reduction in tobacco smoking has been related to decreased lung cancer rates. However, studies have consistently shown that the risk reduction was disproportionately smaller than the reduction in tobacco consumption (50, 51). These same studies also confirm that never smokers and former smokers had decreased rates of smoking-related cancers compared to heavy smokers

Lung Cancer Prevention

113

(defined as ³20 cigarettes smoked per day), and they also reinforce the advantage of complete smoking cessation compared to a decrease in consumption. Successful smoking cessation has been a difficult goal to achieve. Clinical experience has proven that a combination of pharmacologic agents and behavioral counseling leads to the best abstinence rates. Pharmacologic agents approved for smoking cessation include nicotine replacement products (gum, patch, inhaler), bupropion, and varenicline. Meta analysis (52) for the efficiency of those interventions (32,908 patients) showed the following odds ratio for smoking cessation: varenicline ([OR] 2.41, 95% credible interval [CrI] 1.91–3.12), nicotine nasal spray (OR 2.37, 95% CrI 1.12–5.13), bupropion (OR 2.07, 95% CrI 1.73–2.55), transdermal nicotine (OR 2.07, 95% CrI 1.69–2.62), nicotine tablet (OR 2.06, 95% CrI 1.12–5.13) and nicotine gum (OR 1.71, 95% CrI 1.35–2.21). Varenicline is the most recently approved product and is an oral, selective a4b2 nicotinic acetylcholine receptor partial agonist. Phase 3 studies comparing varenicline and bupropion abstinence rates have been completed, and they consistently showed the best continuous abstinence rates after 12 weeks to be in vareniclinetreated subjects (44.0% vs. 29.5% in one study (53) and 43.9% vs. 29.8% in another study (54)). The continuous abstinence rates were also significantly improved in the varenicline group after 52 weeks (53, 54). While smoking cessation does reduce the risk of developing lung cancer, this risk never fully returns to that of never smokers (Fig. 2) (55). For men who stopped at ages 60, 50, 40, and 30, the cumulative risks of lung cancer by age 75 were 10, 6, 3, and 2% (55), as presented in the Fig. 2. This fact, coupled with the epidemiologic finding that the majority of lung cancers in the US are diagnosed in former smokers (56), makes lung cancer chemoprevention an extremely important area of investigation. The rest of this review discusses current and future chemopreventive targets.

18 16

Death Risk (%) from Lung Cancer at age 75

14 12 10 8 6 4 2 0 Never Smoker

30

40

50

60

Never Stopped

Age when stoppted smoking (years)

Fig. 2  Effects of stopping smoking at various ages on the cumulative risk (%) of death from lung cancer up to age 75 (55)

114

N. Peled et al.

Intraepithelial Neoplasia and Atypical Alveolar Hyperplasia Similar to other common cancers, lung cancer develops as the result of predictable histologic and genetic abnormalities. The development of squamous cell lung cancer in the central bronchial epithelium starts with normal epithelium and progresses through hyperplasia, metaplasia, dysplasia, carcinoma in situ to invasive squamous cell lung cancer. The term intraepithelial neoplasia (IEN) has been used to describe the precursor lesions (most often moderate to severe dysplasia) that precede the development of carcinoma in situ and invasive cancer (57, 58). The presence of IEN, which is most reliably detected with fluorescence bronchoscopy (59, 60), can then be used to define high-risk cohorts, and the genetic abnormalities present in IEN can be used to define cancer risk and assist in the selection of agents for trials. The natural history of endobronchial dysplastic lesions is difficult to predict, as some of them may be completely removed at the time of biopsy. Published reports have stated that 37% of severe dysplasias persist or progress (61), and 50% of carcinoma in situ lesions transition to invasive squamous cell lung cancer (62). Most importantly, these severely dysplastic lesions can be targets of chemopreventive trials and, because they typically contain fewer genetic derangements and signaling abnormalities than do established cancers, they may be more amenable to treatment. The premalignant lesions for adenocarcinomas (BAC and invasive adenocarcinomas) are microscopic proliferations of atypical pneumocytes that are termed atypical alveolar hyperplasias (AAH) (63, 64). These lesions may be detected on high-resolution CT as ground glass opacities. AAH contain many of the genetic alterations seen in invasive adenocarcinoma (including Kras mutations, p53 mutations, epidermal growth factor receptor mutations (65), and loss of heterozygosity on chromosomes 3p, 9p, 17p, and 16q) (63). Promoter hypermethylation has also been observed in AAH, and advanced histologic grade was associated with more hypermethylation of tumor suppressor genes (66, 67). It is not known at what frequency AAH progress and how these can be reliably modeled for preclinical testing. This may be better determined, as the number of AAH lesions undergoing longitudinal clinical evaluation increases coinciding with improved thoracic imaging modalities employed in screening trials.

Randomized Clinical Trials Retinoid RCTs As noted briefly above in “Introduction,” several of the completed RCTs in lung cancer prevention have involved retinoids (Table  1) (3, 5, 6, 68–77). Strong epidemiological and laboratory data supported RCTs of retinoids in patients with lung premalignancy, or intraepithelial neoplasia (IEN), or resected lung cancer. The first hints of a role for retinoids – vitamin A and its natural and synthetic

Anethole dithiolethione vs. placebo × 6 months Fenretinide vs. placebo × 6 months

112 (72)

BC beta-carotene; AT alpha-tocopherol

20 (105)

Heavy current smokers

Celecoxib  × 6 months

Iloprost vs. Placebo  × 6 months

Isotretinoin vs. placebo × 6 months

86 (68) 125 (177)

Retinol 6 months

81 (74)

Current and former smokers

Current or former smokers with ³30 pack-year history and bronchial dysplasia Chronic smokers with dysplasia or metaplasia index >15%

82 (71)

150 (69) 112 (75)

Current smokers Current or former smokers with ³30 pack-year history and bronchial dysplasia Current or former smokers with ³30 pack-year history and bronchial dysplasia Current smokers with dysplasia or metaplasia

Isotretinoin vs. placebo × 3 years Retinyl palmitate, N-acetyl acetylcysteine, open label, 2 years BC − retinol vs. placebo × median of 5 years 9-cis-retinoic acid vs. isotretinoin +  AT vs. placebo × 3 months Etretinate vs. placebo × 6 months Budesonide vs. placebo × 6 months

1166 (76) 2,592 (1,023 lung) (6) 755 (70) 226 (73)

BC + retinol vs. placebo × 5 years

18,314 (5)

Former smokers with ³20 pack-years of use

Former asbestos workers

Smokers, former smokers, and workers exposed to asbestos Resected stage I non-small-cell lung cancer Lung, head and neck cancer

Table 1  Randomized Controlled Trials (RCT) in lung cancer chemoprevention Cohort Sample size/ref Intervention and duration SELECT trial; prostate prevention trial 35,533 (77) Oral selenium and/or Vit E × 7 years Male smokers age 50–69 years 29,133 (3) AT, BC vs. placebo × 5–8 years

Serial bronchoscopies with a significant decrease in Ki-67

Decrease in bronchial dysplasia in former smokers

RAR-b expression restored with 9-cis-RA, P = 0.03; no change with isotretinoin Sputum cytologic atypia – no significant effect Bronchial dysplasia regression or progression rates – no significant effect Bronchial dysplasia regression rate – no significant effect Bronchial metaplasia/dysplasia – no significant effect Bronchial dysplasia regression or progression rates – no significant effect Metaplasia index – no significant effect

Sputum cytologic atypia – no significant effect

Second primary tumor – no significant effect Second primary tumor – no significant effect

Primary efficacy Primary lung ca – no significant effect Primary lung cancer incidence – AT (2% reduction); BC (18% increase) Primary lung cancer incidence – 28% increase

Lung Cancer Prevention 115

116

N. Peled et al.

analogs – in lung neoplasia came from pioneering work by Wolbach and Howe reported in 1925, indicating that vitamin A deficiency caused squamous metaplasia in the trachea and other sites that could be reversed by vitamin A replacement (78). Vitamin A deficiency enhances tobacco-induced carcinogenesis in preclinical lung systems. Retinoid effects are mediated through nuclear retinoic-acid receptors (RARs) and retinoid X receptors (RXRs) (each with the three subtypes alpha, beta, and gamma), which are downregulated during lung tumorigenesis (79). The RAR-b subtype may be the primary mediator of retinoid effects (80). RAR-b expression is suppressed by tobacco carcinogens, and is progressively lost (to a greater degree than is any other retinoid receptor) in multistep lung tumorigenesis in association with multiple defects in retinoic acid signaling upstream and downstream of nuclear retinoic acid receptors (81, 82). Retinoid RCTs have been conducted in the increased risk settings of lung IEN, smokers without IEN or cancer, and resected NSCLC (Table 1). In these trials, including one conducted only in patients with lung dysplasia who were evaluated with fluorescence bronchoscopy, retinoids produced no significant effects on histopathologic alterations (74, 83). A large-scale RCT to prevent primary lung cancer in persons at high risk primarily from cigarette smoking, the BetaCarotene and Retinol Efficacy Trial (CARET) (5), involved the retinoid retinyl palmitate (retinol) but was stopped early because of a no-benefit analysis and findings of possible harm that confirmed the observation of an adverse effect of b-carotene in the large-scale Alpha-Tocopherol, Beta-Carotene (ATBC) Study (3). Although the precise influence of retinyl palmitate in CARET could not be determined since this trial tested the retinoid only in combination with b-carotene, another RCT, the European Study on Chemoprevention with Vitamin A and N-Acetylcysteine (EUROSCAN), found that retinyl palmitate (at a much higher dose) had no effect on the rate of lung cancer or second primary tumor (SPT) development in lung and head and neck cancer patients (6). The Lung Intergroup Trial (LIT) was a large-scale RCT of low-dose isotretinoin (vs. placebo) in patients with resected stage-I NSCLC (76). Isotretinoin did not influence SPTs or the prespecified secondary end-points of recurrence or mortality in the overall analysis. Subgroup analyses of LIT suggested that isotretinoin may have increased recurrence and mortality in active smokers, had no effect in former smokers, and decreased recurrence and mortality in never smokers, suggesting a smoking-by-treatment interaction. Factors likely contributing to the lack of a clinical benefit of retinoids in lung cancer prevention are epigenetic silencing (via methylation or histone deacetylation) of RAR-b and cellular retinal binding protein-1, along with other complex signaling aberrations in lung carcinogenesis that are associated with retinoid resistance (81, 82). There are inconsistent reports on the associations between RAR-b expression patterns and lung cancer chemoprevention and prognosis. For example, retinoids both did and did not upregulate RAR-b expression in lung chemoprevention trials (73, 74, 83); RAR-b expression has been associated with both significantly increased (79) and decreased (84) survival in resected NSCLC; and RAR-b has been correlated with the generally poor lung cancer prognosis markers cyclooxygenase-2 (COX-2)

Lung Cancer Prevention

117

(in human NSCLC specimens) and human telomerase reverse transcriptase (in bronchial biopsies of smokers without cancer) (85). The apparent inconsistencies may involve complex interactions of smoking with RAR-b, as suggested by a recent study (in NSCLC patients) of RAR-b promoter methylation, which was associated with a lower SPT rate in active smokers but a higher SPT rate in former smokers (86). The reported inconsistencies also may be due to the relative expressions of different RAR-b isoforms, such as RAR-b2 (which appears to be antineoplastic) and -b4 (which appears to be oncogenic) (87). RAR-b2 expression frequently is lost or reduced in various cancer cells and tissues, and transfecting RAR-b2 suppresses epidermal growth factor receptor (EGFR) and COX-2 expression, cell growth, colony formation, and tumorigenicity (88). Generated by alternative splicing from the same primary transcripts that generate RAR-b2, RAR-b4 may be a dominant-negative form of RAR-b2 and is overexpressed in lung and other cancer cell lines. Recent in  vivo studies in esophageal squamous cancer showed that reduced b2 (and b1) expression was associated with increased b4 and reduced chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) (88). COUP-TFs are important in retinoic acid induction of RAR-b2, growth inhibition, and apoptosis in vitro. Stable expression of COUP-TF can overcome retinoic acid resistance in nur77-positive lung cancer cells. The differential expressions and effects of RAR-b isoforms are a potential new area for molecular-targeted lung cancer chemoprevention. The relationships of RAR-b, RXRs, and retinoic acid signaling with epigenetic events and other signaling pathways/targets (e.g., EGFR, COX-2, and cyclin D1) are discussed later in “Molecular Targeting of Lung Carcinogenesis” section.

ATBC Study The ATBC study tested b-carotene and vitamin E in the form of alpha-tocopherol (AT) in a 2 × 2 factorial design in 29,133 male smokers (3). Despite consistently positive epidemiologic data showing an inverse correlation between dietary estimates of b-carotene intake or serum b-carotene levels and lung cancer risk, b-carotene supplements in the ATBC trial significantly increased lung cancer incidence (the primary end point) and mortality rather than decreasing them (89). This harmful result with an agent considered to be virtually nontoxic was so surprising that it met with substantial doubt (90) until the CARET study (5) subsequently reported similar results with its combination of b-carotene and retinyl palmitate. A post-ATBC-intervention analysis with extended follow-up indicates that the b-carotene effects wore off over time (91). As opposed to the harmful effect seen in smokers, RCTs of b-carotene in nonsmokers produced neutral results (4). AT in the ATBC trial produced no overall effect on lung cancer incidence (3). Other analyses from this trial indicated a beneficial trend in lung cancer rates associated with long-term use of AT, a significant 19% reduction in lung cancer risk associated

118

N. Peled et al.

with the highest versus the lowest quintile of serum AT, and a stronger protective effect of AT in men with fewer years of smoking. The effects of higher-dose, longerterm AT on lung cancer risk in a population that generally smoked far less (compared with the ATBC trial) has been examined in an RCT, the Selenium and Vitamin E Cancer Prevention Trial (SELECT), which failed to show any positive clinical benefit (decreasing prostate, lung or colon cancer) by consuming selenium and/or vitamin E (77, 89, 92).

Selenium RCTs The possible molecular mechanisms underlying selenium effects in carcinogenesis may involve methylation and polyunsaturated fatty acid metabolism. Selenium can inhibit DNA cytosine methyltransferase-1 and suppress COX-2 and 5-lipoxygenase (5-LOX) expression (92, 93). The Nutritional Prevention of Cancer (NPC) trial was designed to test selenium primarily in reducing nonmelanoma skin cancers in 1,312 individuals at a high risk of this disease. The NPC trial did not prevent skin cancer but initially did produce a significant 44% secondary decrease in lung cancer incidence (94). The final result was a 26% (nonsignificant) decrease in lung cancer risk (95). A recent epidemiologic meta-analysis (96) also showed a 26% (significant) overall reduction in lung cancer risk associated with higher selenium exposures (the strongest benefit occurring in populations with low average selenium levels). Selenium has been tested for preventing SPTs in a National Cancer Institute Intergroup RCT in patients with resected early-stage NSCLC. As noted above, selenium has been tested in the SELECT trial, which showed a nonsignificant increase in prostate cancer and no effect on lung cancer incidence with a non significant increase in diabetes mellitus rates (77).

Anethole Dithiolethione and Budesonide RCTs An RCT of the organosulfur compound anethole dithiolethione (ADT) (which can increase glutathione-S-transferase and other phase-II enzymes) in 112 current and former smokers with bronchial dysplasia did not significantly reverse dysplasia (the primary end point) but did reduce dysplasia progression (vs. placebo) (72). An RCT of inhaled budesonide (a corticosteroid) in smokers with dysplasia found no significant effect of this drug on dysplasia regression (the primary end-point) but did find a provocative significant, albeit modest, decrease in the proportion of spiral computed tomography-detected peripheral nodules, which was consistent with data from studies of aerosolized glucocorticoids in mice. These studies suggested a beneficial effect of glucocorticoids on the peripheral adenocarcinoma precursor lesion atypical adenomatous hyperplasia (75).

Lung Cancer Prevention

119

Molecular Targeting of Lung Carcinogenesis Polyunsaturated Fatty Acid Metabolic Pathways Linoleic and arachidonic n-6 polyunsaturated fatty acid metabolic pathways play a key role in tumorigenesis. LOXs and COXs mediate the oxidative metabolism of these fatty acids into the bioactive lipids prostaglandins (PGs), hydroxyeicosatetraenoic acids (HETEs), and hydroxyoctadecadienoic acids (HODEs; Figs.  3 and 4) (97, 98). Growing evidence indicates that there is a dynamic balance between enzymes involved in the metabolism of linoleic acid and arachidonic acid (AA), including recent evidence suggesting that enzymes involved in AA metabolism and their metabolic products have both pro- and anti-tumorigenic effects. The study of the complex positive and negative interactions (e.g., substrate shunting and dynamic balances) involving these signaling pathways is leading to new approaches for lung cancer prevention, including combinatorial chemoprevention targeting different signaling events. Arachidonic Acid Metabolism Cytosolic phospholipase A2 (cPLA2) preferentially hydrolyzes membrane phospholipids at the sn-2 position to release AA, making cPLA2 the rate-limiting enzyme in eicosanoid production (Fig.  4). cPLA2 expression is increased in lung cancer, and

Fig.  3  Polyunsaturated fatty acid (linoleic and arachidonic) metabolic pathways and potential targets within them involved in lung carcinogenesis. 15-LOX-1 15-lipoxygenase-1; 13-S-HODE 13-S-hydroxyoctadecadienoic acid; 15-S-HETE 15-S-hydroxyeicosatetraenoic acid; LTA4H leukotriene A4 hydrolase; COX-1 cyclooxygenase-1; PGH2 prostaglandin H2 (adapted from Hirsch et al. (8), with permission)

120

N. Peled et al.

Fig. 4  Prostanoid biosynthesis. Certain targets within this pathway potentially could be downregulated, for example, prostaglandin E2 (PGE2), or upregulated, e.g., prostacyclin (PGI2), by targeted lung cancer prevention agents and combinations. cPLA2 cytosolic phospholipase A2; COX-1 cyclooxygenase-1; PGDS PGD synthase; mPGES-1 microsomal PGE synthase-1; TxAS thromboxane A2 synthase (adapted from Hirsch et al. (8), with permission)

cPLA2 gene-knockout mice developed significantly fewer tumors in a lung tumorigenesis model (99). Free AA is converted to unstable endoperoxide PGG2 by the two known forms of COX: COX-1, which is constitutively expressed in most cell types and is involved in maintaining cellular homeostasis; and COX-2, which is an immediate early-response gene and is highly regulated by a number of pathways and stimuli, including mitogenic stimuli that induce COX-2 in association with inflammation and cancer. PGG2 is reduced to PGH2, which is isomerized by terminal synthases into the various prostanoids (Fig. 4), which are relevant to lung tumorigenesis. It appears that the profile of the downstream COX metabolites and targets is more relevant to tumorigenesis than is COX-2 protein or activity itself (98). COX-2, PGE2, and PGI2 In NSCLC, COX-2 can enhance a number of tumorigenic events (via a number of COX-2-dependent genes), including angiogenesis (e.g., via CXC ligand 5 and vascular endothelial growth factor), invasion (e.g., via CD44 and matrix metalloproteinase-2), immunosuppression (e.g., via interleukin-10 and -12) and apoptosis

Lung Cancer Prevention

121

resistance (e.g., via survivin and insulin-like growth factor binding protein-3). COX-2 is progressively upregulated in lung carcinogenesis, is frequently overexpressed in human lung IEN and NSCLC, and is reported to be associated with a poor prognosis in stage I NSCLC (100). Increased COX-2 leads to elevated PGE2 levels, and recent work suggests that the PGE2 receptor type 3 is involved in the appearance of the malignant phenotype in lung cells and that Src signaling is involved in PGE2-dependent cell growth in the lung (101). Nonsteroidal antiinflammatory drugs (NSAIDs), including COX-2 inhibitors, are active (e.g., induce apoptosis) in vitro in NSCLC (100). Cigarette smoke activates nuclear-factor kappa B (NF-B) in NSCLC in association with the induction of the NF-B-regulated gene products COX-2, cyclin D1, and matrix metalloproteinase-9. These effects were blocked by the COX-2 inhibitor celecoxib (102). Epidemiologic data are mixed but include case–control studies suggesting that regular aspirin use (in light smokers) or a COX-2 polymorphism is associated with NSCLC risk (100). Animal NSAID studies are mostly positive and include studies showing COX-2 inhibitors preventing nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse lung tumors and suppressing NSCLC xenograft growth and showing that disrupting COX-2 or EP2 reduces Lewis lung carcinoma growth (98, 100, 103). A recent study in 134 advanced NSCLC patients treated by chemotherapy ± COX-2 inhibitors did not produce a survival benefit, but patients whose tumors expressed high COX-2 levels had better survival when treated by celecoxib. The relevance of this finding to chemoprevention should be further assessed (104). Such studies are currently enrolling. Preliminary results from those trials show that using celecoxib for 6 months as a chemopreventive agent in heavy current smokers reduced the Ki-67 index by 35% and increased the expression of nuclear survivin by 23% in the bronchial epithelium (105). A separate Phase II trial evaluating the effects of the non-specific COX inhibitor sulindac on endobronchial histology has also been initiated. COX-2 inhibition has been associated with adverse cardiovascular events, which may be avoided by targeting PGI2, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), and other targets (e.g., thromboxane synthase (106)) related to but downstream of COX-2. Important interactions of COX-2 with EGFR (and other targets/pathways) are discussed in the following sections. PGI2 Increasing evidence suggests that PGI2 in the lung is antineoplastic, and this may be associated more with effects on tumor cell-host interactions than with a direct inhibition of primary tumor growth. Normal lung tissue contains high levels of PGI2 and relatively low levels of PGE2, whereas these levels are reversed in NSCLC. These findings have generated the hypothesis that a high level of PGI2 may protect against lung tumor formation. Therefore, PGI2 may be the antineoplastic flipside of proneoplastic PGE2, and shifts in the balance of production of these prostanoids may play a role in promoting (or reversing) tumor progression (107).

122

N. Peled et al.

The team investigating PGIS overexpression in mice also has found that iloprost (a PGI2 analog) has dose-dependent preventive effects in mouse lung tumorigenesis models. PGI2 analogs decreased the production of proinflammatory cytokines and chemokines and increased the production of anti-inflammatory cytokines IL-10 (108) as well as preventing pulmonary endothelial cell apoptosis induced by cigarette smoke (109). These findings further support the rationale for a recently published RCT of iloprost (vs. placebo) to treat IEN by the US Lung Cancer Biomarker and Chemoprevention Consortium in high-risk smokers with sputum atypia. The primary endpoint for the trial is endobronchial histology, and subjects have fluorescence bronchoscopy performed at study entry and after 6 months of treatment. A total of 125 subjects completed both bronchoscopies (60 patients on iloprost; 65 placebo) and review of endobronchial biopsies showed that former smokers randomized to iloprost treatment had significant improvements in the bronchial dysplasia compared controls. Current smokers did not exhibit similar improvements (177).

15-PGDH Recent data suggest that 15-PGDH, which degrades PGE2 and other PGs, has tumor-suppressor activity. 15-PGDH gene expression is suppressed in NSCLC specimens, and overexpression of 15-PGDH can inhibit tumor growth in A549 lung adenocarcinoma xenografts as a result of the depletion of PGE2, downregulation of Bcl-2 associated with induction of apoptosis, and inhibition of tumor invasion and metastasis (via downregulation of CD44) (110). These exciting lung cancer findings are consistent with recent related data from colon and bladder cancer studies and have initiated novel approaches for inducing 15-PGDH to reverse carcinogenesis. Promising recent data show that 15-PGDH expression was induced by peroxisome proliferator-activated receptor gamma (PPARg) ligands in NSCLC and by an EGFR tyrosine kinase inhibitor (TKI) and transforming growth factor beta in colorectal cancer cells (111). The NSAID indomethacin induced 15-PGDH expression in human medullary thyroid carcinoma cells and in one of four colorectal cancer cell lines (111). In addition, 15-PGDH was shown to be a target of hepatocyte nuclear factor 3 beta and a tumor suppressor in NSCLC (112).

5-LOX and Leukotriene Modifiers Tobacco carcinogens can increase 5-LOX and leukotriene B4 (LTB4) in addition to increasing COX-2. 5-LOX is expressed in lung cancer cell lines and primary tumors, and its product 5-HETE increases lung cancer cell growth. Its expression was neither prognostic nor predictive (104); however, inhibition of 5-LOX, 5-LOX activating protein, and leukotriene synthesis inhibits lung tumorigenesis in  vitro and in  vivo in animals (97, 113, 114). The beneficial effects were much greater when the 5-LOX inhibitor was combined with the nonspecific COX inhibitor aspirin.

Lung Cancer Prevention

123

Results of the combined COX–LOX inhibitors suggest the potential benefit of blocking more than one AA metabolic pathway. Recent data provide evidence for the shunting of AA metabolism from COX-2 to 5-LOX. A clinical study assessed celecoxib effects on PGE2 and LTB4 in alveolar macrophages from bronchoalveolar lavage in heavy smokers. After 1 month of celecoxib treatment, PGE2 was suppressed and LTB4 was increased (compared with baseline measures) in alveolar macrophages stimulated with a calcium ionophore ex vivo. Furthermore, LTB4 generation was markedly increased and then inhibited by a 5-LOX (but not by a COX-2) inhibitor in macrophages exposed to the proinflammatory stimulus of a lipopolysaccharide ex vivo, further supporting combined COX and 5-LOX inhibitors for lung cancer prevention (115–117). Recent data in colon carcinogenesis illustrate the potential complexity of cross talk between 5-LOX and COX-2 and the promise for lung cancer prevention by the natural agents curcumin and green tea, which can inhibit these pathways (118–120). Green tea extract contains multiple polyphenols (including polyphenol E), and the protective effects in cancer chemoprevention arise from the inhibition of cytochrome p450 (thereby blocking the bioactivation of carcinogens) and the activation of phase II detoxifying enzymes via the MAPK pathway (121). Broccoli sprout extract contains phytochemicals that also activate phase II detoxifying enzymes and protect against oxidative-stress-induced DNA damage. Trials of oral supplementation with both agents are also currently being conducted, particularly in premalignant lesions with specific genetic alterations. 12-LOX AA metabolism by 12-LOX results in the stable end product 12-HETE, a signaling molecule that can activate NF-B via protein kinase C-alpha and promote cell proliferation, apoptosis resistance, motility, adhesion, invasiveness, and angiogenesis (122, 123). Lewis lung cancer cells synthesize 12-HETE (which can enhance tumor cell adhesion to endothelial cells and the subendothelial matrix) and are inhibited by a selective 12-LOX inhibitor. A549 lung adenocarcinoma cells also can be inhibited by 12-LOX inhibitors. 15-LOXs 15-LOX products play important roles in lung carcinogenesis. Although not expressed in A549 lung adenocarcinoma cells, 15-LOX-1 is expressed in human bronchial epithelial cells in a differentiation-dependent fashion and in an inverse association with COX-2 expression (97). NSAIDs (including COX-2, broad COX-1 and -2, and non-COX inhibitors) and histone deacetylase (HDAC) inhibitors have been shown to induce 15-LOX-1 expression in colon cancer cell lines, and this 15-LOX-1 expression was mechanistically linked to apoptosis induction and suppression of tumorigenesis (97, 124, 125).

124

N. Peled et al.

15-LOX-2 expression was reported to be variable in NSCLC specimens and to correlate inversely with tumor grade and proliferation (126). 15-HETE is the product of 15-LOX-2, and the growth of A549 cells was shown to be inhibited by 15-HETE-mediated downregulation of PGE2 production (127) and activation of PPARg (128). Many COX-2, and 5- and 12-LOX pathway effects can be antagonized by 15-LOX products in lung cancer and other experimental systems. For example, the 15-LOX-1 product 13-S-HODE competitively inhibits 12-HETE binding to its cell-surface receptor and suppresses LTB4 production (97, 129–131).

EGFR EGFR is upstream of several important lung prevention targets, including COX-2 and cyclin D1 (discussed here) and PI3K and STAT3. High EGFR (ErbB1) gene copy number and protein expression occur in lung IEN and have been associated with a poor prognosis in resected NSCLC, and EGFR inhibitors have activity in a mouse lung cancer prevention model and in NSCLC therapy (in association with high EGFR) (132). In 134 bronchial dysplastic biopsies, there was a linear relationship between severity of the dysplastic changes and increasing marker expression for EGFR, Ki-67, and minichromosome maintenance protein 2 (133). Tobacco carcinogens activate EGFR signaling, which increases PGE2, in vitro and in vivo, in two ways – by inducing COX-2, which makes PGE2, and downregulating 15-PGDH, which degrades PGE2 (111, 134). PGE2 in turn further activates EGFR signaling (Fig.  5). EGFR inhibition (via an antibody or a TKI) can abrogate smoke-induced COX-2 induction. ErbB2 and -3 also appear to be involved in lung tumorigenesis. For example, ErbB2 (HER-2) is overexpressed in lung IEN and is associated with a poor prognosis in lung cancer, and its inhibition reduces COX-2 expression (134). PGE2-stimulated NSCLC cell growth is resistant to EGFR inhibition, suggesting that combined targeting may be more effective than targeting either EGFR or COX-2 alone. Targeting both EGFR and COX-2 has produced promising preclinical results, including recent additive-to-synergistic activity in inhibiting the growth of head and neck cancer in vitro and in vivo (135). EGFR activation can induce (via several pathways) cyclin D1, which is overexpressed in lung IEN and cancer. The cyclin D1/cyclin-dependent kinase-4, -6 complexes can phosphorylate retinoblastoma (Rb), modulate HDAC, and be inhibited by p16. Animal model data also support cyclin D1 as a target for lung cancer chemoprevention. One way to modulate cyclin D1 is indirectly through effects on EGFR. Studies in immortalized human bronchial epithelial cells exposed to the tobacco carcinogen NNK showed that retinoic acid reduced EGFR expression, which led to reductions in cyclin D1 expression, mitogenesis, and cell transformation (136). An EGFR TKI also suppressed cyclin D1 expression and activation in immortalized human bronchial epithelial cells and certain NSCLC cell lines in association with growth inhibition. Limited clinical data in laryngeal and lung carcinogenesis suggest that

Lung Cancer Prevention

125

Fig. 5  Interactions of epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2). EGFR decreases 15-hydroxyprostaglandin dehydrogenase (15-PGDH) expression (as represented by the dotted line terminated by an inhibition bar). 15-PGDH catabolizes and thus decreases PGE2 levels (as represented by the solid line and inhibition bar). EP1–4 prostaglandin E2 receptor types 1–4; PGE2 prostaglandin E2; mPGES microsomal PGE synthase; MAPK mitogen-activated protein kinase; AP-1 activator protein-1 (adapted from Hirsch et al. (8), with permission)

response to an EGFR TKI or retinoic acid treatment may correlate with cyclin D1 suppression (137, 138).

Retinoic Acid Signaling The extensive study of retinoids in tobacco-related carcinogenesis has led to important advances in the understanding of lung carcinogenesis and molecular targeting (e.g., involving EGFR, COX-2, AP-1, cyclin D1, ubiquitin-activating enzyme E1-like protein (139)). In addition, those pathways may be combined with retinoid signaling pathways, e.g., retinoids with methyltransferase or HDAC inhibitors to address the high rate of epigenetic silencing (e.g., of RAR-b) in lung carcinogenesis. Recent RCT data suggest that RXRs may be a target for lung cancer prevention – the retinoid 9-cis-retinoic acid, which binds RARs and RXRs (and activates RXR homo- and heterodimers), increased RAR-b and insulin-like growth factor

126

N. Peled et al.

binding protein-3 (IGFBP-3) in former smokers (73). An RXR agonist (rexinoid) plus an EGFR TKI recently showed promise in a preclinical lung carcinogenesis model (additive activity) and in a phase I trial. The appeal of rexinoids in combinations or alone in this setting includes their activity, lesser toxicity (compared with many other retinoids), and independence of RAR-b (e.g., they trigger proteasomal degradation of cyclin D1) and thus protection from the blocking effects of frequent RAR-b defects occurring in lung carcinogenesis (140). There also are data showing that several synthetic receptor-selective retinoids, such as Targretin, are more potent than is retinoic acid in inhibiting premalignant and NSCLC cell growth in vitro, and combined RXR and RAR ligands have additive activity.

Insulin-Like Growth Factor Axis Insulin-like growth factor (IGF)-mediated signaling is associated with NSCLC growth and metastatic potential. Increased serum IGF-1 and decreased IGFBP-3 levels have been associated with lung cancer risk; reduced IGFBP-3 has been associated with poor survival in stage I NSCLC. Lung cancer cells have high levels of IGFs, and IGFBP-3 and -6 induce apoptosis in NSCLC (141, 142). IGF-COX-2 signaling pathway interactions may be important in lung tumorigenesis. COX-2 has recently been shown to modulate the IGF axis at the level of IGFBP-3. IGF-1 increases COX-2 expression and PGE2 production; celecoxib has been shown to downregulate IGF-1 receptor and suppress IGF-II-related tumor growth. COX-2 suppression of IGFBP-3 increases related neoplastic effects via upregulation of phosphoinositide 3-kinase (PI3K)/Akt signaling (143). Ras-mediated signaling has been implicated in the development of IGFBP-3 resistance and led to studies evaluating farnesyltransferase (FTase) inhibitors, which were designed to block Ras activation. Ras-mediated pathways can be activated by early events in lung tumorigenesis, including EGFR activity. A recent study in NSCLC showed that the apoptotic activity of IGFBP-3 was increased by an FTase inhibitor in vitro and in vivo, possibly via FTase inhibitor effects on the PI3K/Akt pathway, which has important implications for combinatorial prevention and therapy (142).

PI3K/Akt PI3K activation is an early event in lung carcinogenesis, and recent studies indicate that activation of Akt, a downstream kinase, increases in tobacco-carcinogen-induced transformation in vitro (in human lung epithelial cells) and in vivo (in NNK-treated A/J mice) (144). Akt also is highly expressed in human bronchial dysplasia specimens (145). The inhibition of Akt (genetically or pharmacologically) induced apoptosis in premalignant and malignant (NSCLC) human lung cells and was preventive in an animal model of lung carcinogenesis. Deguelin and myo-inositol have preventive

Lung Cancer Prevention

127

activity in animal lung tumorigenesis models, and this activity of both agents is thought to work in part through suppressing the PI3K/Akt pathway (146, 147). mTOR is an important PI3K-pathway target in NSCLC, as potentially are mitogenactivated protein kinase kinase-4 and c-Jun N-terminal kinase.

PPARg Prostacyclin analogues like iloprost selectively increase PPARg activity both in nontransformed epithelial cells and in NSCLC. In human NSCLC cell lines, activation of PPARg by pharmacological agents, or by molecular overexpression, strongly inhibits transformed growth, as assessed by colony formation in soft agar (148, 149). Additionally, NSCLC cells overexpressing PPARg exhibit significantly less invasiveness and metastasis compared to control cells both in vitro and in a rat orthotopic lung xenograft model (148). The thiazolidinediones (TZDs) are oral PPARg agonists, and lung cancer incidence in diabetics on PPARg agonists was decreased 33% when compared to diabetics on non-PPARg modulating medications (150). TZDs have also been shown to induce 15-hydroxyprostaglandin dehydrogenase (15PGDH), the enzyme that inactivates the antiapoptotic and immunosuppressive PGE2 by conversion to 15-keto prostaglandins (151). A recent report has shown that PPARg overexpression chemoprevents murine lung cancer (152). This class of agents will likely be employed in future chemoprevention trials. PPARg is expressed in NSCLC cell lines (and primary lung tumors), and PPARg ligands can induce differentiation and apoptosis in vitro and inhibit tumor progression in NSCLC xenografts via PPARg-dependent and -independent pathways. These effects involve upregulating the cyclin-dependent kinase inhibitor p21, E-cadherin, and gelsolin, and downregulating cyclins D and E, MUC1, MMP2, and 5 integrin expression (153, 154). Overexpression of PPARg promotes differentiation and inhibits invasion and metastasis in NSCLC (148). There are complex interactions between PPARg ligands and polyunsaturated fatty acid metabolism. PPARg ligands can interact with AA metabolism in the lung at the level of 5-LOX, COX-2, and important downstream targets, for example, 15-PGDH and EP2. Furthermore, PGIS overexpression can induce PPARg expression, and some polyunsaturated fatty acid metabolic products (13-HODE and 15-HETE (Fig.  3) and PGJ2 (Fig.  4)) are PPARg ligands (97, 98, 155). Combinations of PPARg ligands with HDAC inhibitors greatly enhanced growth inhibition in NSCLC cells, which occurred in association with marked cyclin D1 suppression in NSCL adenocarcinoma cells (154).

Epigenetic Targets Epigenetic silencing of genes by DNA methylation and histone deacetylation are critical early events in human lung tumorigenesis (156). Extensive methylation studies

128

N. Peled et al.

in multistep lung carcinogenesis involve CDKN2A, which encodes p16 and p14, the methylation of which is associated with smoking, increases progressively in human lung carcinogenesis, and has been identified in precursor lesions of adenocarcinoma in rats. The detection of gene-specific promoter methylation of CDKN2A and/or MGMT in sputum was associated with the subsequent development of squamous cell lung cancer. Methylation also has been shown to inactivate certain other critical genes in the lung, including RAR-b, E-cadherin, laminins, and IGFBP-3. Genetic and pharmacologic DNA methyltransferase 1 (DNMT1) inhibition has been shown to decrease NNK-induced lung tumors. SiRNA knockdown of DNMT1 expression in lung cancer cells reduces promoter methylation of many genes but did not inhibit the malignant phenotype, as indicated by studies of soft agar colony formation (157). Selenium and the tea polyphenol epigallocatechin-3-gallate have been shown to inhibit DNMT activity and reactivate silenced genes, including RAR-b (156). The HDAC inhibitor suberoylanilide hydroxamic acid inhibits NNK-induced lung tumor multiplicity in mice (158). HDAC inhibition (pharmacologic or antisense) inhibits NSCLC growth in vitro, possibly in part by inducing p21 and RhoB expression (159). Studies of RAR-b inducibility by retinoic acid in lung and head and neck premalignancy models suggest that methylation and histone deacetylation are involved in RAR-b suppression and thus are promising preventive targets (160, 161). In several preclinical cancer settings, combined HDAC and methyltransferase inhibitors markedly increased reexpression of silenced genes such as p16. HDAC inhibitor-induced apoptosis is enhanced by methyltransferase inhibition in human lung cancer cells, suggesting that DNA methylation status is an important determinant of apoptotic susceptibility to HDAC inhibitors (162). A more recent study extended this approach to the in vivo setting in the NNK-induced lung tumor A/J mouse model (in which DNMT1 plays a critical role) and found that the combination of an HDAC and methyltransferase inhibitor reduced (163) lung tumor development by more than 50% (164).

Combinatorial Agents Due to the potentially large number of genetic and metabolic derangements in premalignant lesions, combinations of agents may prove to be the most effective route to chemoprevention. This could largely be directed by genetic or proteomic abnormalities detected in the dysplastic lesions. Similar to personalized lung cancer chemotherapy for advanced disease, chemoprevention may also be directed based on specific “signatures” (gene expression, proteomics, etc.) being present in biopsy specimens from high-risk individuals. The most effective chemoprevention may depend on abnormalities found in biologic specimens. A strong rationale exists for the use of combinations of agents that act in an additive or synergistic manner by increasing treatment efficacy and/or decreasing drug toxicity (8). For example, it was previously shown that N-(4-hydroxyphenyl) retinamide (4HPR; fenretinide), a synthetic derivative of retinoic acid, exerts potent

Lung Cancer Prevention

129

proapoptotic effects on a variety of cancer cells (165). Moreover, 4HPR combined with celecoxib inhibited growth and induced apoptosis of non-small cell lung cancer cell lines more efficiently than either agent alone did (166), suggesting further investigations for the treatment of human lung cancer. Preclinical and clinical cancer chemoprevention trials have indicated great promise for 4HPR and celecoxib administered as single agents (167, 168). This raises the question whether the combination of both would enhance their individual effects in an in vitro model of tobacco-induced human lung carcinogenesis. Preliminary results show that celecoxib combined with 4HPR at clinically attainable concentrations inhibited growth and induced apoptosis of premalignant and tumorigenic bronchial epithelial cell lines by activating the mitochondrial apoptosis pathway as well as by suppressing the Akt survival pathway.

Conclusion If this review has given the impression that lung carcinogenesis and its record of clinical and molecular research is extremely complicated, even confusing, this impression is not entirely accidental. Large-scale prevention RCTs of b-carotene (with compelling epidemiologic support), the retinoids (with even more compelling preclinical and early clinical support), and vitamin E (with support from its antioxidant profile) did not bring us any closer to controlling this devastating disease (169). Ongoing RCTs, may be helpful, but their results are as yet unknown. The smoke enshrouding lung cancer chemoprevention is beginning to lift because of promising molecular-targeted research that may lead to combinatorial approaches for preventing this highly complex, thus far refractory disease. Many targets and interactive signaling pathways show potential for developing targeted agents and combinations (Table  2). Targets (e.g., 15-PGDH and PGI2) within the polyunsaturated fatty acid metabolic signaling pathway and downstream of COX-2 are promising for lung cancer prevention. For example, 15-PGDH degrades PGE2, appears to have tumor suppressor activity, and can be induced both by PPARg ligands and an EGFR TKI (independently of their effects on COX-2). Other important targets/pathways include the IGF axis, PI3K pathway, cyclin D and E family members, and epigenetic events. Targets not previously discussed here, but with promising preclinical data for lung cancer prevention, include signal transducer and activator of transcription-3 (STAT-3), mammalian target of rapamycin (mTOR), and angiogenesis targets such as vascular endothelial growth factor and basic fibroblast growth factor (170–175). To succeed where all other approaches have failed thus far, clinical moleculartargeted prevention will depend on developing better preclinical lung models, identifying highest-risk populations (e.g., patients with lung IEN and patients who have undergone resection of early-stage lung cancer), developing novel early clinical trial designs, and resolving other practical concerns of clinical cancer prevention trials, such as determining what levels of toxicity would be acceptable in very high-risk

130

N. Peled et al. Table 2  Molecular Targets in Lung Cancer Prevention Targets COX-2 DNMT cPLA2 HDAC PGI2 RAR-b 15-PGDH RXRs 5-LOX NE-kB 12-LOX PI3K/Akt 15-LOX-1 PPAR-g 15-LOX-2 STAT3 EGFR FTase Cyclin D1 mTOR Cyclin E VEGF IGFBP-3 bFGF Target combination Cox-2 with EGFR, 5-LOX, 12-LOX, 150LOXEGFR with RXRs, PPAR-g, mTOR, STAT3, VEGF Cyclin D1 with other cyclines IGFBP-3 with FTase, PI3K/AKT HDAC with DNMT, RAR-b, PPAR-g DNMT with RAR-b, IGFBP-3 COX-2 cyclooxygenase-2; cPLA2 cytosolic phospholipase A2; PGI2 prostacyclin; 15-PGDH 15-hydroxyprostaglandin dehydrogenase; 5-LOX 5-lipoxygenase; EGFR epidermal growth factor receptor; IGFBP-3 insulin-like growth factor binding protein-3; DNMT DNA methyltransferase; HDAC histone deacetylase; RAR-b retinoic acid receptor-b; RXRs retinoid X receptors; NF-kB nuclear factor kappa B; PI3K phosphoinositide 3-kinase; PPAR-g peroxisome proliferator activated receptor-g ; STAT3 signal transducer and activator of transcription-3; FTase farnesyltransferase; mTOR mammalian target of rapamycin; VEGF vascular endothelial growth factor; bFGF basic fibroblast growth factor

individuals and developing feasible methods (e.g., IEN imaging) for monitoring drug effects (176). In addition, validation of biomarkers for early detection (e.g., Ki-67; EGFR gene) might specify these groups as well. Advances in these and other important areas promise to help molecular-targeted prevention in ultimately reducing the incidence, morbidity, and mortality of lung cancer.

References 1. Spitz MR, Hsu TC, Wu X, Fueger JJ, Amos CI, Roth JA (1995) Mutagen sensitivity as a biological marker of lung cancer risk in African Americans. Cancer Epidemiol Biomarkers Prev 4(2):99–103 2. Sporn MB, Dunlop NM, Newton DL, Smith JM (1976) Prevention of chemical carcinogenesis by vitamin A and its synthetic analogs (retinoids). Fed Proc 35(6):1332–1338 3. (1994) The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. N Engl J Med 330(15):1029–1035 4. Hennekens CH, Buring JE, Manson JE et al (1996) Lack of effect of long-term supplementation with beta carotene on the incidence of malignant neoplasms and cardiovascular disease. N Engl J Med 334(18):1145–1149

Lung Cancer Prevention

131

5. Omenn GS, Goodman GE, Thornquist MD et al (1996) Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. N Engl J Med 334(18):1150–1155 6. van Zandwijk N, Dalesio O, Pastorino U, de Vries N, van Tinteren H (2000) EUROSCAN, a randomized trial of vitamin A and N-acetylcysteine in patients with head and neck cancer or lung cancer. For the EUropean Organization for Research and Treatment of Cancer Head and Neck and Lung Cancer Cooperative Groups. J Natl Cancer Inst 92(12):977–986 7. Muscat JE, Chen SQ, Richie JP Jr et al (2003) Risk of lung carcinoma among users of nonsteroidal antiinflammatory drugs. Cancer 97(7):1732–1736 8. Hirsch FR, Lippman SM (2005) Advances in the biology of lung cancer chemoprevention. J Clin Oncol 23(14):3186–3197 9. Rao CV, Reddy BS (2004) NSAIDs and chemoprevention. Curr Cancer Drug Targets 4(1):29–42 10. Williams CS, Mann M, DuBois RN (1999) The role of cyclooxygenases in inflammation, cancer, and development. Oncogene 18(55):7908–7916 11. Khuri FR, Wu H, Lee JJ et al (2001) Cyclooxygenase-2 overexpression is a marker of poor prognosis in stage I non-small cell lung cancer. Clin Cancer Res 7(4):861–867 12. Hida T, Yatabe Y, Achiwa H et  al (1998) Increased expression of cyclooxygenase 2 occurs frequently in human lung cancers, specifically in adenocarcinomas. Cancer Res 58(17):3761–3764 13. Steinbach G, Lynch PM, Phillips RK et al (2000) The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N Engl J Med 342(26):1946–1952 14. Bardou M, Barkun AN, Ghosn J, Hudson M, Rahme E (2004) Effect of chronic intake of NSAIDs and cyclooxygenase 2-selective inhibitors on esophageal cancer incidence. Clin Gastroenterol Hepatol 2(10):880–887 15. Reddy BS, Hirose Y, Lubet R et  al (2000) Chemoprevention of colon cancer by specific cyclooxygenase-2 inhibitor, celecoxib, administered during different stages of carcinogenesis. Cancer Res 60(2):293–297 16. Hu PJ, Yu J, Zeng ZR et al (2004) Chemoprevention of gastric cancer by celecoxib in rats. Gut 53(2):195–200 17. Diperna CA, Bart RD, Sievers EM, Ma Y, Starnes VA, Bremner RM (2003) Cyclooxygenase-2 inhibition decreases primary and metastatic tumor burden in a murine model of orthotopic lung adenocarcinoma. J Thorac Cardiovasc Surg 126(4):1129–1133 18. Klenke FM, Gebhard MM, Ewerbeck V, Abdollahi A, Huber PE, Sckell A (2006) The selective Cox-2 inhibitor Celecoxib suppresses angiogenesis and growth of secondary bone tumors: an intravital microscopy study in mice. BMC Cancer 6:9 19. Harris RE, Alshafie GA, Abou-Issa H, Seibert K (2000) Chemoprevention of breast cancer in rats by celecoxib, a cyclooxygenase 2 inhibitor. Cancer Res 60(8):2101–2103 20. Li N, Sood S, Wang S et al (2005) Overexpression of 5-lipoxygenase and cyclooxygenase 2 in hamster and human oral cancer and chemopreventive effects of zileuton and celecoxib. Clin Cancer Res 11(5):2089–2096 21. Gupta S, Adhami VM, Subbarayan M et al (2004) Suppression of prostate carcinogenesis by dietary supplementation of celecoxib in transgenic adenocarcinoma of the mouse prostate model. Cancer Res 64(9):3334–3343 22. Grubbs CJ, Lubet RA, Koki AT et al (2000) Celecoxib inhibits N-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder cancers in male B6D2F1 mice and female Fischer-344 rats. Cancer Res 60(20):5599–5602 23. Fischer SM, Lo HH, Gordon GB et al (1999) Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, and indomethacin against ultraviolet light-induced skin carcinogenesis. Mol Carcinog 25(4):231–240 24. Mao JT, Cui X, Reckamp K et al (2005) Chemoprevention strategies with cyclooxygenase-2 inhibitors for lung cancer. Clin Lung Cancer 7(1):30–39 25. Abou-Issa H, Alshafie G (2004) Celecoxib: a novel treatment for lung cancer. Expert Rev Anticancer Ther 4(5):725–734 26. Hirsch FR, Franklin WA, Gazdar AF, Bunn PA Jr (2001) Early detection of lung cancer: clinical perspectives of recent advances in biology and radiology. Clin Cancer Res 7(1):5–22

132

N. Peled et al.

27. Nettesheim P, Marchok A (1983) Neoplastic development in airway epithelium. Adv Cancer Res 39:1–70 28. Brambilla E, Travis WD, Colby TV, Corrin B, Shimosato Y (2001) The new World Health Organization classification of lung tumours. Eur Respir J 18(6):1059–1068 29. Soria JC, Kim ES, Fayette J, Lantuejoul S, Deutsch E, Hong WK (2003) Chemoprevention of lung cancer. Lancet Oncol 4(11):659–669 30. Omenn GS (1998) Chemoprevention of lung cancer: the rise and demise of beta-carotene. Annu Rev Public Health 19:73–99 31. Johnson BE (1998) Second lung cancers in patients after treatment for an initial lung cancer. J Natl Cancer Inst 90(18):1335–1345 32. Peled N, Flex D, Raviv Y et al (2009) The role of routine bronchoscopy for early detection of bronchial stump recurrence of lung cancer-1 year post-surgery. Lung Cancer 65(3):319–323 33. Jemal A, Ward E, Hao Y, Thun M (2005) Trends in the leading causes of death in the United States, 1970–2002. JAMA 294(10):1255–1259 34. Bach PB, Kelley MJ, Tate RC, McCrory DC (2003) Screening for lung cancer: a review of the current literature. Chest 123(1 Suppl):72S–82S 35. Kennedy TC, Proudfoot SP, Franklin WA et al (1996) Cytopathological analysis of sputum in patients with airflow obstruction and significant smoking histories. Cancer Res 56(20): 4673–4678 36. Prindiville SA, Byers T, Hirsch FR et al (2003) Sputum cytological atypia as a predictor of incident lung cancer in a cohort of heavy smokers with airflow obstruction. Cancer Epidemiol Biomarkers Prev 12(10):987–993 37. (2005) Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet 365(9472):1687–717 38. Wilson DO, Weissfeld JL, Balkan A et al (2008) Association of radiographic emphysema and airflow obstruction with lung cancer. Am J Respir Crit Care Med 178(7):738–744 39. Ding L, Getz G, Wheeler DA et  al (2008) Somatic mutations affect key pathways in lung adenocarcinoma. Nature 455(7216):1069–1075 40. Cassidy A, Myles JP, van Tongeren M et al (2008) The LLP risk model: an individual risk prediction model for lung cancer. Br J Cancer 98(2):270–276 41. Sato M, Shames DS, Gazdar AF, Minna JD (2007) A translational view of the molecular pathogenesis of lung cancer. J Thorac Oncol 2(4):327–343 42. Belinsky SA, Liechty KC, Gentry FD et al (2006) Promoter hypermethylation of multiple genes in sputum precedes lung cancer incidence in a high-risk cohort. Cancer Res 66(6):3338–3344 43. Phillips M, Gleeson K, Hughes JM et al (1999) Volatile organic compounds in breath as markers of lung cancer: a cross-sectional study. Lancet 353(9168):1930–1933 44. Phillips M, Altorki N, Austin JH et al (2007) Prediction of lung cancer using volatile biomarkers in breath. Cancer Biomark 3(2):95–109 45. Peng G, Trock E, Haick H (2008) Detecting simulated patterns of lung cancer biomarkers by random network of single-walled carbon nanotubes coated with nonpolymeric organic materials. Nano Lett 8(11):3631–3635 46. Shopland DR, Eyre HJ, Pechacek TF (1991) Smoking-attributable cancer mortality in 1991: is lung cancer now the leading cause of death among smokers in the United States? J Natl Cancer Inst 83(16):1142–1148 47. Perez-Padilla R, Regalado J, Vedal S et  al (1996) Exposure to biomass smoke and chronic airway disease in Mexican women. A case-control study. Am J Respir Crit Care Med 154(3 Pt 1):701–706 48. Delgado J, Martinez LM, Sanchez TT, Ramirez A, Iturria C, Gonzalez-Avila G (2005) Lung cancer pathogenesis associated with wood smoke exposure. Chest 128(1):124–131 49. Anthonisen NR, Skeans MA, Wise RA, Manfreda J, Kanner RE, Connett JE (2005) The effects of a smoking cessation intervention on 14.5-year mortality: a randomized clinical trial. Ann Intern Med 142(4):233–239 50. Song YM, Sung J, Cho HJ (2008) Reduction and cessation of cigarette smoking and risk of cancer: a cohort study of Korean men. J Clin Oncol 26(31):5101–5106

Lung Cancer Prevention

133

51. Tverdal A, Bjartveit K (2006) Health consequences of reduced daily cigarette consumption. Tob Control 15(6):472–480 52. Eisenberg MJ, Filion KB, Yavin D et al (2008) Pharmacotherapies for smoking cessation: a meta-analysis of randomized controlled trials. CMAJ 179(2):135–144 53. Gonzales D, Rennard SI, Nides M et al (2006) Varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist, vs sustained-release bupropion and placebo for smoking cessation: a randomized controlled trial. JAMA 296(1):47–55 54. Jorenby DE, Hays JT, Rigotti NA et al (2006) Efficacy of varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist, vs placebo or sustained-release bupropion for smoking cessation: a randomized controlled trial. JAMA 296(1):56–63 55. Peto R, Darby S, Deo H, Silcocks P, Whitley E, Doll R (2000) Smoking, smoking cessation, and lung cancer in the UK since 1950: combination of national statistics with two case-control studies. BMJ 321(7257):323–329 56. Tong L, Spitz MR, Fueger JJ, Amos CA (1996) Lung carcinoma in former smokers. Cancer 78(5):1004–1010 57. Kelloff GJ, Lippman SM, Dannenberg AJ et  al (2006) Progress in chemoprevention drug development: the promise of molecular biomarkers for prevention of intraepithelial neoplasia and cancer – a plan to move forward. Clin Cancer Res 12(12):3661–3697 58. Kelloff GJ, Sigman CC (2007) Assessing intraepithelial neoplasia and drug safety in cancerpreventive drug development. Nat Rev Cancer 7(7):508–518 59. Lam S, Kennedy T, Unger M et al (1998) Localization of bronchial intraepithelial neoplastic lesions by fluorescence bronchoscopy. Chest 113(3):696–702 60. Lam S, MacAulay C, leRiche JC, Palcic B (2000) Detection and localization of early lung cancer by fluorescence bronchoscopy. Cancer 89(11 Suppl):2468–2473 61. Bota S, Auliac JB, Paris C et al (2001) Follow-up of bronchial precancerous lesions and carcinoma in situ using fluorescence endoscopy. Am J Respir Crit Care Med 164(9):1688–1693 62. Venmans BJ, van Boxem TJ, Smit EF, Postmus PE, Sutedja TG (2000) Outcome of bronchial carcinoma in situ. Chest 117(6):1572–1576 63. Westra WH (2000) Early glandular neoplasia of the lung. Respir Res 1(3):163–169 64. Beasley MB, Brambilla E, Travis WD (2005) The 2004 World Health Organization classification of lung tumors. Semin Roentgenol 40(2):90–97 65. Ikeda K, Nomori H, Ohba Y et al (2008) Epidermal growth factor receptor mutations in multicentric lung adenocarcinomas and atypical adenomatous hyperplasias. J Thorac Oncol 3(5):467–471 66. Licchesi JD, Westra WH, Hooker CM, Herman JG (2008) Promoter hypermethylation of hallmark cancer genes in atypical adenomatous hyperplasia of the lung. Clin Cancer Res 14(9):2570–2578 67. Licchesi JD, Westra WH, Hooker CM, Machida EO, Baylin SB, Herman JG (2008) Epigenetic alteration of Wnt pathway antagonists in progressive glandular neoplasia of the lung. Carcinogenesis 29(5):895–904 68. Lee JS, Lippman SM, Benner SE et al (1994) Randomized placebo-controlled trial of isotretinoin in chemoprevention of bronchial squamous metaplasia. J Clin Oncol 12(5):937–945 69. Lippman SM, Benner SE, Hong WK (1994) Cancer chemoprevention. J Clin Oncol 12(4):851–873 70. McLarty JW, Holiday DB, Girard WM, Yanagihara RH, Kummet TD, Greenberg SD (1995) Beta-Carotene, vitamin A, and lung cancer chemoprevention: results of an intermediate endpoint study. Am J Clin Nutr 62(6 Suppl):1431S–1438S 71. Kurie JM, Lee JS, Khuri FR et al (2000) N-(4-hydroxyphenyl)retinamide in the chemoprevention of squamous metaplasia and dysplasia of the bronchial epithelium. Clin Cancer Res 6(8):2973–2979 72. Lam S, MacAulay C, Le Riche JC et al (2002) A randomized phase IIb trial of anethole dithiolethione in smokers with bronchial dysplasia. J Natl Cancer Inst 94(13):1001–1009 73. Kurie JM, Lotan R, Lee JJ et al (2003) Treatment of former smokers with 9-cis-retinoic acid reverses loss of retinoic acid receptor-beta expression in the bronchial epithelium: results from a randomized placebo-controlled trial. J Natl Cancer Inst 95(3):206–214

134

N. Peled et al.

74. Lam S, Xu X, Parker-Klein H et al (2003) Surrogate end-point biomarker analysis in a retinol chemoprevention trial in current and former smokers with bronchial dysplasia. Int J Oncol 23(6):1607–1613 75. Lam S, leRiche JC, McWilliams A et  al (2004) A randomized phase IIb trial of pulmicort turbuhaler (budesonide) in people with dysplasia of the bronchial epithelium. Clin Cancer Res 10(19):6502–6511 76. Lippman SM, Lee JJ, Karp DD et al (2001) Randomized phase III intergroup trial of isotretinoin to prevent second primary tumors in stage I non-small-cell lung cancer. J Natl Cancer Inst 93(8):605–618 77. Lippman SM, Klein EA, Goodman PJ et al (2009) Effect of selenium and vitamin E on risk of prostate cancer and other cancers: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA 301(1):39–51 78. Lippman SM, Kessler JF, Meyskens FL Jr (1987) Retinoids as preventive and therapeutic anticancer agents (Part II). Cancer Treat Rep 71(5):493–515 79. Brabender J, Metzger R, Salonga D et  al (2005) Comprehensive expression analysis of retinoic acid receptors and retinoid X receptors in non-small cell lung cancer: implications for tumor development and prognosis. Carcinogenesis 26(3):525–530 80. Lotan R, Xu XC, Lippman SM et al (1995) Suppression of retinoic acid receptor-beta in premalignant oral lesions and its up-regulation by isotretinoin. N Engl J Med 332(21):1405–1410 81. Lotan R (2005) A crucial role for cellular retinol-binding protein I in retinoid signaling. J Natl Cancer Inst 97(1):3–4 82. Srinivas H, Juroske DM, Kalyankrishna S et al (2005) c-Jun N-terminal kinase contributes to aberrant retinoid signaling in lung cancer cells by phosphorylating and inducing proteasomal degradation of retinoic acid receptor alpha. Mol Cell Biol 25(3):1054–1069 83. Vourlekis JS, Szabo E (2003) Predicting success in cancer prevention trials. J Natl Cancer Inst 95(3):178–179 84. Khuri FR, Lotan R, Kemp BL et al (2000) Retinoic acid receptor-beta as a prognostic indicator in stage I non-small-cell lung cancer. J Clin Oncol 18(15):2798–2804 85. Soria JC, Xu X, Liu DD et al (2003) Retinoic acid receptor beta and telomerase catalytic subunit expression in bronchial epithelium of heavy smokers. J Natl Cancer Inst 95(2):165–168 86. Kim JS, Lee H, Kim H et al (2004) Promoter methylation of retinoic acid receptor beta 2 and the development of second primary lung cancers in non-small-cell lung cancer. J Clin Oncol 22(17):3443–3450 87. Berard J, Laboune F, Mukuna M, Masse S, Kothary R, Bradley WE (1996) Lung tumors in mice expressing an antisense RARbeta2 transgene. FASEB J 10(9):1091–1097 88. Xu XC, Lee JJ, Wu TT, Hoque A, Ajani JA, Lippman SM (2005) Increased retinoic acid receptor-beta4 correlates in  vivo with reduced retinoic acid receptor-beta2 in esophageal squamous cell carcinoma. Cancer Epidemiol Biomarkers Prev 14(4):826–829 89. Albanes D (2009) Vitamin supplements and cancer prevention: where do randomized controlled trials stand? J Natl Cancer Inst 101(1):2–4 90. Hennekens CH, Buring JE, Peto R (1994) Antioxidant vitamins – benefits not yet proved. N Engl J Med 330(15):1080–1081 91. Virtamo J, Pietinen P, Huttunen JK et al (2003) Incidence of cancer and mortality following alpha-tocopherol and beta-carotene supplementation: a postintervention follow-up. JAMA 290(4):476–485 92. Lippman SM, Goodman PJ, Klein EA et  al (2005) Designing the Selenium and Vitamin E Cancer Prevention Trial (SELECT). J Natl Cancer Inst 97(2):94–102 93. Ghosh J (2004) Rapid induction of apoptosis in prostate cancer cells by selenium: reversal by metabolites of arachidonate 5-lipoxygenase. Biochem Biophys Res Commun 315(3):624–635 94. Clark LC, Combs GF Jr, Turnbull BW et al (1996) Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group. JAMA 276(24):1957–1963 95. Reid ME, Duffield-Lillico AJ, Garland L, Turnbull BW, Clark LC, Marshall JR (2002) Selenium supplementation and lung cancer incidence: an update of the nutritional prevention of cancer trial. Cancer Epidemiol Biomarkers Prev 11(11):1285–1291

Lung Cancer Prevention

135

96. Zhuo H, Smith AH, Steinmaus C (2004) Selenium and lung cancer: a quantitative analysis of heterogeneity in the current epidemiological literature. Cancer Epidemiol Biomarkers Prev 13(5):771–778 97. Shureiqi I, Lippman SM (2001) Lipoxygenase modulation to reverse carcinogenesis. Cancer Res 61(17):6307–6312 98. Muller R (2004) Crosstalk of oncogenic and prostanoid signaling pathways. J Cancer Res Clin Oncol 130(8):429–444 99. Meyer AM, Dwyer-Nield LD, Hurteau GJ et al (2004) Decreased lung tumorigenesis in mice genetically deficient in cytosolic phospholipase A2. Carcinogenesis 25(8):1517–1524 100. Riedl K, Krysan K, Pold M et  al (2004) Multifaceted roles of cyclooxygenase-2 in lung cancer. Drug Resist Updat 7(3):169–184 101. Yamaki T, Endoh K, Miyahara M et al (2004) Prostaglandin E2 activates Src signaling in lung adenocarcinoma cell via EP3. Cancer Lett 214(1):115–120 102. Shishodia S, Aggarwal BB (2004) Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates activation of cigarette smoke-induced nuclear factor (NF)-kappaB by suppressing activation of IkappaBalpha kinase in human non-small cell lung carcinoma: correlation with suppression of cyclin D1, COX-2, and matrix metalloproteinase-9. Cancer Res 64(14):5004–5012 103. Kisley LR, Barrett BS, Dwyer-Nield LD, Bauer AK, Thompson DC, Malkinson AM (2002) Celecoxib reduces pulmonary inflammation but not lung tumorigenesis in mice. Carcinogenesis 23(10):1653–1660 104. Edelman MJ, Watson D, Wang X et al (2008) Eicosanoid modulation in advanced lung cancer: cyclooxygenase-2 expression is a positive predictive factor for celecoxib + chemotherapy – Cancer and Leukemia Group B Trial 30203. J Clin Oncol 26(6):848–855 105. Mao JT, Fishbein MC, Adams B et al (2006) Celecoxib decreases Ki-67 proliferative index in active smokers. Clin Cancer Res 12(1):314–320 106. Nie D, Lamberti M, Zacharek A et al (2000) Thromboxane A(2) regulation of endothelial cell migration, angiogenesis, and tumor metastasis. Biochem Biophys Res Commun 267(1):245–251 107. Keith RL, Miller YE, Hudish TM et al (2004) Pulmonary prostacyclin synthase overexpression chemoprevents tobacco smoke lung carcinogenesis in mice. Cancer Res 64(16):5897–5904 108. Zhou W, Hashimoto K, Goleniewska K et al (2007) Prostaglandin I2 analogs inhibit proinflammatory cytokine production and T cell stimulatory function of dendritic cells. J Immunol 178(2):702–710 109. Nana-Sinkam SP, Lee JD, Sotto-Santiago S et al (2007) Prostacyclin prevents pulmonary endothelial cell apoptosis induced by cigarette smoke. Am J Respir Crit Care Med 175(7):676–685 110. Ding Y, Tong M, Liu S, Moscow JA, Tai HH (2005) NAD+-linked 15-hydroxyprostaglandin dehydrogenase (15-PGDH) behaves as a tumor suppressor in lung cancer. Carcinogenesis 26(1):65–72 111. Backlund MG, Mann JR, Holla VR et al (2005) 15-Hydroxyprostaglandin dehydrogenase is down-regulated in colorectal cancer. J Biol Chem 280(5):3217–3223 112. Huang G, Eisenberg R, Yan M et  al (2008) 15-Hydroxyprostaglandin dehydrogenase is a target of hepatocyte nuclear factor 3beta and a tumor suppressor in lung cancer. Cancer Res 68(13):5040–5048 113. Rioux N, Castonguay A (1998) Prevention of NNK-induced lung tumorigenesis in A/J mice by acetylsalicylic acid and NS-398. Cancer Res 58(23):5354–5360 114. Gunning WT, Kramer PM, Steele VE, Pereira MA (2002) Chemoprevention by lipoxygenase and leukotriene pathway inhibitors of vinyl carbamate-induced lung tumors in mice. Cancer Res 62(15):4199–4201 115. Schroeder CP, Yang P, Newman RA, Lotan R (2007) Simultaneous inhibition of COX-2 and 5-LOX activities augments growth arrest and death of premalignant and malignant human lung cell lines. J Exp Ther Oncol 6(3):183–192 116. Mao JT, Tsu IH, Dubinett SM et al (2004) Modulation of pulmonary leukotriene B4 production by cyclooxygenase-2 inhibitors and lipopolysaccharide. Clin Cancer Res 10(20):6872–6878 117. Mao JT, Roth MD, Serio KJ et al (2003) Celecoxib modulates the capacity for prostaglandin E2 and interleukin-10 production in alveolar macrophages from active smokers. Clin Cancer Res 9(16 Pt 1):5835–5841

136

N. Peled et al.

118. Ye YN, Liu ES, Shin VY, Wu WK, Cho CH (2004) The modulating role of nuclear factor-kappaB in the action of alpha7-nicotinic acetylcholine receptor and cross-talk between 5-lipoxygenase and cyclooxygenase-2 in colon cancer growth induced by 4-(N-methyl-N-nitrosamino)-1(3-pyridyl)-1-butanone. J Pharmacol Exp Ther 311(1):123–130 119. Hong J, Bose M, Ju J et al (2004) Modulation of arachidonic acid metabolism by curcumin and related beta-diketone derivatives: effects on cytosolic phospholipase A(2), cyclooxygenases and 5-lipoxygenase. Carcinogenesis 25(9):1671–1679 120. Ju J, Liu Y, Hong J, Huang MT, Conney AH, Yang CS (2003) Effects of green tea and highfat diet on arachidonic acid metabolism and aberrant crypt foci formation in an azoxymethaneinduced colon carcinogenesis mouse model. Nutr Cancer 46(2):172–178 121. Yu R, Jiao JJ, Duh JL, Gudehithlu K, Tan TH, Kong AN (1997) Activation of mitogen-activated protein kinases by green tea polyphenols: potential signaling pathways in the regulation of antioxidant-responsive element-mediated phase II enzyme gene expression. Carcinogenesis 18(2):451–456 122. Chen YQ, Duniec ZM, Liu B et al (1994) Endogenous 12(S)-HETE production by tumor cells and its role in metastasis. Cancer Res 54(6):1574–1579 123. Grossi IM, Fitzgerald LA, Umbarger LA et al (1989) Bidirectional control of membrane expression and/or activation of the tumor cell IRGpIIb/IIIa receptor and tumor cell adhesion by lipoxygenase products of arachidonic acid and linoleic acid. Cancer Res 49(4):1029–1037 124. Shureiqi I, Jiang W, Zuo X et al (2003) The 15-lipoxygenase-1 product 13-S-hydroxyocta decadienoic acid down-regulates PPAR-delta to induce apoptosis in colorectal cancer cells. Proc Natl Acad Sci USA 100(17):9968–9973 125. Hsi LC, Xi X, Lotan R, Shureiqi I, Lippman SM (2004) The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces apoptosis via induction of 15-lipoxygenase-1 in colorectal cancer cells. Cancer Res 64(23):8778–8781 126. Gonzalez AL, Roberts RL, Massion PP, Olson SJ, Shyr Y, Shappell SB (2004) 15-Lipoxygenase-2 expression in benign and neoplastic lung: an immunohistochemical study and correlation with tumor grade and proliferation. Hum Pathol 35(7):840–849 127. Kudryavtsev IA, Golenko OD, Gudkova MV, Myasishcheva NV (2002) Arachidonic acid metabolism in growth control of A549 human lung adenocarcinoma cells. Biochemistry (Mosc) 67(9):1021–1026 128. Shankaranarayanan P, Nigam S (2003) IL-4 induces apoptosis in A549 lung adenocarcinoma cells: evidence for the pivotal role of 15-hydroxyeicosatetraenoic acid binding to activated peroxisome proliferator-activated receptor gamma transcription factor. J Immunol 170(2):887–894 129. Spanbroek R, Hildner M, Kohler A et  al (2001) IL-4 determines eicosanoid formation in dendritic cells by down-regulation of 5-lipoxygenase and up-regulation of 15-lipoxygenase 1 expression. Proc Natl Acad Sci USA 98(9):5152–5157 130. Profita M, Sala A, Siena L et al (2002) Leukotriene B4 production in human mononuclear phagocytes is modulated by interleukin-4-induced 15-lipoxygenase. J Pharmacol Exp Ther 300(3):868–875 131. Munger KA, Montero A, Fukunaga M et al (1999) Transfection of rat kidney with human 15-lipoxygenase suppresses inflammation and preserves function in experimental glomerulonephritis. Proc Natl Acad Sci USA 96(23):13375–13380 132. Cappuzzo F, Hirsch FR, Rossi E et al (2005) Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst 97(9):643–655 133. Merrick DT, Kittelson J, Winterhalder R et al (2006) Analysis of c-ErbB1/epidermal growth factor receptor and c-ErbB2/HER-2 expression in bronchial dysplasia: evaluation of potential targets for chemoprevention of lung cancer. Clin Cancer Res 12(7 Pt 1):2281–2288 134. Dannenberg AJ, Lippman SM, Mann JR, Subbaramaiah K, DuBois RN (2005) Cyclooxygenase-2 and epidermal growth factor receptor: pharmacologic targets for chemoprevention. J Clin Oncol 23(2):254–266 135. Chen Z, Zhang X, Li M et al (2004) Simultaneously targeting epidermal growth factor receptor tyrosine kinase and cyclooxygenase-2, an efficient approach to inhibition of squamous cell carcinoma of the head and neck. Clin Cancer Res 10(17):5930–5939

Lung Cancer Prevention

137

136. Lonardo F, Dragnev KH, Freemantle SJ et al (2002) Evidence for the epidermal growth factor receptor as a target for lung cancer prevention. Clin Cancer Res 8(1):54–60 137. Izzo JG, Papadimitrakopoulou VA, Liu DD et  al (2003) Cyclin D1 genotype, response to biochemoprevention, and progression rate to upper aerodigestive tract cancer. J Natl Cancer Inst 95(3):198–205 138. Petty WJ, Dragnev KH, Memoli VA et al (2004) Epidermal growth factor receptor tyrosine kinase inhibition represses cyclin D1 in aerodigestive tract cancers. Clin Cancer Res 10(22):7547–7554 139. Pitha-Rowe I, Petty WJ, Feng Q et al (2004) Microarray analyses uncover UBE1L as a candidate target gene for lung cancer chemoprevention. Cancer Res 64(21):8109–8115 140. Dragnev KH, Pitha-Rowe I, Ma Y et  al (2004) Specific chemopreventive agents trigger proteasomal degradation of G1 cyclins: implications for combination therapy. Clin Cancer Res 10(7):2570–2577 141. Sueoka N, Lee HY, Wiehle S et al (2000) Insulin-like growth factor binding protein-6 activates programmed cell death in non-small cell lung cancer cells. Oncogene 19(38):4432–4436 142. Lee HY, Moon H, Chun KH et al (2004) Effects of insulin-like growth factor binding protein-3 and farnesyltransferase inhibitor SCH66336 on Akt expression and apoptosis in nonsmall-cell lung cancer cells. J Natl Cancer Inst 96(20):1536–1548 143. Pold M, Krysan K, Pold A et al (2004) Cyclooxygenase-2 modulates the insulin-like growth factor axis in non-small-cell lung cancer. Cancer Res 64(18):6549–6555 144. West KA, Linnoila IR, Belinsky SA, Harris CC, Dennis PA (2004) Tobacco carcinogeninduced cellular transformation increases activation of the phosphatidylinositol 3¢-kinase/ Akt pathway in vitro and in vivo. Cancer Res 64(2):446–451 145. Tsao AS, McDonnell T, Lam S et al (2003) Increased phospho-AKT (Ser(473)) expression in bronchial dysplasia: implications for lung cancer prevention studies. Cancer Epidemiol Biomarkers Prev 12(7):660–664 146. Chun KH, Kosmeder JW 2nd, Sun S et  al (2003) Effects of deguelin on the phosphatidylinositol 3-kinase/Akt pathway and apoptosis in premalignant human bronchial epithelial cells. J Natl Cancer Inst 95(4):291–302 147. Jyonouchi H, Sun S, Iijima K, Wang M, Hecht SS (1999) Effects of anti-7,8-dihydroxy-9,10epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene on human small airway epithelial cells and the protective effects of myo-inositol. Carcinogenesis 20(1):139–145 148. Bren-Mattison Y, Van Putten V, Chan D, Winn R, Geraci MW, Nemenoff RA (2005) Peroxisome proliferator-activated receptor-gamma (PPAR(gamma)) inhibits tumorigenesis by reversing the undifferentiated phenotype of metastatic non-small-cell lung cancer cells (NSCLC). Oncogene 24(8):1412–1422 149. Wick M, Hurteau G, Dessev C et  al (2002) Peroxisome proliferator-activated receptorgamma is a target of nonsteroidal anti-inflammatory drugs mediating cyclooxygenase-independent inhibition of lung cancer cell growth. Mol Pharmacol 62(5):1207–1214 150. Govindarajan R, Ratnasinghe L, Simmons DL et al (2007) Thiazolidinediones and the risk of lung, prostate, and colon cancer in patients with diabetes. J Clin Oncol 25(12):1476–1481 151. Hazra S, Batra RK, Tai HH, Sharma S, Cui X, Dubinett SM (2007) Pioglitazone and rosiglitazone decrease prostaglandin E2 in non-small-cell lung cancer cells by up-regulating 15-hydroxyprostaglandin dehydrogenase. Mol Pharmacol 71(6):1715–1720 152. Nemenoff R, Meyer AM, Hudish TM et al (2008) Prostacyclin prevents murine lung cancer independent of the membrane receptor by activation of peroxisomal proliferator – activated receptor gamma. Cancer Prev Res (Phila Pa) 1(5):349–356 153. Han S, Rivera HN, Roman J (2005) Peroxisome proliferator-activated receptor-gamma ligands inhibit alpha5 integrin gene transcription in non-small cell lung carcinoma cells. Am J Respir Cell Mol Biol 32(4):350–359 154. Chang TH, Szabo E (2002) Enhanced growth inhibition by combination differentiation therapy with ligands of peroxisome proliferator-activated receptor-gamma and inhibitors of histone deacetylase in adenocarcinoma of the lung. Clin Cancer Res 8(4):1206–1212 155. Blaine SA, Meyer AM, Hurteau G et al (2005) Targeted over-expression of mPGES-1 and elevated PGE2 production is not sufficient for lung tumorigenesis in mice. Carcinogenesis 26(1):209–217

138

N. Peled et al.

156. Belinsky SA (2005) Silencing of genes by promoter hypermethylation: key event in rodent and human lung cancer. Carcinogenesis 26(9):1481–1487 157. Suzuki M, Sunaga N, Shames DS, Toyooka S, Gazdar AF, Minna JD (2004) RNA interferencemediated knockdown of DNA methyltransferase 1 leads to promoter demethylation and gene re-expression in human lung and breast cancer cells. Cancer Res 64(9):3137–3143 158. Desai D, Das A, Cohen L, el-Bayoumy K, Amin S (2003) Chemopreventive efficacy of suberoylanilide hydroxamic acid (SAHA) against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in female A/J mice. Anticancer Res 23(1A):499–503 159. Wang S, Yan-Neale Y, Fischer D et al (2003) Histone deacetylase 1 represses the small GTPase RhoB expression in human nonsmall lung carcinoma cell line. Oncogene 22(40):6204–6213 160. Suh YA, Lee HY, Virmani A et al (2002) Loss of retinoic acid receptor beta gene expression is linked to aberrant histone H3 acetylation in lung cancer cell lines. Cancer Res 62(14):3945–3949 161. Youssef EM, Lotan D, Issa JP et al (2004) Hypermethylation of the retinoic acid receptorbeta(2) gene in head and neck carcinogenesis. Clin Cancer Res 10(5):1733–1742 162. Zhu WG, Lakshmanan RR, Beal MD, Otterson GA (2001) DNA methyltransferase inhibition enhances apoptosis induced by histone deacetylase inhibitors. Cancer Res 61(4):1327–1333 163. Zou CP, Kurie JM, Lotan D, Zou CC, Hong WK, Lotan R (1998) Higher potency of N-(4hydroxyphenyl)retinamide than all-trans-retinoic acid in induction of apoptosis in non-small cell lung cancer cell lines. Clin Cancer Res 4(5):1345–1355 164. Belinsky SA, Klinge DM, Stidley CA et  al (2003) Inhibition of DNA methylation and histone deacetylation prevents murine lung cancer. Cancer Res 63(21):7089–7093 165. Suzuki S, Higuchi M, Proske RJ, Oridate N, Hong WK, Lotan R (1999) Implication of mitochondria-derived reactive oxygen species, cytochrome C and caspase-3 in N-(4-hydroxyphenyl) retinamide-induced apoptosis in cervical carcinoma cells. Oncogene 18(46):6380–6387 166. Sun SY, Schroeder CP, Yue P, Lotan D, Hong WK, Lotan R (2005) Enhanced growth inhibition and apoptosis induction in NSCLC cell lines by combination of celecoxib and 4HPR at clinically relevant concentrations. Cancer Biol Ther 4(4):407–413 167. Sun SY, Hail N Jr, Lotan R (2004) Apoptosis as a novel target for cancer chemoprevention. J Natl Cancer Inst 96(9):662–672 168. Guerrieri-Gonzaga A, Robertson C, Bonanni B et al (2006) Preliminary results on safety and activity of a randomized, double-blind, 2 x 2 trial of low-dose tamoxifen and fenretinide for breast cancer prevention in premenopausal women. J Clin Oncol 24(1):129–135 169. Lippman SM, Lee JJ, Sabichi AL (1998) Cancer chemoprevention: progress and promise. J Natl Cancer Inst 90(20):1514–1528 170. Seki Y, Suzuki N, Imaizumi M et al (2004) STAT3 and MAPK in human lung cancer tissues and suppression of oncogenic growth by JAB and dominant negative STAT3. Int J Oncol 24(4):931–934 171. Liu X, Yue P, Zhou Z, Khuri FR, Sun SY (2004) Death receptor regulation and celecoxibinduced apoptosis in human lung cancer cells. J Natl Cancer Inst 96(23):1769–1780 172. Lubet RA, Zhang Z, Wang Y, You M (2004) Chemoprevention of lung cancer in transgenic mice. Chest 125(5 Suppl):144S–147S 173. Dauer DJ, Ferraro B, Song L et al (2005) Stat3 regulates genes common to both wound healing and cancer. Oncogene 24(21):3397–3408 174. Swanton C (2004) Cell-cycle targeted therapies. Lancet Oncol 5(1):27–36 175. Wislez M, Spencer ML, Izzo JG et al (2005) Inhibition of mammalian target of rapamycin reverses alveolar epithelial neoplasia induced by oncogenic K-ras. Cancer Res 65(8):3226–3235 176. Abbruzzese JL, Lippman SM (2004) The convergence of cancer prevention and therapy in early-phase clinical drug development. Cancer Cell 6(4):321–326 177. Keith R et al. (2009) Oral Iloprost improves endobronchial dysplasia in formers smokers. J Thorac Oncol IASLC meeting; #7753

Adjuvant and Neoadjuvant Therapy of NSCLC Katherine Pisters

Abstract  For patients with operable non-small cell lung cancer (NSCLC), surgery has long been the standard of care. However, despite complete resection, 5-year survival rates have been disappointing with about 50% of patients eventually suffering relapse and death from disease. Efforts at improving survival for patients with operable NSCLC have examined the addition of chemotherapy and/or radiation in the preoperative (neoadjuvant or induction) and postoperative (adjuvant) settings. Thoracic radiation when given after surgery has led to a reduction in local recurrence but has not improved overall survival. A postoperative radiotherapy (PORT) meta-analysis published in 1998 raised significant concerns about the potential hazards of PORT. More recent retrospective studies of PORT have found it safe and not associated with excessive deaths from intercurrent disease. Postoperative radiation is not recommended for completely resected stage I and II NSCLC patients, and its use should be individualized in patients with stage III disease. PORT is currently recommended only for patients at high risk of local relapse (extensive nodal involvement or positive surgical margin). Early trials of postoperative chemotherapy failed to demonstrate a consistent benefit. These trials suffered from poor design with heterogeneous patient populations, inadequate trial size, and less active drug regimens. A meta-analysis examining the role of chemotherapy in NSCLC published in 1995 found a trend in favor of adjuvant cisplatin-based chemotherapy. These findings led to renewed interest in adjuvant chemotherapy for NSCLC, and larger trials with more active chemotherapy regimens were undertaken. Several of these trials have demonstrated a clear survival advantage for patients treated with cisplatin-based adjuvant therapy. Updated meta-analyses have confirmed the benefit of postoperative chemotherapy in NSCLC.

K. Pisters (*) Department of Thoracic/Head & Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center, P.O. Box 301402, Houston, TX 77230-1402, USA e-mail: [email protected] D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_6, © Humana Press, a part of Springer Science+Business Media, LLC 2010

139

140

K. Pisters

Postoperative cisplatin-based chemotherapy is now the standard of care for completely resected stage II and III NSCLC patients with good performance status. Preoperative or induction chemotherapy trials were investigated prior to the positive adjuvant clinical trial data. Support for this concept came from improved survival seen in the locally advanced but inoperable NSCLC patients where chemotherapy administered prior to definitive chest radiation therapy had improved survival. Possible advantages of preoperative administration of chemotherapy include radiographic and pathologic tumor response assessment, earliest treatment of clinically undetectable micrometastatic disease, and improved compliance. Randomized trials in stage IIIA patients found a significant survival benefit but were small studies and their findings remain controversial. Randomized trials in earlier stages have supported the use of chemotherapy in operable NSCLC, but overall survival differences have not achieved statistical significance. Meta-analyses have found significant benefit for preoperative chemotherapy in operable NSCLC. At this time, stronger data exist in support of adjuvant chemotherapy in patients with operable NSCLC. Preoperative chemotherapy should only be administered in the clinical trial setting. Keywords  Non-small cell lung cancer • Neoadjuvant • Induction • Adjuvant • Postoperative • Combined modality therapy • Operable • Resectable

Introduction For patients with operable non-small cell lung cancer (NSCLC), surgery has long been the standard of care. However, despite complete resection, 5-year survival rates have been disappointing with about 50% of patients eventually suffering relapse and death from disease. (1) Survival estimates based on clinical and operative staging are displayed in Table 1. Efforts at improving survival for patients with operable NSCLC have examined the addition of chemotherapy and/or radiation in the preoperative (neoadjuvant or induction) and postoperative (adjuvant) settings.

Adjuvant Radiation Clinical Trials Early efforts to improve survival for operable NSCLC patients focused on the use of postoperative treatments. The value of postoperative radiation was examined in a randomized trial by the Lung Cancer Study Group (2). Two hundred ten eligible patients with stage II and III epidermoid NSCLC were randomized to receive

Adjuvant and Neoadjuvant Therapy of NSCLC

141

Table 1  Expected outcome following complete surgical resection based on clinical (preoperative) and surgical (pathological) staging for resectable NSCLC Estimated 5-year survival rate (%) Stage TNM Clinical staging Surgical staging Stage I   IA T1N0M0 61 67   IB T2N0M0 38 57 Stage II   IIA T1N1M0 38 57   IIB T2N1M0 34 55 T3N0M0 24 39 Stage III   IIIA T3N1M0  9 25 T1-3N2M0 13 23 Adapted from Mountain (1)

postoperative radiotherapy or observation. A decrease in local recurrence rate was observed, but there was no overall survival benefit. (2) The results of the postoperative radiotherapy (PORT) meta-analysis published in 1998 raised significant concerns about the use of PORT (3). A 21% relative increase in the risk of death was associated with postoperative radiation, shortening survival in stage I and II patients, but not in stage III disease. The increase in mortality seen in this meta-analysis may have resulted from the use of older radiation equipment, administration of higher doses of radiation, and lack of recognition or treatment of late toxicities (4). A more recent retrospective study of PORT found its use to be safe and not associated with excessive deaths (5). Moreover, the SEER (Surveillance, Epidemiology, and End Results) database found increased survival rates associated with radiotherapy in N2 disease (HR = 0.855; 95% CI 0.762–0.959) (6) and a subset analysis of the ANITA adjuvant chemotherapy trial comparing 5-year overall survival in N2 patients who did or did not receive postoperative radiation found higher survival rates in stage IIIA patients receiving radiation in both the observation and chemotherapy arms (21% vs. 17%, and 47% vs. 34%, respectively; statistical tests of comparison were not conducted) (7). These results suggest a benefit of PORT in stage IIIA patients, which will be clarified from ongoing randomized trials.

Current Standard of Practice At this time, postoperative radiation should be offered to patients at high risk for local recurrence: those with positive resection margin(s) or extensive lymph node involvement. If indicated, radiotherapy should be delivered after completion of chemotherapy. Most studies have used radiation doses ranging from 30 to 60 Gy (8–10).

142

K. Pisters

Adjuvant Chemotherapy Clinical Trials Early randomized trials of postoperative adjuvant chemotherapy failed to demonstrate a consistent benefit. These trials were underpowered, administered relatively inactive chemotherapy regimens, and employed what would now be considered suboptimal staging techniques (11–13). Renewed interest in postoperative chemotherapy for NSCLC followed the publication of a meta-analysis in 1995. This meta-analysis examined the role of chemotherapy in NSCLC (14). Part of the meta-analysis examined the efficacy of postoperative chemotherapy in patients with completely resected NSCLC. Combining data from eight randomized studies enrolling 1,394 patients, the investigators found a 5% improvement in 5-year overall survival rates or a 13% reduction in the risk of death associated with postoperative cisplatinbased chemotherapy. These differences did not achieve statistical significance, but led to the development and conduct of large randomized trials. These trials are discussed below and summarized in Table 2. The Adjuvant Lung Project Italy (ALPI) trial enrolled NSCLC patients with completely resected stage I, II or III disease (15). A total of 1,088 eligible patients were randomized over a 5-year time period (1994–1999). Patients were randomized to surgery alone or to mitomycin (8 mg/m2, day 1), vindesine (3 mg/m2, days 1 and 8), and cisplatin (100 mg/m2, day 1) every 3 weeks for three cycles. Patients were stratified by center, tumor size, lymph node involvement and intention to perform postoperative radiation. Radiotherapy was administered according to the policy of the individual participating center. Patients in the chemotherapy arm began radiation 3–5 weeks after the last chemotherapy treatment. For patients in the control arm,

Table 2  Recent phase III trials of adjuvant platin-based chemotherapy Pathologic Eligible stages p value Chemotherapy regimen Trial patients included Hazard ratio ALPI (15) 1,088 I–IIIA 0.96   0.589 Mitomycin, vindesine, cisplatin IALT (16, 17) 1867 I–IIIA 0.86 (1st 5 years) <0.03 Vincaa, cisplatin or 1.45 (after 5 years)   0.04 etoposide, cisplatin BLT (18) 381 I–III 1.02   0.9 Cisplatin-based NCIC CTG 482 IB, II 0.69   0.04 Vinorelbine, cisplatin JBR.10 (19) CALGB 344 IB 0.83   0.12 Paclitaxel, carboplatin (20, 21) ANITA (7) 840 IB–IIIA 0.80   0.017 Vinorelbine, cisplatin a Vindesine, vinblastine, or vinorelbine

Adjuvant and Neoadjuvant Therapy of NSCLC

143

radiotherapy was initiated 4–6 weeks after surgery. In both groups, the total radiotherapy dose was 50–54 Gy (2 Gy/day, 5 days/week) over 5–6 weeks. The trial was designed to detect a 20% relative reduction in mortality (5-year survival of 50–57%), with a corresponding hazard ratio of 0.8, two-sided alpha of 0.05, and 80% power. This design called for 1,300 patients; however, the trial closed at 93% of the planned sample size secondary to slow accrual. The two arms of the study were well balanced for stage, age, sex, histology, and type of surgical resection. Of the patients assigned to postoperative chemotherapy, 69% completed the planned three cycles, although nearly half required dose adjustments. Forty-three percent of all patients received postoperative radiotherapy. Treatment-related deaths occurred in seven patients in the control arm, and three in the chemotherapy arm. At a median follow-up of 64.5 months, there was no significant difference in overall survival with a hazard risk of death of 0.96, p = 0.589. Median overall survivals were 55 months (chemotherapy) and 48 months (surgery alone). Progression-free survival differences also did not achieve statistical significance (hazard ratio 0.89, p = 0.128, medians 37 months versus 29 months). The International Adjuvant Lung Cancer Trial Collaborative Group (IALT) randomized completely resected stage I, II, and III NSCLC patients to three to four cycles of cisplatin-based chemotherapy or observation (16). This is the largest adjuvant trial reported to date with 1867 patients randomized. Chemotherapy consisted of cisplatin (80–120 mg/m2, every 3–4 weeks) given with either vindesine, vinblastine, vinorelbine, or etoposide. To facilitate accrual, each center could choose the pathological stages included, dose of cisplatin per cycle, drug administered with cisplatin, and the postoperative radiation therapy policy. Patients were randomized within 60 days of surgery and were stratified by treatment center, type of surgery, and pathological stage. Chemotherapy was to begin within 60 days of surgery and within 14 days of randomization. Postoperative radiotherapy was administered according to the policy of the individual participating center (never, only in pathological stage N2, or in pathological stages N1 and N2) and consisted of 60 Gy or less, delivered to the mediastinal lymph nodes. The trial required 3,300 patients to detect a 5% improvement in 5-year overall survival (50–55%) with 90% power. Enrolment began in February 1995. Secondary to slow accrual, the steering committee decided to discontinue recruitment December 31, 2000. A total of 1,867 patients had been randomized, recruited by 148 centers in 33 countries. Twenty-five patients were ineligible. Patients were well balanced between the two arms of the study. A regimen combining 100 mg/m2 cisplatin for three or four cycles with etoposide was selected for roughly half of the chemotherapy patients and 74% of the chemotherapy patients received at least 240 mg/m2 of cisplatin. Twenty-seven percent of patients received postoperative radiotherapy. Grade 3 or 4 toxicity was experienced by 23% of the chemotherapy patients. Seven patients (0.8%) died of chemotherapy-related toxicity. At the time of initial publication, the median duration of follow-up was 56 months with survival status known in more than 98% of patients (16). The survival rate was significantly higher in the chemotherapy group (p < 0.03). The hazard ratio

144

K. Pisters

for death was 0.86 (95% CI 0.76–0.98) favoring adjuvant chemotherapy. The 5-year survival rates were 44.5 and 40.4% in the chemotherapy and control groups, respectively. Disease-free survival was also significantly improved with chemotherapy (p < 0.003), with a hazard ratio of 0.83 (95% CI 0.74–0.94). Median overall survival was 50.8 months in the chemotherapy arm and 44.4 months in the control arm while median disease-free survivals were 40.2 and 30.5 months, respectively. Five-year disease-free survival rates were 39.4 and 34.3% in the chemotherapy group and in the control group, respectively. An update on the IALT survival data was presented at ASCO 2008 (17). With three additional years of follow-up, the hazard ratio had decreased to 0.91 (95% CI 0.81–1.02, p = 0.19). Disease-free survival remained statistically significant with a hazard ratio of 0.88 (95% CI 0.78–0.98, p = 0.02). However, there was a significant difference between the results of overall survival before and after 5 years, HR: 0.86 (95% CI 0.76–0.97, p = 0.01) versus HR: 1.45 (95% CI 1.02–2.07, p = 0.04); p value for interaction was 0.006. The difference in results between less than and more than 5 years of follow-up may suggest possible late adjuvant chemotherapy-related toxicity and mortality. This information highlights the need for long-term follow-up on adjuvant trials. The BLT (Big Lung Trial) was conducted in Great Britain, and examined the role of cisplatin-based chemotherapy in a variety of treatment settings. Among these, patients with completely resected stage I–III NSCLC were randomized to three cycles of perioperative cisplatin-based chemotherapy (pre-4%, post-96%) or to surgery alone (18). This trial was not designed to specifically answer a surgical adjuvant question and was therefore underpowered to detect a clinically significant survival difference. Cisplatin (50–80 mg/m2 day 1) was given with vindesine (3 mg/mg2, days 1 and 8), mitomycin (6 mg/m2) and ifosfamide (3 mg/m2), or with mitomycin (6 mg/m2) and vinblastine (6 mg/m2) or with vinorelbine (30 mg/m2, days 1 and 8) every 21 days for three cycles. Of a total of 381 patients, 192 were randomized to chemotherapy and 189 to surgery only. An incomplete microscopic resection was reported in 15% of cases. All three cycles of chemotherapy were given to 64% of patients in the chemotherapy group and 40% of those required a dose adjustment. Fourteen percent of patients received postoperative radiation. With a median follow-up of only 2.9 years, there was no significant difference in the two groups with a hazard ratio of 1.02 (p = 0.90). The NCIC CTG (National Cancer Institute of Canada Clinical Trials Group) JBR.10 trial randomized completely resected stage IB and II (excluding T3N0) NSCLC patients to four cycles of vinorelbine and cisplatin chemotherapy or to surgery alone (19). Chemotherapy consisted of vinorelbine (25  mg/m2 weekly – reduced from 30  mg/m2 shortly after study initiation secondary to toxicity) and cisplatin (50 mg/m2, days 1 and 8) every 4 weeks for four cycles, to commence within 6 weeks of surgery. Patients did not receive postoperative thoracic irradiation. Patients were stratified by nodal status (N0 versus N1) and ras mutation (present vs. absent vs. unknown). The study endpoints were overall survival, recurrence-free survival, quality of life, and toxicity.

Adjuvant and Neoadjuvant Therapy of NSCLC

145

Between 1994 and 2001, 482 patients were randomized. The median age was 61 years, all had PS 0 or 1, and 35% were women. Forty-five percent of the patients had T2N0 disease, 40% had T2N1, and 15% had T1N1 disease extent. Slightly more than half (53%) had adenocarcinoma and 24% had ras mutations. There were two chemotherapy-related deaths (febrile neutropenia and pulmonary fibrosis). The most common cause of death was NSCLC (including one patient with second primary NSCLC), while three patients died from toxicity related to later anti-cancer therapy, nine patients died of other primary malignancies, and 21 from other causes. Overall survival was significantly prolonged for the chemotherapy patients (94 months vs. 73 months; hazard ratio = 0.69, 95% CI 0.52–0.91, p = 0.04), as was recurrence-free survival (not reached vs. 47 months; hazard ratio = 0.60, 95% CI 0.45–0.79, p < 0.0001). The 5-year survival rates were 69 and 54% favoring chemotherapy. The Cancer and Leukemia Group B (CALGB) trial 9633 is unique amongst the recent adjuvant trials in that it is the only randomized study to use carboplatin and were randomized to four cycles of postoperative paclitaxel/carboplatin chemotherapy versus surgery alone. Chemotherapy was started within 4–8 weeks of surgery and consisted of paclitaxel 200 mg/m2 over 3 h, followed by carboplatin AUC = 6 every 3 weeks for four cycles. Like the NCIC CTG study, there was no planned thoracic radiotherapy. Between September 1996 and November 2003, 344 patients were accrued. Secondary to slow accrual and accumulation of events over time, the data monitoring committee recommended early closure following accrual of 344 patients when a planned interim efficacy analysis found the p value for the log-rank test of survival was less than the prespecified stopping boundary for a positive impact on survival. The two arms of the study were well balanced with regard to age, gender, race, ethnicity, histology, tumor differentiation, and resection type. Adjuvant chemotherapy was well tolerated with 86% of assigned patients receiving all four cycles of adjuvant chemotherapy. There were no chemotherapy-related deaths. The trial was first reported at ASCO 2004 with a median follow-up of 34 months when only 57% of the deaths required for final analysis had occurred (20). There were 36 deaths from any cause among 173 patients in the chemotherapy group compared to 52 deaths among 171 patients in the observation group (HR 0.62, 95% CI 0.41–0.95, p = 0.028). Overall survival at 4 years was 71% (95% CI 62–81%) and 59% (95% CI 50–69%) in the chemotherapy and observation groups, respectively. There was also a significant advantage in failure-free survival favoring the chemotherapy group (HR = 0.69, 95% CI 0.48–0.98, p = 0.035). Updated survival data were recently published (21). After a median follow-up of 74 months, survival differences were no longer statistically significant. Median overall survival times were 95 and 78 months and the hazard ratio was 0.83 (95% CI 0.64–1.08, p = 0.12). Median disease-free survival times were 89 and 56 months, with a hazard ratio of 0.80 (95% CI 0.62–1.20, p = 0.065). An exploratory analysis demonstrated significant survival benefits for postoperative chemotherapy in patients with tumors greater than or equal to 4 cm in diameter (hazard ratio 0.69, 95% CI 0.48–0.99, p = 0.043).

146

K. Pisters

The ANITA (Adjuvant Navelbine International Trialist Association) trial randomized completely resected stage IB, II, and IIIA NSCLC to four cycles of postoperative vinorelbine (30 mg/m2 weekly × 4) and cisplatin (100 mg/m2 every 4 weeks) or surgery alone (7). This trial enrolled 840 patients. Postoperative radiotherapy was not mandatory and was undertaken according to every center’s policy. The trial was designed to detect an absolute improvement in 2-year overall survival of 10% (30% control), with 90% power, and a one-sided type I error of 5%. This required a sample size of 400 patients per treatment arm. Interim analyses for safety were done at 6 months, 12 months, and when 600 patients had enrolled. Eight hundred and forty patients were enrolled over a 6-year accrual period. Patient characteristics were well balanced between the two study arms. Sixty-one percent completed three of the planned four cycles of chemotherapy, although many required dose modifications. There were seven (2%) treatment-related deaths in the chemotherapy arm, compared to four (1%) in the surgery alone arm. Median overall survival for chemotherapy-treated patients was 65.7 months versus 43.7 for controls, with a hazard ratio of 0.80 (95% CI 0.66–0.96, p = 0.017). The absolute survival benefit at 2 years was 4.7 and 8.6% at 5 years. Disease-free survival also favored adjuvant chemotherapy with a hazard ratio of 0.76 (95% CI – 0.64–0.91, p = 0.002). This benefit was mainly seen in patients with stage II and IIIA disease.

Adjuvant Meta-analyses A meta-analysis examining the role of chemotherapy in NSCLC was published in 1995 (14). Part of this meta-analysis examined randomized trials of adjuvant chemotherapy. Trials which utilized regimens containing alkylating agents were detrimental to overall survival rates. However, data from eight trials randomizing a combined total of 1,394 patients to a postoperative cisplatin-based combination found favorable results. The hazard ratio estimates for these eight trials was 0.87 (95% CI 0.74–1.02, p = 0.08), which corresponded to a 13% reduction in the risk of death, and suggested an absolute benefit from chemotherapy of 3% at 2 years (95% confidence intervals 0.5% detriment to 7% benefit) and 5% at 5 years (95% confidence intervals 1% detriment to 10% benefit). An additional 18 trials were included in an update of this meta-analysis and was presented at ASCO 2007 (22). A total of 8,147 patients were randomized to 30 postoperative chemotherapy trials. A cisplatin-based regimen was used in 15 trials, tegafur or UFT (an oral 5-fluorouracil derivative) was used in eight trials, and seven trials used a combination of tegafur/UFT and cisplatin. Combining data from all trials, the overall hazard ratio was 0.86 (95% CI 0.81–0.93, p < 0.000001), with a 4% improvement in 5-year survival rate for patients receiving adjuvant chemotherapy. There was no subgroup of patients that did not benefit (age, sex or histology) and there was no difference in benefit seen by type of chemotherapy administered. The Lung Adjuvant Cisplatin Evaluation (LACE) meta-analysis (23) combined data from the large cisplatin-based adjuvant trials conducted after the 1995 meta-analysis

Adjuvant and Neoadjuvant Therapy of NSCLC

147

(7, 15, 16, 18, 19). 4,584 patients were included in this meta-analysis comparing postoperative cisplatin-based chemotherapy to no chemotherapy following complete resection. With a median follow-up of 5.2 years, the overall hazard ratio was 0.89 (95% CI 0.82–0.96, p = 0.005), corresponding to a 5.4% improvement in 5-year overall survival. Similar to the findings suggested in the individual trials, the survival benefit of adjuvant chemotherapy varied with stage of disease. The HR for stage IA was 1.40 (95% CI 0.95–2.0), stage IB was 0.93 (95% CI 0.78–1.10), stage II was 0.83 (95% CI 0.73–0.95), and stage III was 0.83 (95% CI 0.72–0.94). The effect of chemotherapy did not vary significantly (test for interaction, p = 0.11) with the associated drugs, including vinorelbine (HR = 0.80, 95% CI 0.70–0.91), etoposide or vinca alkaloid (HR = 0.92, 95% CI 0.80–1.07), or other (HR = 0.97, 95% CI 0.84–1.13). These findings support the use of adjuvant chemotherapy in completely resected stage II and III NSCLC, and confirm the lack of support for the use of adjuvant chemotherapy in stage I disease. In addition to the individual patient data meta-analyses reviewed above, there have been other meta-analyses of adjuvant therapy based on abstracted data from published reports. Meta-analyses based on abstracted data are based only on published trial results or abstracts and do not update patient outcomes, a critical step for interpreting adjuvant data. Although less time-consuming and costly, metaanalyses based on abstracted data are less reliable. An individual patient data (IPD) meta-analysis, involves updating individual patient data from all trials included in the analysis (23). This methodology is considered superior to one based on abstracted or pooled data. A meta-analysis by Bria et al. included 6,494 patients entered onto 12 cisplatinbased adjuvant trials (24). This meta-analysis found a significant benefit of chemotherapy with an absolute survival benefit of 4%. Sedrakyan and colleagues reported a literature-based meta-analysis including 7,200 patients treated on 19 randomized adjuvant trials (25). Cisplatin-based therapy was examined in 12 trials, while seven evaluated UFT. There was an 11% relative reduction in the mortality associated with postoperative cisplatin-based chemotherapy (95% CI, 4–18%, p = 0.004) and a 17% reduction with UFT (95% CI, 5–27%, p = 0.006) compared to surgery alone. A third meta-analysis based on abstracted data by Hotta and colleagues examined 11 trials enrolling a total of 5,716 patients (39). Trials randomizing patients to a cisplatin-based regimen (3,907 patients) showed a beneficial result with a hazard ratio of 0.891, 95% CI 0.815–0.975, p = 0.012, while trials randomizing patients to adjuvant UFT therapy (1,809 patients) also showed a significant survival benefit with treatment, hazard ratio of 0.799 (95% CI 0.668–0.957, p = 0.015).

UFT-Based Adjuvant Trials UFT (Taiho Pharmaceuticals, Japan), an oral fluoropyrimidine, is a combination of tegafur and uracil and is a well-tolerated oral agent. It has been studied extensively in the adjuvant setting in Japan. The 1995 meta-analysis previously discussed (14) examined the effect of adjuvant UFT and found similar benefits to adjuvant cisplatin

148

K. Pisters

chemotherapy, but the number of patients studied was too small to find significant differences. Trials have given adjuvant UFT as a single agent, or following intravenous cisplatin. In all trials, UFT has been administered orally for a prolonged period (6 months to 2 years). Some trials have found benefit (10). The largest trial of adjuvant UFT (26) randomized 979 eligible patients with completely resected stage I adenocarcinoma to either oral UFT, 250 mg/m2 for 2 years or no postoperative treatment. With a median follow-up of over 6 years, the overall survival at 5 years was 88% in the UFT group, and 85% in the control group (p = 0.047). The greatest benefit was in the T2N0 patient subgroup. Of particular concern in this trial was the lack of any difference seen for disease-free survival between the treated and untreated patient groups. A meta-analysis examining the efficacy of adjuvant UFT was presented at ASCO 2004 (27). Data from 2,003 patients enrolled on six different randomized adjuvant trials of single agent oral UFT were analyzed. Adjuvant UFT was found to significantly improve overall survival with a hazard ratio of 0.74 (95% CI 0.61–0.88, p = 0.001). Unfortunately, there are no confirmatory data concerning the use of adjuvant UFT outside of Japan and this agent is not available for use in the United States.

Current Standard of Practice There is strong evidence in support of postoperative cisplatin-based chemotherapy for good performance status patients with completely resected stage II and III NSCLC. Chemotherapy regimens administered in the trials, which confirmed a survival benefit are outlined in Table  3. There is no evidence in support of adjuvant chemotherapy in stage IA NSCLC. The role of adjuvant chemotherapy in stage IB disease is less established. Given the survival differences seen with current chemotherapy in stage IB disease, it has been estimated that in excess of 5,700 stage IB patients would be required to prove statistical significance (9).

Table 3  Chemotherapy regimens used in the positive adjuvant randomized trials Cisplatin plus vindesine 80–120 mg/m2 every 3–4 weeks, for three or four or vinblastine or cycles vinorelbine or 3 mg/m2 weekly × 5, then every 2 weeks etoposide 4 mg/m2 weekly × 5, then every 2 weeks 30 mg/m2 weekly 100 mg/m2 daily × 3 with each cisplatin NCIC CTG Cisplatin plus 50 mg/m2, days 1 and 8, every 4 weeks × 4 JRB.10 (19) vinorelbine 25 mg/m2 weekly × 16 ANITA (7) Cisplatin plus 100 mg/m2 every 4 weeks × 430 mg/m2 weekly × 16 vinorelbine

IALT (17)

Adjuvant and Neoadjuvant Therapy of NSCLC

149

Molecular Markers in NSCLC Biologic markers have the potential to be prognostic and/or predictive in all malignancies. These markers might lead to improved survival rates through many mechanisms. Identification of NSCLC patients at high and low risk of recurrence would lead to selective treatment of those most likely to benefit. Markers of response or resistance to therapy could individualize and optimize selection of therapy. Although this is an area of intensive research, at present, no individual marker or biologic profile has demonstrated a significant relationship with clinical outcome (28–31). Mutations in KRAS were prospectively evaluated in the NCIC JBR.10 trial (19). Over 90% of enrolled patients had KRAS assessment. In the subgroup of patients with KRAS mutations (11%), there was no benefit of adjuvant vinorelbine/cisplatin chemotherapy (32). The effect of expression of the DNA repair gene ERCC1 has been studied (33). Patients with ERCC1-negative tumors appeared to benefit from adjuvant chemotherapy, whereas patients with ERCC1-positive tumors did not. Other biologic markers including cell cycle regulators and Ki-67 have been assessed (34). Expression of p16, cyclin D1, cyclin D3, cyclin E, and Ki-67 did not correlate with benefit from adjuvant therapy (35, 36). However, NSCLC patients with p27-negative tumors appeared to benefit most from adjuvant cisplatin-based chemotherapy following complete resection. Recent reports have detailed efforts to develop molecular profiles of resected NSCLC using gene arrays, which may accurately predict prognosis and identify high-risk patients for adjuvant therapy (37). The lung metagene model provides a potential mechanism to identify patients at high risk of recurrence and target them for adjuvant therapy. Prospective randomized trials to validate this approach are planned.

Induction Chemotherapy Clinical Trials Initial forays into preoperative chemotherapy evaluated cisplatin-based chemotherapy in patients with stage IIIA disease. A preoperative regimen of mitomycin, vinca alkaloid (vindesine or vinblastine), and high-dose cisplatin (MVP) was administered in a phase II trial at Memorial Sloan–Kettering (38) and in a separate confirmatory trial at the University of Toronto (39). Both trials found this approach encouraging with high radiographic response rates, and improved resection and survival rates compared to historical controls. Other phase II trials employing second-generation cisplatin regimens have confirmed these findings (40–42) with radiographic response rates from 39 to 76%, feasible surgical resection, and some pathologic complete responses observed. Patients who have been found to have pathologic complete responses have been noteworthy for significantly prolonged survival (43).

150

K. Pisters

Another important finding of these trials was that radiographic response and surgical pathologic findings did not always correlate (44, 45). Finally, outcome does not appear to be substantially different in studies using combined radiation and chemotherapy as compared to trials using induction chemotherapy alone (46–48). A recent intergroup study comparing induction chemoradiation to induction chemotherapy in stage IIIA/N2 NSCLC sought to clarify the optimal preoperative strategy. Unfortunately, this trial closed secondary to poor accrual. More recent phase II investigations have examined platin-based regimens (cisplatin or carboplatin) in combination with a taxane or gemcitabine (49–58) in stage III NSCLC. Radiographic response rates range from 55 to 74%, complete surgical resections have occurred in 38–74% of patients and the median survival ranges from 15 to 24 months. Although comparison of these phase II trials is hampered by differences in patient characteristics and treatment administered, there are no striking differences between the two- and three-drug regimens, and second vs. third generation agents. Induction chemotherapy has also been evaluated in earlier stages of NSCLC. Phase II trials have utilized either cisplatin or carboplatin in combination with a taxane (45, 59–62) or gemcitabine (63–66). Response rates have ranged from 45 to 75% and complete resection rates from 71 to 95%. Survival rates have been encouraging and have led to phase III trials. Following encouraging phase II results of induction chemotherapy, randomized trials were undertaken. These induction chemotherapy trials with a surgery alone control arm were designed and conducted before the positive findings for postoperative chemotherapy were known. Of the phase III trials conducted in stage III NSCLC, two have been the most influential and controversial. The study conducted at M. D. Anderson Cancer Center randomized potentially resectable stage IIIA NSCLC to perioperative chemotherapy with cyclophosphamide, etoposide, and cisplatin followed by surgery or surgery alone (67, 68). Patients were randomized to three cycles of preoperative chemotherapy and an additional three cycles after surgery if they had evidence of radiographic response. Following an interim analysis, the trial was closed after only 60 patients had been accrued because of a clinically meaningful survival benefit. Long-term follow-up of this trial found median and 5-year survival rates were 21 months and 36% versus 14 months and 15% for surgery alone. A similar phase III trial conducted by Rosell enrolled clinical stage IIIA NSCLC patients (69, 70). Patients were randomized to immediate surgery or surgery preceded by three cycles of mitomycin, ifosfamide, and cisplatin. Both treatment groups received postoperative mediastinal radiation therapy to 50 Gy. Like the Roth trial, an interim analysis after 24-month follow-up with 60 eligible patients found a significant benefit for the combined approach and accrual to the trial was stopped. Re-assessment at 7 years of follow-up found median and 5-year survival rates of 22 months and 17% in the chemotherapy arm compared to 10 months and 0% in the surgery alone arm. These two trials have been criticized because of their small sample size and concerns over imbalance in staging and other important prognostic factors between the two study arms (71).

Adjuvant and Neoadjuvant Therapy of NSCLC

151

Another phase III randomized study of induction chemotherapy was conducted at the National Cancer Institute. This trial randomized stage IIIA/N2 patients to receive two cycles of cisplatin/etoposide chemotherapy prior to surgery (and four postoperative cycles if evidence of radiographic response) or surgery followed by 54–60 Gy of mediastinal radiation. After 4 years, only 27 patients had accrued and the trial was closed. An analysis published in 1992 found a trend toward improved survival in the chemotherapy arm of the study – median survival of 29 versus 16 months, p = 0.095 (72). A large randomized trial of neoadjuvant chemotherapy in stage III NSCLC has been conducted in China (73). This study randomized 724 patients over a 12-year period to preoperative chemotherapy or a control group of surgery alone. Of the 414 patients assigned to chemotherapy, the majority (393) were given intravenous cisplatin-based chemotherapy, while some had novel approaches including intraarterial chemotherapy. The response rate to induction chemotherapy was 73%, and the pathologic complete response rate was 15%. Complete resection rates were similar in the two arms (over 90%) and there were no significant differences in operative complications or mortality. Five- and ten-year survival rates were 34% and 29% versus 24% and 22%, p < 0.01. The Lung Cancer Surgical Study Group of the Japan Clinical Oncology Group has reported their experience of a phase III randomized trial in stage IIIA/N2 NSCLC (74). The trial was designed to accrue 200 patients over a 3-year period. However, the trial was closed secondary to slow accrual after only 62 patients had entered in 5 years. With a median follow-up of 2 years, there was no difference in survival between the two arms in terms of median or 5-year survival rates (17 months and 10% versus 16 months and 22% in the surgery alone arm). Phase III randomized trials of preoperative chemotherapy have been undertaken in earlier stages of NSCLC. These trials are summarized in Table  4. The first randomized study compared induction mitomycin, ifosfamide, cisplatin chemotherapy

Table 4  Recent randomized trials in early stage operable NSCLC Number eligible Clinical stages patients included Results Trial DePierre (75, 76) 355 IB–IIIA (including N2) 41% vs. 32% at 5 years Sorensen (77) 44 IB–IIIA (not N2) 36% vs. 24% at 4 years S9900 (78) 354 IB–IIIA (not N2) HR 0.81, p = 0.19 LU22 (79) 519 I–III HR 1.02, p = 0.86 ChEST (80) 270 IB–IIIA (not N2) HR 0.83, p = 0.053

Chemotherapy regimen Mitomycin, ifosfamide, cisplatin Paclitaxel, carboplatin Paclitaxel, carboplatin Cisplatin or carboplatin based Gemcitabine, cisplatin

152

K. Pisters

to surgery alone in resectable stage IB, II, and IIIA NSCLC (75). Three hundred and fifty-five eligible patients were randomized. Responding patients (radiographically or pathologically) received two additional cycles of the same chemotherapy postoperatively. Postoperative mortality was 6.7% in the chemotherapy arm and 4.5% in the surgery arm (p = 0.38). At the time of initial publication, the median survival was improved by 11 months (37 versus 26 months) and at 4 years, there was an 8.6% increase in survival in the chemotherapy arm, but this was not statistically significant. In a subset analysis, the benefit of chemotherapy was confined to patients with N0 to N1 disease, with a relative risk of death of 0.68, p = 0.027. No difference was seen in local recurrence rates, but there was a significant decrease in distant metastases with chemotherapy (relative risk 0.54, p = 0.01). Follow-up data after 60 months found the 3–5-year survival differences to be stable around 10% (p = 0.04 at 3 years and p = 0.06 at 5 years). Statistically significant benefits in the N0–N1 subgroup were confirmed with 5-year survival rates of 49% versus 34% (p = 0.02) (76). The Scandinavians have reported their randomized trial of neoadjuvant paclitaxel and carboplatin chemotherapy in clinical stage IB, II, and IIIA (excluding N2 patients) NSCLC (77). The study was closed prematurely secondary to slow accrual (90 patients in 6 years). Of the 44 patients randomized to chemotherapy, major radiographic responses were seen in 46 and 79% had complete resection. In the 46 patients treated with surgery alone, complete resection was achieved in 70%. Median and 5-year survival rates were 34 months and 36% compared to 23 months and 24% in the control arm. Although the results were not statistically significant, a beneficial effect for induction chemotherapy was suggested. The Southwest Oncology Group trial, S9900, compared induction paclitaxel/ carboplatin chemotherapy for three cycles followed by surgery to surgery alone in clinical stage IB, II, and IIIA NSCLC (excluding superior sulcus and N2 disease) (78). The trial design called for 600 patients to detect a 33% increase in median survival or a 10% increase in 5-year survival. Accrual to this trial was suspended after data from randomized adjuvant chemotherapy trials in completely resected NSCLC revealed a survival benefit. Total accrual reached 354 patients, and results were presented (78). Patient characteristics were well balanced between the two groups. Of the patients randomized to chemotherapy, 41% had radiographic response and 94% had complete resection. Eighty-nine percent of patients on the control arm had complete resection. With a median follow-up of 53 months, 3- and 5-year survival rates were 62 and 50% versus 57 and 43% for the combined modality and surgery alone arms, respectively. Although the use of chemotherapy was associated with a 19% reduction in the risk of death (hazard ratio 0.81, p = 0.19), this difference did not achieve statistical significance (78). The multicenter, randomized MRC LU22/NVLAT 2/EORTC 08012 trial of preoperative chemotherapy in patients with resectable non-small cell lung cancer did not find a survival benefit of neoadjuvant chemotherapy (79). This study randomized 519 patients from 70 centers. In contrast to other neoadjuvant studies, the majority (61%) were clinical stage I, while 31% had stage II, and 7% were stage III. Seventy-five percent of patients were able to complete the three cycles of platin-based chemotherapy with 49% experiencing a radiographic response. There was no difference

Adjuvant and Neoadjuvant Therapy of NSCLC

153

in the type or completeness of surgery and postoperative complications were not increased. The hazard ratio for overall survival was 1.02 (95% CI 0.80–1.31, p = 0.86). The inclusion of a large number of stage I patients may have led to negative results of this study (71). Results from the Ch.E.S.T. (Chemotherapy in Early Stages in NSCLC Trial) trial were recently presented (80). This phase III randomized trial compared three cycles of induction gemcitabine/cisplatin chemotherapy administered before resection to surgery alone. The primary endpoint of this study was progression-free survival and the original study design required 700 randomized patients. Similar to the S9900 study, the CH.E.S.T. trial was closed early based on the positive adjuvant trials. The 3-year overall survival was 67% in the combined modality arm and 60% in the control arm (p = 0.053). A subset analysis by stage found this survival benefit most pronounced for patients with stage IIB and IIIA disease (3-year overall survival 70% versus 47%, p = 0.001). Finally, a randomized phase II trial comparing induction chemotherapy followed by surgery, to surgery followed by adjuvant chemotherapy, to a third arm of surgery alone has completed accrual. The trial is designed with disease-free survival as the primary endpoint, but may shed some light onto the optimal timing of chemotherapy with respect to resection (81).

Induction Chemotherapy Meta-analyses To date, there have been two meta-analyses examining the efficacy of induction chemotherapy in resectable NSCLC (82, 83). Both of these meta-analyses were not IDP (Individual patient data) meta-analyses, but were based on data extracted from abstracts and manuscripts. Drawbacks to the abstracted data meta-analysis methodology were reviewed earlier in the chapter and the conclusions of these metaanalyses should be interpreted with caution. The meta-analysis by Berghmans et al (82), looked at both induction and adjuvant randomized studies reported between 1965 and June 2004. They found a hazard ratio (HR) of 0.66 (95% CI 0.48–0.93) for the addition of induction chemotherapy and a HR of 0.84 (95% CI 0.78–0.89) for the addition of adjuvant (postoperative chemotherapy). There were six randomized neoadjuvant trials included in this meta-analysis enrolling 590 patients. When examining the effect of induction chemotherapy in the subgroup of patients with clinical stage III NSCLC, the HR became 0.65 (95% CI 0.41–1.04) and although strongly trended in favor of the use of chemotherapy in stage III disease, did not achieve statistical significance. In a subsequent letter to the editor, the statistical methodology used to compare the effects of induction chemotherapy in stages I/II versus stage III NSCLC was questioned and it was suggested that the nonsignificant result may have been related to numbers of patients included or because of the use of the random effects model (84). The meta-analysis by Burdett et  al was also based on data extracted from abstracts and manuscripts from randomized trials (83). Literature searches identified

154

K. Pisters

12 eligible randomized controlled trials. Five of these trials were excluded for insufficient data; the remaining seven trials included 988 patients. The authors found that preoperative chemotherapy improved survival with a hazard ratio of 0.82 (0.69–0.97, p = 0.02). This is equivalent to an absolute benefit of 6% at 5 years. An analysis grouping trials according to the type of chemotherapy administered was also performed. All patients received a platinum-based chemotherapy – either cisplatin or carboplatin – that was combined with other agents. These other agents were split into three groups: vinca alkaloid/etoposide, taxane, or other. There was no clear evidence of a difference of treatment effect shown by chemotherapy group. The authors concluded that the meta-analysis suggested a significant survival benefit for patients with NSCLC who receive preoperative chemotherapy compared to those who do not. The value of this treatment will be further assessed through an ongoing IPD meta-analysis.

Surgical Morbidity and Mortality After Induction Therapy The use of chemotherapy prior to surgery has raised concern that surgical complications may be increased. Data from large series address this issue. Siegenthaler et al. from MDACC have reported on a series of 380 consecutive patients undergoing lobectomy or greater resection for NSCLC from the MDACC Thoracic Surgery database (85). The use of preoperative chemotherapy did not significantly affect morbidity or mortality overall, based on clinical stage, postoperative stage, or extent of resection. No significant differences in overall or subset mortality or morbidity including pneumonia, acute respiratory distress syndrome, reintubation, tracheostomy, wound complication, or length of hospitalization were seen. All patients undergoing thoracotomy after induction chemotherapy from 1993 to 1999 at MSKCC were the subject of a review (86). Four hundred seventy patients treated with induction chemotherapy and surgery were reviewed. Univariate and multivariate methods for logistic regression model were used to identify predictors of adverse events. Overall, the MSKCC group found a surgical mortality rate of 3.8%, which compared favorably to other primary surgery studies. Total morbidity and major complication rates were 38 and 27%, similar to previous primary surgery studies. The authors concluded that overall morbidity rates were not significantly affected by the use of induction therapy. They did find an operative mortality rate of 24% for patients undergoing right pneumonectomy following induction therapy. The authors recommended that right pneumonectomy following induction therapy be performed very selectively and only when no alternative resection is possible. A third series from investigators in France reviewed 114 patients who underwent thoracotomy following induction chemotherapy (87). In this series, there was only one death following pneumonectomy in 55 patients. Overall morbidity rate was 29%, similar to other surgical series. The authors concluded that preoperative chemotherapy did not increase postoperative morbidity and mortality.

Adjuvant and Neoadjuvant Therapy of NSCLC

155

Current Standard of Practice Induction chemotherapy has been extensively evaluated and appears promising based on phase II and phase III trials. The number of patients studied in individual phase III trials has been inadequate to clearly determine the efficacy of this approach. Two meta-analyses, which extracted data from published trials and abstracts, have both shown statistically significant benefits in favor of induction chemotherapy. A meta-analysis employing individual patient data is underway and should yield more reliable results. At present, stronger data exist in support of postoperative administration of chemotherapy for patients with resectable nonsmall cell lung cancer.

Conclusion Recent trials and meta-analyses have found improved survival for the incorporation of systemic chemotherapy into the management of patients with stage II and III resectable NSCLC. At this time, the evidence is stronger for the use of postoperative chemotherapy. The role of chemotherapy in stage IB NSCLC is evolving, and should be individualized at this time. Postoperative radiation is currently recommended only for patients at high risk of local relapse. Ongoing trials should clarify the role of PORT in the near future.

References 1. Mountain CF (1997) Revisions in the international system for staging lung cancer. Chest 111:1710–1717 2. The Lung Cancer Study Group (1986) Effects of postoperative mediastinal radiation on completely resected stage II and stage III squamous cell carcinoma of the lung. N Engl J Med 315:1377–1381 3. PORT Meta-analysis Trialists Group (1988) Postoperative radiotherapy in non-small cell lung cancer: systematic review and meta-analysis of individual patient data from nine randomized controlled trials. Lancet 352:257–263 4. Munro AJ (1998) What now for postoperative radiotherapy for lung cancer? Lancet 352(9124):250–251 5. Machtay M, Lee JH, Shrager JB et al (2001) Risk of death from intercurrent disease is not excessively increased by modern postoperative radiotherapy for high-risk resected non-small cell lung carcinoma. J Clin Oncol 19:3912–3917 6. Lally BE, Zelterman D, Colasanto JM, Haffty GB, Detterbeck FC, Wilson LD (2006) Postoperative radiotherapy for stage II or III non-small-cell lung cancer using the Surveillance, Epidemiology, and End Results Database. J Clin Oncol 24(19):2998–3006 7. Douillard J, Rosell R, De Lena M et  al (2006) Adjuvant vinorelbine plus cisplatin versus observation in patients with completely resected stage IB-IIIA non-small cell lung cancer (Adjuvant Navelbine International Trialist Association [ANITA]): a randomized controlled trial. Lancet Oncol 7:719–727

156

K. Pisters

8. Okawara G, Ung YC, Markman BR et al (2004) Postoperative radiotherapy in stage II or IIIA completely resected non-small cell lung cancer: a systematic review and practice guideline. Lung Cancer 44:1–11 9. Pisters KMW, Evans WK, Azzoli CG et al (2007) Cancer Care Ontario and American Society of Clinical Oncology adjuvant chemotherapy and adjuvant radiation therapy for stages I-IIIA resectable non-small cell lung cancer guidelines. J Clin Oncol 25:5506–5518 10. Pisters KMW, Lechevalier T (2005) Adjuvant chemotherapy in completely resected non-small cell lung cancer. J Clin Oncol 23:3270–3278 11. Holmes EC, Gail M (1986) Surgical adjuvant therapy for stage II and stage III adenocarcinoma and large-cell undifferentiated carcinoma. J Clin Oncol 4:710–715 12. Niiranen A, Niitamo-Korhonen S, Kouri M, Assendelft A, Mattson K, Pyrhönen S (1992) Adjuvant chemotherapy after radical surgery for non-small-cell lung cancer: a randomized study. J Clin Oncol 10(12):1927–1932 13. Feld R, Rubinstein L, Thomas PA, The Lung Cancer Study Group (1993) Adjuvant chemotherapy with cyclophosphamide, doxorubicin, and cisplatin in patients with completely resected stage I non-small-cell lung cancer. J Natl Cancer Inst 85(4):299–306 14. Non-small cell lung cancer collaborative group (1995) Chemotherapy in non-small cell lung cancer: a meta-analysis using updated data on individual patients from 52 randomized clinical trials. BMJ 311:899–909 15. Scagliotti GV, Fossati R, Torri V et al (2003) Randomized study of adjuvant chemotherapy for completely resected stage I, II or IIIA non-small cell lung cancer. J Natl Cancer Inst 95:1453–1461 16. The International Adjuvant Lung Cancer Trial Collaborative Group (2004) Cisplatin-based adjuvant chemotherapy in patients with completely resected non-small cell lung cancer. N Engl J Med 350:351–360 17. Le Chevalier T, Arriagada R, Bergman B et al (2008) Long-term results of the international adjuvant lung cancer trial (IALT) evaluating adjuvant cisplatin-based chemotherapy in resected non-small cell lung cancer (NSCLC). J Clin Oncol 26:398s Abstr 7507 18. Waller D, Peake MD, Stephens RJ et al on behalf of all BLT Participants (2004) Chemotherapy for patients with non-small cell lung cancer. The surgical setting of the Big Lung Trial. Eur J Cardiothorac Surg 26:173–182 19. Winton T, Livingston R, Johnson D et al (2005) Vinorelbine plus cisplatin vs. observation in resected non-small cell lung cancer. N Engl J Med 353:2589–2597 20. Strauss GM, Herndon JE, Maddaus MA, Johnstone DW, Johnson EA, Watson DM et al (2006) Adjuvant chemotherapy in stage IB non-small cell lung cancer (NSCLC): Update of Cancer and Leukemia Group B (CALGB) protocol 9633. [Abstract]. J Clin Oncol 24(Suppl 18):A7007 21. Strauss GM, Herndon JE, Maddaus MA et  al (2008) Adjuvant paclitaxel plus carboplatin compared with observation in stage IB non-small cell lung cancer: CALGB 9633 with the Cancer and Leukemia Group B, Radiation Therapy Oncology Group, and North Central Cancer Treatment Group Study Group. J Clin Oncol 26:5043–5051 22. Stewart LA, Burdett S, Tierney JF, Pignon J, on behalf of The NSCLC Collaborative Group (2007) Surgery and adjuvant chemotherapy compared to surgery alone in non-small cell lung cancer: a meta-analysis using individual patient data from randomized clinical trials. J Clin Oncol ASCO Annual meeting proceedings part I. 25(18S, June 20 Suppl):A7552 23. Pignon JP, Tribodet H, Scagliotti GV et al (2008) Lung Adjuvant Cisplatin Evaluation: A pooled analysis by the LACE collaborative group. J Clin Oncol 26:3552–3559 24. Piedbois P, Byuse M (2004) Meta-analyses based on abstracted data: A step in the right direction, but only a first step. J Clin Oncol 22:3839–3840 25. Bria E, Gralla R, Raftopoulos H et al (2005) Does adjuvant chemotherapy improve survival in non small cell lung (NSCLC)? A pooled-analysis of 6494 patients in 12 studies, examining survival and magnitude of benefit. J Clin Oncol 23(Suppl 16S):abstr7140 26. Sedrakyan A, van Der Meulen J, O’Byrne K et al (2004) Postoperative chemotherapy for nonsmall cell lung cancer: a systematic review and meta-analysis. J Thorac Cardiovasc Surg 128(3):414–419

Adjuvant and Neoadjuvant Therapy of NSCLC

157

27. Hotta K, Matsuo K, Ueoka H, Kiura K, Tabata M, Tanimoto M (2004) Role of adjuvant chemotherapy in patients with resected non-small-cell lung cancer: reappraisal with a metaanalysis of randomized controlled trials. J Clin Oncol 22(19):3860–3867 28. Kato H, Ichinose Y, Ohta M et al (2004) A randomized trial of adjuvant chemotherapy with uracil-tegafur for adenocarcinoma of the lung. N Engl J Med 350:1713–1721 29. HHamada C, Ohta M, Wada H et al (2004) Survival benefit of oral UFT of adjuvant chemotherapy after completely resected non-small cell lung cancer [Abstract]. J Clin Oncol 22(14 Supp):A70 30. Kikuchi T, Carbone DP (2007) Proteomics analysis in lung cancer: challenges and opportunities. Respirology 12(1):22–28 31. Dziadziuszko R, Hirsch F (2008) Advances in genomic and proteomic studies of non-small cell lung cancer: clinical and translational research perspective. Clin Lung Cancer 9(2):78–84 32. Kosaka R, Yatabe Y, Onozato R et  al (2009) Prognostic implication of EGFR, KRAS, and TP53 gene mutations in a large cohort of Japanese patients with surgically treat lung adenocarcinoma. J Thorac Oncol 4:22–29 33. D’Amico TA, Brooks KR, Moore Joshi MB et al (2006) Serum protein expression predicts recurrence in patients with early stage lung cancer after resection. Ann Thorac Surg 81:1982–1987 34. Aviel-Ronen S, Blackhall FH, Shepherd FA, Tsao MS (2006) K-ras mutations in non-smallcell lung carcinoma: a review. Clin Lung Cancer 8(1):30–38 35. Olaussen KA, Dunant A, Fouret P et al (2006) DNA repair by ERCC1 in non-small cell lung cancer and cisplatin-based adjuvant chemotherapy. N Engl J Med 355(10):983–991 36. Filipits M, Pirker R, Dunant A et al (2007) Cell cycle regulators and outcome of adjuvant cisplatin-based chemotherapy in completely resected non-small cell lung cancer: the International Adjuvant Lung Cancer Trial Biologic Program. J Clin Oncol 25(19): 2735–2740 37. Potti A, Mukherjee S, Petersen R et al (2006) A genomic strategy to refine prognosis in early stage non-small cell lung cancer. N Engl J Med 355:570–580 38. Martini N, Kris M, Flehinger B et  al (1993) Preoperative chemotherapy for stage IIIa(N2) lung cancer: The Memorial Sloan-Kettering experience with 136 patients. Ann Thorac Surg 55:1365–1374 39. Burkes R, Shepherd R, Blackstein M et al (2005) Induction chemotherapy with mitomycin, vindesine, and cisplatin for stage IIIA (T1-3N2) unresectable non-small cell lung cancer: final results of the Toronto phase II trial. Lung Cancer 47:103–109 40. Darwish S, Minotti V, Crino L et al (1994) Neoadjuvant cisplatin and etoposide for stage IIIA (clinical N2) non-small cell lung cancer. Am J Clin Oncol 17:64–67 41. Vokes E, Bitran J, Hoffman P et al (1989) Neoadjuvant vindesine, etoposide and cisplatin for locally advanced non-small cell lung cancer. Chest 96:110–113 42. Elias A, Skarin A, Leong T et al (1997) Neoadjuvant therapy for surgically staged IIIA N2 non-small cell lung cancer (NSCLC). Lung Cancer 17:147–161 43. Pisters K, Kris M, Gralla R et al (1993) Pathologic complete response in advanced non-small cell lung cancer following preoperative chemotherapy: Implications for the design of future non-small cell lung cancer combined modality trials. J Clin Oncol 11:1757–1762 44. Patel J, Blum M, Argiris A (2004) Induction chemotherapy for resectable non-small cell lung cancer. Oncology 18:1591–1602 45. Pisters K, Ginsberg R, Giroux D et al (2000) Induction chemotherapy before surgery for earlystage lung cancer: A novel approach. J Thorac Cardiovasc Surg 119:429–439 46. Albain K, Rusch V, Crowley J et al (1995) Concurrent cisplatin/etoposide plus chest radiotherapy followed by surgery for stage IIIA (N2) and IIIB NSCLC: mature results of Southwest Oncology Group phase II study 8805. J Clin Oncol 13:1880–1892 47. Faber LP, Kettle F, Warren WH et al (1989) Preoperative chemotherapy and irradiation for stage III non-small cell lung cancer. Ann Thorac Surg 47:266–272 48. Strauss G, Herndon J, Sherman D et al (1992) Neoadjuvant chemotherapy and radiotherapy followed by surgery in stage IIIA non-small cell lung carcinoma of the lung: Report of a Cancer and Leukemia Group B phase II study. J Clin Oncol 10:1237–1244

158

K. Pisters

49. Betticher D, Hsu Schmitz S, Roth A et  al (2005) Neoadjuvant therapy with docetaxel and cisplatin in patients with non-small cell lung cancer, stage IIIA, pN2: A large phase II study with 59 months of follow-up. Lung Cancer 49:S14 50. Betticher D, Hsu Schmitz S, Totsch M et  al (2003) Mediastinal lymph node clearance after docetaxel-cisplatin neoadjuvant chemotherapy is prognostic of survival in patients with stage IIIA pN2 non-small cell lung cancer: a multicenter phase II trial. J Clin Oncol 21:1752–1759 51. Van Zandwijk N, Smit E, Kramer G et al (2000) Gemcitabine and cisplatin as induction regimen for patients with biopsy-proven stage IIIA N2 non-small cell lung cancer: A phase II study of the European Organization for Research and Treatment of Cancer Lung Cancer Cooperative Group (EORTC 08955). J Clin Oncol 18:2658–2664 52. Migliorino M, De Marinis F, Nelli F et al (2002) A 3-week schedule of gemcitabine plus cisplatin as induction chemotherapy for stage III non-small cell lung cancer. Lung Cancer 35:319–327 53. Choi I, Oh D, Kwon J et al (2005) Paclitaxel/platinum-based perioperative chemotherapy and surgery in stage IIIA non-small cell lung cancer. Jpn J Clin Oncol 35:6–12 54. Garrido P, Rosell R, Torres A et al (2005) Docetaxel, cisplatin and gemcitabine as neoadjuvant treatment in stage IIIAN2 and T4N0-1 non-small cell lung cancer patients. Final results of SLCG phase II trial 9901. Lung Cancer 49:S14 55. Esteban E, Fernancez Y, Vieitez J et  al (2005) Cisplatin plus gemcitabine with or without vinorelbine as neoadjuvant therapy for radically treatable stage III non-small cell lung cancer. Preliminary results of a randomized study of the GON. Lung Cancer 49:S40 56. Lorent N, De Leyn P, Lievens Y et al (2004) Long term survival of surgically staged IIIA-N2 non-small cell lung cancer treated with surgical combined modality approach: analysis of a 7-year prospective experience. Ann Oncol 15:1645–1653 57. Cappuzzo F, De Marinis F, Nelli F et  al (2003) Phase II study of gemcitabine-cisplatinpaclitaxel triplet as induction chemotherapy in inoperable, locally advanced non-small cell lung cancer. Lung Cancer 42:355–361 58. De Marinis F, Nelli F, Migliorino M et  al (2003) Gemcitabine, paclitaxel, and cisplatin as induction chemotherapy for patients with biopsy-proven stage IIIA(N2) non-small cell carcinoma. Cancer 98:1707–1715 59. Brechot J, Saintigny P, Azorin J et al (2005) A phase II randomized study of gemcitabinecisplatin versus mitomycin-ifosfamide-cisplatin as neoadjuvant chemotherapy in resectable stage IIIA non-small cell lung cancer. Lung Cancer 49:S175 60. Marks R, Streitz J, Deschamps C et  al (2001) Response rate and toxicity of preoperative paclitaxel and carboplatin in patients with resectable non-small cell lung cancer: a North Central Cancer Treatment Group study. J Clin Oncol 20:abstr1355 61. Tsuboi M, Ohira T, Hayashi A et  al (2005) Preoperative induction chemotherapy with weekly paclitaxel and carboplatin for clinical-stage IB-IIIA non-small cell lung cancer. Lung Cancer 49:S175 62. Kunitoh H, Hato H, Tsuboi M, et al. (2005) A randomized phase II trial of preoperative docetaxel and cisplatin or docetaxel alone in clinical stage IB/II non-small cell lung cancer: Initial resort of Japan Clinical Oncology Group Trial. J Clin Oncol 23(16S):abstr 7119 63. Abratt R, Lee J, Han J et  al (2005) Phase II trial of gemcitabine-carboplatin-paclitaxel as neoadjuvant chemotherapy for operable non-small cell lung cancer. Lung Cancer 49:S92 64. Lothaire P, Berghmans Th, Lafitte J et al (2003) Resectability after two different chemotherapy regimens: results of the first step of a phase II randomized trial in initially resectable stage I-IIIA non-small cell lung cancer conducted by the European Lung Cancer Working Party. Lung Cancer 41(Suppl 2):S63 65. Socinski M, Detterbeck F, Gralla R et al (2005) Induction chemotherapy with gemcitabinecontaining regimens in stage I-II non-small cell lung cancer: Initial results of the GINEST Project. Lung Cancer 49:S40 66. Sommers E, Ramnath N, Robinson L et al (2005) Neoadjuvant chemotherapy with gemcitabine and vinorelbine in resectable non-small cell lung cancer. Lung Cancer 49:S96–S97 67. Roth J, Fossella F, Komaki R et al (1994) A randomized trial comparing perioperative chemotherapy and surgery with surgery alone in resectable stage IIIA non-small cell lung cancer. J Natl Cancer Inst 86:673–680

Adjuvant and Neoadjuvant Therapy of NSCLC

159

68. Roth J, Atkinson E, Fossella F et al (1998) Long-term followup of patients enrolled in a randomized trial comparing perioperative chemotherapy and surgery with surgery alone in resectable stage IIIA non-small cell lung cancer. Lung Cancer 21:1–6 69. Rosell R, Gomez-Codina J, Camps C et al (1994) A randomized trial comparing preoperative chemotherapy plus surgery with surgery alone in patients with non-small cell lung cancer. N Engl J Med 330:153–158 70. Rosell R, Gomez-Codina J, Camps C et al (1999) Preresectional chemotherapy in stage IIIA nonsmall cell lung cancer: a 7-year assessment of a randomized controlled trial. Lung Cancer 47:7–14 71. Bradbury P, Shepherd FA (2007) Chemotherapy and surgery for operable NSCLC. Lancet 369:1903–1904 72. Pass H, Pogrebniak H, Steinberg S et al (1992) Randomized trial of neoadjuvant therapy for lung cancer: Interim analysis. Ann Thorac Surg 53:992–998 73. Zhou Q, Liu L, Li L et  al (2003) A randomized clinical trial of preoperative neoadjuvant chemotherapy followed by surgery in the treatment of stage III non-small cell lung cancer. Lung Cancer 41(Suppl 2):S45–S46 74. Nagai K, Tsuchiya R, Mori T et al (2003) A randomized trial comparing induction chemotherapy followed by surgery with surgery alone for patients with stage IIIA N2 non-small cell lung cancer (JCOG 9209). J Thorac Cardiovasc Surg 125:254–260 75. Depierre A, Milleron B, Moro-Sibilot D et al (2001) Preoperative chemotherapy followed by surgery compared with primary surgery in resectable stage I (except T1N0), II, and IIIA nonsmall cell lung cancer. J Clin Oncol 20:247–253 76. Depierre A, Westeel V, Milleron B et al (2003) 5 year results of the French Randomized study comparing preoperative chemotherapy followed by surgery and primary surgery in resectable stage I (except T1N0), II and IIIA non-small cell lung cancer. Lung Cancer 41(Suppl 2):S62 77. Sorensen J, Riska H, Ravn J et al (2005) Scandinavian phase III trial of neoadjuvant chemotherapy in NSCLC stages IB-IIIA/T3. J Clin Oncol 23(16S):abstr7146 78. Pisters KM, Vallieres E, Bunn PA et al (2007) S9900: surgery alone or surgery plus induction (ind) paclitaxel/carboplatin (PC) chemotherapy in early stage non-small cell lung cancer (NSCLC): follow-up on a phase III trial. J Clin Oncol 25(18S):abstr 7520 79. Gilligan D, Nicolson M, Smith I et  al (2007) Preoperative chemotherapy in patients with respectable non-small cell lung cancer: results of the MRC LU22/NVALT 2/EORTC 08012 multicentre randomized trial and update of systematic review. Lancet 369:1929–1937 80. Scagliotti GV, Pastorino U, Vansteenkiste JF et  al (2008) A phase III randomized study of surgery alone or surgery plus preoperative gemcitabine-cisplatin in early-stage non-small cell lung cancer (NSCLC): Follow-up data of Ch.E.S.T. J Clin Oncol 26(Suppl):abstr 7508 81. Canela M, Maestre J, Rodriguez-Paniagua M et al (2005) NATCH Trial update. Lung Cancer 49:S93 82. Berghmans T, Paesmans M, Meert A et al (2005) Survival improvement in resectable nonsmall cell lung cancer with (neo)adjuvant chemotherapy: Results of a meta-analysis of the literature. Lung Cancer 49:3–23 83. Burdett S, Stewart L, Rydzewska L et al (2006) A systematic review and meta-analysis of the literature: chemotherapy and surgery versus surgery alone in non-small cell lung cancer. J Thorac Oncol 1:611–621 84. Pignon JP, Burdett S (2005) Letter to the editor. Lung Cancer 51:261–262 85. Siegenthaler M, Pisters K, Merriman K et al (2001) Preoperative chemotherapy for lung cancer does not increase surgical morbidity. Ann Thorac Surg 71:1105–1112 86. Martin J, Ginsberg R, Abolhoda A et  al (2001) Morbidity and mortality after neoadjuvant therapy for lung cancer: The risks of right pneumonectomy. Ann Thorac Surg 72:1149–1154 87. Perrot E, Guibert B, Mulsant P et  al (2005) Preoperative chemotherapy does not increase complications after non-small cell lung cancer resection. Ann Thorac Surg 80:423–427

Chemoradiotherapy for Inoperable Non-small Cell Lung Cancer James D. Cox and David J. Stewart

Abstract  In at least some patients, radiotherapy alone can lead to long-term control of stage III or medically inoperable stage I–II non-small cell lung cancer (NSCLC). Administration of chemotherapy with radiotherapy increases survival rates in stage III NSCLC, and administration of chemotherapy concurrently with radiotherapy is more effective than sequential administration of induction chemotherapy followed later by radiotherapy. Concurrent administration is particularly important for augmenting local control of tumor. Chemotherapy can improve outcome by killing cells that survive radiotherapy, by killing distant micrometastases, and by sensitizing tumor cells to radiation. Radiosensitization has been noted with several classes of systemic agents, including platinums, taxanes, vinca alkaloids, topoisomerase I and II inhibitors, antimetabolites, and agents targeting the epidermal growth factor receptor and HER2/neu. It is anticipated that several other classes of new targeted agents could also play a role in the future. New radiotherapy techniques including intensity-modulated radiation therapy and proton beam therapy have the potential to further improve the outcome, and early clinical trials are underway to optimize the combination of systemic agents with these new radiotherapy options. Keywords  Radiotherapy • Chemoradiotherapy • Radiosensitization • IMRT • Proton beam

Introduction Radiotherapy can provide effective palliation of symptoms from advanced non-small cell lung cancer (NSCLC), can decrease probability of local recurrence following resection of involved mediastinal lymph nodes in stage III disease, and can lead to

J.D. Cox (*) and D.J. Stewart Departments of Thoracic/Head and Neck Medical Oncology, MD Anderson Cancer Center, Houston, TX 77030, USA D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_7, © Humana Press, a part of Springer Science+Business Media, LLC 2010

161

162

J.D. Cox and D.J. Stewart

long-term control in at least some patients with locally advanced (stage III) or medically inoperable stage I–II NSCLC. Over the past two decades, the importance of combining chemotherapy and radiation therapy in the treatment of inoperable NSCLC has become clear. Chemotherapy alone, while effective in the short run in some patients, rarely results in long-term control of the disease. Radiation therapy alone is able to produce long-term progression-free survival in approximately 5% of patients. The first evidence of improvement in outcome by adding chemotherapy to radiotherapy came from the Cancer and Leukemia Group B (CALGB): Dillman et al. (1) reported increased survival with the use of induction chemotherapy with cisplatin and vinblastine followed by radiation therapy (60  Gy in 30 fractions) for patients with NSCLC and favorable performance status compared with radiation therapy alone. Confirmatory trials were carried out in Europe and North America, and meta-analysis of individual patient data supports a role for platinum-based chemotherapy added to radiotherapy in locally advanced NSCLC (2). Chemotherapy given concurrently with radiation therapy proved to be superior to sequential chemotherapy and radiation (3, 4). Thus, concurrent chemotherapy and radiation therapy became the standard of care in North America.

Biological Basis for Radiation Treatment Ionizing radiations produce their lethal effect largely through the production of hydroxyl radicals that interact with DNA. The chemical reactions last microseconds, but the biologic effects can take a much longer time, even years. Radiation damage can be repaired, although the ability of cancer cells to repair is less than that of normal cells. There is some crude relationship between the replication rate of cells and their radiosensitivity. This is one of the reasons why cancer cells are killed selectively in comparison to normal cells. Radiation treatments are usually given in a series, i.e., the total dose is fractionated into smaller doses. For NSCLC, the most common method of delivery is daily treatments, 5  days per week for a period of 6 or 7 weeks. Radiation damage is repaired between fractions, and cells may be redistributed into different phases of the cell cycle (with differing sensitivities for the next dose). A fundamental difference between tumors and normal tissues is the presence of hypoxia. Hypoxia results in decreased sensitivity to radiation effects. Reoxygenation between fractions of radiation results in an overall increase in the sensitivity of residual tumor cells. Radiation effects can result in cell cycle checkpoint arrest that permits repair, or can result in the activation of cell death pathways that lead to apoptosis or mitotic death. While apoptosis may be relevant to radiation cell death with malignant lymphomas, carcinoma cells usually undergo mitotic death. Molecular regulation of repair is critically important to the interaction of radiation and chemotherapy. Several mechanisms affect repair (5), including base-excision repair, nucleotide excision

Chemoradiotherapy for Inoperable Non-small Cell Lung Cancer

163

repair, recombinational repair which may be homologous and error-free or nonhomologous end-joining that is prone to errors. Molecular regulators are specific for each type of repair. Redistribution in the cell cycle results from a single fraction of radiation such that the next fraction may occur during a sensitive phase such as G2/M or a resistant phase such as late S. Since many chemotherapeutic agents affect the cell primarily in the S phase, a favorable interaction of radiation therapy and chemotherapy on the tumor may be realized.

Impact of Chemotherapy with Radiation Therapy As noted above, ionizing radiations produce lethal effects by means of damaging DNA. The most important events leading to cell killing are double strand breaks. These are repaired much less efficiently than single strand breaks, and the subsequent ability of DNA to replicate is impaired or abolished. The most sensitive part of the cell cycle to radiation effects is mitosis or the post S phase pre-mitosis part (G2/M). Cytotoxic chemotherapeutic agents affect the cell at different parts of the cell cycle, often at the S phase, which is the least sensitive part of the cell cycle to radiation. Chemotherapy and radiation therapy interactions are best considered additive or even sub-additive; rarely are they supra additive. A comprehensive review of radiation/chemotherapy interactions can be found in Milas and Cox (6), and we summarize some of these below.

Mechanisms by Which Systemic Therapy May Potentiate Radiation There are several reasons that combining chemotherapy with radiotherapy could prove more effective than using either alone for locally advanced disease. In addition to simple additivity of cell killing of the tumor targeted by the radiotherapy, chemotherapy could potentially kill micrometastases outside the radiotherapy field. The observation of reduced rate of distant metastases in some chemoradiotherapy trials indicates that this may be one important mechanism, and this is supported by the observation that adjuvant chemotherapy also prolongs survival when used before or after surgery in NSCLC. Radiotherapy could also play a role in reducing the development of metastases by decreasing accelerated repopulation in the primary tumor between chemotherapy cycles. The observation in small cell lung cancer (SCLC) that the development of brain metastases is reduced by using early versus delayed thoracic radiotherapy (7) would support this as a mechanism of favorable interaction between the two modalities.

164

J.D. Cox and D.J. Stewart

Platinum-Based Agents Plus Radiotherapy Cisplatin (8) and carboplatin (9) may potentiate radiotherapy effect in addition to being directly toxic to cancer cells, and early clinical trials demonstrated that weekly administration of cisplatin concurrently with radiation was safe (10). Radiopotentiation is seen both in tumor cell lines that are sensitive to cisplatin as well as in cisplatin-resistant lines (11). Cisplatin’s radiation enhancement ratio is higher in hypoxic cells than in the presence of oxygen (12). There are several mechanisms by which cisplatin may potentiate the effect of radiation (8). Cisplatin is electron affinic and may thereby take the place of oxygen in prolonging the half-life of cytotoxic, DNA-damaging hydroxyl radicals generated by ionizing radiation (8). Cisplatin may also inhibit both homologous recombination repair (13) and nonhomologous end-joining repair (13, 14) of radiation-induced DNA lesions, with inhibition of repair of both sublethal (8, 15) and of potentially lethal (8) DNA damage. Platinums may also increase cellular accumulation in the radiation-sensitive G2/M phase of the cell cycle (8) and may potentiate the effect of radiation by depleting cells of protective thiols (8). Conversely, when ionizing radiation hits platinum bound to macromolecules, this may lead to a 1-electron reduction of the bound platinum and the release of toxic ligands (8), and exposure of a cisplatin solution to radiation increases its cytotoxicity (16), presumably by generating toxic metabolites. Relative scheduling of platinum and radiation effect might determine which of these radiopotentiating mechanisms was most relevant. Platinums are tightly bound to human tissues, and may still be detected several months after last cisplatin treatment (17). This long-term bound platinum might in theory still function as a free electron acceptor or generate toxic ligands upon exposure to radiation. Platinums administered several hours before radiation might synchronize cells in G2/M, while platinums administered shortly before radiation might deplete cells of thiols and might be converted by radiation to toxic metabolites. Studies suggest that the optimal schedule for radiopotentiation and for inhibition of repair of sublethal radiation-induced DNA damage may be administration of cisplatin immediately after radiation (15). Since the plasma half-life of free cisplatin is only about 30 min in humans (18) and since cisplatin uptake into human tumors is very rapid (10, 18), this suggests that the optimal schedule for cisplatin-radiation administration if one is targeting DNA repair might be to give the cisplatin immediately before the radiation such that the cisplatin is still circulating in the blood stream and being taken up into the tumor as radiation is administered. A number of studies in lung cancer and other tumor types suggest that this very close timing of cisplatin and radiation administration is feasible, well tolerated, and effective (19–21). While randomized studies indicate that concurrent administration of cisplatin with radiation is superior to sequential administration (3), we do not yet know whether administration of the two modalities within a few minutes of each other is superior to administration within hours or days of each other, but the preclinical data suggest that this should be more fully assessed.

Chemoradiotherapy for Inoperable Non-small Cell Lung Cancer

165

While high doses of cisplatin do not appear to be any more effective than low doses of cisplatin in the treatment of metastatic NSCLC (22), it is unknown how cisplatin dose affects radiopotentiation clinically, and higher platinum doses radiopotentiate somewhat more effectively than do low doses in vitro (11). Higher cisplatin doses are required to achieve radiosensitization in platinum-resistant tumor cells than in platinum-sensitive tumor cells (11), and it is unknown how the many different potential platinum resistance mechanisms (see Chap. 15) would individually affect radiopotentiation. Intra-arterial administration of cisplatin results in higher local blood (23) and tumor (24) platinum concentrations than does intravenous administration, and intra-arterial cisplatin administration with radiation for localized bladder cancer appears to be more effective than intravenous cisplatin administration with radiation (21). On the other hand, daily administration of low dose cisplatin intravenously with radiation appeared to be slightly more effective in locally advanced NSCLC than was a higher dose administered once per week (25), and intracarotid administration of cisplatin did not appear to be any more effective than intravenous cisplatin in potentiating the effect of radiation against glioblastomas (26). Overall, available data suggest that schedule of administration is important in radiopotentiation by cisplatin, with concurrent administration being superior to sequential administration clinically, while the importance of cisplatin dose is unclear. Cisplatin is a somewhat more effective radiopotentiator than is carboplatin in some cell lines (27, 28), while in others, carboplatin is as effective as cisplatin, particularly with long drug exposure times (28). Clinically, cisplatin is somewhat more effective than is carboplatin in advanced NSCLC (29), but there are no direct clinical comparisons between cisplatin and carboplatin as radiopotentiators in human lung cancer.

Anti-tubulin Agents Plus Radiotherapy Both taxanes (30) and vinca alkaloids (31) lead to cell arrest in the radiation-sensitive G2/M phase of the cell cycle, and this could result in schedule-dependent radiosensitization that would require drug administration a few hours to a few days prior to radiation. However, the cytotoxicity of vinca alkaloids is also increased if they are given several hours after radiation during the radiation-induced block of cells in G2/M of the cell cycle (32), indicating that different schedules of drug administration could radiopotentiate in different ways. Radiopotentiation with taxanes is seen predominantly in cell lines that are sensitive to taxanes (33), and taxanes may radiopotentiate p53 mutant cells to a lesser extent than p53 wild-type cells (34), although this has not been noted in all studies (33). Taxanes potentiate radiation’s antiangiogenic properties (35), and may reduce radiation-induced upregulation of the production of proangiogenic factors such as VEGF (30), thereby potentially reducing accelerated tumor cell repopulation between radiation fractions. Taxanes may also increase cytotoxicity of radiation by depleting tumor cells of glutathione (36). Furthermore, taxanes may reduce repair

166

J.D. Cox and D.J. Stewart

of radiation DNA damage by antagonizing the radiation-induced upregulation of expression of DNA repair enzymes such as ERCC1 (30), and vinca alkaloids also reduce repair of radiation-induced DNA damage (37).

Topoisomerase Inhibitors Plus Radiotherapy The topoisomerase I inhibitor irinotecan and its active metabolite SN-38 potentiate the effects of radiation in hypoxic tumor cells, with an enhancement ratio of 1.5–2.1 (38), and radiopotentiation is seen in both p53 mutant and p53 wild-type cells (39). The topoisomerase II inhibitor etoposide also potentiates the effect of radiotherapy, and the enhancement ratio observed when radiation is combined with irinotecan and etoposide together is greater than when given with just one of these agents alone (40). Both irinotecan (40, 41) and etoposide (40, 41) may inhibit repair of radiation-induced sublethal DNA damage, and both irinotecan (42) and etoposide (41) may also inhibit repair potentially lethal radiation DNA damage. Irinotecan increases the proportion of cells in the radiation-sensitive G2/M phase of the cell cycle (39, 43). On the other hand, etoposide is effective as a radiosensitizer if administered after radiotherapy (44, 45), particularly during the G2/M cell cycle block induced by radiotherapy (45), and etoposide radiosensitization correlates with prolongation of this radiation-induced G2 cell cycle arrest (46). Hence, irinotecan might best be given prior to radiotherapy while etoposide might best be given after radiotherapy.

Gemcitabine Plus Radiotherapy The impact of gemcitabine as a radiosensitizer is greater in exponentially growing monolayer cells than in confluent monolayer cells or spheroids (47). By inhibiting ribonucleotide reductase, gemcitabine can inhibit DNA repair by leading to depletion of D-adenosine triphosphate pools (48). In cells that are deficient in DNA mismatch repair, gemcitabine can lead to mismatched nucleotides in DNA following radiation exposure (49). Gemcitabine may also inhibit homologous recombination repair of DNA double strand breaks (50), and it increases chromosome aberrations in response to radiation (51). While S-phase is generally the most radioresistant part of the cell cycle, gemcitabine reverses this S-phase radioresistance, in keeping with its inhibition of potentially lethal damage repair (52). The greatest impact of gemcitabine on radiosensitivity is when it blocks tumor cells in early S-phase (53) or at the G1/S boundary (54) in some cell lines. In other cell lines, radiation increased the ability of gemcitabine to cause cell death during S-phase if the cells had mutant p53 and progressed through S-phase after exposure to radiation and gemcitabine, rather than being blocked at G1, as seen with p53 wild-type cells (48).

Chemoradiotherapy for Inoperable Non-small Cell Lung Cancer

167

The impact of gemcitabine on radiation effect is increased by increasing exposure time to gemcitabine (55) and by decreasing the time interval between exposure to gemcitabine and radiation (52, 56). Its impact on radiation when given concurrently with or following radiotherapy has not been extensively explored in preclinical systems.

EGFR Inhibitors Plus Radiotherapy Each of the epidermal growth factor receptor (EGFR, HER1) tyrosine kinase inhibitor (TKI) gefitinib (57, 58), the EGFR TKI erlotinib (59), the anti-EGFR monoclonal antibody cetuximab (60), and the anti-HER2/neu monoclonal antibody trastuzumab (57) potentiates the effects of radiation in cell lines. Gefitinib increases radiation-induced apoptosis (61) while decreasing repair of DNA double strand breaks (62), and the dose modifying factor is higher if gefitinib and trastuzumab are used together (57). Gefitinib inhibits radiation-induced phosphorylation of EGFR (57, 58) while both gefitinib and trastuzumab inhibit radiation-induced phosphorylation of HER2/neu (57), Akt (57, 58), and MEK1/2 (57). Similarly, the EGFR TKI erlotinib increases radiation-induced apoptosis (59, 63), increases radiation-induced tumor growth delay in xenografts (63), increases proportion of cells in G1 phase of the cell cycle (59, 63), and decreases proportion of cells in S-phase (63). Erlotinib also decreases radiation-induced EGFR autophosphorylation (63) and expression of the DNA repair protein Rad51 (63). Cell lines with high EGFR expression are resistant to radiotherapy but sensitive to erlotinib (59). Erlotinib’s radiation enhancement ratio is highest in cell lines that have highest EGFR expression, highest EGFR autophosphorylation, and lowest intrinsic radiation sensitivity (59).

Clinical Impact of Adding Chemotherapy to Radiotherapy in NSCLC As outlined previously, chemotherapy may make several contributions to combined modality therapy for NSCLC. There is no doubt that it increases local tumor control. This has been demonstrated in virtually every trial that compared radiation therapy alone with chemotherapy and concurrent radiation therapy. Schaake– Koning et al. were the first to show it in NSCLC (25), and it has also been seen with carcinoma of the esophagus (64), nasopharynx (65), larynx (66), and cervix (67). In each of these trials there was also a decrease in distant metastasis, but it is unclear whether that reduction is a direct effect of the chemotherapy or whether it is a derivative of improved local control. Chemotherapy when given concurrently with radiotherapy in some cases is reduced in intensity due to concerns about hematological toxicity. Preradiotherapy induction chemotherapy or postradiotherapy adjuvant chemotherapy is given at full doses, and this could possibly be important

168

J.D. Cox and D.J. Stewart

in the control of distant micrometastases, although the impact of chemotherapy dose on control of micrometastases is not clear in NSCLC, and chemotherapy doseresponse curves tend to flatten at higher doses in advanced NSCLC (22). Concurrent chemotherapy is given with the intent of augmenting the local effects of radiation therapy in addition to having a major systemic impact. The separation of radiation and chemotherapy in time, as with induction chemotherapy, permits spatial cooperation, that is to say control of subclinical metastasis by chemotherapy and control of the gross tumor by radiation therapy. This was certainly the basis of the original demonstration of the benefit of sequential chemotherapy and radiation therapy by Dillman et al. (1). This study was closed early, and there was some doubt about an unexpected number of early deaths in the radiation therapy alone arm, so the Radiation Therapy Oncology Group (RTOG) replicated the study with a larger number of patients in each arm and produced nearly identical results (68). A randomized study from France also supported the value of induction chemotherapy followed by radiation therapy (69). These studies established the use of chemotherapy with radiation therapy for such favorable patients. Schaake–Koning (25) and her colleagues from Europe reported a three-arm trial that compared radiation therapy alone with the same radiation therapy and either daily or weekly cisplatin given concurrently. Both cisplatin arms were found to produce superior survival compared with radiation therapy alone based entirely on increased local control. There was no difference in the rate of distant metastasis. This trial showed convincingly that the local tumor is a major determinant of survival, not just distant metastasis. This trial also demonstrated that it is feasible to administer low dose cisplatin daily (Monday to Friday) with radiation and that, for a given cisplatin dose-intensity, daily low dose administration might be slightly more effective than a higher dose of cisplatin administered once weekly. While the Schaake–Koning study used a split course of radiotherapy (2 weeks followed by a 3 weeks break, then another 2 weeks of radiation, with cisplatin given either daily or weekly the same weeks as the radiation) (25), other nonrandomized studies have demonstrated that daily low dose cisplatin can be given safely during 6 consecutive weeks of radiation in medically inoperable early stage NSCLC patients, with an improvement in survival compared to matched historical controls (19). Still other nonrandomized studies have demonstrated the safety and efficacy of administering daily low dose cisplatin with weekly vinblastine concurrently with a 4-week cycle of twice daily hyperfractionated radiation in NSCLC (20). The question of superiority of induction versus concurrent chemotherapy with radiation therapy became paramount. Furuse et al. (3) had anticipated this question in a study from Western Japan. They found a small but significant improvement with concurrent over induction chemotherapy and radiation therapy. The RTOG conducted a similar trial and produced remarkably similar results (4), and still other studies have also demonstrated either a significant advantage of concurrent over sequential chemoradiotherapy in NSCLC (70) or a strong trend toward a better outcome with concurrent administration (71). Induction chemotherapy had no effect on local tumor control (72). Concurrent radiation therapy permits radiosensitization by chemotherapy or perhaps only an additive effect, but it leads to greater

Chemoradiotherapy for Inoperable Non-small Cell Lung Cancer

169

control of the gross tumor. Schaake–Koning and her colleagues demonstrated this effect with single agent cisplatin (25). As noted above, preclinical studies have shown interactions with a wide variety of chemotherapeutic agents that enhance tumor control with radiation (6). As noted, studies comparing sequential chemotherapy and radiation therapy to concurrent administration of the modalities confirmed the advantage of concurrent treatment (3, 4). The most promising combinations, however, may lie in concurrent chemotherapy and radiation therapy plus a molecular targeting agent. RTOG protocol 0324 was a phase II trial of carboplatin and paclitaxel plus the anti-EGFR antibody cetuximab for locally advanced NSCLC. The median survival of 22.7 months and the 2-year survival rate of 49% are the best reported to date for this disease (73).

Advanced Techniques for Radiation Therapy of NSCLC All of the large, cooperative clinical trials of radiation therapy and chemotherapy reported before 2000 were based on two-dimensional treatment planning. While some participating institutions had the capability to plan and treat using threedimensional conformal radiation therapy (3D CRT), prescriptions written in the radiation therapy sections of protocols used 2D approaches. The important distinction is that 2D was based on chest roentgenograms possibly augmented with information from computed tomography. Relatively simple beam arrangements and relatively large fields were the norm. 3D CRT utilizes the display of tumors and normal tissues in three dimensions to design target volumes that conform to the size and shape of the tumor and avoid critical normal tissues. In addition, dose distributions take into account the contributions of scatter radiation in three dimensions. This approach almost always results in small high-dose volumes with lower doses in normal structures. Tumors are visualized from the perspective of the individual beams. Such “beam’s-eye views” allow treatment from directions rarely considered with 2D, such as oblique anterior/inferior to posterior/superior, for example. It also allows conformal blocking of the beam to encompass the tumor in its entirety from any angle and avoid normal tissues close by. Intensity-modulated radiation therapy (IMRT) uses a relatively large number of beamlets directed at the tumor. Each beamlet does not usually treat the entirety of the tumor. Different dose intensities are delivered with each beamlet such that additional conformality can be achieved. The negative aspect of this treatment is the very large volume of low dose radiation delivered with the entrance and exit of these many beamlets of X-rays. This fact led to hesitancy on the part of many institutions to use IMRT for NSCLC even though it had become well established for carcinomas of the prostate and the head and neck. Planning studies suggested that greater conformity could be achieved with IMRT compared with 3D CRT and a smaller volume of lung would receive 20  Gy (considered to be the threshold for radiation pneumonitis) (74, 75). Clinical studies confirmed the benefit suggested by

170

J.D. Cox and D.J. Stewart

the planning studies with a significantly lower rate of treatment-related pneumonitis (TRP) with IMRT relative to 3D CRT (76). It became quickly apparent that a high degree of conformity with the high dose volume carried the risk that the tumor in the lung might move outside the target volume with respiration. There are several solutions to this problem: gating in which the X-ray beam is only turned on when the tumor is in a specified position; active breath holding, a variation on the theme of gating, where the patients’ breathing is monitored and then stopped at a certain position so that the beam is directed at the tumor only while the respirations are artificially stopped at a certain point in the respiratory cycle; and 4D CT. This latter technique does not require anything special on the part of the patient. It captures the excursion of the tumor and normal anatomy during simulation with the CT data acquisition being performed with multiple slices throughout the respiratory cycle. The entire path of the tumor is encompassed in an “internal target volume” (77) that becomes the target volume for the high dose. Although this may appear to irradiate a larger portion of normal lung than gating, the differences are minimal especially when compared with 3D CRT without 4D CT simulation. As the results of treatment with 3D CRT for carcinoma of the prostate and with IMRT for carcinomas of the prostate and head and neck became available, the potential value of proton beam therapy (PBT) was increasingly compelling. Protons have very similar biologic effects to X-rays, but they deposit doses within the body very differently. Whereas X-ray beams, no matter what energy, deposit the maximum dose near the place of entry, the skin, and penetrate through the body to exit through the opposite skin, protons deposit the maximum dose deep within the body and then they stop. The depth of maximum energy deposition can be determined by the energy given to the proton beam (greater energy, greater depth). Devices can be made to shape the beam so that the final dose deposition has the size and shape of the tumor in three dimensions. PBT can be combined with chemotherapy just as with X-rays and the side effects of the combination can be reduced. For example, most patients treated with 3D CRT and IMRT plus concurrent chemotherapy have at least some degree of esophageal discomfort and for some it may be so intolerable as to necessitate placement of a feeding tube (percutaneous gastrostomy). The frequency and severity of esophageal toxicity can be reduced considerably with PBT. TRP occurs in more than half of patients treated with 3D CRT or IMRT and concurrent chemotherapy, and it may be life threatening or even lethal. The frequency and severity of TRP can greatly be reduced with PBT and chemotherapy. In a study of 67 consecutive patients treated with concurrent chemotherapy and proton therapy, the frequency of severe esophagitis was only 6% (compared with 16% for 3D CRT and 40% for IMRT). There has been no case of severe TRP with PBT (compared with 32% for 3D CRT and 9% for IMRT). More importantly, the median total dose for patients treated with PBT was 74 Cobalt Gray equivalent [CGE] whereas it was only 63 Gy for the patients treated with 3D CRT and IMRT. Our experience with concurrent and PBT is too early to have reliable estimates of tumor control and survival.

Chemoradiotherapy for Inoperable Non-small Cell Lung Cancer

171

Conclusion Radiotherapy is useful in several settings in the treatment of NSCLC, and new radiotherapy methods may both increase its efficacy and reduce its toxicity. Several systemic agents are able to potentiate the effect of radiotherapy, and there are probably several biological mechanisms involved in this radiopotentiation. Concurrent administration of chemotherapy and radiotherapy is now the standard of care for locally advanced NSCLC. It is anticipated that combining new targeted agents with radiotherapy may also eventually play a role in this setting.

References 1. Dillman RO, Seagren SL, Propert KJ et  al (1990) A randomized trial of induction chemotherapy plus high-dose radiation versus radiation alone in stage III non-small-cell lung cancer. N Engl J Med 323(14):940–945 2. Auperin A, Le Pechoux C, Pignon JP et al (2006) Concomitant radio-chemotherapy based on platin compounds in patients with locally advanced non-small cell lung cancer (NSCLC): a meta-analysis of individual data from 1764 patients. Ann Oncol 17(3):473–483 3. Furuse K, Fukuoka M, Kawahara M et al (1999) 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 17(9):2692–2699 4. Curran WJ, Scott C, Langer C, et al (2000) Phase III comparison of sequential vs concurrent chemoradiation for patients (Pts) with unresected stage III non-small cell lung cancer (NSCLC): initial report of Radiation Therapy Oncology Group (RTOG) 9410. Proc Am Soc Clin Oncol Abstract # 1891. 5. Woodward W, Cox J (2008) Molecular basis of radiation therapy. In: Mendelsohn J, Howley P, Israel M (eds) The molecular basis of cancer, 3rd edn. Saunders Elsevier, Philadelphia, PA, pp 593–604 6. Milas L, Cox JD (2010) Principles of combining radiation therapy and chemotherapy. In: Cox JD, Ang KK (eds) Radiation oncology: rationale, technique, results, 9th edn. Mosby, St. Louis, MO, pp 102–117 7. Murray N, Coy P, Pater JL et al (1993) Importance of timing for thoracic irradiation in the combined modality treatment of limited-stage small-cell lung cancer. The National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 11(2):336–344 8. Douple EB, Richmond RC (1980) A review of interactions between platinum coordination complexes and ionizing radiation: implications for cancer therapy. In: Prestayko AW, Crooke ST, Carter SK (eds) Cisplatin: current status and new developments. Academic, New York, pp 125–147 9. Douple EB, Richmond RC, O’Hara JA, Coughlin CT (1985) Carboplatin as a potentiator of radiation therapy. Cancer Treat Rev 12(Suppl A):111–124 10. Stewart DJ, Leavens M, Maor M et al (1982) Human central nervous system distribution of cis-diamminedichloroplatinum and use as a radiosensitizer in malignant brain tumors. Cancer Res 42(6):2474–2479 11. Wilkins DE, Ng CE, Raaphorst GP (1996) Cisplatin and low dose rate irradiation in cisplatin resistant and sensitive human glioma cells. Int J Radiat Oncol Biol Phys 36(1):105–111 12. Richmond RC, Zimbrick JD, Hykes DL (1977) Radiation-induced DNA damage and lethality in E. coli as modified by the antitumor agent cis-dichlorodiammineplatinum (II). Radiat Res 71(2):447–460

172

J.D. Cox and D.J. Stewart

13. Wilson GD, Bentzen SM, Harari PM (2006) Biologic basis for combining drugs with radiation. Semin Radiat Oncol 16(1):2–9 14. Myint WK, Ng C, Raaphorst GP (2002) Examining the non-homologous repair process following cisplatin and radiation treatments. Int J Radiat Biol 78(5):417–424 15. Raaphorst GP, Wang G, Stewart D, Ng CE (1995) A study of cisplatin-radiosensitization through inhibition of repair of sublethal radiation damage in ovarian carcinoma cells sensitive and resistant to cisplatin. Int J Oncol 7:1373–1378 16. Richmond RC (1984) Toxic variability and radiation sensitization by dichlorodiammineplatinum(II) complexes in Salmonella typhimurium cells. Radiat Res 99(3):596–608 17. Stewart DJ, Benjamin RS, Luna M et al (1982) Human tissue distribution of platinum after cis-diamminedichloroplatinum. Cancer Chemother Pharmacol 10(1):51–54 18. Stewart DJ, Molepo JM, Green RM et al (1995) Factors affecting platinum concentrations in human surgical tumour specimens after cisplatin. Br J Cancer 71(3):598–604 19. Cross P, Tomiak E, Abgoola O, et al (2005) Pilot study of daily low cisplatin and radiotherapy for medically inoperable stage I non-small cell lung cancer: long term follow up. Eur J Cancer Suppl 3(2):340 (Abstract #1175). 20. Lochrin C, Goss G, Stewart DJ et  al (1999) Concurrent chemotherapy with hyperfractionated accelerated thoracic irradiation in stage III non-small cell lung cancer. Lung Cancer 23(1):19–30 21. Eapen L, Stewart D, Collins J, Peterson R (2004) Effective bladder sparing therapy with intraarterial cisplatin and radiotherapy for localized bladder cancer. J Urol 172(4 Pt 1):1276–1280 22. Stewart DJ, Chiritescu G, Dahrouge S, Banerjee S, Tomiak EM (2007) Chemotherapy dose– response relationships in non-small cell lung cancer and implied resistance mechanisms. Cancer Treat Rev 33(2):101–137 23. Stewart DJ, Benjamin RS, Zimmerman S et al (1983) Clinical pharmacology of intraarterial cis-diamminedichloroplatinum(II). Cancer Res 43(2):917–920 24. Stewart DJ, Mikhael NZ, Nair RC et  al (1988) Platinum concentrations in human autopsy tumor samples. Am J Clin Oncol 11(2):152–158 25. Schaake-Koning C, van den Bogaert W, Dalesio O et al (1992) Effects of concomitant cisplatin and radiotherapy on inoperable non-small-cell lung cancer. N Engl J Med 326(8):524–530 26. Stewart DJ, Molepo JM, Eapen L et  al (1994) Cisplatin and radiation in the treatment of tumors of the central nervous system: pharmacological considerations and results of early studies. Int J Radiat Oncol Biol Phys 28(2):531–542 27. Begg AC, van der Kolk PJ, Emondt J, Bartelink H (1987) Radiosensitization in vitro by cisdiammine (1, 1-cyclobutanedicarboxylato) platinum(II) (carboplatin, JM8) and ethylenediamminemalonatoplatinum(II) (JM40). Radiother Oncol 9(2):157–165 28. Groen HJ, Sleijfer S, Meijer C et al (1995) Carboplatin- and cisplatin-induced potentiation of moderate-dose radiation cytotoxicity in human lung cancer cell lines. Br J Cancer 72(6):1406–1411 29. Ardizzoni A, Boni L, Tiseo M et al (2007) Cisplatin- versus carboplatin-based chemotherapy in first-line treatment of advanced non-small-cell lung cancer: an individual patient data metaanalysis. J Natl Cancer Inst 99(11):847–857 30. Toiyama Y, Inoue Y, Hiro J et al (2007) The range of optimal concentration and mechanisms of paclitaxel in radio-enhancement in gastrointestinal cancer cell lines. Cancer Chemother Pharmacol 59(6):733–742 31. Fukuoka K, Arioka H, Iwamoto Y et al (2001) Mechanism of the radiosensitization induced by vinorelbine in human non-small cell lung cancer cells. Lung Cancer 34(3):451–460 32. Edelstein MP, Wolfe LA 3rd, Duch DS (1996) Potentiation of radiation therapy by vinorelbine (Navelbine) in non-small cell lung cancer. Semin Oncol 23(2 Suppl 5):41–47 33. Balcer-Kubiczek EK, Attarpour M, Jiang J, Kennedy AS, Suntharalingam M (2006) Cytotoxicity of docetaxel (Taxotere) used as a single agent and in combination with radiation in human gastric, cervical and pancreatic cancer cells. Chemotherapy 52(5):231–240 34. Creane M, Seymour CB, Colucci S, Mothersill C (1999) Radiobiological effects of docetaxel (Taxotere): a potential radiation sensitizer. Int J Radiat Biol 75(6):731–737 35. Dicker AP, Williams TL, Iliakis G, Grant DS (2003) Targeting angiogenic processes by combination low-dose paclitaxel and radiation therapy. Am J Clin Oncol 26(3):e45–e53

Chemoradiotherapy for Inoperable Non-small Cell Lung Cancer

173

36. Nguyen LN, Munshi A, Hobbs ML, Story MD, Meyn RD (2001) Paclitaxel restores radiationinduced apoptosis in a bcl-2-expressing, radiation-resistant lymphoma cell line. Int J Radiat Oncol Biol Phys 49(4):1127–1132 37. Fukuoka K, Arioka H, Iwamoto Y et al (2002) Mechanism of vinorelbine-induced radiosensitization of human small cell lung cancer cells. Cancer Chemother Pharmacol 49(5):385–390 38. Van Rensburg CE, Slabbert JP, Bohm L (2006) Influence of irinotecan and SN-38 on the irradiation response of WHO3 human oesophageal tumour cells under hypoxic conditions. Anticancer Res 26(1A):389–393. 39. Xie X, Sasai K, Shibuya K et al (2000) P53 status plays no role in radiosensitizing effects of SN-38, a camptothecin derivative. Cancer Chemother Pharmacol 45(5):362–368 40. Kim JS, Amorino GP, Pyo H, Cao Q, Choy H (2002) Radiation enhancement by the combined use of topoisomerase I inhibitors, RFS-2000 or CPT-11, and topoisomerase II inhibitor etoposide in human lung cancer cells. Radiother Oncol 62(1):61–67 41. Ng CE, Bussey AM, Raaphorst GP (1994) Inhibition of potentially lethal and sublethal damage repair by camptothecin and etoposide in human melanoma cell lines. Int J Radiat Biol 66(1):49–57 42. Omura M, Torigoe S, Kubota N (1997) SN-38, a metabolite of the camptothecin derivative CPT-11, potentiates the cytotoxic effect of radiation in human colon adenocarcinoma cells grown as spheroids. Radiother Oncol 43(2):197–201 43. Tamura K, Takada M, Kawase I et al (1997) Enhancement of tumor radio-response by irinotecan in human lung tumor xenografts. Jpn J Cancer Res 88(2):218–223 44. Haddock MG, Ames MM, Bonner JA (1995) Assessing the interaction of irradiation with etoposide or idarubicin. Mayo Clin Proc 70(11):1053–1060 45. Giocanti N, Hennequin C, Balosso J, Mahler M, Favaudon V (1993) DNA repair and cell cycle interactions in radiation sensitization by the topoisomerase II poison etoposide. Cancer Res 53(9):2105–2111 46. Minehan KJ, Bonner JA (1993) The interaction of etoposide with radiation: variation in cytotoxicity with the sequence of treatment. Life Sci 53(15):PL237–PL242. 47. Genc M, Castro Kreder N, Barten-van Rijbroek A, Stalpers LJ, Haveman J (2004) Enhancement of effects of irradiation by gemcitabine in a glioblastoma cell line and cell line spheroids. J Cancer Res Clin Oncol 130(1):45–51 48. Ostruszka LJ, Shewach DS (2000) The role of cell cycle progression in radiosensitization by 2’, 2’-difluoro-2’-deoxycytidine. Cancer Res 60(21):6080–6088 49. Flanagan SA, Robinson BW, Krokosky CM, Shewach DS (2007) Mismatched nucleotides as the lesions responsible for radiosensitization with gemcitabine: a new paradigm for antimetabolite radiosensitizers. Mol Cancer Ther 6(6):1858–1868 50. Wachters FM, van Putten JW, Maring JG, Zdzienicka MZ, Groen HJ, Kampinga HH (2003) Selective targeting of homologous DNA recombination repair by gemcitabine. Int J Radiat Oncol Biol Phys 57(2):553–562 51. Rosier JF, Michaux L, Ameye G et al (2003) The radioenhancement of two human head and neck squamous cell carcinomas by 2’-2’ difluorodeoxycytidine (gemcitabine; dFdC) is mediated by an increase in radiation-induced residual chromosome aberrations but not residual DNA DSBs. Mutat Res 527(1–2):15–26 52. Latz D, Fleckenstein K, Eble M, Blatter J, Wannenmacher M, Weber KJ (1998) Radiosensitizing potential of gemcitabine (2’, 2’-difluoro-2’-deoxycytidine) within the cell cycle in vitro. Int J Radiat Oncol Biol Phys 41(4):875–882 53. Pauwels B, Korst AE, Pattyn GG et al (2003) Cell cycle effect of gemcitabine and its role in the radiosensitizing mechanism in vitro. Int J Radiat Oncol Biol Phys 57(4):1075–1083 54. Mose S, Class R, Weber HW, Rahn A, Brady LW, Bottcher HD (2003) Radiation enhancement by gemcitabine-mediated cell cycle modulations. Am J Clin Oncol 26(1):60–69 55. Miao JW, Kong WM, Zhang WH, Niu JW, Deng XH (2004) Mechanistic study of radiosensitization by gemcitabine on human squamous carcinoma cell line of the cervix. Zhonghua Fu Chan Ke Za Zhi 39(5):342–345 56. Pauwels B, Korst AE, Lambrechts HA et al (2006) The radiosensitising effect of difluorodeoxyuridine, a metabolite of gemcitabine, in vitro. Cancer Chemother Pharmacol 58(2):219–228

174

J.D. Cox and D.J. Stewart

57. Fukutome M, Maebayashi K, Nasu S, Seki K, Mitsuhashi N (2006) Enhancement of radiosensitivity by dual inhibition of the HER family with ZD1839 (“Iressa”) and trastuzumab (“Herceptin”). Int J Radiat Oncol Biol Phys 66(2):528–536 58. Miyata H, Sasaki T, Kuwahara K, Serikawa M, Chayama K (2006) The effects of ZD1839 (Iressa), a highly selective EGFR tyrosine kinase inhibitor, as a radiosensitiser in bile duct carcinoma cell lines. Int J Oncol 28(4):915–921 59. Kim JC, Ali MA, Nandi A et al (2005) Correlation of HER1/EGFR expression and degree of radiosensitizing effect of the HER1/EGFR-tyrosine kinase inhibitor erlotinib. Indian J Biochem Biophys 42(6):358–365 60. Nakata E, Hunter N, Mason K, Fan Z, Ang KK, Milas L (2004) C225 antiepidermal growth factor receptor antibody enhances the efficacy of docetaxel chemoradiotherapy. Int J Radiat Oncol Biol Phys 59(4):1163–1173 61. Maddineni SB, Sangar VK, Hendry JH, Margison GP, Clarke NW (2005) Differential radiosensitisation by ZD1839 (Iressa), a highly selective epidermal growth factor receptor tyrosine kinase inhibitor in two related bladder cancer cell lines. Br J Cancer 92(1):125–130 62. Tanaka T, Munshi A, Brooks C, Liu J, Hobbs ML, Meyn RE (2008) Gefitinib radiosensitizes non-small cell lung cancer cells by suppressing cellular DNA repair capacity. Clin Cancer Res 14(4):1266–1273 63. Chinnaiyan P, Huang S, Vallabhaneni G et  al (2005) Mechanisms of enhanced radiation response following epidermal growth factor receptor signaling inhibition by erlotinib (Tarceva). Cancer Res 65(8):3328–3335 64. Herskovic A, Martz K, al-Sarraf M et al (1992) Combined chemotherapy and radiotherapy compared with radiotherapy alone in patients with cancer of the esophagus. N Engl J Med 326(24):1593–1598 65. al-Sarraf M, Pajak TF, Cooper JS, Mohiuddin M, Herskovic A, Ager PJ (1990) Chemoradiotherapy in patients with locally advanced nasopharyngeal carcinoma: a radiation therapy oncology group study. J Clin Oncol 8(8):1342–1351 66. Forastiere AA, Goepfert H, Maor M et al (2003) Concurrent chemotherapy and radiotherapy for organ preservation in advanced laryngeal cancer. N Engl J Med 349(22):2091–2098 67. Eifel PJ, Winter K, Morris M et al (2004) Pelvic irradiation with concurrent chemotherapy versus pelvic and para-aortic irradiation for high-risk cervical cancer: an update of radiation therapy oncology group trial (RTOG) 90–01. J Clin Oncol 22(5):872–880 68. Sause WT, Scott C, Taylor S et al (1995) 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 87(3):198–205 69. Le Chevalier T, Arriagada R, Quoix E et al (1991) Radiotherapy alone versus combined chemotherapy and radiotherapy in nonresectable non-small-cell lung cancer: first analysis of a randomized trial in 353 patients. J Natl Cancer Inst 83(6):417–423 70. Zatloukal P, Petruzelka L, Zemanova M et al (2004) Concurrent versus sequential chemoradiotherapy with cisplatin and vinorelbine in locally advanced non-small cell lung cancer: a randomized study. Lung Cancer 46(1):87–98 71. Fournel P, Robinet G, Thomas P et al (2005) Randomized phase III trial of sequential chemoradiotherapy compared with concurrent chemoradiotherapy in locally advanced non-smallcell lung cancer: Groupe Lyon-Saint-Etienne d’Oncologie Thoracique-Groupe Francais de Pneumo-Cancerologie NPC 95-01 Study. J Clin Oncol 23(25):5910–5917 72. Arriagada R, Le Chevalier T, Quoix E et al (1991) ASTRO (American Society for Therapeutic Radiology and Oncology) plenary: effect of chemotherapy on locally advanced non-small cell lung carcinoma: a randomized study of 353 patients. GETCB (Groupe d’Etude et Traitement des Cancers Bronchiques), FNCLCC (Federation Nationale des Centres de Lutte contre le Cancer) and the CEBI trialists. Int J Radiat Oncol Biol Phys 20(6):1183–1190 73. Komaki R, Moughan J, Ang K (2007) RTOG 0324: A phase II study of cetuximab (C225) in combination with chemoradiation (CRT) in patients (PTS) with stage IIIA/B non-small cell lung cancer (NSCLC): correlation between EGFR expression and the patients outcome. Proc Am Soc Ther Radiol Oncol 69:57S

Chemoradiotherapy for Inoperable Non-small Cell Lung Cancer

175

74. Liu HH, Balter P, Tutt T et al (2007) Assessing respiration-induced tumor motion and internal target volume using four-dimensional computed tomography for radiotherapy of lung cancer. Int J Radiat Oncol Biol Phys 68(2):531–540 75. Murshed H, Liu HH, Liao Z et al (2004) Dose and volume reduction for normal lung using intensity-modulated radiotherapy for advanced-stage non-small-cell lung cancer. Int J Radiat Oncol Biol Phys 58(4):1258–1267 76. Yom SS, Liao Z, Liu HH et al (2007) Initial evaluation of treatment-related pneumonitis in advanced-stage non-small-cell lung cancer patients treated with concurrent chemotherapy and intensity-modulated radiotherapy. Int J Radiat Oncol Biol Phys 68(1):94–102 77. International Commission on Radiation Units and Measurements (1998) Fundamental Quantities and Units for Ionzing Radiation. (ICRU Report 60), International Commission on Radiation Units and Measurements, Bethesda, MD

Management of Advanced Non-small Cell Lung Cancer: Front Line Treatment William N. William Jr. and David J. Stewart

Abstract  Over the past decades, a number of systemic treatment options for incurable, advanced non-small cell lung cancer have been developed. While untreated patients with this disease typically have a median overall survival of 4.5 months, this figure has exceeded the 12-month mark in the latest randomized phase III trials. Progress in drug development, combination chemotherapy, supportive care, and better selection of patients for specific treatments have contributed to improvements in outcome. In this chapter, we will present the key data that support the use of chemotherapy and biologic agents for the first-line treatment of advanced NSCLC. The following aspects will be discussed: selection and number of chemotherapy agents, duration of therapy, use of biologic drugs (i.e., vascular endothelial growth factor and epidermal growth factor receptor inhibitors), and management of elderly and poor performance status patients. Keywords  Non-small cell lung cancer • Frontline • First-line • Chemotherapy • Biologic agents

Introduction Approximately 85% of patients with lung cancer present a non-small cell histology. Of these, 55% are diagnosed, at initial presentation, with stage IIIB or IV disease that is not amenable to curative treatment. More than half of the remaining 45% of patients treated with curative intent will develop distant metastases and will eventually be considered for palliative systemic treatment as well (1, 2).

W.N. William Jr.(*) and D.J. Stewart Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd. Unit 432, Houston, TX 77030, USA e-mail: [email protected] D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_8, © Humana Press, a part of Springer Science+Business Media, LLC 2010

177

178

W.N. William Jr. and D.J. Stewart

Untreated patients with metastatic non-small cell lung cancer (NSCLC) have a median survival of 4.5 months and a 1-year survival rate of 20%, and until the early 1990s, there was limited evidence that chemotherapy could improve survival or quality of life in this population. Cytotoxic treatments available at that time were associated with significant adverse events, and best supportive care was considered the standard approach. Over the past two decades, a number of randomized trials have demonstrated a positive (albeit small) effect of antineoplastic treatment on NSCLC outcomes, leading to the approval of several cytotoxic and biologic agents for the treatment of this disease. As a result, in 2006, the median survival time has, for the first time, exceeded the 12-month mark in a randomized phase III trial for patients with metastatic NSCLC (3). The better toxicity profile of the newer antineoplastic drugs, along with improvements in supportive care medications (e.g., antiemetics) have led to ease of administration and acceptance of cytotoxic treatment as the standard-of-care for patients with advanced NSCLC and good performance status (4). In this chapter, we will present the key data that support the use of chemotherapy and biologic agents for the first-line treatment of advanced NSCLC. The following aspects will be discussed: selection and number of chemotherapy agents, duration of therapy, management of elderly and poor performance status patients, and use of biologic drugs (i.e., vascular endothelial growth factor [VEGF] and epidermal growth factor receptor [EGFR] inhibitors).

Effects of Chemotherapy on Survival and Quality of Life Several randomized studies and meta-analyses have examined the effects of chemotherapy over best supportive care on survival of patients with advanced NSCLC. Most of the trials used platinum-based regimens and demonstrated a hazard ratio (HR) of approximately 0.7 in favor of chemotherapy (5–7). In the following lines,we summarize the results of the most recently published meta-analysis examining this issue: The NSCLC Meta-Analyses Collaborative Group published the updated results (8) of their previous individual patient-data meta-analyses (9) comparing best supportive care ± chemotherapy for patients with advanced NSCLC in 2008. The report included 2,714 patients enrolled in 16 trials (12 platinum-based, 4 nonplatinum single agents). Median and 1-year overall survival increased from 4.5 months and 20%, for the best supportive care group, to 6.0 months and 29% for the chemotherapy group (HR 0.75, 95% confidence interval [CI] 0.67–0.84, P < 0.0001). The beneficial effects of chemotherapy were observed irrespective of age, gender, histology, type of cytotoxic drug [trials with long-term alkylating agents were excluded from the updated meta-analyses, and were associated with a detrimental effect in the earlier 1995 report (9)], or baseline performance status (although less than 25% of the patients had a performance status ³2) (8). In respect to quality of life, three trials using cisplatin-based chemotherapy (10–12), and three trials using single-agent chemotherapy [gemcitabine (13),

Management of Advanced Non-small Cell Lung Cancer: Front Line Treatment

179

vinorelbine (14), or paclitaxel (15)] reported that quality of life was either unchanged or improved in the arms receiving a cytotoxic agent. Particularly relevant is the phase III study of best supportive care ± gemcitabine, the only trial in which quality of life was the primary endpoint. The authors demonstrated a statistically significant sustained improvement of patient-reported symptoms (using the SS14 instrument) in a higher proportion of individuals randomized to the chemotherapy arm versus the supportive care arm. Additionally, the median time to radiotherapy was 29 weeks for the gemcitabine group versus 3.8 weeks for the supportive care alone group (13). In summary, the data provided by these trials support the use of chemotherapy with the goals of increasing survival, while maintaining/improving quality of life for patients with a performance status of 0–1. The results are consistent within the literature and obviate the need for further studies with the best-supportive-care-only arm. For patients with a performance status ³2, use of chemotherapy remains controversial and should be carefully individualized (as will be discussed later in this chapter).

Number of Cytotoxic Agents A number of trials have evaluated the effects of combination chemotherapy compared to a single-drug regimen. These studies varied with respect to the control arm used (i.e., cisplatin alone or a non-platinum agent) and the number of drugs in the combination chemotherapy arm (from one to four). In 1998, Lilenbaum et al. published the first comprehensive meta-analysis examining this issue (16). Twenty-five randomized trials with 5,156 patients were included in the report. Combination chemotherapy was associated with a nearly twofold increase in response rate (relative risk [RR] 1.93, 95% CI 1.54–2.42) and a 22% increase in 1-year survival (RR 1.22, 95% CI 1.03–1.45). When only trials containing a platinum-analogue or vinorelbine as the control group were considered, combination chemotherapy was still associated with an increased response rate (RR 1.79, 95% CI 1.37–2.33) but only a marginally significant improvement in 1-year survival (RR 1.10, 95% CI 0.94–1.43). In 2004, Hotta et al. presented the results of a meta-analysis including only trials evaluating the addition of a platinum-analogue to a non-platinum new single agent (i.e., paclitaxel, docetaxel, irinotecan, gemcitabine, and vinorelbine) (17). Again, there was a statistically significant increase in response rate (odds ratio [OR] 2.32, 95% CI 1.68–3.20, P = 0.01) and survival (HR 0.87, 95% CI 0.80–0.94, P < 0.001) favoring combination chemotherapy. Delbaldo et al. analyzed the effects of doublets versus single-agent chemotherapy and triplets versus doublets in a third meta-analysis published in 2004 (18). Trials with both platinum- and non-platinum-based regimens in the control arm were included. Again, there was a statistically significant 13% increase in response rate (from 13 to 26%, P < 0.001) and a 5% absolute improvement in 1-year survival (30–35%, P < 0.001) in favor of two-drug regimens compared to one-drug regimens. There was also an 8% increase in response rates for doublets versus triplets (23–31%,

180

W.N. William Jr. and D.J. Stewart

P < 0.001), but this did not translate into an improvement in survival (OR 1.01, 95% CI 085–1.21, P = 0.88). All three meta-analyses demonstrated higher toxicity rates associated with increased number of chemotherapy agents (16–18). Taken together, these results demonstrate a small but statistically significant improvement in survival of two-drug regimens over one-drug regimens, at the expense of enhanced toxicity. Although there is an increase in response rate when adding a third drug, triplets also result in higher toxicity rates, yet no improvement in survival, compared to doublets. Currently, a two-drug combination of cytotoxic drugs is accepted as standard of care for the first-line treatment of fit patients with advanced NSCLC. It should be noted that none of the meta-analyses included regimens containing biologic agents. As will be discussed later in this chapter, recent data support the use of combinations containing two cytotoxic drugs plus one biologic agent for selected patients.

Selection of Platinum-Based Doublets Cisplatin is one of the most active agents available to treat NSCLC. Single-agent cisplatin has a response rate in the range of 11–15% (19–21). Several phase III randomized trials attempted to improve the efficacy of cisplatin by adding a second drug. Selected trials of cisplatin-based doublets versus cisplatin alone are listed in Table 1 and discussed below. In 1998, the South West Oncology Group (SWOG) demonstrated a statistically significant increase in response rate and survival for the combination of cisplatin and vinorelbine compared with cisplatin alone (21), rendering this doublet standard of care for the frontline treatment of advanced NSCLC. Similarly, in 2000, the Hoosier Oncology Group (HOG) demonstrated that the addition of gemcitabine to cisplatin yielded a statistically significant increase in response rate and survival Table  1  Selected trials of cisplatin versus platinum-based doublets in the frontline treatment of non-small cell lung cancer Median survival 1-Year (mo) Clinical trial treatment arms N RR (%) survival (%) Southwest Oncology Group (21)   Vinorelbine+cisplatin 206 26a 8a 36a   Cisplatin 209 12 6 20 Hoosier Oncology Group (20)   Gemcitabine+cisplatin 260 30a 9.1a 39 a   Cisplatin 262 11 7.6 28 European (19)   Paclitaxel+cisplatin 207 26a 8.1 30   Cisplatin 207 17 8.6 36 mo months, N number of patients per arm, RR response rates a Statistically significant difference

Management of Advanced Non-small Cell Lung Cancer: Front Line Treatment

181

Table  2  Selected trials of non-platinum single agents versus platinum-based doublets in the frontline treatment of non-small cell lung cancer 1-Year Median survival survival (%) Clinical trial treatment arms N RR (%) (mo) Cancer and Leukemia Group B 9730 (22)   Paclitaxel+carboplatin 284 30a   8.8 37   Paclitaxel 277 17   6.7 32 Greek Cooperative Group (23)   Docetaxel+cisplatin 167 37a 10.5 44   Docetaxel 152 22  8 43 Swedish Lung Cancer Study Group (24)   Gemcitabine+carboplatin 164 30a 10a 40a   Gemcitabine 170 11   8.6 32 mo months, N number of patients per arm, RR response rates a Statistically significant difference

compared to cisplatin alone (20). In 2000, Gatzemeier et al. published the results of the comparison of cisplatin alone versus cisplatin combined with paclitaxel (N = 414). Response rates were higher for the doublet, but there were no differences in overall survival (19). Similar to the trials comparing platinum alone versus a platinum-based doublet, several studies evaluated the addition of a platinum analogue to a non-platinum single agent [i.e., paclitaxel (22), docetaxel (23) or gemcitabine (24)]. In general, these studies also demonstrated an increase in response rate (22, 23) and, in some instances, survival (24) for the combination treatment compared to the singlet (Table 2). A second set of trials evaluated comparisons between different platinum-based doublets (Table  3). The control regimen varied according to each organization sponsoring the trial. The Eastern Cooperative Oncology Group (ECOG), for example, had published in 1986 an analysis of 893 patients enrolled in several phase III trials involving seven different combination chemotherapy regimens. The etoposide/platinum doublet had the highest proportion of 1-year survivors (25%) and became the reference regimen for that cooperative group (25). On the other hand, in the 1990s, the SWOG considered cisplatin/vinorelbine the reference regimen to which new combinations should be compared [based on the aforementioned study of cisplatin versus cisplatin/vinorelbine (21)]. In 2000, the ECOG published a randomized phase III trial demonstrating a small increase in survival associated with the paclitaxel/cisplatin combination compared to cisplatin/etoposide (26). The SWOG, in turn, demonstrated that paclitaxel/carboplatin was as effective as cisplatin/vinorelbine in terms of response rate and survival, but associated with less adverse events and less therapy discontinuation rates. No differences in quality of life were observed between the two groups (27). These trials led to the acceptance of platinum/taxane combinations as the new reference regimens for both cooperative groups. The data presented above led to the design of a landmark study, ECOG 1594, attempting to identify a better regimen among four “modern” platinum-based doublets

182

W.N. William Jr. and D.J. Stewart

Table 3  Selected trials of platinum-based doublets in the frontline treatment of non-small cell lung cancer Median 1-Year Clinical trial treatment arms N RR (%) survival (mo) survival (%) ECOG (26) 12 200   Etoposide+cisplatin 7.6 32 28a 201 10.0b 40b   Paclitaxel (250 mg/m2)+cisplatin 2 a b 198   Paclitaxel (135 mg/m )+cisplatin 25 9.5 37b Southwest Oncology Group (27)   Paclitaxel+carboplatin 206 25 8 38   Vinorelbine+cisplatin 202 28 8 36 ECOG 1594 (28) 34 8.1 17   Paclitaxel+carboplatin 290 31 7.8 21 288   Paclitaxel+cisplatin 31 7.4 17 289   Docetaxel+cisplatin 36 8.1 22 288   Gemcitabine+cisplatin TAX 326 (29) 46 11.3 32 408   Docetaxel+cisplatin 38 9.4 24 406   Docetaxel+carboplatin 40–41 9.9–10.1 25 404   Vinorelbine+cisplatin Italian Lung Cancer Project (67) 43 9.9 32 204   Paclitaxel+carboplatin 37 9.5 30 203   Vinorelbine+cisplatin 37 9.8 30 205   Gemcitabine+cisplatin JMDB (30)   Gemcitabine+cisplatin 830 28 10.3 42   Pemetrexed+cisplatin 839 31 10.3 44 mo months, N number of patients per arm, RR response rates a Statistically significant difference b Statistically significant difference when the two paclitaxel arms were combined

for the frontline treatment of NSCLC. The control arm was the ECOG reference regimen at that time, cisplatin/paclitaxel. The three experimental arms were cisplatin/gemcitabine, cisplatin/docetaxel, and carboplatin/paclitaxel. In this study, 1,155 chemotherapynaïve patients were randomized. The overall response rate was 19%, the median overall survival was 7.9 months, and the 1- and 2-year overall survival rates were 33 and 11%, respectively, with no statistically significant differences between the groups (28). A second large, phase III study (TAX 326) randomized 1,218 patients to receive one of the following regimens: cisplatin/docetaxel, carboplatin/docetaxel, or cisplatin/vinorelbine. The cisplatin/docetaxel arm marginally outperformed cisplatin/ vinorelbine, with a median overall survival of 11.3 months versus 10.1 months (HR 1.183, 97.2% CI 0.989–1.416, P = 0.044) and better quality of life scores (29). Nonetheless, it should be noted that the protocol-determined statistical criteria for superiority was not met by the cisplatin/docetaxel arm; hence, the three regimens should be considered equivalent in terms of efficacy (i.e., overall survival).

Management of Advanced Non-small Cell Lung Cancer: Front Line Treatment

183

Taken together, the randomized phase III trials containing “modern” chemotherapy regimens failed to identify a platinum-based doublet capable of eliciting superior outcomes. As a result, any combination of a platinum agent with paclitaxel, docetaxel, gemcitabine, or vinorelbine constitutes a reasonable treatment option, yielding response rates in the range of 17–32% and a median survival of 7.8–11.3 months. The choice of drug for each individual patient may be determined by the toxicity profile, treatment schedule, cost, and hospital access. Until recently, histology generally did not play a role in selecting the frontline chemotherapy doublet among the available options. In 2008, Scagliotti et al. presented the results of the largest randomized study of frontline chemotherapy performed to date. The investigators assigned 1,725 patients to receive either cisplatin/gemcitabine or cisplatin/pemetrexed. This was a non-inferiority trial with a primary endpoint of overall survival. Median overall survival in both groups was 10.3 months, and the trial met its primary objective. Hematologic toxicity was significantly better in the cisplatin/pemetrexed arm. In a preplanned subgroup analysis, patients with adenocarcinomas and large cell carcinomas treated with pemetrexed had a statistically significant improvement in median overall survival (12.6 and 10.4 months, respectively) compared to gemcitabine (10.9 and 6.7 months, respectively) [P = 0.03]. On the other hand, survival of patients with squamous cell carcinomas was worse in the pemetrexed group compared to gemcitabine (9.4 versus 10.8, respectively, P = 0.05) (30). These results support adding cisplatin/pemetrexed to the list of standard frontline chemotherapy options. However, for the first time, investigators identified a differential effect of a doublet regimen depending on histology. Another frontline phase III study (described in the “Length of Therapy” section in this chapter) and a second-line phase III study (described elsewhere in this book) further confirm a greater benefit from pemetrexed in patients with non-squamous histology (31). These results may be explained by elevated expression of thymidylate synthase messenger RNA and protein in squamous cell carcinomas compared to adenocarcinomas (32). Thymidylate synthase is one of the targets of pemetrexed, and higher levels may confer resistance to this drug. In light of this provocative new finding, cisplatin/ pemetrexed is only approved for the first-line treatment of non-squamous NSCLC in the U.S. and Europe.

Carboplatin Versus Cisplatin Although cisplatin-based regimens are considered the cornerstone for the treatment of NSCLC, administration of cisplatin is not trivial and is associated with several adverse events (including severe nausea and vomiting, nephrotoxicity, neurotoxicity, and ototoxicity, among others). Carboplatin, on the other hand, has generally a more favorable toxicity profile, less emetogenic potential, and a lower incidence of neurologic, kidney and hearing damage. Hence, there has been great interest in substituting carboplatin for cisplatin, particularly in past years in the era of less

184

W.N. William Jr. and D.J. Stewart

effective antiemetics. It is hard to draw any conclusions on the efficacy equivalence of the two platinum drugs examining any single study. Again, a meta-analysis provides insightful information on this topic. The Cisplatin versus Carboplatin (CISCA) Meta-Analysis Group evaluated nine randomized trials containing 2,988 patients who were allocated to either cisplatinor carboplatin-based regimens. Response rates were higher for the patients receiving cisplatin (30 versus 24%, OR 1.37, 95% CI 1.16–1.61, P < 0.001). Carboplatin was associated with a nonstatistically significant increase in mortality (median survival of 8.4 versus 9.1  months for cisplatin, HR 1.07, 95% CI 0.99–1.15, P = 0.1). However, in patients with non-squamous tumors and in patients treated with thirdgeneration chemotherapy (i.e., paclitaxel, docetaxel, or gemcitabine), carboplatin was associated with inferior survival (HR 1.12, 95% CI 1.01–1.23, and HR 1.11, 95% CI 1.01–1.21, respectively). As expected, nausea and vomiting and nephrotoxicity were more frequent in the cisplatin-treated patients, and thrombocytopenia was more common with carboplatin (33). Despite the slight superiority of cisplatin in terms of response rate and survival, carboplatin-based regimens are still accepted and widely used in the community, given the better tolerability and ease of administration, especially in the setting of metastatic disease in which one of the primary goals of treatment is palliation.

Non-platinum Combinations Besides substituting carboplatin for cisplatin, other attempts at reducing cisplatinrelated toxicity have included the use of non-platinum combinations. In the past two decades, several third-generation agents (i.e., a taxane, gemcitabine, vinorelbine, or camptothecin) have become available for the frontline treatment of NSCLC. In general, these drugs tend to be better tolerated and have shown single-agent activity equal to or greater than cisplatin. Phase III studies have, therefore, evaluated combinations of non-platinum agents compared to platinum-based regimens. In 2005, D’Addario et al. reported the results of a meta-analysis including 37 of these trials, the majority of which contained third-generation agents. Response rates were higher for platinumbased chemotherapy compared to non-platinum-based regimens (OR 1.62, 95% CI 1.46–1.8, P < 0.0001). When the analysis was restricted to trials containing only thirdgeneration drugs in the non-platinum arm, cisplatin-based regimens still had an advantage, albeit smaller (OR 1.17, 95% CI 1.01–1.36, P = 0.042). One-year survival also favored cisplatin-based regimens (34% in the platinum arm versus 29% for the non-platinum arm, P = 0.0003). However, when single-agent trials were excluded and platinum-based therapies were compared with third-generation-based combination regimens only, no statistically significant difference could be found in 1-year survival (36% in the platinum arm versus 35% in the non-platinum arm, P = 0.17). The toxicity profile of platinum-based regimens, compared with third-generation-based combination regimens favored the latter, with an excess of anemia, neutropenia, thrombocytopenia, nausea and vomiting, and toxic death rates in the platinum arm (34).

Management of Advanced Non-small Cell Lung Cancer: Front Line Treatment

185

The results of this meta-analysis support the use of non-platinum-based third-generation drug combinations as substitutes for cisplatin-based doublets, especially in populations at higher risk for the aforementioned side effects, and for whom tumor-shrinkage is not the primary goal of treatment.

Length of Therapy The duration of chemotherapy in treatment-naïve NSCLC patients has been explored in three different types of randomized trials: (1) defined number of cycles versus a larger defined number of cycles of the same chemotherapy; (2) defined number of cycles versus treatment until intolerable toxicity or progression, with the same chemotherapy; (3) defined number of induction chemotherapy cycles followed by no maintenance versus maintenance (immediate or delayed), with a different chemotherapy. The most important studies in each category are described below. Smith et al. randomized 380 patients to three versus six cycles of mitomycin, vinblastin, and cisplatin. Median time to progression was 5 months in both arms. Median overall survival was 6 versus 7 months for three versus six cycles, respectively (P = 0.2). Quality of life parameters were either the same or improved for the 3-month arm (35). Subsequently, von Plessen et al. published the results of three versus six cycles of a new-generation, platinum-based chemotherapy (i.e., carboplatin/ vinorelbine) in 297 patients. Median progression-free survival was 16 versus 21 weeks (P = 0.21) and median overall survival was 28 versus 32 weeks (P = 0.75) in the three- and six-cycle arms, respectively. Quality of life was not different between the arms (36). Socinski et al. randomized 230 patients to four cycles of carboplatin/paclitaxel versus the same chemotherapy regimen until disease progression or intolerable toxicity. Median overall survival was 6.6 months for the shorter treatment arm versus 8.5 months for the longer treatment arm (P = 0.63). Again, there were no differences in quality of life between the arms, and the incidence of neuropathy was significantly increased with a higher number of cycles (37). Similarly, trials of maintenance paclitaxel (38) or maintenance gemcitabine (39) following an initial four cycles of chemotherapy containing the same agents failed to demonstrate a statistically significant increase in survival for the prolonged treatment arm. The results of the aforementioned trials led the American Society of Clinical Oncology to recommend treatment for up to four cycles for patients who do not respond to chemotherapy, and the administration of no more than six cycles, even in patients who are responding to treatment (40). In 2007 and 2008, two randomized trials of prolonged chemotherapy addressed a different question: whether maintenance chemotherapy with a different agent (either immediately or delayed), after a fixed number of cycles of induction treatment, would be beneficial. The premise behind these studies was to circumvent frontline chemotherapy resistance by using a drug with a different mechanism of action. Fidias et al. randomized 307 patients who had stable disease or an objective

186

W.N. William Jr. and D.J. Stewart

response to four cycles of induction carboplatin/gemcitabine to receive either docetaxel immediately after induction treatment or upon disease progression. Median progression-free survival was 6.5 versus 2.8  months (P < 0.0001) and median overall survival was 11.9 versus 9.1 months (P = 0.071) for the immediate and delayed treatment arms, respectively (41). Ciuleanu et al. randomized 663 patients who had stable disease or an objective response to four cycles of induction platinum/ taxane or platinum/gemcitabine to receive either immediate maintenance pemetrexed or best supportive care. Second-line treatment was not offered/mandated per protocol after progression on the best supportive care arm. Median progression-free survival was 4.04 versus 1.97 months (P < 0.00001) and median overall survival was 13.01 versus 10.18 months (P = 0.06) for the pemetrexed and best supportive care arms, respectively. Only 50% of the patients in the best supportive care arm eventually received second-line treatment. A preplanned subgroup analysis demonstrated a greater survival advantage for pemetrexed in the non-squamous histology subgroup (median overall survival of 14.4 versus 9.4 months for pemetrexed and best supportive care, respectively, P = 0.005) (31). Of note, in both the Fidias and Ciuleanu trials, progression-free survival and overall survival were measured from time of randomization (i.e., after completion of the induction regimen). The results of maintenance chemotherapy trials are intriguing, especially those pertaining to the use of pemetrexed in patients with non-squamous histology. Nonetheless, a better designed trial with immediate versus delayed maintenance pemetrexed is still warranted to definitively address its role in this subpopulation. Additionally, mature data on overall survival of the Ciuleanu study is eagerly awaited. So far, maintenance chemotherapy should be viewed as an experimental approach, which prolongs progression-free survival but has little, if any, impact in overall survival. These conclusions have been corroborated by a recent meta-analysis (42).

Anti-VEGF Therapy Bevacizumab is a recombinant humanized monoclonal antibody directed to circulating VEGF, and the only anti-VEGF agent currently in use for the treatment of NSCLC. The first phase II trial to evaluate its role (in combination with paclitaxel and carboplatin) in the treatment of patients with chemotherapy-naïve, metastatic NSCLC was published in 2004 and demonstrated encouraging response rates of 31.5% and median survival of 17.7 months (43). Important safety information was obtained from this study: the most prominent drug-related adverse event was bleeding, with 6 out of 67 bevacizumab-treated patients presenting major hemoptysis or hematemesis, resulting in 4 deaths. All six patients had centrally located tumors close to major blood vessels, five had cavitation or necrosis and four were of squamous cell histology. Subsequently, the ECOG conducted a multicenter, randomized, phase III trial of first-line carboplatin and paclitaxel ± bevacizumab in 878 patients with stage IIIB (pleural effusion) or IV or recurrent NSCLC (3). Patients in whom the predominant tumor histology was squamous were not eligible

Management of Advanced Non-small Cell Lung Cancer: Front Line Treatment

187

to participate in this trial. Patients with brain metastases, on therapeutic anticoagulation therapy, with a history of hemoptysis, clinically significant cardiovascular disease, or medically uncontrolled hypertension were also excluded. The response rate in the bevacizumab arm was 35 versus 15% in the chemotherapy-only group. There was a statistically significant difference in the median overall survival favoring the bevacizumab-treated patients (12.3 versus 10.3  months, P = 0.003). The rates of grade 3–5 hypertension, proteinuria, bleeding, neutropenia, febrile neutropenia, and thrombocytopenia were significantly higher in the paclitaxel/carboplatin/bevacizumab arm as compared to the chemotherapy-only arm. There were more deaths in the bevacizumab group (15 versus 2 patients), five of which were due to pulmonary hemorrhage, five due to febrile neutropenia, two due to cerebrovascular events, one due to gastrointestinal hemorrhage, and one due to pulmonary embolism. The results of the ECOG trial led to the approval, in October 2006, of the combination of bevacizumab, paclitaxel, and carboplatin for the frontline treatment of advanced non-squamous, NSCLC. Of note, for the first time, the survival mark of 12 months was exceeded in a randomized, phase III trial of metastatic NSCLC, and a threedrug regimen was proven to be superior to doublet chemotherapy. The results of the three-arm trial BO 17704 comparing cisplatin, gemcitabine ± bevacizumab (7.5 or 15  mg/kg every 3  weeks) in the frontline treatment of nonsquamous, NSCLC have also been presented (44, 45). The response rates in the bevacizumab arms were higher than in the control group (30, 34, and 20%, respectively, for the bevacizumab 15, 7.5 mg/kg, and control arms), and progression-free survival was also improved (HR 0.75, P = 0.003, and HR 0.85, P = 0.046 for the bevacizumab 15, 7.5  mg/kg compared to the control arm, respectively). Median overall survival was 13.1  months in the control arm, 13.6  months in the bevacizumab 7.5 mg/kg arm, and 13.4 months in the 15 mg/kg arm (no statistically significant difference), but heterogeneous second-line treatment could have confounded the results. These data suggest that bevacizumab can be safely combined with other cytotoxic agents in the frontline treatment of NSCLC and that lower doses (i.e., 7.5 mg/kg every 3 weeks) might be as effective as the original doses studied in the pivotal ECOG 4599 phase III trial (15 mg/kg every 3 weeks).

Anti-EGFR Therapy Two strategies for pharmacological blockade of the EGFR and its related network have been evaluated in randomized phase III studies for the frontline treatment of NSCLC: tyrosine kinase inhibitors (e.g., gefitinib and erlotinib) and anti-EGFR antibodies (e.g., cetuximab). Tyrosine kinase inhibitors (TKIs) are small molecules that competitively block adenosine triphosphate (ATP) binding in the intracellular tyrosine kinase domain of the EGFR, thereby inhibiting its autophosphorylation and further intracellular signaling propagation (46). After phase II studies demonstrated single-agent activity of gefitinib in previously treated patients with NSCLC, phase III studies were

188

W.N. William Jr. and D.J. Stewart

launched to assess its efficacy in combination with first-line, platinum-based chemotherapy. The INTACT-1 trial compared first-line cisplatin, gemcitabine and gefitinib (250 or 500 mg/day concurrently and as maintenance after six cycles of chemotherapy) versus cisplatin and gemcitabine alone in 1,093 patients with unresectable stage III or IV NSCLC. Response rate, progression-free survival, and overall survival were not different among the three groups (47). The INTACT-2 trial randomized 1,037 chemo-naïve patients to carboplatin, paclitaxel and gefitinib (250 or 500  mg/day concurrently and as maintenance after six cycles of chemotherapy) versus carboplatin and paclitaxel in a similar design to the INTACT-1 study. There were also no statistically significant differences in response rates, progression-free survival, and overall survival between treatment arms (48). In parallel to the clinical development of gefitinib, erlotinib (another EGFR tyrosine kinase inhibitor) was also tested in two large, phase III randomized trials in chemotherapy-naïve patients with NSCLC. The TRIBUTE trial compared carboplatin, paclitaxel, and erlotinib 150 mg/day (given concurrently and as maintenance after six cycles of chemotherapy) versus carboplatin, paclitaxel, and placebo in 1,079 patients. Response rates, time to progression, and overall survival were not different between the two study arms for the population as a whole. However, prespecified analyses for the following subgroups were undertaken: age, sex, performance status, weight loss, disease stage, and smoking history. Of these, the only subset in which a statistically significant benefit of erlotinib was demonstrated was for the never-smoking patients (overall survival of 22.5  months for erlotinib versus 10.5 months for placebo, P = 0.01) (49). The TALENT trial enrolled 1,172 patients randomized to cisplatin, gemcitabine, and erlotinib 150 mg/day (given concurrently and as maintenance after six cycles of chemotherapy) versus cisplatin, gemcitabine, and placebo (50). Similar to the TRIBUTE trial, there were no statistically significant differences in overall survival and time to progression between the study arms. However, patients with no smoking history in the past also presented a statistically significant benefit from the study drug, as far as overall survival and time to progression were concerned (50). One possible explanation for the lack of efficacy of TKIs given concurrently and continuously with chemotherapy is that these compounds promote G1 cell-cycle arrest, which in turn could render the tumor cells insensitive to chemotherapy and minimize a possible additive effect of the study drugs in the clinical setting. In addition, both chemotherapy and targeted agents may downregulate expression of membrane transporters required for drug uptake, and this in turn might limit drug efficacy (51). As a result, neither gefitinib nor erlotinib are recommended, in combination with chemotherapy, for the frontline treatment of NSCLC. The effects of gefitinib and erlotinib as second- or third-line therapy are discussed elsewhere in this book. In 2008, a provocative phase III study of first-line gefitinib versus carboplatin/ paclitaxel was presented: IPASS enrolled 1,217 Asian, lung adenocarcinoma patients who were never or light smokers to receive gefitinib 250 mg/day or carboplatin and paclitaxel. The primary endpoint was noninferiority (in progression-free survival) of gefitinib versus chemotherapy. Gefitinib-treated patients had a superior progressionfree survival compared to carboplatin/paclitaxel (HR 0.74, 95% CI 0.65–0.85, P < 0.0001).

Management of Advanced Non-small Cell Lung Cancer: Front Line Treatment

189

Preliminary median overall survival was similar in the two groups (18.3 versus 17.3 months for gefitinib and chemotherapy, respectively). Quality of life analyses favored gefitinib (52). These results are intriguing, in the sense that, for the first time, monotherapy with a biologic agent has been shown to be superior to chemotherapy in the first-line setting. The final results of this study may support, in the future, the routine frontline use of an EGFR inhibitor in patients meeting the same characteristics of the subjects enrolled in IPASS. Monoclonal antibodies bind to the extracellular domain of the EGFR and block the binding of other ligands to the receptor (i.e., EGF, TGF-alpha), thus serving as antagonists of ligand-stimulated tyrosine protein kinase activity. Furthermore, due to its bivalent property, the antibodies are capable of inducing EGFR dimerization, forming receptor-containing complexes that result in receptor internalization (downregulation), which also attenuates intracellular signaling. A third possible mechanism of action of antineoplastic effects of the monoclonal antibodies is antibody-dependant cellular cytotoxicity (46). The activity of chemotherapy plus cetuximab as first-line treatment for NSCLC was initially explored in randomized studies: Butts et al. evaluated the addition of cetuximab to gemcitabine/platinum-doublet in a pilot phase II study with 133 patients. Objective responses were seen in 28% of the cetuximabtreated patients and 18% in the chemotherapy-only arm. Median progression-free and overall survivals were 5.1 and 12.0 months for the cetuximab-containing group, and 4.2 and 9.3 for the chemotherapy-only group, respectively (53). Lynch et al. randomized 676 patients to receive paclitaxel and carboplatin ± cetuximab. Response rates were 26 versus 17% for the arms with and without cetuximab, respectively (P < 0.05). Although the investigator-reported progression-free survival was increased with cetuximab, the progression-free survival assessed by an independent committee (the primary endpoint of the trial) was 4.4 versus 4.2 months in the arms with and without cetuximab, respectively (not statistically significant). Overall survival was 9.7 months for the cetuximab arm, compared to 8.4 months with chemotherapy alone (HR 0.89, 95% CI 0.75–1.05, P = 0.17) (54). In 2008, the results of the FLEX trial were presented. In this study, 1,125 chemotherapy-naïve patients with EGFR-positive tumors were randomly assigned to receive cisplatin/vinorelbine ± cetuximab. Although there were no statistically significant differences in progression-free survival between the groups, median overall survival (the primary endpoint of the trial) was prolonged from 10.1 months in the control arm to 11.3 months in the cetuximab-treated patients (HR 0.871, 95% CI 0.762–0.996, P = 0.0441) (55). Taken together, these results suggest that, unlike oral tyrosine kinase inhibitors, the monoclonal antibody cetuximab might confer a small benefit when added to chemotherapy in the first-line treatment of NSCLC. However, it is still unclear whether there is a differential effect depending on the chemotherapy doublet that is combined with cetuximab. Additionally, further studies are necessary to identify which patients should be preferentially treated with anti-VEGF or anti-EGFR therapies upfront, and whether frontline use of cetuximab is still appropriate for patients who subsequently might be treated with gefitinib or erlotinib upon progression on/after cytotoxic chemotherapy.

190

W.N. William Jr. and D.J. Stewart

Treatment of Elderly Patients The median age at diagnosis of patients with metastatic NSCLC is 69 and rising (56). Conversely, this subpopulation is often underrepresented in clinical studies (57). Evidence for the effects of chemotherapy in elderly patients is derived from subgroup analysis of several phase III trials of cytotoxic treatments. In general, these evaluations demonstrate that elderly patients derive the same benefit from chemotherapy as younger patients, at an expense of higher rates of mild toxicity (58–61). Very few randomized trials have been specifically designed focused solely on elderly patients. ELVIS was a pivotal randomized phase III trial of best supportive care ± vinorelbine in patients ³70 years of age. The investigators found an improvement in median overall survival (21–28 weeks, P = 0.03) and quality of life scores for the vinorelbine-treated group (62). The Southern Italian Cooperative Group randomized 120 patients ³70 years of age to vinorelbine or vinorelbine/gemcitabine. The trial was interrupted earlier than planned, after an interim analysis revealed a statistically significant survival advantage and a delay in symptom and quality of life deterioration in favor of the combination regimen (63). MILES was a three-arm study with 698 elderly patients randomly assigned to vinorelbine alone, gemcitabine alone, or vinorelbine/gemcitabine. There were no differences among the three arms in terms of median overall survival and quality of life scores (62). All in all, elderly patients with a good performance status should be offered chemotherapy. Platinum-based doublets may be used in carefully selected patients with adequate organ function, whereas single-agent chemotherapy or non-platinum based combinations are reasonable choices for most elderly individuals. Patients with a poor performance status are best treated with palliative care, exclusively.

Treatment of Poor Performance Status Patients In 1980, the landmark study of the Veterans Administration Lung Group identified performance status as the dominant prognostic factor for survival in patients with inoperable lung cancer (among 77 prognostic factors analyzed in more than 5,000 patients) (64). Despite its importance, there is even less evidence in the literature for the effects of chemotherapy on patients with a performance status £2. To our knowledge, there are no phase III randomized trials targeting exclusively this population. Treatment decisions of patients with poor performance status should carefully take into account the possibility of adverse events. The ECOG 1594 trial originally allowed the inclusion of patients with a performance status of 2. The median overall survival and progression-free survival in this subpopulation were 4.1 and 1.7 months, respectively. Substantial adverse events (treatment related and unrelated), including deaths, were observed, which subsequently led to the exclusion of performance status 2 patients from the trial (65). Conversely, Lilenbaum et al. demonstrated that patients

Management of Advanced Non-small Cell Lung Cancer: Front Line Treatment

191

with a performance status of 2 enrolled in a clinical trial of paclitaxel ± carboplatin enjoyed superior outcome when treated with the combination regimen (22). A recent phase II ECOG study involving only patients with performance status of 2 also reported somewhat encouraging results with dose-attenuated paclitaxel/carboplatin or cisplatin/gemcitabine (66). These data demonstrate that chemotherapy may provide some benefits to poor performance status patients, but is associated with higher incidence of adverse events. Although combination chemotherapy may be considered for carefully selected patients with performance status of 2, single agent chemotherapy or best supportive care are reasonable options. As of now, there is no evidence that patients with performance status ³3 should be offered chemotherapy.

References 1. Bulzebruck H, Bopp R, Drings P et  al (1992) New aspects in the staging of lung cancer. Prospective validation of the International Union Against Cancer TNM classification. Cancer 70(5):1102–1110 2. Mountain CF (1997) Revisions in the international system for staging lung cancer. Chest 111(6):1710–1717 3. Sandler A, Gray R, Perry MC et al (2006) Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 355(24):2542–2550 4. Socinski MA, Morris DE, Masters GA, Lilenbaum R (2003) Chemotherapeutic management of stage IV non-small cell lung cancer. Chest 123(1 Suppl):226S–243S 5. Marino P, Pampallona S, Preatoni A, Cantoni A, Invernizzi F (1994) Chemotherapy vs supportive care in advanced non-small-cell lung cancer. Results of a meta-analysis of the literature. Chest 106(3):861–865 6. Souquet PJ, Chauvin F, Boissel JP et al (1993) Polychemotherapy in advanced non small cell lung cancer: a meta-analysis. Lancet 342(8862):19–21 7. Grilli R, Oxman AD, Julian JA (1993) Chemotherapy for advanced non-small-cell lung cancer: how much benefit is enough? J Clin Oncol 11(10):1866–1872 8. NSCLC Meta-Analyses Collaborative Group (2008) Chemotherapy in addition to supportive care improves survival in advanced non-small-cell lung cancer: a systematic review and metaanalysis of individual patient data from 16 randomized controlled trials. J Clin Oncol 26(28): 4617–4625 9. (1995) Chemotherapy in non-small cell lung cancer: a meta-analysis using updated data on individual patients from 52 randomised clinical trials. Non-small Cell Lung Cancer Collaborative Group. BMJ 311(7010):899–909 10. Brown J, Thorpe H, Napp V et al (2005) Assessment of quality of life in the supportive care setting of the big lung trial in non-small-cell lung cancer. J Clin Oncol 23(30):7417–7427 11. Helsing M, Bergman B, Thaning L, Hero U (1998) Quality of life and survival in patients with advanced non-small cell lung cancer receiving supportive care plus chemotherapy with carboplatin and etoposide or supportive care only. A multicentre randomised phase III trial. Joint Lung Cancer Study Group. Eur J Cancer 34(7):1036–1044 12. Cullen MH, Billingham LJ, Woodroffe CM et al (1999) Mitomycin, ifosfamide, and cisplatin in unresectable non-small-cell lung cancer: effects on survival and quality of life. J Clin Oncol 17(10):3188–3194 13. Anderson H, Hopwood P, Stephens RJ, et  al (2000) Gemcitabine plus best supportive care (BSC) vs BSC in inoperable non-small cell lung cancer – a randomized trial with quality of life as the primary outcome. UK NSCLC Gemcitabine Group. Non-Small Cell Lung Cancer. Br J Cancer 83(4):447–453

192

W.N. William Jr. and D.J. Stewart

14. (1999) Effects of vinorelbine on quality of life and survival of elderly patients with advanced non-small-cell lung cancer. The Elderly Lung Cancer Vinorelbine Italian Study Group. J Natl Cancer Inst 91(1):66–72 15. Ranson M, Davidson N, Nicolson M et  al (2000) Randomized trial of paclitaxel plus supportive care versus supportive care for patients with advanced non-small-cell lung cancer. J Natl Cancer Inst 92(13):1074–1080 16. Lilenbaum RC, Langenberg P, Dickersin K (1998) Single agent versus combination chemotherapy in patients with advanced nonsmall cell lung carcinoma: a meta-analysis of response, toxicity, and survival. Cancer 82(1):116–126 17. Hotta K, Matsuo K, Ueoka H, Kiura K, Tabata M, Tanimoto M (2004) Addition of platinum compounds to a new agent in patients with advanced non-small-cell lung cancer: a literature based meta-analysis of randomised trials. Ann Oncol 15(12):1782–1789 18. Delbaldo C, Michiels S, Syz N, Soria JC, Le Chevalier T, Pignon JP (2004) Benefits of adding a drug to a single-agent or a 2-agent chemotherapy regimen in advanced non-small-cell lung cancer: a meta-analysis. JAMA 292(4):470–484 19. Gatzemeier U, von Pawel J, Gottfried M et al (2000) Phase III comparative study of high-dose cisplatin versus a combination of paclitaxel and cisplatin in patients with advanced non-smallcell lung cancer. J Clin Oncol 18(19):3390–3399 20. Sandler AB, Nemunaitis J, Denham C et al (2000) Phase III trial of gemcitabine plus cisplatin versus cisplatin alone in patients with locally advanced or metastatic non-small-cell lung cancer. J Clin Oncol 18(1):122–130 21. Wozniak AJ, Crowley JJ, Balcerzak SP et  al (1998) Randomized trial comparing cisplatin with cisplatin plus vinorelbine in the treatment of advanced non-small-cell lung cancer: a Southwest Oncology Group study. J Clin Oncol 16(7):2459–2465 22. Lilenbaum RC, Herndon JE 2nd, List MA et al (2005) Single-agent versus combination chemotherapy in advanced non-small-cell lung cancer: the cancer and leukemia group B (study 9730). J Clin Oncol 23(1):190–196 23. Georgoulias V, Ardavanis A, Agelidou A et al (2004) Docetaxel versus docetaxel plus cisplatin as front-line treatment of patients with advanced non-small-cell lung cancer: a randomized, multicenter phase III trial. J Clin Oncol 22(13):2602–2609 24. Sederholm C, Hillerdal G, Lamberg K et al (2005) Phase III trial of gemcitabine plus carboplatin versus single-agent gemcitabine in the treatment of locally advanced or metastatic non-smallcell lung cancer: the Swedish Lung Cancer Study Group. J Clin Oncol 23(33):8380–8388 25. Finkelstein DM, Ettinger DS, Ruckdeschel JC (1986) Long-term survivors in metastatic non-smallcell lung cancer: an Eastern Cooperative Oncology Group Study. J Clin Oncol 4(5):702–709 26. Bonomi P, Kim K, Fairclough D et al (2000) Comparison of survival and quality of life in advanced non-small-cell lung cancer patients treated with two dose levels of paclitaxel combined with cisplatin versus etoposide with cisplatin: results of an Eastern Cooperative Oncology Group trial. J Clin Oncol 18(3):623–631 27. Kelly K, Crowley J, Bunn PA Jr et  al (2001) Randomized phase III trial of paclitaxel plus carboplatin versus vinorelbine plus cisplatin in the treatment of patients with advanced nonsmall-cell lung cancer: a Southwest Oncology Group trial. J Clin Oncol 19(13):3210–3218 28. Schiller JH, Harrington D, Belani CP et al (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 346(2):92–98 29. Fossella F, Pereira JR, von Pawel J et al (2003) Randomized, multinational, phase III study of docetaxel plus platinum combinations versus vinorelbine plus cisplatin for advanced nonsmall-cell lung cancer: the TAX 326 study group. J Clin Oncol 21(16):3016–3024 30. Scagliotti GV, Parikh P, von Pawel J et  al (2008) Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advancedstage non-small-cell lung cancer. J Clin Oncol 26(21):3543–3551 31. Ciuleanu TE, Brodowicz T, Belani CP, et al (2008) Maintenance pemetrexed plus best supportive care (BSC) versus placebo plus BSC: a phase III study. J Clin Oncol 26 (abstract no. 8011) 32. Ceppi P, Volante M, Saviozzi S et al (2006) Squamous cell carcinoma of the lung compared with other histotypes shows higher messenger RNA and protein levels for thymidylate synthase. Cancer 107(7):1589–1596

Management of Advanced Non-small Cell Lung Cancer: Front Line Treatment

193

33. Ardizzoni A, Boni L, Tiseo M et al (2007) Cisplatin- versus carboplatin-based chemotherapy in first-line treatment of advanced non-small-cell lung cancer: an individual patient data metaanalysis. J Natl Cancer Inst 99(11):847–857 34. D’Addario G, Pintilie M, Leighl NB, Feld R, Cerny T, Shepherd FA (2005) Platinum-based versus non-platinum-based chemotherapy in advanced non-small-cell lung cancer: a metaanalysis of the published literature. J Clin Oncol 23(13):2926–2936 35. Smith IE, O’Brien ME, Talbot DC et al (2001) Duration of chemotherapy in advanced nonsmall-cell lung cancer: a randomized trial of three versus six courses of mitomycin, vinblastine, and cisplatin. J Clin Oncol 19(5):1336–1343 36. von Plessen C, Bergman B, Andresen O et al (2006) Palliative chemotherapy beyond three courses conveys no survival or consistent quality-of-life benefits in advanced non-small-cell lung cancer. Br J Cancer 95(8):966–973 37. Socinski MA, Schell MJ, Peterman A et al (2002) Phase III trial comparing a defined duration of therapy versus continuous therapy followed by second-line therapy in advanced-stage IIIB/ IV non-small-cell lung cancer. J Clin Oncol 20(5):1335–1343 38. Belani CP, Barstis J, Perry MC et al (2003) Multicenter, randomized trial for stage IIIB or IV non-small-cell lung cancer using weekly paclitaxel and carboplatin followed by maintenance weekly paclitaxel or observation. J Clin Oncol 21(15):2933–2939 39. Brodowicz T, Krzakowski M, Zwitter M et  al (2006) Cisplatin and gemcitabine first-line chemotherapy followed by maintenance gemcitabine or best supportive care in advanced nonsmall cell lung cancer: a phase III trial. Lung Cancer 52(2):155–163 40. Pfister DG, Johnson DH, Azzoli CG et al (2004) American Society of Clinical Oncology treatment of unresectable non-small-cell lung cancer guideline: update 2003. J Clin Oncol 22(2):330–353 41. Fidias P, Dakhil S, Lyss A, et al (2007) Phase III study of immediate versus delayed docetaxel after induction therapy with gemcitabine plus carboplatin in advanced non-small-cell lung cancer: updated report with survival. J Clin Oncol, 2007 ASCO Annual Meeting Proceedings Part I 25(18S) (Abstract no. LBA7516) 42. Soon Y, Stockler MR, Boyer M, Askie L (2008) Duration of chemotherapy for advanced nonsmall cell lung cancer: an updated systematic review and meta-analysis. J Clin Oncol 26 (Abstract no. 8013) 43. Johnson DH, Fehrenbacher L, Novotny WF et al (2004) Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small-cell lung cancer. J Clin Oncol 22(11):2184–2191 44. Manegold C, Pawel Jv, Zatloukal P, et al (2007) Randomised, double-blind multicentre phase III study of bevacizumab in combination with cisplatin and gemcitabine in chemotherapy-naïve patients with advanced or recurrent non-squamous non-small cell lung cancer (NSCLC): BO17704. J Clin Oncol, 2007 ASCO Annual Meeting Proceedings Part I 25(18S) (Abstract no. LBA7514) 45. Manegold C, Pawel Jv, Zatloukal P, et al (2008) BO17704 (AVAIL): a phase III randomised study of first-line bevacizumab combined with cisplatin/gemcitabine (CG) in patients (pts) with advanced or recurrent non-squamous, non-small cell lung cancer (NSCLC). Ann Oncol 19(suppl 8):viii1 46. Ciardiello F, Tortora G (2008) EGFR antagonists in cancer treatment. N Engl J Med 358(11):1160–1174 47. Giaccone G, Herbst RS, Manegold C et al (2004) Gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer: a phase III trial-INTACT 1. J Clin Oncol 22(5):777–784 48. Herbst RS, Giaccone G, Schiller JH et al (2004) Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: a phase III trial-INTACT 2. J Clin Oncol 22(5):785–794 49. Herbst RS, Prager D, Hermann R et al (2005) TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced nonsmall-cell lung cancer. J Clin Oncol 23(25):5892–5899 50. Gatzemeier U, Pluzanska A, Szczesna A et al (2007) Phase III study of erlotinib in combination with cisplatin and gemcitabine in advanced non-small-cell lung cancer: the Tarceva Lung Cancer Investigation Trial. J Clin Oncol 25(12):1545–1552

194

W.N. William Jr. and D.J. Stewart

51. Stewart DJ (2008) Gefitinib maintenance in stage III non-small-cell lung cancer. J Clin Oncol 26(29):4849–4850; author reply 50–51 52. Mok T, Wu Y-L, Thongprasert S, et  al (2008) Phase III randomised, open-label, first-line study of gefitinib (G) vs carboplatin/paclitaxel (C/P) in clinically selected patients (pts) with advanced non-small cell lung cancer (NSCLC) (IPASS). Ann Oncol 19(suppl 8):viii1 53. Butts CA, Bodkin D, Middleman EL et al (2007) Randomized phase II study of gemcitabine plus cisplatin or carboplatin [corrected], with or without cetuximab, as first-line therapy for patients with advanced or metastatic non small-cell lung cancer. J Clin Oncol 25(36):5777–5784 54. Lynch TJ, Patel T, Dreisbach L, et  al (2007) A randomized multicenter phase III study of cetuximab (Erbitux®) in combination with Taxane/Carboplatin versus Taxane/Carboplatin alone as first-line treatment for patients with advanced/metastatic non-small cell lung cancer (NSCLC). J Thor Oncol 2(8 Suppl 4):S340 55. Pirker R, Szczesna A, Pawel Jv, et al (2007) FLEX: a randomized, multicenter, phase III study of cetuximab in combination with cisplatin/vinorelbine (CV) versus CV alone in the first-line treatment of patients with advanced non-small cell lung cancer (NSCLC). J Clin Oncol 26 (Abstract no. 3) 56. Havlik RJ, Yancik R, Long S, Ries L, Edwards B (1994) The National Institute on Aging and the National Cancer Institute SEER collaborative study on comorbidity and early diagnosis of cancer in the elderly. Cancer 74(7 Suppl):2101–2106 57. Lewis JH, Kilgore ML, Goldman DP et al (2003) Participation of patients 65 years of age or older in cancer clinical trials. J Clin Oncol 21(7):1383–1389 58. Langer CJ, Manola J, Bernardo P et al (2002) Cisplatin-based therapy for elderly patients with advanced non-small-cell lung cancer: implications of Eastern Cooperative Oncology Group 5592, a randomized trial. J Natl Cancer Inst 94(3):173–181 59. Rocha Lima CM, Herndon JE, 2nd, Kosty M, Clamon G, Green MR (2002) Therapy choices among older patients with lung carcinoma: an evaluation of two trials of the Cancer and Leukemia Group B. Cancer 94(1):181–187 60. Belani CP, Fossella F (2005) Elderly subgroup analysis of a randomized phase III study of docetaxel plus platinum combinations versus vinorelbine plus cisplatin for first-line treatment of advanced nonsmall cell lung carcinoma (TAX 326). Cancer 104(12):2766–2774 61. Hensing TA, Peterman AH, Schell MJ, Lee JH, Socinski MA (2003) The impact of age on toxicity, response rate, quality of life, and survival in patients with advanced, Stage IIIB or IV nonsmall cell lung carcinoma treated with carboplatin and paclitaxel. Cancer 98(4):779–788 62. Gridelli C, Perrone F, Gallo C et al (2003) Chemotherapy for elderly patients with advanced non-small-cell lung cancer: the Multicenter Italian Lung Cancer in the Elderly Study (MILES) phase III randomized trial. J Natl Cancer Inst 95(5):362–372 63. Frasci G, Lorusso V, Panza N et al (2000) Gemcitabine plus vinorelbine versus vinorelbine alone in elderly patients with advanced non-small-cell lung cancer. J Clin Oncol 18(13):2529–2536 64. Stanley KE (1980) Prognostic factors for survival in patients with inoperable lung cancer. J Natl Cancer Inst 65(1):25–32 65. Sweeney CJ, Zhu J, Sandler AB et al (2001) Outcome of patients with a performance status of 2 in Eastern Cooperative Oncology Group Study E1594: a Phase II trial in patients with metastatic nonsmall cell lung carcinoma. Cancer 92(10):2639–2647 66. Langer C, Li S, Schiller J, Tester W, Rapoport BL, Johnson DH (2007) Randomized phase II trial of paclitaxel plus carboplatin or gemcitabine plus cisplatin in Eastern Cooperative Oncology Group performance status 2 non-small-cell lung cancer patients: ECOG 1599. J Clin Oncol 25(4):418–423 67. Scagliotti GV, De Marinis F, Rinaldi M et al (2002) Phase III randomized trial comparing three platinum-based doublets in advanced non-small-cell lung cancer. J Clin Oncol 20(21):4285–4291

Chemotherapy in Previously Treated Patients with Non-small Cell Lung Cancer Frank V. Fossella

Abstract  While advanced non-small cell lung cancer often responds to chemotherapy, it eventually develops resistance and remains incurable. Recent studies indicate that each of docetaxel, pemetrexed, and the epidermal growth factor receptor inhibitor erlotinib may prolong survival and improve quality of life for some patients with tumor progression during or after front-line chemotherapy. Keywords  Second line • Docetaxel • Pemetrexed • Erlotinib

Introduction In the past several years, medical oncologists have become increasingly aware that there is a subset of patients with advanced non-small cell lung cancer (NSCLC) for whom second-line chemotherapy and beyond may be appropriate after the failure of first-line therapy. Most of the data in support of the use of second-line chemotherapy have been with docetaxel, pemetrexed, and erlotinib.

Docetaxel Docetaxel is a taxane whose primary mechanism of action is the stabilization of polymerized microtubules, resulting in cell death. It was the first chemotherapeutic agent to undergo systematic evaluation in the second-line setting for patients with NSCLC whose disease had progressed or failed to respond to first-line platinum-based chemotherapy.

F.V. Fossella (*) Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 432, Houston, TX 77030, USA e-mail: [email protected] D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_9, © Humana Press, a part of Springer Science+Business Media, LLC 2010

195

196

F.V. Fossella

Objective responses were consistently seen in four phase II studies of docetaxel in the second-line setting, and response rates ranged from 15 to 22% (1–4). Favorable survival times were noted as well in these trials; median survival ranged from 5.8 to 11 months, and estimated 1-year survival ranged from 25 to 40%. The most compelling evidence to support docetaxel’s activity in the second-line treatment of NSCLC comes from two large randomized phase III trials, which compared docetaxel to either best supportive care (5) (TAX 317) or to a comparator regimen of chemotherapy (6) (TAX 320).

Phase III Trial of Docetaxel vs. Best Supportive Care: TAX 317 TAX 317 was a multicenter international trial reported by Shepherd (5, 7). Eligible patients had advanced NSCLC that had progressed on or after at least one prior platinum-containing chemotherapy regimen. There was no restriction on the number of prior chemotherapy cycles or regimens, and there was no restriction on the prior chemotherapy permitted with the exception that patients with prior paclitaxel exposure were excluded. Patients must have had a performance status of 0–2. Eligible patients were stratified by their best response to prior platinum-based chemotherapy and performance status, and then randomly assigned to receive either docetaxel every 3 weeks vs. best supportive care (BSC). The initial trial design called for a docetaxel dose of 100  mg/m2 (D100) as a 1-h intravenous infusion every 3 weeks. However, because of an unexpectedly high rate of occurrence of adverse events occurring at this dose level, the protocol was subsequently modified to a docetaxel dose of 75 mg/m2 (D75) every 3 weeks. A total of 204 patients were enrolled in this trial: 49 received docetaxel 100 mg/ m2, 55 received docetaxel 75 mg/m2, and 100 received BSC. About 80% of patients had stage IV disease, and 25% of patients had a performance status of 2. The demographics with regard to age and gender were typical for this patient population and were well-balanced between the treatment groups. The predominant histology was adenocarcinoma. One-quarter of patients had received two or more prior chemotherapy regimens before enrollment. Partial response was observed in 6% of patients treated with docetaxel (either dose level), and another 40% of patients had stable disease. The median response duration was 26 weeks. Time-to-progression (TTP) favored treatment with docetaxel vs. BSC. The median TTP was 12.3 weeks with D75 vs. 7 weeks with BSC (P = 0.004). Overall survival (intent-to-treat) also favored treatment with docetaxel. For the entire docetaxel group (i.e., patients treated at both 100 and 75  mg/m2), median survival was 7.2 months (docetaxel) vs. 4.7 months (BSC). Considering only those patients in the D75 group, median survival was 9.0 vs. 4.6 months (P = 0.016), and 1-year survival was 40% vs. 16% (P = 0.016) (Fig. 1). With the exception of neutropenia (grade 3 or 4 in 43% of patients), there were no other significant differences in side effects between the D75 arm compared with BSC.

Chemotherapy in Previously Treated Patients with Non-small Cell Lung Cancer

1.0

BSC 100

0.9 CUMULATIVE PROBABILITY

b

DOCETAXEL 100 mg/m2 (n=49) (n=51)

0.8 0.7 Log-rank test, p=0.780

0.6

1.0 0.9

CUMULATIVE PROBABILITY

a

0.5 0.4 0.3 0.2 0.1

197

DOCETAXEL 75 mg/m2

(n=55)

BSC 75

(n=49)

0.8 0.7 Log-rank test, p=0.010

0.6 0.5 0.4 0.3 0.2 0.1

0.0

0.0 0

15 18 3 6 9 12 SURVIVAL TIME (MONTHS)

21

0

3

6 9 12 15 18 SURVIVAL TIME (MONTHS)

21

Fig. 1  TAX 317: Overall survival analysis for patients treated with docetaxel 100 mg/m2 vs. best supportive care (BSC) (a) and docetaxel 75 mg/m2 vs. BSC (b) (5)

Phase III Trial of Docetaxel vs. a Comparator Chemotherapy Regimen: TAX 320 In support of TAX 317 was TAX 320, which was a multicenter US trial (6, 8). Eligible patients had advanced NSCLC that had progressed on or after at least one prior platinum-containing chemotherapy regimen. There was no restriction on the number of prior chemotherapy cycles or regimens, and in contrast to TAX 317, there was no restriction on the prior chemotherapy permitted. Specifically, patients treated with prior paclitaxel were eligible for the study. Patients must have had a performance status of 0–2. After stratification by best response to prior platinum-based chemotherapy and performance status, patients were randomized to either docetaxel 100 mg/m2 every 3 weeks (D100), docetaxel 75 mg/m2 every 3 weeks (D75), or a comparator arm of either vinorelbine 30 mg/m2/week or ifosfamide 2 gm/m2 × 3 days every 3 weeks (V/I). (For patients who were randomized to arm 3, the choice of either vinorelbine or ifosfamide was left to the treating physician.) A total of 373 patients were enrolled in this trial: 125 received docetaxel 100 mg/m2, 125 received docetaxel 75 mg/m2, and 123 received either the vinorelbine or ifosfamide. About 90% of patients had stage IV disease, and 16% of patients had a performance status of 2. The age and gender were typical for a lung cancer population, and were similar across the three groups. The predominant histology was adenocarcinoma. Close to 40% of patients had received two or more prior chemotherapy regimens before enrollment, and almost 40% of patients had received prior paclitaxel.

198

F.V. Fossella

Partial response was observed in 11% of patients in D100, 7% in D75, and 1% in V/I. The differences in response rated favoring docetaxel over the control group were highly significant, with P-values of 0.002. An additional one-third of patients (in either group) had stable disease. The median response duration was 9 months with D75. TTP favored treatment with docetaxel vs. V/I. Although the median TTP was equivalent between the three groups (at about 8 weeks), the 26-week progression-free survival was 8% in the V/I group, compared with 19% in D100 (P = 0.013) and 17% in D75 (P = 0.031). Overall survival (intent-to-treat) also favored treatment with docetaxel. Although the median survival was equal across the three groups, at about 5.5 months, the 1-year survival was significantly higher in patients treated in the D75 group. Oneyear survival was 32% in patients treated with D75, vs. 21% in D100 and 19% in V/I. The difference in 1-year survival favoring D75 was statistically significant, with a P-value of 0.025 (Fig. 2a). Over one-third of patients in each group received subsequent chemotherapy upon removal from this trial (including taxane therapy in 21% of patients in the control group). Because of the potential impact on survival such subsequent therapy may have had, an intent-to-treat survival analysis was done in which survival observations were censored at the point at which patients received subsequent chemotherapy treatment (Fig. 2b). This analysis showed that 1-year survival significantly favored treatment with either docetaxel arm. The 1-year survival was 32% with either D100 or D75, compared with 10% 1-year survival in the V/I group; these differences were highly statistically significant (P = 0.002). Grade 4 neutropenia and febrile neutropenia occurred with greater frequency in both docetaxel arms compared with the control group; documented infection and grade 4 thrombocytopenia, however, were equivalent across all 3 arms. G-CSF use

b

1.0

CUMULATIVE PROBABILITY

0.9

TAX 100 mg/m2 TAX 75 mg/m2

1.0

VINO/IFOS

0.9

CUMULATIVE PROBABILITY

a

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

TAX 100 mg/m2 TAX 75 mg/m2 VINO/IFOS

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

0.0 0

3

6 9 12 15 18 SURVIVAL TIME (MONTHS)

21

0

3

6 9 12 15 18 SURVIVAL TIME (MONTHS)

21

Fig. 2  TAX 320: Overall survival analysis (a); and overall survival analysis censored for subsequent chemotherapy on removal from study (b) (6). TAX Taxotere (docetaxel); VINO/IFOS vinorelbine or ifosfamide

Chemotherapy in Previously Treated Patients with Non-small Cell Lung Cancer

199

was greatest with D100, at 28% of cycles, but was comparable between D75 and V/I. Severe nonhematologic side effects, including treatment-related death, were equivalent across the 3 groups. As previously noted, many patients in this trial had received paclitaxel prior to enrollment into this study: 31% of patients in D100, 42% of patients in D75, and 41% of patients in V/I. Although this trial was not designed to assess the clinical benefit of docetaxel in this particular subset of patients, the data that are available suggest that prior paclitaxel exposure did not have any bearing on response rate and survival data (8). For example, with regard to objective radiographic response to docetaxel, the partial response rates were equivalent in the cohort of 91 patients who had received prior paclitaxel (10.5%) and the group of 157 patients who had not received prior paclitaxel (8.5%). This suggests that there was no impact on response rate according to prior paclitaxel therapy. In a similar analysis of the survival data, prior paclitaxel therapy had no bearing on the survival advantage seen with docetaxel. In the overall intent-to-treat survival for the entire study population, 1-year survival was 32% in the D75 group compared with 19% in the V/I group (P = 0.025). An intent-to-treat survival curve for the subset of patients with no prior paclitaxel therapy is comparable between the three groups, but the 1-year survival again favors D75: 33% vs. 20% for the V/I group. The P-value of 0.08 is a favorable indication in subgroups of patients with small sample sizes. Similarly, in the cohort of patients who did receive prior paclitaxel treatment, the 1-year survival again favored D75 mg (at 30%) vs. V/I (at 17%). The P-value is 0.13, which again is a favorable indication in subgroups of patients with small sample sizes. Both TAX 317 and TAX 320 prospectively evaluated quality of life parameters (7). Both trials demonstrated clear trends favoring quality of life for the patients treated with docetaxel. TAX 317 employed the Lung Cancer Symptom Scale (LCSS) and the EORTC-QLQ-C30. All of the quality of life parameters that were considered favored the patients treated with docetaxel vs. BSC, including change in performance status and pain, pain control, and fatigue. The TAX 317 patients receiving docetaxel also used significantly fewer medications for control of tumorrelated symptoms. TAX 320 showed similar favorable findings with regard to quality of life using the LCSS. Patients treated in the D75 arm showed a statistically significant improvement in appetite, symptom distress, performance status, and overall quality of life as compared with the V/I control group; and the D75 patients showed a favorable trend (though not statistically significant) with respect to pain, dyspnea, fatigue, and activity compared with the control patients.

Pemetrexed Pemetrexed is a multitargeted antifolate chemotherapy agent that is active in multiple tumor types, including NSCLC (9–12). Its primary mechanism of action is to inhibit the enzyme thymidylate synthase, resulting in decreased thymidine necessary

200

F.V. Fossella

for pyrimidine synthesis. Pemetrexed also inhibits dihydrofolate reductase and glycinamide ribonucleotide formyl transferase, the latter of which is a folatedependent enzyme involved in purine synthesis. Phase II studies of pemetrexed in previously untreated patients with NSCLC have demonstrated single agent response rates of 17–23% (9, 10). A phase II study of pemetrexed in patients with advanced NSCLC, who had progressed during or within 3 months of completing first-line chemotherapy, demonstrated a response rate of 8.9% and median survival time of 5.7 months (11). Based upon these phase II data showing comparable efficacy of pemetrexed with docetaxel in the second line setting, and the expected lower toxicity rates with pemetrexed, a multinational phase III study was done comparing pemetrexed and docetaxel in the second-line treatment of NSCLC (13). In this study, patients with stage III or IV NSCLC who had progressive disease after one prior chemotherapy regimen (or, in the case of patients who had received neoadjuvant or adjuvant therapy, two prior regimens) were randomized to receive either pemetrexed 500 mg/ m2 or docetaxel 75 mg/m2; both treatments were given every 21 days. Patients were stratified based upon performance status (0,1 vs. 2), prior platinum or paclitaxel exposure, number of prior regimens (1 vs. 2), time since last chemotherapy (<3 vs. ³3 months), best response to last chemotherapy (stable disease or response vs. progression), and stage of disease (III vs. IV). The study was designed as a noninferiority trial. Primary endpoint was survival, and secondary endpoints were toxicities, response rate, progression-free survival, and time to progression. This trial enrolled 283 patients in the pemetrexed arm and 288 patients in the docetaxel arm. Both arms were well-balanced with regard to key patient characteristics of age, gender, stage, histology, and performance status. 75% of patients in both arms had stage IV disease, and about half of the patients in each arm had adenocarcinoma. Close to 90% of patients in either arm had performance status of 0–1. Efficacy data between the two treatment groups were comparable. Overall response rates were 9.1% with pemetrexed and 8.8% with docetaxel. Median overall survival was 8.3 months and 7.9 months for pemetrexed and docetaxel, respectively (Fig. 3). Median progression-free survival and 1-year survival were identical for both treatment arms, at 2.9 months and 29.7%, respectively. There was no significant difference in median survival. There was no statistically significant difference between pemetrexed and docetaxel with respect to objective response rate, progressionfree survival, and time-to-progression. The primary study endpoint was survival, and the study did not show an overall survival superiority of pemetrexed. Furthermore, noninferiority of pemetrexed to docetaxel could not be demonstrated because a reliable and consistent survival effect of docetaxel required for a noninferiority analysis could not be estimated from historical trials. It is notable that 47% of patients in the pemetrexed arm and 37% of patients in the docetaxel arm received additional chemotherapy after going off-study. Furthermore, 32% of patients randomized to the pemetrexed arm subsequently received docetaxel (off-protocol). This significant rate of treatment crossover at the time of disease progression may have confounded the survival interpretation.

Chemotherapy in Previously Treated Patients with Non-small Cell Lung Cancer

201

Fig. 3  Pemetrexed vs. docetaxel: Overall survival analysis (13). MST median survival time; Mo months; yr years; Pts patients

Although the efficacy endpoints were similar, there was a statistically significant toxicity advantage noted for patients treated with pemetrexed. The incidence of grade 3 or 4 neutropenia was 5.3% vs. 40.2% for pemetrexed compared with docetaxel. Grade 3 or 4 febrile neutropenia occurred in only 1.9% of patients treated with pemetrexed compared with 12.7% of patients treated with docetaxel. And the incidence of alopecia was 11.3% vs. 42.4% for the two groups.

Erlotinib Erlotinib is an inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) (14–17). Phase II experience with erlotinib, and the related drug gefitinib, demonstrated consistent activity in patients with NSCLC who had previously received platinum-based chemotherapy, with response rates in the range of 10–20% (15–17). Based upon this phase II experience, the National Cancer Institute of Canada conducted a phase III trial of second-line erlotinib for patients with advanced NSCLC; this trial was known as BR.21 (18). BR.21 was a randomized, double-blind placebo-controlled trial in 731 patients with locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen. Patients were randomized 2:1 to receive erlotinib 150 mg or placebo orally, once daily, until disease progression or unacceptable toxicity. Patients were stratified by center of enrollment, performance status, prior response, number of prior regimens, and prior platinum.

202

F.V. Fossella

Key patient characteristics were similar in the two arms of the study. Approximately one-half of patients had EGFR protein expression characterized; however, EGFR status was not a selection criterion for entry in the study. The primary endpoint of this study was overall survival. Secondary endpoints included progression-free survival, response rate, median duration of response, and toxicity. The study demonstrated a significant improvement in overall survival, PFS, and response rate for the patients treated with erlotinib vs. the placebo. The KaplanMeier curve for survival (Fig.  4) showed median survival of 6.7 months for Erlotinib-treated patients vs. 4.7 months for patients receiving placebo. One-year survivals were 31% vs. 22% for the erlotinib vs. placebo arm, respectively. The hazard ratio (HR) for overall survival was 0.73, favoring treatment with erlotinib. Erlotinib also resulted in an improvement in PFS: 9.9 weeks vs. 7.9 weeks with placebo. Response rate was 8.9% with erlotinib vs. 0.9% with placebo. The median duration of response was 7.9 months. An exploratory univariate analysis showed that erlotinib’s survival benefit extended to diverse patient subsets, including patients with differences in such baseline characteristics as tumor type, performance status, and prior chemotherapy. Analyses of patients by performance status and line of therapy demonstrated a significant survival benefit for second-line patients with good performance status (i.e., 0–1). In this subset analysis, median survival was 9.4 months compared with 6.7 months with placebo. EGFR protein expression status was determined in 326 of 731 patients (45%). Patients who never smoked and were EGFR positive had a large erlotinib survival benefit: HR = 0.28).

Fig. 4  Erlotinib vs. placebo: Kaplan–Meier curve for overall survival (18). P values were adjusted for stratification factors (except center) and epidermal growth factor receptor expression

Chemotherapy in Previously Treated Patients with Non-small Cell Lung Cancer

203

Conclusion NSCLC remains a very difficult disease. However, each of docetaxel, pemetrexed, and erlotinib may be effective in palliating some patients with advanced NSCLC suffering tumor progression during or after front-line chemotherapy.

References 1. Fossella FV, Lee JS, Murphy WK et al (1994) Phase II study of docetaxel for recurrent or metastatic non-small cell lung cancer. J Clin Oncol 12:1238 2. Francis PA, Rigas JR, Kris MG et al (1994) Phase II trial of docetaxel in patients with stage III and IV non-small cell lung cancer. J Clin Oncol 12:1232 3. Cerny T, Kaplan S, Pavlidis N et al (1994) Docetaxel (Docetaxel) is active in non-small cell lung cancer: a phase II trial of the EORTC early clinical trials group (ECTG). Br J Cancer 70:384 4. Burris HA, Eckardt J, Fields S et al (1993) Phase II trials of Docetaxel in patients with nonsmall cell lung cancer. Proc Am Soc Clin Oncol 12:335 (abstract) 5. Shepherd FA, Dancey J, Ramlau R et al (2000) Prospective randomized trial of. Docetaxel versus best supportive care in patients with non-small cell lung cancer patients previously treated with platinum-based chemotherapy. J Clin Oncol 18:2095 6. Fossella FV, DeVore R, Kerr RN et al (2000) Randomized phase III trial of docetaxel versus vinorelbine or ifosfamide in patients with advanced non-small cell lung cancer previously treated with platinum-containing chemotherapy. J Clin Oncol 18:2354 7. Shepherd FA, Fossella FV, Lynch T et al (2001) Docetaxel (Docetaxel) shows survival and quality-of-life benefits in the second-line treatment of non-small cell lung cancer: a review of two phase III trials. Semin Oncol 28(Suppl 2):4–9 8. Fossella FV, DeVore R, Kerr RN et al (2001) Randomized phase III trial of docetaxel versus vinorelbine or ifosfamide in patients with advanced non-small cell lung cancer previously treated with platinum-containing chemotherapy. J Clin Oncol Classic Papers and Current Comments 6:97 9. Clarke S, Boyer M, Millward M et  al (1997) Phase II study of LY231514, a multitargeted antifolate, in patients with advanced non-small cell lung cancer. Proc Am Soc Clin Oncol 16:465a (abstract 1670) 10. Rusthoven J, Eisenhauer E, Butts C et al (1999) Multitargeted antifolate LY231514 as firstline chemotherapy for patients with advanced non-small cell lung cancer: a phase II study – National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 17:1194–1199 11. Smit E, Mattson K, von Pawel J et al (2003) Alimta (pemetrexed disodium) as second-line treatment of non-small cell lung cancer: a phase II study. Ann Oncol 14:455–460 12. Vogelzang N, Rusthoven J, Symanowski J et al (2003) Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol 21:2636–2644 13. Hanna N, Shepherd FA, Fossella FV et al (2004) Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small cell lung cancer previously treated with chemotherapy. J Clin Oncol 22:1589–1597 14. Sridhar SS, Seymour L, Shepherd FA (2003) Inhibitors of epidermal-growth-factor receptors: a review of clinical research with a focus on non-small-cell lung cancer. Lancet Oncol 4:397–406 15. Fukuoka M, Yano S, Giaccone G et al (2003) Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer. J Clin Oncol 21:2237–2246

204

F.V. Fossella

16. Kris MG, Natale RB, Herbst RS et al (2003) Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. JAMA 290:2149–2158 17. Perez-Soler R, Chachoua A, Hammond LA et al (2004) Determination of tumor response and survival with erlotinib in patients with non-small cell lung cancer. J Clin Oncol 22:3238–3247 18. Shepherd FA, Pereira JR, Ciuleanu T et al (2005) Erlotinib in previously treated non-smallcell lung cancer. New Engl J Med 353:123–132

Epidermal Growth Factor Receptor Inhibitors in the Treatment of Non-small Cell Lung Cancer Paul Wheatley-Price and Frances A. Shepherd

Abstract  Inhibition of the epidermal growth factor receptor (EGFR) has become a standard target in the treatment of non-small cell lung cancer (NSCLC). This chapter summarizes the clinical trials that have been performed with small molecule tyrosine kinase inhibitors (TKI) and monoclonal antibodies targeting EGFR in NSCLC. Erlotinib has established second- and third-line efficacy following the phase III BR.21 study, results not seen in the corresponding ISEL trial with gefitinib. However, in the INTEREST trial gefitinib demonstrated non-inferiority to docetaxel in the second-line setting, and as first-line therapy may be better than chemotherapy in selected patients. Cetuximab also modestly prolongs survival when combined with chemotherapy as first-line treatment in patients with EGFR expressing tumors. Numerous other TKIs and monoclonal antibodies have demonstrated clinical activity in early phase trials. Novel TKIs may have the ability to overcome resistance to first generation TKI therapy. Furthermore, there are encouraging studies combining EGFR inhibitors and anti-angiogenesis drugs such as bevacizumab. In conclusion, EGFR inhibition, by a range of strategies, remains a central node in the treatment of NSCLC. Keywords  Non-small cell lung cancer • EGFR inhibitor

Introduction Over the past decade, the epidermal growth factor receptor (EGFR) has become a molecular target of increasing importance in the treatment of non-small cell lung cancer (NSCLC). P. Wheatley-Price Division of Medical Oncology, Ottawa Hospital Cancer Centre F.A. Shepherd (*) Scott Taylor Chair in Lung Cancer Research, Department of Medical Oncology, Princess Margaret Hospital, 610 University Avenue, Toronto, ON M5G 2M9, Canada e-mail: [email protected] D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_10, © Humana Press, a part of Springer Science+Business Media, LLC 2010

205

206

P. Wheatley-Price and F.A. Shepherd

The EGFR family consists of four receptors, erb1/EGFR, erb2/HER2, erb3/HER3, and erb4/HER4. The receptors are composed of an extracellular ligand binding domain (except HER2), a transmembrane domain, and an intracellular tyrosine kinase (TK) domain (except HER3). Activation of the receptor leads to an intracellular signaling cascade affecting invasion, apoptosis, and angiogenesis (1, 2). Ligands for these receptors, most importantly epidermal growth factor (EGF) and transforming growth factor-a (TGFa), bind to the extracellular domain resulting in receptor dimerization and autophosphorylation of the intracellular TK domain, leading to downstream signaling, including the activation of ras, raf, mitogenactivated protein kinase (MAPK), phosphatidyl 3-kinase (PI3K), ERK1, and ERK2 (3, 4). These molecules are linked to cell growth, proliferation, motility, and survival (5–7). Overexpression of EGFR is observed in 40–80+% of patients with NSCLC (8–16) and has been associated with poor prognosis in some but not all studies (2, 12–19). EGFR mutations are present in approximately 10% of NSCLC cases in Europe and North America, and in 30–40% in East Asian patients. The most common mutations involve exon 19 deletions or exon 21 point mutations (20). These mutations are associated with the clinical characteristics of female sex, a history of never smoking cigarettes, Asian ethnicity, and adenocarcinoma histology (21, 22); conversely mutations in the KRAS gene are more common in smoking-related NSCLC and seldom occur in tumors with EGFR mutations (23, 24). Targeting the EGFR pathway is a well-established strategy in the treatment of NSCLC. The most common approaches use either small molecules targeting the intracellular TK domain, or monoclonal antibodies that competitively bind to the extracellular domain. This chapter summarizes the current EGFR inhibitors in clinical practice in the treatment of NSCLC, the important trials of EGFR inhibitors, and discusses newer agents in the development pipeline. Randomized clinical trials of the most commonly used anti-EGFR drugs, erlotinib, gefitinib, and cetuximab, are summarized in Tables 1 and 2. Table 3 summarizes EGFR inhibitors in practice or development. Table  4 lists important ongoing clinical trials.

Small Molecule Tyrosine Kinase Inhibitors Tyrosine kinase inhibitors (TKIs) have been the most successful agents to date in targeting EGFR. Erlotinib and gefitinib have received approval for the treatment of advanced NSCLC in the second- and third-line setting, with gefitinib also recently showing phase III trial evidence of activity in the first- and second-line settings and erlotinib in the maintenance setting. Many other TKIs currently are in development.

Pac/Carbo + gefitinib 250 mg Pac/Carbo + gefitinib 500 mg Pac/Carbo + placebo

Pac/Carbo Gefitinib 250 mg

Herbst (50) (INTACT-2)

Mok (46) (IPASS)

Goss (48) (INSTEP)

Hida (51) (WJTOG 0203)

Gefitinib 250 mg Placebo Patients of poor PS

100 101

298 300

118 125

733 733

345 347 345

29% 34% p = 0.19 6% 1%

N/A

Gem/Cis + gefitinib 250 mg Gem/Cis + gefitinib 500 mg Gem/Cis + placebo

Giaccone (49) (INTACT-1)

Kelly (52) (SWOG 0023)

580 579

Gem/Cis + erlotinib 150 mg Gem/Cis + placebo

Gatzemeier (30) (TALENT)

Clinically selected patients (Asian, adenocarcinoma, never/light smokers) Gefitinib 500 mg – 250 mg Placebo Maintenance after definitive chemoradiation (stage III) 6 cycles platinum doublet 3 cycles platinum doublet and maintenance gefitinib

21.5% 19.3% p = 0.36 31.5% 29.9% NS 51.2% 50.3% 47.2% p = NS 30.4% 30.0% 28.7% p = NS 32% 43% p = 0.0001

526 533

365 365 363

ORR

Number

Table 1  Randomized trials of erlotinib and gefitinib Author/study Treatment arms First-line treatment of non-small cell lung cancer Herbst (29) Pac/Carbo + erlotinib (TRIBUTE) Pac/Carbo + placebo 150 mg

4.3 4.6 HR 0.68, p < 0.001 HR 0.82, p = 0.22

812 p = 0.28

5.1 4.9 p = 0.36 5.5 5.7 p = 0.74 5.8 5.5 6.0 p = 0.76 5.3 4.6 5.0 p = 0.06 HR 0.74 p < 0.0001 (favors g efitinib)

PFS (months)

(continued)

23 35 p = 0.01 13.7 12.9 HR 0.86, p = 0.10 HR 0.84, p = 0.27

10.6 10.5 p = 0.95 9.9 10.2 p = 0.49 9.9 9.9 10.9 p = 0.46 9.8 8.7 9.9 p = 0.64 17.3 18.6 HR 0.91

OS (months)

Epidermal Growth Factor Receptor Inhibitors in the Treatment 207

  489

12.2% 12.5% 17.9% 13.2% 13.7% 9.1% 7.6% p = 0.33 22.5% 12.8% p = 0.009

ORR

2.2 2.7 p = 0.47 NS P = 0.34

3.0 4.8 4.4 N/A

PFS (months)

18.4% 2.7 19.0% 2.8 p = NS Kris (41) Gefitinib 250 mg   102 12% N/A (IDEAL-2) Gefitinib 500 mg   114 9% p = 0.51 Shepherd (27) Erlotinib 150 mg   488 8.9% 2.2 (BR.21) Placebo   243 <1% 1.8 p < 0.001 p < 0.001 Thatcher (43) Gefitinib 250 mg 1129 8% 3.0 a (ISEL) Placebo   563 1% 2.6 a p < 0.0001 p < 0.001 ORR overall response rate; PFS progression-free survival; OS overall survival; NS Not significant; N/A Not available a Time to treatment failure

Monotherapy after failure of chemotherapy in advanced non-small cell lung cancer Fukuoka (40) Gefitinib 250 mg   103 (IDEAL-1) Gefitinib 500 mg   106

Gefitinib 250 mg Docetaxel 60 mg/m2

  41   40   39   68   73   733   733

Second-line treatment of advanced non-small cell lung cancer Fehrenbacher (31) Docetaxel (D) or Pemetrexed (P) Bevacizumab + D or P Bevcizumab + erlotinib 150 mg Cufer (55) Gefitinib 250 mg (SIGN) Docetaxel 75 mg/m2 Douillard (56) Gefitinib 250 mg (INTEREST) Docetaxel 75 mg/m2

Niho (57) (V-1532)

Number

Table 1  (continued) Author/study Treatment arms

7.6 8.0 p = NS 7 6 p = 0.40 6.7 4.7 p < 0.001 5.6 5.1 p = 0.09

7.5 7.1 7.6 8.0 HR 1.02 11.5 14.0 HR1.12

N/A

OS (months)

208 P. Wheatley-Price and F.A. Shepherd

Epidermal Growth Factor Receptor Inhibitors in the Treatment

209

Table 2  Randomized trials of cetuximab in first-line treatment of NSCLC Author/study Butts (88) (BMS CA225100) Rosell (89) (LUCAS) Herbst (90) (SWOG 0342) Lynch (91) (BMS CA225099)

Treatment arms Gem/Carbo or Cis + cetuximab Gem/Carbo or Cis Vin/Cis + cetuximab Vin/Cis Pac/Carbo + cetuximab Pac/Carbo followed by cetuximab Pac/Carbo + cetuximab Pac/Carbo

Number   65   66

ORR 27.7% 18.2%

PFS (months) 5.1 4.2

OS (months) 12.0 9.3

  43   43 106 119

31.7% 20.0% 34% 31%

4.7 4.2 4 4

N/A

338 338

25.7% 17.2% p < 0.01 36% 29% p = 0.012

4.4 4.2 p = 0.24 4.8 4.8 p = NS

N/A

11 10

11.3 557 Vin/Cis + cetuximab 10.1 568 Vin/Cis p = 0.044 All patients were EGFR positive ORR Overall response rate; PFS Progression-free survival; OS Overall survival; Gem Gemcitabine; Carbo Carboplatin; Cis Cisplatin; Vin Vinorelbine; Pac Paclitaxel; N/A Not available; NS Not significant Von Pawel (129) (FLEX)

Table 3  EGFR inhibitors in development and in practice Drug name Class of action Target Cetuximab Chimeric MoAb EGFR Panitumumab Humanized MoAb EGFR Matuzumab Humanized MoAb EGFR Nimotuzumab Humanized MoAb EGFR Trastuzumab Humanized MoAb HER2 Pertuzumab Humanized MoAb HER2 Erlotinib Reversible TKI EGFR Gefitinib Reversible TKI EGFR EKB-569 Irreversible TKI EGFR CL-3877785 Irreversible TKI EGFR HKI-272 Irreversible TKI EGFR, HER2 BIBW-2992 Irreversible TKI EGFR, HER2 PKI-166 TKI EGFR, HER2 Lapatinib Reversible TKI EGFR, HER2 CI-1033 Irreversible TKI EGFR, HER2, HER4 PF-00299804 Irreversible TKI EGFR, HER2, HER4 Vandetanib TKI EGFR, VEGF, RET XL647 Reversible TKI EGFR, HER2, EphB4, VEGF AEE788 TKI EGFR, HER2, VEGF MoAb Monoclonal antibody; TKI tyrosine kinase inhibitor

Stage of development (completed) Phase III Phase I/II Phase I Phase I Phase II Phase II In clinical use In clinical use Phase I Pre-clinical Phase I Phase I Phase I Phase II Phase II Phase I Phase II Phase II Phase I

Population treated 1st line maintenance after complete resection of EGFR +ve NSCLC +/− adjuvant chemotherapy 1st line maintenance for advanced NSCLC in patients without disease progression after chemotherapy 1st-line maintenance after chemotherapy + bevacizumab in advanced NSCLC 1st line chemotherapy-naïve advanced NSCLC

TOPICAL 1st line chemotherapy-naïve advanced NSCLC with poor (NCT00275132) performance status (2–3) CALGB-30406 1st line chemotherapy-naïve advanced NSCLC (NCT00126581) TITAN 2nd-line NSCLC after failure of first-line chemotherapy. (RO 50-8231) Stratified by EGFR status NCT00440414 2nd-line NSCLC after failure of first-line chemotherapy BeTa Lung 2nd-line NSCLC after failure of first-line chemotherapy (NCT00130728) NCIC BR.19 1st line maintenance after complete resection of (closed to accrual) stage 1-IIIA NSCLC +/− adjuvant chemotherapy EORTC 08021 1st line maintenance for advanced NSCLC in patients (NCT00091156) without disease progression after chemotherapy ZEAL 2nd-line NSCLC after failure of first-line chemotherapy (NCT00418886) ZEST Advanced NSCLC after failure of at least one line of (NCT00364351) chemotherapy ZEPHYR Advanced NSCLC after failure of chemotherapy and (NCT00404924) 1st-line TKI ZODIAK 2nd-line NSCLC after failure of first-line chemotherapy (NCT00312377) PFS progression-free survival; OS overall survival; RR response rate

Title RADIANT (NCT00373425) SATURN (RO 50-8231) ATLAS (NCT00257608) TORCH (NCT00349219)

Table 4  Important clinical trials currently in progress

PFS OS

Bevacizumab + erlotinib vs. bevacizumab + placebo 1st-line erlotinib followed at progression by 2nd-line chemotherapy vs. 1st-line chemotherapy followed at progression by 2nd-line erlotinib Erlotinib vs. placebo

OS OS OS OS OS PFS PFS OS PFS

Erlotinib vs. docetaxel or pemetrexed Erlotinib vs. pemetrexed Erlotinib + placebo vs. erlotinib + bevacizumab Gefitinib vs. placebo Gefitinib vs. placebo Pemetrexed + vandetanib 100 mg vs. pemetrexed + placebo Vandetanib 300 mg vs. erlotinib Vandetanib 300 mg vs. best supportive care Docetaxel + vandetanib 100 mg vs. docetaxel + placebo

Erlotinib vs. erlotinib, carboplatin and paclitaxel

PFS

Erlotinib vs. placebo

OS, PFS, RR PFS

Primary endpoint DFS

Study arms Erlotinib vs. placebo

210 P. Wheatley-Price and F.A. Shepherd

Epidermal Growth Factor Receptor Inhibitors in the Treatment

211

Erlotinib (Tarceva®, OSI-774; OSI Pharmaceuticals, Melville, NY, USA) Erlotinib is an oral TKI that reversibly binds to the adenosine triphosphate (ATP) binding site of the tyrosine kinase receptor, preventing auto-phosphorylation and down-stream signaling. Efficacy was first demonstrated in a phase II trial in previously treated patients with NSCLC (25, 26). The international BR.21 study subsequently randomized 731 patients with advanced NSCLC, after failure of one or two lines of chemotherapy, to erlotinib or placebo. Median overall survival (OS) was prolonged significantly in the erlotinib arm (hazard ratio [HR] 0.70, 95% confidence interval [CI] 0.58–0.85, p < 0.001), with an 8.9% tumor response rate (Fig. 1) (27). Asian origin, adenocarcinoma histology, female gender, and a history of never smoking were associated with response, but the only clinical factor associated with a differential survival benefit was smoking history. BR.21 also demonstrated that treatment with erlotinib was associated with improved quality-of-life (28). As a result of this landmark trial, erlotinib has become a standard of care for patients in the second- or third-line therapy setting. In the first-line treatment of advanced NSCLC, the addition of erlotinib to standard platinum-based chemotherapy did not result in improved outcomes in the phase III TRIBUTE and TALENT studies (29, 30). However, these trials suggested that survival might be prolonged by the administration of erlotinib to responding and stable patients after chemotherapy. This hypothesis is being tested in the SATURN trial in which, following four cycles of first-line platinumbased chemotherapy, stable and responding patients are randomized to receive erlotinib or placebo. In a second maintenance trial (ATLAS), based on promising phase II results seen in the second-line setting (31, 32), bevacizumab alone is compared to erlotinib plus bevacizumab as maintenance therapy in stable and responding patients after first-line chemotherapy for advanced NSCLC. A phase II trial, comparing bevacizumab plus docetaxel or pemetrexed vs. bevacizumab plus erlotinib vs. chemotherapy alone, reported a trend toward improved survival in the bevacizumab-containing cohorts, and lower toxicity in the bevacizumab/ erlotinib arm (31). The confirmatory phase III BeTa lung trial randomizes previously treated NSCLC patients to erlotinib plus bevacizumab vs. erlotinib plus placebo and remains open to accrual. With concern about chemotherapy toxicity in elderly populations in mind, a phase II trial of erlotinib monotherapy as first-line treatment in the elderly (>70 years of age) reported a response rate of 10% and median survival of 9 months (33). Further, an analysis of erlotinib in the over 70 age cohort from the BR.21 study also demonstrated that efficacy was not dissimilar to that of the younger patients, although higher rates of toxicity and lower dose intensity were also observed (34). A phase II study of erlotinib in patients with bronchoalveolar carcinoma (BAC) reported a response rate of 25%, correlating with a history of never-smoking and

212

P. Wheatley-Price and F.A. Shepherd

Fig. 1  BR.21. Overall survival and progression-free survival (27). Reproduced with permission from: Shepherd FA, Rodrigues Pereira J, Ciuleanu T et al (2005) Erlotinib in previously treated non-small cell lung cancer. N Engl J Med 353:123–132

Epidermal Growth Factor Receptor Inhibitors in the Treatment

213

EGFR mutations, but no responses were seen in patients with KRAS mutations (35–37). A phase II trial of first-line treatment in NSCLC patients with EGFR mutations reported response in 19/21 evaluable patients (38). Finally, the TORCH trial compares erlotinib with chemotherapy in the first-line setting in unselected patients, with crossover upon progression.

Gefitinib (Iressa®, ZD1839; Astra-Zeneca, Macclesfield, United Kingdom) Gefitinib is another oral EGFR TKI (39). The IDEAL-1 (Europe, Australia, and Japan) and IDEAL-2 (North America) phase II studies randomized patients with advanced NSCLC, after one or two previous chemotherapy regimens (irrespective of EGFR status), to gefitinib 250 or 500 mg daily. Response rates ranged from 9 to 19%, with symptomatic improvement reported by 35–43% of patients (40, 41). On the basis of these trials, the FDA approved gefitinib monotherapy for chemotherapyrefractory NSCLC (42). However, the subsequent ISEL study comparing gefitinib to best supportive care in patients who were either refractory to, or intolerant of chemotherapy did not show a significant overall benefit to gefitinib (HR 0.89, 95% CI 0.77–1.02, p = 0.09) (Fig.  2), although a significant survival benefit was seen amongst Asians (HR 0.66, 95% CI 0.48–0.91, p = 0.01) and never-smokers (HR 0.67, 95% CI 0.49–0.92, p = 0.01) (43, 44). In the first-line setting, a phase II trial of gefitinib monotherapy in Korean neversmokers with adenocarcinoma reported a response rate of 69% and an estimated 1-year survival of 73% (45). Subsequently, the phase III IPASS (Iressa Pan-Asia Study) study of gefitinib vs. platinum-based chemotherapy was performed in a clinically selected population of Asians with adenocarcinoma histology and a history of never-smoking or ex-light smokers (46). Significantly, longer progression-free survival (HR 0.74, 95% CI 0.65–0.85, p < 0.0001) and higher response rates (43% vs. 32%, p = 0.0001) were observed in the gefitinib cohort. There was no significant difference in overall survival, but this analysis is immature as the required number of events for the final survival analysis has not yet occurred. The final survival analysis may be confounded as crossover to the alternate treatment is to be expected by a high proportion of patients in both arms. Overall, treatment favored chemotherapy in the first 6 months and subsequently (with approximately 60% of patients progression-free at that point) gefitinib. Of particular interest was the finding of EGFR mutations in approximately 60% of cases tested. In this subset, survival was superior at all time points for patients treated with gefitinib. In the over 70 years age group, gefitinib has been compared to vinorelbine in the first-line setting in a randomized phase II trial (INVITE) (47). No significant differences in response rate, progression-free, or overall survival were observed between treatments, although gefitinib was better tolerated. In an unexpected result, patients whose tumors expressed EGFR benefited more from vinorelbine, a finding that will require further validation. A further randomized phase II study (INSTEP)

214

Fig.  2  ISEL. Survival in the overall population and in patients with adenocarcinoma (43). Reproduced with permission from: Thatcher N, Chang A, Parikh P et al (2005) Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small cell lung cancer: results from a randomized, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet 366:1527–1537

has compared gefitinib with best supportive care as first-line treatment in patients with a poor performance status (48). In this cohort of 201 patients, progression-free and overall survival was not significantly prolonged with gefitinib treatment. In contrast, and as seen with erlotinib, trials of gefitinib in combination with platinum-based chemotherapy (INTACT-1 and 2) showed no survival benefit over

Epidermal Growth Factor Receptor Inhibitors in the Treatment

215

chemotherapy alone (49, 50). However, as with erlotinib, there was a suggestion that treatment with gefitinib after chemotherapy might result in prolonged survival in advanced disease. This hypothesis has been tested in the Japanese WJTOG 0203 phase III trial, and a European Organization for Research and Treatment of Cancer (EORTC 08021) phase III trial of gefitinib or placebo following first-line chemotherapy. Results of the WJTOG 0203 study have been presented in abstract form. In this trial, patients were randomized to either 6 cycles of platinum-based chemotherapy or 3 cycles of chemotherapy followed by maintenance gefitinib. Treatment with gefitinib was associated with a significantly longer progressionfree survival, although there was no advantage in overall survival (51). In stage III NSCLC, maintenance gefitinib failed to prolong survival after definitive chemotherapy and radiotherapy (Table  3) (52). In fact, treatment with gefitinib was associated with significantly shorter survival, although the reasons for this are not clear. Currently, chemotherapy with docetaxel or pemetrexed is the standard of care as second-line treatment after failure of platinum-based chemotherapy for NSCLC (53, 54). Gefitinib has now been compared to docetaxel in the second-line setting in one randomized phase II and two randomized phase III trials (55–57). These studies all demonstrated similar activity and efficacy but an improved toxicity profile in the gefitinib arms. The largest study, the 1,466 patient phase III INTEREST trial, met its primary endpoint of noninferiority and demonstrated lower toxicity rates in patients receiving gefitinib (56).

TKI Molecules in Development EKB-569 (Wyeth, Madison, NJ, USA) (58, 59) and CL-387785 (Calbiochem, Darmstadt, Germany) (60, 61) are irreversible EGFR TKIs currently in development. Molecules that target both EGFR and other receptors of the EGFR family include HKI-272 (Wyeth, Madison, NJ, USA) (62–64), BIBW-2992 (Boehringer Ingelheim, Ingelheim, Germany) (65, 66), PKI-166 (Novartis International AG, Basel, Switzerland) (67, 68), lapatinib (Tykerb®, GW-572016; GlaxoSmithKline, Middlesex, United Kingdom) (69), CI-1033 (Pfizer, Ann Arbor, MI, USA) (70–75), and PF-00299804 (Pfizer, Ann Arbor, MI, USA) (76). Vandetanib (Zactima®, ZD6474; Astra-Zeneca, Macclesfield, United Kingdom) is a small-molecule TKI inhibitor of both EGFR and VEGF. In phase II trials, vandetanib has shown promising efficacy when compared to gefitinib (77), or in combination with docetaxel (78) but showed little activity as monotherapy in the first-line setting (79). Phase III placebo-controlled trials of vandetanib in combination with docetaxel in the second-line setting and single-agent vandetanib in the third- or fourth-line setting are ongoing. XL647 (Exelixis Inc; South San Francisco, CA, USA) (80–82) and AEE788 (Novartis International AG, Basel, Switzerland) (83, 84) are other multitargeted TKIs currently being investigated.

216

P. Wheatley-Price and F.A. Shepherd

Monoclonal Antibodies Cetuximab (Erbitux®, IMC-C225; ImClone Systems Incorporated, New York, NY, USA) is a chimeric IgG1 monoclonal antibody that inhibits EGFR by binding to the extracellular domain, with five times more affinity than the natural ligands (85). Cetuximab has been studied as first-line treatment in combination with chemotherapy. Following phase I/II safety studies in combination with standard chemotherapy (86, 87), two randomized phase II studies in advanced NSCLC patients (irrespective of EGFR status) compared gemcitabine/ platinum (cisplatin or carboplatin) (BMS-CA225-100) (88) or vinorelbine/cisplatin (LUCAS trial) (89) with or without cetuximab. A third randomized phase II trial (SWOG 0342) investigated concurrent and sequential approaches of adding cetuximab to first-line chemotherapy. Both arms demonstrated similar activity and efficacy (90). After improvements in response rate were observed in both the BMS-CA225-100 and LUCAS trials, and longer PFS in the BMS-CA225-100 study, phase III trials were designed to evaluate cetuximab/ chemotherapy combinations. Two randomized phase III trials have now been completed. BMS-CA225099 compared carboplatin and taxane (paclitaxel or docetaxel) chemotherapy with or without cetuximab. There was a significant improvement in response in the cetuximab arm (26% vs. 17%, p < 0.007), but no benefit in progression-free survival (PFS) was observed (4.40 vs. 4.24 months, p = 0.24) and overall survival results were not available at time of writing this chapter (91). The phase III trial of vinorelbine/platinum with or without cetuximab, in EGFR positive patients, FLEX, has however shown a statistically significant median overall survival benefit of 1 month for patients in the cetuximab arm (11.3 vs. 1 months; HR 0.87, 95% CI 0.67–1.00, p = 0.04) (92). While the combination of cetuximab and chemotherapy was relatively well tolerated, the modest survival increment and high cost of the drug may mean that uptake of this combination is limited, at least until the overall survival results from BMS-CA225099 are available. Other monoclonal antibodies targeting EGFR are in development, although only limited efficacy data in NSCLC are available. Antibodies currently in clinical trials include panitumumab (Vectibix®, ABX-EGF; Amgen Inc, Thousand Oaks, CA, USA) (93, 94), matuzumab (EMD72000; Merck, Whitehouse Station, NJ, USA) (95), nimotuzumab (TheraCIM; YM Biosciences Inc, Mississauga, ON, Canada) (96–98), and pertuzumab (Omnitarg®, 2C4; Genentech, South San Francisco, CA, USA) (99, 100). Trastuzumab (Herceptin®; Roche, Basel, Switzerland), a humanized monoclonal antibody that targets HER2, has established efficacy in the treatment of breast cancer (101–103). A randomized phase II trial investigated the addition of trastuzumab to gemcitabine/cisplatin in the first-line treatment of HER2 positive advanced NSCLC. No clinical benefit was observed with the addition of trastuzumab, although in a subgroup of six patients with HER2 3+ or HER2 amplification, the response rate was 83% (104).

Epidermal Growth Factor Receptor Inhibitors in the Treatment

217

Patient Selection for EGFR-Targeted Therapy Clinical Factors Several clinical characteristics (Asian ethnicity, non-smoking history, female gender, and adenocarcinoma or bronchoalveolar carcinoma histology) have been shown to be significantly associated with response to EGFR TKIs (27, 29, 43, 45, 46, 105). However, it is important to note that patients without these characteristics (male, smokers, squamous cell carcinoma) can also respond to EGFR inhibition, as seen in the BR.21, INTEREST, and TARGET studies (27, 56, 106). Furthermore, in BR.21 (27), the only clinical factor that was associated with a differential survival benefit from erlotinib was smoking history.

EGFR Expression, Copy Number, and Mutations At the laboratory level, EGFR protein expression, EGFR gene copy number, and EGFR mutation status have been shown to influence response rates in patients treated with erlotinib and gefitinib (105). In BR.21 and ISEL, overall response rates were higher in patients whose tumors expressed EGFR protein and these patients derived meaningful survival benefit from treatment (HR 0.68, p = 0.02 in BR.21; HR 0.77, p = 0.13 in ISEL), whereas those without EGFR expression did not appear to benefit from treatment (HR 0.93, p = 0.70 in BR.21; HR 1.57, p = 0.14 in ISEL) (107, 108). However, significant interaction was seen only in the ISEL trial. EGFR copy number appears to be both a prognostic and a predictive factor. In both BR.21 (updated analysis) and ISEL, response rates were significantly higher in patients with high EGFR copy (21% vs. 5%, p = 0.02, in BR.21; 16.4% vs. 3.2%, p-value NA, in ISEL) (108, 109). Similarly, in both trials, patients with high EGFR copy number derived meaningful survival benefit from treatment (HR 0.43, p = 0.004 in BR.21; HR 0.77, p = 0.13 in ISEL), whereas those without high EGFR copy number did not appear to benefit from treatment (HR 0.80, p = 0.35 in BR.21; HR 1.57, p = 0.14 in ISEL) Survival curves from the BR.21 study are shown in Fig.  3. In an analysis of the SWOG 0342 phase II trial, investigating concurrent or sequential cetuximab (90), an analysis has been performed of outcomes stratified by EGFR expression in 76 patients for whom tissue was available (110). EGFR positive patients had significantly higher disease control rate (81% vs. 55%, p = 0.02), longer PFS (6 vs. 3 months, p = 0.0008), and longer OS (15 vs. 7 months, p = 0.04) when compared to EGFR lowcopy patients. An association between somatic mutations in EGFR exons 19 and 21 and response to EGFR TKI therapy was first reported by Lynch et al. and Paez et al. (111, 112). BR.21 and ISEL both demonstrated significantly higher response rates to erlotinib or gefitinib in patients with the EGFR mutations (108, 109), and

218

P. Wheatley-Price and F.A. Shepherd

Fig.  3  BR.21. Survival in subgroups according to EGFR and KRAS status (109). Reproduced with permission from: Zhu C-Q, da Cunha Santos G, Ding K et  al (2008) Role of KRAS and EGFR as biomarkers of response to erlotinib in National Cancer Institute of Canada Clinical Trials Group Study BR.21. Journal of Clinical Oncology 26:4268–75

patients with mutations also appeared to derive survival benefit from treatment with erlotinib in BR.21 (HR 0.55 [0.25–1.19], p = 0.12), although a significant interaction was not seen (interaction p = 0.47) (109). In contrast to high copy number that is associated with poorer survival in untreated patients, the presence of EGFR mutations appear to be prognostic of better survival. Sasaki and colleagues reported significantly longer survival for 95 Asian patients with surgically treated NSCLC whose tumors had EGFR mutations (113). Similar findings have been reported from the TRIBUTE and INTACT trials (114, 115). The more recent IPASS study showed a significant correlation between EGFR mutation and PFS in clinically selected patients receiving gefitinib as first-line therapy.

Epidermal Growth Factor Receptor Inhibitors in the Treatment

219

Markers of Resistance A second EGFR point mutation in exon 20 (T790M mutation), in patients who have relapsed following initial successful treatment with erlotinib or gefitinib has now been identified (116–118). Use of novel irreversible TKIs, such as HKI-272, CL-387785, or XL647, may circumvent this pathway of resistance raising the possibility of a subgroup of patients benefiting from sequential EGFR inhibitors (60, 62, 82). KRAS is a downstream effector of the EGFR pathway, and mutations occur in approximately 20–30% of NSCLC cases (119). While data are conflicting as to whether KRAS mutations are a predictive marker for chemo-resistance (120–123), studies have demonstrated KRAS mutations to be associated with lack of sensitivity to EGFR TKIs (114, 124, 125). This is in line with findings in colorectal cancer, where a survival benefit from the addition of cetuximab to chemotherapy is only seen in KRAS-wildtype patients (126). Data from TRIBUTE revealed that survival was significantly worse for patients with KRAS-mutant tumors who received erlotinib with chemotherapy relative to the other patient groups (114). In BR.21, 15% of patients tested had KRAS mutations. Those with wildtype KRAS experienced a survival benefit from erlotinib that was similar to that in the primary analysis (HR: 0.69, p = 0.03), whereas patients with mutant KRAS did not appear to benefit from erlotinib (HR: 1.67, p = 0.31) (127).

Conclusion and Future Directions Combining TKIs with EGFR antibodies is being investigated clinically after showing synergism in preclinical models (128). Numerous further trials are investigating combining EGFR inhibitors with other targeted agents, including anti-angiogenesis and mammalian target of rapamycin (mTOR) inhibitors. In conclusion, erlotinib and gefitinib now have established phase III evidence in the second- and third-line setting. Gefitinib may also be of benefit in the first-line setting in clinically or molecularly selected patients. In first-line therapy, the combination of cetuximab with chemotherapy in EGFR +ve patients has demonstrated modestly improved efficacy. A number of new anti-EGFR antibodies and novel TKI drugs are in development, and new combinations with other targeted agents provide hope for the future.

References 1. Sridhar SS, Seymour L, Shepherd FA (2003) Inhibitors of epidermal-growth-factor receptors: a review of clinical research with a focus on non-small-cell lung cancer. Lancet Oncol 4(7):397–406 2. Bunn PA Jr, Franklin W (2002) Epidermal growth factor receptor expression, signal pathway, and inhibitors in non-small cell lung cancer. Semin Oncol 29(5 Suppl 14):38–44

220

P. Wheatley-Price and F.A. Shepherd

  3. Roskoski R Jr (2004) The ErbB/HER receptor protein-tyrosine kinases and cancer. Biochem Biophys Res Commun 319(1):1–11   4. Burgess AW, Cho HS, Eigenbrot C et al (2003) An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors. Mol Cell 12(3):541–552   5. Alroy I, Yarden Y (1997) The ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand-receptor interactions. FEBS Lett 410(1):83–86   6. Felip E, Baselga J Lung cancer: principles and practice, 3rd edn. Lippincott Williams and Wilkins. Chapter 11, p147–159   7. Lewis TS, Shapiro PS, Ahn NG (1998) Signal transduction through MAP kinase cascades. Adv Cancer Res 74:49–139   8. Berger MS, Gullick WJ, Greenfield C, Evans S, Addis BJ, Waterfield MD (1987) Epidermal growth factor receptors in lung tumours. J Pathol 152(4):297–307   9. Cerny T, Barnes DM, Hasleton P et al (1986) Expression of epidermal growth factor receptor (EGF-R) in human lung tumours. Br J Cancer 54(2):265–269 10. Dazzi H, Hasleton PS, Thatcher N et al (1989) Expression of epidermal growth factor receptor (EGF-R) in non-small cell lung cancer. Use of archival tissue and correlation of EGF-R with histology, tumour size, node status and survival. Br J Cancer 59(5):746–749 11. Sobol RE, Astarita RW, Hofeditz C et al (1987) Epidermal growth factor receptor expression in human lung carcinomas defined by a monoclonal antibody. J Natl Cancer Inst 79(3):403–407 12. Pastorino U, Andreola S, Tagliabue E et al (1997) Immunocytochemical markers in stage I lung cancer: relevance to prognosis. J Clin Oncol 15(8):2858–2865 13. Pfeiffer P, Clausen PP, Andersen K, Rose C (1996) Lack of prognostic significance of epidermal growth factor receptor and the oncoprotein p185HER-2 in patients with systemically untreated nonsmall-cell lung cancer: an immunohistochemical study on cryosections. Br J Cancer 74(1):86–91 14. Rusch V, Klimstra D, Venkatraman E, Pisters PW, Langenfeld J, Dmitrovsky E (1997) Overexpression of the epidermal growth factor receptor and its ligand transforming growth factor alpha is frequent in resectable non-small cell lung cancer but does not predict tumor progression. Clin Cancer Res 3(4):515–522 15. Cox G, Jones JL, O’Byrne KJ (2000) Matrix metalloproteinase 9 and the epidermal growth factor signal pathway in operable non-small cell lung cancer. Clin Cancer Res 6(6):2349–2355 16. Ohsaki Y, Tanno S, Fujita Y et al (2000) Epidermal growth factor receptor expression correlates with poor prognosis in non-small cell lung cancer patients with p53 overexpression. Oncol Rep 7(3):603–607 17. D’Amico TA, Massey M, Herndon JE 2nd, Moore MB, Harpole DH Jr (1999) A biologic risk model for stage I lung cancer: immunohistochemical analysis of 408 patients with the use of ten molecular markers. J Thorac Cardiovasc Surg 117(4):736–743 18. Fontanini G, De Laurentiis M, Vignati S et al (1998) Evaluation of epidermal growth factorrelated growth factors and receptors and of neoangiogenesis in completely resected stage I-IIIA non-small-cell lung cancer: amphiregulin and microvessel count are independent prognostic indicators of survival. Clin Cancer Res 4(1):241–249 19. Volm M, Rittgen W, Drings P (1998) Prognostic value of ERBB-1, VEGF, cyclin A, FOS, JUN and MYC in patients with squamous cell lung carcinomas. Br J Cancer 77(4):663–669 20. Sharma SV, Bell DW, Settleman J, Haber DA (2007) Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 7(3):169–181 21. Shigematsu H, Lin L, Takahashi T et al (2005) Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 97(5):339–346 22. Marchetti A, Martella C, Felicioni L et al (2005) EGFR mutations in non-small-cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment. J Clin Oncol 23(4):857–865 23. Ahrendt SA, Decker PA, Alawi EA et al (2001) Cigarette smoking is strongly associated with mutation of the K-ras gene in patients with primary adenocarcinoma of the lung. Cancer 92(6):1525–1530 24. Husgafvel-Pursiainen K, Hackman P, Ridanpaa M et  al (1993) K-ras mutations in human adenocarcinoma of the lung: association with smoking and occupational exposure to asbestos. Int J Cancer 53(2):250–256

Epidermal Growth Factor Receptor Inhibitors in the Treatment

221

25. Hidalgo M, Siu LL, Nemunaitis J et al (2001) Phase I and pharmacologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies. J Clin Oncol 19(13):3267–3279 26. Perez-Soler R, Chachoua A, Hammond LA et  al (2004) Determinants of tumor response and survival with erlotinib in patients with non-small-cell lung cancer. J Clin Oncol 22(16):3238–3247 27. Shepherd FA, Rodrigues Pereira J, Ciuleanu T et  al (2005) Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 353(2):123–132 28. Bezjak A, Tu D, Seymour L et  al (2006) Symptom improvement in lung cancer patients treated with erlotinib: quality of life analysis of the National Cancer Institute of Canada Clinical Trials Group Study BR.21. J Clin Oncol 24(24):3831–3837 29. Herbst RS, Prager D, Hermann R et al (2005) TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol 23(25):5892–5899 30. Gatzemeier U, Pluzanska A, Szczesna A et al (2007) Phase III study of erlotinib in combination with cisplatin and gemcitabine in advanced non-small-cell lung cancer: the Tarceva Lung Cancer Investigation Trial. J Clin Oncol 25(12):1545–1552 31. Fehrenbacher L, O’Neill V, Belani CP et al (2006) A phase II, multicenter, randomized clinical trial to evaluate the efficacy and safety of bevacizumab in combination with either chemotherapy (docetaxel or pemetrexed) or erlotinib hydrochloride compared with chemotherapy alone for treatment of recurrent or refractory non-small cell lung cancer. J Clin Oncol 24(18S) (abstract 7062) 4743–4750 32. Herbst RS, Johnson DH, Mininberg E et al (2005) Phase I/II trial evaluating the anti-vascular endothelial growth factor monoclonal antibody bevacizumab in combination with the HER-1/ epidermal growth factor receptor tyrosine kinase inhibitor erlotinib for patients with recurrent non-small-cell lung cancer. J Clin Oncol 23(11):2544–2555 33. Jackman DM, Yeap BY, Lindeman NI et al (2007) Phase II clinical trial of chemotherapynaive patients > or  = 70 years of age treated with erlotinib for advanced non-small-cell lung cancer. J Clin Oncol 25(7):760–766 34. Wheatley-Price P, Ding K, Seymour L, Clark GM, Shepherd FA (2008) Erlotinib for advanced non-small-cell lung cancer in the elderly: an analysis of the National Cancer Institute of Canada Clinical Trials Group Study BR.21. J Clin Oncol 26(14):2350–2357 35. Kris MG, Sandler A, Miller V et al (2005) EGFR and KRAS mutations in patients with bronchioloalveolar carcinoma treated with erlotinib in a phase II multicenter trial. J Clin Oncol 23(16S) (abstract 7029) 36. Kris MG, Sandler A, Miller V et al (2004) Cigarette smoking history predicts sensitivity to erlotinib: results of a phase II trial in patients with bronchioloalveolar carcinoma (BAC). J Clin Oncol 22(14S) (abstract 7062) 37. Miller V, Patel J, Shah N et al (2003) The epidermal growth factor receptor tyrosine kinase inhibitor erlotinib (OSI-774) shows promising activity in patients with bronchioloalveolar cell carcinoma (BAC): preliminary results of a phase II trial. Proc Am Soc Clin Onc 22:619 (abstract 2491) 38. Paz-Ares L, Sanchez JM, Garcia-Velasco A et al (2006) A prospective phase II trial of erlotinib in advanced non-small cell lung cancer (NSCLC) patients (p) with mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor. J Clin Oncol 24(18S) (abstract 7020) 39. Herbst RS, Maddox AM, Rothenberg ML et  al (2002) Selective oral epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 is generally well-tolerated and has activity in non-smallcell lung cancer and other solid tumors: results of a phase I trial. J Clin Oncol 20(18):3815–3825 40. Fukuoka M, Yano S, Giaccone G et al (2003) Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial) [corrected]. J Clin Oncol 21(12):2237–2246 41. Kris MG, Natale RB, Herbst RS et al (2003) Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. JAMA 290(16):2149–2158 42. Cohen MH, Williams GA, Sridhara R et al (2004) United States Food and Drug Administration Drug Approval summary: Gefitinib (ZD1839; Iressa) tablets. Clin Cancer Res 10(4):1212–1218

222

P. Wheatley-Price and F.A. Shepherd

43. Thatcher N, Chang A, Parikh P et al (2005) Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet 366(9496):1527–1537 44. Chang A, Parikh P, Thongprasert S et al (2006) Gefitinib (IRESSA) in patients of Asian origin with refractory advanced non-small cell lung cancer: subset analysis from the ISEL study. J Thorac Oncol 1(8):847–855 45. Lee DH, Han JY, Lee HG et al (2005) Gefitinib as a first-line therapy of advanced or metastatic adenocarcinoma of the lung in never-smokers. Clin Cancer Res 11(8):3032–3037 46. Mok T, Wu Y-L, Thongprasert S et  al (2008) Phase III, randomised, open-label, first-line study of gefitinib vs carboplatin/paclitaxel in clinically selected patients with advanced nonsmall cell lung cancer. Ann Oncol 19(8) (abstract LBA2) nejm 361(10):947–957 47. Crino L, Cappuzzo F, Zatloukal P et al (2008) Gefitinib versus vinorelbine in chemotherapynaive elderly patients with advanced non-small-cell lung cancer (INVITE): a randomized, phase II study. J Clin Oncol 26(26):4253–4260 48. Goss G, Ferry D, Laurie S et al (2007) Randomized, double-blind, multicenter, parallel-group, Phase II study of gefitinib (IRESSA) plus best supportive care (BSC) versus placebo plus BSC in chemotherapy-naive patients with advanced non-small-cell lung cancer and poor performance status (INSTEP): B3-02. J Thorac Oncol 2(8S4):S340 49. Giaccone G, Herbst RS, Manegold C et al (2004) Gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer: a phase III trial–INTACT 1. J Clin Oncol 22(5):777–784 50. Herbst RS, Giaccone G, Schiller JH et al (2004) Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: a phase III trial – INTACT 2. J Clin Oncol 22(5):785–794 51. Hida T, Okamoto I, Kashii T et al (2008) Randomized phase III study of platinum-doublet chemotherapy followed by gefitinib versus continued platinum-doublet chemotherapy in patients (pts) with advanced non-small cell lung cancer (NSCLC): results of West Japan Thoracic Oncology Group trial (WJTOG 0203). J Clin Oncol 26(Suppl):LBA8012 52. Kelly K, Chansky K, Gaspar LE et al (2007) Updated analysis of SWOG 0023: a randomized phase III trial of gefitinib versus placebo maintenance after definitive chemoradiation followed by docetaxel in patients with locally advanced stage III non-small cell lung cancer. J Clin Oncol 25(18S) (abstract 7513) 26(15):2450–2456 53. Hanna N, Shepherd FA, Fossella FV et al (2004) Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy. J Clin Oncol 22(9):1589–1597 54. Shepherd FA, Dancey J, Ramlau R et  al (2000) Prospective randomized trial of docetaxel versus best supportive care in patients with non-small-cell lung cancer previously treated with platinum-based chemotherapy. J Clin Oncol 18(10):2095–2103 55. Cufer T, Vrdoljak E, Gaafar R, Erensoy I, Pemberton K (2006) Phase II, open-label, randomized study (SIGN) of single-agent gefitinib (IRESSA) or docetaxel as second-line therapy in patients with advanced (stage IIIb or IV) non-small-cell lung cancer. Anticancer Drugs 17(4):401–409 56. Douillard JY, Kim E, Hirsh V et al (2007) Gefitinib (IRESSA) versus docetaxel in patients with locally advanced or metastatic non-small-cell lung cancer pre-treated with platinumbased chemotherapy: a randomized, open-label Phase III study (INTEREST): PRS-02. J Thorac Oncol 2(8S4):S305–S306 57. Niho S, Ichinose Y, Tamura T et al (2007) Results of a randomized Phase III study to compare the overall survival of gefitinib (IRESSA) versus docetaxel in Japanese patients with non-small-cell lung cancer who failed one or two chemotherapy regimens. J Clin Oncol 25(18S):LBA7509 58. Erlichman C, Hidalgo M, Boni JP et  al (2006) Phase I study of EKB-569, an irreversible inhibitor of the epidermal growth factor receptor, in patients with advanced solid tumors. J Clin Oncol 24(15):2252–2260 59. Yoshimura N, Kudoh S, Kimura T et al (2006) EKB-569, a new irreversible epidermal growth factor receptor tyrosine kinase inhibitor, with clinical activity in patients with non-small cell lung cancer with acquired resistance to gefitinib. Lung Cancer 51(3):363–368

Epidermal Growth Factor Receptor Inhibitors in the Treatment

223

60. Greulich H, Chen TH, Feng W et al (2005) Oncogenic transformation by inhibitor-sensitive and -resistant EGFR mutants. PLoS Med 2(11):e313 61. Kobayashi S, Ji H, Yuza Y et al (2005) An alternative inhibitor overcomes resistance caused by a mutation of the epidermal growth factor receptor. Cancer Res 65(16):7096–7101 62. Kwak EL, Sordella R, Bell DW et al (2005) Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proc Natl Acad Sci USA 102(21):7665–7670 63. Shimamura T, Ji H, Minami Y et al (2006) Non-small-cell lung cancer and Ba/F3 transformed cells harboring the ERBB2 G776insV_G/C mutation are sensitive to the dual-specific epidermal growth factor receptor and ERBB2 inhibitor HKI-272. Cancer Res 66(13):6487–6491 64. Sequist LV, Bell DW, Lynch TJ, Haber DA (2007) Molecular predictors of response to epidermal growth factor receptor antagonists in non-small-cell lung cancer. J Clin Oncol 25(5):587–595 65. Agus DB, Terlizzi E, Stopfer P, Amelsberg A, Gordon MS (2006) A phase I dose escalation study of BIBW 2992, an irreversible dual EGFR/HER2 receptor tyrosine kinase inhibitor, in a continuous schedule in patients with advanced solid tumours. J Clin Oncol 24(18S) (abstract 2074) 66. Spicer J, Calvert H, Vidal L et  al (2007) Activity of BIBW2992, an oral irreversible dual EGFR/HER2 inhibitor, in non-small cell lung cancer (NSCLC) with mutated EGFR: D7-02. J Thorac Oncol 2(8S4):S410 67. Dumez H, Hoekstra R, Eskens F et al (2002) A phase I and pharmacological study of PKI166, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, administered orally 3 times a week to patients with advanced cancer. Proc Am Soc Clin Onc 21 (abstract 341) Clin Canc 6908–6915 68. Murren J, Papadimitrakopoulou VA, Sizer K, Vaidynathan S, Ravera C, Abbruzzese JL (2002) A phase I dose-escalating study to evaluate the biological activity and pharmacokinetics of PKI166, a novel tyrosine kinase inhibitor, in patients with advanced cancers. Proc Am Soc Clin Onc 21 (abstract 377) 69. Smylie M, Blumenschein G, Dowlati A et al (2007) A phase II multicenter trial comparing two schedules of lapatinib (LAP) as first or second line monotherapy in subjects with advanced or metastatic non-small cell lung cancer (NSCLC) with either bronchioloalveolar carcinoma (BAC) or no smoking history. J Clin Oncol 25(18S) (abstract 7611) 70. Calvo E, Tolcher AW, Hammond LA et al (2004) Administration of CI-1033, an irreversible pan-erbB tyrosine kinase inhibitor, is feasible on a 7-day on, 7-day off schedule: a phase I pharmacokinetic and food effect study. Clin Cancer Res 10(21):7112–7120 71. Nemunaitis J, Eiseman I, Cunningham C et  al (2005) Phase 1 clinical and pharmacokinetics evaluation of oral CI-1033 in patients with refractory cancer. Clin Cancer Res 11(10): 3846–3853 72. Zinner RG, Nemunaitis J, Eiseman I et al (2007) Phase I clinical and pharmacodynamic evaluation of oral CI-1033 in patients with refractory cancer. Clin Cancer Res 13(10):3006–3014 73. Simon GR, Garrett CR, Olson SC et al (2006) Increased bioavailability of intravenous versus oral CI-1033, a pan erbB tyrosine kinase inhibitor: results of a phase I pharmacokinetic study. Clin Cancer Res 12(15):4645–4651 74. Chiappori AA, Ellis PM, Hamm JT et al (2006) A phase I evaluation of oral CI-1033 in combination with paclitaxel and carboplatin as first-line chemotherapy in patients with advanced non-small cell lung cancer. J Thorac Oncol 1(9):1010–1019 75. Janne PA, von Pawel J, Cohen RB et  al (2007) Multicenter, randomized, phase II trial of CI-1033, an irreversible pan-ERBB inhibitor, for previously treated advanced non small-cell lung cancer. J Clin Oncol 25(25):3936–3944 76. Schellens JH, Britten CD, Camidge DR et al (2007) First-in-human study of the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of PF-00299804, a small molecule irreversible panHER inhibitor in patients with advanced cancer. J Clin Oncol 25(18S) (abstract 3599) 77. Natale R, Bodkin D, Govindan R et  al (2006) ZD6474 versus gefitinib in patients with advanced NSCLC: final results from a two-part, double-blind, randomized phase II trial. J Clin Oncol 24(18S) (abstract 7000) 27(15):2523–2529 78. Heymach JV, Johnson BE, Prager D et al (2006) A phase II trial of ZD6474 plus docetaxel in patients with previously treated NSCLC: follow-up results. J Clin Oncol 24(18S) (abstract 7016) 25(27):4270–4277

224

P. Wheatley-Price and F.A. Shepherd

79. Heymach JV, Paz-Ares L, De Braud F et al (2007) Randomized phase II study of vandetanib (VAN) alone or in combination with carboplatin and paclitaxel (CP) as first-line treatment for advanced non-small cell lung cancer (NSCLC). J Clin Oncol 25(18S) (abstract 7544) 26(33):5407–5415 80. Wakelee HA, Adjei AA, Halsey J et al (2006) A phase I dose-escalation and pharmacokinetic (PK) study of a novel spectrum selective kinase inhibitor, XL647, in patients with advanced solid malignancies (ASM). J Clin Oncol 24(18S) (abstract 3044) 81. Wakelee HA, Molina JR, Fehling JM, Lensing JL, Sikic BI (2007) A phase I study with exploratory pharmacodynamic endpoints of XL647, a novel spectrum selective kinase inhibitor, administered orally daily to patients (pts) with advanced solid malignancies. J Clin Oncol 25(18S) (abstract 14044) 82. Rizvi N, Kris MG, Miller V et al (2007) A Phase II study of XL647 in non-small cell lung cancer (NSCLC) patients enriched for presence of EGFR mutations. J Thorac Oncol 2(8S4):S737 83. Baselga J, Rojo F, Dumez H et  al (2005) Phase I study of AEE788, a novel multitargeted inhibitor of ErbB and VEGF receptor family tyrosine kinases: A pharmacokinetic (PK)pharmacodynamic (PD) study to identify the optimal therapeutic dose regimen. J Clin Oncol 23(16S) (abstract 3028) 84. Martinelli E, Takimoto C, Van Oosterom A et al (2005) AEE788, a novel multitargeted inhibitor of ErbB and VEGF receptor family tyrosine kinases: preliminary phase 1 results. J Clin Oncol 23(16S) (abstract 3039) 85. Goldstein NI, Prewett M, Zuklys K, Rockwell P, Mendelsohn J (1995) Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model. Clin Cancer Res 1(11):1311–1318 86. Robert F, Blumenschein G, Herbst RS et al (2005) Phase I/IIa study of cetuximab with gemcitabine plus carboplatin in patients with chemotherapy-naive advanced non-small-cell lung cancer. J Clin Oncol 23(36):9089–9096 87. Thienelt CD, Bunn PA Jr, Hanna N et al (2005) Multicenter phase I/II study of cetuximab with paclitaxel and carboplatin in untreated patients with stage IV non-small-cell lung cancer. J Clin Oncol 23(34):8786–8793 88. Butts C, Bodkin D, Middleman EL et al (2007) Gemcitabine/platinum alone or in combination with cetuximab as first-line treatment for advanced non-small cell lung cancer. J Clin Oncol 25(36):5777–5784 89. Rosell R, Daniel C, Ramlau R et al (2004) Randomized phase II study of cetuximab in combination with cisplatin (C) and vinorelbine (V) vs. CV alone in the first-line treatment of patients (pts) with epidermal growth factor receptor (EGFR)-expressing advanced non-small-cell lung cancer (NSCLC). J Clin Oncol 22(14S) (abstract 7012) Anuals of Oncology 19(2):362–369 90. Herbst RS, Chansky K, Kelly K et al (2007) A phase II randomized selection trial evaluating concurrent chemotherapy plus cetuximab or chemotherapy followed by cetuximab in patients with advanced non-small cell lung cancer (NSCLC): Final report of SWOG 0342. J Clin Oncol 25(18S) (abstract 7545) 91. Lynch TJ, Patel T, Dreisbach L et  al (2007) A randomized multicenter phase III study of cetuximab (Erbitux(R)) in combination with Taxane/Carboplatin versus Taxane/Carboplatin alone as first-line treatment for patients with advanced/metastatic non-small cell lung cancer (NSCLC): B3-03. J Thorac Oncol 2(8S4):S340 92. Pirker R, Szczesna A, von Pawel J et al (2008) FLEX: A randomized, multicenter, phase III study of cetuximab in combination with cisplatin/vinorelbine (CV) versus CV alone in the first-line treatment of patients with advanced non-small cell lung cancer (NSCLC). J Clin Oncol 26 (Suppl) (abstract 3) Clin Canc Res 13(20):6175–6181 93. Van Cutsem E, Peeters M, Siena S et al (2007) Open-label phase III trial of panitumumab plus best supportive care compared with best supportive care alone in patients with chemotherapyrefractory metastatic colorectal cancer. J Clin Oncol 25(13):1658–1664 94. Crawford J, Sandler AB, Hammond LA et al (2004) ABX-EGF in combination with paclitaxel and carboplatin for advanced non-small cell lung cancer. J Clin Oncol 22(14S) (abstract 7083)

Epidermal Growth Factor Receptor Inhibitors in the Treatment

225

95. Kollmannsberger C, Schittenhelm M, Honecker F et al (2006) A phase I study of the humanized monoclonal anti-epidermal growth factor receptor (EGFR) antibody EMD 72000 (matuzumab) in combination with paclitaxel in patients with EGFR-positive advanced nonsmall-cell lung cancer (NSCLC). Ann Oncol 17(6):1007–1013   96. Allan DG (2005) Nimotuzumab: evidence of clinical benefit without rash. Oncologist 10(9):760–761   97. Brade AM, Magalhaes J, Siu L et al (2007) A single agent, phase I pharmacodynamic study of nimotuzumab (TheraCIM-h-R3) in patients with advanced refractory solid tumors. J Clin Oncol 25(18S) (abstract 14030)   98. Crombet T, Osorio M, Cruz T et  al (2004) Use of the humanized anti-epidermal growth factor receptor monoclonal antibody h-R3 in combination with radiotherapy in the treatment of locally advanced head and neck cancer patients. J Clin Oncol 22(9):1646–1654   99. Johnson BE, Janne PA (2006) Rationale for a phase II trial of pertuzumab, a HER-2 dimerization inhibitor, in patients with non-small cell lung cancer. Clin Cancer Res 12(14 Pt 2): 4436s–4440s 100. Herbst RS, Davies A, Johnson B et al (2005) Efficacy and safety of single agent pertuzumab (rhuMAb 2C4), a HER dimerization inhibitor, in non-small cell lung cancer (NSCLC) patients after prior chemotherapy. Lung Cancer 49(S2) (abstract O-187) 101. Piccart-Gebhart MJ, Procter M, Leyland-Jones B et al (2005) Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 353(16):1659–1672 102. Romond EH, Perez EA, Bryant J et al (2005) Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 353(16):1673–1684 103. Slamon DJ, Leyland-Jones B, Shak S et al (2001) Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344(11):783–792 104. Gatzemeier U, Groth G, Butts C et al (2004) Randomized phase II trial of gemcitabine-cisplatin with or without trastuzumab in HER2-positive non-small-cell lung cancer. Ann Oncol 15(1):19–27 105. Shepherd FA, Rosell R (2007) Weighing tumor biology in treatment decisions for patients with non-small cell lung cancer. J Thorac Oncol 2(Suppl 2):S68–S76 106. Domine M, Gurdipe LA, Rosillo F et al (2007) Erlotinib as single agent in men with advanced or metastatic NSCLC: a retrospective analysis: P3-080. J Thorac Oncol 2(8S4):S712 107. Tsao MS, Sakurada A, Cutz JC et al (2005) Erlotinib in lung cancer – molecular and clinical predictors of outcome. N Engl J Med 353(2):133–144 108. Hirsch FR, Varella-Garcia M, Bunn PA Jr et al (2006) Molecular predictors of outcome with gefitinib in a phase III placebo-controlled study in advanced non-small-cell lung cancer. J Clin Oncol 24(31):5034–5042 109. Zhu CQ, da Cunha Santos G, Ding K et al (2008) Role of KRAS and EGFR as biomarkers of response to erlotinib in National Cancer Institute of Canada Clinical Trials Group Study BR.21. J Clin Oncol 26(26):4268–4275 110. Hirsch FR, Herbst RS, Olsen C et al (2008) Increased EGFR gene copy number detected by fluorescent in situ hybridization predicts outcome in non-small-cell lung cancer patients treated with cetuximab and chemotherapy. J Clin Oncol 26(20):3351–3357 111. Lynch TJ, Bell DW, Sordella R et al (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350(21):2129–2139 112. Paez JG, Janne PA, Lee JC et al (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304(5676):1497–1500 113. Sasaki H, Shimizu S, Endo K et al (2006) EGFR and erbB2 mutation status in Japanese lung cancer patients. Int J Cancer 118(1):180–184 114. Eberhard DA, Johnson BE, Amler LC et al (2005) Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-smallcell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol 23(25):5900–5909

226

P. Wheatley-Price and F.A. Shepherd

115. Bell DW, Lynch TJ, Haserlat SM et al (2005) Epidermal growth factor receptor mutations and gene amplification in non-small-cell lung cancer: molecular analysis of the IDEAL/ INTACT gefitinib trials. J Clin Oncol 23(31):8081–8092 116. Kobayashi S, Boggon TJ, Dayaram T et al (2005) EGFR mutation and resistance of nonsmall-cell lung cancer to gefitinib. N Engl J Med 352(8):786–792 117. Pao W, Miller VA, Politi KA et al (2005) Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2(3):e73 118. Balak MN, Gong Y, Riely GJ et al (2006) Novel D761Y and common secondary T790M mutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors. Clin Cancer Res 12(21):6494–6501 119. Aviel-Ronen S, Blackhall FH, Shepherd FA, Tsao MS (2006) K-ras mutations in non-smallcell lung carcinoma: a review. Clin Lung Cancer 8(1):30–38 120. Broermann P, Junker K, Brandt BH et al (2002) Trimodality treatment in stage III nonsmall cell lung carcinoma: prrognostic impact of K-ras mutations after neoadjuvant therapy. Cancer 94(7):2055–2062 121. Rosell R, Gomez-Codina J, Camps C et al (1994) A randomized trial comparing preoperative chemotherapy plus surgery with surgery alone in patients with non-small-cell lung cancer. N Engl J Med 330(3):153–158 122. Schiller JH, Adak S, Feins RH et al (2001) Lack of prognostic significance of p53 and K-ras mutations in primary resected non-small-cell lung cancer on E4592: a Laboratory Ancillary Study on an Eastern Cooperative Oncology Group Prospective Randomized Trial of Postoperative Adjuvant Therapy. J Clin Oncol 19(2):448–457 123. Winton T, Livingston R, Johnson D et al (2005) Vinorelbine plus cisplatin vs. observation in resected non-small-cell lung cancer. N Engl J Med 352(25):2589–2597 124. Han SW, Kim TY, Jeon YK et al (2006) Optimization of patient selection for gefitinib in non-small cell lung cancer by combined analysis of epidermal growth factor receptor mutation, K-ras mutation, and Akt phosphorylation. Clin Cancer Res 12(8):2538–2544 125. Pao W, Wang TY, Riely GJ et al (2005) KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med 2(1):e17 126. Van Cutsem E, Lang I, D’haens G et al (2008) KRAS status and efficacy in the first-line treatment of patients with metastatic colorectal cancer (mCRC) treated with FOLFIRI with or without cetuximab: the CRYSTAL experience. J Clin Oncol 26(Suppl) (abstract 2) 127. Shepherd FA, Ding K, Sakurada A et al (2007) Updated molecular analyses of exons 19 and 21 of the epidermal growth factor receptor (EGFR) gene and codons 12 and 13 of the KRAS gene in non-small cell lung cancer (NSCLC) patients treated with erlotinib in National Cancer Institute of Cancer. J Clin Oncol 25(18S) (abstract 7571) 128. Matar P, Rojo F, Cassia R et al (2004) Combined epidermal growth factor receptor targeting with the tyrosine kinase inhibitor gefitinib (ZD1839) and the monoclonal antibody cetuximab (IMCC225): superiority over single-agent receptor targeting. Clin Cancer Res 10(19):6487–6501 129. Von Pawel J, Park K, Pereira JR et al (2006) Phase III study comparing cisplatin/vinorelbine plus cetuximab versus cisplatin/vinorelbine as first-line treatment for patients with epidermal growth factor (EGFR)-expressing advanced non-small cell lung cancer (NSCLC) (FLEX). J Clin Oncol 24(18S) (abstract 7109)

Angiogenesis Inhibitors in Lung Cancer Leora Horn and Alan Sandler

Abstract  Lung cancer is the leading cause of cancer-related mortality in the United States with non-small cell lung cancer (NSCLC) accounting for 80–85% of cases. Regardless of histology, the majority of patients present with advanced disease and have a median survival of approximately 10 months for patients treated with traditional chemotherapy agents. Therefore, novel therapies are required. ECOG4599 was the first trial to demonstrate survival beyond 1 year for patients with NSCLC treated with traditional chemotherapy in combination with an anti-angiogenic agent. Angiogenesis, the growth of new from preexisting vessels, is a fundamental step in the transition of tumors from dormant to malignant. In the last few years, there has been substantial interest in developing novel moleculartargeted agents that inhibit the angiogenic pathway. The aim of this chapter is to summarize the data for agents that have been approved and are in development for the treatment of patients with NSCLC and small cell lung cancer (SCLC). Keywords  Angiogenesis • Bevacizumab • Small molecule inhibitors

Introduction Lung cancer is the leading cause of cancer-related mortality in the United States with 213 380 new cases and 160 390 deaths anticipated in 2008 (1). Non-small cell lung cancer (NSCLC) accounts for 80–85% of cases with approximately 70% of patients presenting with advanced disease (stage IIIB with pleural effusion or stage IV) at the time of diagnosis. Patients with advanced disease have a median survival

L. Horn (*) and A. Sandler Vanderbilt Ingram Cancer Center, Nashville, TN, USA e-mail: [email protected] D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_11, © Humana Press, a part of Springer Science+Business Media, LLC 2010

227

228

L. Horn and A. Sandler

(OS) of approximately 10 months when treated with traditional platinum-based therapy (2). Small cell lung cancer (SCLC) is a highly aggressive disease characterized by its rapid doubling time, high growth fraction, early development of disseminated disease, and dramatic response to first-line chemotherapy and radiation (3). SCLC has a distinct staging system; patients are categorized as limited-stage (LD), defined as disease that is confined to the ipsilateral hemithorax that can be encompassed within a tolerable radiation port, or extensive-stage (ED), defined as the presence of overt metastatic disease by imaging or physical examination (4). Sixty to seventy percent of patients are diagnosed with extensive disease at presentation (5). Despite response rates (RR) to first-line therapy as high as up to 80%, the median survival (OS) ranges from 12 to 20 months for patients with LD and 7 to 11 months for patients with ED (6). Therefore, novel treatments are required for these all too common diseases. As our knowledge of cell biology improves, we are able to identify therapies that more precisely affect the target of interest allowing for a more rational approach to clinical trial design. In this chapter, we discuss the results of recent clinical trials that have evaluated anti-angiogenic agents alone and in combination with standard chemotherapy in the treatment of patients with SCLC and NSCLC. Please refer to Tables 1–3 for complete details on drug regimens, including doses and toxicities.

Role of Angiogenesis in Tumor Development Tumor growth and progression is a complex multi-step process requiring the transformation of a normal cell into a malignant cell through the accumulation of molecular alterations that produce cell populations with augmented growth and invasive potential. In the last two decades, there has been substantial interest in developing molecular-targeted agents that modulate growth factors and signaling cascades that are aberrant in tumor cells. Small tumors, <2  mm in diameter, are dormant receiving oxygen and nutrients via diffusion (7). Tumor growth and progression requires that the tumor develops an independent capillary network. This occurs through two distinct processes – vasculogenesis and angiogenesis (8). During vasculogenesis, endothelial precursor cells (angioblasts) migrate and differentiate in response to stimuli – such as growth factors – to form new blood vessels. These burgeoning vascular trees are then remodeled and extended through angiogenesis. In contrast to vasculogenesis, angiogenesis is the growth of new vessels from preexisting vessels. Angiogenesis is regulated via a dynamic balance of pro- and anti-angiogenic molecules. Tumor progression relies on tipping the balance in favor of molecules that drive angiogenesis. To achieve this, tumors undergo an angiogenic switch, creating an imbalance between pro- and anti-angiogenic factors (9, 10). The secretion of pro-angiogenic factors, during tumor growth and progression, results in cell migration, proliferation, and capillary tube formation (11, 12). Angiogenesis has also been implicated in the growth of distant metastases (13–15).

Table  1  Summary of toxicity profiles of angiogenic agents alone and in combination with chemotherapy Toxicity Drug Alone With chemotherapy Hypertension Neutropenia Bevacizumab alone and in combination with Proteinuria Febrile neutropenia chemotherapy Venous or arterial thrombosis Headache Bleedinga Hyponatremia Diverticulitis Tumor hemorrhage Gastrointestinal perforation Complications with healing Reversible posterior Fatigue leukoencephalopathy syndrome Sorafenib Hand foot syndromeb Dyspnea Elevated ALT Hypertension Neutropenia Fatigue Thrombocytopenia Diarrhea Infection Nausea Thrombosis Bleeding Cardiac ischemia Neutropenia Vandetanib Rash Bleeding Diarrhea Hypertension Asymptomatic QTc prolongation Nausea Headache Dizziness Sunitinib Hypertension Hyperpigmentation and skin discoloration Hypothyroidism Diarrhea Fatigue/asthenia Pain/myalgia Nausea and vomiting Dyspnea Stomatitis/mucosal inflammation Hemorrhage Hair depigmentation Thalidomide Thrombosis Neuropathy Neutropenia Fatigue Nausea Cediranib Diarrhea Rash Dysphonia Abdominal pain Anorexia Hypertension Myalgias Fatigue Neutropenia Hemoptysis Pulmonary emboli Neutropenia (continued)

230

L. Horn and A. Sandler

Table 1  (conitnued) Drug

Toxicity Alone

With chemotherapy

Motesanib

Hypertension Lethargy Fatigue Hypertension Diarrhea Thrombosisc Nausea and vomiting Headache Cholecystitis and gall bladder enlargement Axitinibd Hypertension Fatigue Nausea and vomiting Diarrhea Headache Stomatitis Erythema Hyponatremia Pazopanib Nausea and vomiting Diarrhea Fatigue Hypertension Anorexia Hair depigmentation observed at higher doses BIBF 1120 Nausea Vomiting Diarrhea Elevated liver enzymes Additional toxicities when combined with Fatigue chemotherapy Anorexia Gastrointestinal disorders XL647 Diarrhea Fatigue Rash QTc prolongation Dysgeusia Note: All toxicities are reported to occur in >10% of patients, rare events not mentioned a   Associated with squamous cell histology, tumor cavitation, central tumors, and disease location close to major blood vessels (48) b  Characterized by desquamation of skin on hands and feet, blisters, and parasthesias c  This rate was found to be excessive when motesanib was combined with panitumumab d  Complete toxicity profile is not known

Angiogenesis in tumors is aberrant, and tumor blood vessels have multiple structural and functional abnormalities. Anti-angiogenic agents in addition to inhibiting the growth of tumor vasculature are thought to transiently normalize the tumor vascular supply resulting in improved drug delivery (16, 17).

16.7   8.6   8.6 12.6 13.7

  58   48   41   40   39

II II II

II II II II

I I/II

III

II

Waples(143) Aadjei(62) Herbst(58)

Sorafenib Gutierrez(84) Gatzemeir(82) Aadjei(86) Schiller

Adjei(88) Schiller(89)

Scagliotti(90)

Vandetanib Nakagawa(94)

Vandetanib 100 mg/200 mg/300 mg

Sorafenib 400 mg Sorafenib 400 mg Sorafenib 400 mg  Sorafenib 400 mg Placebo Gefitinib 250 mg + Sorafenib (200–400 mg) Carboplatin AUC 6 + Paclitaxel 225 mg/m2 day 1 + Sorafenib 400 mg twice daily days 2–18 every 3 weeks Carboplatin AUC 6 + Paclitaxel 225 mg/m2 day 1 every 3 weeksCarboplatin AUC 6 + Paclitaxel 225 mg/m2 day 1 + Sorafenib 400 mg twice daily

53

462 464

  15   54   25   51   32   31   15

NR

  22

Oxaliplatin 130 mg/m2 day 1 + Gemcitabine 1,000 mg/m2 days 1 and 8 + Bevacizumab 15 mg/kg day 1 Oxaliplatin 120 mg/m2 + Pemetrexed 500 mg/m2 + bevacizumab 15 mg/kg day 1 Pemetrexed 500 mg/m2+ Bevacizumab 15 mg/kg Docetaxel 75 mg/m2 or Pemetrexed 500 mg/m2 Docetaxel 75 mg/m2 or Pemetrexed 500 mg/m2+ bevacizumab 15 mg/kg Erlotinib 150 mg + Bevacizumab 15 mg/kg

II

Davila(75)

NR

10.7 10.6

NR   7.0   8.8 11.9   9.0 NR NR

14.9 11.6 17.7 10.3 12.3 NR NR NR

13 (continued)

24 30

 3 29

13 29 12 23

26 10 12 13 18

31

19 28 35 35 15 20 34 30

OS (months) RR(%)

  32   35   32 427 440 347 345 351

Pt No:

Table 2  Summary of clinical trials investigating anti-angiogenic therapy in patients with NSCLC Trial Phase Treatment Bevacizumab Johnson(48) II Carboplatin AUC 6+Paclitaxel 200 mg/m2 every 3 weeks Carboplatin AUC 6+Paclitaxel 200 mg/m2 + Bevacizumab 7.5 mg/kg Carboplatin AUC 6 + Paclitaxel 200 mg/m2 + Bevacizumab 15 mg/kg Sandler(49) III Carboplatin AUC 6 + Paclitaxel 200 mg/m2 Carboplatin AUC 6 + Paclitaxel 200 mg/m2 + Bevacizumab 15 mg/kg Manegold(51) III Cisplatin 80 mg/m2 day 1 + Gemcitabine 1,250 mg/m2 days1 and 8 every 3 weeks Cisplatin 80 mg/m2 day 1 + Gemcitabine 1,250 mg/m2 + Bevacizumab 7.5 mg/kg Cisplatin 80 mg/m2 day 1 + Gemcitabine 1,250 mg/m2 + Bevacizumab 15 mg/kg

Angiogenesis Inhibitors in Lung Cancer 231

Phase

II

II

II

II II I

II

III

II

II

I

II II II

Trial

Natale(95)

Heymach(96)

Heymach(98)

Sunitinib Socinski(108) Brahmer(109) Reck(110)

Thalidomide Miller(113)

Lee(114)

Flora(115)

Seidler(116) Other agents Laurie(125)

Schiller(133) Miller(138) Rizvi(139)

Table 2  (continued)

Carboplatin AUC 6 + Paclitaxel 200 mg/m2 day 1 every 3 weeks + Cediranib 30 or 45 mg daily Axitinib 5 mg XL647 XL647

Carboplatin AUC 5 day 1+ Irinotecan 50 mg/m2 days 1 and 8 + Thalidomide 200 mg/day – 1,000 mg/day Carboplatin AUC 5 day 1 + Gemcitabine 1,200 mg/m2 days 1 and 8 every 3 weeks Carboplatin AUC 5 day 1 + Gemcitabine 1,200 mg/m2 days 1 and 8 every 3 weeks + thalidomide 100 mg/day during chemotherapy then 200 mg/day Irinotecan 100 mg/m2 + Gemcitabine 1,000 mg/m2 days 1 and 8 + thalidomide 200–400  mg/day Docetaxel 75 mg/m2 + thalidomide 200 mg/day

Sunitinib 50 mg daily § Sunitinib 37.5 mg daily Cisplatin 80 mg/m2 day 1 + gemcitabine 1,000 or 1,250 mg/m2 days 1 and 8 every 3 weeks + Sunitinib 37.5 mg or 50 mg daily

Docetaxel 75 mg/m2 Docetaxel 75 mg/m2 + Vandetanib 100 mg Docetaxel 75 mg/m2 + Vandetanib 300 mg Carboplatin AUC 6 + Paclitaxel 225 mg/m2 every 3 weeks Carboplatin AUC 6 + Paclitaxel 225 mg/m2 + vandetanib 300 mg daily Vandetanib 300 mg daily

Vandetanib 300 mg dailyGefitinib 250 mg daily

Treatment

32 23 41

12.8 NR NR

NR

5.4

26 20

NR

8.9 8.4

350 372 11

7.3

5.4 8.6 NR

6.1 7.4 13.4 13.1 7.9 12.6 10.2 10.2

9 4 28

45

20

13

42 40

22

11 2 23

12 26 18 25 32 7

81

OS (months) RR(%)

36

63 47 13

83 85 41 44 42 52 56 73

Pt No:

232 L. Horn and A. Sandler

Angiogenesis Inhibitors in Lung Cancer

233

Table 3  Summary of clinical trials investigating anti-angiogenic therapy in patients with SCLC OS Trial Phase Treatment Pt No: (months) RR(%) 64 11.1 80% Sandler (64) II Cisplatin 60 mg/m2 day 1 + Etoposide 120 mg/m2 days 1–3 + bevacizumab 15 mg/kg every 3 weeks 72 11.7 75% Ready (65) II Cisplatin 30 mg/m2 + Irinotecan 85 mg/m2 days 1 and 8 +  bevacizumab 15 mg/kg very 3 weeks 57 15 80% Patton(66) II Carboplatin (AUC 5) day 2 + Irinotecan 50 mg/m2 days 1 and 8 every 3 weeks + TRT (61.2 Gy) → bevacizumab 15 mg/kg every 3 weeks Ramalingam (127) II Cediranib 45 mg daily 25   1.9 4% Arnold (105) II Placebo 54 11.6 NR Vandetanib 300 mg daily 53 11.9 Gitlitz (91) II Sorafenib 400 mg twice daily 81 7/5a 4% Lee (118) II Carboplatin (AUC 5) + Etoposide 26 10.2 65% 120 mg/m2 IV day 1 then 100 mg/m2 po days 2–3 + thalidomide 100 mg daily Cooney (119) II Thalidomide 200 mg daily 22 15.2 NR 259 10.5 NR Lee (120) II Carboplatin (AUC 5) + Etoposide 10.1 NR 120 mg/m2 IV day 1 then 100 mg/m2 po days 2–3 365 Carboplatin (AUC 5) + Etoposide 120 mg/m2 IV day 1 then 100 mg/m2 po days 2–3 + thalidomide 100 mg daily Pujol(121) III 43   8.7 NR 4¢-epidoxorubicin 40 mg/m2 day 49 11.7 1 + cisplatin 100 mg/m2 day 2 + cyclophosphamide 400 mg/m2 day 1–3 4¢-epidoxorubicin 40 mg/m2 day 1 + cisplatin 100 mg/m2 day 2 + cyclophosphamide 400 mg/m2 day 1–3 + thalidomide 100 mg daily a Platinum sensitive/platinum resistant

The Role of Vascular Endothelial Growth Factor (VEGF) in Angiogenesis In the last decade, there has been significant interest in developing agents that target the VEGF family of growth factors and the receptor tyrosine kinases that mediate pro-angiogenic effects (18). VEGF (also referred to as VEGF-A or vascular permeability

234

L. Horn and A. Sandler

factor (19)) belongs to a family of structurally related proteins including VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF) (20). VEGF binds to VEGF receptor-1 (VEGFR-1 or Flt-1) and –2 (VEGFR-2 or KDR/Flk-1), but VEGFR-2 is believed to mediate almost all known cellular response to VEGF (20). VEGFR-2 is expressed predominantly on vascular endothelial cells (21). VEGFR-2 consists of seven extracellular immunoglobulin (Ig)-like domains, a transmembrane region, and an intracellular domain with tyrosine kinase activity (22, 23). VEGF binds to VEGFR-2 resulting in receptor homodimerization that is essential for intracellular signaling. The function of VEGFR-1 is less well defined, although it is thought to modify VEGFR-2 signaling (20). A third receptor (VEGF receptor-3) has been discovered that mediates lymphangiogenesis in response to VEGF-C and VEGF-D binding (20, 24). VEGF is expressed by many solid tumors, including melanoma (25), gastrointestinal (26), breast (27), central nervous system (28), ovarian (29), cervical (30), lung (24), hepatic (31), head and neck (32), and kidney (33) carcinomas. VEGF expression was also found to be elevated in bronchial dysplasia and invasive lung carcinomas compared to normal lung tissue (34). Several studies have demonstrated a significant association between VEGF expression and overall survival in patients with NSCLC; (35–38) prognosis for patients with VEGF-positive tumors has consistently been shown to be significantly worse than that for patients with VEGF-negative tumors (36, 38). VEGF exists in several different isoforms that result from alternative patterns of splicing VEGF mRNA (20). A study evaluating the correlation between the expression of different VEGF mRNA isoforms found that expression of only certain VEGF isoforms may be prognostic indicators for NSCLC (39). Analysis of microvessel density (MVD) can provide an indirect measure of angiogenesis. VEGF levels have been shown to correlate with MVD and the latter has been found to increase with tumor stage (35, 36, 38, 39). MVD has also been shown to be a significant predictor of increased risk of metastatic disease and worse overall survival in patients with NSCLC (40, 41). The median MVD in small cell lung cancer (SCLC) is higher than seen in NSCLC. Elevated MVD and VEGF expression are also associated with worse prognosis in SCLC (42).

Anti-angiogenic Approaches Inhibiting the biological actions of VEGF reduces tumor vascularization thereby inhibiting tumor growth (17). A nonspecific tyrosine kinase inhibitor of the VEGF receptor prevented migration of endothelial cells, blocked capillary-like tubule formation, and prevented tumor blood vessel formation (43). Inhibition also prevented the formation of lung metastases and slowed progression of tumor growth (43). Importantly, VEGF receptor inhibition had minimal effects on established blood vessels or blood flow. Direct blockade of VEGFR-2 leads to vessel regression and normalization as well as stromal maturation, resulting in reversion to a noninvasive tumor phenotype (44). This suggests an essential role for the stromal

Angiogenesis Inhibitors in Lung Cancer

235

microenvironment in regulating tumor phenotype, and thus continued VEGF inhibition may be crucial for prolonged tumor suppression, which in the clinical setting may translate to prolonged progression free survival (44–46). Therefore, VEGF-regulated angiogenesis has become a rationale therapeutic target given the important role this pathway plays in tumor growth and development. The majority of anti-angiogenic approaches to date have focused on the inhibition of key pro-angiogenic factors. Because of their nonoverlapping toxicity with chemotherapy agents, anti-angiogenic agents have been combined with a variety of chemotherapy agents currently used in the treatment of patients with lung cancer. Of these, the most promising approaches are monoclonal antibodies directed at VEGF ligand and small molecule tyrosine kinase inhibitors that block VEGF receptor(s).

Anti-vascular Endothelial Growth Factor Monoclonal Antibodies Vascular endothelial growth factor (VEGF) is an important factor mediating proangiogenic effects. Many human cancer cells, including lung cancer cells, express VEGF receptors (VEGFR) (36, 38). VEGF expression has been found to significantly correlate with new vessel formation, disease-free survival (DFS), and overall survival (OS) in patients with NSCLC (35–37). Bevacizumab, developed from the murine anti-human antibody A4.6.1, is a monoclonal antibody with a high affinity for VEGF (Table 4) (9, 47). A4.6.1 was shown to potently suppress neovascularization and tumor growth and was humanized by site directed mutagenesis to facilitate therapeutic use. Bevacizumab is a recombinant humanized antibody that exerts its anti-angiogenic effects by binding to free, circulating VEGF with similar affinity to that of the original murine antibody. Bevacizumab binds to VEGF thereby inhibiting the binding of VEGF to its receptors, preventing VEGF ligand–receptor downstream signaling (47). Bevacizumab in Patients with Non-small Cell Lung Cancer A randomized phase II trial of 99 patients with advanced or recurrent NSCLC compared carboplatin and paclitaxel with or without bevacizumab (7.5 or 15 mg/ kg) and found the combination of bevacizumab 15  mg/kg with carboplatin and paclitaxel increased response rate (RR) (31.5% vs. 18.8%) and prolonged time to progression (7.4 vs. 4.2 months; p = 0.023) compared to chemotherapy alone. There was also a nonsignificant improvement in OS, 17.7 months vs. 14.9 months. Although there were no objective responses, 1-year survival was 47% for patients (n = 19) who progressed and went on to receive single-agent bevacizumab 15 mg/kg. A higher incidence of bleeding was noted in the bevacizumab-treated patients. Severe pulmonary hemorrhage, which was observed in six patients (9.1%) and led to four fatalities, was associated with squamous cell histology, tumor cavitation,

236

L. Horn and A. Sandler

Table  4  Anti-angiogenic agents approved or in development for the treatment of patients with NSCLC Route of Frequency of Drug Target administration administration Clinical status Bevacizumab VEGF ligand IV Every 3 weeks Approved Sorafenib Raf, Kit, Flt-3, VEGFR-2, PO Twice daily Phase III VEGFR-3, PDGFR-b Vandetanib VEGFR-2, VEGFR-3, RET, PO Daily Phase III EGFR PO Twice daily Phase III Sunitinib VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-a, PDGFR-b, Flt-3, c-kit Cediranib VEGFR-2, VEGFR-1, PO Daily Phase III VEGFR-3, c-kit, Flt-3 PO Daily Phase III Motesanib VEGFR-1, VEGFR-2, VEGFR-3, PDGFR, Ret, kit Thalidomide Unknown PO Daily Approved BIBF 1120 VEGFR, PDGFR and FGFR,PO Daily Phase II Src, Lck, Lyn Axitinib VEGFR-1, VEGFR-2, PO Twice daily Phase II VEGFR-3, PDGFR-b, kit PO Daily Phase II Pazopanib VEGFR-2, VEGFR-2, VEGFR-3, PDGFR-a, PDGFR-b, c-kit Daily/twice Phase II XL647 VEGFR-2, EGFR, erbB2, PO EphB4 daily

central tumors, and disease location close to major blood vessels (48). Based on these results, ECOG conducted a phase III trial (E4599) comparing carboplatin and paclitaxel with or without bevacizumab (15 mg/kg) in 878 patients with recurrent or advanced nonsquamous NSCLC. There was a significant improvement in RR, OS, and progression-free survival (PFS) for patients treated with bevacizumab plus chemotherapy compared to chemotherapy alone, 35% vs. 15% (p < 0.001), 12.3 vs. 10.3 months (HR 0.79; p = 0.003), and 6.2 vs. 4.5 months (HR, 0.66; p < 0.001), respectively. In an unspecified subset analysis, the benefit of bevacizumab plus chemotherapy on OS was evident among men (11.1 vs. 8.7 months) but not women (13.3 vs. 13.1 months), while RR and PFS were significantly improved for both genders. There was a significantly higher incidence of toxicities for bevacizumabtreated patients, although the incidence of grade 3–5 pulmonary hemorrhage was only 2.1% vs. 0.5% in the chemotherapy alone arm (49). An unspecified retrospective subset analysis of elderly patients (³70 years of age) found a trend toward higher RR and PFS, but no improvement in OS for paclitaxel and carboplatin plus bevacizumab compared to paclitaxel and carboplatin alone (50). A second phase III randomized trial compared cisplatin and gemcitabine with or without bevacizumab 7.5 mg/kg or 15 mg/kg in 1,043 patients with recurrent or

Angiogenesis Inhibitors in Lung Cancer

237

advanced nonsquamous NSCLC (51). Following the positive survival results of the ECOG trial, the primary endpoint of AVAiL was amended from OS to PFS. A significant improvement in PFS was seen at both bevacizumab doses, 6.7 months for 7.5  mg/kg (HR 0.75; p = 0.003) and 6.5 months for 15  mg/kg (HR 0.82; p = 0.03), compared to 6.1 months for the placebo group. However, OS was not significantly different between treatment groups but has not been formally presented. Objective response rates were 34.1%, 30.4%, and 20.1% for bevacizumab 7.5 mg/kg, 15 mg/kg, and placebo, respectively. Although the trial was not powered to directly compare the two doses of bevacizumab, efficacy and safety data were similar for both doses. The difference in survival between E4599 and AVAiL may be explained by a recent publication that found paclitaxel induces circulating endothelial progenitor cells (CEPs) while gemcitabine does not, and the addition of an anti-VEGFR-2 antibody acts synergistically only in combination with CEPmobilizing chemotherapy agents (52). Therefore, it may be that the combination of anti-angiogenic agents and chemotherapy is only beneficial in combination with certain chemotherapy treatments. The VEGF and EGFR pathways share common downstream signaling pathways (53). Inhibition of VEGF is believed to contribute to the mechanism of action of agents targeting EGFR (54). Increased VEGF activity is a mechanism via which tumors develop resistance to EGFR inhibitors (55). Dual EGFR and VEGF inhibition has been shown to have additive effects and overcome resistance to EGFR inhibition (53). A phase I/II trial of 40 patients with advanced, nonsquamous NSCLC who had failed at least one prior chemotherapy regimen demonstrated a 20% RR, 6.2 months PFS, 12.6 months OS, and no grade 3/4 toxicities when bevacizumab (15 mg/kg) was combined with erlotinib (56). Retrospective proteomic profiling of 37 patients enrolled in this study identified protein/peptide expression patterns that appear to discriminate between responders and nonresponders to treatment. Prospective studies are planned to validate these results (57). The efficacy of this regimen led to a randomized phase II trial in previously treated NSCLC patients comparing chemotherapy alone (docetaxel or pemetrexed) to chemotherapy plus bevacizumab or bevacizumab plus erlotinib. The RR was highest for the erlotinib plus bevacizumab combination 17.9% vs. 12.2% for chemotherapy alone and 12.5% for chemotherapy plus bevacizumab. The median PFS, OS, and 1-year survival were superior for patients receiving bevacizumab (3.0 vs. 4.8 vs. 4.4 months and 8.6 vs. 12.6 vs. 13.7 months and 33% vs. 53.8% vs. 57.4% for chemotherapy vs. chemotherapy plus bevacizumab vs. erlotinib plus bevacizumab, respectively). Grade 3/4 toxicities were greater in the chemotherapy treatment arms, with a higher percentage of patients discontinuing treatment due to adverse events (24% for chemotherapy alone vs. 28% for chemotherapy plus bevacizumab and 13% for erlotinib plus bevacizumab) (58). Based on the results of this study, two pharmaceutical-sponsored phase III trials were opened for previously treated NSCLC patients; the BeTa Lung trial is comparing treatment with erlotinib to erlotinib plus bevacizumab and the ATLAS trial, that is now closed to patient accrual, is evaluating the efficacy and safety of maintenance bevacizumab with or without erlotinib following four cycles of chemotherapy with bevacizumab.

238

L. Horn and A. Sandler

Several large trials have reported on the safety of bevacizumab in combination with chemotherapy in patients with advanced NSCLC. These trials have included patients with brain metastases, hypertension, and on anticoagulation. The data to date suggest that it may be safe to administer bevacizumab to NSCLC patients with treated brain metastases, that there is less of a safety concern for patients on anticoagulation than previously believed, and that hypertension can be managed. However, we await the final results of these trials before treatment recommendations can be made (59–63). Bevacizumab in Patients with Small Cell Lung Cancer Three phase II trials have evaluated bevacizumab in combination with chemotherapy in patients with ED-SCLC. The first study, conducted by ECOG (E3501) enrolled 64 patients with newly diagnosed ED-SCLC. Bevacizumab (15 mg/kg) was combined with standard first-line therapy of cisplatin and etoposide. Patients without disease progression continued on single-agent bevacizumab until disease progression or unacceptable toxicity. This study met its primary endpoint of increased PFS at 6 months from 16 to 33%. A median of 6 cycles of bevacizumab treatment was administered. RR was 80%, PFS was 4.7 months, 34.8% of patients were progressionfree at 6 months, and OS was 11.1 months (64). However, ECOG has elected not to evaluate this regimen any further. Genentech is sponsoring a larger randomized phase II trial comparing the combination of cisplatin and etoposide with or without bevacizumab in ED-SCLC. CALGB 30306 enrolled 72 patients from the same population and employed bevacizumab plus cisplatin and irinotecan (65). Although RR and PFS were higher (at 75% and 7.1 months, respectively) with this combination than in the etoposide– cisplatin plus bevacizumab study, OS was similar at 11.7 months (64, 65). A phase II trial evaluated the role of maintenance bevacizumab following combined modality treatment in patients with LD-SCLC. In this trial, 57 patients with LD-SCLC received 61.2 Gy of radiation therapy plus chemotherapy with carboplatin and irinotecan. Those patients without progressive disease went on to receive additional treatment with bevacizumab every 2 weeks for 10 doses. Although the 1- and 2-year PFS rates were slightly higher (63 and 54%), the response rate of 80%, and the 1- and 2-year OS rates of 71 and 29%, respectively are similar to what is seen with traditional chemotherapy with cisplatin/etoposide and radiation alone (66, 67). Therefore, bevacizumab is the first anti-angiogenic agent approved for the treatment of patients with advanced NSCLC. Two large randomized phase III studies have shown an improvement in PFS when bevacizumab was combined with chemotherapy compared to chemotherapy alone. The ECOG 4599 trial showed the addition of bevacizumab to standard chemotherapy – carboplatin and paclitaxel – also results in an improvement in OS compared to chemotherapy alone. A subset analysis suggests there may be limited benefit in elderly patients. A multitude of phase II trials combining bevacizumab with platinum-based therapy in chemotherapy-naïve patients with

Angiogenesis Inhibitors in Lung Cancer

239

advanced NSCLC are currently ongoing (68–76). There are currently no data to support a role for bevacizumab as adjuvant therapy in patients with NSCLC outside of a clinical trial; ECOG is conducting a randomized phase III trial (E1505) of platinum-based chemotherapy with or without bevacizumab in patients with completely resected NSCLC. There are currently no data to support the use of bevacizumab in patients with SCLC.

Small Molecule Receptor Tyrosine Kinase Inhibitors Sorafenib in Patients with Non-small Cell Lung Cancer Sorafenib (Bay 43-9006; Nexavar®) is an oral multi-kinase inhibitor (Table 4). It directly targets tumor proliferation via inhibition of Raf, stem cell factor receptor (c-kit), and fms-like tyrosine kinase-3 (Flt-3), and also inhibits angiogenesis by targeting VEGFR-2 and VEGFR-3 and platelet-derived growth factor beta (PDGFR-b) (77–79). In preclinical models, sorafenib has been found to inhibit tumor growth, including NSCLC cells, when administered alone and/or in combination with vinorelbine, cisplatin, and gefitinib (80, 81). A phase II trial treated 54 patients with relapsed or refractory NSCLC with single-agent sorafenib. Patients with squamous cell histology and asymptomatic brain metastases were included and those with significant bleeding in the prior month were excluded. Median PFS was 11.9 weeks and OS was 29.3 weeks. Although there were no confirmed PRS, tumor shrinkage was observed in 29% of patients, while 59% of patients had SD (82). Preliminary results from a second study involving 15 patients with relapsed NSCLC treated with sorafenib reported 2 PR (13%) and 7 patients with SD (46%). Patients were evaluated by DCE-MRI, which showed a reduction in tumor permeability associated with reductions in tumor size (83, 84). Accrual to this trial is ongoing. ECOG conducted a phase II randomized discontinuation trial (E2501) of single-agent sorafenib in 342 patients with NSCLC who had progressed following two or more chemotherapy regimens. In this trial, all patients were initially treated with sorafenib. Patients were evaluated after two cycles and those who were responding continued on treatment with sorafenib, those with SD were randomized to sorafenib or placebo and those with progression were taken off study. One hundred and seven patients with SD were randomized and 83 were evaluable for response. Eight of 30 patients on placebo crossed over to treatment with sorafenib. An error in randomization was discovered by ECOG after 55 patients had been randomized. This was thought not to affect the primary endpoint of the study – the proportion of patients with disease control 2 months after randomization. There were a significantly higher number of patients with disease control (SD/PR/CR) at two months in the sorafenib treatment group compared to placebo (47% vs. 19%, P = 0.01). Median PFS was also significantly longer (3.6 vs. 2.0 months, P = 0.018) and there was a trend toward improvement in OS (11.9 months vs. 9.0 months) (85).

240

L. Horn and A. Sandler

In a first-line study, 25 patients with advanced NSCLC were treated with sorafenib prior to receiving standard chemotherapy. Although the study did not meet stage I efficacy criteria (only one confirmed PR in the first 20 patients), the authors concluded that the median survival of 8.8 months and objective response rate of 12% suggest single-agent sorafenib achieves similar activity compared to two-drug combinations and should be considered for combination studies with standard chemotherapy regimens (86). One study documented that single-agent sorafenib does not appear to adversely affect health-related quality of life in patients with advanced NSCLC (87). In a phase I dose escalation trial, 31 patients, 12 with locally advanced or recurrent NSCLC, were treated with sorafenib (200 or 400  mg) plus gefitinib. An additional 20 patients were treated with single-agent sorafenib vs. single-agent gefitinib followed by the combination of sorafenib and gefitinib. There was 1 PR and 20 patients with SD of ³4 months duration (88). In a phase I/II trial, carboplatin and paclitaxel were combined with sorafenib in patients with advanced NSCLC. An encouraging median PFS of 8.5 months was achieved, 29% of patients had a PR and 50% had SD (89). However, a randomized phase III trial (ESCAPE) of 926 chemotherapy-naïve patients with advanced NSCLC comparing carboplatin and paclitaxel with or without sorafenib was closed early after a planned interim analysis showed no difference in RR (30% vs. 24%), PFS (5.1 vs. 5.4 months) (HR 1.0, P = 0.514), or OS (10.7 vs. 10.6 months) (HR 1.16, P = 0.930) for patients receiving chemotherapy with sorafenib compared to chemotherapy alone. In subset analysis, patients with squamous cell histology appeared to have a worse OS when treated with the combination of chemotherapy and sorafenib compared to chemotherapy alone (8.9 months vs. 13.6 months, HR 1.81). This was true for PFS as well (90). A second large phase III trial, NExUS, remains open evaluating chemotherapy with gemcitabine and cisplatin with or without sorafenib in the same patient population; however, patients with squamous cell histology are no longer eligible for this study based on the data from the ESCAPE trial. Sorafenib in Patients with Small Cell Lung Cancer SWOG evaluated single-agent sorafenib (400  mg twice daily for 28 days) in 81 patients with SCLC who had progressed following first-line platinum-based chemotherapy. Eighteen patients (22%) discontinued therapy due to adverse events or side effects. Three patients achieved a PR (4%) and 25 patients had SD (32%). Median survival was 7 months for platinum sensitive and 5 months for platinum refractory patients, similar to treatment with current cytotoxic therapy. Despite the high rate of toxicity resulting in treatment discontinuation (>20%), SWOG investigators have recommended further evaluation of this agent in SCLC (91). Therefore, it appears that although single-agent sorafenib may have activity in patients with previously treated NSCLC, there are no data to suggest that combination chemotherapy with sorafenib improves survival for patients with advanced SCLC or NSCLC.

Angiogenesis Inhibitors in Lung Cancer

241

Vandetanib in Patients with Non-small Cell Lung Cancer Vandetanib (AZD6474, Zactima®) is an oral agent that targets angiogenesis through inhibition of VEGFR-2, VEGFR-3 and that also targets tumor growth through inhibition of RET, and epidermal growth factor receptor (EGFR/HER1) (Table  4) (92, 93). Therapy with vandetanib (100, 200, or 300  mg daily) was evaluated in a randomized phase II dose-finding study of 53 Japanese patients with previously treated NSCLC. Overall there was a 13.2% PR rate. Time to progression was 8.3 weeks at the 100 mg dose and 12.3 weeks at both the 200 and 300 mg dose (94). In a randomized phase II trial of 168 patients with NSCLC, single-agent vandetanib was compared to gefitinib in patients after failure of first- or second-line platinum-based therapy. Of note, patients with hemoptysis, thromboses, squamous cell carcinoma, and brain metastases were permitted to enter this trial. Median PFS was 11 weeks for vandetanib compared to 8.1 weeks for patients receiving gefitinib. Objective responses were seen in 8% of vandetanib-treated patients compared to 1% in gefitinib-treated patients. On progression, patients were permitted to switch to the alternative regimen. In patients who switched therapies, disease control >8 weeks was seen in 16/37 (43%) patients who switched to vandetanib and 7/29 (24%) patients who switched to gefitinib. OS was not significantly different between treatment arms (95). Vandetanib (100 or 300 mg) plus docetaxel was compared to docetaxel alone in a phase II randomized, placebo-controlled trial of 127 patients with advanced NSCLC who had failed platinum-based chemotherapy. Patients with squamous cell histology were permitted to enter the trial, as were patients with clinically stable, treated brain metastases. A PR was seen in 12% of patients receiving docetaxel alone, 26% of patients receiving vandetanib 100 mg and docetaxel, and 18% of patients receiving vandetanib 300 mg and docetaxel. There was no statistically significant difference in OS (13.4 vs. 13.1 vs. 7.9 months, respectively) (96). The results of this study have led to a phase III trial comparing vandetanib plus docetaxel to docetaxel alone as a second-line therapy in patients with advanced NSCLC. In a second randomized phase II trial, 181 chemotherapy-naïve patients with advanced NSCLC were randomized to one of three treatments – single-agent vandetanib, vandetanib plus paclitaxel and carboplatin, or paclitaxel and carboplatin alone. Patients with CNS metastases and squamous-cell histology were permitted to enter this trial. The objective response rates were 7% for vandetanib alone, 32% for combination therapy, and 25% for chemotherapy alone. Combination therapy with vandetanib plus carboplatin and paclitaxel prolonged PFS compared to carboplatin and paclitaxel alone (24 vs. 23 weeks, HR 0.75, 95% CI 0.05–01.15, P = 0.098). The vandetanib monotherapy arm was stopped early after a planned interim analysis of PFS met criteria for discontinuation. OS was not significantly different for vandetanib plus chemotherapy vs. chemotherapy alone. Contrary to the ECOG 4599 trial, an unplanned exploratory analysis suggested a benefit for female patients receiving vandetanib with chemotherapy compared to chemotherapy alone (97, 98). The preferential benefit in female patients with NSCLC may be due to vandetanib’s dual inhibition of both EGFR and VEGF pathways. Gender has been

242

L. Horn and A. Sandler

shown to correlate with response to EGFR inhibitors (99, 100). However, the lower dose of vandetanib when combined with chemotherapy is not thought to affect the EGFR axis. A phase II trial is currently underway evaluating adjuvant vandetanib plus carboplatin and paclitaxel in patients with resected stage I–III NSCLC (101). Heymach et  al. conducted an exploratory analysis measuring baseline VEGF levels in 351 patients (82% of trial participants) who participated in the three trials involving vandetanib in combination with chemotherapy listed above. In all three trials, vandetanib appeared to have a beneficial impact on progression-free in patients with low baseline levels of VEGF but not in patients with high VEGF levels (102).

Vandetanib in Patients with Small Cell Lung Cancer In a phase II study, maintenance therapy with vandetanib (300  mg daily) was compared to placebo in 107 patients with both LD- and ED-SCLC who achieved a CR or PR to induction therapy. There was no difference in PFS (2.7 vs. 2.6 months) or OS (11.9 vs. 10.6 months) for vandetanib-treated patients compared to placebo. A significant interaction for vandetanib-treated patients was noted for stage: limited stage patients had longer overall survival when treated with vandetanib (HR 0.45; one-sided, P = 0.07) and extensive stage patients had shorter survival (HR 2.27; one-sided, P = 0.996). Similar to the aforementioned trial in patients with NSCLC (103), a nonsignificant trend toward improved OS was seen in female patients (104, 105). Quality of life (QoL) was comparable between treatment arms. Vandetanibtreated patients had improved QoL symptoms for hemoptysis and pain and worse for diarrhea and sore mouth compared to placebo (104, 105). Therefore, it appears single-agent vandetanib has shown promising activity in patients with advanced NSCLC. Trials in patients with SCLC have been disappointing. Ongoing phase II and III clinical trials are evaluating single-agent vandetanib and vandetanib in combination with chemotherapy in the first-, second- and thirdline treatment of patients with NSCLC.

Sunitinib Sunitinib (SU11248, Sutent®) is a novel, multi-targeted, small molecule inhibitor of the receptor tyrosine kinases (RTKs) involved in tumor proliferation and angiogenesis, including VEGFR-1, VEGFR-2, and -3, PDGFR-a and VEGFR-b, Flt3, c-kit and the receptor encoded by the ret proto-oncogene (RET; rearranged during transfection), and fms-like tyrosine kinase 3 (Flt3) (Table 4) (106). Of note, it has been suggested that combined inhibition of PDGFR and VEGFR-2-mediated signaling may be particularly potent in the inhibition of angiogenesis (107). Two phase II trials have evaluated sunitinib in patients with previously treated NSCLC and reported RR of 2% and 11%, SD in 19% and 27% of patients, PFS of 12 weeks and OS of 5.4 and 8.6 months, respectively (108, 109).

Angiogenesis Inhibitors in Lung Cancer

243

A phase I study in untreated patients with advanced NSCLC evaluated sunitinib (37.5 or 50 mg) in combination with cisplatin and gemcitabine. No dose-limiting toxicities were seen at the sunitinib 37.5 mg dose, while two were noted (neutropenia and infection) at the 50 mg dose. Three patients achieved a PR at the 50-mg dose level. Sunitinib 37.5  mg appears to be the recommended dose with this chemotherapy schedule (110). A second phase I study in 37 patients with advanced solid tumors, including 13 patients with NSCLC evaluated sunitinib in combination with docetaxel. Neutropenia (with or without fever) was seen in five patients. One patient had pulseless electrical activity and pulmonary hemorrhage. With greater than 50% SD seen, studies are ongoing (111). Trials are investigating the addition of sunitinib to platinum-based chemotherapy in untreated NSCLC patients. A phase III trial is evaluating sunitinib in combination with erlotinib vs. erlotinib alone in previously treated NSCLC patients. Thalidomide in Patients with Non-small Cell Lung Cancer Thalidomide, a glutamic acid derivative, is an immunomodulatory agent with antiinflammatory and purported anti-angiogenic effects (Table  4) (112). Preliminary results from a phase II trial of thalidomide in combination with carboplatin and irinotecan in 36 patients with advanced NSCLC reported a response rate of 22% and a median survival of 7.3 months (113). A large phase III randomized, controlled trial of 722 patients with advanced NSCLC demonstrated no benefit for carboplatin and gemcitabine with thalidomide compared to placebo, with OS 8.4 vs. 8.9 months (HR 1.13, 95% CI 0.96–1.32, P = 0.14) (114). A small phase II trial evaluated thalidomide in combination with gemcitabine and irinotecan and included biological correlates. With only eight patients evaluable for response, the authors reported a correlation between serum hypoxia markers (including VEGF) and disease status, and also noted a reduction in these markers in a patient who had a response to therapy (115). Accrual to this trial is ongoing. A phase I–II trial combined thalidomide and docetaxel in 26 patients with previously treated NSCLC and reported a 19% RR and 5.4 months OS, which is similar to treatment with docetaxel alone (116, 117). Despite ongoing trials, there is currently no evidence to support the use of thalidomide in patients with advanced NSCLC. Thalidomide in Patients with Small Cell Lung Cancer Thalidomide was combined with carboplatin and etoposide in 26 chemotherapynaïve patients with LD- and ED-SCLC. There was a 65% RR in the 23 patients evaluable for response. Median OS was 10.2 months and 1-year survival was 42% (118). In a second phase II trial, maintenance thalidomide was administered after chemotherapy to 22 patients with ED-SCLC who had responded to induction chemotherapy. OS was 15.7 months and 1-year survival was 60% (119). Two phase III trials have evaluated thalidomide in combination with chemotherapy in patients with SCLC. The first trial evaluated thalidomide in combination

244

L. Horn and A. Sandler

with carboplatin and etoposide in 724 patients with LD- and ED-SCLC. Thalidomide treatment was associated with an increase in thrombotic events (RR 1.68, 95% CI 1.16–2.44) and there was no significant difference in OS or 2-year survival (10.2 months and 13% for both treatment groups) (120). The second phase III trial combined thalidomide with 4¢-epidoxorubicin plus cisplatin and cyclophosphamide in patients with ED-SCLC. In this study, 92 patients who had responded to 2 cycles of conventional chemotherapy were randomly assigned to four more cycles of chemotherapy with or without thalidomide. A higher number of patients treated with thalidomide discontinued therapy due to toxicity 33% vs. 19%. Patients receiving thalidomide had a longer median OS and 1-year survival compared to the placebo, 11.7 vs. 8.7 months and 49% vs. 30% respectively, although this was not statistically significantly different (HR 0.74, 95% CI 0.49–1.12, P = 0.16). PFS was similar at 6.6 and 6.4 months for patients receiving thalidomide and placebo, respectively. Performance status appeared to be important, as patients with a PS of 1 or 2 had a significantly longer survival (HR 0.59, 95% CI 0.37–0.92, P = 0.02) and slower disease progression (HR 0.54, 95% CI 0.36–0.87, P = 0.02) compared to the control. The number of patients receiving second line therapy was significantly higher in thalidomide-treated patients 67% vs. 46% (121). Therefore, there is currently no data to support the use of thalidomide in the treatment of patients with SCLC or NSCLC. Cediranib Cediranib (AZD2171, Recentin) is a potent inhibitor of both VEGFR-1 and VEGFR-2. It also has activity against c-kit, PDGFR-b, and Flt-4 at nanomolar concentrations, but is not active against other serine/threonine kinases studied (Table 4) (122). Cediranib (20, 30, or 45 mg daily) has been evaluated in phase I studies in combination with four chemotherapy regimens, including pemetrexed and docetaxel two agents commonly employed in treatment of patients with NSCLC. Response rates were promising and cediranib did not appear to affect the pharmacokinetic profile of either agent (123). Three phase I studies have evaluated cediranib in combination with erlotinib or chemotherapy (carboplatin and paclitaxel or cisplatin and gemcitabine) and found the combinations to be well tolerated (124–126). When cediranib was combined with carboplatin and paclitaxel in 20 patients with chemotherapy-naïve NSCLC, there were 9 PR (45%) and 11 patients with SD. Median time to progression was 7.6 months (125). These results led to a trial by the National Cancer Institute of Canada (NCI-C), BR-24, a randomized, double-blind, placebo-controlled trial of cediranib in combination with carboplatin and paclitaxel. This trial has recently been closed due to excessive toxicities, raising questions about the use of cediranib in combination with chemotherapy in the treatment of patients with advanced NSCLC. A phase II trial evaluated cediranib in 25 patients with recurrent SCLC who had progressed following platinum-based chemotherapy. The starting dose was cediranib 45 mg daily; however, 7 of 12 patients failed to complete their first cycle of therapy due to toxicities and the starting dose

Angiogenesis Inhibitors in Lung Cancer

245

was amended to 30 mg daily. Median PFS was 8 weeks, there was one unconfirmed PR and eight patients with SD. Circulating endothelial cells (CEC) and VEGF levels were also assessed. There was no correlation between treatment effects and VEGF level; however, CECs were noted to rise in several patients on disease progression. The response rate did not meet the predefined target of 20% and therefore the study was closed (127).

Other Agents Several other small molecule inhibitors are currently in phase I or II testing in patients with NSCLC including motesanib (AMG 706) (128–131), axitinib (AG013736), (132, 133) pazopanib (GW786034) (134), BIBF 1120 (135–137), and XL647 (Table 4) (138, 139). AE-941 (Neovastat) is a shark cartilage extract that binds to VEGFR (140) as well as inhibiting matrix metalloproteinases (MMPs) and stimulating apoptosis and tissue plasminogen activating enzymes (141). A phase I/II trial of 80 patients with advanced NSCLC evaluated AE-941 at two doses (£2.6 or >2.6 mL/kg/day). While both doses were well tolerated, the median survival was higher at the higher dose (6.1 months vs. 4.6 months) (142). Trials with neovastat in combination with chemotherapy and radiation therapy in patients with NSCLC are ongoing.

Conclusion Anti-angiogenic agents now have an established role in the treatment of NSCLC. Bevacizumab has been documented to be of benefit in selected patients with advanced disease, and new oral VEGFR antagonists show promise. We may anticipate increasing benefit from this new class of compounds as we gain further insights into the biology of tumor angiogenesis.

References 1. Jemal A, Siegel R, Ward E et al (2008) Cancer statistics, 2008. CA Cancer J Clin 58:71–96 2. Schiller JH, Harrington D, Belani CP et al (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 346:92–98 3. Elias AD (1997) Small cell lung cancer: state-of-the-art therapy in 1996. Chest 112:251S–258S 4. Simon GR, Turrisi A (2007) Management of small cell lung cancer: ACCP evidence-based clinical practice guidelines (2nd edition). Chest 132:324S–339S 5. Clark R, Ihde DC (1998) Small-cell lung cancer: treatment progress and prospects. Oncology (Williston Park) 12:647–658; discussion 661-643 6. Janne PA, Freidlin B, Saxman S et al (2002) Twenty-five years of clinical research for patients with limited-stage small cell lung carcinoma in North America. Cancer 95:1528–1538 7. Folkman J (1974) Proceedings: tumor angiogenesis factor. Cancer Res 34:2109–2113

246

L. Horn and A. Sandler

8. Carmeliet P (2005) Angiogenesis in life, disease and medicine. Nature 438:932–936 9. Ferrara N, Hillan KJ, Gerber HP et al (2004) Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov 3:391–400 10. Bergers G, Benjamin LE (2003) Tumorigenesis and the angiogenic switch. Nat Rev Cancer 3:401–410 11. Folkman J (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med 285:1182–1186 12. Hanahan D, Folkman J (1996) Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86:353–364 13. Yano S, Shinohara H, Herbst RS et al (2000) Expression of vascular endothelial growth factor is necessary but not sufficient for production and growth of brain metastasis. Cancer Res 60:4959–4967 14. Macchiarini P, Fontanini G, Dulmet E et al (1994) Angiogenesis: an indicator of metastasis in non-small cell lung cancer invading the thoracic inlet. Ann Thorac Surg 57:1534–1539 15. Holmgren L, O’Reilly MS, Folkman J (1995) Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nat Med 1:149–153 16. Kerbel RS (2006) Antiangiogenic therapy: a universal chemosensitization strategy for cancer? Science 312:1171–1175 17. Baluk P, Hashizume H, McDonald DM (2005) Cellular abnormalities of blood vessels as targets in cancer. Curr Opin Genet Dev 15:102–111 18. Ferrara N (2002) VEGF and the quest for tumour angiogenesis factors. Nat Rev Cancer 2:795–803 19. Dvorak HF (2002) Vascular permeability factor/vascular endothelial growth factor: a critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy. J Clin Oncol 20:4368–4380 20. Ferrara N, Gerber HP, LeCouter J (2003) The biology of VEGF and its receptors. Nat Med 9:669–676 21. Shibuya M, Ito N, Claesson-Welsh L (1999) Structure and function of vascular endothelial growth factor receptor-1 and -2. Curr Top Microbiol Immunol 237:59–83 22. Shibuya M, Yamaguchi S, Yamane A et al (1990) Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family. Oncogene 5:519–524 23. Terman BI, Carrion ME, Kovacs E et  al (1991) Identification of a new endothelial cell growth factor receptor tyrosine kinase. Oncogene 6:1677–1683 24. O’Byrne KJ, Koukourakis MI, Giatromanolaki A et al (2000) Vascular endothelial growth factor, platelet-derived endothelial cell growth factor and angiogenesis in non-small-cell lung cancer. Br J Cancer 82:1427–1432 25. Ugurel S, Rappl G, Tilgen W et al (2001) Increased serum concentration of angiogenic factors in malignant melanoma patients correlates with tumor progression and survival. J Clin Oncol 19:577–583 26. Brown LF, Berse B, Jackman RW et al (1993) Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in adenocarcinomas of the gastrointestinal tract. Cancer Res 53:4727–4735 27. Brown LF, Berse B, Jackman RW et  al (1995) Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer. Hum Pathol 26:86–91 28. Huang H, Held-Feindt J, Buhl R et al (2005) Expression of VEGF and its receptors in different brain tumors. Neurol Res 27:371–377 29. Boocock CA, Charnock-Jones DS, Sharkey AM et al (1995) Expression of vascular endothelial growth factor and its receptors flt and KDR in ovarian carcinoma. J Natl Cancer Inst 87:506–516 30. Guidi AJ, Abu-Jawdeh G, Berse B et  al (1995) Vascular permeability factor (vascular endothelial growth factor) expression and angiogenesis in cervical neoplasia. J Natl Cancer Inst 87:1237–1245 31. Suzuki K, Hayashi N, Miyamoto Y et al (1996) Expression of vascular permeability factor/vascular endothelial growth factor in human hepatocellular carcinoma. Cancer Res 56:3004–3009 32. Eisma RJ, Spiro JD, Kreutzer DL (1997) Vascular endothelial growth factor expression in head and neck squamous cell carcinoma. Am J Surg 174:513–517

Angiogenesis Inhibitors in Lung Cancer

247

33. Jacobsen J, Rasmuson T, Grankvist K et  al (2000) Vascular endothelial growth factor as prognostic factor in renal cell carcinoma. J Urol 163:343–347 34. Merrick DT, Haney J, Petrunich S et al (2005) Overexpression of vascular endothelial growth factor and its receptors in bronchial dypslasia demonstrated by quantitative RT-PCR analysis. Lung Cancer 48:31–45 35. Fontanini G, Faviana P, Lucchi M et al (2002) A high vascular count and overexpression of vascular endothelial growth factor are associated with unfavourable prognosis in operated small cell lung carcinoma. Br J Cancer 86:558–563 36. Imoto H, Osaki T, Taga S et al (1998) Vascular endothelial growth factor expression in nonsmall-cell lung cancer: prognostic significance in squamous cell carcinoma. J Thorac Cardiovasc Surg 115:1007–1014 37. Fontanini G, Vignati S, Boldrini L et al (1997) Vascular endothelial growth factor is associated with neovascularization and influences progression of non-small cell lung carcinoma. Clin Cancer Res 3:861–865 38. Volm M, Koomagi R, Mattern J (1997) Prognostic value of vascular endothelial growth factor and its receptor Flt-1 in squamous cell lung cancer. Int J Cancer 74:64–68 39. Yuan A, Yu CJ, Kuo SH et al (2001) Vascular endothelial growth factor 189 mRNA isoform expression specifically correlates with tumor angiogenesis, patient survival, and postoperative relapse in non-small-cell lung cancer. J Clin Oncol 19:432–441 40. Fontanini G, Bigini D, Vignati S et al (1995) Microvessel count predicts metastatic disease and survival in non-small cell lung cancer. J Pathol 177:57–63 41. Fontanini G, Lucchi M, Vignati S et al (1997) Angiogenesis as a prognostic indicator of survival in non-small-cell lung carcinoma: a prospective study. J Natl Cancer Inst 89:881–886 42. Lucchi M, Mussi A, Fontanini G et al (2002) Small cell lung carcinoma (SCLC): the angiogenic phenomenon. Eur J Cardiothorac Surg 21:1105–1110 43. Osusky KL, Hallahan DE, Fu A et al (2004) The receptor tyrosine kinase inhibitor SU11248 impedes endothelial cell migration, tubule formation, and blood vessel formation in vivo, but has little effect on existing tumor vessels. Angiogenesis 7:225–233 44. Vosseler S, Mirancea N, Bohlen P et al (2005) Angiogenesis inhibition by vascular endothelial growth factor receptor-2 blockade reduces stromal matrix metalloproteinase expression, normalizes stromal tissue, and reverts epithelial tumor phenotype in surface heterotransplants. Cancer Res 65:1294–1305 45. Kamba T, Tam BY, Hashizume H et al (2006) VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature. Am J Physiol Heart Circ Physiol 290:H560–H576 46. Mancuso MR, Davis R, Norberg SM et al (2006) Rapid vascular regrowth in tumors after reversal of VEGF inhibition. J Clin Invest 116:2610–2621 47. Presta LG, Chen H, O’Connor SJ et al (1997) Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 57:4593–4599 48. Johnson DH, Fehrenbacher L, Novotny WF et al (2004) Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small-cell lung cancer. J Clin Oncol 22:2184–2191 49. Sandler A, Gray R, Perry MC et al (2006) Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 355:2542–2550 50. Ramalingam SS, Dahlberg SE, Langer CJ et al (2008) Outcomes for elderly, advanced-stage non small-cell lung cancer patients treated with bevacizumab in combination with carboplatin and paclitaxel: analysis of Eastern Cooperative Oncology Group Trial 4599. J Clin Oncol 26:60–65 51. Manegold C, von Pawel J, Zatloukal P et al (2007) Randomised, double-blind multicentre phase III study of bevacizumab in combination with cisplatin and gemcitabine in chemotherapy-naive patients with advanced or recurrent non-squamous non-small cell lung cancer (NSCLC): BO17704. ASCO Meet Abstr 25:LBA7514 52. Shaked Y, Henke E, Roodhart JM et al (2008) Rapid chemotherapy-induced acute endothelial progenitor cell mobilization: implications for antiangiogenic drugs as chemosensitizing agents. Cancer Cell 14:263–273

248

L. Horn and A. Sandler

53. Tabernero J (2007) The role of VEGF and EGFR inhibition: implications for combining anti-VEGF and anti-EGFR agents. Mol Cancer Res 5:203–220 54. Ellis LM (2004) Epidermal growth factor receptor in tumor angiogenesis. Hematol Oncol Clin North Am 18:1007–1021, viii 55. Vallbohmer D, Zhang W, Gordon M et al (2005) Molecular determinants of cetuximab efficacy. J Clin Oncol 23:3536–3544 56. Herbst RS, Johnson DH, Mininberg E et al (2005) Phase I/II trial evaluating the anti-vascular endothelial growth factor monoclonal antibody bevacizumab in combination with the HER-1/epidermal growth factor receptor tyrosine kinase inhibitor erlotinib for patients with recurrent non-small-cell lung cancer. J Clin Oncol 23:2544–2555 57. Salmon JS, Sandler A, Billheimer D et al (2005) MALDI-TOF mass spectrometry proteomic profiling to discriminate response to the combination of bevacizumab and erlotinib in nonsmall cell lung cancer (NSCLC). ASCO Meet Abstr 23:7022 58. Herbst RS, O’Neill VJ, Fehrenbacher L et al (2007) Phase II study of efficacy and safety of bevacizumab in combination with chemotherapy or erlotinib compared with chemotherapy alone for treatment of recurrent or refractory non small-cell lung cancer. J Clin Oncol 25:4743–4750 59. Akerley WL, Langer CJ, Oh Y et  al (2008) Acceptable safety of bevacizumab therapy in patients with brain metastases due to non-small cell lung cancer. ASCO Meet Abstr 26:8043 60. Dansin E, Mezger J, Isla D et al (2008) Safety of bevacizumab-based therapy as first-line treatment of patients with advanced or recurrent non-squamous non-small cell lung cancer (NSCLC): MO19390 (SAiL). ASCO Meet Abstr 26:8085 61. Griesinger F, Laskin JJ, Pavlakis N et  al (2008) Safety of first-line bevacizumab-based therapy with concomitant cardiovascular or anticoagulation medication in advanced or recurrent non-squamous non-small cell lung cancer (NSCLC) in MO19390 (SAiL). ASCO Meet Abstr 26:8049 62. Adjei AA, Mandrekar SJ, Dy GK et al (2008) A phase II second-line study of pemetrexed (pem) in combination with bevacizumab (bev) in patients with advanced non-small cell lung cancer (NSCLC): an NCCTG and SWOG study. J Clin Oncol (Meet Abstr) 26:8080 63. Lynch TJ, Brahmer J, Fischbach N et  al (2008) Preliminary treatment patterns and safety outcomes for non-small cell lung cancer (NSCLC) from ARIES, a bevacizumab treatment observational cohort study (OCS). ASCO Meet Abstr 26:8077 64. Sandler A, Szwaric S, Dowlati A et al (2007) A phase II study of cisplatin (P) plus etoposide (E) plus bevacizumab (B) for previously untreated extensive stage small cell lung cancer (SCLC) (E3501): a trial of the Eastern Cooperative Oncology Group. J Clin Oncol (Meet Abstr) 25:7564 65. Ready N, Dudek AZ, Wang XF et al (2007) CALGB 30306: a phase II study of cisplatin (C), irinotecan (I) and bevacizumab (B) for untreated extensive stage small cell lung cancer (ES-SCLC). ASCO Meet Abstr 25:7563 66. Patton JF, Spigel DR, Greco FA et al (2006) Irinotecan (I), carboplatin (C), and radiotherapy (RT) followed by maintenance bevacizumab (B) in the treatment (tx) of limited-stage small cell lung cancer (LS-SCLC): update of a phase II trial of the Minnie Pearl Cancer Research Network. ASCO Meet Abstr 24:7085 67. Evans WK, Shepherd FA, Feld R et al (1985) VP-16 and cisplatin as first-line therapy for small-cell lung cancer. J Clin Oncol 3:1471–1477 68. Reynolds C, Barrera D, Vu DQ et al (2007) An open-label, phase II trial of nanoparticle albumin bound paclitaxel (nab-paclitaxel), carboplatin, and bevacizumab in first-line patients with advanced non-squamous non-small cell lung cancer (NSCLC). ASCO Meet Abstr 25:7610 69. William WN Jr, Kies MS, Fossella FV et al (2007) Phase II study of bevacizumab in combination with docetaxel and carboplatin in patients with metastatic non-small cell lung cancer (NSCLC). ASCO Meet Abstr 25:18098 70. Ferrer N, Paredes A, Munoz-Langa JM et al (2008) Bevacizumab in combination with cisplatin and docetaxel as first line treatment of patients (pts) with advanced or metastatic, non squamous, non-small-cell lung cancer (NSCLC). ASCO Meet Abstr 26:19109

Angiogenesis Inhibitors in Lung Cancer

249

71. Dalsania CJ, Hageboutros A, Harris E et al (2007) Phase II trial of bevacizumab plus pemetrexed and carboplatin in previously untreated advanced nonsquamous non-small cell lung cancer. ASCO Meet Abstr 25:18163 72. Patel JD, Hensing TA, Villafor V et al (2007) Pemetrexed and carboplatin plus bevacizumab for advanced non-squamous non-small cell lung cancer (NSCLC): preliminary results. ASCO Meet Abstr 25:7601 73. Heist RS, Fidias P, Huberman M et al (2007) Phase II trial of oxaliplatin, pemetrexed, and bevacizumab in previously-treated advanced non-small cell lung cancer (NSCLC). ASCO Meet Abstr 25:7700 74. Waples JM, Auerbach M, Boccia R et al (2007) A phase II study of oxaliplatin and pemetrexed plus bevacizumab in advanced non-squamous non-small cell lung cancer. ASCO Meet Abstr 25:18025 75. Davila E, Lilenbaum R, Raez L et al (2006) Phase II trial of oxaliplatin and gemcitabine with bevacizumab in first-line advanced non-small cell lung cancer (NSCLC). ASCO Meet Abstr 24:17009 76. Kraut MJ (2006) Phase II study of gemcitabine and carboplatin plus bevacizumab for stage III/IV non-small cell lung cancer: preliminary safety data. ASCO Meet Abstr 24:17091 77. Ahmad T, Eisen T (2004) Kinase inhibition with BAY 43-9006 in renal cell carcinoma. Clin Cancer Res 10:6388S–6392S 78. Wilhelm SM, Carter C, Tang L et al (2004) BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 64:7099–7109 79. Murphy DA, Makonnen S, Lassoued W et al (2006) Inhibition of tumor endothelial ERK activation, angiogenesis, and tumor growth by sorafenib (BAY43-9006). Am J Pathol 169:1875–1885 80. Carter C, Chen C, Brink C et al (2007) Sorafenib is efficacious and tolerated in combination with cytostatic agents in preclinical models of human non-small cell lung carcinoma. Cancer Chemother Pharmacol 59:183–195 81. Wilhelm S, Chien DS (2002) BAY 43-9006: preclinical data. Curr Pharm Des 8:2255–2257 82. Gatzemeier U, Blumenschein G, Fosella F et al (2006) Phase II trial of single-agent sorafenib in patients with advanced non-small cell lung carcinoma. ASCO Meet Abstr 24:7002 83. Liu B, Barrett T, Choyke P et  al (2006) A phase II study of BAY 43-9006 (Sorafenib) in patients with relapsed non-small cell lung cancer (NSCLC). ASCO Meet Abstr 24:17119 84. Gutierrez M, Kummar S, Allen D et al (2008) A phase II study of multikinase inhibitor sorafenib in patients with relapse non-small cell lung cancer (NSCLC). ASCO Meet Abstr 26:19084 85. Schiller JH, Lee JW, Hanna NH et al (2008) A randomized discontinuation phase II study of sorafenib versus placebo in patients with non-small cell lung cancer who have failed at least two prior chemotherapy regimens: E2501. ASCO Meet Abstr 26:8014 86. Adjei AA, Molina JR, Hillman SL et al (2007) A front-line window of opportunity phase II study of sorafenib in patients with advanced non-small cell lung cancer: a North Central Cancer Treatment Group study. ASCO Meet Abstr 25:7547 87. Bukowski R, Cella D, Gondek K et al (2007) Effects of sorafenib on symptoms and quality of life: results from a large randomized placebo-controlled study in renal cancer. Am J Clin Oncol 30:220–227 88. Adjei AA, Molina JR, Mandrekar SJ et al (2007) Phase I trial of sorafenib in combination with gefitinib in patients with refractory or recurrent non-small cell lung cancer. Clin Cancer Res 13:2684–2691 89. Schiller JH, Flaherty KT, Redlinger M et  al (2006) Sorafenib combined with carboplatin/ paclitaxel for advanced non-small cell lung cancer: a phase I subset analysis. ASCO Meet Abstr 24:7194 90. Scagliotti G, Von Pawel J, Reck M et  al (2008) Phase III trial comparing carboplatin and paclitaxel with or without sorafenib in chemonaive patients with stage IIIB (with effusion) or IV non-small cell lung cancer. 1st IASLC-ESMO European Lung Cancer Conference 2008, Geneva, 23–26 April 2008, p 48

250

L. Horn and A. Sandler

  91. Gitlitz BJ, Glisson BS, Moon J et al (2008) Sorafenib in patients with platinum (plat) treated extensive stage small cell lung cancer (E-SCLC): a SWOG (S0435) phase II trial. ASCO Meet Abstr 26:8039   92. Carlomagno F, Vitagliano D, Guida T et al (2002) ZD6474, an orally available inhibitor of KDR tyrosine kinase activity, efficiently blocks oncogenic RET kinases. Cancer Res 62:7284–7290   93. Wedge SR, Ogilvie DJ, Dukes M et al (2002) ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration. Cancer Res 62:4645–4655   94. Kiura K, Nakagawa K, Shinkai T et al (2008) A randomized, double-blind, phase IIa dosefinding study of Vandetanib (ZD6474) in Japanese patients with non-small cell lung cancer. J Thorac Oncol 3:386–393   95. Natale RB, Bodkin D, Govindan R et  al (2006) ZD6474 versus gefitinib in patients with advanced NSCLC: final results from a two-part, double-blind, randomized phase II trial. ASCO Meet Abstr 24:7000   96. Heymach JV, Johnson BE, Prager D et al (2007) Randomized, placebo-controlled phase II study of vandetanib plus docetaxel in previously treated non small-cell lung cancer. J Clin Oncol 25:4270–4277   97. Johnson BE, Ma P, West H et al (2005) Preliminary phase II safety evaluation of ZD6474, in combination with carboplatin and paclitaxel, as 1st-line treatment in patients with NSCLC. ASCO Meet Abstr 23:7102   98. Heymach J, Paz-Ares L, De Braud F et al (2007) Randomized phase II study of vandetanib (VAN) alone or in combination with carboplatin and paclitaxel (CP) as first-line treatment for advanced non-small cell lung cancer (NSCLC). ASCO Meet Abstr 25:7544   99. Shepherd FA, Rodrigues Pereira J, Ciuleanu T et al (2005) Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 353:123–132 100. Perez-Soler R, Chachoua A, Hammond LA et al (2004) Determinants of tumor response and survival with erlotinib in patients with non-small-cell lung cancer. J Clin Oncol 22:3238–3247 101. http://www.cancer.gov 102. Heymach JV, Hanrahan EO, Mann H et al (2008) Baseline VEGF as a potential predictive biomarker of vandetanib clinical benefit in patients with advanced NSCLC. ASCO Meet Abstr 26:8009 103. Heymach J, Paz-Ares L, De Braud F et al (2007) Randomized phase II study of vandetanib (VAN) alone or in combination with carboplatin and paclitaxel (CP) as first-line treatment for advanced non-small cell lung cancer (NSCLC). J Clin Oncol (Meet Abstr) 25:7544 104. Arnold AM, Smylie M, Ding K et al (2007) Randomized phase II study of maintenance vandetanib (ZD6474) in small cell lung cancer (SCLC) patients who have a complete or partial response to induction therapy: NCIC CTG BR.20. J Clin Oncol (Meet Abstr) 25:7522 105. Arnold AM, Seymour L, Smylie M et al (2007) Phase II study of vandetanib or placebo in small-cell lung cancer patients after complete or partial response to induction chemotherapy with or without radiation therapy: National Cancer Institute of Canada Clinical Trials Group Study BR.20. J Clin Oncol 25:4278–4284 106. Abrams TJ, Lee LB, Murray LJ et  al (2003) SU11248 inhibits KIT and platelet-derived growth factor receptor beta in preclinical models of human small cell lung cancer. Mol Cancer Ther 2:471–478 107. Cabebe E, Wakelee H (2007) Role of anti-angiogenesis agents in treating NSCLC: focus on bevacizumab and VEGFR tyrosine kinase inhibitors. Curr Treat Options Oncol 8:15–27 108. Socinski MA, Novello S, Brahmer JR et al (2008) Multicenter, phase II trial of sunitinib in previously treated, advanced non-small-cell lung cancer. J Clin Oncol 26:650–656 109. Brahmer JR, Govindan R, Novello S et  al (2007) Efficacy and safety of continuous daily sunitinib dosing in previously treated advanced non-small cell lung cancer (NSCLC): results from a phase II study. ASCO Meet Abstr 25:7542 110. Reck M, Frickhofen N, Gatzemeier U et al (2007) A phase I dose escalation study of sunitinib in combination with gemcitabine + cisplatin for advanced non-small cell lung cancer (NSCLC). ASCO Meet Abstr 25:18057

Angiogenesis Inhibitors in Lung Cancer

251

111. Robert F, Sandler A, Schiller JH et al (2007) A phase I dose-escalation and pharmacokinetic (PK) study of sunitinib (SU) plus docetaxel (D) in patients (pts) with advanced solid tumors (STs). ASCO Meet Abstr 25:3543 112. Franks ME, Macpherson GR, Figg WD (2004) Thalidomide. Lancet 363:1802–1811 113. Miller AA, Case D, Atkins J et al (2004) Phase II study of carboplatin, irinotecan, and thalidomide in patients with advanced non-small cell lung cancer (NSCLC). ASCO Meet Abstr 22:7132 114. Lee S, Rudd RM, Woll PJ et  al (2008) Two randomised phase III, double blind, placebo controlled trials of thalidomide in patients with advanced non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). ASCO Meet Abstr 26:8045 115. Flora DB, Kleykamp B, Knapp M et al (2004) A phase II trial of thalidomide (T), irinotecan (I) and gemcitabine (G) in chemonaive patients (pts) with advanced non-small cell lung cancer (NSCLC). ASCO Meet Abstr 22:7258 116. Seidler CW, Scepansky E, Khanani S et  al (2006) Phase I-II trial of daily thalidomide in combination with docetaxel in patients with relapsed non-small cell lung cancer: a final analysis. ASCO Meet Abstr 24:17060 117. Hanna N, Shepherd FA, Fossella FV et al (2004) Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy. J Clin Oncol 22:1589–1597 118. Lee SM, James LE, Mohamed-Ali V et al (2002) A phase II study of carboplatin/etoposide with thalidomide in small cell lung cancer (SCLC). Proc Am Soc Clin Oncol 21:2002 (abstr 1251) 119. Cooney MM, Subbiah S, Chapman R et al (2005) Phase II trial of maintenance daily oral thalidomide in patients with extensive-stage small cell lung cancer (ES-SCLC) in remission. J Clin Oncol (Meet Abstr) 23:7166 120. Lee S, Rudd RM, Woll PJ et  al (2008) Two randomised phase III, double blind, placebo controlled trials of thalidomide in patients with advanced non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). J Clin Oncol (Meet Abstr) 26:8045 121. Pujol JL, Breton JL, Gervais R et al (2007) Phase III double-blind, placebo-controlled study of thalidomide in extensive-disease small-cell lung cancer after response to chemotherapy: an intergroup study FNCLCC cleo04 IFCT 00–01. J Clin Oncol 25:3945–3951 122. Wedge SR, Kendrew J, Hennequin LF et al (2005) AZD2171: a highly potent, orally bioavailable, vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor for the treatment of cancer. Cancer Res 65:4389–4400 123. Lorusso PM, Heath E, Valdivieso M et al (2006) Phase I evaluation of AZD2171, a highly potent and selective inhibitor of VEGFR signaling, in combination with selected chemotherapy regimens in patients with advanced solid tumors. ASCO Meet Abstr 24:3034 124. van Cruijsen H, Voest EE, van Herpen CML et  al (2005) Phase I clinical evaluation of AZD2171 in combination with gefitinib, in patients with advanced tumors. ASCO Meet Abstr 23:3030 125. Laurie SA, Gauthier I, Arnold A et al (2008) Phase I and pharmacokinetic study of daily oral AZD2171, an inhibitor of vascular endothelial growth factor tyrosine kinases, in combination with carboplatin and paclitaxel in patients with advanced non-small-cell lung cancer: the National Cancer Institute of Canada clinical trials group. J Clin Oncol 26:1871–1878 126. Goss GD, Laurie S, Shepherd F et al (2007) IND.175: phase I study of daily oral AZD2171, a vascular endothelial growth factor receptor inhibitor (VEGFRI), in combination with gemcitabine and cisplatin (G/C) in patients with advanced non-small cell lung cancer (ANSCLC): a study of the NCIC Clinical Trials Group. ASCO Meet Abstr 25:7649 127. Ramalingam SS, Mack PC, Vokes EE et al (2008) Cediranib (AZD2171) for the treatment of recurrent small cell lung cancer (SCLC): a California Consortium phase II study. J Clin Oncol 26:2008 (May 20 suppl; abstr 8078) 128. Polverino A, Coxon A, Starnes C et al (2006) AMG 706, an oral, multikinase inhibitor that selectively targets vascular endothelial growth factor, platelet-derived growth factor, and kit receptors, potently inhibits angiogenesis and induces regression in tumor xenografts. Cancer Res 66:8715–8721

252

L. Horn and A. Sandler

129. Rosen L, Kurzrock R, Jackson E et al (2005) Safety and pharmacokinetics of AMG 706 in patients with advanced solid tumors. ASCO Meet Abstr 23:3013 130. Blumenschein G Jr, Sandler A, O’Rourke T et al (2006) Safety and pharmacokinetics (PK) of AMG 706, panitumumab, and carboplatin/paclitaxel (CP) for the treatment of patients (pts) with advanced non-small cell lung cancer (NSCLC). ASCO Meet Abstr 24:7119 131. Crawford J, Burris H, Stephenson J Jr et  al (2007) Safety and pharmacokinetics (PK) of AMG 706 in combination with panitumumab and gemcitabine-cisplatin in patients (pts) with advanced cancer. ASCO Meet Abstr 25:14057 132. Rugo HS, Herbst RS, Liu G et  al (2005) Phase I trial of the oral antiangiogenesis agent AG-013736 in patients with advanced solid tumors: pharmacokinetic and clinical results. J Clin Oncol 23:5474–5483 133. Schiller JH, Larson T, Ou SI et al (2007) Efficacy and safety of axitinib (AG-013736; AG) in patients (pts) with advanced non-small cell lung cancer (NSCLC): a phase II trial. ASCO Meet Abstr 25:7507 134. Nikolinakos P, Altorki N, Guarino M et al (2008) Analyses of plasma cytokine/angiogenic factors (C/AFs) profile during preoperative treatment with pazopanib (GW786034) in earlystage non-small cell lung cancer. J Clin Oncol (Meet Abstr) 26:7568 135. Hilberg F, Roth GJ, Krssak M et  al (2008) BIBF 1120: triple angiokinase inhibitor with sustained receptor blockade and good antitumor efficacy. Cancer Res 68:4774–4782 136. Von Pawel J, Kaiser R, Eschbach C et al (2007) A double blind phase II study of BIBF 1,120 in patients suffering from relapsed advanced non-small cell lung cancer (NSCLC). J Clin Oncol (Meet Abstr) 25:7635 137. Hanna N, Ellis P, Stopfer P et al (2007) A phase I study of continuous oral treatment with the triple angiokinase inhibitor BIBF 1120 together with pemetrexed in previously treated patients with non-small cell lung cancer: P3–091. J Thorac Oncol 2:S717–S718 138. Miller VA, Wakelee HA, PN LARA et al (2008) Activity and tolerance of XL647 in NSCLC patients with acquired resistance to EGFR-TKIs: preliminary results of a phase II trial. ASCO Meet Abstr 26:8028 139. Rizvi NA, Kris MG, Miller VA et al (2008) Activity of XL647 in clinically selected NSCLC patients (pts) enriched for the presence of EGFR mutations. Result from phase 2. ASCO Meet Abstr 26:8053 140. Beliveau R, Gingras D, Kruger EA et al (2002) The antiangiogenic agent neovastat (AE-941) inhibits vascular endothelial growth factor-mediated biological effects. Clin Cancer Res 8:1242–1250 141. Boivin D, Gendron S, Beaulieu E et al (2002) The antiangiogenic agent Neovastat (AE-941) induces endothelial cell apoptosis. Mol Cancer Ther 1:795–802 142. Latreille J, Batist G, Laberge F et  al (2003) Phase I/II trial of the safety and efficacy of AE-941 (Neovastat) in the treatment of non-small-cell lung cancer. Clin Lung Cancer 4:231–236 143. Waples JM, Auerbach M, Steis R et al (2008) A phase II study of oxaliplatin and pemetrexed plus bevacizumab in advanced non-squamous non-small cell lung cancer (An International Oncology Network study, #I-04-015). ASCO Meet Abstr 26:19018

Other Molecular Targeted Agents in Non-small Cell Lung Cancer Benjamin Besse and Jean-Charles Soria

Abstract  The successes of EGFR targeted drugs and antiangiogenic agents in non-small cell lung cancer (NSCLC) have speed up the development of molecular agents targeting other pathways. Promising new targets are reviewed on this chapter with a special focus on (1) mTOR/PI3K and IGF-1R that might modulate the activity of cytotoxic drugs or EGFR targeted agents, (2) the RAS/Erk pathway that has an appealing new interest giving the recent advances on K-RAS predictive and prognostic value, (3) Met, recently highlighted as a candidate for EGFR inhibitor resistance, (4) protein kinase C or integrins, and their role in angiogenesis, (5) Bcl-2 and death receptors in the apoptotic field. We discuss the challenges that we are facing to implement these new drugs among the approved drugs and validated strategies. Keywords  Non-small cell lung cancer • Molecular targeted agents

Introduction Recent clinical developments in non-small cell lung cancer (NSCLC) have mainly focused on EGFR targeted drugs and antiangiogenic agents. As we increase our knowledge of these drugs, we may not be far from reaching a new therapeutic plateau with these agents. Recent advances in lung cancer molecular research have lead to (1) the rediscovery of old stars of the molecular field (K-RAS, apoptosis pathway, acetylation, etc.) and (2) the rise of new potential target (c-met, mTOR/PI3K, IGF-1R, Integrins, etc.). Emerging therapies may overcome resistance to approved agents, potentiate their efficacy, or become a novel therapeutic class active as a single agent, opening new perspectives in lung cancer treatment. B. Besse (*) and J.-C. Soria Department of Medicine, Institut Gustave Roussy, Villejuif, France e-mail: [email protected]

D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_12, © Humana Press, a part of Springer Science+Business Media, LLC 2010

253

254

B. Besse and J.-C. Soria

In this chapter, we summarize the main molecular targeted therapies in lung cancer by following the proposed classification of the hallmarks of cancer cells (1). EGFR targeted therapies and antiangiogenic agents are treated in separate chapters and are therefore excluded. We offer an integrated view of agents under investigation in Fig.  1. We will focus on cell growth pathways, programmed cell death (apoptosis), local invasion, and distant metastatic processes. Current status of a selection of molecular targeted agents in NSCLC is provided in Table 1.

Fig. 1  Molecular-targeted agents under investigation in NSCLC

Other Molecular Targeted Agents in Non-small Cell Lung Cancer

255

Table 1  Current status of selected targeted agents in NSCLC Agent Targets Phase Everolimus mTOR II CP-751,871 IGF-1R III R1507 IGF-1R II Bortezomib Proteasome III – Stopped AMG951 Death receptors Phase Ib/II DR4 and DR5 CI 1040 MEK II AZD6244 MEK II AMG102 HGF/Met Ph I ARQ 197 MET Ph II Enzastaurin PKCß II – Stopped aprinocarsen PKC-a III – Stopped Etaracizumab aVb3 I Cilengitide aVb3 II Volociximab a5b1 IB I Mapatumumab Tumor necrosis factor-related apoptosisinducing ligand receptor 1

Cancer Cell Growth Pathways Schematically, cell growth signaling can be divided into three distinct, though closely connected parts. First, there are the upstream growth factors and their receptors at the cell membrane. In this category, EGFR and VEGF/VEGFR1–3 are the most frequently targeted pathways, but other candidates are being actively explored such as IGF-1R. From the cell membrane receptors, molecular mechanisms of signal transduction and intracellular messengers inform the nucleus of a given stimulus. Finally, an effector pathway leads the cell to eventual cell division and proliferation. Many molecules are involved in cell transduction pathways, each of them being a potential target. We will focus here on the most advanced agents developed in this setting.

Insulin-Like Growth Factor Receptor – IGF-1R The IGF-1R axis is one of the signaling pathways which plays a key role in the regulation of cellular activities, including cell proliferation, differentiation, and apoptosis (Fig.  2). The IGF system includes the IGF ligands (IGF-I and IGF-II), the corresponding cell surface receptors (IGF-1R and IGF-2R), and the IGF binding proteins (IGFBP1-6) (2). IGF-1R is a tyrosine kinase cell surface receptor that shares ~50% overall homology with the insulin receptor. IGF-1R ligand binding leads to receptor tyrosine kinase activity and resultant phosphorylation of tyrosine residues. Activated IGF-1R recruits and phosphorylates adaptor proteins belonging to the insulin receptor

256

B. Besse and J.-C. Soria

Fig. 2  Insulin-like growth factor 1 receptor (IGF-1R) pathway

substrate (IRS) family or Src homology 2 domain-containing transforming protein 1 (SHC), which leads to the activation of phosphoinositide-3-phosphate (PI3K)/AKT and Ras-protein/mitogen-activated protein kinases (MAPK) pathways (2). Experimental and clinical studies have demonstrated that the IGF-1R is overexpressed in cancer cells compared with normal tissues (3). In a cohort of 79 NSCLC specimens, IGF-1R staining has been considered positive in 39% of the tumors (4). In vivo, xenograft models of Lewis lung cancer cell lines (M-27), transfected to overexpress IGF-1R (threefold increase in binding sites), exhibit significant development of tumor nodules compared with mock transfected cells which do not give rise to tumors (5). In a model of A459 NSCLC cells, inhibition of IGF-1R by antiIGF-1R antibody demonstrated reduction of tumor volume by 55 and 94% growth inhibition when compared with controls when anti-IGF-1R antibody was combined with vinorelbine. Phase I monotherapy study results reported for the monoclonal antibodies CP-751,871, R1507, AMG 479, or AVE1642 have shown limited toxicities, apart from some hyperglycemia and, for some of the antibodies, thrombocytopenia (6–9). In contrast, dramatic single agent activity has been noted in Ewing sarcoma patients, fast-tracking a range of subsequent sarcoma-specific phase II studies (7, 10). To date, in lung cancer, preliminary phase II results of only one anti-IGF-1R agent have been reported. In a randomized first-line advanced NSCLC phase II study of paclitaxel and carboplatin plus/minus CP-751,871, 46% of patients in the experimental arm achieved objective responses (22/48 patients) vs. 32% (8/25 patients) in the control arm (11). An unplanned subgroup analysis by histology has suggested a greater benefit in patients with squamous histology within this trial, but the results

Other Molecular Targeted Agents in Non-small Cell Lung Cancer

257

of additional patient numbers, including planned enrichment for those with squamous histology, are awaited. In an in  vitro model of lung cancer, IGF-1R kinases were more phosphorylated in cells expressing mutant EGFR than in cells expressing wild type EGFR or mutant RAS (12). Trials are thus ongoing in NSCLC, in the front line setting in non-adenocarcinoma tumors with carboplatin-paclitaxel, and in combination with erlotinib in 2nd/3rd line (summarized in Table 2).

Targeting mTOR The mammalian target of rapamycin (mTOR) is a protein kinase of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway with a central role in the control of cell growth, survival, mobility, and angiogenesis. In more than 50% of lung carcinomas, AKT and mTOR are activated (13). In response to growth factor stimulation, PI3K is activated leading to the recruitment of AKT to the plasma membrane where it is activated by phosphorylation (14). AKT is a serine/threonine kinase that phosphorylates many effectors, including TSC (tuberous sclerosis complex)-1/2, leading to its inactivation. The inactivation of TSC2 leads to the activation of mTOR. Mutations in these components or in PTEN, a negative regulator of PI3K, may result in the upregulation of this pathway (15). Furthermore, AKT activity is frequent in preneoplastic lesions, suggesting that the activation of this pathway is a very early event in lung oncogenesis. In vitro studies have shown that in situ preneoplasia can be reversed after inhibition of the AKT pathway (16). These and other data suggest a possible synergy with either classical chemotherapy (17) or other molecular targeted agents (18–20), which raises great expectations for possible applications in preventive chemotherapy or even in advanced stages. mTOR inhibitors are a class of signal transduction inhibitors with anticancer activity that were initially developed as immunosuppressive agents. The mTOR inhibitor rapamycin (sirolimus), a macrocyclic lactone produced by Streptomyces hydroscipus, was the first inhibitor to be used in the clinical setting, followed afterward by derivatives: temsirolimus, everolimus, and tacrolimus. These agents bind to immunophilin FKMP-12 to form a complex that interacts with the mTOR kinase, thereby blocking its activity. This in turn results in the inhibition of key transduction pathways, including those regulated by p70s6 kinase and the eukaryotic initiation factor 4E-binding protein (4E-BP1), leading eventually to cell cycle arrest in G1. Temsirolimus (CCI-779) in the first-line setting of metastatic renal cancer enhanced the overall survival (hazard ratio for death, 0.73; 95% confidence interval [CI], 0.58–0.92; P = 0.008) compared to interferon alpha alone in a large phase III trial (21). After failure of VEGR inhibitors, the orally available everolimus (RAD001) improved progression-free survival (PFS) from 1.9 to 4 months compared to placebo in metastatic renal cancer (22). Based on these studies, temsirolimus and everolimus have been recently approved in metastatic renal cancer. Of them, only everolimus has been actively developed in lung cancer.

CP-751,871 Combination study of CP-751,871 with paclitaxel and carboplatin In advanced lung cancer (NCT00147537) CP-751,871 Study of CP-751,871 in combination with carboplatin and paclitaxel in advanced lung Cancer (NCT00603538) R1507 A study of the effect of R1507 in combination with tarceva (erlotinib) on progression-free survival in patients with stage IIIb/IV non-small cell lung cancer (NSCLC) (NCT00760929) R1507 A study of the effect of R1507 in combination with tarceva (erlotinib) on progression-free survival in patients with stage IIIb/IV non-small cell lung cancer (NSCLC) having received tarceva monotherapy (NCT00773383) AMG 479 Phase 1b/2 study of AMG 479 in combination with paclitaxel and carboplatin for 1st line treatment of advanced squamous non-small cell lung cancer (NCT00807612) AMG 479 AMG 655 in combination with AMG 479 in advanced, refractory solid tumors (NCT00819169) Japanmetastatic (stage IIIB or IV) NSCLC Metastatic (stage IIIB or IV) NSCLC

Metastatic (stage IIIB or IV) NSCLCReceived at least 3 months of erlotinib

Metastatic (stage IIIB or IV) NSCLCSquamous cell only

Metastatic (stage IIIB or IV) NSCLC

IIR

II

Ib/II

I/II

Population Metastatic (stage IIIB or IV) NSCLC predominantly squamous cell, large cell or adenosquamous carcinoma Metastatic (stage IIIB or IV) NSCLC predominantly squamous cell, large cell or adenosquamous carcinoma Metastatic (stage IIIB or IV) NSCLC

I

I/II

Table 2  Summary of ongoing studies with IGF-1R inhibitors Drug Study Phase CP-751,871 Carboplatin and paclitaxel with or without CP-751,871 III (an IGF-1R inhibitor) for advanced NSCLC of squamous, large cell and adenosquamous carcinoma histology (NCT00596830) CP-751,871 Trial of CP-751,871 and erlotinib in refractory lung III cancer (NCT00673049)

2nd or 3rd line

1st line

3rd or 4th line

2nd or 3rd line

1st line

1st line

Ongoing

AMG 655 with AMG 479

Ongoing

ApprovedCarboplatin and not yet paclitaxel with active AMG 479

Erlotinib with R1507

Ongoing Carboplatin and paclitaxel with CP-751,871 Ongoing Carboplatin and paclitaxel with CP-751,871 ApprovedErlotinib with not yet or without active R1507

Treatment arm Status Ongoing Carboplatin and paclitaxel with or without CP-751,871 Ongoing 2nd line Erlotinib with or without or CP-751,871 more

Setting 1st line

258 B. Besse and J.-C. Soria

Other Molecular Targeted Agents in Non-small Cell Lung Cancer

259

In NSCLC, everolimus has limited efficacy as a single agent (10  mg/d). In a phase I study, 4 disease stabilizations and one partial response (PR) was reported from 14 NSCLC patients. In a phase II trial, 85 patients with previously treated NSCLC were included (2nd–4th line therapy), 43 of whom were pretreated with an EGFR tyrosine kinase inhibitor (arm 2) (23). A PR was seen in three patients (1 in arm 2), stable disease (SD) ³12 weeks was reported in 34.2 and 41.7% of the cases and median PFS was 11.3 weeks (95% CI: 8.1–12.4) and 9.7 weeks (7.3–13.0) in arms 1 and 2, respectively. The most frequent side effect was stomatitis in 43 patients. Surprisingly, no infectious events related to immunomodulation by everolimus were reported. Due to the limited activity of everolimus alone, combination therapy with either gefitinib or erlotinib was implemented. Everolimus has been combined at a dose of 5  mg/day with gefitinib (250  mg/d) in a phase I trial which encompassed ten NSCLC patients. Dose-limiting toxicity (DLT) was stomatitis and hypotension, and two patients experienced a PR (24). In a phase II trial, ever-smoker patients with previously untreated (n = 17) or treated (n=18) NSCLC were included (25). Respectively, 3 (18%) and 2 (13%) untreated and previously treated patients experienced a partial response, but median PFS was not reported. Grade 3 diarrhea or mucositis was seen in 11 and 3% of the patients. Definitive results are awaited. In a phase I/II trial, daily or weekly everolimus has been combined with 150 mg/d erlotinib (26). All 93 patients were treated in the second or third line setting for advanced NSCLC. The final dose selected for phase II study was 5 mg/d of everolimus and 150 mg of erlotinib. Dose-limiting toxicities were stomatitis, diarrhea, and skin rash. No PR was seen in the weekly cohort (n = 20), but 65% had SD. In the daily cohorts (n = 72), 14% of the patients experienced a PR, and 47% had SD. A phase II trial is currently ongoing. Phase I/II trials are ongoing with everolimus in combination with docetaxel or paclitaxel/carboplatin/bevacizumab. Temsirolimus and everolimus are currently being evaluated in association with radiation therapy in NSCLC in phase I studies. mTOR inhibitor studies in NSCLC are summarized in Table 3.

Targeting Ras Pathway The RAS–RAF–MEK–ERK pathway (or ERK pathway) is an important signal transduction system involved in the control of cell proliferation, survival, and differentiation. RAS protein is a guanine nucleotide binding protein that is a key intermediate in signal transduction pathways (27). The mammalian genome encodes three RAS genes that give rise to four ubiquitously expressed gene products: H-RAS, N-RAS, K-RAS 4A and K-RAS 4B (K-RAS 4A, and K-RAS 4B are splice variants of a single gene). RAS is mutationally activated in 30% of all cancers. Prevalence of RAS mutations in lung cancer depends on histology and smoking status: mutations are rare in squamous-cell carcinoma, and in adenocarcinomas, they are seen predominantly in current or former smokers

Temsirolimus

Temsirolimus and radiation for non-small cell lung cancer(NCT00796796)

I

Table 3  Summary of ongoing studies with mTOR inhibitors Drug Study Phase I/II Everolimus Phase I/II trial of RAD001 plus (RAD001) docetaxel in patients with metastatic or recurrent non-small cell lung cancer (NCT00406276) I/II Everolimus Combination of RAD001 and (RAD001) erlotinib in patients with advanced non-small cell lung cancer previously treated only with chemotherapy (NCT00456833) I/II Everolimus Combination of RAD001 with (RAD001) carboplatin, paclitaxel and bevacizumab in Non-Small-Cell Lung Cancer (NSCLC) patients not treated previously with systemic therapy (NCT00457119) Everolimus Phase 1b trial of RAD001 in patients I (RAD001) with operable non-small cell lung cancer (NSCLC)(NCT00401778) Everolimus Everolimus and radiation for nonI (RAD001) small cell lung cancer Neo-adjuvant Everolimus

Stage I-IIIA operable NSCLC

Metastatic (stage IIIB or IV)Indication Locally advanced for thoracic radiation disease Not be candidates for definitive chemoradiation with curative intent Metastatic (stage IIIB or IV)Indication Locally advanced for thoracic radiation disease Not be candidates for definitive chemoradiation with curative intent

1st line

Metastatic (stage IIIB or IV)Eligible for bevacizumab

ongoing

Status Ongoing

Ongoing

Approved – not yet active

Temsirolimus and radiation

Temsirolimus and radiation

Ongoing

Ongoing Carboplatin, paclitaxel, and bevacizumab with everolimus

Erlotinib or erlotinib with everolimus

2nd or 3rd line

Metastatic (stage IIIB or IV)

Treatment arm Docetaxel with everolimus

Setting 2nd line or more

Population Metastatic (stage IIIB or IV)PS 0–2

260 B. Besse and J.-C. Soria

Other Molecular Targeted Agents in Non-small Cell Lung Cancer

261

(10–30% of whom have mutated K-RAS) (28). K-RAS mutations are uncommon in never-smokers and are almost mutually exclusive with EGFR mutations (29). RAS mutations lead to a protein that remains locked in an active state, thereby relaying uncontrolled proliferative signals and generally confer a poor prognosis (30). Targeting RAS can be achieved by targeting the posttranslational modifications, which determine RAS localization and mediate its attachment to the membrane, or by targeting the complex regulatory network that controls the activation of the RAS super family. Farnesyl Transferase Inhibitors (FTIs) The enzyme farnesyl transferase is involved in the posttranslational modification of RAS proteins by linking covalently a farnesyl group onto RAS. After farnesylation, RAS can be translocated to the cell membrane and then activate signal transduction pathways involved in proliferation and inhibition of apoptosis. Farnesyl transferase inhibitors (FTIs) aim to block farnesylation and RAS signaling. However, given that more than a hundred molecules can be processed by farnesyl transferase, FTIs do have other multiple targets. Nevertheless, FTIs have been evaluated in cancer patients, and tipifarnib (R115777) induced a PR in a patient treated in a phase I trial in advanced NSCLC (31). In further testing, tipifarnib showed no significant antitumor activity as a single agent either in untreated advanced NSCLC (no PR, but SD >6 months in 16% of the patients) or in sensitive-relapse small cell lung cancer (SCLC) (32, 33). Lonafarnib (SCH6636) seemed to restore sensitivity to paclitaxel in patients resistant or refractory to that agent (progression during or within 3 months of paclitaxel): 3 out of 29 patients experienced a PR and PFS was 16 weeks (34). Surprisingly, few investigations have been directed to explore relationships between RAS mutational status and antitumor activity in clinical trials. In preclinical research, it was shown that mutation in the RAS gene is not a prerequisite for activity of FTIs, and clinical responses, particularly in patients with hematological malignancies, were seen irrespective of RAS mutational status (35). Thus, antitumor activity of FTIs may not be dependent on the presence of a RAS mutation. The root of the problem lies in the fact that, although H-RAS is exclusively modified by farnesyltransferase, K-RAS and, to a lesser extent, N-RAS can also be modified by gernylgeranyltransferase (GGT). Attempting to inhibit the function of K-RAS and N-RAS by using FTIs and GGTIs together has failed because of the very high toxicity that is associated with this combination (36). Assessment of a dual inhibitor of FPTase and GGPTase-I, such as L-778,123 in humans indicated that the intended target of the drug, K-RAS, was not inhibited (37). In 19 patients with advanced NSCLC, no objective responses were seen (38). As activation of the RAS/Erk pathway through mutations in the RAS gene contributes to resistance of NSCLC cells to EGFR inhibitors, FTIs could overcome EGFR inhibitor resistance in K-RAS mutated tumors. Preclinical studies in A549 and H622 cell lines indicated that the EGFR tyrosine kinase inhibitor erlotinib

262

B. Besse and J.-C. Soria

showed additive cytotoxic effects in combination with the FTI tipifarnib. A single phase I trial (that did not included lung cancer patients) has combined tipifarnib and erlotinib. Diarrhea was the dose-limiting toxicity. A preliminary report of this study indicated that no response were observed, but 8 out of 15 patients were stabilized (39). Tipifarnib has also been combined with other tyrosine kinase inhibitors, such as sorafenib, and cutaneous rash was dose-limiting. Signs of activity were seen in thyroid cancers (but no data were available for NSCLC) (40, 41). RAF/MAPK Pathway B-RAF is recruited by activated RAS to further activate mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK). Although B-RAF is the kinase most frequently mutated in human tumors (frequently V600E mutation), the reported frequency of B-RAF mutations in NSCLC is low (2–3%) (28). B-RAF mutated tumors may have a distinct clinical profile since NSCLC cells with both V600E and non-V600E B-RAF mutations are selectively sensitive to MEK inhibition compared with those harboring mutations in EGFR or K-RAS (42). CI 1040 is a highly potent, orally active selective MEK inhibitor that showed promising results in phase I clinical trials in patients with advanced cancer (43). In contrast to this, phase II trials in patients with various cancer types, including NSCLC patients treated in first (n = 1) or second (n = 17) line, showed negative results (44). Immunohistochemical assessment of archived tumor specimens revealed that pERK expression was generally elevated, but not predictive of the CI 1040 benefit. B-RAF mutational status was not evaluated. AZD6244 is another orally available selective inhibitor of MEK1/2 that has been developed in a phase I setting and that demonstrated a reduced ERK phosphorylation (geometric mean, 79%) in 24 paired tumor biopsies. RAF or RAS mutations were detected in 10 of 26 assessable tumor samples. Nine of 57 patients had SD for at least 5 months (45). In a randomized phase II study, AZD6244 was compared to pemetrexed in second or third line in 84 NSCLC patients (46). Two PRs were reported in each group and the PFS was 67 days in the AZD6244 arm, and 90 days in the pemetrexed arm. An acneiform rash (43%), diarrhea (33%), nausea (20%), and vomiting (20%) were the most common side effects with AZD6244. XL518, an orally bioavailable inhibitor of MEK1, has been evaluated in a phase I trial in which a NSCLC patient was treated for at least 7 months (47). In comparison of pre and postdose samples, XL518 inhibited phosphorylation of ERK in tumor by 29% in one patient and by 52% in another patient, and similarly inhibited ERK phosphorylation in hair bulbs by 26% in one patient and by 42% in another. In contrast, XL518 did not cause consistent inhibition of MEK in peripheral blood cells. Other MEK inhibitors are being developed as single agents (RDEA119, AZD8330, GSK1120212, and RO5126766) or in combination with other drugs (RDEA119/sorafenib).

Other Molecular Targeted Agents in Non-small Cell Lung Cancer

263

Targeting MET The MET protein consists of a 50 kD extracellular alpha chain and a 140 kD transmembrane beta chain, which are linked by disulfide bonds. It contains phosphorylation sites in the JM (juxtamembrane), the TK (tyrosine kinase), and the tail domains (48–53). MET’s ligand is HGF (hepatocyte growth factor), also known as scatter factor, which is secreted by fibroblasts and smooth muscle cells and induces MET dimerization, autophosphorylation, and activation of tyrosine kinase catalytic activity (54). Tyrosine phosphorylation of JM, TK, and tail domains, respectively, regulate internalization, catalytic activity, and docking of substrates, such as Gab-1, Grb2, Shc, c-Cbl, which subsequently activate signal transducers such as PI3K, PLC-gamma, STAT, ERK1, ERK2, and FAK (55). The regulation of MET may be altered via overexpression, gene amplification, mutation, or epigenetic mechanisms (55). Met expression varies among the different cancer types (56). In NSCLC, the reported rates of Met expression or overexpression are 40–100% (56–60). Some studies have suggested a prognostic role of Met in NSCLC, but this remains uncertain, and additional studies using wellcharacterized and specific antibodies are warranted (61). Expression of HGF in non-neoplastic tissues is frequent (56). Tokunou et al. found Met stromal overexpression in 53% of lung adenocarcinomas, and this staining was associated with shorter patient survival (62). The HGF/c-MET system may then constitute an autocrine activation loop in cancer-stromal myofibroblasts. MET amplification or HGF expression has been observed in NSCLC, and it is associated with EGFRTKI resistance (63–65). MET amplification incidence is about 21% (9 out of 43) among patients with acquired resistance. Among untreated patients, it occurs much less frequently (3–21%) (63, 66, 67). MET amplification is able to activate the HER3-dependent PI3K/Akt pathway, and ultimately lead to gefitinib resistance (65). Its occurrence is independent of the T790M mutation (63). Inversely, HGF induces gefitinib resistance of lung adenocarcinoma cells with EGFRactivating mutations by restoring the PI3K/AKT signaling pathway via phosphorylation of MET, but not by phosphorylation of EGFR or HER3 (64). MET mutations have also been documented in NSCLC (56, 58). Ma et  al. described seven mutations from four NSCLC cell lines and 127 adenocarcinoma tissues (58). The mutations were found to be predominantly located in the non-kinase domain, namely the extracellular Sema domain and the short cytoplasmic juxtamembrane domain. Anti-HGF antibodies are being developed, such as AMG102 (Amgen, Inc.), a fully human IgG2 monoclonal antibody that binds and neutralizes SF/HGF, preventing its binding to c-MET and activation of this pathway (68). In an interim report of a single AMG102 phase I (n = 31), all grade toxicities were fatigue (52%), nausea (45%), vomiting (32%), and oedema (26%) (69). Stabilizations were reported but not specifically in NSCLC patients. A phase I trial is ongoing in SCLC.

264

B. Besse and J.-C. Soria

The inhibition of MET tyrosine kinase by small molecules is in early clinical development with either MET specific inhibitors (JNJ-38877605, ARQ-197, SGX523, and K252a) or multitargeted tyrosine kinase inhibitors that target both MET and other factors (XL880, XL184, PF2341066, MP-470, and MGCD265). Other agents undergoing early phase development include drugs from SGX, Inc., from MethylGene, Inc., and the agent HPK-56 (MP-470) from Supergen, Inc. ARQ197 (Arqule, Inc.) has demonstrated preclinical activity against several human xenografts, and is currently in clinical trials. A phase I study in 38 patients was reported with this orally administered, selective small molecule inhibitor of MET (70). Of 33 patients evaluable for efficacy; two achieved a PR and 19 achieved SD. There was no dose-limiting toxicity. Adverse events included fatigue (24%) and GI symptoms such as constipation (21%) and diarrhea (21%). Grade 3 toxicities included elevation in liver function enzymes seen in 3% of treated patients. The optimal dose was determined to be 120 mg PO BID. A randomized phase 2 study of erlotinib + ARQ 197 vs. erlotinib + placebo in previously treated patients with locally advanced or metastatic NSCLC is ongoing. XL880 (Exelixis, Inc.) is another orally bioavailable small molecule targeted to multiple receptor tyrosine kinases, including MET, VEGFR, c-Kit, FLT3, PDGRF, Ron, and Tie-2. Results of two phase I clinical trials were recently reported (71). Toxicities were similar to those with anti-VEGFR agents (asymptomatic elevation in liver function enzymes, palmar-plantar erythrodysesthesia, tumor hemorrhage hypertension, and dehydration). Activity of the agent was seen in tumors sensitive to antiangiogenic agents (e.g., papillary or clear cell renal carcinoma, colorectal carcinoma) and in MET amplified poorly differentiated gastric cancer (72, 73). XL184 (Exelixis, Inc.) also targets multiple receptor tyrosine kinases other than MET, including VEGFR2/KDR, KIT, FLT3, and Tie-2. Results from a phase I trial in 55 patients reported 6 DLTs including grade 3 palmar/plantar erythema and grade 3 elevation in liver function enzymes and lipase. Prominent non-DLT toxicities included diarrhea and hypopigmentation of the hair. No NSCLC patient was included, but a phase II trial is ongoing in this population, alone and in combination with erlotinib.

Antiangiogenic-Related Agents Protein kinase C inhibitors and agents targeting integrins were first developed as antiangiogenic agents. Two major observations raise the possibility that the main mechanism of action of these drugs may be more than anti-angiogenesis alone. First, in vitro studies have revealed a direct effect of these drugs on tumor cell proliferation. Second, the toxicity profile radically differs from the classical vascular effects of antiangiogenics, with a low incidence of hypertension, thrombosis, and bleeding. Thus, these agents will probably be reclassified in the near future, in the light of upcoming fundamental discoveries.

Other Molecular Targeted Agents in Non-small Cell Lung Cancer

265

Protein Kinase C (PKC) The protein kinase C (PKC) family comprises 12 different isozymes (74). Remarkable heterogeneity exists within the PKC signal transduction pathway. Clinical research in lung cancer has focused, until now, on cPKC isoforms (PKCa, PKCßI, PKCßII, and PKCg) that may be referred to as “conventional isoforms” and that are diacylglycerol (DAG) sensitive and Ca2+ responsive (responding through an archetypal C2 domain). PKCa has an increased expression in tumor compared to normal tissue, and has been implicated in malignant transformation and proliferation (75). Preclinical evidence of antitumor activity by inhibition of PKC-a was demonstrated in a lung cancer model in a variety of in vitro and in vivo experiments (76). The mechanism of action of aprinocarsen (ISIS 3521) and other antisense oligonucleotides depends on their binding to the cognate sequence within the mRNA, resulting in the inhibition of mRNA translation and in RNase H-mediated mRNA degradation. In untreated NSCLC patients, aprinocarsen has been tested in combination with cisplatin-based chemotherapy (77). The combination toxicity was very similar to what is generally expected for the standard cisplatin regimen. In a randomized phase III study, aprinocarsen did not prolong survival in advanced NSCLC when combined with carboplatin/paclitaxel (78) or with cisplatin–gemcitabine (79). PKCß plays a role in the regulation of tumor-induced angiogenesis, cell cycle progression, tumor cell proliferation, survival, and tumor invasiveness (80, 81). Using a standard immunohistochemistry technique, PKCßII was expressed in 71% (45/63) of NSCLC patients (82). Enzastaurin, an orally available PKCß inhibitor, was originally evaluated in human tumor xenograft-bearing mice for its anti-angiogenic activity upon PKCß inhibition, as it induced reduction of plasma VEGF levels together with a significant decrease in intratumoral vessel density (83). However, several studies have shown that enzastaurin exhibits direct growth inhibiting effects on a wide array of cultured human tumor cells and that its antitumor effects are mediated through interference with the PI3K/AKT pathway (84, 85). In 55 metastatic NSCLC patients, enzastaurin had minor activity as a single agent in secondor third-line therapy (86). In this cohort, median PFS was 1.8 months (95% CI, 1.7–1.9) and median overall survival (OS) was 8.4 months (95% CI, 6.0–13.6 months). Nineteen patients (35%) had SD (no objective responses were observed). The most common toxicity was fatigue. In the A549 NSCLC cell line, enzastaurin exposure resulted in G2/M checkpoint abrogation and apoptosis induction in pemetrexed-damaged cells. Enzastaurin also significantly reduced pemetrexedinduced upregulation of TS expression, thus providing an experimental basis to their evaluation in combination (87). In vivo, enzastaurin appeared to increase the anemia and thrombocytopenia of the pemetrexed–carboplatin combination in a randomized phase II study (88). Efficacy data are pending for this latter study and for a randomized phase II study of pemetrexed–placebo vs. pemetrexed– enzastaurin. The PKC family represents an interesting and challenging target for the development of new therapeutic agents but results to date in the clinic have been disappointing.

266

B. Besse and J.-C. Soria

Improvements have to be made to design more specific inhibitors (given the complexity of the functions and interactions of the PKC isozymes), to develop predictive biomarkers, and to examine the combination of PKC inhibitors with conventional cytotoxics or molecular targeted agents.

Integrins Integrins are heterodimer transmembrane receptors for the extracellular matrix composed of an alpha and beta subunit. A particular feature of integrins is their tight regulation of ligand binding activity. Transition from a low to a high-affinity state (“affinity maturation”) can be induced by intracellular signaling events (“inside-out” signaling) or by high-affinity ligands (89). Natural integrin ligands include laminin, fibronectin, and vitronectin, but they also encompass fibrinogen and fibrin, thrombospondin, MMP-2, and fibroblast growth factor 2. Ligand binding induces allosteric changes in the receptor conformation, leading to the activation of intracellular signaling pathways, including the RAS-MAPK, PI3K-PKB-mTOR, and small GTPases (e.g., Rho, Rac) pathways (“outside-in” signaling) (89). In preclinical studies, pharmacologic inhibition of integrin function efficiently suppressed angiogenesis and inhibited tumor progression (90). Of the 24 known integrin heterodimers, aVß3 and aVß5 were the first vascular integrins targeted to suppress tumor angiogenesis. The differential expression of integrin aVb3 (present at low levels on quiescent endothelial cells, but strongly induced on tumoral angiogenic endothelial cells) is of particular interest to inhibit pathological angiogenesis. Three classes of integrin inhibitors are currently in preclinical and clinical development: monoclonal antibodies targeting the extracellular domain of the heterodimer (Vitaxin, volociximab), synthetic peptides containing an RGD sequence (cilengitide), and peptidomimetics (S247), which are orally bioavailable non-peptidic molecules mimicking the RGD sequence. MEDI-523 (Vitaxin) was the first monoclonal antibody directed against aVb3 that was developed in clinical trials. Due to a lack of efficacy, an IgG1 humanized monoclonal antibody (etaracizumab, MEDI-522, Abegrin) was genetically engineered as an affinity-matured derivative of MEDI-523. In vivo, tumor growth of avß3-expressing tumor cells is inhibited by etaracizumab in a dose-dependent manner (91). Interestingly, the antitumor activity of etaracizumab steadily increased when animals were treated with 1–10 mg/kg, but the biological activity of etaracizumab decreased at doses >20 mg/kg. In a phase I study, the toxicity profile was favorable. Frequently reported adverse events were grades 1–2 asthenia and infusion reactions (92). Phase II studies are ongoing in metastatic renal cancer since disease stabilization was seen in renal cancer in phase I trials. Cilengitide is an IV agent that binds with high affinity to aVb3 (IC50 of 0.6 nM) and inhibits aVb3 and aVb5-dependent adhesion (93). The pharmacokinetic (PK) profile revealed a short elimination half-life of 4 h, and the most common

Other Molecular Targeted Agents in Non-small Cell Lung Cancer

267

grade 1 or 2 adverse events, included fatigue, nausea, and headache. As for all the integrin inhibitors, the toxicity profile is significantly different from that of usual antiangiogenic agents (that induce hypertension and hemorrhage). It shows modest activity in patients with recurrent gliomas (94). Phase II studies are planned in NSCLC patients. Volociximab, a function-blocking, human/mouse chimeric antibody against integrin a5b1, inhibits endothelial cell proliferation, at least in part by promoting cell death (95). In a rabbit model, volociximab was found to significantly inhibit the growth of tumors (96). In a phase I study, mild (grade 1 or 2), reversible fatigue was the principal toxicity of volociximab at the highest dose levels of 10 and 15 mg/kg (97). Nausea, fever, anorexia, headache, vomiting, and myalgias were mild and infrequent, and there was no hematologic toxicity. Phase II trials have been performed in melanoma and ovarian cancer. A phase Ib study is ongoing in NSCLC in combination with paclitaxel and carboplatin.

Apoptosis The ability to resist and bypass programmed cell death, a process that is normally induced as a response to some external physiological signal or internal stress, is one of the main features of cancer cells. Apoptotic effectors are interesting candidates as anticancer targets.

Targeting Bcl-2 The Bcl-2 family consists of a homologous network of genes that regulate apoptosis or programmed cell death. Bcl-2 protects mitochondrial membrane activity, thereby preventing the release of cytochrome c and the formation of the apoptosome and caspase-9 activation. Thus, it is an antiapoptotic protein. The mutation and overexpression of Bcl-2 by a specific translocation (t(14,18)) is a major oncogenetic event in follicular lymphoma and is possibly an important prognostic factor in large B cell lymphoma (98). Surprisingly, according to a recent review, Bcl-2 expression could confer a favorable prognosis to patients with NSCLC (99). Obatoclax (GX15-070) is an antagonist of the BH3-binding groove of the Bcl-2 family of antiapoptotic proteins. It induced apoptosis in a subset of NSCLC cell lines, and it has been evaluated and combined with docetaxel in a phase I study (100, 101). Eighteen advanced NSCLC patients that received a median of one prior regimen (range, 1–6) were included. Reported side effects are somnolence and euphoria. There have been two PRs (11%) and four patients confirmed to have SD (22%). The combination is currently being evaluated in a phase II trial in relapsed NSCLC.

268

B. Besse and J.-C. Soria

High levels of Bcl-2 and Bcl-xL have been described in SCLC (102). ABT-263 is an oral inhibitor of Bcl-2, Bcl-xL, and Bcl-w. Strong early signs of antitumor activity against lymphomas and specific platelet toxicity have been reported in phase I studies (103).

Proteasome Inhibitors The 26S proteasome degrades ubiquinated proteins; it then plays a central role in the regulation of a wide variety of proteins involved in cell cycle regulation and apoptosis, such as cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors, c-myc, or nuclear factor kappa B. Bortezomib is a proteasome inhibitor approved by the FDA for use in the treatment of relapsed and refractory multiple myeloma patients. Proteasome inhibition with bortezomib has also shown activity and manageable toxicity in mantle cell and other lymphomas, and in solid malignancies, including NSCLC (104, 105). It has been tested in 155 pretreated patients with advanced NSCLC in a randomized phase II study (106). Bortezomib alone induced a PR in 8% of cases, while in combination with docetaxel, PR was reported in 9% of cases. A similar study with pemetrexed has been conducted and results are awaited. Bortezomib has been combined with erlotinib and compared to erlotinib alone in 50 pretreated NSCLC patients (107). Among 24 response-evaluable patients in each arm, there were three PRs and one complete response in the single agent arm, and two PRs in the combination arm. Median PFS was 2.7 and 1.4 months, respectively. The study was halted as required at the planned interim analysis due to insufficient clinical activity of the combination. Adverse-event profiles were as expected in both arms, with no significant additivity. Bortezomib has been evaluated in front line therapy in combination with gemcitabine and a platinum compound in two phase I studies (108). With carboplatin, this combination demonstrated encouraging activity in patients with advanced NSCLC, with a 35% response rate. The combination of bortezomib, gemcitabine, and carboplatin was associated with manageable toxicities, of which the most common was thrombocytopenia. Surprisingly, neurotoxicity was not a DLT with the triplet. There was no grade 3/4 neuropathy. This regimen was evaluated in a phase II study that included 114 patients (52% adenocarcinoma, 85% stage IV) (109). The median OS was 11 months and the median PFS was 5 months. Response rate was 23%. The most common grade 3/4 toxicities were thrombocytopenia (63%) and neutropenia (52%). Grade 3/4 neuropathy occurred in 4%, and a further 6% experienced grade 2 sensory neuropathy. The tolerance seemed to be quite equivalent with cisplatin in a phase I study in 34 patients (110). Most common grade 3 treatment-related toxicities were thrombocytopenia and neutropenia. No grade 3 treatment-related sensory neuropathy was reported. All patients had evaluable disease, and 13 achieved PRs, 17 had SD, and 4 had progressive disease. In contrast, single agent activity in a front line study was disappointing in two phase II studies that both closed early due to insufficient activity. In the first study,

Other Molecular Targeted Agents in Non-small Cell Lung Cancer

269

bortezomib was administrated intravenously at the dose of 1.5 mg/m2/day on days 1, 4, 9, and 11 for every 21 days (defined as one cycle) in 17 untreated NSCLC patients (111). Twelve patients (71%) had grade 3 toxicity: four hematological, one infection, three anorexia, one nausea, one stomatitis, nine fatigue. Thirteen patients developed grade 1/2 neuropathy. The non-progression rate was 59% [33–82%] at 6 weeks (10/17 patients) and the median PFS was 2.4 m. The median OS was 9.8 m. No objective responses were seen, and no PET responses were seen. The second study used a lower dose of bortezomib (1.3  mg/m2/day) and was terminated after 14 patients enrolled (112). Twelve patients (median age 70 years, range 50–84; female/male 6/6; median ECOG performance status 1) received at least one dose and a median of two cycles of bortezomib. There was no objective response observed. Three patients (25%) had SD and received eight, six and four cycles of treatment; the duration of response was 11.0, 2.8, and 2.0 months, respectively. Median time to progression was 1.4 months (range, 0.6–11.5 months). Grade 3 toxicities included fatigue (8%), deep vein thrombosis (8%), and thrombocytopenia (8%). Hence, bortezomib monotherapy is not active in chemotherapy-naïve, advanced NSCLC patients. Further study in combination therapy may be warranted.

Death Receptors Death Receptors belong to the Tumor Necrosis Factor Receptor (TNFR) gene superfamily. They contain a homologous cytoplasmic sequence termed the “death domain.” Adapter-molecules like FADD, TRADD, or DAXX themselves contain death domains so that they can interact with the death receptors and transmit the apoptotic signal to the death-machinery when death receptors are activated. AMG 951 consists of amino acids 114–281 of the native Apo2L/TRAIL that triggers apoptosis through activation of two specific receptors – death receptor 4 (DR4) and death receptor 5 (DR5) – that belong to the TNF receptor superfamily (113). In the first-in-human study, rhApo2L/TRAIL was well tolerated and showed antitumor efficacy, including an objective PR in a patient with a chondrosarcoma (114). In NSCLC, AMG 951 has been evaluated in a phase I study, combined with paclitaxel, carboplatin, and bevacizumab as a front line treatment for metastatic or advanced non-squamous lung carcinomas (115). Twenty-four patients received ³1 dose of rhApo2L/TRAIL plus paclitaxel/carboplatin/bevacizumab. Of these, 18 completed six cycles of therapy, and six discontinued treatment: three due to disease progression and three withdrew from treatment (1 after cycle 3 due to grade 2 bowel perforation, 1 after cycle 4 due to grade 1 arthralgia, and 1 after cycle 5 due to Gilbert’s syndrome). There were no DLTs. Of 18 patents in the first three cohorts, ten had confirmed responses (nine partial, one complete) with an overall response rate of 56%. A phase II study is ongoing. The same pathway is targeted by AMG 655, a fully human monoclonal agonist antibody that binds human TRAIL receptor 2 (TR-2/ DR5). In the first, in human phase I, there was no maximal tolerated dose identified. Fatigue and elevated serum lipase were the only grade 3 or higher events (8).

270

B. Besse and J.-C. Soria

Mapatumumab (TRM-1, HGS-ETR1) is a fully human agonistic monoclonal antibody that binds and activates the tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1) (116). A phase 1 study was performed to evaluate the safety, tolerability and PKs of mapatumumab in combination with gemcitabine and cisplatin. Of the 49 patients treated, six had NSCLC. The DoseLimiting Toxicity included elevations of transaminases, neutropenic fever, thrombocytopenia, and fatigue, but there was no apparent evidence that mapatumumab exacerbates adverse events associated with chemotherapy. None of the nine patients experiencing a PR had a NSCLC.

Perspectives The proven success of EGFR and angiogenesis inhibitors in advanced NSCLC has quickly led to their evaluation in early-stage disease in the adjuvant setting. The RADIANT trial is evaluating erlotinib as adjuvant treatment in early-stage disease, whereas a non-randomized phase II trial (NCT00406302) evaluated the neoadjuvant effect of cetuximab (in combination with docetaxel and oxaliplatin) in resectable NSCLC. Similarly, for antiangiogenic agents, a large phase III Intergroup trial (E1505) is underway to evaluate the addition of bevacizumab to adjuvant platinumbased chemotherapy, whereas pazopanib (an oral VEGFR inhibitor) is being evaluated as a single agent in stage I patients (French intergroup trial IFCT 0703). None of the new molecular targeted agents reviewed in this chapter has reached an approval in NSCLC, but some are good potential candidates due to their tolerance profile and early signs of activity. Even without clear efficacy in the metastatic setting, efficacy in the adjuvant setting cannot be ruled out. As an example, a benefit of single-agent uracil-tegafur has been demonstrated in the adjuvant setting, whereas it is not a validated regimen in patients with metastatic disease (117). Clinical trials must continue to investigate the efficacy of biologic agents in different settings knowing the potential pitfalls that have been highlighted by agents targeting EGFR or VEGFR:

Pitfalls in Combination with Chemotherapy The risk of a lack of benefit has to be strongly considered even if the drug is effective as a single agent, as for erlotinib, active in second or third line, but ineffective if combined with a platinum combination in front line therapy (118, 119). Dose and sequence may be two of the key issues of the “biochemotherapy” combination, as suggested recently in a randomized phase II trial (120). In chemotherapy-naive stage IIIB or IV NSCLC patients, a short course of high dose erlotinib followed by chemotherapy seems more effective than the opposite schema.

Other Molecular Targeted Agents in Non-small Cell Lung Cancer

271

Pitfalls in Combination with other Biologic Agents Those combinations may lead to elevated rates of side effects, but also unexpected ones, in particular, if both drugs target the same pathway. For example, the phase I trial reported by Feldman et al. (combining sunitinib and bevacizumab) is striking because of the high degree of vascular, renal, and hematologic toxicities reported: the most frequently reported grade 3/4 adverse events in that trial included hypertension (60%), proteinuria (36%), and thrombocytopenia (24%). Furthermore, five patients had laboratory and clinical features consistent with microangiopathic hemolytic anemia (MAHA). In terms of efficacy, poor activity of a single agent does not preclude high activity in combination therapy, if the drug reverses the resistance of an active drug. For example, loss of PTEN and AKT/mTOR activation have been reported to mediate resistance to trastuzumab. Indeed, everolimus (an mTOR inhibitor) reversed trastuzumab resistance in a phase I trial (121).

Pitfalls in Combination with Radiation Therapy No cytotoxic agent or molecular targeted agent has significantly increased radiosensitivity in NSCLC in the past decade. Potential candidates are, however, in clinical development. For example, the in vitro rationale for combining EGFR inhibition and concurrent ionizing radiation is well established, but to date, scant clinical data are available in lung cancer. The harmful effect of the EGFR inhibitor, gefitinib, given after radio-chemotherapy for locally advanced NSCLC, is a strong signal to carefully evaluate those new drugs in combination with radiotherapy (122). Radiation therapy may have a strong impact on tumor biology and modify tumor response to targeted agents.

Conclusion In their seminal paper on cancer biology, Hanahan and Weinberg listed six hallmarks for cancer cells. More than 500 molecular targeted therapy products are currently being developed, covering the entire range of those six hallmarks. In lung cancer, two targeted therapies have already been approved by the FDA in advanced NSCLC: the EGFR TKI erlotinib and the antiangiogenic bevacizumab; while a third one (cetuximab) is expected to be approved shortly. Orally available antiangiogenic compounds and pan-HER inhibitors may be the next generation of approved agents, given their activity in advanced NSCLC. The field of predictive markers has to be actively clarified given the significant toxicities and costs of these new agents. Adjuvant trials integrating

272

B. Besse and J.-C. Soria

these agents are also awaited in order to potentially cure more resected patients. Fewer studies have been conducted in patients with SCLC, which is sometimes considered to be an orphan disease, whereas it still concerns nearly 15% of our lung cancer patients. Acknowledgment  Cedric Verjat (figure design)

References 1. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100(1):57–70 2. Pollak M (2008) Insulin and insulin-like growth factor signalling in neoplasia. Nat Rev Cancer 8(12):915–928 3. Tao Y, Pinzi V, Bourhis J, Deutsch E (2007) Mechanisms of disease: signaling of the insulinlike growth factor 1 receptor pathway – therapeutic perspectives in cancer. Nat Clin Prac Oncol 4(10):591–602 4. Cappuzzo F, Toschi L, Tallini G, Ceresoli GL, Domenichini I, Bartolini S et al (2006) Insulinlike growth factor receptor 1 (IGFR-1) is significantly associated with longer survival in nonsmall-cell lung cancer patients treated with gefitinib. Ann Oncol 17(7):1120–1127 5. Long L, Rubin R, Brodt P (1998) Enhanced invasion and liver colonization by lung carcinoma cells overexpressing the type 1 insulin-like growth factor receptor. Exp Cell Res 238(1):116–121 6. Haluska P, Shaw HM, Batzel GN, Yin D, Molina JR, Molife LR et al (2007) Phase I dose escalation study of the anti insulin-like growth factor-I receptor monoclonal antibody CP-751,871 in patients with refractory solid tumors. Clin Cancer Res 13(19):5834–5840 7. Rodon J, Patnaik A, Stein M, Tolcher A, Ng C, Dias C et al (2007) A phase I study of q3W R1507, a human monoclonal antibody IGF-1R antagonist in patients with advanced cancer. J Clin Oncol (Meet Abstr) 25(18 suppl):3590 8. Tolcher AW, Rothenberg ML, Rodon J, Delbeke D, Patnaik A, Nguyen L et al (2007) A phase I pharmacokinetic and pharmacodynamic study of AMG 479, a fully human monoclonal antibody against insulin-like growth factor type 1 receptor (IGF-1R), in advanced solid tumors. J Clin Oncol (Meet Abstr) 25(18 suppl):3002 9. Tolcher AW, Patnaik A, Till E, Takimoto CH, Papadopoulos KP, Massard C et al (2008) A phase I study of AVE1642, a humanized monoclonal antibody IGF-1R (insulin like growth factor1 receptor) antagonist, in patients (pts) with advanced solid tumor (ST). J Clin Oncol (Meet Abstr) 26(15 suppl):3582 10. Olmos D, Okuno S, Schuetze SM, Paccagnella ML, Yin D, Gualberto A et al (2008) Safety, pharmacokinetics and preliminary activity of the anti-IGF-IR antibody CP-751,871 in patients with sarcoma. J Clin Oncol (Meet Abstr) 26(15 suppl):10501 11. Karp DD, Paz-Ares LG, Novello S, Haluska P, Garland L, Cardenal F et al (2008) High activity of the anti-IGF-IR antibody CP-751,871 in combination with paclitaxel and carboplatin in squamous NSCLC. J Clin Oncol (Meet Abstr) 26(15 suppl):8015 12. Guha U, Chaerkady R, Marimuthu A, Patterson AS, Kashyap MK, Harsha HC et al (2008) Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS. Proc Natl Acad Sci 105(37):14112–14117 13. Balsara BR, Pei J, Mitsuuchi Y, Page R, Klein-Szanto A, Wang H et al (2004) Frequent activation of AKT in non-small cell lung carcinomas and preneoplastic bronchial lesions. Carcinogenesis 25(11):2053–2059 14. Bjornsti MA, Houghton PJ (2004) The tor pathway: a target for cancer therapy. Nat Rev Cancer 4(5):335–348 15. Cully M, You H, Levine AJ, Mak TW (2006) Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat Rev Cancer 6(3):184–192

Other Molecular Targeted Agents in Non-small Cell Lung Cancer

273

16. Wislez M, Spencer ML, Izzo JG, Juroske DM, Balhara K, Cody DD et al (2005) Inhibition of mammalian target of rapamycin reverses alveolar epithelial neoplasia induced by oncogenic K-ras. Cancer Res 65(8):3226–3235 17. Wu C, Wangpaichitr M, Feun L, Kuo M, Robles C, Lampidis T et  al (2005) Overcoming cisplatin resistance by mTOR inhibitor in lung cancer. Mol Cancer 4(1):25 18. Marsit CJ, Zheng S, Aldape K, Hinds PW, Nelson HH, Wiencke JK et al (2005) PTEN expression in non-small-cell lung cancer: evaluating its relation to tumor characteristics, allelic loss, and epigenetic alteration. Hum Pathol 36(7):768–776 19. Kokubo Y, Gemma A, Noro R, Seike M, Kataoka K, Matsuda K et al (2005) Reduction of PTEN protein and loss of epidermal growth factor receptor gene mutation in lung cancer with natural resistance to gefitinib (IRESSA). Br J Cancer 92(9):1711–1719 20. Janmaat ML, Rodriguez JA, Gallegos-Ruiz M, Kruyt FA, Giaccone G (2006) Enhanced cytotoxicity induced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol-3 kinase pathways in non-small cell lung cancer cells. Int J Cancer 118(1):209–214 21. Hudes G, Carducci M, Tomczak P, Dutcher J, Figlin R, Kapoor A et al (2007) Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med 356(22): 2271–2281 22. Motzer RJ, Escudier B, Oudard S, Hutson TE, Porta C, Bracarda S et al (2008) Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled phase III trial. The Lancet 372(9637):449–456 23. Papadimitrakopoulou V, Soria JC, Douillard JY, Giaccone G, Wolf J, Crino L et al (2007) A phase II study of RAD001 (R) (everolimus) monotherapy in patients (pts) with advanced nonsmall cell lung cancer (NSCLC) failing prior platinum-based chemotherapy (C) or prior C and EGFR inhibitors (EGFR-I). J Clin Oncol (Meet Abstr) 25(18 suppl):7589 24. Milton DT, Riely GJ, Azzoli CG, Gomez JE, Heelan RT, Kris MG et al (2007) Phase 1 trial of everolimus and gefitinib in patients with advanced nonsmall-cell lung cancer. Cancer 110(3):599–605 25. Kris MG, Riely GJ, Azzoli CG, Heelan RT, Krug LM, Pao W et al (2007) Combined inhibition of mTOR and EGFR with everolimus (RAD001) and gefitinib in patients with non-small cell lung cancer who have smoked cigarettes: a phase II trial. J Clin Oncol (Meet Abstr) 25(18 suppl):7575 26. Papadimitrakopoulou V, Blumenschein GR Jr, Leighl NB, Bennouna J, Soria JC, Burris HA III et al (2008) A phase 1/2 study investigating the combination of RAD001 (R) (everolimus) and erlotinib (E) as 2nd and 3rd line therapy in patients (pts) with advanced non-small cell lung cancer (NSCLC) previously treated with chemotherapy (C): phase 1 results. J Clin Oncol (Meet Abstr) 26(15 suppl):8051 27. Boguski MS, McCormick F (1993) Proteins regulating Ras and its relatives. Nature 366(6456):643–654 28. Herbst RS, Heymach JV, Lippman SM (2008) Lung cancer. N Engl J Med 359(13):1367–1380 29. Riely GJ, Kris MG, Rosenbaum D, Marks J, Li A, Chitale DA et al (2008) Frequency and distinctive spectrum of KRAS mutations in never smokers with lung adenocarcinoma. Clin Cancer Res 14(18):5731–5734 30. Mascaux C, Iannino N, Martin B, Paesmans M, Berghmans T, Dusart M et al (2004) The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis. Br J Cancer 92(1):131–139 31. Crul M, de Klerk GJ, Swart M, van’t Veer LJ, de Jong D, Boerrigter L et al (2002) Phase I clinical and pharmacologic study of chronic oral administration of the farnesyl protein transferase inhibitor R115777 in advanced cancer. J Clin Oncol 20(11):2726–2735 32. Heymach JV, Johnson DH, Khuri FR, Safran H, Schlabach LL, Yunus F et al (2004) Phase II study of the farnesyl transferase inhibitor R115777 in patients with sensitive relapse small-cell lung cancer. Ann Oncol 15(8):1187–1193 33. Adjei AA, Mauer A, Bruzek L, Marks RS, Hillman S, Geyer S et al (2003) Phase II study of the farnesyl transferase inhibitor R115777 in patients with advanced non-small-cell lung cancer. J Clin Oncol 21(9):1760–1766

274

B. Besse and J.-C. Soria

34. Kim ES, Kies MS, Fossella FV, Glisson BS, Zaknoen S, Statkevich P et al (2005) Phase II study of the farnesyltransferase inhibitor lonafarnib with paclitaxel in patients with taxanerefractory/resistant nonsmall cell lung carcinoma. Cancer 104(3):561–569 35. Appels NMGM, Beijnen JH, Schellens JHM (2005) Development of farnesyl transferase inhibitors: a review. Oncologist 10(8):565–578 36. Lobell RB, Omer CA, Abrams MT, Bhimnathwala HG, Brucker MJ, Buser CA et al (2001) Evaluation of farnesyl:protein transferase and geranylgeranyl:protein transferase inhibitor combinations in preclinical models. Cancer Res 61(24):8758–8768 37. Lobell RB, Liu D, Buser CA, Davide JP, DePuy E, Hamilton K et al (2002) Preclinical and clinical pharmacodynamic assessment of L-778, 123, a dual inhibitor of farnesyl:protein transferase and geranylgeranyl:protein transferase type-I. Mol Cancer Ther 1(9):747–758 38. Evans TL, Fidias P, Skarin AT, Johnson BE, Moore M, Pramanik B et al (2002) A phase II study of efficacy and tolerability of the farnesyl-protein transferase inhibitor L-778, 123 as first-line therapy in patients with advanced non-small cell lung cancer (NSCLC). J Clin Oncol (Meet Abstr) 21:1861 39. Ma CX, Croghan G, Reid J, Hanson L, Mandrekar S, Marks R et al (2005) A phase I trial of the combination of erlotinib and tipifarnib in patients with advanced solid tumors. J Clin Oncol (Meet Abstr) 23(16 suppl)):3000 40. Chintala L, Kurzrock R, Fu S, Naing A, Wheler JJ, Moulder SL et al (2008) Phase I study of tipifarnib and sorafenib in patients with biopsiable advanced cancer (NCI protocol 7156). J Clin Oncol (Meet Abstr) 26(15 suppl):3593 41. Hong D, Ye L, Gagel R, Chintala L, El Naggar AK, Wright J et al (2008) Medullary thyroid cancer: targeting the RET kinase pathway with sorafenib/tipifarnib. Mol Cancer Ther 7(5):1001–1006 42. Pratilas CA, Hanrahan AJ, Halilovic E, Persaud Y, Soh J, Chitale D et al (2008) Genetic predictors of MEK dependence in non-small cell lung cancer. Cancer Res 68(22):9375–9383 43. LoRusso PM, Adjei AA, Varterasian M, Gadgeel S, Reid J, Mitchell DY et al (2005) Phase I and pharmacodynamic study of the oral MEK inhibitor CI-1040 in patients with advanced malignancies. J Clin Oncol 23(23):5281–5293 44. Rinehart J, Adjei AA, LoRusso PM, Waterhouse D, Hecht JR, Natale RB et  al (2004) Multicenter phase II study of the oral MEK inhibitor, CI-1040, in patients with advanced nonsmall-cell lung, breast, colon, and pancreatic cancer. J Clin Oncol 22(22):4456–4462 45. Adjei AA, Cohen RB, Franklin W, Morris C, Wilson D, Molina JR et al (2008) Phase I pharmacokinetic and pharmacodynamic study of the oral, small-molecule mitogen-activated protein kinase kinase 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancers. J Clin Oncol 26(13):2139–2146 46. Tzekova V, Cebotaru C, Ciuleanu TE, Damjanov D, Ganchev H, Kanarev V et al (2008) Efficacy and safety of AZD6244 (ARRY-142886) as second/third-line treatment of patients (pts) with advanced non-small cell lung cancer (NSCLC). J Clin Oncol (Meet Abstr) 26(15 suppl):8029 47. Rosen LS, Galatin P, Fehling JM, Laux I, Dinolfo M, Frye J et  al (2008) A phase 1 doseescalation study of XL518, a potent MEK inhibitor administered orally daily to subjects with solid tumors. J Clin Oncol (Meet Abstr) 26(15 suppl):14585 48. Gandino L, Longati P, Medico E, Prat M, Comoglio PM (1994) Phosphorylation of serine 985 negatively regulates the hepatocyte growth factor receptor kinase. J Biol Chem 269(3): 1815–1820 49. Abella JV, Peschard P, Naujokas MA, Lin T, Saucier C, Urbe S et al (2005) Met/Hepatocyte growth factor receptor ubiquitination suppresses transformation and is required for Hrs phosphorylation. Mol Cell Biol 25(21):9632–9645 50. Petrelli A, Gilestro GF, Lanzardo S, Comoglio PM, Migone N, Giordano S (2002) The endophilin-CIN85-Cbl complex mediates ligand-dependent downregulation of c-Met. Nature 416(6877):187–190 51. Fournier TM, Kamikura D, Teng K, Park M (1996) Branching tubulogenesis but not scatter of madin-darby canine kidney cells requires a functional Grb2 binding site in the Met receptor tyrosine kinase. J Biol Chem 271(36):22211–22217

Other Molecular Targeted Agents in Non-small Cell Lung Cancer

275

52. Rodrigues GA, Park M (1994) Autophosphorylation modulates the kinase activity and oncogenic potential of the Met receptor tyrosine kinase. Oncogene 9(7):2019–2027 53. Weidner KM, Sachs M, Riethmacher D, Birchmeier W (1995) Mutation of juxtamembrane tyrosine residue 1001 suppresses loss-of-function mutations of the Met receptor in epithelial cells. Proc Natl Acad Sci USA 92(7):2597–2601 54. Peruzzi B, Bottaro DP (2006) Targeting the c-Met signaling pathway in cancer. Clin Cancer Res 12(12):3657–3660 55. Cipriani NA, Abidoye OO, Vokes E, Salgia R (2009) MET as a target for treatment of chest tumors. Lung Cancer 63(2):169–179 56. Ma PC, Tretiakova MS, MacKinnon AC, Ramnath N, Johnson C, Dietrich S et  al (2008) Expression and mutational analysis of MET in human solid cancers. Genes Chromosomes Cancer 47(12):1025–1037 57. Masuya D, Huang C, Liu D, Nakashima T, Kameyama K, Haba R et al (2004) The tumour-stromal interaction between intratumoral c-Met and stromal hepatocyte growth factor associated with tumour growth and prognosis in non-small-cell lung cancer patients. Br J Cancer 90(8): 1555–1562 58. Ma PC, Jagadeeswaran R, Jagadeesh S, Tretiakova MS, Nallasura V, Fox EA et  al (2005) Functional expression and mutations of c-Met and its therapeutic inhibition with SU11274 and small interfering RNA in non-small cell lung cancer. Cancer Res 65(4):1479–1488 59. Tsao MS, Liu N, Chen JR, Pappas J, Ho J, To C et al (1998) Differential expression of Met/hepatocyte growth factor receptor in subtypes of non-small cell lung cancers. Lung Cancer 20(1):1–16 60. Takanami I, Tanana F, Hashizume T, Kikuchi K, Yamamoto Y, Yamamoto T et  al (1996) Hepatocyte growth factor and c-Met/hepatocyte growth factor receptor in pulmonary adenocarcinomas: an evaluation of their expression as prognostic markers. Oncology 53(5):392–397 61. Zhu CQ, Shih W, Ling CH, Tsao MS (2006) Immunohistochemical markers of prognosis in non-small cell lung cancer: a review and proposal for a multiphase approach to marker evaluation. J Clin Pathol 59(8):790–800 62. Tokunou M, Niki T, Eguchi K, Iba S, Tsuda H, Yamada T et al (2001) c-MET expression in myofibroblasts: role in autocrine activation and prognostic significance in lung adenocarcinoma. Am J Pathol 158(4):1451–1463 63. Bean J, Brennan C, Shih JY, Riely G, Viale A, Wang L et al (2007) MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci 104(52):20932–20937 64. Yano S, Wang W, Li Q, Matsumoto K, Sakurama H, Nakamura T et al (2008) Hepatocyte growth factor induces gefitinib resistance of lung adenocarcinoma with epidermal growth factor receptor-activating mutations. Cancer Res 68(22):9479–9487 65. Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO et  al (2007) MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316(5827):1039–1043 66. Beau-Faller M, Ruppert AM, Voegeli AC, Neuville A, Meyer N, Guerin E et al (2008) MET gene copy number in non-small cell lung cancer: molecular analysis in a targeted tyrosine kinase inhibitor naive cohort. J Thorac Oncol 3(4):331–339 67. Cappuzzo F, Janne PA, Skokan M, Finocchiaro G, Rossi E, Ligorio C et  al (2009) MET increased gene copy number and primary resistance to gefitinib therapy in non-small-cell lung cancer patients. Ann Oncol 20(2):298–304 68. Burgess T, Coxon A, Meyer S, Sun J, Rex K, Tsuruda T et al (2006) Fully human monoclonal antibodies to hepatocyte growth factor with therapeutic potential against hepatocyte growth factor/c-Met-dependent human tumors. Cancer Res 66(3):1721–1729 69. Gordon MS, Mendelson DS, Sweeney C, Erbeck N, Patel R, Kakkar T et al (2007) Interim results from a first-in-human study with AMG102, a fully human monoclonal antibody that neutralizes hepatocyte growth factor (HGF), the ligand to c-Met receptor, in patients (pts) with advanced solid tumors. J Clin Oncol (Meet Abstr) 25(18 suppl):3551 70. Yap TA, Harris D, Barriuso J, Wright M, Riisnaes R, Clark J et  al (2008) Phase I trial to determine the dose range for the c-Met inhibitor ARQ 197 that inhibits c-Met and FAK

276

B. Besse and J.-C. Soria

phosphorylation, when administered by an oral twice-a-day schedule. J Clin Oncol (Meet Abstr) 26(15 suppl):3584 71. Eder JP, Heath E, Appleman L, Shapiro G, Wang D, Malburg L et al (2007) Phase I experience with c-MET inhibitor XL880 administered orally to patients (pts) with solid tumors. J Clin Oncol (Meet Abstr) 25(18 suppl):3526 72. Srinivasan R, Choueiri TK, Vaishampayan U, Rosenberg JE, Stein MN, Logan T et al (2008) A phase II study of the dual MET/VEGFR2 inhibitor XL880 in patients (pts) with papillary renal carcinoma (PRC). J Clin Oncol (Meet Abstr) 26(15 suppl):5103 73. Jhawer MP, Kindler HL, Wainberg ZA, Hecht JR, Kerr RO, Ford JM et al (2008) Preliminary activity of XL880, a dual MET/VEGFR2 inhibitor, in MET amplified poorly differentiated gastric cancer (PDGC): interim results of a multicenter phase II study. J Clin Oncol (Meet Abstr) 26(15 suppl):4572 74. Dempsey EC, Newton AC, Mochly-Rosen D, Fields AP, Reyland ME, Insel PA et al (2000) Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol 279(3):L429–L438 75. Nishizuka Y (1988) The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334(6184):661–665 76. Dean NM, McKay R, Condon TP, Bennett CF (1994) Inhibition of protein kinase C-alpha expression in human A549 cells by antisense oligonucleotides inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters. J Biol Chem 269(23):16416–16424 77. Villalona-Calero MA, Ritch P, Figueroa JA, Otterson GA, Belt R, Dow E et al (2004) A phase I/II study of LY900003, an antisense inhibitor of protein kinase C-alpha, in combination with cisplatin and gemcitabine in patients with advanced non-small cell lung cancer. Clin Cancer Res 10(18):6086–6093 78. Lynch TJ, Raju R, Lind M, Riviere A, Gatzemeier U, Drorr A et al (2003) Randomized phase III trial of chemotherapy and antisens oligonucleotide LY900003 (ISIS 3521) in patients with advanced NSCLC: initial report. Proc Am Soc Clin Oncol 22:623 79. Paz-Ares L, Douillard JY, Koralewski P, Manegold C, Smit EF, Reyes JM et al (2006) Phase III study of gemcitabine and cisplatin with or without aprinocarsen, a protein kinase C-alpha antisense oligonucleotide, in patients with advanced-stage non-small-cell lung cancer. J Clin Oncol 24(9):1428–1434 80. Jirousek MR, Goekjian PG (2001) Protein kinase C inhibitors as novel anticancer drugs. Expert Opin Investig Drugs 10(12):2117–2140 81. Liu Y, Su W, Thompson EA, Leitges M, Murray NR, Fields AP (2004) Protein kinase CbetaII regulates its own expression in rat intestinal epithelial cells and the colonic epithelium in vivo. J Biol Chem 279(44):45556–45563 82. Lahn M, McClelland P, Ballard D, Mintze K, Thornton D, Sandusky G (2006) Immunohistochemical detection of protein kinase C-beta (PKC-beta) in tumour specimens of patients with non-small cell lung cancer. Histopathology 49(4):429–431 83. Keyes K, Mann L, Sherman M, Galbreath E, Schirtzinger L, Ballard D et al (2004) LY317615 decreases plasma VEGF levels in human tumor xenograft-bearing mice. Cancer Chemother Pharmacol 53(2):133–140 84. Graff JR, McNulty AM, Hanna KR, Konicek BW, Lynch RL, Bailey SN et  al (2005) The protein kinase Cbeta-selective inhibitor, enzastaurin (LY317615.HCl), suppresses signaling through the AKT pathway, induces apoptosis, and suppresses growth of human colon cancer and glioblastoma xenografts. Cancer Res 65(16):7462–7469 85. Lee KW, Kim SG, Kim HP, Kwon E, You J, Choi HJ et al (2008) Enzastaurin, a protein kinase Cbeta inhibitor, suppresses signaling through the ribosomal S6 kinase and bad pathways and induces apoptosis in human gastric cancer cells. Cancer Res 68(6):1916–1926 86. Oh Y, Herbst RS, Burris H, Cleverly A, Musib L, Lahn M et al (2008) Enzastaurin, an oral serine/threonine kinase inhibitor, as second- or third-line therapy of non-small-cell lung cancer. J Clin Oncol 26(7):1135–1141 87. Tekle C, Giovannetti E, Sigmond J, Graff JR, Smid K, Peters GJ (2008) Molecular pathways involved in the synergistic interaction of the PKC[beta] inhibitor enzastaurin with the antifolate pemetrexed in non-small cell lung cancer cells. Br J Cancer 99(5):750–759

Other Molecular Targeted Agents in Non-small Cell Lung Cancer

277

  88. Kocs DM, Raju RN, Socinski MA, Stinchcombe TE, Rousey SR, Barrera D et  al (2008) Preliminary results of a randomized phase II trial of pemetrexed (P) + carboplatin (Cb) {+/−} enzastaurin (ENZ) versus docetaxel (D) + Cb as first-line treatment of patients with stage IIIB/IV non-small cell lung cancer (NSCLC). J Clin Oncol (Meet Abstr) 26(15 suppl):8061   89. Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110(6):673–687   90. Alghisi GC, Rüegg C (2006) Vascular integrins in tumor angiogenesis: mediators and therapeutic targets. Endothelium 13(2):113–135   91. Mulgrew K, Kinneer K, Yao XT, Ward BK, Damschroder MM, Walsh B et al (2006) Direct targeting of alphavbeta3 integrin on tumor cells with a monoclonal antibody, AbegrinTM. Mol Cancer Ther 5(12):3122–3129   92. Delbaldo C, Raymond E, Vera K, Hammershaimb L, Kaucic K, Lozahic S et al (2008) Phase I and pharmacokinetic study of etaracizumab (AbegrinGäó), a humanized monoclonal antibody against alphavbeta3 integrin receptor, in patients with advanced solid tumors. Invest New Drugs 26(1):35–43   93. Dechantsreiter MA, Planker E, Matha B, Lohof E, Holzemann G, Jonczyk A et al (1999) N-methylated cyclic RGD peptides as highly active and selective aVb3 integrin antagonists. J Med Chem 42(16):3033–3040   94. Reardon DA, Fink KL, Mikkelsen T, Cloughesy TF, O’Neill A, Plotkin S et  al (2008) Randomized phase II study of cilengitide, an integrin-targeting arginine-glycine-aspartic acid peptide, in recurrent glioblastoma multiforme. J Clin Oncol 26(34):5610–5617   95. Ramakrishnan V, Bhaskar V, Law DA, Wong MH, Dubridge RB, Breinberg D et al (2006) Preclinical evaluation of an anti-alpha5beta1 integrin antibody as a novel anti-angiogenic agent. J Exp Ther Oncol 5(4):273–286   96. Bhaskar V, Fox M, Breinberg D, Wong M, Wales P, Rhodes S et al (2008) Volociximab, a chimeric integrin alpha5beta1 antibody, inhibits the growth of VX2 tumors in rabbits. Invest New Drugs 26(1):7–12   97. Ricart AD, Tolcher AW, Liu G, Holen K, Schwartz G, Albertini M et al (2008) Volociximab, a chimeric monoclonal antibody that specifically binds alpha5beta1 integrin: a phase I, pharmacokinetic, and biological correlative study. Clin Cancer Res 14(23):7924–7929   98. Tsujimoto Y, Ikegaki N, Croce CM (1987) Characterization of the protein product of bcl-2, the gene involved in human follicular lymphoma. Oncogene 2(1):3–7   99. Martin B, Paesmans M, Berghmans T, Branle F, Ghisdal L, Mascaux C et al (2003) Role of Bcl-2 as a prognostic factor for survival in lung cancer: a systematic review of the literature with meta-analysis. Br J Cancer 89(1):55–64 100. Haura EB, Williams CA, Chiappori AA, Adams J, Northfelt DW, Malik SM et al (2008) A phase I trial of the small molecule pan-bcl-2 inhibitor obatoclax (GX15-070) in combination with docetaxel in patients with relapsed non-small cell lung cancer (NSCLC). J Clin Oncol (Meet Abstr) 26(15 suppl):19035 101. Li J, Viallet J, Haura EB (2008) A small molecule pan-Bcl-2 family inhibitor, GX15–070, induces apoptosis and enhances cisplatin-induced apoptosis in non-small cell lung cancer cells. Cancer Chemother Pharmacol 61(3):525–534 102. Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S et  al (2008) ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res 68(9):3421–3428 103. Roberts A, Gandhi L, O’Connor OA, Rudin CM, Khaira D, Xiong H et al (2008) Reduction in platelet counts as a mechanistic biomarker and guide for adaptive dose-escalation in phase I studies of the Bcl-2 family inhibitor ABT-263. J Clin Oncol (Meet Abstr) 26(15 suppl)):3542 104. O’Connor OA, Wright J, Moskowitz C, Muzzy J, MacGregor-Cortelli B, Stubblefield M et al (2005) Phase II clinical experience with the novel proteasome inhibitor bortezomib in patients with indolent non-Hodgkin’s lymphoma and mantle cell lymphoma. J Clin Oncol 23(4):676–684 105. Aghajanian C, Soignet S, Dizon DS, Pien CS, Adams J, Elliott PJ et al (2002) A phase I trial of the novel proteasome inhibitor PS341 in advanced solid tumor malignancies. Clin Cancer Res 8(8):2505–2511

278

B. Besse and J.-C. Soria

106. Fanucchi MP, Fossella FV, Belt R, Natale R, Fidias P, Carbone DP et al (2006) Randomized phase II study of bortezomib alone and bortezomib in combination with docetaxel in previously treated advanced non-small-cell lung cancer. J Clin Oncol 24(31):5025–5033 107. Lynch TJ, Fenton DW, Hirsh V, Bodkin DJ, Middleman E, Chiappori A et  al (2007) Randomized phase II study of erlotinib alone and in combination with bortezomib in previously treated advanced non-small cell lung cancer (NSCLC). J Clin Oncol (Meet Abstr) 25(18 suppl):7680 108. Davies AM, Ruel C, Lara PN, Lau DH, Gumerlock PH, Bold R et al (2008) The proteasome inhibitor bortezomib in combination with gemcitabine and carboplatin in advanced non-small cell lung cancer: a California Cancer Consortium Phase I study. J Thorac Oncol 3(1):68–74 109. Davies AM, Chansky K, Lara PN Jr, Gumerlock PH, Crowley J, Albain KS et  al (2009) Bortezomib plus gemcitabine/carboplatin as first-line treatment of advanced non-small cell lung cancer: a phase II Southwest Oncology Group Study (S0339). J Thorac Oncol 4(1):87–92 110. Voortman J, Smit EF, Honeywell R, Kuenen BC, Peters GJ, van de Velde H et al (2007) A parallel dose-escalation study of weekly and twice-weekly bortezomib in combination with gemcitabine and cisplatin in the first-line treatment of patients with advanced solid tumors. Clin Cancer Res 13(12):3642–3651 111. Planchard D, Besse B, Meric JB, Veillard AS, Pignon JP, Chan K et al (2008) A phase II study of first-line bortezomib (B) in patients (pts) with stage IIIB/IV non-small-cell lung cancer (NSCLC). Ann Oncol 19(9):viii96 112. Ho L, Li T, Piperdi B, Macapinlac M, Rigas JR, Camacho F et al (2008) Phase II study to evaluate the efficacy and safety of bortezomib (PS-341) in chemotherapy-naive patients with advanced stage non-small cell lung cancer (NSCLC). J Clin Oncol (Meet Abstr) 26(15 suppl):19096 113. Leblanc HN, Ashkenazi A (2003) Apo2L/TRAIL and its death and decoy receptors. Cell Death Differ 10(1):66–75 114. Herbst RS, Mendolson DS, Ebbinghaus S, Gordon MS, O’Dwyer P, Lieberman G et  al (2006) A phase I safety and pharmacokinetic (PK) study of recombinant Apo2L/TRAIL, an apoptosis-inducing protein in patients with advanced cancer. J Clin Oncol (Meet Abstr) 24(18 suppl):3013 115. Soria J, Smit EF, Khayat D, Besse B, Burton J, Yang X et al (2008) Phase Ib study of recombinant human (rh)Apo2L/TRAIL in combination with paclitaxel, carboplatin, and bevacizumab (PCB) in patients (pts) with advanced non-small cell lung cancer (NSCLC). J Clin Oncol (Meet Abstr) 26(15 suppl):3539 116. Oldenhuis C, Mom C, Sleijfer S, Gietema JA, Fox NL, Corey A et al (2008) A phase I study with the agonistic TRAIL-R1 antibody, mapatumumab, in combination with gemcitabine and cisplatin. J Clin Oncol (Meet Abstr) 26(15 suppl):3540 117. Hamada C, Tanaka F, Ohta M, Fujimura S, Kodama K, Imaizumi M et  al (2005) Metaanalysis of postoperative adjuvant chemotherapy with tegafur-uracil in non-small-cell lung cancer. J Clin Oncol 23(22):4999–5006 118. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, Tan EH, Hirsh V, Thongprasert S et al (2005) Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 353(2):123–132 119. Herbst RS, Prager D, Hermann R, Fehrenbacher L, Johnson BE, Sandler A et  al (2005) TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol 23(25): 5892–5899 120. Riely GJ, Rizvi NA, Kris MG, Milton DT, Solit DB, Rosen N et al (2009) Randomized phase II study of pulse erlotinib before or after carboplatin and paclitaxel in current or former smokers with advanced non-small-cell lung cancer. J Clin Oncol 27(2):264–270 121. Andre F, Campone M, Hurvitz SA, Vittori L, Pylvaenaeinen I, Sahmoud T et  al (2008) Multicenter phase I clinical trial of daily and weekly RAD001 in combination with weekly paclitaxel and trastuzumab in patients with HER2-overexpressing metastatic breast cancer with prior resistance to trastuzumab. J Clin Oncol (Meet Abstr) 26(15 suppl):1003 122. Kelly K, Chansky K, Gaspar LE, Albain KS, Jett J, Ung YC et al (2008) Phase III trial of maintenance gefitinib or placebo after concurrent chemoradiotherapy and docetaxel consolidation in inoperable stage III non-small-cell lung cancer: SWOG S0023. J Clin Oncol 26(15):2450–2456

Vaccine Therapy for Lung Cancer John Nemunaitis and Jack Roth

Abstract  Evidence of lung cancer sensitivity to immune reactivity continues to accumulate. However, consistent therapeutic opportunities remain limited. Recently, as a result of increased awareness of immunoreactive components and new technological development, a new crop of therapeutic vaccines are being explored for the purpose of modulating immunity against lung cancer. This review summarizes the key investigative opportunities. Keywords  Vaccine • Gene • Cancer • Lung

Introduction Evidence of an endogenous immune modulating effect in NSCLC is suggested based on heterogeneity of clinical progression, which is observed among patients with the same histological type of malignancy (1, 2). Rarely, improved survival of lung cancer patients who develop empyema has been observed (3). Furthermore, biopsy of responsive disease has occasionally demonstrated the isolation of tumor-infiltrating lymphocytes within the cancer parenchyma, suggestive of endogenous immune affect (4). There is also evidence for shared antigens in lung cancers (5–12), as is seen in other tumor types (13, 14). Dendritic cells, which are responsible for the induction of antitumor immunity in tumor-bearing hosts by a process of antigenic cross-presentation (15, 16),

J. Nemunaitis (*) and J. Roth Mary Crowley Cancer Research Centers, 1700 Pacific Avenue, Suite 1100, Dallas, TX, 75201, USA Texas Oncology PA, Dallas, TX, USA Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, USA e-mail: [email protected]

D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_13, © Humana Press, a part of Springer Science+Business Media, LLC 2010

279

280

J. Nemunaitis and J. Roth

have been shown to be activated in NSCLC. A great deal has been learned over the past 30 years regarding the potential application of immune-directed therapeutics in lung cancer. Furthermore, recent advances in molecular biology have allowed us to identify new antigens, cytokines, and mechanisms that enhance our understanding toward the development of immunotherapeutic approaches. The role of Dendritic Cells (DCs) in cell-mediated immunity has been extensively investigated (17–21). DCs have been found to play a central role in the induction of antitumor immunity in tumor-bearing hosts by a process of antigenic cross-presentation and have displayed activity in NSCLC (22). They efficiently display antigens on major histocompatability complexes (MHC II) ultimately stimulating proliferation and activation of CD4+ and CD8+ T cells. CD4+ cells further augment the activity of natural killer cells and macrophages, in addition to amplifying antigen-specific immunity by local secretion of cytokines (23–27). These attributes make DCs a pivotal component in the therapeutic strategies of many current immune based therapies in NSCLC. However, previous approaches to immunotherapy in lung cancer have failed to realize the potential of this promising strategy. There are several hypotheses to explain potential lack of activity, including ineffective priming of tumor-specific T cells, lack of high-avidity of primed tumor-specific T cells, and physical or functional disabling of primed tumor-specific T cells by the primary host and/or tumor-related mechanism. For example, in NSCLC, a high proportion of the tumorinfiltrating lymphocytes are immunosuppressive T regulatory cells (CD4+ CD25+) that secrete transforming growth factor-b (TFG-b) and express a high level of cytotoxic T lymphocyte (CTL) antigen-4 (28, 29). These cells have been shown to impede immune activation by facilitating T cell tolerance to tumor associated antigens rather than cross-priming CD8+ T cells. This results in the nonproliferation of killer T cells that recognize the tumor and will not attack it (28–34). Elevated levels of IL-10 and TFG-b are found in patients with NSCLC. Animal models have shown immune suppression that is mediated by these cytokines, serving as a defense for malignant cells against the body’s immune system (35–44). These factors are dealt with in different ways with more recent vaccine therapeutics described in this review.

Non-Small Cell Lung Cancer Vaccine Development Belagenpumatucel Belagenpumatucel (45) is a nonviral gene-based allogeneic vaccine that incorporates the TGF-b2 antisense gene into a cocktail of four different NSCLC cell lines. Elevated levels of TGF-b2 are linked to immunosuppression in cancer patients (46–51), and the level of TGF-b2 is inversely correlated with prognosis in patients with NSCLC (52). TGF-b2 has antagonistic effects on natural killer cells, lymphokine-activated killer cells, and dendritic cells (35, 40, 41, 53–55).

Vaccine Therapy for Lung Cancer

281

Using an antisense gene to inhibit TGF-b2, several groups have demonstrated an inhibition of cellular TGF-b2 expression resulting in an increased immunogenicity of gene-modified cancer cells (12–16, 56–59). In a recent Phase II study involving 75 early- (n = 14) and late-stage (n = 61) NSCLC patients, a doserelated effect of belagenpumatucel was defined (Table  1) (45). Patients were randomized to one of the three dose cohorts. In 41 advanced-stage (IIIB, IV) patients, the investigators found no adverse toxicity and an impressive survival advantage at dose levels ³2.5 × 107 cells/injection, with an estimated 2-year survival of 47% in response to Lucanix™. This compared favorably with the historical 2-year survival rate of <20% of stage IIIB/IV NSCLC patients (5–8, 60, 61). Furthermore, there was a correlation of positive outcome with induction of immune enhancement of tumor antigen recognition. Immune function was explored in the 61 advanced stage (IIIB/IV) patients. Cytokine production (IFN-g, p = 0.006; IL-6, p = 0.004; and IL-4, p = 0.007) was induced, an antibody-mediated response to vaccine HLA antigen was observed (p = 0.014), and there was a trend toward the correlation between a cell-mediated response and achievement of stable disease or better (p = 0.086). Subsequent phase II investigation in stage IIIB/IV NSCLC patients further validated survival results observed in the initial phase I/II trial. Median survival in the subsequent trial was 19 months. There was a suggestion that circulating tumor cells may predict for a worse response (62).

GVAX Vaccines transduced with granulocyte–macrophage colony-stimulating factor (GM–CSF) gene were potent inducers of tumor immunity in animal models (63). Secretion of GM–CSF by genetically modified tumor cells induced local tumor antigen expression and stimulated cytokine release at the vaccine site, which activated and attracted antigen-presenting cells, thereby inducing a tumor-specific cellular immune response (64). Preclinical studies conducted with GVAX showed no significant local and systemic toxicities at clinically relevant doses (63, 65–67). Several phase I/II human trials using GM–CSF-secreting autologous or allogeneic tumor cell vaccines have been performed (Table 1) (68–73). One multicenter phase I/II trial involving patients with early stage and advanced-stage NSCLC evaluated an autologous GVAX vaccine (10). For vaccine preparation, tumor tissue was obtained surgically or by thoracentesis in the case of malignant effusions. Cells were exposed overnight to an adenoviral vector supernatant (Ad-GM). GVAX was administered intradermally. A total of 43 NSCLC patients (10 early stage, 33 latestage) were vaccinated. The most common vaccine-related adverse events were local vaccine injection site reactions (93%), followed by fatigue (16%) and nausea (12%). Three advanced-stage patients achieved durable, complete tumor regressions. Two remain without disease more than 5 years following vaccine. Both had failed prior frontline and second-line therapy prior to vaccination and had multisite disease. One complete responder showed an in vitro T cell response to autologous

Stage IIIB, IV

IA–IIIB

IIIB, IV

III, IV

IIIB, IV

IV

IIIB/IV

II, III, IV

IIIB, IV (I, III A)

# Patients 19

16

13

40

43

35

33

75

26

Vaccine Allogenic Ad B7.1, HLA-A modified cell vaccine Dendritic cells pulsed with Allo NSCLC line 1650 Dexosome (auto derived DC-exosomes loaded with MAGE from NSCLC) Recombinant EGF protein

Recombinant EGF protein

GVAX (Ad-GMCSF gene transfected auto NSCLC vaccine) GVAX (Ad-GMCSF gene transfected auto NSCLC vaccine) Belagenpumatucel (TGFb2 AS gene transfected Allo NSCLC vaccine) Telomerase peptide (GY 1001 and HR 2822) vaccine

ND

ND

ND

8.2 mo

441 days (IIIB/IV only) 8.5 mo

Mild induration and erythema at inj site, chills, fever

12 mo

36%

ND

(152)

(194)

(10)

(72)

5SD 1MR

3 CR 6 SD

(122)

(124)

(159)

(22)

Reference (117)

ND

12/40 SD

6/16 ag specific responses 3/9 induced response to MAGE

Response 1 PR/5SD

54% (IIIB/ 6 PR IV only)

44%

ND Low dose: 6.43 mo High dose: 8.4 mo ND ND

N/A

1-Year survival 52%

N/A

Median survival 18 months

Grade 3; arm swelling (n = 1)

Grade 2; chills, fever, vomiting, nausea, hypertension, cephalgea, dizziness, flushing, pain at inj site Grade 1–2; fever, chills, nausea, vomiting, tremors, anorexia, pain Grade 1–2; erythema and indurations, fatigue, flulike symptoms Grade 1–2 reaction at inj site, fatigue, nausea, dyspnea

Minor skin erythema and fatigue Grade 1–2; inj site reaction, flu-like symptoms, edema and pain

Side effects Minor skin erythema

Table 1  Update of demographics and responses for recent vaccine trials in NSCLC

282 J. Nemunaitis and J. Roth

7

IV

ND 4SD (161) Grade 1–2: inj pain, erythema, ND fatigue, hypertension, bradycardia cough, diarrhea, dyspnea, headache, nausea, vomiting, pleural effusion MAGE 3 protein vaccine 17 I/II ND ND ND ND (138) ND 100% 2 PD £ 1 yr (121) L523S protein vaccine 13 IB/II Grade 1,2: erythema, site pain, nausea, flu-like, hypertension GVAX Bystander vaccine 49 IIIB/IV Grade 1,2: inj pain, fatigue, 7 mo 31% 7SD ³ 12 wks (74) dyspnea, nausea, fever GVAX 10 IB/II Grade 1,2 inj site reaction, ND 100% 4 SD ³ 20 mo (10) fatigue, nausea, dysprea 17.4 mo ND ND (81) L BLP25 88 IIIB/IV Grade 1,2 inj site reaction, fatigue, nausea Ad Adenovirus; Allo allogenic; NSCLC Non small cell lung cancer; Auto autologous; inj injection; N/A not applicable; ND not done; mo months; PR partial response; SD stable disease; ag antigen; MR minor response; CR complete response; PD progressive disease; yr year; wks weeks

Allogenic NSCLC murine (1,3) galactosyltransferase transfected cell vaccine

Vaccine Therapy for Lung Cancer 283

284

J. Nemunaitis and J. Roth

tumor-pulsed dendritic cells after vaccination. Survival at 1 year was 44% for all advanced stage treated patients and median survival was 12 months. Median survival among patients receiving vaccines secreting GM–CSF at a rate of ³40 ng/24 h/106 cells was 17 months, when compared with 7 months for those receiving vaccines secreting less GM–CSF. A subsequent trial in advanced NSCLC using a vaccine composed of autologous tumor cells mixed with an allogeneic GM–CSF-secreting cell line (K562 cells) failed to demonstrate evidence of clinical efficacy (74). Evidence of vaccineinduced immune activation was demonstrated; however, objective tumor responses were not seen despite a 25-fold higher GM-CSF secretion concentration with the bystander GVAX vaccine.

L-BLP-25 Mucin (MUC)-1 is a high molecular weight protein containing large amounts of O-linked sugars and is expressed on the apical borders of most normal secretory epithelial cells (75). It is expressed in many cancers, including NSCLC (76). Tumor-associated MUC1 is antigenically distinct from normal MUC1 (77). Recent studies have identified that MUC1 is associated with cellular transformation, as demonstrated by tumorigenicity (78), and can confer resistance to genotoxic agents (79). Both the oligosaccharide portion and the tandem repeat of the MUC extracellular domain have potential for immunotherapeutic activity. L-BLP-25 vaccine has been tested in three NSCLC trials (Table  1) (80). Three doses and two regimens were tested, including one regime using liposomal IL-2 as an adjuvant. Recently, results of a phase III study (81) of L-BLP-25 in 171 advanced stage NSCLC patients were reported (74). Patients with stable or responding stage IIIB or IV NSCLC following standard first-line chemotherapy were randomized to either L-BLP-25 (88 patients) or best supportive care (83 patients). There was a 4.4 month longer median survival for patients on the L-BLP-25 arm (17.4 vs. 13 months), although this did not reach statistical significance. The median survival for a subset of 35 stage IIIB patients who received vaccine was 30 months versus 13.3 months for the 30 who received best supportive care (p = 0.09). There were no major toxicities. The clinically meaningful survival advantages seen for stage IIIB patients is encouraging. A phase III randomized trial of L-BLP-25 for unresectable stage III NSCLC patients with response or stable disease after chemoradiation is now ongoing.

IDM-2101 IDM-2101 is a peptide-based vaccine designed to induce CTLs against five tumor associated antigens (TAAs) frequently overexpressed in NSCLC [i.e., carcinoembryonic antigen (CEA) (82), p53 (83, 84), HER-2/neu (85, 86), and melanoma antigens (MAGE) 2 and 3 (87)]. These TAAs have been used in previous vaccine studies involving patients with NSCLC (88–107) and have been extensively characterized

Vaccine Therapy for Lung Cancer

285

in the literature. IDM-2101 is composed of ten synthetic peptides from these TAAs. Nine of the peptides represent CTL epitopes and each CTL epitope is restricted by HLA-A2.1 and at least one other member of the HLA-A2 superfamily of major histocompatibility complex class I molecules, providing coverage of approximately 45% of the general population. The tenth synthetic peptide is the pan-DR epitope (PADRE), a rationally designed helper T-lymphocyte (HTL) epitope included to augment the magnitude and duration of CTL responses (108). IDM-2101 was tested in an open label phase II study involving 63 HLA-A2-positive stage IIIB/IV NSCLC patients who had failed prior chemotherapy. No significant adverse events were noted. Low-grade erythema and pain at the injection site were the most common side effects. One-year survival in the treated patients was 60%, and median survival was 17.3 months. One complete and one partial response were identified. Survival was longer in patients demonstrating an immune response to epitope peptides (P < 0.001). Overall, treated patients appeared to do well when compared to historical controls. Immune responses in 33 patients collectively showed induction of CTLs to all of the vaccine epitopes. Although patient to patient variability was observed with respect to the frequency and magnitude of the CTL responses, 85% of tested patients responded to at least two epitopes. These data are consistent with results from an earlier phase I trial (109). Moreover, longer survival was shown in patients achieving responses to two or more epitopes (P < 0.001).

B7.1 Vaccine B7.1 (CD80+) is a costimulating molecule associated with induction of a T and NK cell response (93, 110–112). Tumor cells transfected with B7.1 and HLA molecules have been shown to stimulate an avid immune response by direct antigen presentation and direct activation of T cells, in addition to allowing cross-presentation (113–116). In a Phase I trial, Raez et al. (117) used an allogeneic NSCLC tumor cell line (AD100) transfected with B7.1 (CD80) and HLA-A1 or -A2 to generate CD8 CTL responses (Table 1). Patients who were HLA-A1 or -A2 allotype received the corresponding HLA-matched vaccine. A total of 19 patients with stage IIIB/IV NSCLC were treated, and most had received prior chemotherapy. Patients who were neither HLA-A1 nor -A2 received the HLA-A1-transfected vaccine. A total of 18 patients received at least one full course of treatment. One patient was removed before the completion of the first course because of a serious adverse event not associated with the vaccine. Three more patients experienced serious adverse events, which were also not associated with the vaccine. Side effects included minimal skin erythema for four patients. One patient showed a partial response for 13 months and five patients had stable disease ranging from 1.6 to >52 months (117, 118). The Kaplan–Meier estimate for the survival for the 19 patients was 18 months. One-year survival was estimated at 52%. The low toxicity and good survival in this study suggested benefit from clinical vaccination.

286

J. Nemunaitis and J. Roth

L523S Vaccine L523S is a lung cancer antigen originally identified through screening of genes differentially expressed in cancer versus normal tissue (119, 120). L523S is expressed in ~80% of NSCLC cells (119, 120). The immunogenicity of L523S in humans was initially shown by detecting the presence of existent antibody and helper T cell responses to L523S in patients with lung cancer (BB-IND-#10833 public FDA database for this protocol IND). Subsequent studies further validated L532S immunogenicity by demonstrating that human CTLs could specifically recognize and kill cells that express L523S. In preclinical studies, the gene proved safe when injected intramuscularly as an expressive plasmid (pVAX/L523S) and when delivered following incorporation into an EIB-deleted adenovirus (Ad/L523S). In a phase I clinical trial in 13 stage IB, IIA, and IIB NSCLC patients, both delivery vehicles (pVAX/L523S and Ad/L523S) were used to administer the gene to three patients in each of three cohorts (121) (Table 1). No significant toxic effect was identified. All but 1 patient demonstrated at least twofold elevation in antiadenovirus antibodies; however, despite the positive preclinical studies, vaccination induced an immune response in only one patient in the phase I study. The reasons for a lack of significant detectable immune response are unknown. The use of alternative formulations and/or regimens and the assessment of other surrogate immune function parameters might be considered. Two patients developed disease recurrence and all remained alive after a median of 290 days follow up.

Epidermal Growth Factor Vaccine Overexpression of epidermal growth factor receptor (EGFR) and its ligand, epidermal growth factor (EGF), has been linked with the promotion of cell proliferation, survival and motility. EGF transduces signaling through EGFR following binding to this cell surface receptor, ultimately resulting in the stimulation of cell proliferation. The immunotherapy developed by Ramos et al. (122) induces an immune response against selfproduced EGF. This vaccine is a human recombinant EGF linked to a P64K recombinant carrier protein from Neisseria meningitides. Several pilot trials have been completed (122–124). Results from these studies have demonstrated that vaccination with EGF is immunogenic and appears to be well-tolerated (Table 1). In one study, 43 patients with stage IIIB/IV NSCLC randomly received either a single dosage or a double dose (122). Immune response against EGF was measured in 38 of the 43 patients, and 15 achieved a good antibody response (GAR) against EGF following vaccination. Kaplan–Meyer analysis separating patients by dose predicted a median estimated life expectancy of 6.4 months for patients who received the single dose, and 8.4 months for the patients who received the double dose. Based on immune response, however, patients classified as GARs had a life expectancy estimated

Vaccine Therapy for Lung Cancer

287

at 12 months, whereas those who had a less favorable GAR had a life expectancy of 7 months. Two other studies conducted by Gonzalez and colleagues compared the effect of different adjuvants on patients’ antibody response (40). The patients were treated each time when antibody titers decreased to at least 50% of their induction phase peak titer. The pooled data of the two trials suggested that higher antibody responses were obtained when the vaccine was emulsified in adjuvant montanide ISA 51 or when low-dose cyclophosphamide was administered before the vaccination; however, the difference was not statistically significant. Median survival of GAR patients was 9.1 months, whereas poor antibody responding patients had a survival of 4.5 months.

Melanoma-Associated Antigen E-3 Vaccine MAGE-3 is the most commonly expressed cancer testis antigen and is expressed in testicular germ cells, but no other normal tissue (125). It is aberrantly expressed in a wide variety of tumors, including NSCLC (125). Several CD8+ T cell epitopes of MAGE-3 have been identified in  vitro (126–134), including HLA-A1-restricted epitope 168–176 (135), and HLA-A2-restricted epitope 271–279 (136). Based on these findings, synthetic peptides corresponding to these epitopes have been introduced into clinical vaccination studies, in which they were associated with regression of melanoma in individual cases (137). Clinical vaccination studies using full-length recombinant proteins have the advantage that this antigen potentially includes the full range of epitopes for CD4+ and CD8+ T cells. In addition, it is likely that protein vaccination leads to the presentation of epitopes in the context of various HLA alleles, and therefore, this type of vaccine should be applicable to any patient regardless of HLA restriction (138). Atanackovic et al. (138) used a MAGE-3 protein as a vaccine to induce CD4+ T cells in patients with stage I or II NSCLC (Table  1). All patients had undergone surgical resection of the primary lung tumor and had no evidence of disease at the onset of the study. Of the nine patients who received only the MAGE-3 protein, three developed an increase in antibodies against MAGE-3 protein and one had a CD8+ T cell response. By comparison, of the eight patients who received MAGE 3 antigen combined with the adjuvant ASO2B, seven showed an increase in serum concentrations of anti-MAGE-3 and four had a CD4+ response to HLA-DP4restricted peptide. Based on these results, further testing in a larger randomized phase II trial was recently reported (139), involving 182 (122 vaccine and 60 placebo) early stage (IB, II) NSCLC MAGE-A3+ patients. No significant toxicity issues were identified and preliminary analysis revealed a 33% disease free survival improvement in the vaccinated arm when compared with the placebo arm. Results trended toward significant in the stage II patients.

288

J. Nemunaitis and J. Roth

Transcriptase Catalytic Subunit Antigen Vaccine It is well established that T cells of the human immune system can recognize telomerase (140–148). Although telomerase is also expressed in some normal cells, such as bone marrow stem cells (149) and epithelial cells in gastrointestinal tract crypts (150), it is highly expressed in virtually all cancer cells. GV1001 is a unique peptide corresponding to a sequence derived from the active site of the catalytic subunit of human telomerase reverse transcriptase (hTERT). It contains the 611–626 sequence of hTERT and is capable of binding to molecules encoded by multiple alleles of all three loci of HLA class II (151). HR2822 is a second peptide corresponding to sequences 540–548 of hTERT. Brunsvig et  al. (152) initiated a phase I/II trial (Table  1) involving 26 patients with late-stage NSCLC. No clinically significant toxic events related to the treatment were reported. Importantly, no bone marrow or severe gastrointestinal toxicities were observed. Side effects were mild and included flu-like symptoms, chills and fever. Eleven patients demonstrated an immune response against GV1001, and only two patients demonstrated a response to HR2822. After receiving booster shots, two patients were converted to immune responders. One patient with stage IIIA NSCLC showed a complete tumor response and developed GV1001-specific CTLs that could be cloned from peripheral blood. The median survival time for all 26 patients was 8.5 months.

Dexosome Vaccine Exosomes are cell-derived lipid vesicles that express high levels of a narrow spectrum of cell proteins (153–155). Vesicles released from dendritic cells (dexosomes) have been demonstrated to play a role in the activation of the immune response (156, 157). In vitro, dexosomes have the capacity to present antigen to naïve CD8+ cytolytic T cells and CD4+ T cells (154, 158). Purified dexosomes were shown to be effective in both suppressing tumor growth and eradicating an established tumor in murine models (153). Morse et  al. developed a vaccine using dendritic cellderived exosomes loaded with MAGE tumor antigens (159). The phase I trial enrolled 13 patients with stage IIIB or IV NSCLC demonstrating MAGE-A3 or -A4 expression. Autologous dendritic cells were harvested to produce dexosomes. They were loaded with MAGE-A3, -A4, -A10 and -3DPG4 peptides. Dexosome therapy was administered to nine patients (Table 1). Patients experienced grade 1–2 toxicities, including injection site reactions, flu-like symptoms, edema, and pain. Three patients exhibited delayed type hypersensitivity reactions against MAGE peptides. Survival ranged from 52 to 665 days.

Vaccine Therapy for Lung Cancer

289

a (1,3)-Galactosyltransferase a(1,3)-Galactosyltransferase (agal) epitopes are present on the surface of most non human mammalian cells and are the primary antigen source inductive of hyperactive xenograft rejection. Expression of agal epitopes after gene transfer (using a retroviral vector) in human A375 melanoma cells prevented tumor formation in nude mice (160). Preliminary results by Morris et al. (161) using three irradiated lung cancer cell lines genetically altered to express xenotransplantation antigens by retroviral transfer of the murine agal gene, were recently described in seven patients with stage IV, recurrent or refractory NSCLC (Table 1). Toxicity involved grade 1–2 pain at the injection site, local skin reaction, fatigue and hypertension. Four patients had stable disease for >16 months.

Non-Small Cell Lung Cancer Dendritic Cell Vaccines Dendritic cells are potent antigen-presenting cells. As part of a phase II study (22), Hirshowitz et  al. (17–21) recently generated dendritic cell vaccines from CD14+ precursors, which were pulsed with apoptic bodies of an allogeneic NSCLC cell line that overexpressed Her2/neu, CEA, WT1, MAGE-2 and survivin. A total of 16 patients with stage IA-IIIB NSCLC were vaccinated (Table 1). There were ten patients who experienced skin erythema at the injection site and four patients experienced minor fatigue. No patients experienced a serious adverse event. Five patients showed a tumor antigen-independent response, and six patients showed an antigen-specific response. The study concluded that the vaccine was safe and demonstrated biological activity.

Cyclophilin B Cyclophilin-B (CypB) is a ubiquitous protein playing an important role in protein folding (162, 163), and is expressed in both normal and cancerous cells. CypBderived peptides are recognized by HLA-A24 restricted cytotoxic lymphocytes (CTL) isolated from lung adenocarcinoma. CypB peptides induce CTLs from leukemic patients, but failed to induce an immune response in cells isolated from patients with epithelial cancer or normal donors. Modification of a single amino acid of the CypB gene increases its immunogenicity and results in CTL activation in both cancer patients and healthy donors (164).

290

J. Nemunaitis and J. Roth

Gohara et al. investigated the immune response in advanced-stage lung cancer patients treated with CypB vaccine. Sixteen HLA-A24+ patients, fifteen with NSCLC, and one with SCLC were treated with CypB or modified CypB peptide vaccine following the completion of chemotherapy (165). All patients had stable disease at 5-week follow-up. Following vaccination, IFN-g production by peripheral blood mononuclear cells isolated from patient sera were elevated in 3 of 12 patients. Overall survival for NSCLC patients receiving CypB or modified CypB vaccine was 67+ and 28+ weeks, respectively (Table 1). One patient with SCLC was not evaluable for response.

Small Cell Lung Cancer Vaccine Development Fucosyl-GM1 The ganglioside fucosyl-GM1 is a carbohydrate molecule present in most cases of SCLC (166, 167), but absent in normal lung tissue. Immunostaining has demonstrated the presence of fucosyl-GM1 in culture media from SCLC cell lines, in tumor extracts, and in serum of mouse xenografts (168). Fucosyl-GM1 was detected in the serum of 4 of 20 SCLC patients with extensive-stage disease, but was not present in the serum of 12 patients with non-SCLC or in 20 healthy volunteers (168). The specificity of fucosyl-GM1 to SCLC makes it a potential target for immunotherapy. Dickler et  al. treated 13 patients with Fuc-GM1 isolated from bovine thyroid tissue; ten patients completed the study and were evaluable (169). All ten patients demonstrated high titers of IgM and IgG antibodies to Fuc-GM1. The most common toxicity was local skin reaction, lasting 2–5 days. Three of six patients who completed the entire course of vaccinations remained relapse free at 18, 24, and 30 months from diagnosis. Subsequently, Krug et  al. administered synthetic fucosyl-GM1 after conventional chemotherapy to 17 patients (170). Five of six patients at the high dose demonstrated increased levels of antifucosyl GM1 IgM. Three of six patients receiving the middle dose showed antifucosyl GM1 IgM production, and none of five patients at the low dose showed elevated IgM levels. Toxicities were minimal (Table 2).

BEC2 Ganglioside GD3 is a cell surface glycosphingolipid with differential expression limited to cells of neuroectodermal origin and a subset of T lymphocytes (171–173). High levels of expression have been demonstrated in SCLC tumors and cell lines (174). Because GD3 is present at low levels in normal tissues, it is poorly immunogenic. BEC2, an anti-idiotypic IgG2b mouse antibody that is structurally similar to GD3, demonstrated strong immunogenic properties in patients with melanoma (175).

29

p53

11.8 months from 1st vaccination

ES extensive stage; LS limited stage; N/A not available; PR partial response; SD stable disease

ES

8ES, 5LS

11%

61%

13

PolySA

8ES, 7LS

22 months from 1st vaccination

15

BEC2

6ES, 10LS

NA

69%

17.5 months from 1st vaccination

16

Fucosyl GM1

20.5 months from diagnosis

NA

NA

Grade 1–3: local skin reaction, flu-like symptoms, sensory neuropathy Grade 1–2: local skin reaction, myalgia, sensory neuropathy Grade 1–3: local skin reaction, fever Grade 1–4: local skin reaction, peripheral neuropathy Grade 2: fatigue, arthralgia

13

Fucosyl GM1

9ES, 4LS

1-Year survival

Median survival

Table 2  Demographics and results of recent vaccine trials in SCLC Number of Vaccine patients Stage Side effects

1 PR, 7 SD

NA

NA

NA

NA

Response

Reference

(196)

(184)

(176)

(170)

(169)

Vaccine Therapy for Lung Cancer 291

292

J. Nemunaitis and J. Roth

Grant et al. treated 15 SCLC patients, 8 with extensive-stage and 7 with limited stage disease, with BEC2 vaccination (176). Thirteen patients were evaluable for response; all developed IgM antibodies to BEC2, and three developed IgG antibodies. Duration of antibody production was variable, with at least one patient demonstrating measurable antibody production 1 year following treatment. Median survival was 20.5 months from diagnosis, and patients with measurable anti-GD3 antibodies showed the longest relapse-free intervals (Table  2). When compared to SCLC patients treated with conventional therapy alone, the authors found patients treated with BEC2 vaccine to have longer than expected survival time, though not statistically significant. Significant toxicity was minimized to local skin irritation.

PolySA Polysialic acid (polySA) is found on the surface of Gram-negative bacteria (such as group B meningococcus), embryonic neural crest cells, and some malignancies of neural crest origin (177, 178). The large size and negative charge of this molecule inhibit binding of cell adhesion molecules, and it is this property that is believed to contribute to its role in neural crest cell migration and early metastasis of malignant cells (179). PolySA has been shown to be expressed abundantly by SCLC tissues (180–183), making it a potentially viable target for SCLC vaccine therapy. Krug et  al. investigated the immunogenicity of polySA vaccination in 11 SCLC patients following conventional therapy (184). Two forms of polySA were administered to patients. Five patients received vaccination with polySA, and six patients received polySA manipulated by N-propionylation (NP-polySA), which has been shown to boost the IgG response in mice (185). One of five patients treated with unmodified polySA demonstrated an IgM response. Of the six patients vaccinated with NP-polySA, all produced measurable IgM antibody responses. In five of the six cases, these antibodies cross-reacted with unmodified polySA. Flow cytometry confirmed the presence of IgM antibodies reactive to SCLC cell lines. Despite the demonstrable production of IgM antibodies to polySA, complement-dependent lysis of polySApositive tumor cells with human complement could not be demonstrated. Common adverse effects were minimal and included injection-site reaction and flu-like symptoms lasting 2–4 days (Table 2). Four patients reported sensory neuropathy.

WT1 The Wilm’s tumor gene (WT1) is responsible for Wilm’s tumor, a pediatric renal cancer, and encodes a protein involved in cell proliferation and differentiation, apoptosis, and organ development (186–188). WT1 is overexpressed in several hematological malignancies as well as various solid tumors, including lung, breast, thyroid and colorectal cancers (189, 190). WT1-specific cytotoxic lymphocytes

Vaccine Therapy for Lung Cancer

293

(CTL) lyse WT1 expressing tumor cells in vitro without damaging normal tissues that express WT1 physiologically (191, 192). Oka et al. treated 26 patients, including 10 lung cancer patients (histological type not specified), with WT1 vaccine following the completion of conventional therapy (193). Three NSCLC patients showed decreased serum levels of tumor markers (CEA or SLX) following vaccination; one patient also showed a decrease in tumor size radiographically. One NSCLC patient had stable disease at follow-up; four patients developed progressive disease, and two were unevaluable. Three patients demonstrated increased activity of WT1-specific CTL activity. A correlation (p = 0.0397) between immunological and clinical response was observed for all study patients.

Conclusion In conclusion, several vaccine opportunities demonstrate evidence of activity (195). All appear remarkably safe. Limitations, however, involve identification of sensitive subset patient populations and surrogate measures to indicate relevant immune reactivity. Vaccines described in this review focus on different elements of immune reactivity (i.e., antigen exposure, dendritic activation, T cell activation, inhibition of T regulatory cells, inhibition of TGF beta expression). Each of these approaches has demonstrated evidence of activity in subsets of patients. However, phase III trials are required to conclusively determine relevance to lung cancer. Data appear encouraging, particularly in the setting of minimal disease early in the therapeutic course and at earlier stages of disease. It is also enticing to consider the combination of vaccines, particularly those with varied mechanisms of action. Future trials will undoubtedly explore combination vaccine approaches or products with multiple immune component stimulation.

References 1. Shankaran V, Ikeda H, Bruce AT et  al (2001) IFNgamma and lymphocytes prevent primary tumor development and shape tumour immunogenicity. Nature 410:1107–1111 2. Bell JW (1970) Possible immune factors in spontaneous regression of bronchogenic carcinoma. Ten year survival in a patient treated with minimal (1, 200 r) radiation alone. Am J Surg 120(6): 804–806 3. Ruckdeschel JC, Codish SD, Stranahan A, McKneally MF (1972) Postoperative empyema improves survival in lung cancer. Documentation and analysis of a natural experiment. N Engl J Med 287(20):1013–1017 4. Wei YQ, Hang ZB (1989) In situ observation of lymphocyte-tumor cell interaction in human lung carcinoma. Immunol Invest 18(9–10):1095–1105 5. Hanna N, Shepherd FA, Fossella FV et  al (2004) Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy. J Clin Oncol 22(9):1589–1597 6. Tsao MS, Sakurada A, Cutz JC et al (2005) Erlotinib in lung cancer – molecular and clinical predictors of outcome. N Engl J Med 353(2):133–144

294

J. Nemunaitis and J. Roth

7. Shepherd FA, Rodrigues Pereira J, Ciuleanu T et  al (2005) Erlotinib in previously treated nonsmall-cell lung cancer. N Engl J Med 353(2):123–132 8. Kris MG, Natale RB, Herbst RS et al (2003) Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. JAMA 290(16):2149–2158 9. Nemunaitis J (2005) Vaccines in cancer: GVAX, a GM-CSF gene vaccine. Expert Rev Vaccines 4(3):259–274 10. Nemunaitis J, Sterman D, Jablons D et al (2004) Granulocyte-macrophage colony-stimulating factor gene-modified autologous tumor vaccines in non-small-cell lung cancer. J Natl Cancer Inst 96(4):326–331 11. Fakhrai H, Gramatikova S, Safaei R (2001) Down-regulation of TGF-beta 2 as a therapeutic approach. In: Brain tumor immunotherapy. Humana Press, Totowa, New Jersey, pp. 289–305. 12. Dorigo O, Shawler DL, Royston I, Sobol RE, Berek JS, Fakhrai H (1998) Combination of transforming growth factor beta antisense and interleukin-2 gene therapy in the murine ovarian teratoma model. Gynecol Oncol 71(2):204–210 13. Tzai TS, Shiau AL, Liu LL, Wu CL (2000) Immunization with TGF-beta antisense oligonucleotide-modified autologous tumor vaccine enhances the antitumor immunity of MBT-2 tumor-bearing mice through upregulation of MHC class I and Fas expressions. Anticancer Res 20(3A):1557–1562 14. Tzai TS, Lin CI, Shiau AL, Wu CL (1998) Antisense oligonucleotide specific for transforming growth factor-beta 1 inhibit both in vitro and in vivo growth of MBT-2 murine bladder cancer. Anticancer Res 18(3A):1585–1589 15. Marzo AL, Fitzpatrick DR, Robinson BW, Scott B (1997) Antisense oligonucleotides specific for transforming growth factor beta2 inhibit the growth of malignant mesothelioma both in vitro and in vivo. Cancer Res 57(15):3200–3207 16. Park JA, Wang E, Kurt RA, Schluter SF, Hersh EM, Akporiaye ET (1997) Expression of an antisense transforming growth factor-beta1 transgene reduces tumorigenicity of EMT6 mammary tumor cells. Cancer Gene Ther 4(1):42–50 17. Gilboa E, Nair SK, Lyerly HK (1998) Immunotherapy of cancer with dendritic-cell-based vaccines. Cancer Immunol Immunother 46(2):82–87 18. Timmerman JM, Levy R (1999) Dendritic cell vaccines for cancer immunotherapy. Annu Rev Med 50:507–529 19. Conrad C, Nestle FO (2003) Dendritic cell-based cancer therapy. Curr Opin Mol Ther 5(4):405–412 20. Keilholz U, Weber J, Finke JH et  al (2002) Immunologic monitoring of cancer vaccine therapy: results of a workshop sponsored by the Society for Biological Therapy. J Immunother 25(2):97–138 21. Cranmer LD, Trevor KT, Hersh EM (2004) Clinical applications of dendritic cell vaccination in the treatment of cancer. Cancer Immunol Immunother 53(4):275–306 22. Hirschowitz EA, Foody T, Kryscio R, Dickson L, Sturgill J, Yannelli J (2004) Autologous dendritic cell vaccines for non-small-cell lung cancer. J Clin Oncol 22(14):2808–2815 23. Banchereau J, Briere F, Caux C et  al (2000) Immunobiology of dendritic cells. Annu Rev Immunol 18:767–811 24. Germain RN (1994) MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell 76(2):287–299 25. McAdam AJ, Schweitzer AN, Sharpe AH (1998) The role of B7 co-stimulation in activation and differentiation of CD4+ and CD8+ T cells. Immunol Rev 165:231–247 26. Pulendran B, Smith JL, Caspary G et al (1999) Distinct dendritic cell subsets differentially regulate the class of immune response in vivo. Proc Natl Acad Sci USA 96(3):1036–1041 27. Akbari O, DeKruyff RH, Umetsu DT (2001) Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen. Nat Immunol 2(8):725–731 28. Woo EY, Yeh H, Chu CS et  al (2002) Cutting edge: regulatory T cells from lung cancer patients directly inhibit autologous T cell proliferation. J Immunol 168(9):4272–4276 29. Woo EY, Chu CS, Goletz TJ et al (2001) Regulatory CD4(+)CD25(+) T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer. Cancer Res 61(12):4766–4772

Vaccine Therapy for Lung Cancer

295

30. Neuner A, Schindel M, Wildenberg U, Muley T, Lahm H, Fischer JR (2002) Prognostic significance of cytokine modulation in non-small cell lung cancer. Int J Cancer 101(3):287–292 31. Neuner A, Schindel M, Wildenberg U, Muley T, Lahm H, Fischer JR (2001) Cytokine secretion: clinical relevance of immunosuppression in non-small cell lung cancer. Lung Cancer 34(Suppl 2):S79–S82 32. Dohadwala M, Luo J, Zhu L et al (2001) Non-small cell lung cancer cyclooxygenase-2-dependent invasion is mediated by CD44. J Biol Chem 276(24):20809–20812 33. Schwartz RH (1996) Models of T cell anergy: is there a common molecular mechanism? J Exp Med 184(1):1–8 34. Lombardi G, Sidhu S, Batchelor R, Lechler R (1994) Anergic T cells as suppressor cells in vitro. Science 264(5165):1587–1589 35. Ruffini PA, Rivoltini L, Silvani A, Boiardi A, Parmiani G (1993) Factors, including transforming growth factor beta, released in the glioblastoma residual cavity, impair activity of adherent lymphokine-activated killer cells. Cancer Immunol Immunother 36(6):409–416 36. Roszman T, Elliott L, Brooks W (1991) Modulation of T-cell function by gliomas. Immunol Today 12(10):370–374 37. Smith KA (1988) Interleukin-2: inception, impact, and implications. Science 240(4856): 1169–1176 38. Smith KA (1993) Lowest dose interleukin-2 immunotherapy. Blood 81(6):1414–1423 39. Tigges MA, Casey LS, Koshland ME (1989) Mechanism of interleukin-2 signaling: mediation of different outcomes by a single receptor and transduction pathway. Science 243(4892):781–786 40. Rook AH, Kehrl JH, Wakefield LM et al (1986) Effects of transforming growth factor beta on the functions of natural killer cells: depressed cytolytic activity and blunting of interferon responsiveness. J Immunol 136(10):3916–3920 41. Tsunawaki S, Sporn M, Ding A, Nathan C (1988) Deactivation of macrophages by transforming growth factor-beta. Nature 334(6179):260–262 42. Fontana A, Frei K, Bodmer S et al (1989) Transforming growth factor-beta inhibits the generation of cytotoxic T cells in virus-infected mice. J Immunol 143(10):3230–3234 43. Hirte HW, Clark DA, O’Connell G, Rusthoven J, Mazurka J (1992) Reversal of suppression of lymphokine-activated killer cells by transforming growth factor-beta in ovarian carcinoma ascitic fluid requires interleukin-2 combined with anti-CD3 antibody. Cell Immunol 142(1):207–216 44. Ranges GE, Figari IS, Espevik T, Palladino MA Jr (1987) Inhibition of cytotoxic T cell development by transforming growth factor beta and reversal by recombinant tumor necrosis factor alpha. J Exp Med 166(4):991–998 45. Nemunaitis J, Dillman RO, Schwarzenberger PO et al (2006) Phase II study of belagenpumatucel-L, a transforming growth factor beta-2 antisense gene-modified allogeneic tumor cell vaccine in non-small-cell lung cancer. J Clin Oncol 24(29):4721–4730 46. Sporn MB, Roberts AB, Wakefield LM, Assoian RK (1986) Transforming growth factor-beta: biological function and chemical structure. Science 233(4763):532–534 47. Massague J (1987) The TGF-beta family of growth and differentiation factors. Cell 49(4):437–438 48. Border WA, Ruoslahti E (1992) Transforming growth factor-beta in disease: the dark side of tissue repair. J Clin Invest 90(1):1–7 49. Bodmer S, Strommer K, Frei K et  al (1989) Immunosuppression and transforming growth factor-beta in glioblastoma. Preferential production of transforming growth factor-beta 2. J Immunol 143(10):3222–3229 50. Jakowlew SB, Mathias A, Chung P, Moody TW (1995) Expression of transforming growth factor beta ligand and receptor messenger RNAs in lung cancer cell lines. Cell Growth Differ 6(4):465–476 51. Constam DB, Philipp J, Malipiero UV, ten Dijke P, Schachner M, Fontana A (1992) Differential expression of transforming growth factor-beta 1, -beta 2, and -beta 3 by glioblastoma cells, astrocytes, and microglia. J Immunol 148(5):1404–1410 52. Kong F, Jirtle RL, Huang DH, Clough RW, Anscher MS (1999) Plasma transforming growth factor-beta1 level before radiotherapy correlates with long term outcome of patients with lung carcinoma. Cancer 86(9):1712–1719

296

J. Nemunaitis and J. Roth

53. Kasid A, Bell GI, Director EP (1988) Effects of transforming growth factor-beta on human lymphokine-activated killer cell precursors. Autocrine inhibition of cellular proliferation and differentiation to immune killer cells. J Immunol 141(2):690–698 54. Hirte H, Clark DA (1991) Generation of lymphokine-activated killer cells in human ovarian carcinoma ascitic fluid: identification of transforming growth factor-beta as a suppressive factor. Cancer Immunol Immunother 32(5):296–302 55. Naganuma H, Sasaki A, Satoh E et  al (1996) Transforming growth factor-beta inhibits interferon-gamma secretion by lymphokine-activated killer cells stimulated with tumor cells. Neurol Med Chir (Tokyo) 36(11):789–795 56. Fakhrai H, Mantil JC, Liu L et al (2006) Phase I clinical trial of TGF-b antisense-modified tumor cell vaccine in patients with advanced glioma. Cancer Gene Ther 13(12):1052–1060 57. Liau LM, Fakhrai H, Black KL (1998) Prolonged survival of rats with intracranial C6 gliomas by treatment with TGF-beta antisense gene. Neurol Res 20(8):742–747 58. Kettering JD, Mohamedali AM, Green LM, Gridley DS (2003) IL-2 gene and antisense TGFbeta1 strategies counteract HSV-2 transformed tumor progression. Technol Cancer Res Treat 2(3):211–221 59. Fakhrai H, Dorigo O, Shawler DL et  al (1996) Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy. Proc Natl Acad Sci USA 93(7):2909–2914 60. Shepherd FA, Dancey J, Ramlau R et  al (2000) Prospective randomized trial of docetaxel versus best supportive care in patients with non-small-cell lung cancer previously treated with platinum-based chemotherapy. J Clin Oncol 18(10):2095–2103 61. Fossella FV, DeVore R, Kerr RN et al (2000) Randomized phase III trial of docetaxel versus vinorelbine or ifosfamide in patients with advanced non-small-cell lung cancer previously treated with platinum-containing chemotherapy regimens. The TAX 320 Non-Small Cell Lung Cancer Study Group. J Clin Oncol 18(12):2354–2362 62. Nemunaitis J, Nemunaitis M, Senzer N et al (2009) Phase II trial of belagenpumatucel-L, a TGF-b2 antisense gene modified allogeneic tumor vaccine in advanced non small cell lung cancer (NSCLC) patients. Cancer Gene Ther 16(8):620–624 63. Dranoff G, Jaffee E, Lazenby A et  al (1993) Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci USA 90(8):3539–3543 64. Scheffer SR, Nave H, Korangy F et al (2003) Apoptotic, but not necrotic, tumor cell vaccines induce a potent immune response in vivo. Int J Cancer 103(2):205–211 65. Borrello I, Pardoll D (2002) GM-CSF-based cellular vaccines: a review of the clinical experience. Cytokine Growth Factor Rev 13(2):185–193 66. Jaffee EM, Thomas MC, Huang AY, Hauda KM, Levitsky HI, Pardoll DM (1996) Enhanced immune priming with spatial distribution of paracrine cytokine vaccines. J Immunother Emphasis Tumor Immunol 19(3):176–183 67. Couch M, Saunders JK, O’Malley BW Jr, Pardoll D, Jaffee E (2003) Genetically engineered tumor cell vaccine in a head and neck cancer model. Laryngoscope 113(3):552–556 68. Simons JW, Mikhak B, Chang JF et al (1999) Induction of immunity to prostate cancer antigens: results of a clinical trial of vaccination with irradiated autologous prostate tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer. Cancer Res 59(20):5160–5168 69. Soiffer R, Lynch T, Mihm M et al (1998) Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte-macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma. Proc Natl Acad Sci USA 95(22):13141–13146 70. Simons JW, Jaffee EM, Weber CE et al (1997) Bioactivity of autologous irradiated renal cell carcinoma vaccines generated by ex vivo granulocyte-macrophage colony-stimulating factor gene transfer. Cancer Res 57(8):1537–1546 71. Jaffee EM, Hruban RH, Biedrzycki B et al (2001) Novel allogeneic granulocyte-macrophage colony-stimulating factor-secreting tumor vaccine for pancreatic cancer: a phase I trial of safety and immune activation. J Clin Oncol 19(1):145–156

Vaccine Therapy for Lung Cancer

297

72. Salgia R, Lynch T, Skarin A et  al (2003) Vaccination with irradiated autologous tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor augments antitumor immunity in some patients with metastatic non-small-cell lung carcinoma. J Clin Oncol 21(4):624–630 73. Soiffer R, Hodi FS, Haluska F et al (2003) Vaccination with irradiated, autologous melanoma cells engineered to secrete granulocyte-macrophage colony-stimulating factor by adenoviralmediated gene transfer augments antitumor immunity in patients with metastatic melanoma. J Clin Oncol 21(17):3343–3350 74. Nemunaitis J, Jahan T, Ross H et al (2006) Phase 1/2 trial of autologous tumor mixed with an allogeneic GVAX vaccine in advanced-stage non-small-cell lung cancer. Cancer Gene Ther 13(6):555–562 75. Kufe D, Inghirami G, Abe M, Hayes D, Justi-Wheeler H, Schlom J (1984) Differential reactivity of a novel monoclonal antibody (DF3) with human malignant versus benign breast tumors. Hybridoma 3(3):223–232 76. Burchell J, Gendler S, Taylor-Papadimitriou J et al (1987) Development and characterization of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin. Cancer Res 47(20):5476–5482 77. Gendler SJ, Lancaster CA, Taylor-Papadimitriou J et al (1990) Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem 265(25):15286–15293 78. Li Y, Liu D, Chen D, Kharbanda S, Kufe D (2003) Human DF3/MUC1 carcinoma-associated protein functions as an oncogene. Oncogene 22(38):6107–6110 79. Ren J, Agata N, Chen D et  al (2004) Human MUC1 carcinoma-associated protein confers resistance to genotoxic anticancer agents. Cancer Cell 5(2):163–175 80. MacLean GD, Reddish MA, Koganty RR, Longenecker BM (1996) Antibodies against mucin-associated sialyl-Tn epitopes correlate with survival of metastatic adenocarcinoma patients undergoing active specific immunotherapy with synthetic STn vaccine. J Immunother Emphasis Tumor Immunol 19(1):59–68 81. Butts C, Murray N, Maksymiuk A et al (2005) Randomized phase IIB trial of BLP25 liposome vaccine in stage IIIB and IV non-small-cell lung cancer. J Clin Oncol 23(27):6674–6681 82. Slodkowska J, Szturmowicz M, Rudzinski P et al (1998) Expression of CEA and trophoblastic cell markers by lung carcinoma in association with histological characteristics and serum marker levels. Eur J Cancer Prev 7(1):51–60 83. Fijolek J, Wiatr E, Rowinska-Zakrzewska E et al (2006) p53 and HER2/neu expression in relation to chemotherapy response in patients with non-small cell lung cancer. Int J Biol Markers 21(2):81–87 84. Tsao MS, Aviel-Ronen S, Ding K et al (2007) Prognostic and predictive importance of p53 and RAS for adjuvant chemotherapy in non small-cell lung cancer. J Clin Oncol 25(33):5240–5247 85. Brabender J, Danenberg KD, Metzger R et al (2001) Epidermal growth factor receptor and HER2-neu mRNA expression in non-small cell lung cancer is correlated with survival. Clin Cancer Res 7(7):1850–1855 86. Vallbohmer D, Brabender J, Yang DY et al (2006) Sex differences in the predictive power of the molecular prognostic factor HER2/neu in patients with non-small-cell lung cancer. Clin Lung Cancer 7(5):332–337 87. Sienel W, Varwerk C, Linder A et  al (2004) Melanoma associated antigen (MAGE)-A3 expression in Stages I and II non-small cell lung cancer: results of a multi-center study. Eur J Cardiothorac Surg 25(1):131–134 88. Marshall JL, Hoyer RJ, Toomey MA et al (2000) Phase I study in advanced cancer patients of a diversified prime-and-boost vaccination protocol using recombinant vaccinia virus and recombinant nonreplicating avipox virus to elicit anti-carcinoembryonic antigen immune responses. J Clin Oncol 18(23):3964–3973 89. Fong L, Hou Y, Rivas A et al (2001) Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy. Proc Natl Acad Sci USA 98(15):8809–8814 90. Knutson KL, Schiffman K, Disis ML (2001) Immunization with a HER-2/neu helper peptide vaccine generates HER-2/neu CD8 T-cell immunity in cancer patients. J Clin Invest 107(4):477–484

298

J. Nemunaitis and J. Roth

  91. Rosenberg SA, Yang JC, Schwartzentruber DJ et  al (1998) Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med 4(3):321–327   92. Arlen P, Tsang KY, Marshall JL et al (2000) The use of a rapid ELISPOT assay to analyze peptide-specific immune responses in carcinoma patients to peptide vs. recombinant poxvirus vaccines. Cancer Immunol Immunother 49(10):517–529   93. Horig H, Lee DS, Conkright W et al (2000) Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule. Cancer Immunol Immunother 49(9):504–514   94. Vierboom MP, Nijman HW, Offringa R et  al (1997) Tumor eradication by wild-type p53-specific cytotoxic T lymphocytes. J Exp Med 186(5):695–704   95. Vierboom MP, Bos GM, Ooms M, Offringa R, Melief CJ (2000) Cyclophosphamide enhances anti-tumor effect of wild-type p53-specific CTL. Int J Cancer 87(2):253–260   96. Rosenwirth B, Kuhn EM, Heeney JL et  al (2001) Safety and immunogenicity of ALVAC wild-type human p53 (vCP207) by the intravenous route in rhesus macaques. Vaccine 19(13–14):1661–1670   97. van der Burg SH, de Cock K, Menon AG et  al (2001) Long lasting p53-specific T cell memory responses in the absence of anti-p53 antibodies in patients with resected primary colorectal cancer. Eur J Immunol 31(1):146–155   98. Ferries E, Connan F, Pages F et  al (2001) Identification of p53 peptides recognized by CD8(+) T lymphocytes from patients with bladder cancer. Hum Immunol 62(8):791–798   99. Tartaglia J, Bonnet MC, Berinstein N, Barber B, Klein M, Moingeon P (2001) Therapeutic vaccines against melanoma and colorectal cancer. Vaccine 19(17–19):2571–2575 100. http://www.clinicaltrials.gov (2002) 101. van der Bruggen P, Traversari C, Chomez P et al (1991) A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 254(5038):1643–1647 102. Thurner B, Haendle I, Roder C et al (1999) Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J Exp Med 190(11):1669–1678 103. Weber JS, Hua FL, Spears L, Marty V, Kuniyoshi C, Celis E (1999) A phase I trial of an HLA-A1 restricted MAGE-3 epitope peptide with incomplete Freund’s adjuvant in patients with resected high-risk melanoma. J Immunother 22(5):431–440 104. Coulie PG, Karanikas V, Colau D et  al (2001) A monoclonal cytolytic T-lymphocyte response observed in a melanoma patient vaccinated with a tumor-specific antigenic peptide encoded by gene MAGE-3. Proc Natl Acad Sci USA 98(18):10290–10295 105. Banchereau J, Palucka AK, Dhodapkar M et  al (2001) Immune and clinical responses in patients with metastatic melanoma to CD34(+) progenitor-derived dendritic cell vaccine. Cancer Res 61(17):6451–6458 106. Slamon DJ, Godolphin W, Jones LA et al (1989) Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244(4905):707–712 107. Disis ML, Calenoff E, McLaughlin G et al (1994) Existent T-cell and antibody immunity to HER-2/neu protein in patients with breast cancer. Cancer Res 54(1):16–20 108. Alexander J, Sidney J, Southwood S et  al (1994) Development of high potency universal DR-restricted helper epitopes by modification of high affinity DR-blocking peptides. Immunity 1(9):751–761 109. Ishioka GY, Disis ML, Morse MA et al (2004) Multi-epitope CTL responses induced by a peptide vaccine (EP-2101) in colon and non-small cell lung cancer patients [Abstract]. J Immunother 27(6):S23–S24 110. Antonia SJ, Seigne J, Diaz J et al (2002) Phase I trial of a B7–1 (CD80) gene modified autologous tumor cell vaccine in combination with systemic interleukin-2 in patients with metastatic renal cell carcinoma. J Urol 167(5):1995–2000 111. Hull GW, McCurdy MA, Nasu Y et al (2000) Prostate cancer gene therapy: comparison of adenovirus-mediated expression of interleukin 12 with interleukin 12 plus B7–1 for in situ gene therapy and gene-modified, cell-based vaccines. Clin Cancer Res 6(10):4101–4109

Vaccine Therapy for Lung Cancer

299

112. von Mehren M, Arlen P, Tsang KY et al (2000) Pilot study of a dual gene recombinant avipox vaccine containing both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas. Clin Cancer Res 6(6):2219–2228 113. Johnston JV, Malacko AR, Mizuno MT et  al (1996) B7-CD28 costimulation unveils the hierarchy of tumor epitopes recognized by major histocompatibility complex class I-restricted CD8+ cytolytic T lymphocytes. J Exp Med 183(3):791–800 114. Liu B, Podack ER, Allison JP, Malek TR (1996) Generation of primary tumor-specific CTL in vitro to immunogenic and poorly immunogenic mouse tumors. J Immunol 156(3): 1117–1125 115. Nabel GJ, Gordon D, Bishop DK et al (1996) Immune response in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA-liposome complexes. Proc Natl Acad Sci USA 93(26):15388–15393 116. Yamazaki K, Spruill G, Rhoderick J, Spielman J, Savaraj N, Podack ER (1999) Small cell lung carcinomas express shared and private tumor antigens presented by HLA-A1 or HLAA2. Cancer Res 59(18):4642–4650 117. Raez LE, Cassileth PA, Schlesselman JJ et  al (2004) Allogeneic vaccination with a B7.1 HLA-A gene-modified adenocarcinoma cell line in patients with advanced non-small-cell lung cancer. J Clin Oncol 22(14):2800–2807 118. Raez LE, Santos ES, Mudad R, Podack ER (2005) Clinical trials targeting lung cancer with active immunotherapy: the scope of vaccines. Expert Rev Anticancer Ther 5(4):635–644 119. Mueller-Pillasch F, Pohl B, Wilda M et al (1999) Expression of the highly conserved RNA binding protein KOC in embryogenesis. Mech Dev 88(1):95–99 120. Aguiar JC, Hedstrom RC, Rogers WO et al (2001) Enhancement of the immune response in rabbits to a malaria DNA vaccine by immunization with a needle-free jet device. Vaccine 20(1–2):275–280 121. Nemunaitis JMT, Senzer N, Cunningham C, Anthony S, Vukelja S, Berman B, Sarmiento S, Arzaga R, Cheever M (2006) Phase I trial of sequential administration of recombinant DNA and adenoviral expressing L523S protein in early stage non small cell lung cancer (NSCLC). Mol Ther 13(6):1185–1191 122. Ramos TC, Vinageras E, Ferrer MC et al (2006) Treatment of NSCLC patients with an EGFbased cancer vaccine: report of a phase I trial. Cancer Biol Ther 5:145–149 123. Gonzalez G, Crombet T, Catala M et al (1998) A novel cancer vaccine composed of humanrecombinant epidermal growth factor linked to a carrier protein: report of a pilot clinical trial. Ann Oncol 9(4):431–435 124. Gonzalez G, Crombet T, Torres F et al (2003) Epidermal growth factor-based cancer vaccine for non-small-cell lung cancer therapy. Ann Oncol 14:461–466 125. Van den Eynde BJ, van der Bruggen P (1997) T cell defined tumor antigens. Curr Opin Immunol 9(5):684–693 126. Herman J, van der Bruggen P, Luescher IF et al (1996) A peptide encoded by the human MAGE3 gene and presented by HLA-B44 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE3. Immunogenetics 43(6):377–383 127. Fleischhauer K, Fruci D, Van Endert P et al (1996) Characterization of antigenic peptides presented by HLA-B44 molecules on tumor cells expressing the gene MAGE-3. Int J Cancer 68(5):622–628 128. Tanaka F, Fujie T, Tahara K et al (1997) Induction of antitumor cytotoxic T lymphocytes with a MAGE-3-encoded synthetic peptide presented by human leukocytes antigen-A24. Cancer Res 57(20):4465–4468 129. Kawashima I, Hudson SJ, Tsai V et al (1998) The multi-epitope approach for immunotherapy for cancer: identification of several CTL epitopes from various trumor-associated antigens expressed on solid epithelial tumors. Hum Immunol 59:1–14 130. Oiso M, Eura M, Katsura F et al (1999) A newly identified MAGE-3-derived epitope recognized by HLA-A24-restricted cytotoxic T lymphocytes. Int J Cancer 81(3):387–394 131. Tanzarella S, Russo V, Lionello I et al (1999) Identification of a promiscuous T-cell epitope encoded by multiple members of the MAGE family. Cancer Res 59(11):2668–2674

300

J. Nemunaitis and J. Roth

132. Russo V, Tanzarella S, Dalerba P et al (2000) Dendritic cells acquire the MAGE-3 human tumor antigen from apoptotic cells and induce a class I-restricted T cell response. Proc Natl Acad Sci USA 97(5):2185–2190 133. Keogh E, Fikes J, Southwood S, Celis E, Chesnut R, Sette A (2001) Identification of new epitopes from four different tumor-associated antigens: recognition of naturally processed epitopes correlates with HLA-A*0201-binding affinity. J Immunol 167(2):787–796 134. Schultz ES, Chapiro J, Lurquin C et al (2002) The production of a new MAGE-3 peptide presented to cytolytic T lymphocytes by HLA-B40 requires the immunoproteasome. J Exp Med 195(4):391–399 135. Gaugler B, Van den Eynde B, van der Bruggen P et al (1994) Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes. J Exp Med 179(3):921–930 136. van der Bruggen P, Bastin J, Gajewski T et al (1994) A peptide encoded by human gene MAGE-3 and presented by HLA-A2 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE-3. Eur J Immunol 24(12):3038–3043 137. Marchand M, van Baren N, Weynants P et al (1999) Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1. Int J Cancer 80(2):219–230 138. Atanackovic D, Altorki NK, Stockert E et al (2004) Vaccine-induced CD4+ T cell responses to MAGE-3 protein in lung cancer patients. J Immunol 172:3289–3296 139. Halmos BH (2006) Lung cancer II. ASCO Annu Meet Summ:156–160 140. Vonderheide RH, Hahn WC, Schultze JL, Nadler LM (1999) The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes. Immunity 10(6):673–679 141. Minev B, Hipp J, Firat H, Schmidt JD, Langlade-Demoyen P, Zanetti M (2000) Cytotoxic T cell immunity against telomerase reverse transcriptase in humans. Proc Natl Acad Sci USA 97(9):4796–4801 142. Vonderheide RH, Anderson KS, Hahn WC, Butler MO, Schultze JL, Nadler LM (2001) Characterization of HLA-A3-restricted cytotoxic T lymphocytes reactive against the widely expressed tumor antigen telomerase. Clin Cancer Res 7(11):3343–3348 143. Arai J, Yasukawa M, Ohminami H, Kakimoto M, Hasegawa A, Fujita S (2001) Identification of human telomerase reverse transcriptase-derived peptides that induce HLA-A24-restricted antileukemia cytotoxic T lymphocytes. Blood 97(9):2903–2907 144. Hernandez J, Garcia-Pons F, Lone YC et  al (2002) Identification of a human telomerase reverse transcriptase peptide of low affinity for HLA A2.1 that induces cytotoxic T lymphocytes and mediates lysis of tumor cells. Proc Natl Acad Sci USA 99(19):12275–12280 145. Scardino A, Gross DA, Alves P et  al (2002) HER-2/neu and hTERT cryptic epitopes as novel targets for broad spectrum tumor immunotherapy. J Immunol 168(11):5900–5906 146. Gross DA, Graff-Dubois S, Opolon P et al (2004) High vaccination efficiency of low-affinity epitopes in antitumor immunotherapy. J Clin Invest 113(3):425–433 147. Schroers R, Huang XF, Hammer J, Zhang J, Chen SY (2002) Identification of HLA DR7restricted epitopes from human telomerase reverse transcriptase recognized by CD4+ T-helper cells. Cancer Res 62(9):2600–2605 148. Schroers R, Shen L, Rollins L et al (2003) Human telomerase reverse transcriptase-specific T-helper responses induced by promiscuous major histocompatibility complex class II-restricted epitopes. Clin Cancer Res 9(13):4743–4755 149. Uchida N, Otsuka T, Shigematsu H et al (1999) Differential gene expression of human telomerase-associated protein hTERT and TEP1 in human hematopoietic cells. Leuk Res 23(12): 1127–1132 150. Tahara H, Yasui W, Tahara E et  al (1999) Immuno-histochemical detection of human telomerase catalytic component, hTERT, in human colorectal tumor and non-tumor tissue sections. Oncogene 18(8):1561–1567 151. Shepherd F, Carney D (2000) Treatment of NSCLC: Chemotherapy. In: Textbook of lung cancer. Martin Dunitz, Hansen HH (ed.), London; pp. 213–242

Vaccine Therapy for Lung Cancer

301

152. Brunsvig PF, Aamdal S, Gjertsen MK et al (2006) Telomerase peptide vaccination: a phase I/II study in patients with non-small cell lung cancer. Cancer Immunol Immunother 55(12): 1553–1564 153. Zitvogel L, Regnault A, Lozier A et  al (1998) Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med 4:594–600 154. Théry C, Duban L, Sergura E et al (2002) Indirect activation of naive CD4+ T cells by dendritic cell-derived exosomes. Nat Immunol 3:1156–1162 155. Théry C, Regnault A, Garin J et al (1999) Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73. J Cell Biol 147:599–610 156. Raposo G, Nijman H, Stoorvogel W et al (1996) B lymphocytes secrete antigen-presenting vesicles. J Exp Med 183:1161–1172 157. Denzer K, van Eijk M, Kleijmeer MJ et al (2000) Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface. J Immunol 165:1259–1265 158. Hsu DH, Paz P, Villaflor G et al (2003) Exosomes as a tumor vaccine: enhancing potency through direct loading of antigenic peptides. J Immunother 26:440–450 159. Morse MA, Garst J, Osada T et al (2005) A phase I study of dexosome immunotherapy in patients with advanded non-small cell lung cancer. J Transl Med 3:9 160. Link CJ, Seregina T, Atchison R et al (1998) Eliciting hyperacute xenograft response to treat human cancer: a(1-3)galactosyltransferase gene therapy. Anticancer Res 18:2301–2308 161. Morris JC, Vahanian N, Janik JE (2005) Phase I study of an antitumor vaccination using a(1,3)galactosyltransferase expressing allogeneic tumor cells in patients (Pts) with refractory or recurrent non-small cell lung cancer (NSCLC). J Clin Oncol, ASCO Annu Meet Proc 23(No. 16S, Part I of II (June 1 Suppl)):2586 162. Price ER, Zydowsky LD, Jin MJ, Baker CH, McKeon FD, Walsh CT (1991) Human cyclophilin B: a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence. Proc Natl Acad Sci USA 88(5):1903–1907 163. Bergsma DJ, Eder C, Gross M et al (1991) The cyclophilin multigene family of peptidylprolyl isomerases. Characterization of three separate human isoforms. J Biol Chem 266(34):23204–23214 164. Gomi S, Nakao M, Niiya F et  al (1999) A cyclophilin B gene encodes antigenic epitopes recognized by HLA-A24-restricted and tumor-specific CTLs. J Immunol 163(9):4994–5004 165. Gohara R, Imai N, Rikimaru T et al (2002) Phase 1 clinical study of cyclophilin B peptide vaccine for patients with lung cancer. J Immunother 25(5):439–444 166. Brezicka FT, Olling S, Nilsson O et al (1989) Immunohistological detection of fucosyl-GM1 ganglioside in human lung cancer and normal tissues with monoclonal antibodies. Cancer Res 49(5):1300–1305 167. Brezicka T, Bergman B, Olling S, Fredman P (2000) Reactivity of monoclonal antibodies with ganglioside antigens in human small cell lung cancer tissues. Lung Cancer 28(1):29–36 168. Vangsted AJ, Clausen H, Kjeldsen TB et al (1991) Immunochemical detection of a small cell lung cancer-associated ganglioside (FucGM1) antigen in serum. Cancer Res 51(11):2879–2884 169. Dickler MN, Ragupathi G, Liu NX et al (1999) Immunogenicity of a fucosyl-GM1-keyhole limpet hemocyanin conjugate vaccine in patients with small cell lung cancer. Clin Cancer Res 5(10):2773–2779 170. Krug LM, Ragupathi G, Hood C et al (2004) Vaccination of patients with small-cell lung cancer with synthetic fucosyl GM-1 conjugated to keyhole limpet hemocyanin. Clin Cancer Res 10(18 Pt 1):6094–6100 171. Graus F, Cordon-Cardo C, Houghton AN, Melamed MR, Old LJ (1984) Distribution of the ganglioside GD3 in the human nervous system detected by R24 mouse monoclonal antibody. Brain Res 324(1):190–194 172. Dippold WG, Dienes HP, Knuth A (1985) Meyer zum Buschenfelde KH. Immunohistochemical localization of ganglioside GD3 in human malignant melanoma, epithelial tumors, and normal tissues. Cancer Res 45(8):3699–3705 173. Welte K, Miller G, Chapman PB et al (1987) Stimulation of T lymphocyte proliferation by monoclonal antibodies against GD3 ganglioside. J Immunol 139(6):1763–1771

302

J. Nemunaitis and J. Roth

174. Fuentes R, Allman R, Mason MD (1997) Ganglioside expression in lung cancer cell lines. Lung Cancer 18(1):21–33 175. McCaffery M, Yao TJ, Williams L, Livingston PO, Houghton AN, Chapman PB (1996) Immunization of melanoma patients with BEC2 anti-idiotypic monoclonal antibody that mimics GD3 ganglioside: enhanced immunogenicity when combined with adjuvant. Clin Cancer Res 2(4):679–686 176. Grant SC, Kris MG, Houghton AN, Chapman PB (1999) Long survival of patients with small cell lung cancer after adjuvant treatment with the anti-idiotypic antibody BEC2 plus Bacillus Calmette-Guerin. Clin Cancer Res 5(6):1319–1323 177. Rutishauser U (1998) Polysialic acid at the cell surface: biophysics in service of cell interactions and tissue plasticity. J Cell Biochem 70(3):304–312 178. Finne J, Finne U, Deagostini-Bazin H, Goridis C (1983) Occurrence of alpha 2–8 linked polysialosyl units in a neural cell adhesion molecule. Biochem Biophys Res Commun 112(2):482–487 179. Rutishauser U, Landmesser L (1996) Polysialic acid in the vertebrate nervous system: a promoter of plasticity in cell-cell interactions. Trends Neurosci 19(10):422–427 180. Daniel L, Trouillas J, Renaud W et  al (2000) Polysialylated-neural cell adhesion molecule expression in rat pituitary transplantable tumors (spontaneous mammotropic transplantable tumor in Wistar-Furth rats) is related to growth rate and malignancy. Cancer Res 60(1):80–85 181. Komminoth P, Roth J, Lackie PM, Bitter-Suermann D, Heitz PU (1991) Polysialic acid of the neural cell adhesion molecule distinguishes small cell lung carcinoma from carcinoids. Am J Pathol 139(2):297–304 182. Lantuejoul S, Moro D, Michalides RJ, Brambilla C, Brambilla E (1998) Neural cell adhesion molecules (NCAM) and NCAM-PSA expression in neuroendocrine lung tumors. Am J Surg Pathol 22(10):1267–1276 183. Zhang S, Cordon-Cardo C, Zhang HS et  al (1997) Selection of tumor antigens as targets for immune attack using immunohistochemistry: I. Focus on gangliosides. Int J Cancer 73(1):42–49 184. Krug LM, Ragupathi G, Ng KK et al (2004) Vaccination of small cell lung cancer patients with polysialic acid or N-propionylated polysialic acid conjugated to keyhole limpet hemocyanin. Clin Cancer Res 10(3):916–923 185. Jennings HJ, Roy R, Gamian A (1986) Induction of meningococcal group B polysaccharidespecific IgG antibodies in mice by using an N-propionylated B polysaccharide-tetanus toxoid conjugate vaccine. J Immunol 137(5):1708–1713 186. Drummond IA, Madden SL, Rohwer-Nutter P, Bell GI, Sukhatme VP, Rauscher FJ 3rd (1992) Repression of the insulin-like growth factor II gene by the Wilms tumor suppressor WT1. Science 257(5070):674–678 187. Goodyer P, Dehbi M, Torban E, Bruening W, Pelletier J (1995) Repression of the retinoic acid receptor-alpha gene by the Wilms’ tumor suppressor gene product, wt1. Oncogene 10(6):1125–1129 188. Hewitt SM, Hamada S, McDonnell TJ, Rauscher FJ 3rd, Saunders GF (1995) Regulation of the proto-oncogenes bcl-2 and c-myc by the Wilms’ tumor suppressor gene WT1. Cancer Res 55(22):5386–5389 189. Oji Y, Miyoshi S, Maeda H et al (2002) Overexpression of the Wilms’ tumor gene WT1 in de novo lung cancers. Int J Cancer 100(3):297–303 190. Miyoshi Y, Ando A, Egawa C et al (2002) High expression of Wilms’ tumor suppressor gene predicts poor prognosis in breast cancer patients. Clin Cancer Res 8(5):1167–1171 191. Oka Y, Elisseeva OA, Tsuboi A et al (2000) Human cytotoxic T-lymphocyte responses specific for peptides of the wild-type Wilms’ tumor gene (WT1) product. Immunogenetics 51(2):99–107 192. Ohminami H, Yasukawa M, Fujita S (2000) HLA class I-restricted lysis of leukemia cells by a CD8(+) cytotoxic T-lymphocyte clone specific for WT1 peptide. Blood 95(1):286–293 193. Oka Y, Tsuboi A, Taguchi T et al (2004) Induction of WT1 (Wilms’ tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression. Proc Natl Acad Sci USA 101(38):13885–13890

Vaccine Therapy for Lung Cancer

303

194. Nemunaitis J, Dillman RO, Schwarzenberger PO et al (2006) Phase II study of belagenpumatucel-L, a transforming growth factor beta-2 antisense gene-modified allogeneic tumor cell vaccine in non-small-cell lung cancer. J Clin Oncol 24(29):4721–4730. 195. Nemunaitis J, Nemunaitis J (2003) Granulocyte-macrophage colony-stimulating factor genetransfected autologous tumor cell vaccine: focus[correction to fcous] on non-small-cell lung cancer. Clin Lung Cancer 5(3):148–157 196. Antonia SJ, Mirza N, Fricke I et al (2006) Combination of p53 cancer vaccine with chemotherapy in patients with extensive stage small cell lung cancer. Clin Cancer Res 12(3 Pt 1):878–887

Gene-Based Therapies for Lung Cancer John Nemunaitis and Jack Roth

Abstract  Recent advances in genetics, molecular biology, molecular pharmacology, and biomolecular technology have brought targeted therapeutic opportunities to the forefront of clinical development. Physician and patient communities are highly attracted to lung cancer management opportunities that may involve a personalized approach based on utilizing a unique cancer signal with a targetspecific therapy. In this chapter, we will review several advanced clinical developments involving gene-based targeted therapies in lung cancer. Discussion will focus on replacement therapies for abnormal p53 function, FUS1 mediated molecular therapy, antisense technologies, and early developments with RNA interference technology. Keywords  Gene • Molecular • Lung • Cancer therapy

Introduction Non-small cell lung cancer (NSCLC) management over the last 10 years has significantly improved with the successful development of angiogenesis inhibitors and EGFR inhibitors. However, despite these recent additions to our oncology armament, metastatic disease patients receiving frontline treatment with doublet platinum based chemotherapy in combination with angiogenesis inhibition still have a median survival of less than 1 year. Survival of second line patients is approximately 8 months. Survival of small cell lung cancer patients treated with etoposide based chemotherapy regimes is similar to the

J. Nemunaitis (*) Mary Crowley Cancer Research Centers, 1700 Pacific Avenue, Suite 1100, Dallas, TX 75201, USA e-mail: [email protected] D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_14, © Humana Press, a part of Springer Science+Business Media, LLC 2010

305

306

J. Nemunaitis and J. Roth

survival of patients with advanced NSCLC. In patients with advanced disease, both histologic types of lung cancer have 5-year-survival rates of 1–2% regardless of treatment. Understanding of the biomolecular basis of cancer has exploded over the last 10 years. Developments in genetics, molecular biology, molecular pharmacology, and biomolecular technology promise to dramatically alter strategies of cancer treatment. Like other cancers, NSCLC and SCLC are driven by a complex adaptive network of dynamic evolving spatial–temporal biomolecular interactions. Six essential alterations in the neoplastic physiolome collectively dictate malignant growth. These include self sufficiency, insensitivity to growth inhibition (including immune “escape”), independence from programmed cell death, unlimited replicative potential, sustained angiogenesis, and local and metastatic invasiveness (1). Although it appears intuitive that disruption of any one of these global physiologic capabilities would provide a therapeutic opportunity, each cancer is a robust system capable of maintaining its functional characteristics following internal or external perturbation (2, 3). Cancer cells are able to buffer the impact of genetic modification by virtue of having redundant functional pathways in which different structural elements have overlapping functions, termed “degeneracy” (4). Positive and negative feedback controls allow for stochastic robustness by dampening natural noise. Multileveled functional complementation results from self-contained modules at each organizational level (genome → transcriptosome → proteome → metabolome) which interrelate in a functional organizational hierarchy (5). Work is now underway to integrate theoretical and experimental programs to map out and model in quantifiable terms topological and dynamic properties of networks which control the behavior of one cell. Development of high throughput data collection techniques (i.e., microarrays) allows for simultaneous interrogation of the status of a cell’s components at any given time. New technology platforms, such as protein chips and yeast two hybrid screens, help define how proteins interact with each other and will enable us to determine various types of interactive networks (protein to protein interaction, metabolic, signaling, and transcription/regulatory networks) (6, 7). Interestingly, the modulation of pathways that produce “robustness” in certain insults are generally associated with enhanced “fragility” of other perturbations (2), thereby exposing an “Achilles heel” of cancer and potentially permitting a reasoned coordinated multitarget lethal attack on the cancer (8, 9). Specifically, technologies have been developed that enable the systematic discovery of the molecular pathways thereby setting the stage for targeted therapeutics which focus on driving reduction in proliferation and tumor growth following transcriptional and translational modulation. Efforts to improve these statistics recently have centered around a number of innovative approaches involving immune mediated anticancer effect and/or molecular inhibitory approaches. The purpose of this chapter is to summarize key molecular directed approaches in lung cancer, specifically, p53 gene therapy, antisense technology, and RNA interference technology.

Gene-Based Therapies for Lung Cancer

307

Gene Therapy to Replace Genes Including Missing/Defective Tumor Suppressor Genes Mechanism of p53 Tumor Suppression and Rationale for p53 Gene Therapy Many studies over the past 20 years have established a genetic basis for lung cancer. Genes that suppress tumors and repair DNA can be damaged by more than 100 carcinogens contained in tobacco smoke (10). Lung cancers show multiple genetic lesions even in histologically normal bronchial mucosa from people with a smoking history. These genetic abnormalities provide an array of targets for therapy. The p53 tumor suppressor gene appears to play a central role in lung cancer development and was the initial focus of gene therapy approaches to lung cancer. Two tumor suppressor genes, Rb (retinoblastoma gene) and p53, which are both regulated at the protein level by oncogenes and other tumor suppressor genes, regulate cell proliferation. The Rb protein regulates the maintenance of, and release from, the G1 phase. The p53 protein monitors cellular stress and DNA damage, either causing growth arrest to facilitate DNA repair or inducing apoptosis if DNA damage is extensive (11). When a cell is stressed by oncogene activation, hypoxia, or DNA damage, an intact p53 pathway may determine whether the cell will receive a signal to arrest at the G1 stage of the cell cycle, whether DNA repair will be attempted, or whether the cell will self-destruct via apoptosis (programmed cell death). Apoptosis plays a key role in numerous normal cellular mechanisms, from embryogenesis to destruction of cells that have sustained irreparable DNA damage due to random mutations, ionizing radiation, or DNA damaging chemicals including chemotherapeutic agents. The observation that expression of a wild-type p53 gene in a cancer cell triggers apoptosis provided the rationale for gene therapy approaches (12). Previously, it was believed that gene therapy could not replace all the damaged genes in a cancer cell, and thus would not have a significant effect. The fact that restoration of only one of the defective genes is enough to trigger apoptosis suggests that the DNA damage present in a cancer cell may prime it for an apoptotic event that can be provided through a single pathway. The p53 gene product is a transcription factor that plays a major role in regulating the apoptosis genes (13). p53 also downregulates the prosurvival (or antiapoptotic) genes, including the antiapoptotic genes bcl-2 and bcl-XL, and upregulates the proapoptotic genes bax, bad, bid, puma, and noxa (14). Available transcripts of each of the pro and antiapoptotic genes with bcl2 homology-3 domains interact with one another to form heterodimers, and the relative ratio of proapoptotic to prosurvival proteins in these heterodimers determines the activity of the resulting molecule, thereby determining whether the cell lives or undergoes apoptosis. p53 also targets the death-receptor signaling pathway, including DR5 and Fas/CD95, and the apoptosis machinery, including caspase-6, Apaf-1, and PIDD. It may also directly mediate cytochrome c release.

308

J. Nemunaitis and J. Roth

The p53 pathway is regulated at the protein level by other tumor suppressor genes and by several oncogenes (11). For example, mdm2 normally binds to the N-terminal transactivating domain of p53, prohibiting p53 activation and leading to its rapid degradation. In normal cells, mdm2 is inhibited by expression of p14ARF, a tumor suppressor gene encoded by the same gene locus as p16INK4a but expressed as an alternate reading frame (15). Deletion or mutation of the tumor suppressor gene p14ARF, which has been noted in some cancers, results in increased levels of mdm2 and subsequent inactivation of p53, resulting in an inappropriate progression through the cell cycle. The expression of p14ARF is induced by hyperproliferative signals from oncogenes such as ras and myc, thus indicating an important role for p53 in protecting cells from oncogene activation. Importantly, p53 also plays a central role in mediating cell cycle arrest. This function is significant, as prolonged tumor stability has often been observed in clinical trials of p53 gene replacement, suggesting that this effect may be predominant over apoptosis in some tumors. p53 is involved in regulating cell cycle checkpoints, and p53 expression can promote cell senescence through its control of cell cycle effectors such as p21CIP1/WAF1. Loss of function in the p53 pathway is the most common alteration identified in human cancer to date. About 50% of common epithelial cancers have p53 mutations (16–18). In some cancers, loss of p53 also appears to be linked to resistance to conventional DNA damaging therapies that require functional cellular apoptosis to accomplish cell death. Preclinical Studies of p53 Gene Replacement The studies described above suggest that expressing a wild-type p53 gene in cancer cells defective in p53 function could mediate either apoptosis or cell growth arrest, both of which would be of therapeutic benefit to a cancer patient. Initial studies showed that restoration of functional p53 using a retroviral vector suppressed the growth of some, but not all, human lung cancer cell lines (19). Because of limitations inherent in the use of retroviruses, subsequent studies of p53 gene replacement in lung cancer made use of an adenoviral vector (Ad-p53) (20). The original adenoviral vector was a serotype 5 replication-defective vector with a deleted E1 region, which has been used in all p53 clinical trials. The first published study of p53 gene therapy showed suppression of tumor growth in an orthotopic human lung cancer model using a retroviral expression vector (21). This was the first study to show that restoring the function of a single tumor suppressor gene could result in the regression of human cancer cells in vivo. Ad-p53 also induced apoptosis in cancer cells with nonfunctional p53 without significantly affecting the proliferation of normal cells (22). Subsequent studies with Ad-p53 demonstrated inhibition of tumor growth in a mouse model of human orthotopic lung cancer (23) and induction of apoptosis and suppression of proliferation in various other cancer cell lines and in vivo mouse xenograft tumor models (24–26). Bystander killing (killing of nontransduced cells by transduced cells), now known to be an important phenomenon in the success of gene therapy, appears to

Gene-Based Therapies for Lung Cancer

309

involve regulation of angiogenesis (27, 28), immune upregulation (29–31), and secretion of soluble proapoptotic proteins (32). Clinical Trials of p53 Gene Replacement The first clinical trial protocol for p53 gene-replacement utilized a replicationdefective retroviral vector expressing wild-type p53 driven by a beta-actin promoter (33). The gene/vector construct was injected into tumors of nine patients with unresectable NSCLC that had progressed after conventional therapy. Three of the nine patients showed evidence of tumor regression with no vector-related toxicity, demonstrating the feasibility and safety of p53 gene therapy. Subsequent p53 clinical trials were conducted with the adenovirus p53 vector described above. A phase I trial enrolled 28 NSCLC patients whose cancers had not responded to conventional treatments. Successful gene transfer was demonstrated in 80% of evaluable patients (34). Expression of p53 was detected in 46% of patients, apoptosis was seen in all but one of the patients expressing the gene, and, importantly, no significant toxicity was observed. More than a 50% reduction in tumor size was observed in two patients, with one patient remaining free of tumor more than a year after concluding therapy and another experiencing nearly complete regression of a chemotherapy- and radiation-resistant upper lobe endobronchial tumor. Additional studies in patients with head and neck cancer helped to establish Ad-p53 gene transfer as a clinically feasible strategy resulting in successful gene transfer and gene expression, low toxicity, and strong evidence of tumor regression. Gene Replacement in Combination with Conventional DNA Damaging Agents in NSCLC Preclinical studies of p53 gene therapy combined with cisplatin in cultured NSCLC cells and in human xenografts in nude mice showed that sequential administration of cisplatin and p53 gene therapy resulted in enhanced expression of the p53 gene product (35, 36), and similar studies of Ad-p53 gene transfer combined with radiation therapy indicated that delivery of Ad-p53 increases the sensitivity of p53deficient tumor cells to external beam radiation (26). Many tumors are resistant to chemotherapy and radiation therapy and, therefore, fail initial therapeutic interventions. P53, often missing or nonfunctional in radiationand chemotherapy-resistant tumors, is known to play a key role in detecting damage to DNA and either directing repair or inducing apoptosis. Once apoptosis was implicated as a mechanism of cell killing in response to these DNA damaging agents, it followed that a defect in the normal apoptotic pathway might confer resistance to some tumor cells. Due to Ad-p53’s low toxicity (less than a 5% incidence of serious adverse events) in initial trials, therapeutic strategies combining Ad-p53 gene replacement and conventional DNA damaging therapies were logical extensions of earlier studies (37).

310

J. Nemunaitis and J. Roth

Clinical Trials of Tumor Suppressor Gene Replacement Combined with Chemotherapy Twenty-four NSCLC patients with tumors previously unresponsive to conventional treatment were enrolled in a phase I trial of p53 in sequence with cisplatin (38). Seventy-five percent of the patients had previously experienced tumor progression on cisplatin- or carboplatin-containing regimens. Up to six monthly courses of intravenous cisplatin, each followed 3 days later by intratumoral injection of Ad-p53, resulted in 17 patients remaining stable for at least 2 months, two patients achieving partial responses, four patients continuing to exhibit progressive disease, and one patient unevaluable due to progressive disease. Seventy-nine percent of tumor biopsies showed an increase in the number of apoptotic cells, 7% showed a decrease in apoptosis, and 14% showed no change. A phase II clinical trial evaluated two comparable metastatic lesions in each NSCLC patient enrolled in the study (39). All patients received chemotherapy, either three cycles of carboplatin plus paclitaxel or three cycles of cisplatin plus vinorelbine, and then Ad-p53 was injected directly into one lesion. Ad-p53 treatment resulted in minimal vector-related toxicity and no overall increase in chemotherapy-related adverse events. Detailed statistical analysis of the data indicated that patients receiving carboplatin plus paclitaxel, the combination of drugs that provided the greatest benefit on its own, did not realize additional benefit from Ad-p53 gene transfer. However, patients treated with the lesssuccessful cisplatin and vinorelbine regimen experienced significantly greater mean local tumor regression, as measured by size, in the Ad-p53-injected lesion than in the control lesion. Clinical Trials of p53 Gene Replacement Combined with Radiation Therapy Preclinical studies suggesting that p53 gene replacement might confer radiation sensitivity to some tumors (26, 40–43) led to a phase II clinical trial of p53 gene transfer in conjunction with radiation therapy (44). Patients with a poor performance status who could not undergo surgery and would be at high risk for combined chemotherapy and radiation received 60  Gy over 6 weeks with Ad-p53 injected on days 1, 18, and 32. Nineteen patients with localized NSCLC were treated, resulting in a complete response in one patient (5%), partial response in 11 patients (58%), stable disease in three patients (16%), and progressive disease in two patients (11%). Two patients (11%) were not evaluable due to tumor progression or early death. Three months after the completion of therapy, biopsies revealed no viable tumor in 12 patients (63%) and viable tumor in three (16%). Tumors of four patients (21%) were not biopsied because of tumor progression, early death, or weakness. The 1-year progression-free survival rate was 45.5%. Among 13 evaluable patients after 1 year, five (39%) had a complete response and three (23%) had a partial response or disease stabilization. Most treatment failures were caused by metastatic disease without local progression.

Gene-Based Therapies for Lung Cancer

311

In that study, biopsies of the tumor were performed before and after treatment so that detailed studies of gene expression were possible. Ad-p53 vector-specific DNA was detected in biopsy specimens from 9 of 12 patients with paired biopsies (days 18 and 19). The ratio of copies of Ad-p53 vector DNA to copies of actin DNA was 0.15 or higher in eight of nine patients (range, 0.05–3.85), with four patients having a ratio >0.5. For 11 patients with adequate samples for both vector DNA and mRNA analysis, eight showed a postinjection increase in mRNA expression associated with detectable vector DNA. Postinjection increases in p53 mRNA were detected in 11 of 12 paired biopsies obtained 24 h after Ad-p53 injection, with 10 of 11 increasing threefold or more. Preinjection biopsy specimens that were shown by immunohistochemistry to be negative for p53 protein expression were stained for p53 protein expression after Ad-p53 injection. Staining results confirmed that the p53 protein was expressed in the posttreatment samples in the nuclei of cancer cells. For p21 (CDKN1A) mRNA, increases of statistical significance were noted 24 h after Ad-p53 injection and during treatment, as compared with the pretreatment biopsy. MDM2 mRNA levels were higher during treatment than before treatment. Levels of FAS mRNA did not change significantly during treatment. BAK mRNA expression increased significantly 24 h after injection of Ad-p53 and thus appeared to be the marker most acutely upregulated by Ad-p53 injection. The safety profile for intratumoral injection of Ad-p53 has been excellent. The most frequently reported adverse events related to treatment with Ad-p53 injection were fever and chills, asthenia, injection site pain, nausea, and vomiting. The vast majority of these events were mild to moderate. To date, no maximum tolerated dose for Ad-p53 injection has been established. Systemic Gene Therapy for Metastases Local control of cancers is important, but most patients with lung cancer die from systemic metastases. The development of a cancer vaccine to p53 is one approach. Although the p53 protein is expressed by normal cells, it has a short half-life and is thus present at low levels. Mutant p53 is conformationally altered and resists degradation in cancer cells. Thus, it has a prolonged half-life and is expressed at high levels in cancer cells. These differences in expression between normal and cancer cells suggest that p53 could function as a tumor antigen and vaccine target (45–48). Several studies have shown in cultured cells and mouse models induction of antip53 cytotoxic lymphocytes that killed cancer cells but not normal cells. A strategy was developed using dendritic cells, which are the most effective antigen-presenting cells, transduced with Ad-p53 (49). Patients with extensive-stage small-cell lung cancer (SCLC) were entered into a trial. SCLC patients with extensive stage disease have a median survival of 2–4 months untreated or 6–8 months with chemotherapy. In that trial, the patients’ autologous dendritic cells were treated ex vivo with Ad-p53, which activates the cells and results in the expression of high levels of p53 protein. Patients were first treated with conventional chemotherapy. Those who achieved at least stable disease

312

J. Nemunaitis and J. Roth

received the vaccine biweekly for a total of three to six injections. If patients progressed, they were treated with chemotherapy. Of the 29 patients treated, one had a partial response, seven had stable disease, and 21 had progression. Patients having progression then received second-line chemotherapy. Clinical follow-up was completed for 21 patients. Complete or partial responses to the second-line chemotherapy were observed in 61.9% of the 21 patients treated. Eleven of the patients were alive 1 year after the first vaccine treatment. These clinical responses were correlated with induction of immune responses to the vaccine. Published objective response rates for second-line chemotherapy in extensive-stage SCLC patients ranges from 5 to 30%. Gene delivery to distant sites of cancer is essential for successful cancer gene therapy. Recently, the development of nanoscale synthetic particles that can encapsulate plasmid DNA and deliver it to cells after intravenous injection has been reported. This has been studied in mouse xenograft models of disseminated human lung cancer. In addition to p53, other tumor suppressor genes have been delivered using this technique. Multiple 3p21.3 genes show different degrees of tumor suppression activities in various human cancers in  vitro and in preclinical animal models. One of the tumor suppressor genes at this locus is FUS1, which is not expressed in most lung cancers. When wild-type FUS1 is expressed in a lung cancer cell, apoptosis occurs. To translate these findings to clinical applications for molecular cancer therapy, we recently developed a systemic treatment strategy by using a novel FUS1-expressing plasmid vector complexed with DOTAP:cholesterol (DOTAP:Chol) liposome, termed FUS1 nanoparticle, for treating lung cancer and lung metastases (50, 51). In a preclinical trial, we showed that intratumoral administration of FUS1 nanoparticles to subcutaneous NSCLC H1299 and A549 tumor xenografts resulted in significant inhibition of tumor growth. Intravenous injections of FUS1 nanoparticles into mice bearing experimental A549 lung metastasis significantly decreased the number of metastatic tumor nodules. Lung tumor-bearing animals treated with FUS1 nanoparticles survived longer (median survival time: 80 days) than control animals. These results demonstrate the potent tumor suppressive activity of the FUS1 gene, making it a promising therapeutic agent for treatment of primary and disseminated human lung cancer (50, 51). Based on these studies, a phase I clinical trial with FUS1-mediated molecular therapy by systemic administration of FUS1 nanoparticles is now under way in stage IV lung cancer patients at The University of Texas M. D. Anderson Cancer Center in Houston, Texas. Summary and Conclusions Current therapy such as radiation and chemotherapy controls less than 50% of lung cancers, and overall 5-year survival is only 15%. Combining existing treatments has reached a plateau of efficacy, and the addition of conventional cytotoxic agents is limited because of toxicity. The clinical trials summarized in this article clearly demonstrate that, contrary to initial predictions that gene therapy would not be suitable for cancer, gene replacement therapy targeted to a tumor

Gene-Based Therapies for Lung Cancer

313

suppressor gene can cause cancer regression by activation of known pathways with minimal toxicity. Gene expression has been documented and occurs even in the presence of an antiadenovirus immune response, clinical trials have demonstrated that direct intratumoral injection can cause tumor regression or prolonged stabilization of local disease, and the low toxicity associated with gene transfer indicates that tumor suppressor gene replacement can be readily combined with existing and future treatments. Initial concerns that the wide diversity of genetic lesions in cancer cells would prevent the application of gene therapy to cancer appear unfounded; on the contrary, correction of a single genetic lesion has resulted in significant tumor regression. Studies using the transfer of tumor suppressor genes in combination with conventional DNA damaging treatments indicate that correction of a defect in apoptosis induction can restore sensitivity to radiation and chemotherapy in some resistant tumors, and indications that sensitivity to killing might be enhanced in already sensitive tumors may eventually lead to reduced toxicity from chemotherapy and radiation therapy. The most recent laboratory data demonstrating damage to tumor suppressor genes in normal tissue and premalignant lesions suggests that these genes could someday be useful in early intervention, diagnosis, and even prevention of cancer. Preclinical studies have shown that systemic delivery for treatment of metastases can be achieved. The ready availability of gene libraries, the ability to administer genes without the extensive reformulation required of small molecules, and their specificity make this an attractive therapeutic approach. Despite the obvious promise evident in the results of these studies, though, it is critical to recognize that there are still gaps in knowledge and technology to address. The major issues for the future development of gene therapy include: 1. Development of more efficient and less toxic gene delivery vectors for systemic gene delivery. 2. Identification of the optimal genes for various tumor types. 3. Optimizing combination therapy. 4. Monitoring gene uptake and expression by cancer cells. 5. Overcoming resistance pathways. However, given the rapid progress in the field, it is likely that many of these technological problems will be solved in the near future.

Antisense Technology in NSCLC Antisense oligonucleotides (AS ODNs) are unmodified or chemically modified single-stranded DNA molecules of 13–25 nucleotides in length that are designed to specifically hybridize to corresponding RNA by Watson–Crick binding. They inhibit mRNA function by several mechanisms, one, through inhibition of protein translation by disrupting ribosome assembly, and two, through utilization of endogenous RNase H enzymes that cleave the mRNA strand (52–56). The specificity of

314

J. Nemunaitis and J. Roth

hybridization of an AS ODN to the target mRNA makes the AS strategy attractive for selective modulation of expression of genes involved in the pathogenesis of malignant disease. One AS ODN has been approved for local therapy of cytomegalovirus (CMV) retinitis, and a number of AS ODN’s are currently being tested in clinical trials. These include ODN’s that target C-Raf kinase, C-Raf 1, H ras, protein kinase A-Type I, protein kinase C-alpha, bcl-2, survivin, and DNA methyltransferase (57, 58). Most oligonucleotides being clinically tested have a phosphorothioate backbone in which one of the oxygens on the phosphate moiety is replaced with a sulfur. Phosphorothioate oligonucleotides enable mRNA degradation through enzymatic cleavage via activation of RNase H, and they carry a negative charge which has been shown to bind plasma proteins in a manner similar to heparin (59, 60). This characteristic protects them from filtration thereby prolonging product half-life. However, the negative charge has also been correlated with side effects, including thrombocytopenia and activation of the complement cascade (61). Phosphorothioates accumulate predominantly in the liver but also in the kidneys (62–64).

Protein Kinase C-a: ISIS 3521 Protein Kinase C (PKC) is a family of phospholipid-dependent cytoplasmic serine threonine kinases that comprises distinct isoenzymes which differ in their biochemical properties, tissue-specific expression, and intracellular localization (65, 66). PKC isoenzymes provide signals that lead to proliferation or differentiation (66, 67) PKC-a activity specifically appears to be involved in signaling (68) malignant transformation and proliferation. Overexpression of the PKC-a gene in breast cancer cells results in increased proliferation, anchorage-independent growth, and enhanced tumorigenicity (69). PKC-a expression is also elevated in human breast cancers (70). Inhibition of PKC-a limits growth of hepatoma (71) and medulloblastoma (72). ISIS 3521 (also designated ISI 641A) is a 20-mer phosphorothioate oligodeoxynucleotide that hybridizes to the 3¢-untranslated region of the human PKC-a mRNA, resulting in a site amenable to degradation by RNase H (73). Phase I testing demonstrated acceptable safety and evidence of clinical activity (two patients with lymphoma had complete response to ISIS 3521) (74). Early evaluation of ISIS 3521 in NSCLC involved combination with carboplatin and paclitaxel. Forty-eight evaluable patients with advanced stage IIIB or IV NSCLC were entered into trial (75). Minimal toxicity consisting of neutropenia and thrombocytopenia lead to treatment delays in less than 15% of patients. Patients received a median of six cycles and achieved a response rate of 48%, with 2% (one patient) obtaining complete response and 46% (22 patients) partial response. The median time to progression and the median survival were 6.3 and 15.9 months, respectively. A second phase II trial tested ISIS 3521 in combination with cisplatin and gemcitabine. Forty-four chemotherapy-naïve patients with advanced NSCLC were entered into trial. Toxicity was moderate but included thrombocytopenia,

Gene-Based Therapies for Lung Cancer

315

neutropenia, anemia, fatigue, dehydration, sepsis and neutropenic fever (76). In the updated analysis of the trial, the response rate was 37%, including one complete remission and 11 partial remissions. Based on these phase II data, two large randomized phase III trials were initiated as first-line treatment in patients with NSCLC. The first enrolled 600 patients with stage IV NSCLC using ISIS 3521 in combination with carboplatin and paclitaxel. Results were disappointing. No difference was observed in time to progression or overall survival between treatment and control groups. There were, however, indications of antitumor activity, as a subset of patients who completed the prescribed course of ISIS 3521 (six cycles) had a median survival of 17.4 months when compared with 14.3 months in patients who did not (p = 0.048). Negative results were also obtained in the second phase III trial involving advanced NSCLC patients testing ISIS 3521 in combination with gemcitabine and cisplatin. Therapy was well tolerated, but median survival was roughly 10 months in both groups.

Clusterin: OGX-011 Overexpression of clusterin prolongs cell survival and leads to enhanced metastatic potential of cancer cells in vitro (77). AS against clusterin significantly enhanced chemosensitivity in prostate and renal carcinoma cells in vitro (78). A phase I trial using OGX-011 for patients with localized prostate cancer has been published (79). The most frequently reported side effects were mild (grade 1 or 2) and included fevers, rigors, fatigue, and transient elevations of aspartate aminotransferase and alanine aminotransferase. A second phase I study was designed to determine the recommended dose of OGX-011 in combination with docetaxel (Taxotere™) in various solid tumors (80, 81). OGX-011 is currently in phase II development for patients with prostate, breast, and lung cancer. Combination of OGX-011 and docetaxel in 38 patients with different solid tumors reveals a linear dose-dependent pharmacokinetics of OGX-011, with no apparent interaction with docetaxel. Similar results with OGX-011 were found in combination with cisplatin and gemcitabine. A dose-dependent increase in OGX011 Cmax and AUC was noted, with no apparent interaction with either chemotherapeutic (82). In another trial, OGX-011 was administered in combination with docetaxel (80). The study enrolled 38 patients with a variety of solid tumors (including NSCLC, and prostate, ovarian, renal cell, and breast cancer). A significant decrease in serum clusterin levels was observed in relation to dose of OGX011. Of 24 patients with measurable disease, there was one patient with a partial response (PR) and eight patients with stable disease (SD). In a subsequent clinical trial involving ten chemotherapy-naïve patients with advanced NSCLC OGX-011 was administered in combination with cisplatin and gemcitabine. Two of nine patients with stable disease to prior therapy achieved a PR to OGX-011, cisplatin and gemcitabine. Toxicity primarily occurred within the first week of therapy and diminished with continued dosing. Hematological adverse effects included grade 1

316

J. Nemunaitis and J. Roth

leukopenia, thrombocytopenia, and anemia. Other self-limiting common adverse events were fever, fatigue and rigors occurring several hours after infusion, and grade 1 and 2 elevations in hepatic transaminase levels. No apparent dose-dependent induction of serum complement was observed (79).

H-ras: ISIS 2503 ISIS 2503 is a 20-base phosphorothioate AS ODN that binds to the translation initiation region of human H-ras mRNA (ISIS 2503) and that selectively reduced the expression of H-ras mRNA and protein in cell culture. In a phase I trial, ISIS 2503 administration was not associated with any dose-limiting toxicity. Out of 23 patients, four had stabilization of their disease for six to ten cycles of therapy. No consistent decreases in H-ras mRNA levels were observed in peripheral blood lymphocytes (83). A subsequent multicenter phase II trial analyzed ISIS 2503 in stage IIIB/IV NSCLC. Out of 20 evaluable patients, 7 achieved SD and 13 progressed within the first three cycles. There were no partial or complete responses (84). Given that limited activity was seen and most relevant mutations involving ras oncogene in NSCLC0 are K-ras rather than H-ras, further development if ISIS 2503 in NSCLC has not been done.

C-Raf-1: ISIS 5132 Raf kinases are serine/threonine kinases that regulate mitotic signaling pathways, most notably those involving the mitogen-activated protein kinase pathway signals from ras. This regulation of ras-dependent pathways by raf is potentially important since the ras oncogene is dysregulated or mutated more frequently than any other oncogene studied in human cancer (85, 86). In several tumors, including breast and NSCLC, the presence of a ras mutation is a negative prognostic factor (87). C-raf has also been reported to bind to Bcl-2 and to be involved in the regulation of apoptosis. An AS ODN directed to the 3¢ untranslated region of the c-raf mRNA (ISIS 5132) inhibits growth of human tumor cell lines in vitro and in vivo in association with specific downregulation of target message expression. In a phase I trial, changes in c-raf-1 mRNA expression were analyzed in peripheral blood mononuclear cells (PBMC) collected from patients with advanced cancers treated with ISIS 1532. Significant reductions of c-raf-1 expression from baseline were detected in 13 of 14 patients. Clinical toxicities included fever and fatigue, neither of which were dose limiting. Two patients experienced prolonged disease stabilization for more than 7 months. In both of these cases, this was associated with reduction in c-raf-1 expression in PBMC. Initial results of a phase I trial testing continuous IV infusion of ISIS 5132 in 34 patients with a variety of solid tumors refractory to standard therapy reported one patient at high dose with fever as a dose-limiting toxicity

Gene-Based Therapies for Lung Cancer

317

(88), three patients had grade 3 or 4 thrombocytopenia, and one patient had grade 3 leukopenia. One patient with refractory ovarian cancer had a dramatic reduction in her CA-125 level (97%), and two other patients had prolonged disease stabilization for 9 and 10 months, respectively. No objective responses were seen in a phase II trial for 22 patients with progressive lung cancer (18 NSCLC, 4 SCLC) (89). Hematological toxicity did not exceed grade 2. Nonhematological toxicity was mild to moderate. More recently, a different Raf-1 AS ODN has been developed in a new formulation called LErafAON (NeoPharm, Lake Forest, IL) (90). To avoid the need to chemically protect the oligonucleotide from degradation and to improve intracellular delivery, LErafAON has been encapsulated in a cationic liposome (90). LErafAON is undergoing phase I testing in patients with advanced solid tumors (91).

Bcl-2: Oblimersen Oblimersen is an AS ODN which downregulates Bcl-2 protein expression. Animal studies validated mechanism, safety, and clinical opportunity (92–98). Phase I, II, and III studies have been and are being performed testing oblimersen in patients with multiple advanced cancers including lymphoma, melanoma, breast cancer, hormonerefractory prostate cancer, and a small number of lung cancer patients (99, 100). Phase I and II trial investigation in non Hodgkin’s lymphoma (101, 102) demonstrated dose-related safety. Two patients achieved complete remissions. Reduction in Bcl-2 protein as predicted was able to be demonstrated in a subset of patients (102). Fever and transient grade 3 increases in hepatic enzymes were observed. Phase III investigation involving melanoma (103) did demonstrate limited efficacy; however, it was not sufficient for the Food and Drug Administration (FDA) approval. In melanoma, the overall response rate of the combination of dacarbazine and oblimersen was 12.4% vs. 6.8% for dacarbazine alone (p = 0.0007) (103). The median progression-free survival was 2.4 months vs. 1.6 months (p = 0.0003), but there was only a trend toward improvement in overall median survival (9.0 months vs. 7.8 months, p = 0.077). Oblimersen has been tested in combination with paclitaxel and in combination with carboplatin and etoposide in advanced small cell lung cancer patients, but limited efficacy has been demonstrated (100).

Survivin: LY2181308 Survivin is a member of the IAP gene family, and has an important role in both cell division and apoptosis inhibition (104–106). Survivin is expressed at a high level in a wide range of human cancer types, including lung, colon, pancreas, breast and prostate cancers (105, 107). However, survivin is generally not expressed in normal tissue. Survivin expression levels correlate with lower apoptotic index in tumor

318

J. Nemunaitis and J. Roth

cells and poor prognosis in cancer patients, and serial analysis of gene expression studies have indicated that survivin is the fourth most common gene that is uniformly expressed in cancer cells but not in normal tissues (108). A novel 2¢-MOE ASO (called LY2181308) has been constructed. It specifically downregulates survivin expression in a broad range of human cancer cells and has produced potent antitumor activity in human tumor xenograft models (109, 110). Antitumor activity displayed by LY2181308 in these models is oligonucleotide-sequence specific, and is associated with reduced survivin levels in tumor tissue. Clinical development of LY2181308 is moving forward.

RNA Interference RNA interference (RNAi) is an evolutionarily conserved gene-silencing mechanism which functions during vertebrate embryonic development and is incorporated as an additional layer in the immune defense mechanism (111) whereby small sequences of intrinsic antisense RNA or extrinsic dsRNA (i.e., viral) trigger translational suppression. In cells that endogenously express a gene, introduction of siRNA molecules that target the gene triggers mRNA degradation. The degradation process occurs following interaction of siRNA with ATP dependent helicase and with the ATP dependent RNase enzyme Dicer through the formation of a “RNA interfering silencing complex” (RISC) (112). Endonucleolytic cleavage of the target mRNA occurs at a single site at the center of the target mRNA-siRNA antisense strand duplex (113) and is mediated by Slicer (Ago2) (114). The use of synthetic siRNA molecules has gained wide acceptance as a laboratory tool for target validation, but clinical trials in oncology patients have not yet commenced. Nevertheless RNAi has gained greater acceptance in 2 years than traditional antisense oligonucleotides (ASO) and ribozymes (RBZ) achieved in 20 years (115). Unlike single-stranded RNA, duplex RNA is quite stable and does not require chemical modifications to achieve a satisfactory half-life in cell-culture media (116, 117). While antisense oligonucleotides have been tested clinically (74, 83, 88), the backbone modifications required for oligonucleotide stability increased the risk of toxicity thereby limiting administration at dose levels sufficient to induce significant tumor response, and siRNA methods could potentially avoid this problem. Progress in the development of RNAi technology benefited from previous research aimed at optimizing traditional ASO and RBZ nucleotides. For example, cellular uptake was a major obstacle for efficient gene inhibition inside cells and lessons learned from difficulties in transfecting cells with ASO and RBZ’s were applied to RNAi (118). Wide varieties of efficient delivery systems for nucleic acids have now been developed and are commercially available. In addition, researchers using traditional ASOs had already described potential pitfalls and developed criteria for the essential control experiments needed to validate preliminary data (119).

Gene-Based Therapies for Lung Cancer

319

Finally, biodistribution studies of single-stranded ASOs had been performed, providing suggestions about potential target requirements for siRNA (120–122).

Antitumor Effects of RNAi The exquisite specificity of RNAi has been utilized in multiple studies to exploit phenotypic differences between cancer cells and normal cells. The early work of Martinez and coworkers (123) demonstrated that the p53 mutant-specific RNAi molecule can knockdown the mutant message that differed from wild type by only a single nucleotide, and restored wild type p53 function in heterozygous tumor cells. Similarly, mutant ras silencing by RNAi produced an antitumor effect that nullified the oncogenic phenotype (124). Kawasaki showed that mutant ras decreased by 90% through RNA silencing without altering wild type messages in  vitro and in  vivo (125, 126). Mutant K-ras knockdown also produced ~70% reduction of cancer cell growth in the human colon carcinoma cell line SW480. Retroviral delivery of an RNAi molecule specifically inhibited the mutant K-rasv-12 allele in the human pancreatic carcinoma cell line CAPAN-1 without affecting wild type K-ras level, and collaterally led to a loss of anchor independent growth and tumorigenicity. Similar success has also been attained by targeting the mutant H-ras oncogene (126–128). The targeting of p53 and ras reflect widely different requirements for siRNA reagent design. Insofar as p53 point mutations are located throughout the 11 exon sequences, custom reagents have to be designed for each mutation. By contrast, ras mutations are primarily limited to “hotspots,” thereby allowing a limited number of reagents to cover the most mutated messages. In vitro cancer growth inhibition has been achieved by targeting unique cancer oncogenetic messages that are derived from novel gene fusions (e.g., bcr-abl in myelogenous leukemia) (129), virally-expressed genes (HPV E6/E7 in cervical cancer cells) (130), or overexpressed messages (including HER-2/neu in human breast and ovarian cancer cells (131, 132), protein kinase A in pancreatic cancer cells (133), multidrug resistance genes (134), telomerase (135), and the antiapoptotic bcl-2 gene (136)). In vivo studies have also led to favorable outcomes by RNAi targeting of critical components for tumor cell growth (124, 137–140), metastasis (141–143), angiogenesis (144, 145), and chemoresistance (146, 147). As with ASOs and RBZ’s, efficacy of siRNA depends on the cell type as well as the level of expression of the targeted gene (148). Nonetheless, RNAi has repeatedly proven to be more robust in terms of consistency of transcript knockdowns at threshold concentrations that are several orders of magnitude below typically-used ASOs (113, 148–151). Theoretically, approximately 1–3 molecules of duplexed RNA per cell are effective at knocking down gene expression (112), although most studies in mammalian cells require an intracellular concentration at the nanomolar range. At these concentrations, more prolonged knockdown activity has been observed in vivo as compared with ASO and RBZ (152).

320

J. Nemunaitis and J. Roth

RNAi Delivery Building on the premise that RNAi molecules may have a higher therapeutic index than ASO and RBZ’s, a markedly lower intracellular concentration of RNAi is needed for the desired effect of targeted gene knockdown. Hence the success of RNAi therapy, which requires effective and global delivery of RNAi to target cells, is more likely to be attained. This is particularly applicable to cancer therapy, where multiorgan metastatic foci are largely responsible for the morbidity and mortality of advanced cancer. Murine studies show that RNAi can be administered “hydrodynamically” (153) by rapidly injecting duplex RNAi molecules through the tail vein. This strategy is not feasible clinically and produces severe cardiovascular side effects (152). RNAi molecule delivery by lipid-based technologies (cationic liposomes, liposome-protamine/DNA) (154–156) or viral vectors (157, 158) have also been explored in animal models. Liposomal-based technologies allow for up to 90% transfection efficiency in vitro, but they are costly, difficult to generate and associated with induction of clinically toxic cytokines (IL6, TNFa) (154). Cell-specific immunoliposomes have been used successfully to deliver chemotherapy drugs to target cells and may serve as a viable alternative for cationic-based RNAi delivery. In a recent study by Zhang et al. (159)., weekly intravenous injection of pegylated immunoliposomes effectively delivered epidermal growth factor receptor siRNA to xenografts of intracranial gliomas, resulting in 95% suppression of EGFR function and an 88% increase in survival time. A number of studies have documented stable transduction of siRNA-expressing constructs with viral vectors. Retroviral delivered siRNA effectively targeted p53 in both cell lines and primary fibroblasts (160). Lentiviral vectors were similarly effective, with a lasting effect of >25 days (112). Lentiviral delivery of antiviral siRNA inhibited HIV production from primary human T cells and macrophages (161, 162) in vitro, and silenced target genes in vivo in transgenic mice (163). However, concern over the risk of insertional mutagenesis with retroviruses precludes their clinical use for cancer therapy at this time. Theoretically, bacterial vectors could also be utilized (164), but to date, most cancer gene transfer trials, whether intratumoral, intravenous or intra-arterial, involve adenoviral delivery vehicles.

Potential Hurdles for siRNA Cancer Therapeutics siRNA faces unique hurdles as a cancer gene therapeutic in addition to common concerns that it shares with ASO- and RBZ-based therapies. There are concerns regarding the specificity of siRNA gene silencing with respect to interferon (IFN) induction and “off-target” activity. Contrary to the initial observations of Elbashir et al. (113), Sledz et al. identified JAK–STAT pathway activation and global upregulation of IFN-stimulated genes following PKR activation by a 21-bp siRNA molecule (165). However, nonspecific lFN induction or toxicity was not observed

Gene-Based Therapies for Lung Cancer

321

in various in vivo studies (112, 166). Other contributing elements of IFN activation include the plasmid vector used for siRNA delivery, which may cause formation of long hairpin RNA duplexes, or chemical modifications (e.g., 3¢ triphosphates on the duplex) at the 3¢ end of siRNA (167). The liposomal transfecting agent may also contribute to nonspecific toxicity (168). Hence, each siRNA construct and its delivery system should be carefully scrutinized with respect to its likelihood of soliciting a nonspecific IFN response that would negatively impact therapeutic outcome. In a recent gene expression profile analysis, Jackson and coworkers suggested that siRNAs may exhibit silencing activities on unintended target sequences having less than 18-nucleotide homology with the intended target sequence (169). This apparent lack of fidelity may be explained by an inadequacy of transcriptome search (170). A more extensive evaluation of siRNA’s that had been designed for specific targets revealed multiple examples of other nucleotide sequence homology in addition to the intended target sequence (170). In fact, Snove and Holen identified unintended target sequences with three or fewer mismatches in 75% of 359 published siRNA sequences (170), highlighting the potential risk of siRNA design based on limited sequence analysis. The recent discovery of endogenous microRNAs (miRNAs) furthered misgivings regarding the off-target activity of siRNA. miRNAs are single-stranded RNAs of 21–25 nucleotides found in all multicellular organisms (171–173). In humans, 200–255 genes in the human genome encode miRNAs (172). miRNAs are generated from genome hairpin RNAs through processing by Drosher, and are believed to serve a regulatory function. miRNA inhibits the translation of mRNAs into proteins through imperfect base pairing with the target mRNA, but does not impede transcription or destroy mRNAs. It appears, however, that siRNAs “acting as miRNA” contribute minimally to off-target activity, as synergism between multiple, partially complementarity-bound miRNAs are needed for effective translational silencing (171). Furthermore, unique, target sequence-independent signatures of individual siRNAs remain a laboratory manifestation defined by gene array analyses. The impact of such off-target activity has not been evident in animal studies (152). Preliminary evidence suggests that in vitro off-target activity may be reduced further through chemical modifications, such as nucleotide selection in key positions, and the intentional introduction of mismatches at defined positions between the siRNA sense and antisense strands (174). Development of siRNA technology is moving forward. Initial delivery vehicles will include nonviral strategies (i.e., cationic liposomes). Some of the initial targets will likely include similar genes identified for ASO development.

Concluding Remarks In conclusion, a broad array of targeted gene-based therapies are under active clinical development in NSCLC. Common attributes of these therapies include remarkable safety with virtually no evidence of clinically significant off target effect

322

J. Nemunaitis and J. Roth

outside of the target specificity. Tolerable toxicity, however, is observed in relation to delivery components. Further development is ongoing to reduce toxicity attributed to delivery of gene-based targeted therapeutics. Evidence of clinical activity has been demonstrated and further phase II and a phase III investigation is moving forward. At the same time quantitative proteomic and genomic technology is becoming more accessible, thereby enabling personalized attempts to match a particular targeted therapy with a unique cancer specific molecular signal.

References 1. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100(1):57–70 2. Carlson JM, Doyle J (2002) Complexity and robustness. Proc Natl Acad Sci U S A 99(Suppl 1):2538–2545 3. Stelling J, Sauer U, Szallasi Z, Doyle FJ III, Doyle J (2004) Robustness of cellular functions. Cell 118(6):675–685 4. Edelman GM, Gally JA (2001) Degeneracy and complexity in biological systems. Proc Natl Acad Sci U S A 98(24):13763–13768 5. Laub MT, McAdams HH, Feldblyum T, Fraser CM, Shapiro L (2000) Global analysis of the genetic network controlling a bacterial cell cycle. Science 290(5499):2144–2148 6. Barabasi AL, Oltvai ZN (2004) Network biology: understanding the cell’s functional organization. Nat Rev 5(2):101–113 7. Papin JA, Hunter T, Palsson BO, Subramaniam S (2005) Reconstruction of cellular signalling networks and analysis of their properties. Nat Rev Mol Cell Biol 6(2):99–111 8. Hartwell LH, Szankasi P, Roberts CJ, Murray AW, Friend SH (1997) Integrating genetic approaches into the discovery of anticancer drugs. Science 278(5340):1064–1068 9. Jeong H, Mason SP, Barabasi AL, Oltvai ZN (2001) Lethality and centrality in protein networks. Nature 411(6833):41–42 10. Denissenko MF, Pao A, Tang M, Pfeifer GP (1996) Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53. Science 274(5286):430–432 11. Burns TF, El-Deiry WS (1999) The p53 pathway and apoptosis. J Cell Physiol 181(2):231–239 12. Fujiwara T, Grimm EA, Mukhopadhyay T, Cai DW, Owen-Schaub LB, Roth JA (1993) A retroviral wild-type p53 expression vector penetrates human lung cancer spheroids and inhibits growth by inducing apoptosis. Cancer Res 53(18):4129–4133 13. Raycroft L, Wu HY, Lozano G (1990) Transcriptional activation by wild-type but not transforming mutants of the p53 anti-oncogene. Science 249(4972):1049–1051 14. Adams JM, Cory S (1998) The Bcl-2 protein family: arbiters of cell survival. Science 281(5381):1322–1326 15. Kamijo T, Zindy F, Roussel MF et al (1997) Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell 91(5):649–659 16. Isobe T, Hiyama K, Yoshida Y, Fujiwara Y, Yamakido M (1994) Prognostic significance of p53 and ras gene abnormalities in lung adenocarcinoma patients with stage I disease after curative resection. Jpn J Cancer Res 85(12):1240–1246 17. Martin HM, Filipe MI, Morris RW, Lane DP, Silvestre F (1992) p53 Expression and prognosis in gastric carcinoma. Int J Cancer 50(6):859–862 18. Quinlan DC, Davidson AG, Summers CL, Warden HE, Doshi HM (1992) Accumulation of p53 protein correlates with a poor prognosis in human lung cancer. Cancer Res 52(17):4828–4831

Gene-Based Therapies for Lung Cancer

323

19. Cai DW, Mukhopadhyay T, Roth J (1993) A novel ribozyme for modification of mutated p53 pre-mRNA in non-small cell lung cancer cell lines. In: 3rd antisense workshop, 13 Nov 1993 20. Zhang WW, Fang X, Mazur W, French BA, Georges RN, Roth JA (1994) High-efficiency gene transfer and high-level expression of wild-type p53 in human lung cancer cells mediated by recombinant adenovirus. Cancer Gene Ther 1(1):5–13 21. Fujiwara T, Cai DW, Georges RN, Mukhopadhyay T, Grimm EA, Roth JA (1994) Therapeutic effect of a retroviral wild-type p53 expression vector in an orthotopic lung cancer model. J Natl Cancer Inst 86(19):1458–1462 22. Wang J, Bucana CD, Roth JA, Zhang WW (1995) Apoptosis induced in human osteosarcoma cells is one of the mechanisms for the cytocidal effect of Ad5CMV-p53. Cancer Gene Ther 2(1):9–17 23. Georges RN, Mukhopadhyay T, Zhang Y, Yen N, Roth JA (1993) Prevention of orthotopic human lung cancer growth by intratracheal instillation of a retroviral antisense K-ras construct. Cancer Res 53(8):1743–1746 24. Bouvet M, Fang B, Ekmekcioglu S et al (1998) Suppression of the immune response to an adenovirus vector and enhancement of intratumoral transgene expression by low-dose etoposide. Gene Ther 5(2):189–195 25. Nielsen LL, Dell J, Maxwell E, Armstrong L, Maneval D, Catino JJ (1997) Efficacy of p53 adenovirus-mediated gene therapy against human breast cancer xenografts. Cancer Gene Ther 4(2):129–138 26. Spitz FR, Nguyen D, Skibber JM, Meyn RE, Cristiano RJ, Roth JA (1996) Adenoviralmediated wild-type p53 gene expression sensitizes colorectal cancer cells to ionizing radiation. Clin Cancer Res 2(10):1665–1671 27. Dameron KM, Volpert OV, Tainsky MA, Bouck N (1994) Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 265(5178):1582–1584 28. Miyashita T, Reed JC (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80(2):293–299 29. Carroll JL, Nielsen LL, Pruett SB, Mathis JM (2001) The role of natural killer cells in adenovirus-mediated p53 gene therapy. Mol Cancer Ther 1(1):49–60 30. Molinier-Frenkel V, Le Boulaire C, Le Gal FA et al (2000) Longitudinal follow-up of cellular and humoral immunity induced by recombinant adenovirus-mediated gene therapy in cancer patients. Hum Gene Ther 11(13):1911–1920 31. Yen N, Ioannides CG, Xu K et al (2000) Cellular and humoral immune responses to adenovirus and p53 protein antigens in patients following intratumoral injection of an adenovirus vector expressing wild-type. P53 (Ad-p53). Cancer Gene Ther 7(4):530–536 32. Owen-Schaub LB, Zhang W, Cusack JC et al (1995) Wild-type human p53 and a temperaturesensitive mutant induce Fas/APO-1 expression. Mol Cell Biol 15(6):3032–3040 33. Roth JA, Nguyen D, Lawrence DD et  al (1996) Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer. Nat Med 2(9):985–991 34. Swisher SG, Roth JA, Nemunaitis J et al (1999) Adenovirus-mediated p53 gene transfer in advanced non-small-cell lung cancer. J Natl Cancer Inst 91(9):763–771 35. Fujiwara T, Grimm EA, Mukhopadhyay T, Zhang WW, Owen-Schaub LB, Roth JA (1994) Induction of chemosensitivity in human lung cancer cells in  vivo by adenovirus-mediated transfer of the wild-type p53 gene. Cancer Res 54(9):2287–2291 36. Nguyen DM, Spitz FR, Yen N, Cristiano RJ, Roth JA (1996) Gene therapy for lung cancer: enhancement of tumor suppression by a combination of sequential systemic cisplatin and adenovirus-mediated p53 gene transfer. J Thorac Cardiovasc Surg 112(5):1372–1376 discussion 6–7 37. Yver A, Dreiling LK, Mohanty S et  al (1999) Tolerance and safety of RPR/INGN 201, an adeno-viral vector containing a p53 gene, administered intratumorally in 309 patients with advanced cancer enrolled in phase I and II studies world-wide. Proc Am Soc Clin Oncol 19:460a

324

J. Nemunaitis and J. Roth

38. Nemunaitis J, Swisher SG, Timmons T et al (2000) Adenovirus-mediated p53 gene transfer in sequence with cisplatin to tumors of patients with non-small-cell lung cancer. J Clin Oncol 18(3):609–622 39. Schuler M, Herrmann R, De Greve JL et al (2001) Adenovirus-mediated wild-type p53 gene transfer in patients receiving chemotherapy for advanced non-small-cell lung cancer: results of a multicenter phase II study. J Clin Oncol 19(6):1750–1758 40. Broaddus WC, Liu Y, Steele LL et al (1999) Enhanced radiosensitivity of malignant glioma cells after adenoviral p53 transduction. J Neurosurg 91(6):997–1004 41. Feinmesser M, Halpern M, Fenig E et al (1999) Expression of the apoptosis-related oncogenes bcl-2, bax, and p53 in Merkel cell carcinoma: can they predict treatment response and clinical outcome? Hum Pathol 30(11):1367–1372 42. Jasty R, Lu J, Irwin T, Suchard S, Clarke MF, Castle VP (1998) Role of p53 in the regulation of irradiation-induced apoptosis in neuroblastoma cells. Mol Genet Metab 65(2):155–164 43. Sakakura C, Sweeney EA, Shirahama T et al (1996) Overexpression of bax sensitizes human breast cancer MCF-7 cells to radiation-induced apoptosis. Int J Cancer 67(1):101–105 44. Swisher S, Roth JA, Komaki R et al (2000) A phase II trial of adenoviral mediated p53 gene transfer (RPR/INGN 201) in conjunction with radiation therapy in patients with localized non-small cell lung cancer (NSCLC). Am Soc Clin Oncol 19:461a 45. Chada S, Mhashilkar A, Roth JA, Gabrilovich D (2003) Development of vaccines against self-antigens: the p53 paradigm. Curr Opin Drug Discov Devel 6(2):169–173 46. Ishida T, Chada S, Stipanov M et al (1999) Dendritic cells transduced with wild-type p53 gene elicit potent anti-tumour immune responses. Clin Exp Immunol 117(2):244–251 47. Mayordomo JI, Loftus DJ, Sakamoto H et al (1996) Therapy of murine tumors with p53 wildtype and mutant sequence peptide-based vaccines. J Exp Med 183(4):1357–1365 48. Nikitina EY, Clark JI, Van Beynen J et al (2001) Dendritic cells transduced with full-length wild-type p53 generate antitumor cytotoxic T lymphocytes from peripheral blood of cancer patients. Clin Cancer Res 7(1):127–135 49. Antonia SJ, Mirza N, Fricke I et al (2006) Combination of p53 cancer vaccine with chemotherapy in patients with extensive stage small cell lung cancer. Clin Cancer Res 12(3 Pt 1):878–887 50. Ito I, Ji L, Tanaka F et al (2004) Liposomal vector mediated delivery of the 3p FUS1 gene demonstrates potent antitumor activity against human lung cancer in vivo. Cancer Gene Ther 11(11):733–739 51. Uno F, Sasaki J, Nishizaki M et al (2004) Myristoylation of the fus1 protein is required for tumor suppression in human lung cancer cells. Cancer Res 64(9):2969–2976 52. Clark RE (2000) Antisense therapeutics in chronic myeloid leukaemia: the promise, the progress and the problems. Leukemia 14(3):347–355 53. Baker BF, Monia BP (1999) Novel mechanisms for antisense-mediated regulation of gene expression. Biochim Biophys Acta 1489(1):3–18 54. Crooke ST (1999) Molecular mechanisms of action of antisense drugs. Biochim Biophys Acta 1489(1):31–44 55. Gewirtz AM (2000) Oligonucleotide therapeutics: a step forward. J Clin Oncol 18(9):1809–1811 56. Koller E, Gaarde WA, Monia BP (2000) Elucidating cell signaling mechanisms using antisense technology. Trends Pharmacol Sci 21(4):142–148 57. Coppelli FM, Grandis JR (2005) Oligonucleotides as anticancer agents: from the benchside to the clinic and beyond. Curr Pharm Des 11(22):2825–2840 58. Tamm I, Wagner M (2006) Antisense therapy in clinical oncology: preclinical and clinical experiences. Mol Biotechnol 33(3):221–238 59. Kurreck J (2003) Antisense technologies. Improvement through novel chemical modifications. Eur J Biochem/FEBS 270(8):1628–1644 60. Brown DA, Kang SH, Gryaznov SM et al (1994) Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding. J Biol Chem 269(43):26801–26805

Gene-Based Therapies for Lung Cancer

325

61. Levin AA (1999) A review of the issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides. Biochim Biophys Acta 1489(1):69–84 62. Zhang R, Iyer RP, Yu D et al (1996) Pharmacokinetics and tissue disposition of a chimeric oligodeoxynucleoside phosphorothioate in rats after intravenous administration. J Pharmacol Exp Ther 278(2):971–979 63. Zhang R, Lu Z, Zhang X et  al (1995) In vivo stability and disposition of a self-stabilized oligodeoxynucleotide phosphorothioate in rats. Clin Chem 41(6 Pt 1):836–843 64. Zhang R, Diasio RB, Lu Z et al (1995) Pharmacokinetics and tissue distribution in rats of an oligodeoxynucleotide phosphorothioate (GEM 91) developed as a therapeutic agent for human immunodeficiency virus type-1. Biochem Pharmacol 49(7):929–939 65. Nishizuka Y (1988) The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334(6184):661–665 66. Basu A (1993) The potential of protein kinase C as a target for anticancer treatment. Pharmacol Ther 59(3):257–280 67. Blobe GC, Obeid LM, Hannun YA (1994) Regulation of protein kinase C and role in cancer biology. Cancer Metastasis Rev 13(3–4):411–431 68. Yuspa SH (1994) The pathogenesis of squamous cell cancer: lessons learned from studies of skin carcinogenesis – thirty-third G. H. A. Clowes Memorial Award Lecture. Cancer Res 54(5):1178–1189 69. Ways DK, Kukoly CA, de Vente J et al (1995) MCF-7 breast cancer cells transfected with protein kinase C-alpha exhibit altered expression of other protein kinase C isoforms and display a more aggressive neoplastic phenotype. J Clin Invest 95(4):1906–1915 70. O’Brian C, Vogel VG, Singletary SE, Ward NE (1989) Elevated protein kinase C expression in human breast tumor biopsies relative to normal breast tissue. Cancer Res 49(12):3215–3217 71. Perletti GP, Smeraldi C, Porro D, Piccinini F (1994) Involvement of the alpha isoenzyme of protein kinase C in the growth inhibition induced by phorbol esters in MH1C1 hepatoma cells. Biochem Biophys Res Commun 205(3):1589–1594 72. Adesina AM, Dooley N, Yong VW, Nalbantoglu J (1998) Differential role for protein kinase C-mediated signaling in the proliferation of medulloblastoma cell lines. Int J Oncol 12(4):759–768 73. Dean N, McKay R, Miraglia L et al (1996) Inhibition of growth of human tumor cell lines in nude mice by an antisense of oligonucleotide inhibitor of protein kinase C-alpha expression. Cancer Res 56(15):3499–3507 74. Nemunaitis J, Holmlund JT, Kraynak M et  al (1999) Phase I evaluation of ISIS 3521, an antisense oligodeoxynucleotide to protein kinase C-alpha, in patients with advanced cancer. J Clin Oncol 17(11):3586–3595 75. Yuen AR, Halsey J, Fisher GA et al (1999) Phase I study of an antisense oligonucleotide to protein kinase C-alpha (ISIS 3521/CGP 64128A) in patients with cancer. Clin Cancer Res 5(11):3357–3363 76. Villalona-Calero MA, Ritch P, Figueroa JA et al (2004) A phase I/II study of LY900003, an antisense inhibitor of protein kinase C-alpha, in combination with cisplatin and gemcitabine in patients with advanced non-small cell lung cancer. Clin Cancer Res 10(18 Pt 1):6086–6093 77. Miyake H, Chi KN, Gleave ME (2000) Antisense TRPM-2 oligodeoxynucleotides chemosensitize human androgen-independent PC-3 prostate cancer cells both in vitro and in vivo. Clin Cancer Res 6(5):1655–1663 78. Zellweger T, Miyake H, July LV, Akbari M, Kiyama S, Gleave ME (2001) Chemosensitization of human renal cell cancer using antisense oligonucleotides targeting the antiapoptotic gene clusterin. Neoplasia 3(4):360–367 79. Chi KN, Eisenhauer E, Fazli L et al (2005) A phase I pharmacokinetic and pharmacodynamic study of OGX-011, a 2¢-methoxyethyl antisense oligonucleotide to clusterin, in patients with localized prostate cancer. J Natl Cancer Inst 97(17):1287–1296

326

J. Nemunaitis and J. Roth

80. Chi KN, Siu LL, Hirte H et  al (2008) A phase I study of OGX-011, a 2¢-methoxyethyl phosphorothioate antisense to clusterin, in combination with docetaxel in patients with advanced cancer. Clin Cancer Res 14(3):833–839 81. Gleave M, Miyake H (2005) Use of antisense oligonucleotides targeting the cytoprotective gene, clusterin, to enhance androgen- and chemo-sensitivity in prostate cancer. World J Urol 23(1):38–46 82. Laskin J, Chi KN, Melosky B et al (2006) Phase I study of OGX-011, a second generation antisense oligonucleotide (ASO) to clusterin, combined with cisplatin and gemcitabine as first-line treatment for patients with stage IIB/IV non-small cell lung cancer (NSCLC). J Clin Oncol 24(18S):17078 83. Cunningham CC, Holmlund JT, Geary RS et al (2001) A Phase I trial of H-ras antisense oligonucleotide ISIS 2503 administered as a continuous intravenous infusion in patients with advanced carcinoma. Cancer 92(5):1265–1271 84. Dang T, Johnson DH, Kelly K, Rizvi N, Holmlund J, Dorr A (2001) Multicenter phase II trial of an antisense inhibitor of H-ras (ISIS-2503) in advanced non-small cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 20:1325a 85. Bollag G, McCormick F (1991) Regulators and effectors of ras proteins. Annu Rev Cell Biol 7:601–632 86. Bos JL (1989) ras Oncogenes in human cancer: a review. Cancer Res 49(17):4682–4689 87. Eckhardt SG, Rizzo J, Sweeney KR et  al (1999) Phase I and pharmacologic study of the tyrosine kinase inhibitor SU101 in patients with advanced solid tumors. J Clin Oncol 17(4):1095–1104 88. Cunningham CC, Holmlund JT, Schiller JH et al (2000) A phase I trial of c-Raf kinase antisense oligonucleotide ISIS 5132 administered as a continuous intravenous infusion in patients with advanced cancer. Clin Cancer Res 6(5):1626–1631 89. Coudert B, Anthoney A, Fiedler W et al (2001) Phase II trial with ISIS 5132 in patients with small-cell (SCLC) and non-small cell (NSCLC) lung cancer. A European Organization for Research and Treatment of Cancer (EORTC) Early Clinical Studies Group report. Eur J Cancer 37(17):2194–2198 90. Gokhale PC, Zhang C, Newsome JT et al (2002) Pharmacokinetics, toxicity, and efficacy of ends-modified raf antisense oligodeoxyribonucleotide encapsulated in a novel cationic liposome. Clin Cancer Res 8(11):3611–3621 91. Rudin CM, Marshall JL, Huang CH et  al (2004) Delivery of a liposomal c-raf-1 antisense oligonucleotide by weekly bolus dosing in patients with advanced solid tumors: a phase I study. Clin Cancer Res 10(21):7244–7251 92. Reed JC, Stein C, Subasinghe C et al (1990) Antisense-mediated inhibition of BCL2 protooncogene expression and leukemic cell growth and survival: comparisons of phosphodiester and phosphorothioate oligodeoxynucleotides. Cancer Res 50(20):6565–6570 93. Kitada S, Miyashita T, Tanaka S, Reed JC (1993) Investigations of antisense oligonucleotides targeted against bcl-2 RNAs. Antisense Res Dev 3(2):157–169 94. Kitada S, Takayama S, De Riel K, Tanaka S, Reed JC (1994) Reversal of chemoresistance of lymphoma cells by antisense-mediated reduction of bcl-2 gene expression. Antisense Res Dev 4(2):71–79 95. Cotter FE, Johnson P, Hall P et  al (1994) Antisense oligonucleotides suppress B-cell lymphoma growth in a SCID-hu mouse model. Oncogene 9(10):3049–3055 96. Gleave ME, Miayake H, Goldie J, Nelson C, Tolcher A (1999) Targeting bcl-2 gene to delay androgen-independent progression and enhance chemosensitivity in prostate cancer using antisense bcl-2 oligodeoxynucleotides. Urology 54(6A Suppl):36–46 97. Miyake H, Tolcher A, Gleave ME (1999) Antisense Bcl-2 oligodeoxynucleotides inhibit progression to androgen-independence after castration in the Shionogi tumor model. Cancer Res 59(16):4030–4034 98. Cotter FE, Corbo M, Raynaud F et al (1996) Bcl-2 antisense therapy in lymphoma: in vitro and in vivo mechanisms, efficacy, pharmacokinetics and toxicity studies. Ann Oncol 7:32

Gene-Based Therapies for Lung Cancer

327

99. Tolcher AW, Chi K, Kuhn J et al (2005) A phase II, pharmacokinetic, and biological correlative study of oblimersen sodium and docetaxel in patients with hormone-refractory prostate cancer. Clin Cancer Res 11(10):3854–3861 100. Rudin CM, Otterson GA, Mauer AM et al (2002) A pilot trial of G3139, a bcl-2 antisense oligonucleotide, and paclitaxel in patients with chemorefractory small-cell lung cancer. Ann Oncol 13(4):539–545 101. Webb A, Cunningham D, Cotter F et  al (1997) BCL-2 antisense therapy in patients with non-Hodgkin lymphoma. Lancet 349(9059):1137–1141 102. Waters JS, Webb A, Cunningham D et al (2000) Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin’s lymphoma. J Clin Oncol 18(9):1812–1823 103. Bedikian AY, Millward M, Pehamberger H et al (2006) Bcl-2 antisense (oblimersen sodium) plus dacarbazine in patients with advanced melanoma: the Oblimersen Melanoma Study Group. J Clin Oncol 24(29):4738–4745 104. Li F, Ambrosini G, Chu EY et al (1998) Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396(6711):580–584 105. Ambrosini G, Adida C, Altieri DC (1997) A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med 3(8):917–921 106. Tamm I, Wang Y, Sausville E et al (1998) IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res 58(23):5315–5320 107. Lu CD, Altieri DC, Tanigawa N (1998) Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res 58(9):1808–1812 108. Lal A, Lash AE, Altschul SF et al (1999) A public database for gene expression in human cancers. Cancer Res 59(21):5403–5407 109. Li F, Ackermann EJ, Bennett CF et al (1999) Pleiotropic cell-division defects and apoptosis induced by interference with survivin function. Nat Cell Biol 1(8):461–466 110. Chen J, Wu W, Tahir SK et al (2000) Down-regulation of survivin by antisense oligonucleotides increases apoptosis, inhibits cytokinesis and anchorage-independent growth. Neoplasia 2(3):235–241 111. Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75(5):843–854 112. Ichim TE, Li M, Qian H et al (2004) RNA interference: a potent tool for gene-specific therapeutics. Am J Transplant 4(8):1227–1236 113. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411(6836):494–498 114. Liu J, Carmell MA, Rivas FV et al (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305(5689):1437–1441 115. Paroo Z, Corey DR (2004) Challenges for RNAi in vivo. Trends Biotechnol 22(8):390–394 116. Braasch DA, Jensen S, Liu Y et al (2003) RNA interference in mammalian cells by chemically-modified RNA. Biochemistry 42(26):7967–7975 117. Hough SR, Wiederholt KA, Burrier AC, Woolf TM, Taylor MF (2003) Why RNAi makes sense. Nat Biotechnol 21(7):731–732 118. Hogrefe RI (1999) An antisense oligonucleotide primer. Antisense Nucleic Acid Drug Dev 9(4):351–357 119. Crooke ST (2000) Evaluating the mechanism of action of antiproliferative antisense drugs. Antisense Nucleic Acid Drug Dev 10(2):123–126 discussion 7 120. Sands H, Gorey-Feret LJ, Cocuzza AJ, Hobbs FW, Chidester D, Trainor GL (1994) Biodistribution and metabolism of internally 3H-labeled oligonucleotides. I. Comparison of a phosphodiester and a phosphorothioate. Mol Pharmacol 45(5):932–943

328

J. Nemunaitis and J. Roth

121. Geary RS, Watanabe TA, Truong L et  al (2001) Pharmacokinetic properties of 2¢-O-(2methoxyethyl)-modified oligonucleotide analogs in rats. J Pharmacol Exp Ther 296(3):890–897 122. Geary RS, Yu RZ, Levin AA (2001) Pharmacokinetics of phosphorothioate antisense oligodeoxynucleotides. Curr Opin Investig Drugs 2(4):562–573 123. Martinez LA, Naguibneva I, Lehrmann H et  al (2002) Synthetic small inhibiting RNAs: efficient tools to inactivate oncogenic mutations and restore p53 pathways. Proc Natl Acad Sci U S A 99(23):14849–14854 124. Brummelkamp TR, Bernards R, Agami R (2002) Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer cell 2(3):243–247 125. Kawasaki H, Suyama E, Iyo M, Taira K (2003) siRNAs generated by recombinant human Dicer induce specific and significant but target site-independent gene silencing in human cells. Nucleic Acids Res 31(3):981–987 126. Kawasaki H, Taira K (2003) Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Res 31(2):700–707 127. Yang G, Thompson JA, Fang B, Liu J (2003) Silencing of H-ras gene expression by retrovirus-mediated siRNA decreases transformation efficiency and tumorgrowth in a model of human ovarian cancer. Oncogene 22(36):5694–5701 128. Yin JQ, Gao J, Shao R, Tian WN, Wang J, Wan Y (2003) siRNA agents inhibit oncogene expression and attenuate human tumor cell growth. J Exp Ther Oncol 3(4):194–204 129. Scherr M, Battmer K, Winkler T, Heidenreich O, Ganser A, Eder M (2003) Specific inhibition of bcr-abl gene expression by small interfering RNA. Blood 101(4):1566–1569 130. Yoshinouchi M, Yamada T, Kizaki M et al (2003) In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by E6 siRNA. Mol Ther 8(5):762–768 131. Choudhury A, Charo J, Parapuram SK et al (2004) Small interfering RNA (siRNA) inhibits the expression of the Her2/neu gene, upregulates HLA class I and induces apoptosis of Her2/ neu positive tumor cell lines. Int J Cancer 108(1):71–77 132. Yang G, Cai KQ, Thompson-Lanza JA, Bast RC Jr, Liu J (2004) Inhibition of breast and ovarian tumor growth through multiple signaling pathways by using retrovirus-mediated small interfering RNA against Her-2/neu gene expression. J Biol Chem 279(6):4339–4345 133. Farrow B, Rychahou P, Murillo C, O’Connor KL, Iwamura T, Evers BM (2003) Inhibition of pancreatic cancer cell growth and induction of apoptosis with novel therapies directed against protein kinase A. Surgery 134(2):197–205 134. Yague E, Higgins CF, Raguz S (2004) Complete reversal of multidrug resistance by stable expression of small interfering RNAs targeting MDR1. Gene Ther 11(14):1170–1174 135. Kosciolek BA, Kalantidis K, Tabler M, Rowley PT (2003) Inhibition of telomerase activity in human cancer cells by RNA interference. Mol Cancer Ther 2(3):209–216 136. Cioca DP, Aoki Y, Kiyosawa K (2003) RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines. Cancer Gene Ther 10(2):125–133 137. Aharinejad S, Paulus P, Sioud M et al (2004) Colony-stimulating factor-1 blockade by antisense oligonucleotides and small interfering RNAs suppresses growth of human mammary tumor xenografts in mice. Cancer Res 64(15):5378–5384 138. Li K, Lin SY, Brunicardi FC, Seu P (2003) Use of RNA interference to target cyclin E-overexpressing hepatocellular carcinoma. Cancer Res 63(13):3593–3597 139. Uchida H, Tanaka T, Sasaki K et al (2004) Adenovirus-mediated transfer of siRNA against survivin induced apoptosis and attenuated tumor cell growth in vitro and in vivo. Mol Ther 10(1):162–171 140. Verma UN, Surabhi RM, Schmaltieg A, Becerra C, Gaynor RB (2003) Small interfering RNAs directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells. Clin Cancer Res 9(4):1291–1300

Gene-Based Therapies for Lung Cancer

329

141. Duxbury MS, Ito H, Zinner MJ, Ashley SW, Whang EE (2004) CEACAM6 gene silencing impairs anoikis resistance and in vivo metastatic ability of pancreatic adenocarcinoma cells. Oncogene 23(2):465–473 142. Duxbury MS, Ito H, Zinner MJ, Ashley SW, Whang EE (2004) RNA interference targeting the M2 subunit of ribonucleotide reductase enhances pancreatic adenocarcinoma chemosensitivity to gemcitabine. Oncogene 23(8):1539–1548 143. Salisbury AJ, Macaulay VM (2003) Development of molecular agents for IGF receptor targeting. Horm Metab Res 35(11–12):843–849 144. Filleur S, Courtin A, Ait-Si-Ali S et  al (2003) SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. Cancer Res 63(14):3919–3922 145. Takei Y, Kadomatsu K, Yuzawa Y, Matsuo S, Muramatsu T (2004) A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics. Cancer Res 64(10):3365–3370 146. Chen J, Wall NR, Kocher K et  al (2004) Stable expression of small interfering RNA sensitizes TEL-PDGFbetaR to inhibition with imatinib or rapamycin. J Clin Invest 113(12):1784–1791 147. Lakka SS, Gondi CS, Yanamandra N et al (2004) Inhibition of cathepsin B and MMP-9 gene expression in glioblastoma cell line via RNA interference reduces tumor cell invasion, tumor growth and angiogenesis. Oncogene 23(27):4681–4689 148. Bass BL (2001) RNA interference. The short answer. Nature 411(6836):428–429 149. Aoki Y, Cioca DP, Oidaira H, Kamiya J, Kiyosawa K (2003) RNA interference may be more potent than antisense RNA in human cancer cell lines. Clin Exp Pharmacol Physiol 30(1–2):96–102 150. Coma S, Noe V, Lavarino C et  al (2004) Use of siRNAs and antisense oligonucleotides against survivin RNA to inhibit steps leading to tumor angiogenesis. Oligonucleotides 14(2):100–113 151. Miyagishi M, Hayashi M, Taira K (2003) Comparison of the suppressive effects of antisense oligonucleotides and siRNAs directed against the same targets in mammalian cells. Antisense Nucleic Acid Drug Dev 13(1):1–7 152. Lieberman J, Song E, Lee SK, Shankar P (2003) Interfering with disease: opportunities and roadblocks to harnessing RNA interference. Trends Mol Med 9(9):397–403 153. McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA (2002) RNA interference in adult mice. Nature 418(6893):38–39 154. Sioud M, Sorensen DR (2003) Cationic liposome-mediated delivery of siRNAs in adult mice. Biochem Biophys Res Commun 312(4):1220–1225 155. Sorensen DR, Leirdal M, Sioud M (2003) Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J Mol Biol 327(4):761–766 156. Tan Y, Zhang JS, Huang L (2002) Codelivery of NF-kappaB decoy-related oligodeoxynucleotide improves LPD-mediated systemic gene transfer. Mol Ther 6(6):804–812 157. Cao X, Daniel J, Ozvaran M et al (2004) Bcl-XL silencing in thoracic malignancies using short interfering RNA (siRNA) (Abstract). Cancer Gene Ther (in press) 158. Xia H, Mao Q, Paulson HL, Davidson BL (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 20(10):1006–1010 159. Zhang Y, Zhang YF, Bryant J, Charles A, Boado RJ, Pardridge WM (2004) Intravenous RNA interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer. Clin Cancer Res 10(11):3667–3677 160. Barton GM, Medzhitov R (2002) Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci U S A 99(23):14943–14945 161. Lee MT, Coburn GA, McClure MO, Cullen BR (2003) Inhibition of human immunodeficiency virus type 1 replication in primary macrophages by using Tat- or CCR5-specific small interfering RNAs expressed from a lentivirus vector. J Virol 77(22):11964–11972

330

J. Nemunaitis and J. Roth

162. Qin XF, An DS, Chen IS, Baltimore D (2003) Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci U S A 100(1):183–188 163. Tiscornia G, Singer O, Ikawa M, Verma IM (2003) A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci U S A 100(4):1844–1848 164. Lin J, Lin E, Nemunaitis J (2004) Bacteria in the treatment of cancer. Curr Opin Mol Ther 6(6):629–639 165. Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR (2003) Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5(9):834–839 166. Hill JA, Ichim TE, Kusznieruk KP et al (2003) Immune modulation by silencing IL-12 production in dendritic cells using small interfering RNA. J Immunol 171(2):691–696 167. Kim DH, Longo M, Han Y, Lundberg P, Cantin E, Rossi JJ (2004) Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Nat Biotechnol 22(3):321–325 168. Khvorova A, Reynolds A, Jayasena SD (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115(2):209–216 169. Jackson AL, Bartz SR, Schelter J et al (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 21(6):635–637 170. Snove O Jr, Holen T (2004) Many commonly used siRNAs risk off-target activity. Biochem Biophys Res Commun 319(1):256–263 171. Novina CD, Sharp PA (2004) The RNAi revolution. Nature 430(6996):161–164 172. Lim LP, Glasner ME, Yekta S, Burge CB, Bartel DP (2003) Vertebrate microRNA genes. Science 299(5612):1540 173. Moss EG (2003) Silencing unhealthy alleles naturally. Trends Biotechnol 21(5):185–187 174. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22(3):326–330

Lung Cancer Resistance to Chemotherapy David J. Stewart

Abstract  Metastatic lung cancer remains incurable by chemotherapy. Several factors contribute to resistance to chemotherapy, including many factors that are adaptations of systems that evolved to protect normal cells from a hostile environment. Tumor cell characteristics, tumor cell interactions with extracellular matrix and stromal cells, and tumor physical characteristics all contribute to resistance. Resistance may arise from gene upregulation or downregulation as a downstream consequence of the oncogene mutations or tumor suppressor gene deletions that underlie tumorigenesis or may also arise due to tumor hypoxia or due to exposure to therapy. Host gene polymorphisms may alter resistance by determining the half-life or enzymatic activity of upregulated resistance factors. Resistance may arise from decreased drug delivery to tumor, impact of extracellular pH on drug uptake, altered drug uptake transporters or cell membrane characteristics, increased drug efflux or detoxification, decreased drug binding, altered drug targets, increased DNA repair, decreased proapoptotic factors, increased antiapoptotic factors, altered cell cycling or mitotic checkpoints, or altered transcription factors. This diversity of resistance mechanisms magnifies the challenges facing us in predicting patient prognosis and in overcoming resistance. Keywords  Lung cancer • Chemotherapy • Resistance

Lung Cancer and Resistance As outlined elsewhere in this text, despite 20–50% of patients with advanced non-small cell lung cancer (NSCLC) and 60–80% of patients with extensive small cell lung cancer (SCLC) initially responding to chemotherapy, widely metastatic

D.J. Stewart (*) Department of Thoracic/Head & Neck Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA e-mail: [email protected]

D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_15, © Humana Press, a part of Springer Science+Business Media, LLC 2010

331

332

D.J. Stewart

disease cannot be cured since almost all tumors, that are not intrinsically resistant, rapidly develop acquired broad cross-resistance to therapy. The mechanisms by which tumors become resistant to chemotherapy are generally adaptations of mechanisms that have developed through evolution to protect normal tissues from a hostile environment. The observation that many genes often concurrently have altered expression within the same resistant lung cancer (1, 2) suggests that resistance is generally due to the cumulative effect of several factors acting together, rather than being due to the effect of just one or a few factors. The fact that tumor gene expression arrays (3) and in vitro sensitivity testing (4–7) are highly accurate in predicting clinical resistance but less accurate in predicting sensitivity in lung cancer suggests that tumor cellular factors alone are sufficient to cause resistance, but that in  vivo tumor physical characteristics and host factors may preclude response despite the presence of intrinsically sensitive tumor cells. In addition, while drug efficacy differs somewhat across types of lung cancer preclinically (8) and clinically (9), there are also substantial similarities, and the broad crossresistance seen between chemotherapy agents in both preclinical (10–12) and clinical (13) studies suggests that factors that render a tumor resistant to one agent will also often render it resistant to most other agents.

Types of Resistance Resistance is often classified as “intrinsic” vs. “acquired.” As outlined in Table 1, it can also be classified as “active” (due to excess of a resistance factor) vs. “nonsaturable passive” (due to mutation or alteration of a factor) vs. “saturable passive” (due to deficiency or saturation of a factor required for drug efficacy) (14). Flattening of dose–response curves at higher chemotherapy doses (15) suggests that resistance due to deficiency or saturation of factors required for drug efficacy (e.g., as a result of gene silencing through drug-induced DNA hypermethylation (16)) may be particularly important in NSCLC and other epithelial tumors. Resistance may also be “accelerated” (due to rapid tumor cell repopulation) or “quiescent” (from insufficient cycling through sensitive phases of the cell cycle, with the quiescent resistance being related in some cases to broad downregulation of

Table 1  Examples of ways to classify resistance Intrinsic vs. acquired Active vs. non-saturable passive vs. saturable passive Quiescent vs. accelerated Due to:   Mutation vs. epigenetic   Host factors vs. tumor factors   Tumor cell factors vs. microenvironment/stromal factors vs. abscopal effects of a distant resistant tumor

Lung Cancer Resistance to Chemotherapy

333

membrane transporters (17), to reversible senescence (18), or to autophagy (19)). Furthermore, resistance may be “genetic” (due to resistance-generating mutations) or “epigenetic” (due to upregulation or downregulation of expression of relevant genes), and it may be related to tumor cell characteristics, stromal characteristics, or host factors. Tumors in one part of the body may render tumors at a distance resistant (20), possibly through mobilization of protective mesenchymal stem cells from the bone marrow. All of these mechanisms probably play a role in rendering advanced lung cancer incurable.

Importance of the Host Genotype Tumors inherit the genotype of the host, in addition to having tumor-specific mutations. Gene polymorphisms inherited from the host may modulate resistance by altering the enzymatic activity or protein half-life of a resistance factor, such as a DNA repair protein (Table 2). The correlation of chemotherapy-induced leukopenia with tumor response in SCLC (21) is in keeping with a link between host genotype and tumor sensitivity to therapy. Little is known regarding the relative importance of host-derived factors vs. tumor-specific factors in resistance, and it is likely that both play a role. The number of copies of a gene for a resistance factor may be higher in tumor than in normal cells (due to gene amplification or polyploidy), and tumor-specific factors could also affect gene transcription and posttranscriptional modifications of the resistance factor, but the protein expression of the resistance factor could also be increased or decreased if the host polymorphisms are associated

Table 2  Genes for which host genetic polymorphisms have been reported to contribute to resistance Factor Agents affected MRP2 Cisplatin + irinotecan MDR1/p-glycoprotein Cisplatin + etoposide or vinorelbinea Glutathione-S-transferase-p Cisplatin regimens Deoxycytidine deaminase Gemcitabine ERCC1 Platinum regimens Xeroderma pigmentosum C Platinum regimensb Xeroderma pigmentosum D Platinum regimensb Xeroderma pigmentosum G Platinum regimens XRCC1 Platinum regimensb NQO1 Platinum regimens p53 Platinum regimens Cyclin D1 Platinum regimens a No association with outcome in patients treated with cisplatin–docetaxel b Data were equivocal or negative in some individual trials

334

D.J. Stewart

with increased or decreased half-life of the protein, and for a given degree of expression, the effect of the resistance factor could vary with polymorphisms that alter the enzymatic activity of the factor. Host gene polymorphisms could also affect drug efficacy by altering drug metabolism. In NSCLC patients, efficacy correlated with cytochrome p450 polymorphisms in patients receiving vinorelbine-based chemotherapy (22), and efficacy and toxicity correlated with uridine diphosphate-glucuronosyltransferase polymorphisms in patients receiving irinotecan plus cisplatin (23).

Chemotherapy as “Targeted” Therapy Chemotherapy agents may hit a variety of targets, including DNA, tubulin, topoisomerases, a variety of other enzymes, etc. However, little is known about how chemotherapy agents achieve the selectivity that permits major tumor shrinkage with relatively little damage to most normal organs. An early concept was that the major selectivity factor was the rapid growth of tumor cells. However, the observation that agents like cisplatin can shrink cancers while causing minimal damage to bone marrow, gastrointestinal mucosa, skin, or other rapidly proliferating normal tissues indicates that tumor growth rate is not necessarily the factor conferring selectivity. While substantial attention has been paid to the investigation of factors that render tumors resistant, much less attention has been paid to characteristics or “targets” that are required in order for a tumor to be sensitive to a chemotherapy agent. Below, we discuss mechanisms of acquisition of resistance or loss of sensitivity that have been investigated in lung cancer cell lines and xenografts (Table 3), and that pertain to commonly used standard chemotherapy agents. We also outline factors that have been assessed in human lung cancer samples and that appeared to alter drug efficacy (Table 4), as well as presenting factors that did not correlate with treatment efficacy clinically despite modulating resistance in preclinical systems (Table 5).

Drug and Oxygen Delivery Drug delivery to tumor cells may be limited primarily by tumor blood flow (“flowlimited” drugs) or may be limited primarily by cell membrane characteristics (“membrane-limited” drugs) (24). Tumor blood flow may be reduced by high tissue pressure, high serum fibrinogen, decreased red blood cell membrane deformability, and impaired blood flow autoregulation (24). Gemcitabine delivery appeared to be flow-limited in a SCLC model (25), while various observations suggest that cisplatin delivery may be membrane-limited (24), and relatively little is known about other agents.

Lung Cancer Resistance to Chemotherapy

335

Table 3  Tumor factors contributing to lung cancer resistance in cell lines or xenografts Factor (expression or activity) Agents affected Decreased tumor blood flow   ↓ Drug delivery All?   ↓ Oxygen delivery Etoposide, paclitaxel (not cisplatin, topotecan)   ↑ HIF-1a Cisplatin, doxorubicin, paclitaxel Alterations of tumor extracellular pH   ↓ pH Weak bases (doxorubicin, vinca alkaloids)   ↑ pH Weak acids (platinums, alkylating agents) Decreased drug uptake   ↑ Cell membrane rigidity/sphingomyelin/ Platinums, etoposide, paclitaxel cholesterol   ↓ Long chain and unsaturated fatty acids Platinums   ↓ CTR1 Platinums   ↓ Multiple membrane transporters Platinums   ↓ Na+, K+ ATPase/↑ thromboxane A2/↑ sorbitol Platinums   ↓ Human equilibrative nucleoside transporter 1 Gemcitabine Increased drug efflux   ↑ MRP/GS-X Platinumsa, anthracyclines, vincas, etoposide, taxanes, gemcitabinea   ↑ MDR1/p-glycoproteinb Anthracyclines, vincas, etoposide, taxanes   ↑ RLIP76/RALBP1 Vinorelbine, doxorubicin   ↑ Lung resistance proteinc Cisplatina, etoposidea   ↑ P-type adenosine triphosphatase 7B Cisplatin Increased drug detoxification   ↑ Glutathione Cisplatin, etoposidea, anthracyclinesa, vincasa, camptothecins, mitomycin, alkylating agents, methotrexate, radiation   ↑ Glutamate-cysteine ligase Cisplatin   ↑ Glutathione peroxidase/glutathione reductase Cisplatin   ↑ Glutathione-S-transferase-p Cisplatina   ↑ Metallothioneins Cisplatin, etoposide   ↑ Dihydrodiol dehydrogenase Cisplatin, doxorubicin, taxanes, vincas, melphalan   ↑ Thymidine and folate pools Pemetrexed   ↑ Peroxiredoxin V Doxorubicin, etoposide   ↑ Deoxycytidine deaminase Gemcitabine Decreased drug activation   ↓ Deoxycytidine kinase activity Gemcitabine Decreased drug binding/↑ intracellular pH Cisplatin Increased, decreased or altered target   ↑ Folate pathway enzymes Pemetrexed   ↑ Stathmin (oncoprotein 18) Vincas   ↑ Class III b tubulin (+/ − a tubulin) Taxanes, vincasa, cisplatin, doxorubicin, etoposide   ↓ or mutated Topoisomerase II-a Etoposide, anthracyclines   ↑ Fragile histidine triad gene Etoposide, camptothecins (continued)

336 Table 3  (continued) Factor (expression or activity) Increased DNA damage repair   ↑ Topoisomerase II-a   ↑ Nucleotide excision repair    ↑ ERCC1    ↑ Xeroderma pigmentosum A   ↑ Ribonucleotide reductase M1   ↑ Rad51 (homologous combination repair)   ↑ DNA-dependent protein kinase   ↑ Hus1   ↑ BRCA1   ↓ High mobility group box 2   ↓ Fragile histidine triad gene   ↑ Thymidylate synthase   ↑ Dihydropyrimidine dehydrogenase Decreased apoptotic response   ↓ DNA mismatch repair   Mutant p53   ↓ p53-Binding protein 2   ↓ GML protein   ↓ Caspase-8 activity   ↓ Caspase-9 activity   ↓ FUS1   ↓ SAPK/c-Jun N-terminal kinase   ↓ Bak   ↓ Baxa   ↓ Apoptosis signal transduction Increased apoptosis inhibitors   ↑ Cyclooxygenase-2                      

↑ Telomerase ↑ Heat shock protein 90 ↑ PPARg splice variant Altered membrane gangliosides ↑ Caveolin-1/caveolae organelles ↑ Clusterin ↑ Attachment to extracellular matrix/stromal cells ↓ big-h3 ↑ Stromal-cell-derived factor-1/CXCL12 ↓ Connexin 32 ↑ Epidermal growth factor receptor

D.J. Stewart

Agents affected Cisplatin, radiation, vincas Platinums Platinumsa Platinums Gemcitabine, cisplatin Platinums, etoposide Etoposide Cisplatin Platinumsd Cisplatin Cisplatin Platinums Platinums Platinums Cisplatin, etoposide, camptothecin, methotrexate, anthracyclines, radiation, taxanesa, others Cisplatin, radiation Cisplatin Cisplatin, topotecan, radiation Cisplatin Cisplatin Platinums, gemcitabine Cisplatin, etoposide, radiation, Fas ligand Cisplatin, etoposide, taxanes, doxorubicin Cisplatin, taxanes Cisplatin, anthracyclines, etoposide, vincas, taxanes, gemcitabine Cisplatin, docetaxel, etoposide Taxanes Cisplatin Cisplatin Etoposide, paclitaxel Paclitaxel, gemcitabine Cisplatin, doxorubicin, taxanes, etoposide, others Etoposide Etoposide Vinorelbine Cisplatina, doxorubicin, etoposide, vincas, taxanes, camptothecin, pemetrexed, gemcitabine, others (continued)

Lung Cancer Resistance to Chemotherapy Table 3  (continued) Factor (expression or activity)   ↑ HER-2/neu (erbB-2, p185)        

↑ STAT3 ↓ ERK1/2 and MAPK/ERK kinase ↑ Hepatocyte growth factor ↑ PI3K/Akt pathway activation

       

↑ p70S6K and S6 phosphorylation ↑ PKC-e ↑ PKC-d ↑ PKC-a, PKC-h

                 

↓ PKC-b ↑ IGF-1R ↑ c-myc ↑ MAPK phosphatase-1 ↑ Growth hormone releasing hormone ↑ Fibroblast growth factor 2 ↑ Annexin IV ↑ Hyaluronan ↑ Bcl-2a,f

  ↑ Bcl-xL ↑ Mcl-1 ↑ Survivin ↑ Livin ↑ X-linked inhibitor of apoptosis protein (XIAP)   ↑ Inhibitor of apoptosis proteins (IAPs)   ↑ Nrf2/heme oxygenase-1   ↑ P21WAF1/CIP1        

  ↑ TRAIL decoy receptors DcR1 and DcR2 Altered cell cycling   Cell cycle phase   ↓ Mitotic slippage/↓ aneuploidy   ↑ Aneuploidy   ↑ RB/↓ pRB   ↑ SKP2   ↑ E2F4/↓ E2F1   ↓ CHK2 kinase   Mitotic spindle checkpoint abnormalities   ↑ 14-3-3z

337

Agents affected Cisplatin, etoposide, doxorubicin, taxanes, gemcitabinea,e, others Cisplatin Taxanesa, cisplatin Cisplatin Cisplatin, etoposide, taxanes, gemcitabine, others Cisplatin Etoposide, doxorubicin Etoposide, cisplatin Platinumsa, vincas, taxanesa, doxorubicin, others Cisplatin, etoposide Platinums, etoposide Cisplatin Cisplatin Taxanes Etoposide Taxanes Cisplatin Cisplatin, camptothecin, doxorubicin, etoposide, vincas Cisplatin, gemcitabine, doxorubicin, vincas, taxanes, etoposide, others Cisplatin, etoposide, taxanes, radiation Cisplatin, gemcitabine, taxanes Etoposide Cisplatin, etoposide Gemcitabine Cisplatin Cisplatin, camptothecin, doxorubicin, etoposide Doxorubicin, etoposide Varies with drug Taxanes Etoposide, topotecan, gemcitabine Cisplatin, etoposide, taxanes, 5-FU Cisplatin, camptothecin, others Cisplatin, etoposide Cisplatin Vinorelbine, taxanes Cisplatin (continued)

338 Table 3  (continued) Factor (expression or activity) Increased transcription factors   ↑ NF-kB

D.J. Stewart

Agents affected Cisplatin, doxorubicina, etoposidea, gemcitabine, taxanes Cisplatin Cisplatin Cisplatin, etoposide Cisplatin, etoposide Cisplatin

↑ TWIST ↑ SNAIL ↑ Clock ↑ Activating transcription factor 4 ↑ HIV-1 Tat interacting protein 60 (Tip60) a Not consistent across all studies b No association with resistance to platinums; may sensitize to gemcitabine c No association with resistance to anthracyclines, vinca alkaloids, bleomycin, irinotecan/SN-38 d BRCA1 expression sensitized cells to antimicrotubule agents e Paradoxical increase in sensitivity to gemcitabine–cisplatin combination in one study f Paradoxical increase in sensitivity to taxanes in some studies

         

Reduced tumor blood flow also decreases oxygen delivery. Hypoxia may directly reduce efficacy of etoposide (26) and paclitaxel (27), it may increase efficacy of topotecan (28), and it has little impact on efficacy of cisplatin (28, 29) in lung cancer cells. Hypoxia may also have indirect effects on drug efficacy by upregulating the expression of resistance-associated antiapoptotic factors (26) and by increasing expression of hypoxia inducible factor-1a (HIF-1a). HIF-1a in turn may render NSCLC cells more resistant to cisplatin (30), doxorubicin (30), and paclitaxel (27), and after initial tumor cell killing by chemotherapy, HIF-1a may support accelerated repopulation of tumors by upregulating the expression of the vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) that support angiogenesis (31). The role of tumor blood flow and hypoxia in resistance remains uncertain in lung cancer patients. By (18)F-fluoromisonidazole imaging studies, the hypoxic cell fraction is low in NSCLC (32), HIF-1a expression in NSCLC tumors resected after neoadjuvant cisplatin–gemcitabine did not correlate with patient survival (33), and prechemotherapy serum VEGF levels (34) and tumor VEGF expression by immunohistochemistry (IHC) (35) did not predict outcome in advanced NSCLC patients receiving cisplatin-based combinations.

Extracellular pH Low extracellular pH augments cellular uptake and cytotoxicity of weak acids such as cisplatin (36) and alkylating agents (37), while high extracellular pH augments cellular uptake and cytotoxicity of weak bases such as doxorubicin (37) and vinca alkaloids (38), and pH has little net effect on zwitterions like paclitaxel (37). The role of pH clinically remains unknown, but both dietary factors and concurrent medications may alter tumor extracellular pH and hence might alter resistance (38).

Lung Cancer Resistance to Chemotherapy

339

Table 4  Factors for which some available clinical data support a role in lung cancer resistance Factor Agents affected Decreased drug uptake   ↓ Na+, K+ ATPase Platinumsa   ↓ Human equilibrative Gemcitabinea nucleoside transporter 1 Increased drug efflux   ↑ MRP/GS-X Multiple platinum regimensa; vindesine + etoposide   ↑ MDR1/p-glycoprotein Multiple regimensa   ↑ Breast cancer resistance Platinum regimens protein   ↑ Lung resistance protein Platinum regimensa Increased drug detoxification   ↑ Glutathione-S-transferase-p Platinum regimensa   ↑ Metallothioneins Cisplatin–etoposide/CAV Increased, decreased or altered target   ↓ Stathmin (oncoprotein 18) Cisplatin–vinorelbineb   ↑/Mutated class III b tubulin Taxanes, cisplatin–vinorelbinea   ↓/Mutated topoisomerase II-a Etoposide Increased damage repair   ↑ Topoisomerase II-a Cisplatin regimens   ↑ Nucleotide excision repair/↑ Platinum regimensa ERCC1   ↑ Ribonucleotide reductase M1 Gemcitabine regimens   ↑ BRCA1 Cisplatin + gemcitabinea Decreased apoptotic response   ↓ DNA mismatch repair Platinum regimensa   Mutant p53    By sequencing Platinum regimens    By IHC positivity Platinum regimensa,c, CAV   ↓ GML protein Cisplatin Increased apoptosis inhibitors   ↑ Cyclooxygenase-2 Carboplatind, gemcitabined, vinorelbinee, docetaxel   ↑ Heat shock protein 27 Vinorelbinee   ↑ Caveolin-1 Gemcitabine/cisplatin, gemcitabine/epirubicin   ↓ p-ERK Gemcitabine   Mutant K-ras Taxanes   ↑ c-Kit Cisplatin + etoposide   ↑ PC cell-derived growth factor Platinum regimens   ↑ Survivin Cisplatin/etoposide   ↑ P21WAF1/CIP1 Platinum regimens Altered cell cycling   ↑ RB Cisplatin regimensa   ↑ p27Kip1 Cisplatin regimensa   ↓ Cyclin B1 Platinums + antimitotic agents   ↑ 14-3-3s Cisplatin + gemcitabine   ↓ Eg5 Cisplatin + antimitotic agent (continued)

340

D.J. Stewart

Table 4  (continued) Factor Agents affected a Data were equivocal or negative in some individual trials b Effect clinically was opposite from preclinical effect c Paradoxical increase in efficacy in p53 IHC positive patients in occasional studies d Celecoxib improved outcome in patients whose tumors expressed COX-2 e Trend present

Table 5  Factors for which available clinical data fail to strongly support a role in lung cancer resistance despite a role in preclinical resistance Factor Agents assessed Cisplatin combinations Tumor blood flow/HIF-1a/ VEGF ↑ Lung resistance protein Taxanes, CAV, some cisplatin regimens ↑ Glutathione-S-transferase-p Vinorelbine regimens, some platinum regimens ↑ Nucleotide excision repair Gemcitabine/docetaxel, gemcitabine/epirubicin (ERCC1) ↑ Ribonucleotide reductase M1 Platinum + etoposide ↑ Rad51 (homologous Cisplatin + gemcitabine recombination repair) ↑ BRCA1 Gemcitabine + epirubicin ↑ FANCD2 Platinum regimens Mutant p53 Taxanes or vincas without platinums ↑ Epidermal growth factor receptor Platinums, taxane, gemcitabine, vinorelbine, radiation by IHC or FISHa ↑ HER-2/neu (erbB-2, p185) Platinum regimensb ↓ p-ERK Platinum regimens, taxane regimens ↑ p-AKT Platinum regimens, taxanes K-ras mutations Platinum regimensb ↑ PKC-a Cisplatin + gemcitabine ↑ Bcl-2 Platinum regimens (multiple), vincas, taxanes, etoposide regimens ↑ Bcl-xL Vinorelbine ↓ Bak, Bad, Bid Vinorelbine ↓ Bax Cisplatin regimens, Vinorelbine/docetaxel a Patients with EGFR mutations, particularly with exon 19 deletions did benefit more from chemotherapy than did patients with EGFR wild type tumors in some studies Data are equivocal or not consistent across all clinical studies

b

Drug Uptake Tumor uptake of drugs may be by passive diffusion, by active transport or by both. Mechanisms of cellular uptake of taxanes and vinca alkaloids in lung cancer cells are uncertain. For cisplatin, reduced uptake has been reported in a high proportion

Lung Cancer Resistance to Chemotherapy

341

of resistant NSCLC (39–43) and SCLC (42–44) cell lines. Reduced cisplatin uptake may be accompanied by membrane changes that might alter either passive diffusion or membrane transporter activity. Membrane changes include increases in rigidity (45), density of lipid packing (46) and sphingomyelin content (45), and decreased long chain and unsaturated fatty acids (46). Incorporation of exogenous long chain fatty acids into membrane phospholipids augmented cisplatin uptake and reduced resistance (47). Cisplatin-resistant lung cancer cell lines may have reduced expression of the copper/platinum uptake transporter CTR1 (48). Platinum-resistant cells may also have broad cross-resistance and decreased expression of a wide spectrum of membrane transporters (17), and conversely, exposure to many types of chemotherapy and targeted agents could potentially render tumors cross-resistant to platinums through temporary downregulation of CTR1 expression (49, 50). Na+, K+ ATPase may also be important in cisplatin uptake and efficacy in lung cancer cell lines, particularly with NSCLC (51, 52). The Na+, K+ ATPase antagonists thromboxane A2 (52, 53) and sorbitol (54) decrease cisplatin uptake and efficacy, and the antagonism of N+, K+ ATPase by the glucose metabolite sorbitol could potentially augment cisplatin resistance in poorly controlled diabetes (54). Thallium-201 (T201) retention on SPECT scanning may reflect Na+, K+ ATPase activity. In clinical trials, pretreatment tumor T201 retention predicted outcome with chemotherapy in one SCLC study (55), but did not correlate in another SCLC study (56), nor in a NSCLC study (57). It is unknown if the lack of correlation is due to a lack of importance clinically of N+, K+ ATPase, to an inaccurate prediction of N+, K+ ATPase by T201 retention, or to a modifying effect on outcome by the agents used in combination with cisplatin. With respect to other agents, etoposide uptake into resistant NSCLC tumor cells is lower than uptake into sensitive SCLC tumor cells (58). It is not known how etoposide enters lung cancer cells, but detergents that may increase membrane fluidity increased etoposide efficacy in NSCLC cell lines (59), while increased cellular content of cholesterol (which increases cell membrane rigidity) augmented resistance (60). Human equilibrative nucleoside transporter 1 (hENT1) plays a role in cellular uptake of gemcitabine (61), and hENT1 deficiency was associated with gemcitabine resistance (61, 62), particularly with intrinsic resistance (62). While some clinical studies suggested a role for hENT1 in NSCLC resistance to gemcitabine (63), others did not (61). Overall, preclinical data suggest that reduced drug uptake may be an important cause of resistance in lung cancer, but clinical data are very limited.

Drug Efflux Efflux pumps may also render cells resistant by pumping drugs out of cells after they enter.

342

D.J. Stewart

Multidrug Resistance Protein NSCLC cell lines tend to have greater expression of multidrug resistance protein (MRP) (which may function as a glutathione S-conjugate (GS-X) pump (64)) than do SCLC cell lines (65, 66). MRP expression was associated with decreased accumulation of cisplatin (44), paclitaxel, (67) and other agents (68) in lung cancer cell lines. Protein or mRNA expression of MRP in SCLC (44, 65–67, 69, 70) and NSCLC (65–68, 70–72) cell lines or heterotransplants (73) may be associated with resistance to anthracyclines (44, 64–66, 68–70), vinca alkaloids (44, 64, 65, 68), etoposide (64–66, 70), taxanes (67, 71, 73), gemcitabine, (72) and cisplatin (44, 65), although an association with resistance has not been seen in all cell lines or with all drugs assessed (74–76) (particularly for cisplatin (68, 70) and gemcitabine (76)). MRP1 (65), MRP3 (65), and MRP7 (67) may be particularly important. Impact of MRP on resistance may be decreased by 5-fluorouracil (77) (5-FU), by verapamil, (66) or by glutathione depletion (64). Clinically, MRP expression is common in both NSCLC (70, 78–80) and SCLC (69), and MRP expression increased after exposure to platinum-based regimens (81–83), suggesting that it may be upregulated as a protective response. High MRP expression was associated with decreased response rates (79–81, 84, 85) or survival (79, 80, 86) in SCLC (81, 84, 85) and NSCLC (79, 80, 86) patients receiving platinum-based combinations (79–81, 84–86) or with vindesine plus etoposide (79, 80). Outcome in advanced NSCLC patients treated with cisplatin plus irinotecan also varied significantly with MRP2 host genotype (87). However, no correlation was seen between MRP expression and response (86, 88) or survival (89) with platinum-based combinations in some other NSCLC studies, and impact of MRP expression on clinical outcome may be greater in adenocarcinomas than in squamous cell carcinomas (90). Overall, the available data suggest that MRP may play a role in resistance in lung cancer.

MDR1/p-Glycoprotein In SCLC (67, 91–93) and NSCLC (10, 94–97) cell lines, increased IHC expression of the efflux pump p-glycoprotein (P-gp) or increased mRNA expression for its gene MDR1 was associated with increased resistance to anthracyclines (10, 91, 94), vinca alkaloids (10, 94, 95), etoposide (92–94), and taxanes (10, 67, 94, 96, 97), but was not usually associated with the uptake of (41, 74) or resistance to (10, 41, 74, 94) platinums (which may inhibit P-gp (98)). P-gp may actually sensitize cells to gemcitabine (76). In lung cancer cell lines (10, 60, 99) and tumor samples (100), P-gp expression was occasionally associated with MDR1 gene amplification (10) or correlated with hypoxia (99) or with expression of HIF-1a (99, 100) or caveolin 1 (60). Expression of the gap junction protein and tumor suppressor CX32 in NSCLC cells downregulated MDR1 and sensitized cells to vinorelbine (95).

Lung Cancer Resistance to Chemotherapy

343

Clinically, MDR1 mRNA or P-gp was expressed in 11–32% of NSCLC tumor samples (101–105) and in 13–60% of SCLC tumor samples (69, 104, 105). Expression increased after chemotherapy treatment (81, 103, 105). In SCLC clinical studies (81, 84, 104, 106–109) and in some NSCLC clinical studies, (90, 110, 111) high tumor MDR1 or P-gp expression was significantly associated with decreased response (81, 84, 104, 106–108, 110, 111) and/or survival (90, 108, 109) in patients treated with chemotherapy, including cisplatin–etoposide (81, 84, 104, 106–108), paclitaxel plus a platinum (110, 111), cyclophosphamide–doxorubicin– vincristine (CAV) (104, 108), or other doxorubicin or etoposide regimens (109). However, in other NSCLC trials, tumor P-gp expression did not correlate significantly with response to a variety of platinum-based regimens (86, 104, 112, 113) that also included vinca alkaloids (86, 104, 113), irinotecan (86), taxanes (86), gemcitabine (86) or radiation (113). In pharmacogenetic studies, the MDR1 3435 CC host genotype was associated with significantly better response to cisplatin–etoposide (114) and cisplatin–vinorelbine (115) in SCLC (114) and NSCLC (115) patients than were other genotypes, but was not associated with the outcome in NSCLC patients treated with cisplatin–docetaxel (116). High tumor expression of MDR1/P-gp or MRP was associated with reduced uptake or retention of Tc-99m methoxyisobutyl isonitrile (MIBI) and technetium99m tetrofosmin (Tc-TF) on SPECT scanning in some studies (106, 110, 117–119), but not in others (120, 121). Tumor MIBI uptake was significantly lower in NSCLC than in SCLC (122). In SCLC (56, 106, 107, 118, 123, 124) and NSCLC (57, 110, 119, 121, 125, 126) clinical trials, there was a significant correlation between tumor MIBI (56, 57, 106, 110, 119, 121, 123) or Tc-TF (107, 118, 124–126) uptake/retention on SPECT and response to cisplatin–etoposide-based regimens (106, 107, 118, 123, 124, 126, 127), to cisplatin, mitomycin-C plus vindesine (57, 121), to paclitaxel-based regimens (110, 119), or to other nonplatinum regimens (56), or there was a trend toward an association (128, 129). One small MIBI study including both NSCLC and SCLC patients was negative (117). The calcium channel blocker verapamil (75, 130) and the epidermal growth factor receptor (EGFR) inhibitor gefitinib (97) augmented chemotherapy uptake (75) or efficacy (97, 130) in P-gp expressing lung cancer cell lines (75, 97) and xenografts (130), but in randomized lung cancer clinical trials, neither verapamil (131) nor gefitinib (132, 133) nor the hormonal agent/P-gp antagonist megestrol acetate (134) improved outcome when added to chemotherapy. Overall, available evidence suggests that MDR1/P-gp is associated with resistance to some chemotherapy agents in SCLC, and much (but not all) of the available evidence also suggests a role for MDR1/P-gp in resistance in NSCLC.

Breast Cancer Resistance Protein High tumor IHC expression (86, 135) and blood concentrations (136) of the efflux transporter breast cancer resistance protein (BCRP) were associated with lower

344

D.J. Stewart

response rates (135, 136) and shorter survival (86) in NSCLC patients receiving platinum-based chemotherapy.

Ral-Interacting Protein (RLIP76) (RALBP1) With vinorelbine (137) and doxorubicin (138, 139), increased efflux, decreased cellular concentrations and resistance in SCLC and NSCLC cell lines was seen with overexpression of the glutathione-conjugate transporter RLIP76, the transport activity of which is regulated by protein kinase C (PKC)-a-mediated phosphorylation (139). The differential phosphorylation of RLIP76 in NSCLC vs. SCLC may contribute to the greater resistance to doxorubicin in NSCLC cells (139).

Lung Resistance Protein Lung resistance protein (LRP) expression correlated with resistance to cisplatin (140, 141) and etoposide (142) in some NSCLC cell lines, but in other NSCLC cell lines, LRP expression did not correlate with resistance to cisplatin (41, 74), etoposide (140, 141, 143), anthracyclines (140, 141), vinca alkaloids (140, 141), bleomycin (140, 141), or the irinotecan metabolite SN-38 (141). In some clinical studies, NSCLC response to platinum-based chemotherapy was decreased in patients whose tumors expressed LRP (90, 103, 144), while there was no significant link between efficacy and LRP expression in other NSCLC (88, 145, 146) and SCLC (81, 84, 145) studies using platinums combined with taxanes (88, 145) or epipodophyllotoxins (84, 88, 145), or using taxane-based chemotherapy (146), or CAV (81, 145). Exposure to platinums did not upregulate expression of LRP (147). Overall, LRP does not appear to play a major role in lung cancer resistance.

P-Type Adenosine Triphosphatase (ATP 7B) In NSCLC xenografts, cisplatin resistance correlated with expression of the copper transporter ATP 7B which may play a role in cisplatin efflux (148).

Drug Detoxification Glutathione (GSH) GSH may bind and inactivate cisplatin, augment repair of platinum–DNA adducts (149), and potentiate drug efflux via GS-X pumps (including MRP (64)). In NSCLC (41, 42, 150, 151) and SCLC (42, 43, 152, 153) cell lines, increased GSH

Lung Cancer Resistance to Chemotherapy

345

content was associated with resistance to cisplatin (41–43, 150, 152, 153) (with reduced platinum–DNA binding (42, 153) and reduced intracellular platinum accumulation (43)), etoposide (43, 151), anthracyclines (43, 151), vinca alkaloids (153), camptothecins (43), mitomycin-C (43), alkylating agents (43), methotrexate (43), and radiation (150). However, there were also examples where GSH content did not correlate with cisplatin resistance in NSCLC cell lines (39), NSCLC xenografts (154), or SCLC cell lines (155, 156), or did not correlate with resistance to anthracyclines (153), epipodophyllotoxins (153), vinca alkaloids (43), or 5-FU (43). Expression (157, 158) or activity (159) of the glutamate-cysteine ligase/gammaglutamylcysteine synthetase gene responsible for GSH synthesis correlated with cisplatin resistance, and expression of this gene was higher in NSCLC than in SCLC (157). The enzymes GSH peroxidase and GSH reductase also may play a role in cisplatin resistance (160). While results vary across cell lines, the bulk of available preclinical evidence suggests that GSH may play a role in resistance to cisplatin and perhaps other agents. Clinical data in lung cancer remain limited.

Glutathione-S-Transferase-pi The binding of GSH to drugs may be catalyzed by GST and the expression of the GST isoenzyme glutathione-S-transferase-pi (GSTp) was higher in NSCLC than in SCLC cell lines (161, 162). As with GSH, the association between GST and chemotherapy resistance varied across studies. GST inhibitors increased sensitivity to cisplatin (156, 163), and cisplatin resistance correlated with GST activity (155) or with GSTp expression (161, 162, 164) in NSCLC explants (164), in some NSCLC cell lines (161), and in some SCLC cell lines (155, 156, 161–163). However, in a number of other NSCLC (39, 41, 42, 94, 165) and SCLC (42, 44, 153) cell lines, resistance to cisplatin (39, 41, 42, 44, 153, 165) and several other chemotherapy agents (44, 94, 153, 165) did not correlate with GSTp expression. Clinically, up to 70% of NSCLC tumors have high GSTp IHC expression (164, 166, 167), and high tumor GSTp expression was associated with decreased response to various platinum-based regimens (166, 167) or short time to relapse after platinum-based adjuvant therapy (164). However, in some other NSCLC clinical studies, there was no significant correlation between tumor GSTp expression and outcome with platinum-based (112, 113, 168) or vinorelbine-based (113) therapy. GSTp host genotype correlated with the outcome with cisplatin-based therapy in one NSCLC study (169), and there was a trend toward an association in a second pharmacogenetic study (170).

Metallothioneins Metallothioneins inactivate chemotherapy, and their expression was increased in some NSCLC (41, 171) and SCLC (156) cell lines resistant to cisplatin (41, 156)

346

D.J. Stewart

and etoposide (171). Clinically, tumor metallothionein expression (which was less common in SCLC than in NSCLC (172)) correlated negatively with survival in SCLC patients treated with cisplatin–etoposide/CAV (173) and increased after chemotherapy (172).

Dihydrodiol Dehydrogenase In NSCLC cells, overexpression of the aldo–keto oxidoreductase enzyme dihydrodiol dehydrogenase (DDH) was induced by interleukin-6 and blocked by PKC inhibitors (174), and high DDH expression increased resistance to cisplatin (174–176), doxorubicin (175, 176), paclitaxel (176), vincristine (176), melphalan (176), and radiation (175). DDH expression correlated inversely with apoptosis-inducing factor and with the DNA repair factor Nijmegen breakage syndrome 1 (NBS1) (174). Clinically, high tumor DDH expression in NSCLC tumors was associated with increased risk of recurrence (175).

Cytotoxicity Bypass Pemetrexed cytotoxicity in NSCLC and other cell lines was decreased by thymidine (unless its transport was blocked by dipyridamole) (177) and by high extracellular folate pools (178).

Peroxiredoxin V Redox reactions and the redox factor peroxiredoxin V (PrxV) increased resistance to doxorubicin and etoposide in lung cancer cells (179).

Deoxycytidine Deaminase Gemcitabine resistance in lung cancer cell lines correlated with activity of the enzyme deoxycytidine deaminase (which catabolizes gemcitabine triphosphate) (76). Clinically, efficacy and toxicity of gemcitabine combinations in NSCLC varied with host cytidine deaminase polymorphisms (180).

Lung Cancer Resistance to Chemotherapy

347

Drug Activation: Deoxycytidine Kinase Activity of deoxycytidine kinase (which converts gemcitabine to the active triphosphate) was decreased in some gemcitabine-resistant NSCLC (62, 76, 181) and SCLC (76) cell lines, particularly in lines with acquired rather than intrinsic resistance (62). Corticosteroids reduced gemcitabine efficacy through an effect on deoxycytidine kinase activity in NSCLC cell lines (182), and the MRP antagonist verapamil paradoxically decreased deoxycytidine kinase activity and gemcitabine efficacy in cells overexpressing MRP1 (76, 182). Pemetrexed potentiated the effect of gemcitabine by upregulating expression of deoxycytidine kinase and of the gemcitabine transporter (hENT1), while decreasing Akt phosphorylation (181).

Drug Binding: Platinums Cisplatin resistance in NSCLC (42, 183) and SCLC (42, 47, 156) cell lines is often associated with decreased platinum–DNA adduct formation. Decreased cisplatin DNA binding, cellular uptake and efficacy (183, 184), and increased efflux (183) were associated with higher intracellular pH, possibly since low pH favors conversion of platinums to positively charged, highly reactive aquated species. Cisplatin also concentrated predominantly in acidic organelles in resistant lines with increased intracellular pH (184).

Increased, Decreased, or Altered Target Folate Pathway Increased expression of the pemetrexed targets thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase was associated with decreased pemetrexed efficacy in NSCLC cell lines (181).

Stathmin (Oncoprotein 18) Stathmin helps regulate tubulin dynamics. Transfection of lung cancer cells with the stathmin gene augmented vinca alkaloid efficacy (185), but high tumor expression of stathmin in NSCLC patients receiving vinorelbine–cisplatin was associated with shortened time to progression (186).

348

D.J. Stewart

Tubulin Expression of class III b-tubulin (187–189) (and to a lesser extent a-tubulin (189)) was increased in some taxane-resistant NSCLC cell lines. Findings were less consistent with vinca alkaloid resistance (187, 190). Taxane-resistant NSCLC cell lines with high microtubule dynamic instability (191) and SCLC cell lines with increased a-tubulin acetylation (192) may exhibit partial dependence on taxanes for growth. Inhibiting bIII-tubulin expression in NSCLC cell lines increased efficacy of both tubulin-binding agents (taxanes, vinca alkaloids) and of other agents (cisplatin, doxorubicin, etoposide) (193). Hypoxia and HIF-1a expression increased taxane resistance and altered b-tubulin distribution and cellular morphology without altering b-tubulin expression levels (27). Taxane exposure enhanced the copolymerization of microtubule-associated proteins (MAPs) with a and b tubulins in taxane-sensitive (but not in resistant) SCLC cell lines by indirectly inhibiting MAPkinase and p34cdc2 kinase activity (194), while MAP kinase activity was substantially increased in etoposide-resistant SCLC cell lines (195). Inhibiting polo-like kinase 1 (expression of which is increased in NSCLC) increased sensitivity to vinorelbine while inducing apoptosis, abrogating microtubule polymerization, and arresting cells in G2/M (196). Clinically, most tested NSCLC tumors had at least some cells IHC-positive for class II and III tubulins, all were positive for pan-b tubulin and class I tubulin, and most were negative for d2 a tubulin (197). NSCLC b-tubulin mutations were uncommon in Japanese patients (198) but were seen in 33% of European patients and were associated with decreased response to taxane regimens (199). High class III b-tubulin expression was associated with poor outcome after surgery alone (200), and with lower response rates (186, 201), more rapid tumor progression (186, 197, 201), and shorter survival (201) in patients with advanced NSCLC treated with taxanes. High b-tubulin III (186, 202) or d2 a-tubulin (202) expression was also associated with poor outcome in advanced NSCLC patients treated with vinorelbine/cisplatin, but conversely, the greatest benefit of adjuvant vinorelbine/ cisplatin in resected NSCLC was associated with high tumor class III b-tubulin expression (200). b-Tubulin expression did not correlate with outcome in patients receiving non-tubulin-binding regimens (201). Overall, current data suggest that class III b-tubulin mutation or overexpression confers resistance to taxanes (and possibly to vinca alkaloids).

Topoisomerase (Topo) II-a Topo II inhibitors (e.g., etoposide, doxorubicin and other anthracyclines) block rejoining of DNA strands after topo II breaks DNA to permit unwinding. When compared to NSCLC cell lines, SCLC lines had increased sensitivity to topo II inhibitors (58, 203), modestly higher cellular topo II-a levels and topo II catalytic

Lung Cancer Resistance to Chemotherapy

349

activity, (203) and substantially higher nuclear topo II activity (58). SCLC (43, 93, 153, 204–208) and NSCLC (209) cell lines resistant to topo II inhibitors had decreased topo II-a expression (153, 204, 205, 209) or activity, (93, 206), mutated topo IIa (205, 207), a shift of topo II from nucleus to cytoplasm (207, 208), increased topo I activity (43) or decreased expression of topo II-b (204). Conversely, since topo II-a may play a role in repair of DNA damage, SCLC cell lines with increased expression of topo II-a were resistant to cisplatin (44, 153), radiation (44) and vinca alkaloids (44, 153). Clinically, SCLC tumors had higher topo II-a expression than did NSCLC samples (105, 210). After therapy with etoposide, some SCLC tumors had decreased topo II-a expression (105) or topo II-a mutations (105). Conversely, in patients with NSCLC (88) or SCLC (211) treated with a platinum combined with etoposide or other agents, high tumor topo II-a (88, 211) or topo II-b (211) expression was associated with worse outcome. Overall, cell line data suggest that topo II-a mutation or reduced expression may cause resistance to topo II inhibitors, while high expression may increase resistance to platinums. Clinical data are inconclusive.

DNA Repair Several DNA repair pathways may protect tumor cells from damage induced by cisplatin and carboplatin, including nucleotide excision repair (NER), base excision repair (which has not been assessed in lung cancer), Fanconi anemia and homologous recombination repair pathways (24).

Excision Repair Cross-Complementation Group 1 Protein NER pathway activity (212) and expression of the NER component excision repair cross-complementation group 1 protein (ERCC1) (213) correlated with cisplatin resistance in NSCLC cells, although no correlation between platinum resistance and ERCC1 expression was seen in other NSCLC and SCLC cell lines (214), and ERCC1 expression did not correlate with resistance to paclitaxel, vinorelbine, etoposide, irinotecan, 5-FU (213) or gemcitabine (213, 214). Clinically, 40–46% of NSCLC tumor samples were IHC positive for ERCC1 (215–218). Association between ERCC1 mRNA or IHC expression and outcome with chemotherapy was assessed in several NSCLC studies in advanced disease (216, 218–223), or in the neoadjuvant (217, 224) or postoperative adjuvant (215) setting. In some studies involving platinums combined with taxanes (224), gemcitabine (216, 222) or vinca alkaloids (216), there was a significantly lower response

350

D.J. Stewart

rate (224) or a trend toward a lower response rate (216, 222) in ERCC1-positive tumors than in negative tumors, and response rate was improved in ERCC1positive patients by combining gemcitabine instead of cisplatin with docetaxel (225). Similarly, survival was significantly shorter with high vs. low tumor ERCC1 expression in patients treated with platinums combined with taxanes (217, 219, 220), etoposide (215, 217, 220), vinca alkaloids (215, 216, 219, 220), gemcitabine (216, 220, 221, 223), or mitomycin-C plus ifosfamide plus radiation (217), or there was a trend toward shorter survival (216, 219), and host ERCC1 genetic polymorphisms also correlated with response (226) or survival (116, 227–230) in some assessments. Only NSCLC patients with ERCC1-negative tumors derived benefit from platinum-based adjuvant chemotherapy (215). Significant correlations between tumor ERCC1 expression and response (218–221) or survival (218, 224) were not seen in a few other studies using platinum-based regimens, nor in a study combing epirubicin with gemcitabine (218). High tumor ERCC1 expression also correlated with poor survival after therapy with platinums combined with etoposide (231), etoposide plus ifosfamide (232) or irinotecan (232) in limited SCLC (but not in extensive SCLC (231)). Overall, available clinical data support a role for ERCC1 in lung cancer resistance to platinum regimens.

Other NER Pathway Components In NSCLC cells, inhibiting expression of the xeroderma pigmentosum (XP) group A (XPA) gene increased cisplatin efficacy (233). Clinically, while some pharmacogenetic studies have been negative (116, 180, 227), in other pharmacogenetic analyses, host polymorphisms for XPC (234), XPD (235, 236), and XRCC1 (235, 237, 238) correlated significantly with response (234–237) or survival (238) in NSCLC (234–238) and SCLC (238) patients treated with platinum-based regimens. Significant interactions were seen between XPC, XPD, and ERCC1 (234) and ERCC6 (230) or between XPD and XRCC1 polymorphisms (237), and the correlation with survival was further strengthened by including polymorphisms for XPG, NQO1, p53, and GSTp (230). Overall, available data suggest a role for XPC, XPD, and XRCC1 in lung cancer resistance to platinum regimens.

Ribonucleotide Reductase M1 The gemcitabine target ribonucleotide reductase M1 (RRM1) encodes the RR regulatory subunit, and RR plays a major role in cell growth and DNA repair (222). RRM1 expression was significantly higher in SCLC than in NSCLC cell lines (214). RRM1 expression was associated with gemcitabine and cisplatin resistance in NSCLC cell lines (222), and gemcitabine resistance was also associated with

Lung Cancer Resistance to Chemotherapy

351

RRM1 gene polymorphisms (239) and gene amplification (240) in cell lines. Clinically in NSCLC, high tumor RRM1 expression was associated with significantly decreased response rates (222, 241), time to progression (186, 242) and overall survival (186, 223, 241–243) in patients treated with gemcitabine alone (223) or gemcitabine combined with platinums (186, 222, 223, 241, 243) or docetaxel (242). RRM1 host gene polymorphisms also correlated with response to gemcitabine-based therapy (244). The related factor RRM2 may also contribute to resistance (242). RRM1 expression correlated strongly with ERCC1 expression in NSCLC (223, 241, 243, 245) and SCLC (231), and patients whose tumors had high expression of both RRM1 and ERCC1 had a particularly bad outcome with therapy (223, 243). RRM1 host gene polymorphisms in NSCLC patients treated with cisplatin plus docetaxel (116) and tumor RRM1 expression in SLCC patients treated with a platinum plus etoposide (231) did not correlate with outcome. Overall, available data support a role for RRM1 in lung cancer resistance to gemcitabine regimens.

Rad51 In cell lines, expression of Rad51, a component of the homologous recombination repair pathway, correlated with etoposide resistance in SCLC (246) and with resistance to platinums (213, 247) (but not to gemcitabine, 5-FU, irinotecan, taxane, etoposide, or vinca alkaloids (213)) in NSCLC. Platinum resistance was most marked if Rad51 was coexpressed with ERCC1 (213). Platinum exposure increased Rad51 expression, but administration of EGFR tyrosine kinase inhibitors decreased this Rad51 induction and augmented cisplatin efficacy by blocking ERK1/2 signaling (247). ERK1/2 blockage reduced Rad51 mRNA expression and destabilized Rad51 protein (247). Clinically, Rad51 expression varied with tumor type, and was noted in 12–41% of NSCLC tumors (213, 218), but did not correlate with outcome in patients treated with cisplatin plus gemcitabine (218). Overall, preclinical data suggest a role for Rad51 in lung cancer resistance to platinums and etoposide, but this has not yet been confirmed clinically.

Breast Cancer 1 In association with Rad51, the Fanconi Anemia (FAN) DNA repair pathway component breast cancer 1 (BRCA1) repairs DNA double strand breaks. High BRCA1 expression conferred resistance to platinums but sensitivity to antimicrotubule agents in NSCLC cell lines (248) and was associated with shortened survival in NSCLC patients after surgery alone (249) or after neoadjuvant cisplatin plus gemcitabine (250). However, BRCA1 expression did not correlate with outcome in advanced NSCLC patients treated with gemcitabine combined with cisplatin or epirubicin (218), and tumor expression of another FAN pathway protein, FANCD2, also did not correlate with efficacy of platinum-based chemotherapy in NSCLC (251).

352

D.J. Stewart

DNA Mismatch Repair Mismatch repair (MMR) may trigger apoptosis, and MMR-deficient cells are resistant to platinums (24). Increasing expression of the MMR-related NPRL2 gene in NSCLC cell lines or in orthotopic lung cancer model significantly decreased resistance to cisplatin (252). Clinically, efficacy of vinorelbine ± cisplatin plus radiotherapy in NSCLC patients correlated with expression of the MMR protein hMSH2, while it did not correlate with hMLH1 expression (113). However, outcome in NSCLC patients treated with gemcitabine plus cisplatin did not correlate with expression of the MMR components hMLH1 and hMSH2 (253). Hence, the role of MMR in lung cancer chemotherapy resistance is uncertain.

Other Potentially Relevant DNA Repair Factors Several other factors involved in DNA repair also may induce resistance. Examples include DNA-dependent protein kinase proteins (which are involved in nonhomologous end-joining repair and are associated with repair of etoposide-induced double strand breaks and with etoposide resistance in SCLC cell lines (246)), Hus1 (which plays a role in DNA repair, in G2/M cell cycle checkpoint control pathways, and in cisplatin resistance in NSCLC cell lines (254)), and the nucleic acid synthesis enzymes thymidylate synthase and dihydropyrimidine dehydrogenase which have links to platinum resistance in NSCLC (255). Paradoxically, transfecting cells with high mobility group box  2 (HMG2) (a chromatin-associated protein that may be involved in DNA repair) increased NSCLC cell sensitivity to cisplatin and decreased DNA repair (256). Transfecting cells with fragile histidine triad (FHIT) gene increased sensitivity to cisplatin in p53 wildtype NSCLC cells (257) (possibly by blocking DNA homologous recombination repair (258) and by down regulating topo I and II expression (257)) but reduced sensitivity to topo I and II inhibitors (257).

Damage Tolerance Decreased proapoptotic factors or augmented apoptosis inhibitors may increase resistance by increasing cell tolerance to DNA damage (259).

Reduced Apoptotic Response p53 p53 Activation may induce either apoptosis or cell cycle arrest, and hypoxia may favor cell cycle arrest rather than apoptosis by altering downstream transcriptional

Lung Cancer Resistance to Chemotherapy

353

events after p53 activation (260). p53 Mutations (which are common in NSCLC cell lines (261)) or transfection of mutant (MT) p53 into wild type (WT) p53 NSCLC cell lines increased resistance to cisplatin (262–265), cyclophosphamide (263), 5-FU (262), etoposide (262, 264, 265), camptothecin (265), methotrexate (262), anthracyclines (262), bleomycin (262), and radiation (261), and the ability to induce resistance to chemotherapy (264) and radiation (261) varied with p53 mutation type. Conversely, introduction of WTp53 into cells with MTp53 reversed resistance (262, 265), and transfection of WTp53 into NSCLC cell lines that were p53 null (266, 267) or that were WTp53 (266) increased sensitivity to platinums (266, 267), 5-FU (266), doxorubicin (266), irinotecan (266), and etoposide (266), and led to cellular senescence (267, 268). Transfection of the p53 homologue p73 into WTp53 and p53 null NSCLC cells also increased cisplatin-induced apoptosis (269), and efficacy of cisplatin and radiation correlated with expression of p53binding protein 2 (270). Taxanes may interact with p53 somewhat differently than do other agents. In some studies, introduction of WTp53 into p53 null cells increased sensitivity to taxanes (266), p53 siRNA increased taxane resistance (96), and WTp53 prevented a compensatory increase in b-tubulin gene transcription following taxane exposure (188), but WTp53 introduction into other cell lines instead decreased taxane sensitivity (268), and MTp53 was not still associated with taxane resistance in still other studies (73, 262, 271). Clinically, p53 mutations are present in 40–90% of NSCLC tumors (263, 272, 273), and 40–63% express p53 by IHC (35, 104, 112). IHC positivity for p53 is often used as an indicator of the presence of MTp53 since the MTp53 protein is more stable than is the WTp53 protein, and tumors with MTp53 are more likely than WTp53 tumors to be IHC positive. However, there is only 70% concordance (263), concordance has not been seen consistently (274), and correlations of tumor p53 IHC staining with in vitro chemosensitivity of resected tumors are inconsistent and do not mirror cell line correlations of MTp53 with resistance (275, 276). In clinical NSCLC studies assessing MTp53 by DNA sequencing, presence of MTp53 in tumors of patients treated with platinum-based therapy was associated with a significant decrease in response rate and survival (274, 277), while MTp53 was not significantly associated with response in patients treated with a taxane without a platinum (273). In clinical NSCLC studies assessing p53 by IHC, p53 positivity was associated with reduced response rate (35, 104, 112, 113, 278–280), with a trend toward a reduced response rate (281), or with shortened survival (35, 112, 113, 220, 278, 282) in patients receiving platinums combined with vinca alkaloids (104, 220, 278), taxanes (220), etoposide (35, 278), gemcitabine (35, 220), ifosfamide (278), mitomycin-C/ifosfamide (35), mitomycin-C/vindesine (279), 5FU/folinic acid (278), or vinca alkaloids plus radiotherapy (with or without a platinum) (113). Response rate to platinum-based regimens also varied with host p53 genotype (283) and with normal lymphocyte p53 and p21waf1 expression (284). In gene therapy studies involving NSCLC patients treated with a platinum plus paclitaxel or vinorelbine, tumors injected with adenovirus/WTp53 shrank more than did noninjected tumors (285).

354

D.J. Stewart

Conversely, p53 IHC positivity did not correlate with reduced response (274, 286–288) or survival (274, 286, 287, 289) in other studies using vinca alkaloids alone (288), or using platinums combined with etoposide (144, 289), gemcitabine (289), radiotherapy (287, 290), vinca alkaloids (144, 286, 287), taxanes (144), irinotecan (144), mitomycin-C (144), mitomycin-C/ifosfamide (289), or unspecified concurrent agents (274, 291), and in two of these studies, p53 positivity was associated with a paradoxical augmentation of therapy efficacy (144, 291). As with NSCLC, SCLC clinical results have been inconsistent. In SCLC, 48–63% of tumors are p53 IHC positive (104, 173). In SCLC patients treated with platinums combined with etoposide (104, 173, 232, 282, 292), irinotecan (232), ifosfamide (232), cyclophosphamide (173), doxorubicin (173), or vincristine (173), or treated with CAV without a platinum (104, 282) or with unspecified regimens (85), p53 IHC positivity was associated with significantly shortened survival (292) or with a trend toward shortened survival (173) in some trials, but did not correlate with the outcome in other studies (85, 104, 232, 282). Overall, available preclinical data suggest that MTp53 causes NSCLC resistance to many agents, but taxane data are inconclusive, and SCLC data are limited. Clinical data suggest a possible p53 role in resistance, but remain inconclusive and more studies specifically assessing p53 mutations (rather than IHC) might be helpful.

Caspases Caspase-9 inhibition by XIAP did not block chemotherapy-induced apoptosis in NSCLC cell lines, but caspase-8 inhibition by cytokine response modifier A blocked apoptosis induction by cisplatin, topotecan and gemcitabine (293), and transfection of caspases-8 and -9 into NSCLC xenografts augmented cisplatin efficacy (294). Glucocorticoids downregulate caspase-3, -8, and -9 activity, and transfection of caspase-8 or -9 into lung cancer cell lines or xenografts that had been rendered resistant by glucocorticoids restored sensitivity to cisplatin (295).

SAPK/c-Jun N-Terminal Kinase and c-Jun Cisplatin (296–298), gemcitabine (299), tumor suppressive anti-GD2-monoclonal antibodies (298), and other anticancer agents (298) induced apoptosis in NSCLC (296, 299) and SCLC (297, 298) cell lines by activating c-Jun N-terminal kinase (JNK). JNK transfection restored gemcitabine sensitivity in resistant NSCLC cell lines, while JNK inhibitors rendered cells resistant to gemcitabine (299). The JNK inhibitor curcumin (298) and overexpression of the JNK target, c-Jun (297) reduced sensitivity of SCLC cells to platinums. Clinically, JNK’s role in lung cancer remains undefined.

Lung Cancer Resistance to Chemotherapy

355

Apoptosis Signal Transduction Failure of some NSCLC cell lines to cleave PARP (300), to relocalize caspase-3 from cytosol to nucleus (300), or to demonstrate other downstream aspects of apoptosis (301) despite normal activation of caspases-3 (300, 301), -8 (301), and -9 (300, 301), and despite normal release of cytochrome c (300, 301) in response to cisplatin (301), etoposide (300), radiation (300), or Fas ligand (300) suggests that apoptosis inhibition in NSCLC may occur downstream of mitochondrial events.

Other Proapoptotic Factors Glycosylphosphatidylinositol-anchored molecule-like (GML) protein plays a role in apoptosis, is induced by p53, and is expressed in 30% of NSCLC tumor samples (280). GML expression correlated with cisplatin efficacy in  vitro and clinically (280). Restoration of FUS1 (a frequently deleted tumor suppressor gene) in NSCLC cells and xenografts activated Apaf-1-dependent apoptosis and augmented cisplatin efficacy (302). Clinically in SCLC, patient survival with cisplatin plus etoposide was worse in patients with tumors negative for c-kit IHC expression than in those with positive tumors (303).

Apoptosis Inhibitors Several factors may augment resistance by nonspecifically opposing apoptosis.

Cyclooxygenase-2 Cyclooxygenase-2 (COX-2) is constitutively expressed in many NSCLC cell lines (304) and nonsteroidal anti-inflammatory drugs (NSAIDs) may affect lung cancer cells both through their ability to inhibit COX-2 and through other mechanisms (305). COX-2 inhibitors and various other NSAIDs inhibited growth of NSCLC (304, 306) and SCLC (304) cell lines, particularly those with high COX-2 expression (306). In NSCLC cell lines (304–308) and xenografts (307), NSAIDs and COX-2 inhibitors also increased the cytotoxicity of anthracyclines (305), epipodophyllotoxins (305, 306), vincristine (305), taxanes (304, 306, 307), cisplatin (304, 306), gemcitabine (308), and 13-cisretinoic acid (304), although an effect has not been seen with all drugs in some studies (305). Clinically, COX-2 expression was a negative prognostic factor in NSCLC (309), and there was a trend toward an association between host COX-2 genotype and outcome in NSCLC patients receiving vinorelbine (115). In randomized trials, adding celecoxib to carboplatin plus gemcitabine prolonged median survival in

356

D.J. Stewart

patients whose tumors expressed COX-2 (309), and adding rofecoxib to gemcitabine significantly improved the response rate (310). In a nonrandomized trial in patients with previously treated NSCLC, adding celecoxib to docetaxel gave a response rate (24%) and median survival (11 months) higher than would be expected with docetaxel alone (311). Overall, available data suggest that COX-2 inhibition is worthy of further study in NSCLC.

Telomeres and Telomerase Telomerase inhibition increased induction of apoptosis by cisplatin, docetaxel, and etoposide in NSCLC cell lines (312), suggesting that telomerase increased resistance. Growth of SCLC cells that were resistant to etoposide, irinotecan, and cisplatin was suppressed by a histone deacetylase inhibitor that inhibited hTERT mRNA expression (313).

Heat Shock Proteins (HSPs) Hsp90 inhibitors promoted degradation of Hsp90-interacting proteins (including mutant EGFR) and sensitized NSCLC xenografts to paclitaxel (314). Conversely, HSP27 inhibition did not potentiate efficacy of various agents in lung carcinoma cell lines (315), but there was a trend toward worse outcome in NSCLC patients treated with single agent vinorelbine if tumors were Hsp27-positive (288).

Caveolin-1 The caveolae membrane component caveolin inhibits cell signaling molecules (316). In sensitive NSCLC cells, caveolin-1 expression was upregulated by paclitaxel (316). NSCLC cell lines resistant to etoposide (60) and paclitaxel (316) had increased expression of caveolin-1 (60, 316), caveolae organelles (316), and the caveolae component, cholesterol (60). Clinically, 16.4% of NSCLCs expressed caveolin-1, and expression was associated with decreased response and survival following gemcitabine ± cisplatin or epirubicin (317).

Cell Attachment to Extracellular Matrix Attachment of NSCLC (318) and SCLC (195, 319–322) cells to the extracellular matrix (ECM) proteins fibronectin (318, 322), laminin (322) or collagen IV (322) or to stromal cells (321) increased resistance to cisplatin (318, 322), doxorubicin (322),

Lung Cancer Resistance to Chemotherapy

357

cyclophosphamide (195), paclitaxel (318), mitomycin-C (318), etoposide (195, 319–322), and radiation (318). NSCLC cell attachment also promoted clustering of b(1) integrins after chemotherapy exposure (318). In SCLC cells, attachment upregulated expression of cell adhesion molecules (CD 44 and integrin subunits a2, b3 and b4) (195) and epithelial differentiation markers (cyclin D1 and endothelin) (195), activated phosphoinositide-3-OH kinase (PI3K) (319), Akt (195), MAP kinase (195), other protein tyrosine kinases (322), Bad protein (195) and nuclear factor-kB (NF-kB) (195), suppressed caspase-3 activation (319), blocked etoposide-induced p21Cip1/WAF1 and p27Kip1 upregulation and G2/M cell cycle arrest (319), downregulated cyclins E, A and B (319) and neuroendocrine markers (195), and altered cell morphology (195). Decreased expression of the protein big-h3 (which may modulate cell adhesion) increased etoposide resistance, and low expression was found in 35% of lung cancers (323). The chemokine stromal-cellderived factor-1 (SDF-1/CXCL12) increased SCLC cell adhesion to ECM components by activating the CXCL12 receptor CXCR4 and a2, a4, a5, and b1 integrins (321). The impact of cell adhesion on chemotherapy resistance was abrogated by inhibiting CXCR4 (321) or PI3K (319).

Epidermal Growth Factor Receptor NSCLC cells with high EGFR expression were resistant to cisplatin (160, 324), doxorubicin (324), etoposide (324), vinorelbine (325), paclitaxel (325), camptothecin (325), and 5-FU (325). EGFR downregulation or inhibition reduced NSCLC resistance to platinums (324, 326–328), doxorubicin (324), etoposide (324), taxanes (326–330), pemetrexed (331, 332), and gemcitabine (327). This sensitization did not depend on the presence of an EGFR mutation (326, 331, 332), although in some studies it was seen only in cell lines with high EGFR expression (324) or in cell lines in which pEGFR increased after exposure to chemotherapy (326). In some studies (328) but not others, (332) potentiation of chemotherapy by EGFR antagonists was seen only in cell lines sensitive to the EGFR antagonist alone. EGFR tyrosine kinase inhibitors (TKIs) optimally potentiated chemotherapy if they were administered following (329–332) or concurrently with (331, 332) chemotherapy, with antagonism due to TKI-induced G1 block seen when they preceded chemotherapy (330, 332). EGFR TKIs potentiated pemetrexed activity by decreasing activity of the pemetrexed target thymidylate synthase (331) and by inhibiting pemetrexed-induced activation of EGFR (331) and PI3-kinase/AKT (332). Clinically, EGFR expression was seen in 49–54% of NSCLC tumors, and survival correlated inversely with EGFR IHC expression, but not with expression by Northern blot, PCR, or ligand competition methods (333). However, EGFR IHC expression (113, 334, 335) and gene copy number (335) did not correlate with response rate (113, 334, 335), progression-free survival, (335) or overall survival (113, 335) in patients treated with chemotherapy (334, 335) or chemoradiotherapy (113). Tumors with EGFR exon 19 deletions were more likely to respond to chemotherapy

358

D.J. Stewart

than were tumors with other EGFR mutations (334). Survival was longer with chemotherapy in NSCLC patients with EGFR mutant tumors than in those with EGFR wild type tumors in one study (336), but not in another study (337). Addition of EGFR inhibitors to chemotherapy did not improve outcome in patients with advanced NSCLC (132, 133, 338) except in patients who had never smoked (339). Overall, preclinical evidence suggests that EGFR antagonizes chemotherapy and that schedule-dependent EGFR inhibition may increase chemotherapy efficacy, but EGFR expression clinically is not associated with resistance, tumors with EGFR mutations may be more sensitive to chemotherapy than are EGFR wild type tumors, and EGFR inhibitors do not improve outcome when added to chemotherapy except in never smokers.

HER-2/neu (erbB-2, p185) In cell lines, HER-2/neu expression is common in NSCCL but uncommon in SCLC (340). High p185 (212, 272, 341) or HER-2/neu mRNA expression (342) in NSCLC cell lines (212, 272, 341, 342) and heterotransplants (73) was associated with a low S-phase fraction (272), a long doubling time (272), high NER pathway activity (212), and resistance to etoposide (272, 341, 342), doxorubicin (272, 342), cisplatin (212, 272, 341, 342), paclitaxel (73), and other agents (342), but did not confer resistance to gemcitabine (341). NSCLC cell lines with higher p185 expression paradoxically had augmented sensitivity to the gemcitabine–cisplatin combination (341). Depletion or inhibition of HER-2/neu in NSCLC cell lines expressing it potentiated the effect of cisplatin (340, 343, 344), gemcitabine (340), paclitaxel (340), vinorelbine (340), doxorubicin, (344) and etoposide (344). Clinically, HER-2/neu overexpression was associated with poor prognosis in NSCLC (345), but HER-2 gene copy number (334) and IHC expression (113, 281, 287, 346, 347) did not correlate significantly with response (113, 281, 287, 334, 346, 347) or survival (113, 287, 346, 347) in patients receiving platinums combined with vinca alkaloids (113, 281, 287, 347) or etoposide (346) plus radiation (113, 287, 346) in a palliative or adjuvant setting, although there was a trend toward a poor outcome in HER-2/neu positive tumors in some studies (281, 347), particularly if tumors were also positive for p53 (281). Overall, while preclinical data suggest that HER-2/neu increases resistance, clinical data are largely negative.

Extracellular Signal-Regulated Kinase (ERK1/2) and MAPK/ERK Kinase (MEK) In NSCLC cell lines, ERK inhibition reduced the efficacy of paclitaxel (348), cisplatin markedly induced ERK activity (349), and MEK inhibition reduced cisplatin

Lung Cancer Resistance to Chemotherapy

359

efficacy (349), suggesting a role for the ERK/MEK pathway in chemotherapy cytotoxicity. However, constitutive ERK1/2 activity was associated with resistance to paclitaxel (350), and ERK pathway activation by hypoxia was associated with resistance to UV light and etoposide (26). Clinically, tumor p-ERK IHC expression did not correlate with efficacy of platinum-based and taxane-based regimens in NSCLC and did not correlate with time to progression in patients treated with gemcitabine, but it did correlate positively with response to gemcitabine (337). Overall, it remains unclear whether the ERK/MEK pathway plays a role in chemotherapy sensitivity in lung cancer.

Phosphatase and Tensin Homolog (PTEN)/Phosphoinositide 3-Kinase (PI3K)/Protein Kinase B (Akt)/Mammalian Target of Rapamycin (mTOR) Pathway The PI3K/Akt pathway may be constitutively activated due to activating mutations of PI3K (which are common) or due to mutation, deletion or hypermethylation of the PI3K inhibitor PTEN. As outlined below, available preclinical data support a role for the PTEN/PI3K/Akt/mTOR pathway in lung cancer resistance to chemotherapy, but clinical data remain sparse.

PTEN/PI3K In NSCLC (26, 96) and SCLC (319, 351) cell lines, the PI3K/Akt pathway was activated by inactivation of PTEN (96), by hypoxia (26), by b1 integrin expression following adhesion to ECM (319), by stem cell factor (351) and by insulin-like growth factor (351). This PI3K/Akt pathway activation was associated with increased resistance to paclitaxel (96), UV light, (26) and etoposide (26, 319), and PI3K inhibition sensitized NSCLC cell lines (26, 352, 353) or xenografts (354) and SCLC cell lines (351) to etoposide (26, 351–353), paclitaxel (352, 354), cisplatin (352), gemcitabine (352), trastuzumab, (352) and radiation (352).

Akt Akt raises the threshold for therapy-induced apoptosis (355) and this is mediated in part through mTOR (356). Many NSCLC cell lines have Akt1 overexpression (356), gene amplification (356), or PI3K-dependent Akt1 constitutive activation (352, 355). In SCLC cell lines, Akt activity was increased by adhesion to ECM components (195, 357). Increased expression or activation of Akt in NSCLC (352, 355, 356) and SCLC (195, 357) cell lines was associated with augmented resistance to several agents (195, 352, 355–357), including cisplatin (355–357), doxorubicin

360

D.J. Stewart

(355), mitoxantrone (355), paclitaxel (355), etoposide (352, 357), 5-FU (355), and radiation (352). Blockade of Akt’s downstream effects via mTOR inhibition (358) or suppression of Akt activity by PI3K inhibitors (352, 357), by transfection with dominant negative Akt (352, 356), by siRNA (349), by growth of cells on uncoated surfaces (195), or by growth hormone releasing hormone antagonists (359) sensitized NSCLC (349, 352, 356, 357, 359) and SCLC (358) cells to etoposide (352, 357), cisplatin (349, 352, 356–358), and taxanes (352, 359). Ability of PI3K inhibitors to increase chemotherapy sensitivity was reversed by expression of constitutively activated Akt (351). Despite the impact of Akt on chemotherapy efficacy in cell lines, response of NSCLC patients to chemotherapy did not correlate with p-Akt IHC expression (334, 337).

P70 S6 Kinase (p70S6K) and S6 mTOR phosphorylates p70S6K which in turn phosphorylates the S6 ribosomal protein. P70S6K is activated by cisplatin exposure, phosphorylated p70S6K and S6 are increased in cisplatin-resistant SCLC cells, and downregulation or inhibition of p70S6K sensitized cells to cisplatin (360).

K-ras and the RasGTPase Regulator RasGAP K-ras mutations did not correlate with resistance to doxorubicin, cisplatin, melphalan, carmustine, etoposide, or mitomycin-C in NSCLC cell lines (342). At low level caspase activity, RasGAP was cleaved into resistance-associated antiapoptotic fragments that activated the Ras/PI3K/Akt pathway, while high caspase activity further cleaved RasGAP into proapoptotic factors (361). SCLC cell lines had lower RasGAP expression and higher RasGAP spontaneous cleavage than did NSCLC lines (361). Clinically, K-ras mutations (present in approximately 21% of all NSCLC tumors (336) and in 26% of lung adenocarcinomas (362)) are associated with poor prognosis (363). K-ras mutations did not correlate with response or survival in NSCLC patients treated with a platinum combined with paclitaxel (336) or with ifosfamide and etoposide (362), but were associated with resistance in patients treated with paclitaxel alone (364). Patients with resected wild type K-ras tumors had significantly improved survival with adjuvant cisplatin plus vinorelbine (hazard ratio = 0.69), while survival was not significantly improved by the adjuvant therapy in patients with mutant K-ras (hazard ratio = 0.95), but in multivariate analysis, the presence of a K-ras mutation did not emerge as a significant predictor of chemotherapy effect (p = 0.27), and mutation status did not predict survival (365). Patients with K-ras mutant tumors receiving postoperative cisplatin, etoposide, and radiotherapy had a trend toward worse outcome than did patients with wild type tumors, while K-ras mutation status did not correlate with outcome in patients receiving

Lung Cancer Resistance to Chemotherapy

361

postoperative radiation without chemotherapy (366). Patients with K-ras mutations had significantly worse survival if an EGFR TKI was added to chemotherapy than if they received chemotherapy alone (336). Addition to paclitaxel of the farnesyltransferase inhibitor lonafarnib (which inhibits mutant K-ras) in taxane-refractory, NSCLC patients gave a response rate (10%) and stability rate (38%) higher than would have been expected with paclitaxel alone (367). Currently available data suggest that K-ras mutations may be important prognostically and may confer resistance to EGFR TKIs. Data are suggestive but inconclusive with respect to a role for K-ras in chemotherapy resistance.

PKC In SCLC cell lines, resistance to etoposide (368, 369) and doxorubicin (368) was increased by PKC-e (which may increase expression of the antiapoptotic proteins XIAP and Bcl-xL after complexing with B-raf and S6K2 (369)). PKC-e expression also correlated with chemotherapy resistance in NSCLC cell lines (368). Similarly, high PKC-d expression was associated with SCLC cell line resistance to cisplatin and etoposide (370). While PKC-a (371) and PKC-h (372) expression was associated with resistance to carboplatin (371), vincristine (371, 372), paclitaxel (372), doxorubicin (371), and other agents (371) in NSCLC cell lines, high PKC-a expression was instead associated with cisplatin sensitivity in SCLC cell lines, and high PKC-b expression and PKC activity were associated with increased SCLC cell line sensitivity to cisplatin and etoposide (370). In SCLC cell lines, resistance did not correlate with PKC-z or PKC-i expression (370), and paclitaxel resistance did not correlate with expression of any PKC isozymes or with PKC activity (370). Clinically, PKC-a inhibitors did not improve efficacy of cisplatin plus gemcitabine in phase II trials in NSCLC patients (373, 374). Overall, the role of PKC in lung cancer resistance remains unclear, but may possibly vary with type of lung cancer and with PKC isozyme.

Mitogen-Activated Protein Kinase (MAPK) phosphatase-1 (MKP1) MKP1 downregulates MAPK signaling (375), ERK (376), the apoptosis mediator JNK (296, 376), and p38 (376). MKP1 is more frequently expressed in NSCLC than in SCLC cell lines (376), and is often expressed in NSCLC tumor samples (296, 376). In lung cancer cell lines, cisplatin induced MKP1 expression, thereby rendering them resistant to cisplatin (375). MKP1 inhibition markedly augmented cisplatin-induced expression of JNK and p38 (296), and reversed cisplatin resistance in lung cancer cell lines (296, 375) and xenografts (296).

362

D.J. Stewart

Bcl-2 and Related Proteins Bcl-2 Expression of the antiapoptotic protein Bcl-2 in NSCLC cell lines (377–380) and orthotopic xenografts (381) and in SCLC cell lines (382) correlated with resistance to cisplatin (377, 378, 382), camptothecin (378), doxorubicin (378, 382), and etoposide (382). Bcl-2 antagonists sensitized resistant cells to cisplatin (378, 382), etoposide (379, 382), anthracyclines (378, 379, 382), camptothecin (378), and vinorelbine (381). However, Bcl-2 expression did not correlate with drug resistance in some other NSCLC (94) and SCLC (383) cell lines, and Bcl-2 antagonists paradoxically reduced sensitivity to chemotherapy in one SCLC cell line with low baseline Bcl-2 expression (382). Furthermore, Bcl-2 interaction with taxanes may differ from that with other drugs, in that, there was no correlation between taxane resistance and Bcl-2 expression in some lung cancer cell lines (94, 383), in vitro sensitivity to taxanes was increased rather than decreased for NSCLC tumor samples with high Bcl-2 expression (102), efficacy of taxanes was higher in Bcl-2positive tumors than in Bcl-2-negative tumors heterotransplanted from patients into nude mice (73), Bcl-2 antagonists did not increase taxane efficacy in NSCLC cell lines (380), and taxanes inactivated Bcl-2 by phosphorylation, while platinums dephosphorylated Bcl-2 (380). (Bcl-2 may be constitutively phosphorylated in cisplatin-sensitive but not in cisplatin-resistant lung cancer cell lines (383).) In the process of inducing apoptosis through the mitochondrial pathway in sensitive NSCLC cells, cisplatin generates reactive oxygen species including peroxide that lead to Bcl-2 dephosphorylation, ubiquitination, and proteosomal degradation (384), and this Bcl-2 degradation may be prevented by antioxidant enzymes such as catalase and glutathione peroxidase (384) or by nitric-oxide-induced Bcl-2Snitrosylation (377). In one clinical study, tumor Bcl-2 IHC expression did decrease following treatment with etoposide–cisplatin (385). Clinically, 5–37% of resected NSCLC tumors expressed Bcl-2 (35, 102, 386). Across all stages, Bcl-2 expression correlated significantly with improved (rather than worsened) survival in NSCLC patients (387). In NSCLC (35, 113, 144, 288, 290, 386) and in SCLC (232), there was no correlation between tumor Bcl-2 IHC expression and response (113, 144, 288, 386) or survival (113, 232, 290, 386) in patients treated with a variety of regimens including with vinorelbine (288), docetaxel plus vinorelbine (386), etoposide or irinotecan ± ifosfamide (232), or with platinums combined with vinca alkaloids (144), taxanes (144), etoposide (35, 144), irinotecan (144), gemcitabine (35), mitomycin-C (144), mitomycin-C/ifosfamide (35), radiation (290) or radiation plus vinorelbine (113). In one NSCLC study, high tumor Bcl-2 expression was actually associated with improved survival (35), and addition of the Bcl-2 antisense oligonucleotide oblimersen to carboplatin plus etoposide in SCLC did not improve response rates and unexpectedly worsened survival (388). However, in one SCLC study involving therapy with multiple agents, high tumor Bcl-2 expression was associated with shortened survival in multivariate

Lung Cancer Resistance to Chemotherapy

363

analysis (211). Bcl-2 serum levels did not correlate with survival in NSCLC patients treated with platinum regimens, and serum Bcl-2 levels did not change with therapy (34). Overall, while preclinical studies suggest that Bcl-2 confers resistance to chemotherapy, most clinical data do not support this, and high tumor Bcl-2 expression may be a favorable prognostic marker across lung cancer stages. Bcl-xL While decreased expression of the antiapoptotic protein Bcl-xL was noted in one cisplatin-resistant SCLC cell line (383), a gemcitabine-resistant NSCLC cell line had Bcl-xL upregulation following gemcitabine exposure (389), and Bcl-xL overexpression was noted in NSCLC cell lines (94, 390) and xenografts (390) resistant to gemcitabine (390), doxorubicin (94), vincristine (94), etoposide (94), paclitaxel (94), and 5-FU (94). Bcl-xL antagonists reduced resistance of NSCLC cell lines to gemcitabine (389), cisplatin (391, 392), paclitaxel (372, 392), etoposide (379, 392, 393), doxorubicin (379, 392), and vincristine (372), while FGF2-induced upregulation of Bcl-xL increased resistance in an SCLC cell line (369). Tumor Bcl-xL expression did not predict clinical response of NSCLC to vinorelbine (288). Hence, preclinical data suggest that Bcl-xL contributes to lung cancer resistance, but more clinical studies are needed. Myeloid Cell Leukemia-1 Protein Expression of the Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) is common in NSCLC cell lines and resected NSCLC tumors, and epidermal growth factor exposure increases Mcl-1 expression (394). Mcl-1 overexpression was associated with resistance to cisplatin, etoposide, paclitaxel, and gefitinib, and Mcl-1 depletion reduced resistance to chemotherapy and radiation (394). The Bcl-2 inhibitor GX15-070 potentiated cisplatin efficacy in NSCLC cells by disrupting Mcl-1:Bak interactions (395). Other Bcl-2 Family Members The proapoptotic Bcl-2 family members Bak and Bax may also be expressed in lung cancer cell lines. Activation of transfected Bak in NSCLC cell lines increased efficacy of etoposide, paclitaxel, doxorubicin, and cisplatin (396). Bax expression was associated with trends toward increased paclitaxel efficacy in NSCLC heterotransplants (73) and increased cisplatin sensitivity in SCLC cell lines (383), although some resistant lines paradoxically demonstrated increased (not decreased) Bax expression (94). Clinically, the outcome did not correlate with tumor expression of Bak, Bad, or Bid in vinorelbine-treated NSCLC patients (288) or with Bax expression in NSCLC patients treated with vinorelbine/docetaxel (386) or SCLC patients treated with platinum combinations (232).

364

D.J. Stewart

Survivin Inhibiting expression of the antiapoptotic protein survivin potentiated cisplatin effect in resistant NSCLC cells (397) and xenografts (398), while nicotine augmented NSCLC resistance to gemcitabine, cisplatin, and paclitaxel by inducing expression of XIAP and survivin (399). Clinically, response and survival in NSCLC patients treated with cisplatin plus etoposide correlated inversely with tumor survivin expression (320).

Inhibitor of Apoptosis Proteins (IAPs), X-Linked Inhibitor of Apoptosis Protein (XIAP), and Livin (ML-IAP or KIAP) Following NSCLC cell line exposure to gemcitabine, increased expression of the caspase inhibitor IAP-1 was mediated by downregulation of IkB-a/upregulation of NF-kB (400). Blocking NF-kB activation prevented the IAP-1 increase and sensitized the cells to gemcitabine (400). The antiapoptotic protein XIAP increased NSCLC (401, 402) and SCLC (402) cell line resistance to cisplatin (401) and etoposide (402). The apoptogenic protein Smac/DIABLO (401, 402), Smac mimics (401), and an IAP-binding peptide (402) antagonized XIAP and restored sensitivity. XIAP expression was higher and cIAP-2 expression was lower in SCLC than NSCLC cell lines (403). Apoptosis was blocked downstream of caspase activation in NSCLC, unlike in SCLC cells (403). Efficacy of etoposide and UV-irradiation was increased by inhibition of expression of the antiapoptotic protein Livin in NSCLC cells (404). Hence, in preclinical systems, each of IAPs, XIAP, and Livin are associated with resistance, but clinical data are limited.

Nrf2/Heme Oxygenase-1 Keap1 inactivation by mutation, etc., may result in Nrf2 constitutive activation, and activated Nrf2 reduced sensitivity to cisplatin by upregulating expression of efflux pumps, phase II detoxifying enzymes, and antioxidative stress enzymes (405), including the antioxidative, antiapoptotic enzyme heme oxygenase-1 (HO-1) (406). HO-1 inhibition (by siRNA or by MAPK inhibitors) reduced cisplatin resistance in NSCLC cells by augmenting cisplatin-induced production of reactive oxygen species (406).

P21WAF1/CIP1 In NSCLC cell lines, p21Waf1/Cip1 overexpression or transfection with p21Waf1/ Cip1 inhibited apoptosis (378) and was associated with resistance to camptothecin (378), doxorubicin (378), etoposide (267), and cisplatin (267, 378). Clinically, time

Lung Cancer Resistance to Chemotherapy

365

to failure was shorter in NSCLC patients receiving neoadjuvant platinum-based chemotherapy if their tumors had high baseline p21Waf1/Cip1 expression (407).

Other Antiapoptotic Factors Several other antiapoptotic factors have also been linked to resistance. Cisplatin resistance in lung cancer cell lines has been associated with peroxisome proliferator-activated receptor gamma (PPARg) dominant-negative splice variants (408), some cell membrane ganglioside expression patterns (409), constitutive activation of signal transducer and activator of transcription 3 (STAT3) (410), the c-Met ligand hepatocyte growth factor (HGF) (which is often overexpressed in NSCLC) (411), and hyalouronan/CD44 and its receptor, hyaluronate (412). Insulin-like growth factor-1 receptor (IGF-1R) activation blocks apoptosis by activating the PI3K/Akt pathway and is associated with resistance to platinums (413, 414) and etoposide (414). In SCLC cell lines, resistance to cisplatin (415, 416) (but not to doxorubicin or vincristine (416)) was associated with amplification of N-myc (415) or with expression of c-myc (416), and SCLC patients with amplified or overexpressed c-myc had a poor prognosis (417). Platelet derived growth factor receptora (PDGFRa) expression is very common in clinical NSCLC and SCLC tumor samples, and use of imatinib to inhibit PDGFRa phosphorylation potentiated cisplatin cytotoxicity in NSCLC cell lines (418). Tumor (IHC) (135) or blood (419) expression of the growth factor PC cell-derived growth factor (PCDGF) was associated with resistance to platinum-based regimens in patients with NSCLC. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) decoy receptors 1 and 2 are associated with resistance to TRAIL, doxorubicin, and etoposide (420, 421). Etoposide resistance is also increased by glucose-regulated protein78 (GRP78) in cell lines (422). Fibroblast growth factor-2 (FGF-2) complexes with B-Raf, PKC-e and S6K2 to augment expression of the antiapoptotic proteins, XIAP and Bcl-X(L), and to render SCLC cells resistant to etoposide (369). The stress-associated cytoprotective protein clusterin (which is frequently expressed in NSCLC clinical tumor samples) is associated with resistance to paclitaxel and gemcitabine (423). Growth hormone releasing hormone (GHRH) was associated with expression of K-Ras, COX-2 and pAkt, and augmented docetaxel resistance in NSCLC xenografts (359), while annexin IV rendered NSCLC cell lines resistant to paclitaxel (424).

Cell Cycling Cell Cycle Phase Quiescent cells are resistant to many chemotherapy drugs, but may remain sensitive to platinums (29). NSCLC sensitivity to cisplatin is highest with exposure during G1 and lowest with exposure during G2 (425). Conversely, murine lung cancer

366

D.J. Stewart

cells are most resistant to doxorubicin and etoposide during G1, possibly since topoisomerase II expression is reduced during G1 (426). Blockage of NSCLC cells in G0-1 by tumor necrosis factor (427) or erlotinib (332) antagonized pemetrexed (332) and doxorubicin (427), but not cisplatin (427). NSCLC cells in G2 had increased sensitivity to taxanes (428), while transfection of WT p53 gene into p53null NSCLC cells induced senescence, G1 arrest and paclitaxel resistance (268).

Mitotic Slippage/Aneuploidy The presence of abnormal mitoses will induce G2-M arrest, but such cells may subsequently undergo apoptosis that is triggered by an escape from G2-M arrest (“mitotic slippage”) and the formation of aneuploid G1 cells (429). NSCLC cell lines that are sensitive to taxanes and other microtubule-stabilizing agents had increased aneuploidy when compared with resistant lines, suggesting that factors that reduce mitotic slippage may increase taxane resistance (429). Conversely, microtubule-destabilizing agents such as vinca alkaloids did not initiate aneuploidy (430). Aneuploid tumors tended to have increased resistance to etoposide and topotecan, with less resistance to gemcitabine, and no increase in resistance to platinums (275).

Cell Cycle Regulators Retinoblastoma Protein Retinoblastoma protein (RB) blocks cell cycle progression. Progression through the cell cycle may occur if RB is inactivated by phosphorylation (forming pRB). In NSCLC cell lines, increasing RB function by RB transfection into RB-deficient cells (431) or by using dexamethasone to dephosphorylate pRB (348) increased both the G1 checkpoint response to chemotherapy (431) and resistance to cisplatin (431), etoposide (431), 5-FU (431), and paclitaxel (348), with a similar trend toward increased resistance in RB-transfected SCLC cells (432). Clinically, RB was expressed in 38% of NSCLC tumor samples (35) and in 10% of SCLC tumors (173) by IHC. Response rates with cisplatin-based chemotherapy were lower in NSCLC patients with Rb positive vs. negative tumors in one study, but survival did not correlate with Rb status (35), and in other studies neither response (432), nor tumor cell sensitivity (432), nor patient survival (173) correlated with NSCLC (432) or SCLC (173, 432) Rb expression. S-Phase Kinase-Associated Protein 2 and p27Kip1 S-Phase kinase-associated protein 2 (SKP2) controls the stability of cell cyclerelated proteins such as the cyclin dependent kinase inhibitor p27Kip1 (433).

Lung Cancer Resistance to Chemotherapy

367

p27Kip1 arrests cells in G1. SKP2 overexpression in NSCLC cells increased S-phase cells by decreasing expression of p27Kip1, cyclin E, and p21Cip1, and increased resistance to camptothecin, cisplatin, and other agents (433). Clinically, p27Kip1 IHC expression in NSCLC tumors was associated with decreased adjuvant benefit from cisplatin combined with a vinca alkaloid or etoposide (434). Expression of cell cycle regulators (p16INK4A, cyclin D1, cyclin D3, cyclin E, and Ki-67) did not predict benefit from adjuvant cisplatin-based chemotherapy (434). Conversely, p27 expression correlated with improved outcome in advanced NSCLC treated with platinum-based regimens (435). E2F1 and E2F4 Proliferation-associated genes are suppressed by E2F4 and potentiated by E2F1. In NSCLC cell lines, E2F4 expression is decreased by exposure to cisplatin or etoposide (436). E2F4 expression is associated with increased resistance, while E2F1 is associated with increased sensitivity (436). CHK2 Cisplatin-resistant NSCLC cell lines had promoter hypermethylation and decreased expression of the enzyme CHK2 kinase (which is involved in DNA-damageinduced apoptosis and in cell cycle regulation) (437). Decreased expression and promoter hypermethylation of CHK2 was also seen in a high proportion of clinical NSCLC tumor samples (437). Mitotic Spindle Checkpoint Vinca alkaloids, taxanes, and other anti-microtubule agents activated the mitotic spindle checkpoint (MSC), which blocks segregation of abnormal chromosomes (438). MSC abnormalities (which are common in NSCLC cell lines) may contribute to chromosomal instability, and conferred resistance to vinorelbine and docetaxel, but were not associated with cisplatin resistance (438). 14-3-3 The 14-3-3 regulatory proteins play a role in cell cycle control and signaling and in apoptosis. Lung cancer cell sensitivity to cisplatin was increased by 14-3-3z downregulation, and high 14-3-3z expression in clinical NSCLC tumor samples was associated with advanced stage and high grade (439). In NSCLC patients receiving cisplatin/gemcitabine, 14-3-3s gene methylation was associated with improved survival and with a trend (p = 0.06) toward an increased response probability (440).

368

D.J. Stewart

Other Cell Cycle Regulators Clinically, expression of Cdc2/Cdk1 is increased in lung cancer cells surviving induction chemotherapy, suggesting that Cdc2/Cdk1 may be associated with resistance (441), while cyclin D1 host gene polymorphisms correlated with response (but not survival) in NSCLC patients treated with platinum-based chemotherapy (442). NSCLC patients receiving platinums plus antimitotic agents were more likely to respond if their tumors were IHC positive for cyclin B1 (which is involved in the G2/M transition) (443). NSCLC expression of the microtubule motor/bipolar spindle assembly protein Eg5 correlated directly with response rate in patients receiving antimitotic agents plus platinums (443).

Transcription Factors Nuclear Factor-k B In NSCLC cells, cisplatin (444), taxanes (444), doxorubicin (421), etoposide (421), and gemcitabine (400, 445), increased nuclear factor-kB (NF-kB) activity, and NF-kB increased expression of IAPs (400), Bcl-XL (379) and Bcl-2 (379) antiapoptotic proteins. Factors or manipulations promoting expression of the NF-k B antagonist IkBa in resistant NSCLC cells decreased NF-kB activation (379, 444, 446) and expression of NF-kB-regulated antiapoptotic proteins (379, 400), while increasing efficacy of cisplatin (444), doxorubicin (379, 444), etoposide (379), gemcitabine (400, 445) and taxanes (444, 446, 447). Manipulations increasing IkBa expression also blocked paclitaxel-induced (446) or gemcitabine-induced (445) upregulation of expression of the NF-kB heterodimerization partner p65 (446), NF-kB binding to DNA (445) and transcription of NF-kBregulated genes (445), and augmented gemcitabine efficacy in NSCLC xenografts (445). However, NF-kB inhibition also increased resistance to doxorubicin and etoposide in NSCLC xenografts by blocking chemotherapy induction of the NF-kB-dependent expression of the proapoptotic proteins TRAIL and DR5 (421), suggesting that the impact of NF-kB inhibition on clinical resistance could be unpredictable.

Other Transcription Factors Expression of the Kruppel-related zinc finger protein-1(HKR1) transcription factor is induced by platinums in lung-cancer cell lines and in clinical lung cancer tissues (448), but its role (if any) in resistance remains uncertain. The zinc finger transcription factors TWIST (249, 449) and SNAIL (450) may induce epithelial mesenchymal

Lung Cancer Resistance to Chemotherapy

369

transition, and increases cisplatin resistance by blocking activation of the JNK/ mitochondrial apoptosis pathway. Thyroid Transcription Factor-1 (TTF-1) expression correlated with good prognosis in NSCLC (451), but its role (if any) in NSCLC resistance is unknown. Overexpression of the circadian transcription factor Clock (452) and its target Activating Transcription Factor 4 (ATF4) (452, 453) correlated with resistance to cisplatin (452, 453) and etoposide (452) in NSCLC cell lines, and ATF4 promoted expression of glutathione metabolism genes (452). The histone acetyltransferase gene HIV-1 Tat interacting protein (Tip60) (which is regulated by Clock and is involved in chromatin remodeling, transcription and signal transduction) regulates several DNA repair genes and was associated with resistance to cisplatin (but not with resistance to oxaliplatin, vincristine, or etoposide) in lung cancer cell lines (454).

Summary There are numerous factors that directly or indirectly affect sensitivity of lung cancer to chemotherapy. Upregulation of resistance factors or downregulation of factors required for drug efficacy may arise as a downstream consequence of oncogene mutation or amplification, tumor suppressor gene deletion or silencing, tumor physical characteristics and microenvironment, and interactions of tumor cells with extracellular matrix, with stromal cells, and with cytokines produced by tumor-infiltrating host cells. While there may also be mutations of resistanceassociated genes, most tumor genes reflect the host genotype, and host gene polymorphisms may largely determine the stability and activity of tumor proteins encoded by these genes. Protein expression of a resistance factor will depend both on upregulation or downregulation of the amount of mRNA produced as well as on the stability of the resulting protein encoded by the mRNA. Protein function, in turn, will depend both on the amount of protein present and on its enzymatic efficiency. While enzymatic efficiency will be determined primarily by the polymorphisms in the gene encoding it, it may also be influenced by local factors such as pH and by coexpression of factors that inhibit or potentiate the enzyme. Drug metabolism will be influenced by host factors, drug delivery will vary with tumor blood flow, drug uptake may vary with tumor pH and tumor tissue pressure, and activity of drug entering tumor cells may vary with pH, with cell cycle phase, and with expression of a wide range of factors designed to detoxify drugs, to repair damage, or to permit cells to survive damage. Hence, numerous factors influence drug activity, including mutations that drive tumor development, tumor size and physical characteristics, tumor mRNA, tumor protein expression of various factors and host gene polymorphisms. This helps explain the discrepancy between in  vitro sensitivity testing and clinical drug activity and underlines the fact that looking at mRNA or protein expression of just a few resistance factors in isolation is unlikely to be very helpful clinically.

370

D.J. Stewart

References 1. Kikuchi T, Daigo Y, Katagiri T et al (2003) Expression profiles of non-small cell lung cancers on cDNA microarrays: identification of genes for prediction of lymph-node metastasis and sensitivity to anti-cancer drugs. Oncogene 22(14):2192–2205 2. Dong M, Feng FY, Lin C et al (2004) Mechanisms of the drug resistance of a 2¢, 2-difluorodeoxycytide (gemcitabine)-resistant variant of the human lung adenocarcinoma cell line. Zhonghua Yi Xue Za Zhi 84(4):323–328 3. Hsu DS, Balakumaran BS, Acharya CR et al (2007) Pharmacogenomic strategies provide a rational approach to the treatment of cisplatin-resistant patients with advanced cancer. J Clin Oncol 25(28):4350–4357 4. Yamada Y, Kubota T, Asanuma F et al (1993) The predictability of clinical antitumor effects using two distinctive in vitro chemosensitivity tests: an analysis of true positive cases. Surg Today 23(3):193–199 5. Ohnoshi T, Hiraki S (1985) In vitro drug-sensitivity test using human tumor clonogenic assay in lung cancer patients. Gan To Kagaku Ryoho 12(8):1582–1587 6. Yoshimasu T, Oura S, Hirai I et al (2007) Data acquisition for the histoculture drug response assay in lung cancer. J Thorac Cardiovasc Surg 133(2):303–308 7. Kawamura M, Gika M, Abiko T et al (2007) Clinical evaluation of chemosensitivity testing for patients with unresectable non-small cell lung cancer (NSCLC) using collagen gel droplet embedded culture drug sensitivity test (CD-DST). Cancer Chemother Pharmacol 59(4):507–513 8. Loprevite M, Favoni RE, de Cupis A et al (1999) Pre-clinical evaluation of new antineoplastic agents in NSCLC cell lines: evidence of histological subtype-dependent cytotoxicity. Int J Oncol 15(4):787–792 9. Scagliotti GV, Parikh P, von Pawel J et  al (2008) Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advancedstage non-small-cell lung cancer. J Clin Oncol 26(21):3543–3551 10. Yabuki N, Sakata K, Yamasaki T et  al (2007) Gene amplification and expression in lung cancer cells with acquired paclitaxel resistance. Cancer Genet Cytogenet 173(1):1–9 11. Chen GY, Yang ZY, Hong X, Wang M, Lu L, Zhang CH (2007) Establishment of a multi drug-resistant human lung adenocarcinoma cell line and biological characteristics there of. Zhonghua Yi Xue Za Zhi 87(13):924–926 12. Stordal B, Davey M (2007) Understanding cisplatin resistance using cellular models. IUBMB Life 59(11):696–699 13. Stewart DJ, Tomiak E, Shamji FM, Maziak DE, MacLeod P (2004) Phase II study of alternating chemotherapy regimens for advanced non-small cell lung cancer. Lung Cancer 44(2):241–249 14. Stewart DJ, Raaphorst GP, Yau J, Beaubien AR (1996) Active vs. passive resistance, dose– response relationships, high dose chemotherapy, and resistance modulation: a hypothesis. Invest New Drugs 14(2):115–130 15. Stewart DJ, Chiritescu G, Dahrouge S, Banerjee S, Tomiak EM (2007) Chemotherapy dose– response relationships in non-small cell lung cancer and implied resistance mechanisms. Cancer Treat Rev 33(2):101–137 16. Nyce J (1989) Drug-induced DNA hypermethylation and drug resistance in human tumors. Cancer Res 49(21):5829–5836 17. Shen DW, Su A, Liang XJ, Pai-Panandiker A, Gottesman MM (2004) Reduced expression of small GTPases and hypermethylation of the folate binding protein gene in cisplatin-resistant cells. Br J Cancer 91(2):270–276 18. Puig PE, Guilly MN, Bouchot A et al (2008) Tumor cells can escape DNA-damaging cisplatin through DNA endoreduplication and reversible polyploidy. Cell Biol Int 32(9):1031–1043 19. Carew JS, Nawrocki ST, Cleveland JL (2007) Modulating autophagy for therapeutic benefit. Autophagy 3(5):464–467

Lung Cancer Resistance to Chemotherapy

371

20. Teicher BA, Chatterjee D, Liu JT, Holden SA, Ara G (1993) Protection of bone-marrow granulocyte-macrophage colony-forming units in mice bearing in  vivo alkylating-agentresistant EMT-6 tumors. Cancer Chemother Pharmacol 32(4):315–319 21. Jereczek-Fossa B, Jassem J, Karnicka-Mlodkowska H et  al (1998) Does chemotherapyinduced leukopenia predict a response in small-cell lung cancer? J Cancer Res Clin Oncol 124(2):106–112 22. Pan JH, Han JX, Wu JM, Sheng LJ, Huang HN (2007) CYP450 polymorphisms predict clinic outcomes to vinorelbine-based chemotherapy in patients with non-small-cell lung cancer. Acta Oncol 46(3):361–366 23. Han JY, Lim HS, Shin ES et al (2006) Comprehensive analysis of UGT1A polymorphisms predictive for pharmacokinetics and treatment outcome in patients with non-small-cell lung cancer treated with irinotecan and cisplatin. J Clin Oncol 24(15):2237–2244 24. Stewart DJ (2007) Mechanisms of resistance to cisplatin and carboplatin. Crit Rev Oncol Hematol 63(1):12–31 25. Kristjansen PE, Brown TJ, Shipley LA, Jain RK (1996) Intratumor pharmacokinetics, flow resistance, and metabolism during gemcitabine infusion in ex vivo perfused human small cell lung cancer. Clin Cancer Res 2(2):359–367 26. Lee SM, Lee CT, Kim YW, Han SK, Shim YS, Yoo CG (2006) Hypoxia confers protection against apoptosis via PI3K/Akt and ERK pathways in lung cancer cells. Cancer Lett 242(2):231–238 27. Zeng L, Kizaka-Kondoh S, Itasaka S et al (2007) Hypoxia inducible factor-1 influences sensitivity to paclitaxel of human lung cancer cell lines under normoxic conditions. Cancer Sci 98(9):1394–1401 28. Lund EL, Hansen LT, Kristjansen PE (2005) Augmenting tumor sensitivity to topotecan by transient hypoxia. Cancer Chemother Pharmacol 56(5):473–480 29. Knight LA, Conroy M, Fernando A, Polak M, Kurbacher CM, Cree IA (2005) Pilot studies of the effect of zoledronic acid (Zometa) on tumor-derived cells ex vivo in the ATP-based tumor chemosensitivity assay. Anticancer Drugs 16(9):969–976 30. Song X, Liu X, Chi W et al (2006) Hypoxia-induced resistance to cisplatin and doxorubicin in non-small cell lung cancer is inhibited by silencing of HIF-1alpha gene. Cancer Chemother Pharmacol 58(6):776–784 31. Weinberg RA (2007) The biology of cancer. Garland Science, New York, NY 32. Cherk MH, Foo SS, Poon AM et al (2006) Lack of correlation of hypoxic cell fraction and angiogenesis with glucose metabolic rate in non-small cell lung cancer assessed by 18F-Fluoromisonidazole and 18F-FDG PET. J Nucl Med 47(12):1921–1926 33. Felip E, Taron M, Rosell R et al (2005) Clinical significance of hypoxia-inducible factor-1a messenger RNA expression in locally advanced non-small-cell lung cancer after platinum agent and gemcitabine chemotherapy followed by surgery. Clin Lung Cancer 6(5):299–303 34. Tas F, Duranyildiz D, Oguz H, Camlica H, Yasasever V, Topuz E (2006) Serum vascular endothelial growth factor (VEGF) and bcl-2 levels in advanced stage non-small cell lung cancer. Cancer Invest 24(6):576–580 35. Ludovini V, Gregorc V, Pistola L et  al (2004) Vascular endothelial growth factor, p53, Rb, Bcl-2 expression and response to chemotherapy in advanced non-small cell lung cancer. Lung Cancer 46(1):77–85 36. Laurencot CM, Kennedy KA (1995) Influence of pH on the cytotoxicity of cisplatin in EMT6 mouse mammary tumor cells. Oncol Res 7(7–8):371–379 37. Mahoney BP, Raghunand N, Baggett B, Gillies RJ (2003) Tumor acidity, ion trapping and chemotherapeutics. I. Acid pH affects the distribution of chemotherapeutic agents in  vitro. Biochem Pharmacol 66(7):1207–1218 38. Raghunand N, Martinez-Zaguilan R, Wright SH, Gillies RJ (1999) pH and drug resistance. II. Turnover of acidic vesicles and resistance to weakly basic chemotherapeutic drugs. Biochem Pharmacol 57(9):1047–1058 39. Matsumura T, Takigawa N, Kiura K et  al (2005) Determinants of cisplatin and irinotecan activities in human lung adenocarcinoma cells: evidence of cisplatin accumulation and topoisomerase I activity. In Vivo 19(4):717–721

372

D.J. Stewart

40. Shimura M, Saito A, Matsuyama S et al (2005) Element array by scanning X-ray fluorescence microscopy after cis-diamminedichloro-platinum(II) treatment. Cancer Res 65(12):4998–5002 41. Kawai H, Kiura K, Tabata M et al (2002) Characterization of non-small-cell lung cancer cell lines established before and after chemotherapy. Lung Cancer 35(3):305–314 42. Shellard SA, Fichtinger-Schepman AM, Lazo JS, Hill BT (1993) Evidence of differential cisplatin–DNA adduct formation, removal and tolerance of DNA damage in three human lung carcinoma cell lines. Anticancer Drugs 4(4):491–500 43. Moritaka T, Kiura K, Ueoka H et al (1998) Cisplatin-resistant human small cell lung cancer cell line shows collateral sensitivity to vinca alkaloids. Anticancer Res 18(2A):927–933 44. Henness S, Davey MW, Harvie RM, Davey RA (2002) Fractionated irradiation of H69 smallcell lung cancer cells causes stable radiation and drug resistance with increased MRP1, MRP2, and topoisomerase IIalpha expression. Int J Radiat Oncol Biol Phys 54(3):895–902 45. Popovic P, Wong PTT, Kates M et al (1994) Membrane fluidity and lipids in cisplatin resistant cells with low cisplatin uptake. Proc AACR 35:440 46. Liang XJ, Shen DW, Gottesman MM (2004) A pleiotropic defect reducing drug accumulation in cisplatin-resistant cells. J Inorg Biochem 98(10):1599–1606 47. Timmer-Bosscha H, Hospers GA, Meijer C et al (1989) Influence of docosahexaenoic acid on cisplatin resistance in a human small cell lung carcinoma cell line. J Natl Cancer Inst 81(14): 1069–1075 48. Song IS, Savaraj N, Siddik ZH et  al (2004) Role of human copper transporter Ctr1 in the transport of platinum-based antitumor agents in cisplatin-sensitive and cisplatin-resistant cells. Mol Cancer Ther 3(12):1543–1549 49. Stewart DJ, Nunez M, Jelinek J et al (2008) Tumor CTR1 copper transporter modulation by decitabine (DAC) and relationship to global DNA methylation and time from prior therapy. Proc ASCO 26(15S):599s (Abstract # 11088) 50. Stewart DJ (2008) Gefitinib maintenance in stage III non-small-cell lung cancer. J Clin Oncol 26(29):4849–4850 author reply 50–51 51. Bando T, Fujimura M, Kasahara K, Matsuda T (1998) Significance of Na+, K(+)-ATPase on intracellular accumulation of cis-diamminedichloroplatinum(II) in human non-small-cell but not in small-cell lung cancer cell lines. Anticancer Res 18(2A):1085–1089 52. Bando T, Fujimura M, Kasahara K, Matsuda T (1998) Role of thromboxane receptor on the intracellular accumulation of cis-diamminedichloroplatinum(II) in non-small-cell but not in small-cell lung cancer cell lines. Anticancer Res 18(2A):1079–1084 53. Fujimura M, Kasahara K, Shirasaki H et al (1999) Up-regulation of ICH-1L protein by thromboxane A2 antagonists enhances cisplatin-induced apoptosis in non-small-cell lung-cancer cell lines. J Cancer Res Clin Oncol 125(7):389–394 54. Bando T, Fujimura M, Kasahara K et al (1997) Exposure to sorbitol induces resistance to cisplatin in human non-small-cell lung cancer cell lines. Anticancer Res 17(5A):3345–3348 55. Tokuchi Y, Isobe H, Takekawa H et al (1998) Predicting chemotherapeutic response to smallcell lung cancer of platinum compounds by thallium-201 single-photon emission computerized tomography. Br J Cancer 77(8):1363–1368 56. Yamamoto Y, Nishiyama Y, Satoh K et al (1998) Comparative study of technetium-99m-sestamibi and thallium-201 SPECT in predicting chemotherapeutic response in small cell lung cancer. J Nucl Med 39(9):1626–1629 57. Nishiyama Y, Yamamoto Y, Satoh K et  al (2000) Comparative study of Tc-99m MIBI and TI-201 SPECT in predicting chemotherapeutic response in non-small-cell lung cancer. Clin Nucl Med 25(5):364–369 58. Kasahara K, Fujiwara Y, Sugimoto Y et  al (1992) Determinants of response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in human lung cancer cell lines. J Natl Cancer Inst 84(2):113–118 59. Tsujino I, Yamazaki T, Masutani M, Sawada U, Horie T (1999) Effect of Tween-80 on cell killing by etoposide in human lung adenocarcinoma cells. Cancer Chemother Pharmacol 43(1):29–34 60. Belanger MM, Gaudreau M, Roussel E, Couet J (2004) Role of caveolin-1 in etoposide resistance development in A549 lung cancer cells. Cancer Biol Ther 3(10):954–959

Lung Cancer Resistance to Chemotherapy

373

61. Seve P, Mackey JR, Isaac S et al (2005) cN-II Expression predicts survival in patients receiving gemcitabine for advanced non-small cell lung cancer. Lung Cancer 49(3):363–370 62. Achiwa H, Oguri T, Sato S, Maeda H, Niimi T, Ueda R (2004) Determinants of sensitivity and resistance to gemcitabine: the roles of human equilibrative nucleoside transporter 1 and deoxycytidine kinase in non-small cell lung cancer. Cancer Sci 95(9):753–757 63. Oguri T, Achiwa H, Muramatsu H et al (2007) The absence of human equilibrative nucleoside transporter 1 expression predicts nonresponse to gemcitabine-containing chemotherapy in non-small cell lung cancer. Cancer Lett 256(1):112–119 64. Zaman GJ, Lankelma J, van Tellingen O et  al (1995) Role of glutathione in the export of compounds from cells by the multidrug-resistance-associated protein. Proc Natl Acad Sci U S A 92(17):7690–7694 65. Young LC, Campling BG, Cole SP, Deeley RG, Gerlach JH (2001) Multidrug resistance proteins MRP3, MRP1, and MRP2 in lung cancer: correlation of protein levels with drug response and messenger RNA levels. Clin Cancer Res 7(6):1798–1804 66. Narasaki F, Oka M, Fukuda M et al (1997) Multidrug resistance-associated protein gene expression and drug sensitivity in human lung cancer cells. Anticancer Res 17(5A):3493–3497 67. Oguri T, Ozasa H, Uemura T et  al (2008) MRP7/ABCC10 expression is a predictive biomarker for the resistance to paclitaxel in non-small cell lung cancer. Mol Cancer Ther 7(5):1150–1155 68. Gonzalez Manzano R, Versanvoort C, Wright K, Twentyman PR (1996) Rapid recovery of a functional MDR phenotype caused by MRP after a transient exposure to MDR drugs in a revertant human lung cancer cell line. Eur J Cancer 32A(12):2136–2141 69. Campling BG, Young LC, Baer KA et al (1997) Expression of the MRP and MDR1 multidrug resistance genes in small cell lung cancer. Clin Cancer Res 3(1):115–122 70. Giaccone G, van Ark-Otte J, Rubio GJ et al (1996) MRP is frequently expressed in human lung-cancer cell lines, in non-small-cell lung cancer and in normal lungs. Int J Cancer 66(6):760–767 71. Liang Y, O’Driscoll L, McDonnell S et  al (2004) Enhanced in  vitro invasiveness and drug resistance with altered gene expression patterns in a human lung carcinoma cell line after pulse selection with anticancer drugs. Int J Cancer 111(4):484–493 72. Oguri T, Achiwa H, Sato S et al (2006) The determinants of sensitivity and acquired resistance to gemcitabine differ in non-small cell lung cancer: a role of ABCC5 in gemcitabine sensitivity. Mol Cancer Ther 5(7):1800–1806 73. Perez-Soler R, Kemp B, Wu QP et  al (2000) Response and determinants of sensitivity to paclitaxel in human non-small cell lung cancer tumors heterotransplanted in nude mice. Clin Cancer Res 6(12):4932–4938 74. Ikuta K, Takemura K, Sasaki K et al (2005) Expression of multidrug resistance proteins and accumulation of cisplatin in human non-small cell lung cancer cells. Biol Pharm Bull 28(4):707–712 75. van Ark-Otte J, Samelis G, Rubio G, Lopez Saez JB, Pinedo HM, Giaccone G (1998) Effects of tubulin-inhibiting agents in human lung and breast cancer cell lines with different multidrug resistance phenotypes. Oncol Rep 5(1):249–255 76. Bergman AM, Pinedo HM, Talianidis I et al (2003) Increased sensitivity to gemcitabine of P-glycoprotein and multidrug resistance-associated protein-overexpressing human cancer cell lines. Br J Cancer 88(12):1963–1970 77. Zhan M, Liu X (1999) Schedule-dependent reversion of cisplatin resistance by 5-fluorouracil in a cisplatin-resistant human lung adenocarcinoma cell line A549DDP. Chin Med J (Engl) 112(4):336–339 78. Xu M, Li J, Xia Q (1999) Expression of multidrug resistance-associated protein gene in nonsmall cell lung cancer. Zhonghua Jie He He Hu Xi Za Zhi 22(5):268–270 79. Oshika Y, Nakamura M, Tokunaga T et al (1998) Multidrug resistance-associated protein and mutant p53 protein expression in non-small cell lung cancer. Mod Pathol 11(11):1059–1063 80. Ota E, Abe Y, Oshika Y et al (1995) Expression of the multidrug resistance-associated protein (MRP) gene in non-small-cell lung cancer. Br J Cancer 72(3):550–554

374

D.J. Stewart

81. Triller N, Korosec P, Kern I, Kosnik M, Debeljak A (2006) Multidrug resistance in small cell lung cancer: expression of P-glycoprotein, multidrug resistance protein 1 and lung resistance protein in chemo-naive patients and in relapsed disease. Lung Cancer 54(2):235–240 82. Oguri T, Isobe T, Fujitaka K, Ishikawa N, Kohno N (2001) Association between expression of the MRP3 gene and exposure to platinum drugs in lung cancer. Int J Cancer 93(4):584–589 83. Oguri T, Isobe T, Suzuki T et al (2000) Increased expression of the MRP5 gene is associated with exposure to platinum drugs in lung cancer. Int J Cancer 86(1):95–100 84. Yeh JJ, Hsu NY, Hsu WH, Tsai CH, Lin CC, Liang JA (2005) Comparison of chemotherapy response with P-glycoprotein, multidrug resistance-related protein-1, and lung resistancerelated protein expression in untreated small cell lung cancer. Lung 183(3):177–183 85. Ushijima R, Takayama K, Izumi M et al (2007) Immunohistochemical expression of MRP2 and clinical resistance to platinum-based chemotherapy in small cell lung cancer. Anticancer Res 27(6C):4351–4358 86. Yoh K, Ishii G, Yokose T et al (2004) Breast cancer resistance protein impacts clinical outcome in platinum-based chemotherapy for advanced non-small cell lung cancer. Clin Cancer Res 10(5):1691–1697 87. Han JY, Lim HS, Yoo YK et al (2007) Associations of ABCB1, ABCC2, and ABCG2 polymorphisms with irinotecan-pharmacokinetics and clinical outcome in patients with advanced non-small cell lung cancer. Cancer 110(1):138–147 88. Dingemans AC, van Ark-Otte J, Span S et al (2001) Topoisomerase IIalpha and other drug resistance markers in advanced non-small cell lung cancer. Lung Cancer 32(2):117–128 89. Filipits M, Haddad V, Schmid K et  al (2007) Multidrug resistance proteins do not predict benefit of adjuvant chemotherapy in patients with completely resected non-small cell lung cancer: International Adjuvant Lung Cancer Trial Biologic Program. Clin Cancer Res 13(13):3892–3898 90. Wang J, Liu X, Jiang W (2000) Expression of LRP, MRP and MDR1 in non-small-cell lung cancer and its clinical significance. Zhonghua Zhong Liu Za Zhi 22(4):304–307 91. Bergman AM, Munch-Petersen B, Jensen PB et al (2001) Collateral sensitivity to gemcitabine (2¢, 2¢-difluorodeoxycytidine) and cytosine arabinoside of daunorubicin- and VM-26-resistant variants of human small cell lung cancer cell lines. Biochem Pharmacol 61(11):1401–1408 92. Glisson BS, Alpeter MD (1992) Multidrug resistance in a small cell lung cancer line: rapid selection with etoposide and differential chemosensitization with cyclosporin A. Anticancer Drugs 3(4):359–366 93. Takigawa N, Ohnoshi T, Ueoka H, Kiura K, Kimura I (1992) Establishment and characterization of an etoposide-resistant human small cell lung cancer cell line. Acta Med Okayama 46(3):203–212 94. NicAmhlaoibh R, Heenan M, Cleary I et al (1999) Altered expression of mRNAs for apoptosis-modulating proteins in a low level multidrug resistant variant of a human lung carcinoma cell line that also expresses mdr1 mRNA. Int J Cancer 82(3):368–376 95. Sato H, Fukumoto K, Hada S et al (2007) Enhancing effect of connexin 32 gene on vinorelbine-induced cytotoxicity in A549 lung adenocarcinoma cells. Cancer Chemother Pharmacol 60(3):449–457 96. Ji D, Deeds SL, Weinstein EJ (2007) A screen of shRNAs targeting tumor suppressor genes to identify factors involved in A549 paclitaxel sensitivity. Oncol Rep 18(6):1499–1505 97. Kitazaki T, Oka M, Nakamura Y et al (2005) Gefitinib, an EGFR tyrosine kinase inhibitor, directly inhibits the function of P-glycoprotein in multidrug resistant cancer cells. Lung Cancer 49(3):337–343 98. Bogush TA, Konukhova AV, Ravcheeva AB et al (2003) Inhibition of ABC-transporter(s)’ function in non-small cell lung cancer cells by platinum drugs. Antibiot Khimioter 48(10):11–15 99. Xia S, Yu SY, Yuan XL, Xu SP (2004) Effects of hypoxia on expression of P-glycoprotein and multidrug resistance protein in human lung adenocarcinoma A549 cell line. Zhonghua Yi Xue Za Zhi 84(8):663–666 100. Yasuda H, Nakayama K, Watanabe M et  al (2006) Nitroglycerin treatment may enhance chemosensitivity to docetaxel and carboplatin in patients with lung adenocarcinoma. Clin Cancer Res 12(22):6748–6757

Lung Cancer Resistance to Chemotherapy

375

101. Oka M, Fukuda M, Sakamoto A et al (1997) The clinical role of MDR1 gene expression in human lung cancer. Anticancer Res 17(1B):721–724 102. Inoue Y, Gika M, Abiko T et al (2005) Bcl-2 overexpression enhances in vitro sensitivity against docetaxel in non-small cell lung cancer. Oncol Rep 13(2):259–264 103. Peng ZM, Luo J, Wang WB, Wang XH, Chen JH, Lan SM (2004) Predictive value of drug resistance-related genes expression in neoadjuvant chemotherapy in patients with non-small cell lung cancer of stage III. Ai Zheng 23(8):963–967 104. Kawasaki M, Nakanishi Y, Kuwano K, Takayama K, Kiyohara C, Hara N (1998) Immunohistochemically detected p53 and P-glycoprotein predict the response to chemotherapy in lung cancer. Eur J Cancer 34(9):1352–1357 105. Kreisholt J, Sorensen M, Jensen PB, Nielsen BS, Andersen CB, Sehested M (1998) Immunohistochemical detection of DNA topoisomerase IIalpha, P-glycoprotein and multidrug resistance protein (MRP) in small-cell and non-small-cell lung cancer. Br J Cancer 77(9):1469–1473 106. Kao A, Shiun SC, Hsu NY, Sun SS, Lee CC, Lin CC (2001) Technetium-99m methoxyisobutylisonitrile chest imaging for small-cell lung cancer. Relationship to chemotherapy response (six courses of combination of cisplatin and etoposide) and p-glycoprotein or multidrug resistance related protein expression. Ann Oncol 12(11):1561–1566 107. Yeh JJ, Hsu WH, Huang WT, Wang JJ, Ho ST, Kao A (2003) Technetium-99m tetrofosmin SPECT predicts chemotherapy response in small cell lung cancer. Tumour Biol 24(3):151–155 108. Savaraj N, Wu CJ, Xu R et al (1997) Multidrug-resistant gene expression in small-cell lung cancer. Am J Clin Oncol 20(4):398–403 109. Tabata M, Ohnoshi T, Ueoka H, Kiura K, Kimura I (1993) MDR1 gene expression and treatment outcome in small cell lung cancer: MDR1 gene expression as an independent prognostic factor. Acta Med Okayama 47(4):243–248 110. Hsu WH, Yen RF, Kao CH et  al (2002) Predicting chemotherapy response to paclitaxelbased therapy in advanced non-small-cell lung cancer (stage IIIb or IV) with a higher T stage (>T2). Technetium-99m methoxyisobutylisonitrile chest single photon emission computed tomography and P-glycoprotein express ion. Oncology 63(2):173–179 111. Yeh JJ, Hsu WH, Wang JJ, Ho ST, Kao A (2003) Predicting chemotherapy response to paclitaxel-based therapy in advanced non-small-cell lung cancer with P-glycoprotein expression. Respiration 70(1):32–35 112. Miyatake K, Gemba K, Ueoka H et al (2003) Prognostic significance of mutant p53 protein, P-glycoprotein and glutathione S-transferase-pi in patients with unresectable non-small cell lung cancer. Anticancer Res 23(3C):2829–2836 113. Brooks KR, To K, Joshi MB et  al (2003) Measurement of chemoresistance markers in patients with stage III non-small cell lung cancer: a novel approach for patient selection. Ann Thorac Surg 76(1):187–193 discussion 93 114. Sohn JW, Lee SY, Lee SJ et al (2006) MDR1 polymorphisms predict the response to etoposide–cisplatin combination chemotherapy in small cell lung cancer. Jpn J Clin Oncol 36(3):137–141 115. Pan JH, Han JX, Wu JM, Sheng LJ, Huang HN, Yu QZ (2008) MDR1 single nucleotide polymorphisms predict response to vinorelbine-based chemotherapy in patients with nonsmall cell lung cancer. Respiration 75(4):380–385 116. Isla D, Sarries C, Rosell R et al (2004) Single nucleotide polymorphisms and outcome in docetaxel–cisplatin-treated advanced non-small-cell lung cancer. Ann Oncol 15(8):1194–1203 117. Dirlik A, Burak Z, Goksel T et al (2002) The role of Tc-99m sestamibi imaging in predicting clinical response to chemotherapy in lung cancer. Ann Nucl Med 16(2):103–108 118. Kuo TH, Liu FY, Chuang CY, Wu HS, Wang JJ, Kao A (2003) To predict response chemotherapy using technetium-99m tetrofosmin chest images in patients with untreated small cell lung cancer and compare with p-glycoprotein, multidrug resistance related protein-1, and lung resistance-related protein expression. Nucl Med Biol 30(6):627–632 119. Shih CM, Hsu WH, Huang WT, Wang JJ, Ho ST, Kao A (2003) Usefulness of chest single photon emission computed tomography with technetium-99m methoxyisobutylisonitrile to

376

D.J. Stewart

predict taxol based chemotherapy response in advanced non-small cell lung cancer. Cancer Lett 199(1):99–105 120. Sasaki M, Kuwabara Y, Ichiya Y et al (1999) Prediction of the chemosensitivity of lung cancer by 99mTc-hexakis-2-methoxyisobutyl isonitrile SPECT. J Nucl Med 40(11):1778–1783 121. Yuksel M, Cermik TF, Doganay L et  al (2002) 99mTc-MIBI SPET in non-small cell lung cancer in relationship with Pgp and prognosis. Eur J Nucl Med Mol Imaging 29(7):876–881 122. Ceriani L, Giovanella L, Bandera M, Beghe B, Ortelli M, Roncari G (1997) Semi-quantitative assessment of 99Tcm-sestamibi uptake in lung cancer: relationship with clinical response to chemotherapy. Nucl Med Commun 18(11):1087–1097 123. Bom HS, Lim SC, Kim YC et al (1999) Dipyridamole modulated Tc-99m sestamibi lung SPECT in small cell lung cancer. Clin Nucl Med 24(2):97–101 124. Kao CH, Ho YJ, Shen YY, Lee JK (1999) Evaluation of chemotherapy response in patients with small cell lung cancer using Technetium-99m-tetrofosmin. Anticancer Res 19(3B):2311–2315 125. Shih CM, Shiau YC, Wang JJ, Ho ST, Kao A (2003) Using technetium-99m tetrofosmin chest imaging to predict taxol-based chemotherapy response in non-small cell lung cancer but not related to lung resistance protein expression. Lung 181(2):103–111 126. Fukumoto M, Yoshida D, Hayase N, Kurohara A, Akagi N, Yoshida S (1999) Scintigraphic prediction of resistance to radiation and chemotherapy in patients with lung carcinoma: technetium 99m-tetrofosmin and thallium-201 dual single photon emission computed tomography study. Cancer 86(8):1470–1479 127. Bom HS, Kim YC, Song HC, Min JJ, Kim JY, Park KO (1998) Technetium-99m-MIBI uptake in small cell lung cancer. J Nucl Med 39(1):91–94 128. Changlai SP, Tsai CS, Ding HJ, Huang WT, Kao A, Hsu WH (2003) Using technetium-99m methoxyisobutylisonitrile lung single-photon-emission computed tomography to predict response to chemotherapy and compare with P-glycoprotein expression in patients with untreated small cell lung cancer. Med Oncol 20(3):247–253 129. Akgun A, Cok G, Karapolat I, Goksel T, Burak Z (2006) Tc-99m MIBI SPECT in prediction of prognosis in patients with small cell lung cancer. Ann Nucl Med 20(4):269–275 130. Arvelo F, Poupon MF, Bichat F et  al (1995) Adding a reverser (verapamil) to combined chemotherapy overrides resistance in small cell lung cancer xenografts. Eur J Cancer 31A(11):1862–1868 131. Milroy R (1993) A randomised clinical study of verapamil in addition to combination chemotherapy in small cell lung cancer. West of Scotland Lung Cancer Research Group, and the Aberdeen Oncology Group. Br J Cancer 68(4):813–818 132. Giaccone G, Herbst RS, Manegold C et al (2004) Gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer: a phase III trial – INTACT 1. J Clin Oncol 22(5):777–784 133. Herbst RS, Giaccone G, Schiller JH et al (2004) Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: a phase III trial – INTACT 2. J Clin Oncol 22(5):785–794 134. Wood L, Palmer M, Hewitt J et al (1998) Results of a phase III, double-blind, placebo-controlled trial of megestrol acetate modulation of P-glycoprotein-mediated drug resistance in the first-line management of small-cell lung carcinoma. Br J Cancer 77(4):627–631 135. Hu Y, Lin DM, Cheng SJ, Liu YN, Feng FY (2006) Influences of PC cell-derived growth factor and breast cancer resistance protein on the curative effects of platinum-based chemotherapeutic regimens for advanced non-small cell lung cancer. Zhonghua Yi Xue Za Zhi 86(37):2611–2614 136. Hu Y, Feng FY, Cheng SJ, Gao YN, Xiao T, Liu YN (2006) Expression of serum breast drug-resistance protein in predicting chemosensitivity of NSCLC. Zhonghua Zhong Liu Za Zhi 28(10):750–752 137. Stuckler D, Singhal J, Singhal SS, Yadav S, Awasthi YC, Awasthi S (2005) RLIP76 transports vinorelbine and mediates drug resistance in non-small cell lung cancer. Cancer Res 65(3):991–998

Lung Cancer Resistance to Chemotherapy

377

138. Singhal SS, Yadav S, Singhal J, Zajac E, Awasthi YC, Awasthi S (2005) Depletion of RLIP76 sensitizes lung cancer cells to doxorubicin. Biochem Pharmacol 70(3):481–488 139. Singhal SS, Wickramarachchi D, Singhal J, Yadav S, Awasthi YC, Awasthi S (2006) Determinants of differential doxorubicin sensitivity between SCLC and NSCLC. FEBS Lett 580(9):2258–2264 140. Berger W, Elbling L, Micksche M (2000) Expression of the major vault protein LRP in human non-small-cell lung cancer cells: activation by short-term exposure to antineoplastic drugs. Int J Cancer 88(2):293–300 141. Ikeda K, Oka M, Narasaki F et al (1998) Lung resistance-related protein gene expression and drug sensitivity in human gastric and lung cancer cells. Anticancer Res 18(4C):3077–3080 142. Lee E, Lim SJ (2006) The association of increased lung resistance protein expression with acquired etoposide resistance in human H460 lung cancer cell lines. Arch Pharm Res 29(11):1018–1023 143. Trussardi A, Poitevin G, Gorisse MC et al (1998) Sequential overexpression of LRP and MRP but not P-gp 170 in VP16-selected A549 adenocarcinoma cells. Int J Oncol 13(3):543–548 144. Harada T, Ogura S, Yamazaki K et al (2003) Predictive value of expression of P53, Bcl-2 and lung resistance-related protein for response to chemotherapy in non-small cell lung cancers. Cancer Sci 94(4):394–399 145. Dingemans AM, van Ark-Otte J, van der Valk P et al (1996) Expression of the human major vault protein LRP in human lung cancer samples and normal lung tissues. Ann Oncol 7(6):625–630 146. Chiou JF, Liang JA, Hsu WH, Wang JJ, Ho ST, Kao A (2003) Comparing the relationship of Taxol-based chemotherapy response with P-glycoprotein and lung resistance-related protein expression in non-small cell lung cancer. Lung 181(5):267–273 147. Oguri T, Fujiwara Y, Ochiai M et al (1998) Expression of lung-resistance protein gene is not associated with platinum drug exposure in lung cancer. Anticancer Res 18(6A):4159–4162 148. Nakagawa T, Inoue Y, Kodama H et  al (2008) Expression of copper-transporting P-type adenosine triphosphatase (ATP7B) correlates with cisplatin resistance in human non-small cell lung cancer xenografts. Oncol Rep 20(2):265–270 149. Meijer C, Mulder NH, Hospers GA, Uges DR, de Vries EG (1990) The role of glutathione in resistance to cisplatin in a human small cell lung cancer cell line. Br J Cancer 62(1):72–77 150. Oshita F, Fujiwara Y, Saijo N (1992) Radiation sensitivities in various anticancer-drugresistant human lung cancer cell lines and mechanism of radiation cross-resistance in a cisplatin-resistant cell line. J Cancer Res Clin Oncol 119(1):28–34 151. Curtin NJ, Turner DP (1999) Dipyridamole-mediated reversal of multidrug resistance in MRP over-expressing human lung carcinoma cells in vitro. Eur J Cancer 35(6):1020–1026 152. Meijer C, Mulder NH, Timmer-Bosscha H, Sluiter WJ, Meersma GJ, de Vries EG (1992) Relationship of cellular glutathione to the cytotoxicity and resistance of seven platinum compounds. Cancer Res 52(24):6885–6889 153. Jain N, Lam YM, Pym J, Campling BG (1996) Mechanisms of resistance of human small cell lung cancer lines selected in VP-16 and cisplatin. Cancer 77(9):1797–1808 154. D’Incalci M, Bonfanti M, Pifferi A et al (1998) The antitumour activity of alkylating agents is not correlated with the levels of glutathione, glutathione transferase and O6-alkylguanineDNA-alkyltransferase of human tumour xenografts. EORTC SPG and PAMM Groups. Eur J Cancer 34(11):1749–1755 155. Sharma R, Singhal SS, Srivastava SK, Bajpai KK, Frenkel EP, Awasthi S (1993) Glutathione and glutathione linked enzymes in human small cell lung cancer cell lines. Cancer Lett 75(2):111–119 156. Kasahara K, Fujiwara Y, Nishio K et al (1991) Metallothionein content correlates with the sensitivity of human small cell lung cancer cell lines to cisplatin. Cancer Res 51(12):3237–3242 157. Nishi M, Abe Y, Fujimori S et al (2005) The modifier subunit of glutamate cysteine ligase relates to cisplatin resistance in human small cell lung cancer xenografts in vivo. Oncol Rep 14(2):421–424

378

D.J. Stewart

158. Fujimori S, Abe Y, Nishi M et al (2004) The subunits of glutamate cysteine ligase enhance cisplatin resistance in human non-small cell lung cancer xenografts in  vivo. Int J Oncol 25(2):413–418 159. Inoue Y, Tomisawa M, Yamazaki H et al (2003) The modifier subunit of glutamate cysteine ligase (GCLM) is a molecular target for amelioration of cisplatin resistance in lung cancer. Int J Oncol 23(5):1333–1339 160. Ogawa J, Iwazaki M, Inoue H, Koide S, Shohtsu A (1993) Immunohistochemical study of glutathione-related enzymes and proliferative antigens in lung cancer. Relation to cisplatin sensitivity. Cancer 71(7):2204–2209 161. Hida T, Ariyoshi Y, Kuwabara M et al (1993) Glutathione S-transferase pi levels in a panel of lung cancer cell lines and its relation to chemo-radiosensitivity. Jpn J Clin Oncol 23(1):14–19 162. Nakagawa K, Yokota J, Wada M et al (1988) Levels of glutathione S transferase pi mRNA in human lung cancer cell lines correlate with the resistance to cisplatin and carboplatin. Jpn J Cancer Res 79(3):301–304 163. Awasthi S, Sharma R, Singhal SS, Herzog NK, Chaubey M, Awasthi YC (1994) Modulation of cisplatin cytotoxicity by sulphasalazine. Br J Cancer 70(2):190–194 164. Inoue T, Ishida T, Sugio K, Maehara Y, Sugimachi K (1995) Glutathione S transferase Pi is a powerful indicator in chemotherapy of human lung squamous-cell carcinoma. Respiration 62(4):223–227 165. Miyara H, Hida T, Nishida K et al (1996) Modification of chemo-radiosensitivity of a human lung cancer cell line by introduction of the glutathione S-transferase pi gene. Jpn J Clin Oncol 26(1):1–5 166. Nakanishi Y, Kawasaki M, Bai F et  al (1999) Expression of p53 and glutathione S-transferase-pi relates to clinical drug resistance in non-small cell lung cancer. Oncology 57(4):318–323 167. Arai T, Yasuda Y, Takaya T et  al (2000) Immunohistochemical expression of glutathione transferase-pi in untreated primary non-small-cell lung cancer. Cancer Detect Prev 24(3):252–257 168. Unsal M, Akpolat I, Kandemir B (2003) Glutathione-S transferase-pi expression in non small cell lung cancer in the assessment of response to chemotherapy. Saudi Med J 24(5):493–498 169. Lu C, Spitz MR, Zhao H et al (2006) Association between glutathione S-transferase pi polymorphisms and survival in patients with advanced nonsmall cell lung carcinoma. Cancer 106(2):441–447 170. Booton R, Ward T, Heighway J, Ashcroft L, Morris J, Thatcher N (2006) Glutathione-Stransferase P1 isoenzyme polymorphisms, platinum-based chemotherapy, and non-small cell lung cancer. J Thorac Oncol 1(7):679–683 171. Shimoda R, Achanzar WE, Qu W et al (2003) Metallothionein is a potential negative regulator of apoptosis. Toxicol Sci 73(2):294–300 172. Matsumoto Y, Oka M, Sakamoto A et al (1997) Enhanced expression of metallothionein in human non-small-cell lung carcinomas following chemotherapy. Anticancer Res 17(5B):3777–3780 173. Joseph MG, Banerjee D, Kocha W, Feld R, Stitt LW, Cherian MG (2001) Metallothionein expression in patients with small cell carcinoma of the lung: correlation with other molecular markers and clinical outcome. Cancer 92(4):836–842 174. Wang HW, Lin CP, Chiu JH et al (2007) Reversal of inflammation-associated dihydrodiol dehydrogenases (AKR1C1 and AKR1C2) overexpression and drug resistance in nonsmall cell lung cancer cells by wogonin and chrysin. Int J Cancer 120(9):2019–2027 175. Hung JJ, Chow KC, Wang HW, Wang LS (2006) Expression of dihydrodiol dehydrogenase and resistance to chemotherapy and radiotherapy in adenocarcinoma cells of lung. Anticancer Res 26(4B):2949–2955 176. Deng HB, Adikari M, Parekh HK, Simpkins H (2004) Ubiquitous induction of resistance to platinum drugs in human ovarian, cervical, germ-cell and lung carcinoma tumor cells over-

Lung Cancer Resistance to Chemotherapy

379

expressing isoforms 1 and 2 of dihydrodiol dehydrogenase. Cancer Chemother Pharmacol 54(4):301–307 177. Smith PG, Marshman E, Newell DR, Curtin NJ (2000) Dipyridamole potentiates the in vitro activity of MTA (LY231514) by inhibition of thymidine transport. Br J Cancer 82(4):924–930 178. Chattopadhyay S, Tamari R, Min SH, Zhao R, Tsai E, Goldman ID (2007) Commentary: a case for minimizing folate supplementation in clinical regimens with pemetrexed based on the marked sensitivity of the drug to folate availability. Oncologist 12(7):808–815 179. Kropotov A, Gogvadze V, Shupliakov O et al (2006) Peroxiredoxin V is essential for protection against apoptosis in human lung carcinoma cells. Exp Cell Res 312(15):2806–2815 180. Tibaldi C, Giovannetti E, Vasile E et al (2008) Correlation of CDA, ERCC1, and XPD polymorphisms with response and survival in gemcitabine/cisplatin-treated advanced non-small cell lung cancer patients. Clin Cancer Res 14(6):1797–1803 181. Giovannetti E, Mey V, Nannizzi S et al (2005) Cellular and pharmacogenetics foundation of synergistic interaction of pemetrexed and gemcitabine in human non-small-cell lung cancer cells. Mol Pharmacol 68(1):110–118 182. Bergman AM, Pinedo HM, Peters GJ (2001) Steroids affect collateral sensitivity to gemcitabine of multidrug-resistant human lung cancer cells. Eur J Pharmacol 416(1–2):19–24 183. Chau Q, Stewart DJ (1999) Cisplatin efflux, binding and intracellular pH in the HTB56 human lung adenocarcinoma cell line and the E-8/0.7 cisplatin-resistant variant. Cancer Chemother Pharmacol 44(3):193–202 184. Huang Z, Huang Y (2005) The change of intracellular pH is involved in the cisplatin-resistance of human lung adenocarcinoma A549/DDP cells. Cancer Invest 23(1):26–32 185. Nishio K, Nakamura T, Koh Y, Kanzawa F, Tamura T, Saijo N (2001) Oncoprotein 18 overexpression increases the sensitivity to vindesine in the human lung carcinoma cells. Cancer 91(8):1494–1499 186. Rosell R, Scagliotti G, Danenberg KD et al (2003) Transcripts in pretreatment biopsies from a three-arm randomized trial in metastatic non-small-cell lung cancer. Oncogene 22(23):3548–3553 187. Wehbe H, Kearney CM, Pinney KG (2005) Combretastatin A-4 resistance in H460 human lung carcinoma demonstrates distinctive alterations in beta-tubulin isotype expression. Anticancer Res 25(6B):3865–3870 188. Chang JT, Chang GC, Ko JL et al (2006) Induction of tubulin by docetaxel is associated with p53 status in human non small cell lung cancer cell lines. Int J Cancer 118(2):317–325 189. Han EK, Gehrke L, Tahir SK et al (2000) Modulation of drug resistance by alpha-tubulin in paclitaxel-resistant human lung cancer cell lines. Eur J Cancer 36(12):1565–1571 190. Chan MW, Chiang CD, Song EJ, Yang VC (1998) Effects of cytoskeletal inhibitors on the accumulation of vincristine in a resistant human lung cancer cell line with high level of polymerized tubulin. Cancer Biochem Biophys 16(4):347–363 191. Goncalves A, Braguer D, Kamath K et  al (2001) Resistance to taxol in lung cancer-cells associated with increased microtubule dynamics. Proc Natl Acad Sci U S A 98(20): 11737–11742 192. Ohta S, Nishio K, Kubota N et  al (1994) Characterization of a taxol-resistant human small-cell lung cancer cell line. Jpn J Cancer Res 85(3):290–297 193. Gan PP, Pasquier E, Kavallaris M (2007) Class III beta-tubulin mediates sensitivity to chemotherapeutic drugs in non small cell lung cancer. Cancer Res 67(19):9356–9363 194. Nishio K, Arioka H, Ishida T et al (1995) Enhanced interaction between tubulin and microtubule-associated protein 2 via inhibition of MAP kinase and CDC2 kinase by paclitaxel. Int J Cancer 63(5):688–693 195. Kraus AC, Ferber I, Bachmann SO et al (2002) In vitro chemo- and radio-resistance in small cell lung cancer correlates with cell adhesion and constitutive activation of AKT and MAP kinase pathways. Oncogene 21(57):8683–8695 196. Zhou Q, Bai M, Su Y (2004) Effect of antisense RNA targeting polo-like kinase 1 on cell cycle and proliferation in A549 cells. Chin Med J (Engl) 117(11):1642–1649

380

D.J. Stewart

197. Dumontet C, Isaac S, Souquet PJ et al (2005) Expression of class III beta tubulin in nonsmall cell lung cancer is correlated with resistance to taxane chemotherapy. Bull Cancer 92(2):E25–E30 198. Tsurutani J, Komiya T, Uejima H et al (2002) Mutational analysis of the beta-tubulin gene in lung cancer. Lung Cancer 35(1):11–16 199. Rosell R, Felip E (2001) Predicting response to paclitaxel/carboplatin-based therapy in nonsmall cell lung cancer. Semin Oncol 28(4 Suppl 14):37–44 200. Seve P, Lai R, Ding K et al (2007) Class III beta-tubulin expression and benefit from adjuvant cisplatin/vinorelbine chemotherapy in operable non-small cell lung cancer: analysis of NCIC JBR.10. Clin Cancer Res 13(3):994–999 201. Seve P, Mackey J, Isaac S et al (2005) Class III beta-tubulin expression in tumor cells predicts response and outcome in patients with non-small cell lung cancer receiving paclitaxel. Mol Cancer Ther 4(12):2001–2007 202. Seve P, Isaac S, Tredan O et al (2005) Expression of class III b-tubulin is predictive of patient outcome in patients with non-small cell lung cancer receiving vinorelbine-based chemotherapy. Clin Cancer Res 11(15):5481–5486 203. Yamazaki K, Isobe H, Hanada T et  al (1997) Topoisomerase II alpha content and topoisomerase II catalytic activity cannot explain drug sensitivities to topoisomerase II inhibitors in lung cancer cell lines. Cancer Chemother Pharmacol 39(3):192–198 204. Evans CD, Mirski SE, Danks MK, Cole SP (1994) Reduced levels of topoisomerase II alpha and II beta in a multidrug-resistant lung-cancer cell line. Cancer Chemother Pharmacol 34(3):242–248 205. Giaccone G, Gazdar AF, Beck H, Zunino F, Capranico G (1992) Multidrug sensitivity phenotype of human lung cancer cells associated with topoisomerase II expression. Cancer Res 52(7):1666–1674 206. de Jong S, Zijlstra JG, de Vries EG, Mulder NH (1990) Reduced DNA topoisomerase II activity and drug-induced DNA cleavage activity in an adriamycin-resistant human small cell lung carcinoma cell line. Cancer Res 50(2):304–309 207. de Lucio B, Manuel V, Barrera-Rodriguez R (2005) Characterization of human NSCLC cell line with innate etoposide-resistance mediated by cytoplasmic localization of topoisomerase II alpha. Cancer Sci 96(11):774–783 208. Wessel I, Jensen PB, Falck J, Mirski SE, Cole SP, Sehested M (1997) Loss of amino acids 1490Lys-Ser-Lys1492 in the COOH-terminal region of topoisomerase IIalpha in human small cell lung cancer cells selected for resistance to etoposide results in an extranuclear enzyme localization. Cancer Res 57(20):4451–4454 209. Eijdems EW, de Haas M, Timmerman AJ et al (1995) Reduced topoisomerase II activity in multidrug-resistant human non-small cell lung cancer cell lines. Br J Cancer 71(1):40–47 210. Guinee DG Jr, Holden JA, Benfield JR et al (1996) Comparison of DNA topoisomerase II alpha expression in small cell and nonsmall cell carcinoma of the lung. In search of a mechanism of chemotherapeutic response. Cancer 78(4):729–735 211. Dingemans AM, Witlox MA, Stallaert RA, van der Valk P, Postmus PE, Giaccone G (1999) Expression of DNA topoisomerase IIalpha and topoisomerase IIbeta genes predicts survival and response to chemotherapy in patients with small cell lung cancer. Clin Cancer Res 5(8):2048–2058 212. Tsai CM, Chang KT, Li L, Perng RP, Yang LY (2000) Interrelationships between cellular nucleotide excision repair, cisplatin cytotoxicity, HER-2/neu gene expression, and epidermal growth factor receptor level in non-small cell lung cancer cells. Jpn J Cancer Res 91(2):213–222 213. Takenaka T, Yoshino I, Kouso H et al (2007) Combined evaluation of Rad51 and ERCC1 expressions for sensitivity to platinum agents in non-small cell lung cancer. Int J Cancer 121(4):895–900 214. Shimizu J, Horio Y, Osada H et al (2008) mRNA expression of RRM1, ERCC1 and ERCC2 is not associated with chemosensitivity to cisplatin, carboplatin and gemcitabine in human lung cancer cell lines. Respirology 13(4):510–517 215. Olaussen KA, Dunant A, Fouret P et al (2006) DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy. N Engl J Med 355(10):983–991

Lung Cancer Resistance to Chemotherapy

381

216. Huang PY, Liang XM, Lin SX, Luo RZ, Hou JH, Zhang L (2004) Correlation analysis among expression of ERCC-1, metallothionein, p53 and platinum resistance and prognosis in advanced non-small cell lung cancer. Ai Zheng 23(7):845–850 217. Hwang IG, Ahn MJ, Park BB et  al (2008) ERCC1 expression as a prognostic marker in N2(+) nonsmall-cell lung cancer patients treated with platinum-based neoadjuvant concurrent chemoradiotherapy. Cancer 113(6):1379–1386 218. Wachters FM, Wong LS, Timens W, Kampinga HH, Groen HJ (2005) ERCC1, hRad51, and BRCA1 protein expression in relation to tumour response and survival of stage III/IV NSCLC patients treated with chemotherapy. Lung Cancer 50(2):211–219 219. Booton R, Ward T, Ashcroft L, Morris J, Heighway J, Thatcher N (2007) ERCC1 mRNA expression is not associated with response and survival after platinum-based chemotherapy regimens in advanced non-small cell lung cancer. J Thorac Oncol 2(10):902–906 220. Azuma K, Komohara Y, Sasada T et al (2007) Excision repair cross-complementation group 1 predicts progression-free and overall survival in non-small cell lung cancer patients treated with platinum-based chemotherapy. Cancer Sci 98(9):1336–1343 221. Lord RV, Brabender J, Gandara D et  al (2002) Low ERCC1 expression correlates with prolonged survival after cisplatin plus gemcitabine chemotherapy in non-small cell lung cancer. Clin Cancer Res 8(7):2286–2291 222. Bepler G, Kusmartseva I, Sharma S et  al (2006) RRM1 modulated in  vitro and in  vivo efficacy of gemcitabine and platinum in non-small-cell lung cancer. J Clin Oncol 24(29):4731–4737 223. Ceppi P, Volante M, Novello S et  al (2006) ERCC1 and RRM1 gene expressions but not EGFR are predictive of shorter survival in advanced non-small-cell lung cancer treated with cisplatin and gemcitabine. Ann Oncol 17(12):1818–1825 224. Fujii T, Toyooka S, Ichimura K et al (2008) ERCC1 protein expression predicts the response of cisplatin-based neoadjuvant chemotherapy in non-small-cell lung cancer. Lung Cancer 59(3):377–384 225. Cobo M, Isla D, Massuti B et al (2007) Customizing cisplatin based on quantitative excision repair cross-complementing 1 mRNA expression: a phase III trial in non-small-cell lung cancer. J Clin Oncol 25(19):2747–2754 226. Su D, Ma S, Liu P et al (2007) Genetic polymorphisms and treatment response in advanced non-small cell lung cancer. Lung Cancer 56(2):281–288 227. Ryu JS, Hong YC, Han HS et al (2004) Association between polymorphisms of ERCC1 and XPD and survival in non-small-cell lung cancer patients treated with cisplatin combination chemotherapy. Lung Cancer 44(3):311–316 228. Park SY, Hong YC, Kim JH et al (2006) Effect of ERCC1 polymorphisms and the modification by smoking on the survival of non-small cell lung cancer patients. Med Oncol 23(4):489–498 229. Zhou W, Gurubhagavatula S, Liu G et  al (2004) Excision repair cross-complementation group 1 polymorphism predicts overall survival in advanced non-small cell lung cancer patients treated with platinum-based chemotherapy. Clin Cancer Res 10(15):4939–4943 230. Wu X, Lu C, Ye Y et al (2008) Germline genetic variations in drug action pathways predict clinical outcomes in advanced lung cancer treated with platinum-based chemotherapy. Pharmacogenet Genomics 18(11):955–965 231. Ceppi P, Longo M, Volante M et al (2008) Excision repair cross complementing-1 and topoisomerase IIalpha gene expression in small-cell lung cancer patients treated with platinum and etoposide: a retrospective study. J Thorac Oncol 3(6):583–589 232. Lee HW, Han JH, Kim JH et al (2008) Expression of excision repair cross-complementation group 1 protein predicts poor outcome in patients with small cell lung cancer. Lung Cancer 59(1):95–104 233. Fan W, Zhang HL, Wu XM (2005) Enhancement effect of nucleotide excision repair gene xeroderma pigmentosun group a antisense RNA on sensitivity of human lung adenocarcinoma cell line A549 to cisplatin. Ai Zheng 24(4):403–407

382

D.J. Stewart

234. Yuan P, Miao XP, Zhang XM et al (2005) Correlation of genetic polymorphisms in nucleotide excision repair system to sensitivity of advanced non-small cell lung cancer patients to platinum-based chemotherapy. Ai Zheng 24(12):1510–1513 235. Gurubhagavatula S, Liu G, Park S et al (2004) XPD and XRCC1 genetic polymorphisms are prognostic factors in advanced non-small-cell lung cancer patients treated with platinum chemotherapy. J Clin Oncol 22(13):2594–2601 236. Booton R, Ward T, Heighway J et  al (2006) Xeroderma pigmentosum group D haplotype predicts for response, survival, and toxicity after platinum-based chemotherapy in advanced nonsmall cell lung cancer. Cancer 106(11):2421–2427 237. Yuan P, Miao XP, Zhang XM et al (2006) XRCC1 and XPD genetic polymorphisms predict clinical responses to platinum-based chemotherapy in advanced non-small cell lung cancer. Zhonghua Zhong Liu Za Zhi 28(3):196–199 238. Giachino DF, Ghio P, Regazzoni S et al (2007) Prospective assessment of XPD Lys751Gln and XRCC1 Arg399Gln single nucleotide polymorphisms in lung cancer. Clin Cancer Res 13(10):2876–2881 239. Kwon WS, Rha SY, Choi YH et al (2006) Ribonucleotide reductase M1 (RRM1) 2464G > A polymorphism shows an association with gemcitabine chemosensitivity in cancer cell lines. Pharmacogenet Genomics 16(6):429–438 240. Tooker P, Yen WC, Ng SC, Negro-Vilar A, Hermann TW (2007) Bexarotene (LGD1069, Targretin), a selective retinoid X receptor agonist, prevents and reverses gemcitabine resistance in NSCLC cells by modulating gene amplification. Cancer Res 67(9):4425–4433 241. Rosell R, Felip E, Taron M et al (2004) Gene expression as a predictive marker of outcome in stage IIB-IIIA-IIIB non-small cell lung cancer after induction gemcitabine-based chemotherapy followed by resectional surgery. Clin Cancer Res 10(12 Pt 2):4215s–4219s 242. Souglakos J, Boukovinas I, Taron M et al (2008) Ribonucleotide reductase subunits M1 and M2 mRNA expression levels and clinical outcome of lung adenocarcinoma patients treated with docetaxel/gemcitabine. Br J Cancer 98(10):1710–1715 243. Rosell R, Danenberg KD, Alberola V et al (2004) Ribonucleotide reductase messenger RNA expression and survival in gemcitabine/cisplatin-treated advanced non-small cell lung cancer patients. Clin Cancer Res 10(4):1318–1325 244. Kim SO, Jeong JY, Kim MR et al (2008) Efficacy of gemcitabine in patients with non-small cell lung cancer according to promoter polymorphisms of the ribonucleotide reductase M1 gene. Clin Cancer Res 14(10):3083–3088 245. Simon G, Sharma A, Li X et  al (2007) Feasibility and efficacy of molecular analysisdirected individualized therapy in advanced non-small-cell lung cancer. J Clin Oncol 25(19):2741–2746 246. Hansen LT, Lundin C, Spang-Thomsen M, Petersen LN, Helleday T (2003) The role of RAD51 in etoposide (VP16) resistance in small cell lung cancer. Int J Cancer 105(4):472–479 247. Ko JC, Ciou SC, Cheng CM et al (2008) Involvement of Rad51 in cytotoxicity induced by epidermal growth factor receptor inhibitor (gefitinib, IressaR) and chemotherapeutic agents in human lung cancer cells. Carcinogenesis 29(7):1448–1458 248. Rosell R, Cuello M, Cecere F et al (2006) Usefulness of predictive tests for cancer treatment. Bull Cancer 93(8):E101–E108 249. Rosell R, Skrzypski M, Jassem E et al (2007) BRCA1: a novel prognostic factor in resected non-small-cell lung cancer. PLoS ONE 2(11):e1129 250. Taron M, Rosell R, Felip E et al (2004) BRCA1 mRNA expression levels as an indicator of chemoresistance in lung cancer. Hum Mol Genet 13(20):2443–2449 251. Ferrer M, Span SW, Vischioni B et al (2005) FANCD2 expression in advanced non-smallcell lung cancer and response to platinum-based chemotherapy. Clin Lung Cancer 6(4):250–254 252. Ueda K, Kawashima H, Ohtani S et al (2006) The 3p21.3 tumor suppressor NPRL2 plays an important role in cisplatin-induced resistance in human non-small-cell lung cancer cells. Cancer Res 66(19):9682–9690

Lung Cancer Resistance to Chemotherapy

383

253. Scartozzi M, Franciosi V, Campanini N et al (2006) Mismatch repair system (MMR) status correlates with response and survival in non-small cell lung cancer (NSCLC) patients. Lung Cancer 53(1):103–109 254. Kinzel B, Hall J, Natt F, Weiler J, Cohen D (2002) Downregulation of Hus1 by antisense oligonucleotides enhances the sensitivity of human lung carcinoma cells to cisplatin. Cancer 94(6):1808–1814 255. Takizawa M, Kawakami K, Obata T et  al (2006) In vitro sensitivity to platinum-derived drugs is associated with expression of thymidylate synthase and dihydropyrimidine dehydrogenase in human lung cancer. Oncol Rep 15(6):1533–1539 256. Arioka H, Nishio K, Ishida T et  al (1999) Enhancement of cisplatin sensitivity in high mobility group 2 cDNA-transfected human lung cancer cells. Jpn J Cancer Res 90(1):108–115 257. Andriani F, Perego P, Carenini N, Sozzi G, Roz L (2006) Increased sensitivity to cisplatin in non-small cell lung cancer cell lines after FHIT gene transfer. Neoplasia 8(1):9–17 258. Hu B, Wang H, Wang X et al (2005) Fhit and CHK1 have opposing effects on homologous recombination repair. Cancer Res 65(19):8613–8616 259. Almeida GM, Duarte TL, Farmer PB, Steward WP, Jones GD (2008) Multiple end-point analysis reveals cisplatin damage tolerance to be a chemoresistance mechanism in a NSCLC model: implications for predictive testing. Int J Cancer 122(8):1810–1819 260. Corn PG, El-Deiry WS (2007) Microarray analysis of p53-dependent gene expression in response to hypoxia and DNA damage. Cancer Biol Ther 6(12):1858–1866 261. Bergqvist M, Brattstrom D, Gullbo J, Hesselius P, Brodin O, Wagenius G (2003) p53 Status and its in  vitro relationship to radiosensitivity and chemosensitivity in lung cancer. Anticancer Res 23(2B):1207–1212 262. Wang T, Xu J, Zhong NS (2005) Relationship between the acquired multi-drug resistance of human large cell lung cancer cell line NCI-H460 by cisplatin selection and p53 mutation. Zhonghua Jie He He Hu Xi Za Zhi 28(2):102–107 263. Brattstrom D, Bergqvist M, Lamberg K et al (1998) Complete sequence of p53 gene in 20 patients with lung cancer: comparison with chemosensitivity and immunohistochemistry. Med Oncol 15(4):255–261 264. Blandino G, Levine AJ, Oren M (1999) Mutant p53 gain of function: differential effects of different p53 mutants on resistance of cultured cells to chemotherapy. Oncogene 18(2):477–485 265. Lai SL, Perng RP, Hwang J (2000) p53 Gene status modulates the chemosensitivity of non-small cell lung cancer cells. J Biomed Sci 7(1):64–70 266. Inoue A, Narumi K, Matsubara N et al (2000) Administration of wild-type p53 adenoviral vector synergistically enhances the cytotoxicity of anti-cancer drugs in human lung cancer cells irrespective of the status of p53 gene. Cancer Lett 157(1):105–112 267. Wang Y, Blandino G, Oren M, Givol D (1998) Induced p53 expression in lung cancer cell line promotes cell senescence and differentially modifies the cytotoxicity of anti-cancer drugs. Oncogene 17(15):1923–1930 268. Ling YH, Zou Y, Perez-Soler R (2000) Induction of senescence-like phenotype and loss of paclitaxel sensitivity after wild-type p53 gene transfection of p53-null human non-small cell lung cancer H358 cells. Anticancer Res 20(2A):693–702 269. He Y, Fan SZ, Jiang YG et al (2004) Effect of p73 gene on chemosensitivity of human lung adenocarcinoma cells H1299. Ai Zheng 23(6):645–649 270. Mori T, Okamoto H, Takahashi N, Ueda R, Okamoto T (2000) Aberrant overexpression of 53BP2 mRNA in lung cancer cell lines. FEBS Lett 465(2–3):124–128 271. Vogt U, Zaczek A, Klinke F, Granetzny A, Bielawski K, Falkiewicz B (2002) p53 Gene status in relation to ex vivo chemosensitivity of non-small cell lung cancer. J Cancer Res Clin Oncol 128(3):141–147 272. Tsai CM, Chang KT, Wu LH et al (1996) Correlations between intrinsic chemoresistance and HER-2/neu gene expression, p53 gene mutations, and cell proliferation characteristics in non-small cell lung cancer cell lines. Cancer Res 56(1):206–209

384

D.J. Stewart

273. Safran H, King T, Choy H et al (1996) p53 Mutations do not predict response to paclitaxel/ radiation for nonsmall cell lung carcinoma. Cancer 78(6):1203–1210 274. Kandioler-Eckersberger D, Kappel S, Mittlbock M et al (1999) The TP53 genotype but not immunohistochemical result is predictive of response to cisplatin-based neoadjuvant therapy in stage III non-small cell lung cancer. J Thorac Cardiovasc Surg 117(4):744–750 275. d’Amato TA, Landreneau RJ, Ricketts W et al (2007) Chemotherapy resistance and oncogene expression in non-small cell lung cancer. J Thorac Cardiovasc Surg 133(2):352–363 276. Higashiyama M, Kodama K, Yokouchi H et  al (1998) Immunohistochemical p53 protein status in nonsmall cell lung cancer is a promising indicator in determining in vitro chemosensitivity to some anticancer drugs. J Surg Oncol 68(1):19–24 277. Kandioler D, Stamatis G, Eberhardt W et al (2008) Growing clinical evidence for the interaction of the p53 genotype and response to induction chemotherapy in advanced non-small cell lung cancer. J Thorac Cardiovasc Surg 135(5):1036–1041 278. Gajra A, Tatum AH, Newman N et al (2002) The predictive value of neuroendocrine markers and p53 for response to chemotherapy and survival in patients with advanced non-small cell lung cancer. Lung Cancer 36(2):159–165 279. Rusch V, Klimstra D, Venkatraman E et al (1995) Aberrant p53 expression predicts clinical resistance to cisplatin-based chemotherapy in locally advanced non-small cell lung cancer. Cancer Res 55(21):5038–5042 280. Higashiyama M, Miyoshi Y, Kodama K et al (2000) p53-Regulated GML gene expression in non-small cell lung cancer. A promising relationship to cisplatin chemosensitivity. Eur J Cancer 36(4):489–495 281. Fijolek J, Wiatr E, Rowinska-Zakrzewska E et al (2006) p53 and HER2/neu expression in relation to chemotherapy response in patients with non-small cell lung cancer. Int J Biol Markers 21(2):81–87 282. Kawasaki M, Nakanishi Y, Kuwano K, Yatsunami J, Takayama K, Hara N (1997) The utility of p53 immunostaining of transbronchial biopsy specimens of lung cancer: p53 overexpression predicts poor prognosis and chemoresistance in advanced non-small cell lung cancer. Clin Cancer Res 3(7):1195–1200 283. Yuan P, Miao XP, Zhang XM et  al (2006) Association of the responsiveness of advanced non-small cell lung cancer to platinum-based chemotherapy with p53 and p73 polymorphisms. Zhonghua Zhong Liu Za Zhi 28(2):107–110 284. Shih CM, Chen K, Wang YC, Lee PJ, Wang YC (2007) Elevated p53 and p21waf1 mRNA expression in blood lymphocytes from lung cancer patients with chemoresistance. Cancer Detect Prev 31(5):366–370 285. Schuler M, Herrmann R, De Greve JL et al (2001) Adenovirus-mediated wild-type p53 gene transfer in patients receiving chemotherapy for advanced non-small-cell lung cancer: results of a multicenter phase II study. J Clin Oncol 19(6):1750–1758 286. Johnson EA, Klimstra DS, Herndon JE II et al (2002) Aberrant p53 staining does not predict cisplatin resistance in locally advanced non-small cell lung cancer. Cancer Invest 20(5–6):686–692 287. Graziano SL, Tatum A, Herndon JE II et al (2001) Use of neuroendocrine markers, p53, and HER2 to predict response to chemotherapy in patients with stage III non-small cell lung cancer: a Cancer and Leukemia Group B study. Lung Cancer 33(2–3):115–123 288. Berrieman HK, Cawkwell L, O’Kane SL, Smith L, Lind MJ (2006) Hsp27 may allow prediction of the response to single-agent vinorelbine chemotherapy in non-small cell lung cancer. Oncol Rep 15(1):283–286 289. Gregorc V, Darwish S, Ludovini V et al (2003) The clinical relevance of Bcl-2, Rb and p53 expression in advanced non-small cell lung cancer. Lung Cancer 42(3):275–281 290. van de Vaart PJ, Belderbos J, de Jong D et al (2000) DNA-adduct levels as a predictor of outcome for NSCLC patients receiving daily cisplatin and radiotherapy. Int J Cancer 89(2):160–166 291. Oshita F, Nishio K, Kameda Y et al (2000) Increased expression levels of p53 correlate with good response to cisplatin-based chemotherapy in non-small cell lung cancer. Oncol Rep 7(6):1225–1228

Lung Cancer Resistance to Chemotherapy

385

292. Oshita F, Kameda Y, Hamanaka N et al (2004) High expression of integrin beta1 and p53 is a greater poor prognostic factor than clinical stage in small-cell lung cancer. Am J Clin Oncol 27(3):215–219 293. Ferreira CG, Span SW, Peters GJ, Kruyt FA, Giaccone G (2000) Chemotherapy triggers apoptosis in a caspase-8-dependent and mitochondria-controlled manner in the non-small cell lung cancer cell line NCI-H460. Cancer Res 60(24):7133–7141 294. Okouoyo S, Herzer K, Ucur E et al (2004) Rescue of death receptor and mitochondrial apoptosis signaling in resistant human NSCLC in vivo. Int J Cancer 108(4):580–587 295. Herr I, Ucur E, Herzer K et al (2003) Glucocorticoid cotreatment induces apoptosis resistance toward cancer therapy in carcinomas. Cancer Res 63(12):3112–3120 296. Chattopadhyay S, Machado-Pinilla R, Manguan-Garcia C et al (2006) MKP1/CL100 controls tumor growth and sensitivity to cisplatin in non-small-cell lung cancer. Oncogene 25(23):3335–3345 297. Levresse V, Marek L, Blumberg D, Heasley LE (2002) Regulation of platinum-compound cytotoxicity by the c-Jun N-terminal kinase and c-Jun signaling pathway in small-cell lung cancer cells. Mol Pharmacol 62(3):689–697 298. Yoshida S, Kawaguchi H, Sato S, Ueda R, Furukawa K (2002) An anti-GD2 monoclonal antibody enhances apoptotic effects of anti-cancer drugs against small cell lung cancer cells via JNK (c-Jun terminal kinase) activation. Jpn J Cancer Res 93(7):816–824 299. Teraishi F, Zhang L, Guo W et al (2005) Activation of c-Jun NH2-terminal kinase is required for gemcitabine’s cytotoxic effect in human lung cancer H1299 cells. FEBS Lett 579(29):6681–6687 300. Joseph B, Ekedahl J, Lewensohn R, Marchetti P, Formstecher P, Zhivotovsky B (2001) Defective caspase-3 relocalization in non-small cell lung carcinoma. Oncogene 20(23):2877–2888 301. Ikuta K, Takemura K, Kihara M et  al (2005) Defects in apoptotic signal transduction in cisplatin-resistant non-small cell lung cancer cells. Oncol Rep 13(6):1229–1234 302. Deng WG, Wu G, Ueda K, Xu K, Roth JA, Ji L (2008) Enhancement of antitumor activity of cisplatin in human lung cancer cells by tumor suppressor FUS1. Cancer Gene Ther 15(1):29–39 303. Camps C, Sirera R, Bremnes RM et al (2006) Analysis of c-kit expression in small cell lung cancer: prevalence and prognostic implications. Lung Cancer 52(3):343–347 304. Soriano AF, Helfrich B, Chan DC, Heasley LE, Bunn PA Jr, Chou TC (1999) Synergistic effects of new chemopreventive agents and conventional cytotoxic agents against human lung cancer cell lines. Cancer Res 59(24):6178–6184 305. Duffy CP, Elliott CJ, O’Connor RA et  al (1998) Enhancement of chemotherapeutic drug toxicity to human tumour cells in vitro by a subset of non-steroidal anti-inflammatory drugs (NSAIDs). Eur J Cancer 34(8):1250–1259 306. Hida T, Kozaki K, Muramatsu H et al (2000) Cyclooxygenase-2 inhibitor induces apoptosis and enhances cytotoxicity of various anticancer agents in non-small cell lung cancer cell lines. Clin Cancer Res 6(5):2006–2011 307. Hida T, Kozaki K, Ito H et al (2002) Significant growth inhibition of human lung cancer cells both in vitro and in vivo by the combined use of a selective cyclooxygenase 2 inhibitor, JTE522, and conventional anticancer agents. Clin Cancer Res 8(7):2443–2447 308. Chen XJ, Xiao W, Qu X, Zhou SY (2008) NS-398 enhances the efficacy of gemcitabine against lung adenocarcinoma through up-regulation of p21WAF1 and p27KIP1 protein. Neoplasma 55(3):200–204 309. Edelman MJ, Watson D, Wang X et al (2008) Eicosanoid modulation in advanced lung cancer: cyclooxygenase-2 expression is a positive predictive factor for celecoxib + chemotherapy – Cancer and Leukemia Group B Trial 30203. J Clin Oncol 26(6):848–855 310. Gridelli C, Gallo C, Ceribelli A et al (2007) Factorial phase III randomised trial of rofecoxib and prolonged constant infusion of gemcitabine in advanced non-small-cell lung cancer: the GEmcitabine-COxib in NSCLC (GECO) study. Lancet Oncol 8(6):500–512 311. Gasparini G, Meo S, Comella G et al (2005) The combination of the selective cyclooxygenase-2 inhibitor celecoxib with weekly paclitaxel is a safe and active second-line therapy for non-small cell lung cancer: a phase II study with biological correlates. Cancer J 11(3):209–216

386

D.J. Stewart

312. Misawa M, Tauchi T, Sashida G et al (2002) Inhibition of human telomerase enhances the effect of chemotherapeutic agents in lung cancer cells. Int J Oncol 21(5):1087–1092 313. Tsurutani J, Soda H, Oka M et al (2003) Antiproliferative effects of the histone deacetylase inhibitor FR901228 on small-cell lung cancer lines and drug-resistant sublines. Int J Cancer 104(2):238–242 314. Sawai A, Chandarlapaty S, Greulich H et  al (2008) Inhibition of Hsp90 down-regulates mutant epidermal growth factor receptor (EGFR) expression and sensitizes EGFR mutant tumors to paclitaxel. Cancer Res 68(2):589–596 315. Ekedahl J, Joseph B, Marchetti P et al (2003) Heat shock protein 72 does not modulate ionizing radiation-induced apoptosis in U1810 non-small cell lung carcinoma cells. Cancer Biol Ther 2(6):663–669 316. Yang CP, Galbiati F, Volonte D, Horwitz SB, Lisanti MP (1998) Upregulation of caveolin-1 and caveolae organelles in taxol-resistant A549 cells. FEBS Lett 439(3):368–372 317. Ho CC, Kuo SH, Huang PH, Huang HY, Yang CH, Yang PC (2008) Caveolin-1 expression is significantly associated with drug resistance and poor prognosis in advanced non-small cell lung cancer patients treated with gemcitabine-based chemotherapy. Lung Cancer 59(1):105–110 318. Cordes N, Beinke C, Plasswilm L, van Beuningen D (2004) Irradiation and various cytotoxic drugs enhance tyrosine phosphorylation and beta(1)-integrin clustering in human A549 lung cancer cells in a substratum-dependent manner in  vitro. Strahlenther Onkol 180(3):157–164 319. Hodkinson PS, Mackinnon AC, Sethi T (2007) Extracellular matrix regulation of drug resistance in small-cell lung cancer. Int J Radiat Biol 83(11–12):733–741 320. Hodkinson PS, Elliott T, Wong WS et al (2006) ECM overrides DNA damage-induced cell cycle arrest and apoptosis in small-cell lung cancer cells through beta1 integrin-dependent activation of PI3-kinase. Cell Death Differ 13(10):1776–1788 321. Hartmann TN, Burger JA, Glodek A, Fujii N, Burger M (2005) CXCR4 chemokine receptor and integrin signaling co-operate in mediating adhesion and chemoresistance in small cell lung cancer (SCLC) cells. Oncogene 24(27):4462–4471 322. Rintoul RC, Sethi T (2002) Extracellular matrix regulation of drug resistance in small-cell lung cancer. Clin Sci (Lond) 102(4):417–424 323. Zhao Y, El-Gabry M, Hei TK (2006) Loss of Betaig-h3 protein is frequent in primary lung carcinoma and related to tumorigenic phenotype in lung cancer cells. Mol Carcinog 45(2):84–92 324. Lei W, Mayotte JE, Levitt ML (1999) Enhancement of chemosensitivity and programmed cell death by tyrosine kinase inhibitors correlates with EGFR expression in non-small cell lung cancer cells. Anticancer Res 19(1A):221–228 325. Ando K, Ohmori T, Inoue F et al (2005) Enhancement of sensitivity to tumor necrosis factor alpha in non-small cell lung cancer cells with acquired resistance to gefitinib. Clin Cancer Res 11(24 Pt 1):8872–8879 326. Van Schaeybroeck S, Kyula J, Kelly DM et  al (2006) Chemotherapy-induced epidermal growth factor receptor activation determines response to combined gefitinib/chemotherapy treatment in non-small cell lung cancer cells. Mol Cancer Ther 5(5):1154–1165 327. Steiner P, Joynes C, Bassi R et  al (2007) Tumor growth inhibition with cetuximab and chemotherapy in non-small cell lung cancer xenografts expressing wild-type and mutated epidermal growth factor receptor. Clin Cancer Res 13(5):1540–1551 328. Raben D, Helfrich B, Chan DC et al (2005) The effects of cetuximab alone and in combination with radiation and/or chemotherapy in lung cancer. Clin Cancer Res 11(2 Pt 1):795–805 329. Rosetti M, Zoli W, Tesei A et al (2007) Iressa strengthens the cytotoxic effect of docetaxel in NSCLC models that harbor specific molecular characteristics. J Cell Physiol 212(3):710–716 330. Mahaffey CM, Davies AM, Lara PN Jr et al (2007) Schedule-dependent apoptosis in K-ras mutant non-small-cell lung cancer cell lines treated with docetaxel and erlotinib: rationale for pharmacodynamic separation. Clin Lung Cancer 8(9):548–553

Lung Cancer Resistance to Chemotherapy

387

331. Giovannetti E, Lemos C, Tekle C et al (2008) Molecular mechanisms underlying the synergistic interaction of erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor, with the multitargeted antifolate pemetrexed in non-small-cell lung cancer cells. Mol Pharmacol 73(4):1290–1300 332. Li T, Ling YH, Goldman ID, Perez-Soler R (2007) Schedule-dependent cytotoxic synergism of pemetrexed and erlotinib in human non-small cell lung cancer cells. Clin Cancer Res 13(11):3413–3422 333. Meert AP, Martin B, Delmotte P et al (2002) The role of EGF-R expression on patient survival in lung cancer: a systematic review with meta-analysis. Eur Respir J 20(4):975–981 334. Cappuzzo F, Ligorio C, Toschi L et  al (2007) EGFR and HER2 gene copy number and response to first-line chemotherapy in patients with advanced non-small cell lung cancer (NSCLC). J Thorac Oncol 2(5):423–429 335. Dziadziuszko R, Holm B, Skov BG et al (2007) Epidermal growth factor receptor gene copy number and protein level are not associated with outcome of non-small-cell lung cancer patients treated with chemotherapy. Ann Oncol 18(3):447–452 336. Eberhard DA, Johnson BE, Amler LC et al (2005) Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol 23(25):5900–5909 337. Lee KH, Han SW, Hwang PG et al (2006) Epidermal growth factor receptor mutations and response to chemotherapy in patients with non-small-cell lung cancer. Jpn J Clin Oncol 36(6):344–350 338. Gatzemeier U, Pluzanska A, Szczesna A et al (2007) Phase III study of erlotinib in combination with cisplatin and gemcitabine in advanced non-small-cell lung cancer: the Tarceva Lung Cancer Investigation Trial. J Clin Oncol 25(12):1545–1552 339. Herbst RS, Prager D, Hermann R et al (2005) TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol 23(25):5892–5899 340. Bunn PA Jr, Helfrich B, Soriano AF et  al (2001) Expression of Her-2/neu in human lung cancer cell lines by immunohistochemistry and fluorescence in situ hybridization and its relationship to in  vitro cytotoxicity by trastuzumab and chemotherapeutic agents. Clin Cancer Res 7(10):3239–3250 341. Tsai CM, Chang KT, Chen JY, Chen YM, Chen MH, Perng RP (1996) Cytotoxic effects of gemcitabine-containing regimens against human non-small cell lung cancer cell lines which express different levels of p185neu. Cancer Res 56(4):794–801 342. Tsai CM, Chang KT, Perng RP et al (1993) Correlation of intrinsic chemoresistance of nonsmall-cell lung cancer cell lines with HER-2/neu gene expression but not with ras gene mutations. J Natl Cancer Inst 85(11):897–901 343. You XL, Yen L, Zeng-Rong N, Al Moustafa AE, Alaoui-Jamali MA (1998) Dual effect of erbB-2 depletion on the regulation of DNA repair and cell cycle mechanisms in non-small cell lung cancer cells. Oncogene 17(24):3177–3186 344. Zhang L, Hung MC (1996) Sensitization of HER-2/neu-overexpressing non-small cell lung cancer cells to chemotherapeutic drugs by tyrosine kinase inhibitor emodin. Oncogene 12(3):571–576 345. Meert AP, Martin B, Paesmans M et  al (2003) The role of HER-2/neu expression on the survival of patients with lung cancer: a systematic review of the literature. Br J Cancer 89(6):959–965 346. Graziano SL, Kern JA, Herndon JE et al (1998) Analysis of neuroendocrine markers, HER2 and CEA before and after chemotherapy in patients with stage IIIA non-small cell lung cancer: a Cancer and Leukemia Group B study. Lung Cancer 21(3):203–211 347. Junker K, Stachetzki U, Rademacher D et al (2005) HER2/neu expression and amplification in non-small cell lung cancer prior to and after neoadjuvant therapy. Lung Cancer 48(1):59–67 348. Morita M, Suyama H, Igishi T et  al (2007) Dexamethasone inhibits paclitaxel-induced cytotoxic activity through retinoblastoma protein dephosphorylation in non-small cell lung cancer cells. Int J Oncol 30(1):187–192

388

D.J. Stewart

349. Lee MW, Kim DS, Min NY, Kim HT (2008) Akt1 inhibition by RNA interference sensitizes human non-small cell lung cancer cells to cisplatin. Int J Cancer 122(10):2380–2384 350. Brognard J, Dennis PA (2002) Variable apoptotic response of NSCLC cells to inhibition of the MEK/ERK pathway by small molecules or dominant negative mutants. Cell Death Differ 9(9):893–904 351. Krystal GW, Sulanke G, Litz J (2002) Inhibition of phosphatidylinositol 3-kinase-Akt signaling blocks growth, promotes apoptosis, and enhances sensitivity of small cell lung cancer cells to chemotherapy. Mol Cancer Ther 1(11):913–922 352. Brognard J, Clark AS, Ni Y, Dennis PA (2001) Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res 61(10):3986–3997 353. Hemstrom TH, Sandstrom M, Zhivotovsky B (2006) Inhibitors of the PI3-kinase/Akt pathway induce mitotic catastrophe in non-small cell lung cancer cells. Int J Cancer 119(5):1028–1038 354. Yu K, Lucas J, Zhu T et  al (2005) PWT-458, a novel pegylated-17-hydroxywortmannin, inhibits phosphatidylinositol 3-kinase signaling and suppresses growth of solid tumors. Cancer Biol Ther 4(5):538–545 355. Hovelmann S, Beckers TL, Schmidt M (2004) Molecular alterations in apoptotic pathways after PKB/Akt-mediated chemoresistance in NCI H460 cells. Br J Cancer 90(12):2370–2377 356. Liu LZ, Zhou XD, Qian G, Shi X, Fang J, Jiang BH (2007) AKT1 amplification regulates cisplatin resistance in human lung cancer cells through the mammalian target of rapamycin/ p70S6K1 pathway. Cancer Res 67(13):6325–6332 357. Tsurutani J, West KA, Sayyah J, Gills JJ, Dennis PA (2005) Inhibition of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway but not the MEK/ERK pathway attenuates laminin-mediated small cell lung cancer cellular survival and resistance to imatinib mesylate or chemotherapy. Cancer Res 65(18):8423–8432 358. Wu C, Wangpaichitr M, Feun L et  al (2005) Overcoming cisplatin resistance by mTOR inhibitor in lung cancer. Mol Cancer 4(1):25 359. Hohla F, Schally AV, Szepeshazi K et  al (2006) Synergistic inhibition of growth of lung carcinomas by antagonists of growth hormone-releasing hormone in combination with docetaxel. Proc Natl Acad Sci U S A 103(39):14513–14518 360. Dhar R, Basu A (2008) Constitutive activation of p70 S6 kinase is associated with intrinsic resistance to cisplatin. Int J Oncol 32(5):1133–1137 361. Bartling B, Yang JY, Michod D, Widmann C, Lewensohn R, Zhivotovsky B (2004) RasGTPase-activating protein is a target of caspases in spontaneous apoptosis of lung carcinoma cells and in response to etoposide. Carcinogenesis 25(6):909–921 362. Rodenhuis S, Boerrigter L, Top B et al (1997) Mutational activation of the K-ras oncogene and the effect of chemotherapy in advanced adenocarcinoma of the lung: a prospective study. J Clin Oncol 15(1):285–291 363. Mascaux C, Iannino N, Martin B et  al (2005) The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis. Br J Cancer 92(1):131–139 364. Rosell R, Gonzalez-Larriba JL, Alberola V et  al (1995) Single-agent paclitaxel by 3-hour infusion in the treatment of non-small cell lung cancer: links between p53 and K-ras gene status and chemosensitivity. Semin Oncol 22(6 Suppl 14):12–18 365. Tsao MS, Aviel-Ronen S, Ding K et al (2007) Prognostic and predictive importance of p53 and RAS for adjuvant chemotherapy in non small-cell lung cancer. J Clin Oncol 25(33):5240–5247 366. Schiller JH, Adak S, Feins RH et al (2001) Lack of prognostic significance of p53 and K-ras mutations in primary resected non-small-cell lung cancer on E4592: a Laboratory Ancillary Study on an Eastern Cooperative Oncology Group Prospective Randomized Trial of Postoperative Adjuvant Therapy. J Clin Oncol 19(2):448–457 367. Kim ES, Kies MS, Fossella FV et al (2005) Phase II study of the farnesyltransferase inhibitor lonafarnib with paclitaxel in patients with taxane-refractory/resistant nonsmall cell lung carcinoma. Cancer 104(3):561–569

Lung Cancer Resistance to Chemotherapy

389

368. Ding L, Wang H, Lang W, Xiao L (2002) Protein kinase C-epsilon promotes survival of lung cancer cells by suppressing apoptosis through dysregulation of the mitochondrial caspase pathway. J Biol Chem 277(38):35305–35313 369. Pardo OE, Wellbrock C, Khanzada UK et al (2006) FGF-2 protects small cell lung cancer cells from apoptosis through a complex involving PKCepsilon, B-Raf and S6K2. EMBO J 25(13):3078–3088 370. Basu A, Weixel K, Saijo N (1996) Characterization of the protein kinase C signal transduction pathway in cisplatin-sensitive and -resistant human small cell lung carcinoma cells. Cell Growth Differ 7(11):1507–1512 371. Wang XY, Liu HT (1998) Antisense expression of protein kinase C alpha improved sensitivity to anticancer drugs in human lung cancer LTEPa-2 cells. Zhongguo Yao Li Xue Bao 19(3):265–268 372. Sonnemann J, Gekeler V, Ahlbrecht K et al (2004) Down-regulation of protein kinase Ceta by antisense oligonucleotides sensitises A549 lung cancer cells to vincristine and paclitaxel. Cancer Lett 209(2):177–185 373. Villalona-Calero MA, Ritch P, Figueroa JA et al (2004) A phase I/II study of LY900003, an antisense inhibitor of protein kinase C-alpha, in combination with cisplatin and gemcitabine in patients with advanced non-small cell lung cancer. Clin Cancer Res 10(18 Pt 1):6086–6093 374. Paz-Ares L, Douillard JY, Koralewski P et  al (2006) Phase III study of gemcitabine and cisplatin with or without aprinocarsen, a protein kinase C-alpha antisense oligonucleotide, in patients with advanced-stage non-small-cell lung cancer. J Clin Oncol 24(9):1428–1434 375. Wang Z, Xu J, Zhou JY, Liu Y, Wu GS (2006) Mitogen-activated protein kinase phosphatase-1 is required for cisplatin resistance. Cancer Res 66(17):8870–8877 376. Vicent S, Garayoa M, Lopez-Picazo JM et al (2004) Mitogen-activated protein kinase phosphatase-1 is overexpressed in non-small cell lung cancer and is an independent predictor of outcome in patients. Clin Cancer Res 10(11):3639–3649 377. Chanvorachote P, Nimmannit U, Stehlik C et al (2006) Nitric oxide regulates cell sensitivity to cisplatin-induced apoptosis through S-nitrosylation and inhibition of Bcl-2 ubiquitination. Cancer Res 66(12):6353–6360 378. Zhang Y, Fujita N, Tsuruo T (1999) p21Waf1/Cip1 acts in synergy with bcl-2 to confer multidrug resistance in a camptothecin-selected human lung-cancer cell line. Int J Cancer 83(6):790–797 379. Linardopoulos S (2007) Aurora-A kinase regulates NF-kappaB activity: lessons from combination studies. J BUON 12(Suppl 1):S67–S70 380. Losert D, Pratscher B, Soutschek J et al (2007) Bcl-2 downregulation sensitizes nonsmall cell lung cancer cells to cisplatin, but not to docetaxel. Anticancer Drugs 18(7):755–761 381. Hu Y, Bebb G, Tan S et  al (2004) Antitumor efficacy of oblimersen Bcl-2 antisense oligonucleotide alone and in combination with vinorelbine in xenograft models of human non-small cell lung cancer. Clin Cancer Res 10(22):7662–7670 382. Zangemeister-Wittke U, Schenker T, Luedke GH, Stahel RA (1998) Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etoposide, doxorubicin and cisplatin on small-cell lung cancer cell lines. Br J Cancer 78(8):1035–1042 383. Kumar Biswas S, Huang J, Persaud S, Basu A (2004) Down-regulation of Bcl-2 is associated with cisplatin resistance in human small cell lung cancer H69 cells. Mol Cancer Ther 3(3):327–334 384. Wang L, Chanvorachote P, Toledo D et al (2008) Peroxide is a key mediator of Bcl-2 downregulation and apoptosis induction by cisplatin in human lung cancer cells. Mol Pharmacol 73(1):119–127 385. Mandziuk S, Dudzisz-Sledz M, Korszen-Pilecka I, Milanowski J, Wojcierowski J, Korobowicz E (2003) Expression of p21 and bcl-2 proteins in paraffin-embedded preparations of non-small cell lung cancer in stage IIIA after etoposide and cisplatin induced chemotherapy. Ann Univ Mariae Curie Sklodowska Med 58(1):149–153 386. Krug LM, Miller VA, Filippa DA, Venkatraman E, Ng KK, Kris MG (2003) Bcl-2 and bax expression in advanced non-small cell lung cancer: lack of correlation with chemotherapy response or survival in patients treated with docetaxel plus vinorelbine. Lung Cancer 39(2):139–143

390

D.J. Stewart

387. Martin B, Paesmans M, Berghmans T et al (2003) Role of Bcl-2 as a prognostic factor for survival in lung cancer: a systematic review of the literature with meta-analysis. Br J Cancer 89(1):55–64 388. Rudin CM, Salgia R, Wang X et al (2008) Randomized phase II Study of carboplatin and etoposide with or without the bcl-2 antisense oligonucleotide oblimersen for extensive-stage small-cell lung cancer: CALGB 30103. J Clin Oncol 26(6):870–876 389. Takemura A, Gemma A, Shibuya M et  al (2007) Gemcitabine resistance in a highly metastatic subpopulation of a pulmonary adenocarcinoma cell line resistant to gefitinib. Int J Oncol 31(6):1325–1332 390. Kurdow R, Schniewind B, Zoefelt S et al (2005) Apoptosis by gemcitabine in non-small cell lung cancer cell line KNS62 is induced downstream of caspase 8 and is profoundly blocked by Bcl-xL over-expression. Langenbecks Arch Surg 390(3):243–248 391. Lei X, Huang Z, Zhong M, Zhu B, Tang S, Liao D (2007) Bcl-XL small interfering RNA sensitizes cisplatin-resistant human lung adenocarcinoma cells. Acta Biochim Biophys Sin (Shanghai) 39(5):344–350 392. Shoemaker AR, Oleksijew A, Bauch J et  al (2006) A small-molecule inhibitor of Bcl-XL potentiates the activity of cytotoxic drugs in vitro and in vivo. Cancer Res 66(17):8731–8739 393. Hajji N, Wallenborg K, Vlachos P, Nyman U, Hermanson O, Joseph B (2008) Combinatorial action of the HDAC inhibitor trichostatin A and etoposide induces caspase-mediated AIFdependent apoptotic cell death in non-small cell lung carcinoma cells. Oncogene 27(22):3134–3144 394. Song L, Coppola D, Livingston S, Cress D, Haura EB (2005) Mcl-1 regulates survival and sensitivity to diverse apoptotic stimuli in human non-small cell lung cancer cells. Cancer Biol Ther 4(3):267–276 395. Li J, Viallet J, Haura EB (2008) A small molecule pan-Bcl-2 family inhibitor, GX15-070, induces apoptosis and enhances cisplatin-induced apoptosis in non-small cell lung cancer cells. Cancer Chemother Pharmacol 61(3):525–534 396. Wesarg E, Hoffarth S, Wiewrodt R et al (2007) Targeting BCL-2 family proteins to overcome drug resistance in non-small cell lung cancer. Int J Cancer 121(11):2387–2394 397. Zhang MC, Hu CP, Chen Q (2006) Effect of down-regulation of survivin gene on apoptosis and cisplatin resistance in cisplatin resistant human lung adenocarcinoma A549/CDDP cells. Zhonghua Zhong Liu Za Zhi 28(6):408–412 398. Zhang MC, Hu CP, Chen Q, Xia Y (2006) Experimental study of antisense oligodeoxynucleotide targeting survivin gene for cisplatin resistant human lung adeno-carcinoma xenograft in nude mice. Zhong Nan Da Xue Xue Bao Yi Xue Ban 31(5):717–722 399. Dasgupta P, Kinkade R, Joshi B, Decook C, Haura E, Chellappan S (2006) Nicotine inhibits apoptosis induced by chemotherapeutic drugs by up-regulating XIAP and survivin. Proc Natl Acad Sci U S A 103(16):6332–6337 400. Bandala E, Espinosa M, Maldonado V, Melendez-Zajgla J (2001) Inhibitor of apoptosis-1 (IAP-1) expression and apoptosis in non-small-cell lung cancer cells exposed to gemcitabine. Biochem Pharmacol 62(1):13–19 401. Checinska A, Hoogeland BS, Rodriguez JA, Giaccone G, Kruyt FA (2007) Role of XIAP in inhibiting cisplatin-induced caspase activation in non-small cell lung cancer cells: a small molecule Smac mimic sensitizes for chemotherapy-induced apoptosis by enhancing caspase-3 activation. Exp Cell Res 313(6):1215–1224 402. Bartling B, Lewensohn R, Zhivotovsky B (2004) Endogenously released Smac is insufficient to mediate cell death of human lung carcinoma in response to etoposide. Exp Cell Res 298(1):83–95 403. Ekedahl J, Joseph B, Grigoriev MY et al (2002) Expression of inhibitor of apoptosis proteins in small- and non-small-cell lung carcinoma cells. Exp Cell Res 279(2):277–290 404. Crnkovic-Mertens I, Muley T, Meister M et  al (2006) The anti-apoptotic livin gene is an important determinant for the apoptotic resistance of non-small cell lung cancer cells. Lung Cancer 54(2):135–142

Lung Cancer Resistance to Chemotherapy

391

405. Ohta T, Iijima K, Miyamoto M et  al (2008) Loss of Keap1 function activates Nrf2 and provides advantages for lung cancer cell growth. Cancer Res 68(5):1303–1309 406. Kim HR, Kim S, Kim EJ et al (2008) Suppression of Nrf2-driven heme oxygenase-1 enhances the chemosensitivity of lung cancer A549 cells toward cisplatin. Lung Cancer 60(1):47–56 407. Morero JL, Poleri C, Martin C, Van Kooten M, Chacon R, Rosenberg M (2007) Influence of apoptosis and cell cycle regulator proteins on chemotherapy response and survival in stage IIIA/IIIB NSCLC patients. J Thorac Oncol 2(4):293–298 408. Kim HJ, Hwang JY, Kim HJ et al (2007) Expression of a peroxisome proliferator-activated receptor gamma 1 splice variant that was identified in human lung cancers suppresses cell death induced by cisplatin and oxidative stress. Clin Cancer Res 13(9):2577–2583 409. Kiura K, Watarai S, Ueoka H et  al (1998) An alteration of ganglioside composition in cisplatin-resistant lung cancer cell line. Anticancer Res 18(4C):2957–2960 410. Ikuta K, Takemura K, Kihara M et al (2005) Overexpression of constitutive signal transducer and activator of transcription 3 mRNA in cisplatin-resistant human non-small cell lung cancer cells. Oncol Rep 13(2):217–222 411. Chen JT, Huang CY, Chiang YY et al (2008) HGF increases cisplatin resistance via downregulation of AIF in lung cancer cells. Am J Respir Cell Mol Biol 38(5):559–565 412. Ohashi R, Takahashi F, Cui R et  al (2007) Interaction between CD44 and hyaluronate induces chemoresistance in non-small cell lung cancer cell. Cancer Lett 252(2):225–234 413. Dong A, Kong M, Ma Z, Qian J, Cheng H, Xu X (2008) Knockdown of insulin-like growth factor 1 receptor enhances chemosensitivity to cisplatin in human lung adenocarcinoma A549 cells. Acta Biochim Biophys Sin (Shanghai) 40(6):497–504 414. Warshamana-Greene GS, Litz J, Buchdunger E, Garcia-Echeverria C, Hofmann F, Krystal GW (2005) The insulin-like growth factor-I receptor kinase inhibitor, NVP-ADW742, sensitizes small cell lung cancer cell lines to the effects of chemotherapy. Clin Cancer Res 11(4):1563–1571 415. Mizushima Y, Kashii T, Kobayashi M (1996) Effect of cisplatin exposure on the degree of N-myc amplification in small cell lung carcinoma cell lines with N-myc amplification. Oncology 53(5):417–421 416. Van Waardenburg RC, Meijer C, Burger H et  al (1997) Effects of an inducible anti-sense c-myc gene transfer in a drug-resistant human small-cell-lung-carcinoma cell line. Int J Cancer 73(4):544–550 417. Van Waardenburg RC, Prins J, Meijer C, Uges DR, De Vries EG, Mulder NH (1996) Effects of c-myc oncogene modulation on drug resistance in human small cell lung carcinoma cell lines. Anticancer Res 16(4A):1963–1970 418. Zhang P, Gao WY, Turner S, Ducatman BS (2003) Gleevec (STI-571) inhibits lung cancer cell growth (A549) and potentiates the cisplatin effect in vitro. Mol Cancer 2:1 419. Hu Y, Feng FY, Chen SJ, Gao YN, Xiao T, Liu YN (2006) Correlation between the expression of PCDGF in serum and the chemotherapeutic sensitivity in NSCLC. Zhonghua Zhong Liu Za Zhi 28(8):603–605 420. Liu X, Yue P, Khuri FR, Sun SY (2005) Decoy receptor 2 (DcR2) is a p53 target gene and regulates chemosensitivity. Cancer Res 65(20):9169–9175 421. Spalding AC, Jotte RM, Scheinman RI et al (2002) TRAIL and inhibitors of apoptosis are opposing determinants for NF-kappaB-dependent, genotoxin-induced apoptosis of cancer cells. Oncogene 21(2):260–271 422. Wang Q, Wang T, Wang Y et al (2007) VP-16 resistance in the NCI-H460 human lung cancer cell line is significantly associated with glucose-regulated protein78 (GRP78) induction. Anticancer Res 27(4B):2359–2364 423. July LV, Beraldi E, So A et  al (2004) Nucleotide-based therapies targeting clusterin chemosensitize human lung adenocarcinoma cells both in  vitro and in  vivo. Mol Cancer Ther 3(3):223–232 424. Han EK, Tahir SK, Cherian SP, Collins N, Ng SC (2000) Modulation of paclitaxel resistance by annexin IV in human cancer cell lines. Br J Cancer 83(1):83–88

392

D.J. Stewart

425. Demarcq C, Bastian G, Remvikos Y (1992) BrdUrd/DNA flow cytometry analysis demonstrates cis-diamminedichloroplatinum (II)-induced multiple cell-cycle modifications on human lung carcinoma cells. Cytometry 13(4):416–422 426. Holdaway KM, Finlay GJ, Baguley BC (1992) Relationship of cell cycle parameters to in vitro and in vivo chemosensitivity for a series of Lewis lung carcinoma lines. Eur J Cancer 28A(8–9):1427–1431 427. Prewitt TW, Matthews W, Chaudhri G, Pogrebniak HW, Pass HI (1994) Tumor necrosis factor induces doxorubicin resistance to lung cancer cells in  vitro. J Thorac Cardiovasc Surg 107(1):43–49 428. Wahl AF, Donaldson KL, Mixan BJ, Trail PA, Siegall CB (2001) Selective tumor sensitization to taxanes with the mAb–drug conjugate cBR96-doxorubicin. Int J Cancer 93(4):590–600 429. Chen JG, Yang CP, Cammer M, Horwitz SB (2003) Gene expression and mitotic exit induced by microtubule-stabilizing drugs. Cancer Res 63(22):7891–7899 430. Chen JG, Horwitz SB (2002) Differential mitotic responses to microtubule-stabilizing and -destabilizing drugs. Cancer Res 62(7):1935–1938 431. Reed MF, Zagorski WA, Knudsen ES (2007) RB activity alters checkpoint response and chemosensitivity in lung cancer lines. J Surg Res 142(2):364–372 432. Shimizu E, Coxon A, Otterson GA et  al (1994) RB protein status and clinical correlation from 171 cell lines representing lung cancer, extrapulmonary small cell carcinoma, and mesothelioma. Oncogene 9(9):2441–2448 433. Ishii T, Matsuse T, Masuda M, Teramoto S (2004) The effects of S-phase kinase-associated protein 2 (SKP2) on cell cycle status, viability, and chemoresistance in A549 lung adenocarcinoma cells. Exp Lung Res 30(8):687–703 434. Filipits M, Pirker R, Dunant A et  al (2007) Cell cycle regulators and outcome of adjuvant cisplatin-based chemotherapy in completely resected non-small-cell lung cancer: the International Adjuvant Lung Cancer Trial Biologic Program. J Clin Oncol 25(19):2735–2740 435. Oshita F, Kameda Y, Nishio K et al (2000) Increased expression levels of cyclin-dependent kinase inhibitor p27 correlate with good responses to platinum-based chemotherapy in nonsmall cell lung cancer. Oncol Rep 7(3):491–495 436. Ma Y, Freeman SN, Cress WD (2004) E2F4 deficiency promotes drug-induced apoptosis. Cancer Biol Ther 3(12):1262–1269 437. Zhang P, Wang J, Gao W, Yuan BZ, Rogers J, Reed E (2004) CHK2 kinase expression is down-regulated due to promoter methylation in non-small cell lung cancer. Mol Cancer 3:14 438. Masuda A, Maeno K, Nakagawa T, Saito H, Takahashi T (2003) Association between mitotic spindle checkpoint impairment and susceptibility to the induction of apoptosis by anti-microtubule agents in human lung cancers. Am J Pathol 163(3):1109–1116 439. Fan T, Li R, Todd NW et al (2007) Up-regulation of 14-3-3zeta in lung cancer and its implication as prognostic and therapeutic target. Cancer Res 67(16):7901–7906 440. Ramirez JL, Rosell R, Taron M et al (2005) 14-3-3sigma methylation in pretreatment serum circulating DNA of cisplatin-plus-gemcitabine-treated advanced non-small-cell lung cancer patients predicts survival: The Spanish Lung Cancer Group. J Clin Oncol 23(36):9105–9112 441. Roberson RS, Kussick SJ, Vallieres E, Chen SY, Wu DY (2005) Escape from therapyinduced accelerated cellular senescence in p53-null lung cancer cells and in human lung cancers. Cancer Res 65(7):2795–2803 442. Gautschi O, Hugli B, Ziegler A et  al (2006) Cyclin D1 (CCND1) A870G gene polymorphism modulates smoking-induced lung cancer risk and response to platinum-based chemotherapy in non-small cell lung cancer (NSCLC) patients. Lung Cancer 51(3):303–311 443. Saijo T, Ishii G, Ochiai A et al (2006) Eg5 expression is closely correlated with the response of advanced non-small cell lung cancer to antimitotic agents combined with platinum chemotherapy. Lung Cancer 54(2):217–225 444. Li Y, Ahmed F, Ali S, Philip PA, Kucuk O, Sarkar FH (2005) Inactivation of nuclear factor kappaB by soy isoflavone genistein contributes to increased apoptosis induced by chemotherapeutic agents in human cancer cells. Cancer Res 65(15):6934–6942

Lung Cancer Resistance to Chemotherapy

393

445. Denlinger CE, Rundall BK, Keller MD, Jones DR (2004) Proteasome inhibition sensitizes non-small-cell lung cancer to gemcitabine-induced apoptosis. Ann Thorac Surg 78(4):1207– 1214 discussion 1214 446. Ni J, Takayama K, Ushijima R et  al (2008) Adenovirus-mediated inhibitor kappaB gene transfer improves the chemosensitivity to anticancer drugs in human lung cancer in vitro and in vivo. Anticancer Res 28(2A):601–608 447. Oyaizu H, Adachi Y, Okumura T et al (2001) Proteasome inhibitor 1 enhances paclitaxel-induced apoptosis in human lung adenocarcinoma cell line. Oncol Rep 8(4):825–829 448. Oguri T, Katoh O, Takahashi T et  al (1998) The Kruppel-type zinc finger family gene, HKR1, is induced in lung cancer by exposure to platinum drugs. Gene 222(1):61–67 449. Zhuo WL, Wang Y, Zhuo XL, Zhang YS, Chen ZT (2008) Short interfering RNA directed against TWIST, a novel zinc finger transcription factor, increases A549 cell sensitivity to cisplatin via MAPK/mitochondrial pathway. Biochem Biophys Res Commun 369(4):1098–1102 450. Zhuo W, Wang Y, Zhuo X, Zhang Y, Ao X, Chen Z (2008) Knockdown of Snail, a novel zinc finger transcription factor, via RNA interference increases A549 cell sensitivity to cisplatin via JNK/mitochondrial pathway. Lung Cancer 62(1):8–14 451. Berghmans T, Paesmans M, Mascaux C et al (2006) Thyroid transcription factor 1 – a new prognostic factor in lung cancer: a meta-analysis. Ann Oncol 17(11):1673–1676 452. Igarashi T, Izumi H, Uchiumi T et al (2007) Clock and ATF4 transcription system regulates drug resistance in human cancer cell lines. Oncogene 26(33):4749–4760 453. Tanabe M, Izumi H, Ise T et al (2003) Activating transcription factor 4 increases the cisplatin resistance of human cancer cell lines. Cancer Res 63(24):8592–8595 454. Miyamoto N, Izumi H, Noguchi T et al (2008) Tip60 is regulated by circadian transcription factor clock and is involved in cisplatin resistance. J Biol Chem 283(26):18218–18226

Small Cell Carcinoma of the Lung Emer O. Hanrahan and Bonnie Glisson

Abstract  Small cell lung cancer (SCLC) is a malignant epithelial tumor that occurs almost exclusively in smokers. It accounts for approximately 13% of lung cancer diagnoses in the United States annually. For treatment planning, SCLC is divided into either limited stage disease (LS-SCLC– that is, disease is confined to the chest and can be encompassed within a tolerable radiation field)– or extensive stage disease (ES-SCLC). Approximately 60–70% of patients with SCLC have extensive stage disease (ES-SCLC) at diagnosis. With treatment, the median survival times are about 18–30 months in LS-SCLC and 8–12 months in ES-SCLC. ES-SCLC is treated with systemic chemotherapy with palliative intent. LS-SCLC is treated with concurrent chemotherapy and thoracic radiation therapy (TRT) with curative intent. The standard-of-care chemotherapy regimen is an etoposide–platinum doublet, largely due to a more favorable toxicity profile than older alkylating-agent and anthracycline-based regimens, particularly when administered with TRT. Cisplatin is the platinum of choice in LS-SCLC as it may be more efficacious. In ES-SCLC, carboplatin is often used because of its greater tolerability and ease of administration. Although about 80% of patients respond to initial chemotherapy or chemoradiation, most relapse and die of their disease within 2 years. Many attempts to improve on the outcomes with conventional chemotherapy over the past 30 years have been unsuccessful, including dose dense, dose intense and maintenance chemotherapy, the addition of other cytotoxins to the etoposide–platinum base, and the replacement of etoposide with other cytotoxins in a platinum-doublet. The advances with the greatest impact on patient outcome in the first-line treatment of SCLC over the past decade have been in the field of radiation oncology. Considerable clinical evidence has been accumulated through phase III trials and meta-analyses that the earlier initiation of TRT in combination with cisplatin-based chemotherapy, particularly during cycle 1, improves survival in LS-SCLC. Although clinical trials continue to evaluate the optimal dose and schedule of concurrent TRT in

E.O. Hanrahan (*) and B. Glisson MD Anderson Cancer Center, Unit 432, 1515 Holcombe Blvd, Houston, TX 77030, USA e-mail: [email protected] D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_16, © Humana Press, a part of Springer Science+Business Media, LLC 2010

395

396

E.O. Hanrahan and B. Glisson

LS-SCLC, the current gold standard is accelerated hyperfractionated TRT, with 45  Gy administered twice daily (1.5  Gy per fraction) over a period of 3 weeks. While the administration of prophylactic cranial irradiation (PCI) to patients with LS-SCLC who responded to first-line treatment has been long-accepted, it has only recently been shown that PCI also improves survival in patients with ES-SCLC who have responded to chemotherapy. Patients with relapsed SCLC generally have a very poor prognosis. Patients who respond to first-line chemotherapy and have a treatment free interval of at least 3 months before progression are considered to have “sensitive disease,” and they are most likely to benefit from second-line chemotherapy. There is no accepted standard regimen for relapsed SCLC. Commonly employed approaches include reinduction with the regimen that was used first-line, the use of a “non-cross resistant” multi-drug regimen (such as CAV: cyclophosphamide, doxorubicin, and vincristine), or singleagent topotecan. Topotecan is the only drug with an FDA indication for treatment of relapsed SCLC. Amrubicin and picoplatin are new cytotoxic agents in phase III clinical trials for SCLC. Although the data from phase II studies of these agents are encouraging, significantly improving the survival rate of patients with SCLC will require the development of more novel therapeutic agents based on our increasing understanding of the molecular biology of SCLC. The most promising therapeutic targets at present include regulators of apoptosis (such as BCL-2) and angiogenesis (such as VEGF and its receptors), and the c-Met/HGF, IGF-1/IGF-1R, and PI3K/Akt/mTOR pathways. Patient participation in clinical trials of agents targeting these pathways will be crucial. Keywords  Small cell lung cancer • Etoposide • Cisplatin • Irinotecan • Topotecan • Thoracic radiation therapy • Prophylactic cranial irradiation

Introduction Small cell lung cancer (SCLC) is a malignant epithelial tumor that occurs almost exclusively in smokers. In comparison with non-small cell lung cancer, SCLC generally has a more rapid doubling time, a higher growth fraction, and a greater propensity for the early development of widespread metastases. Although SCLC is usually highly sensitive to chemotherapy and radiotherapy at initial treatment, most patients ultimately die from recurrent disease.

Epidemiology and Etiology It is projected that the number of new lung cancer cases and lung cancer deaths in the United States in 2009 will be 219,440 and 159,390, respectively (1). SCLC is expected to account for approximately 13% of these cases. The incidence of SCLC

Small Cell Carcinoma of the Lung

397

in the U.S. is declining, as illustrated by a report that SCLC constituted 17% of lung cancer diagnoses in 1986 (2). These epidemiologic changes may be due, at least in part, to a decrease in smoking rates, increased use of filtered cigarettes, and to changes in the pathologic criteria for SCLC, such that some cases are now classified as large cell neuroendocrine carcinoma (3). SCLC occurs almost exclusively in patients with a history of tobacco exposure, and SCLC is the lung cancer that has the strongest association with tobacco smoking (4).

Pathology Classification of SCLC Malignant lung tumors are classified according to the 1999 WHO/IASLC classification of lung and pleural tumors (5). This recognizes small cell carcinoma (SCLC) and only one variant: combined small cell carcinoma, where at least 10% of the tumor bulk consists of an associated non-small cell component (6). A new variant of large cell carcinoma, large cell neuroendocrine carcinoma (LCNEC), is described in the WHO/IASLC classification system as a subtype of non-small cell carcinoma. Nevertheless, the biologic and clinical behavior of SCLC and LCNEC are very similar, and these tumor types are generally managed in a similar fashion.

Histology A diagnosis of SCLC is made primarily on light microscopy. SCLC is characterized by small “blue” round, oval, or spindle-shaped cells with scant cytoplasm, illdefined borders, finely granular nuclear chromatin, and absent or inconspicuous nucleoli (5, 7). Nuclear molding is considered characteristic in well-preserved specimens, but “crush” artifacts due to the soft texture of the tumor and mechanical alterations are more frequently seen (7). The mitotic rate is high, with necrosis of tumor cells commonly seen. The neoplastic cells are typically arranged in clusters, sheets, or trabeculae, separated by a delicate fibrovascular stroma. Histomorphological grading is not applicable. Complementary to light microscopy, immunohistochemical documentation of epithelial and neuroendocrine origin are routine (7). Expression of keratin and one or more neuroendocrine markers, such as chromogranin, synaptophysin, and CD56, are usually assessed. Demonstration of neuroendocrine markers can help with the differential diagnosis of other small blue round cell tumors, such as lymphomas, small cell sarcomas, and poorly differentiated non-small-cell lung cancer and lymphoma. However, it must be noted that expression of neuroendocrine markers has been reported in up to 10% of NSCLC.

398

E.O. Hanrahan and B. Glisson

Molecular Pathology Over the past 20 years, substantial knowledge of the molecular abnormalities involved in the pathogenesis of SCLC has been acquired. Molecular abnormalities implicated in the pathogenesis of SCLC include chromosomal aberrations, loss of tumor suppressor genes, expression of proto-oncogenes, dysregulation of autocrine growth loops, and constitutive activation of cell signaling pathways. This growing knowledge base is being exploited for the development of new therapies for SCLC, and is described further in the section on “Molecular Biology and Novel Targeted Therapies.”

Clinical Presentation SCLC typically arises in the central airways, infiltrates the submucosa, and gradually obstructs the bronchial lumen through extrinsic or endobronchial spread. Patients commonly present with symptoms of dyspnea and persistent cough, and chest imaging studies will usually show a large hilar mass and bulky mediastinal adenopathy. This disease distribution may lead to postobstructive pneumonia at presentation. Hemoptysis can occur, but is relatively uncommon due to the submucosal growth pattern of the tumor. Patients may also present with superior vena cava syndrome, with SCLC being the most common malignant cause of superior vena caval obstruction. Approximately 60–70% of patients have metastatic disease at presentation. Bone, liver, central nervous system, adrenal glands, and pleura are the common sites of metastases. Therefore, symptoms of metastatic disease may include bone or right upper quadrant abdominal pain, headache, seizures, fatigue, anorexia, and weight loss. Occasionally, patients with SCLC present with a paraneoplastic syndrome.

Paraneoplastic Syndromes The paraneoplastic syndromes most commonly associated with SCLC can be broadly classified as endocrine or neurologic, and these are outlined in Table  1. Generally, the endocrine syndromes related to peptide production by the tumor usually abate with effective treatment of cancer, but the neurologic syndromes, which are autoimmune in basis, are usually progressive, and their course is generally independent of the outcome of cancer therapy. A number of dermatologic paraneoplastic syndromes have also been reported to occur in patients with SCLC, including dermatomyositis, acquired tylosis, trip palms, and erythema gyratum repens. Of note, hypercalcemia, which is often seen in patients with NSCLC, is very uncommon in SCLC.

Cerebellar degeneration

Neurologic Lambert–Eaton myasthenic syndrome (3%)

Cushing syndrome (3–7% (15, 188))

SIADH (10–15% (187))

Symptoms/signs: weakness of proximal muscles of lower and upper extremities, with relative sparing of respiratory and bulbar muscles. In contrast to patients with myasthenia gravis, motor strength initially improves after exercise (postexercise facilitation), but weakens if activity is sustained. Symptoms/signs: loss of coordination, truncal and limb ataxia, dysarthria, and nystagmus

Biochemical: hyponatremia, serum hypoosmolality, urine osmolality >100 mosmol/kg, urine sodium concentration >40 meq/LSymptoms/ signs: anorexia, nausea, vomiting, cerebral edema with agitation, personality changes, confusion, coma, seizures, and respiratory arrest Biochemical: hypokalemic alkalosis and hyperglycemiaSymptoms/signs: muscle weakness, weight loss, hypertension, hirsutism

Table 1  Paraneoplastic syndromes associated with small cell lung cancer Syndrome (frequencya) Features Endocrine

Anti-Hu, anti-CV2/CRMP5, anti-Zic4

Autoantibodiesb P/Q type voltage-gated calcium channel antibodies

Ectopic ACTH

Mechanism Ectopic hormone production by tumor cells Ectopic ADH

(continued)

Often remits or improves with anticancer treatment. Symptomatic therapies include guanidine, 3,4-diaminopyridine, acetylcholinesterase inhibitors, plasma exchange, intravenous immunoglobulin (IVIG), immunosuppressives. May improve or stabilize with early anticancer treatment

Patients with SCLC and Cushing’s syndrome appear to have a worse prognosis than those without Cushing’s syndrome (189, 190). Opportunistic infections may contribute to this worse outcome.

Usually resolves within a few weeks of starting chemotherapy

Treatment/prognosis

Small Cell Carcinoma of the Lung 399

Features

Symptoms/signs depend on part of CNS that is involved Symptoms/signs: acute/subacute behavioral/ mood changes, short-term memory impairment, seizures, hypothalamic dysfunction Symptoms/signs: hypothermia, hypoventilation, sleep apnea, intestinal pseudo-obstruction, cardiac arrhythmias, sudden death Parasthesia, pain, sensory ataxia and pseudoathetoid movement Anti-Hu, anti-CV2

Usually does not improve with anticancer treatment or immune therapy Usually does not improve with anticancer treatment or immune therapy

Usually does not improve with anticancer treatment or immune therapy

Anti-Hu, anti-CV2

Treatment/prognosis May improve or stabilize with early anticancer treatment (191) May improve with anti-cancer treatment (most likely with anti-Hu negative cases) (192)

Mechanism Anti-Hu, anti-amphiphysin, anti-CV2 Anti-Hu, anti-CV2, antiamphiphysin

Retinopathy

Anti-recoverin Symptoms/signs: photosensitivity, abnormal visual acuity, color vision abnormalities, scotomata, night blindness, attenuated retinal arteriole caliber Voltage-gated potassium May improve with plasma exchange Myotonia/Isaac’s syndrome Symptoms/signs: muscle stiffness caused channels antibodies or IVIG by continuous muscle fiber activity, undulating muscle twitching during rest (myokymia), cramps, pseudomyotonia (delayed relaxation), increased sweating, motor weaknessBiochemical: serum creatinine kinase concentration may be elevated aPercentage of SCLC patients affected b Cross-reactivity between tumor-associated antigens and antigens normally expressed exclusively by the nervous system

Subacute sensory neuropathy

Autonomic neuropathy

Limbic encephalitis

Encephalomyelitis

Table 1  (continued) Syndrome (frequencya)

400 E.O. Hanrahan and B. Glisson

Small Cell Carcinoma of the Lung

401

Staging When the TNM (tumor, nodes, metastases) system was first developed for NSCLC in the 1960s, it was not prognostic when applied to a population of patients with SCLC. This was most likely explained by the very low incidence of stage I or II SCLC and the fact that without chemotherapy, all patients with SCLC had very short survival. In a placebo-controlled trial of intravenous cyclophosphamide, the Veterans Administration Lung Study Group (VALG) developed a two-stage system for SCLC (8). They separated patients into limited or extensive disease groups, based on whether or not their disease could be encompassed by a radiation port. This classification was prognostic in patients on both arms of the trial, with median survival rates considerably longer in patients with limited disease as compared with those with extensive disease. This trial also established a role for chemotherapy in the management of SCLC. The median survival times of extensive and limited disease patients treated with placebo were 6 and 12 weeks, respectively. The median survival times for cyclophosphamide-treated extensive and limited disease patients were 17 and 24 weeks, respectively (9). A modified version of the VALG classification system for SCLC remains in use today, and is described in Table 2. The “limited-stage disease” classification has been refined to identify those who are candidates for curative-intent chemoradiation. The current International Union Against Cancer (UICC)/American Joint Committee on Cancer (AJCC) TNM system (sixth edition) is presently only applied in evaluating resectability of the unusual patient with SCLC who presents with a T1-2 primary and appears clinically node-negative (approximately 5% of SCLC). However, there is re-emerging interest in using the TNM system for SCLC. The International Association for the Study of Lung Cancer has proposed a new TNM stage and grouping system for lung cancer. Among the proposed changes, pleural or pericardial effusion would be designated M1 rather than T4 disease and metastatic nodules in a different lobe of the ipsilateral lung would be T4 rather than M1

Table 2  Staging of small cell lung cancer Stage Extent of disease Limited-stage Disease that is confined disease to one hemithorax and that can be encompassed within a tolerable radiation port. Includes patients with mediastinal and ipsilateral lymph node involvement. Extensive-stage Disease extending disease beyond the ipsilateral hemithorax

Comments Excludes patients with malignant pleural and pericardial effusions. Excludes patients with contralateral supraclavicular or contralateral hilar nodes (due to size of radiation port). Includes malignant pleural and pericardial effusions, as well as hematogenous spread

Treatment goal Multimodality with curative intent

Palliative therapy

402

E.O. Hanrahan and B. Glisson

disease. In a study of approximately 10,000 patients identified between 1991 and 2005 in the California Cancer registry, the IASLC-proposed TNM staging was shown to better differentiate stage-specific survival in SCLC than the current UICC/AJCC staging system (10). Approximately 60–70% of patients with SCLC have extensive stage disease (ES-SCLC) at diagnosis. The process of staging of SCLC is key to determining both prognosis and therapy. A complete history and physical examination, as well as routine blood counts and chemistries, often can give clues to the extent of disease. Standard chest radiography with posterior–anterior and lateral views can assess the primary tumor and concurrent parenchymal disease. Mediastinal lymph node involvement often can be suggested by mediastinal widening. Computed tomography (CT) scan of the thorax is essential to further evaluate the primary tumor and local regional disease, and is critical to planning the ports for administration of radiation therapy in patients with limited stage disease (LS-SCLC). At this point in the staging evaluation, patients who do not have findings consistent with ES-SCLC should have further investigations to confirm disease stage. Patients who have conclusive evidence of ES-SCLC, do not require further thorough staging for all potential sites of spread. The common sites for metastatic disease and the respective studies to evaluate these sites are shown in Table 3. PET imaging, and more so the integrated PET-CT, may provide a combination of anatomical and functional assessment of the primary tumor and suspected areas of metastasis. Although PET has been widely evaluated in the staging of NSCLC, Table 3  Common metastatic sites in SCLC Common metastatic Frequency at sites in SCLC presentation (193–198) Bone 19–38%

Bone marrow

17–23% But isolated bone marrow metastases in < 5%

Brain

At least 10%, and most are symptomatic (199)

Liver

17–34%

Adrenal glands Extrathoracic lymph nodes Soft tissue

31% 7–25% 3–11%

Staging studies Radionuclide bone scan. If bone scan suggests metastatic disease, further imaging recommended to confirm, e.g., site-specific bone radiographs Routine bone marrow aspiration and biopsy are no longer part of the staging work-up for SCLC. Currently, bone marrow examination is indicated if a patient has unexplained cytopenias. All patients should have brain imaging at diagnosis. MRI brain is more sensitive than CT brain (200). Positive findings are an indication for brain radiation. CT abdomen. Liver function tests and liver enzymes are neither sensitive nor specific as a screening tool. CT Abdomen Physical examination, CT thorax and abdomen Physical examination, CT thorax and abdomen

Small Cell Carcinoma of the Lung

403

somewhat limited data are available on its role in patients with SCLC. There is some evidence that PET and PET-CT can influence the staging of SCLC (11, 12). In one study with 120 patients with SCLC, 8% of patients were upstaged and 2.3% were downstaged (12). The potential for PET imaging to guide radiation plans and minimize normal tissue exposure in LS-SCLC is also being evaluated (13). However, PET/PET-CT are not currently considered routine for the staging of SCLC or for radiation planning.

Natural History and Prognosis Without treatment, SCLC has a very aggressive clinical course, with median survival from diagnosis of only 2–4 months (9). SCLC is very responsive to chemotherapy, with response rates to first-line, combination chemotherapy in the order of 60–70%, and median survival times of about 18–30 months in LS-SCLC and 8–12 months in ES-SCLC (14). Unfortunately, the majority of patients relapse and die of their disease within 2 years. The 5-year survival rates are approximately 10–15% for patients with LS-SCLC and 1–2% for patients with ES-SCLC based on SEER data in the U.S. Many studies of prognostic factors in SCLC have been conducted. Poor performance status and extensive disease are almost uniformly found to be the most important adverse clinical prognosticators (15). Poor performance status also increases the risk of early treatment-related death during initial chemotherapy. Several retrospective analyses have found different sets of hematologic/biochemical laboratory results and serum markers to be of prognostic value. Elevated LDH seems to be the most consistent adverse laboratory prognostic factor, and may be most prognostic in patients with LS-SCLC (15).

Initial Management of SCLC Treatment for ES-SCLC consists primarily of systemic chemotherapy. Initial treatment for LS-SCLC usually consists of systemic chemotherapy and early integration of concurrent thoracic radiation. Prophylactic cranial irradiation is considered after completion of chemotherapy in patients with LS- or ES-SCLC that has responded to chemotherapy.

First-Line Chemotherapy for Extensive Stage SCLC The combination of etoposide and cisplatin (EP) has become over the past 25 years the standard of care for initial therapy of SCLC. This regimen is usually administered for 4–6 cycles. In patients with ES-SCLC, the EP regimen achieves response

404

E.O. Hanrahan and B. Glisson

rates in the range of 60–80%, but median survival times are approximately 8–12 months, and 5-year survival rates are less than 5%. Unfortunately, there has been little progress in improving upon these survival outcomes. In an overview of ES-SCLC outcomes in 21 North American phase III clinical trials and in the SEER database between 1972 and 1990, median survival times for patients diagnosed between 1972 and 1981 were compared with those diagnosed between 1982 and 1990, and an approximate 2-month improvement was revealed (increased from 7 to 9 months, approximately) (16). Much of this modest improvement in survival has been attributed to better supportive care measures, rather than anti-cancer therapeutic advances. The first compelling evidence for a role for chemotherapy in the management of SCLC came from the VALG study of cyclophosphamide reported in 1969, as already described above (9). Cytotoxins in a variety of other drug classes have been shown to have single agent activity of 30% or greater in chemotherapy-naïve SCLC, including anthracyclines, camptothecin derivatives, epipodophyllotoxins, platinum compounds, vinca alkaloids, and taxanes (17). Combination chemotherapy regimens for SCLC were shown to be superior to single agents in terms of response rates and survival in a number of clinical trials in the 1970s (17–21). During that time, cyclophosphamide-based regimens, such as the combination of cyclophosphamide, doxorubicin, and vincristine (CAV), emerged as the standard treatment for SCLC (22). Interest in the combination of etoposide and cisplatin (EP) emerged after it was shown by Evans and colleagues in 1978 to be a tolerable and effective (response rate 55%) combination chemotherapy regimen for SCLC resistant to CAV (23). In 1985, the same group also reported a response rate of 86% with EP as a first-line therapy for SCLC, and median survival times for responding patients compared favorably with those reported with CAV (24). A number of studies subsequently compared EP with CAV, and although most failed to demonstrate superiority for EP, it emerged as the standard of care because of its relatively favorable toxicity profile, its consistent performance in clinical trials and, most relevant to the combined-modality treatment of LS-SCLC, its tolerability with radiotherapy. EP is usually administered for 4–6 cycles. In their overview of data from 21 North American, phase III trials for patients with ES-SCLC registered between 1972 and 1990, Chute et al. reported that the median survival of patients treated with cisplatin-based regimens was greater than those treated with non-cisplatin-based regimens (9.5 months vs. 7.1 months) (16). Similarly, a meta-analysis of data from 19 trials published between 1981 and 1999 reported significantly greater response rates and survival with cisplatincontaining regimens, and no increase in the risk of treatment-related deaths (25). A recent randomized trial in Europe compared 5 cycles of EP vs. cyclophosphamide, epirubicin, and vincristine among 436 patients, and found that EP was superior in terms of survival (10.2 months vs. 7.8 months), although this survival benefit was only significant in patients with LS-SCLC on subset analysis (LS-SCLC, 14.5 months vs. 9.7 months, P = 0.001; ES-SCLC, 8.4 months vs. 6.5 months, P > 0.05) (26).

Small Cell Carcinoma of the Lung

405

The administration of cisplatin requires fluid hydration, which is time-consuming and can be problematic in patients with subnormal cardiac function. In addition, cisplatin is more nephrotoxic, neurotoxic, and ototoxic than carboplatin (Cbp). Cisplatin is generally considered to be a more active anti-cancer agent in solid tumors, but comparative data in SCLC are sparse. A randomized, phase III trial involving 147 patients with LS-SCLC or ES-SCLS directly compared 6 cycles of EP with the same number of cycles of ECbp and found no difference in response rate or survival between the two regimens, but this trial was not powered to prove noninferiority of ECbp to EP (27). ECb was associated with less toxicity than EP. In a German multicenter study, reported in abstract form only, Wolf et  al. randomized 350 patients to receive doxorubicin, ifosfamide, and vincristine alternating with either EP or ECbp (28). The median survival with EP was higher than with ECbp (14 months vs. 12 months), but no difference was seen in patients with ES-SCLC on subgroup analysis. Thus, while EP remains the gold standard regimen in SCLC, carboplatin is widely used in lieu of cisplatin in patients with ES-SCLC. Many strategies have been investigated in an effort to improve on EP and, with few exceptions, these have not been successful in prolonging survival. The strategies which have been investigated are reviewed below, and include alternating or sequential “non-cross-resistant” regimens, addition of other cytotoxins to the EP base, the substitution of etoposide for another cytotoxin in the platinum doublet, dose-dense or dose-escalated therapy with growth factor support, high-dose therapy with marrow or stem cell rescue, and maintenance or prolonged chemotherapy.

Alternating or Sequential Regimens Alternating or sequential chemotherapy regimens have been tested in a series of trials in SCLC in the 1980s. These studies used varying combinations and sequences of etoposide, cisplatin, cyclophosphamide, doxorubicin, and vincristine, but there was no consistent evidence of a survival benefit. The rationale for this approach arose from the hypothesis of Goldie and Coldman, who provided a mathematical model of the spontaneous development of drug-resistant clones at a mutation rate proportional to the number of actively dividing tumor cells. This model implies that multiple active agents should be given at full doses to maximize the chance of eradication of the entire tumor cell population. Because patients who were resistant to CAV responded to EP, it was thought that these regimens were at least partly non-cross resistant and that exposure to all of these drugs would improve patient outcome. In a trial by the National Cancer Institute of Canada Clinical Trials Group (NCIC), the alternating schedule of CAV/EP for 6 cycles prolonged the overall survival in patients with ES-SCLC compared with 6 cycles of CAV (9.6 months vs. 8.0 months, P = 0.03) (29). However, the survival difference did not reach statistical significance when patients with LS-SCLC were excluded from the analysis. Furthermore, because EP alone was not tested in this trial, it was unclear whether

406

E.O. Hanrahan and B. Glisson

the benefit of CAV/EP over CAV was truly due to the alternating schedule rather than a superior drug regimen. Two subsequent three-arm, randomized studies attempted to address this issue by comparing the three regimens of CAV, EP and alternating CAV/EP. The trial by Fukuoka et al. involved 288 patients with SCLC in Japan between 1985 and 1988 (30). The response rates for EP (78%) and CAV/ EP (76%) were significantly greater than with CAV (55%), and the median survival time with CAV/EP (11.8 months) was superior to that with CAV (9.9 months) or with EP (9.9 months). However, the difference was only significant for the comparison with CAV, and was only seen in LS-SCLC patients on subgroup analysis. The Southeastern Cancer Study Group conducted a similar study comparing 4 cycles of EP, 6 cycles of CAV, and 6 cycles of alternating CAV/EP among patients with ES-SCLC only, and found the regimens to be equivalent in terms of response rate (61, 51, 59%, respectively), and median survival (8.6, 8.3, 8.1 months, respectively) (31). It was this trial that established 4–6 cycles of chemotherapy as the standard duration of first-line therapy for SCLC. Of note, two further trials confined to patients with LS-SCLC have also failed to show a survival benefit for alternating schedules: A NCIC study that compared alternating CAV and EP for 6 cycles vs. 3 cycles of CAV followed by 3 cycles of EP, and a Southwest Oncology Group (SWOG) trial that compared alternating CAV/EP with a regimen of concurrent etoposide, vincristine, doxorubicin, and cyclophosphamide (32, 33).

Etoposide–Platinum-Based Triplet Regimens The addition of other cytotoxic agents to the backbone of EP has generally yielded greater toxicity and no consistent improvement in survival. Although the addition of ifosfamide to EP (VIP) improved survival relative to EP (median 9.0 months vs. 7.3 months, P = 0.045) in a phase III trial involving 171 patients with ES-SCLC, VIP did not supersede EP as the regimen of choice for ES-SCLC in the USA (34). The reasons for this include the modest extent of the absolute survival benefit, the greater toxicities, logistical difficulties associated with ifosfamide administration, and the non-compatibility of this regimen with radiation therapy in LS-SCLC (35). The addition of paclitaxel to the EP base for SCLC has also been studied in two randomized, phase III studies. However, the addition of paclitaxel to EP did not improve survival and significantly increased treatment-related toxicities (36, 37). Another randomized phase III trial compared carboplatin, etoposide, and vincristine (the control arm) vs. carboplatin, etoposide, and paclitaxel (TEC) among 614 patients with both LS- or ES-SCLC (38). Patients with LS-SCLC were treated with sequential thoracic radiotherapy upon completion of chemotherapy. Median survival for patients in the TEC arm was superior to that in the control arm (12.7 months vs. 11.7 months), but on subgroup analysis, the survival advantage appeared to be confined to LS-SCLC. Excessive toxicities with the use of paclitaxel in this trial were not reported, most likely because carboplatin was substituted for cisplatin.

Small Cell Carcinoma of the Lung

407

However, the sequential use of chemotherapy and radiation for LS-SCLC in this trial is now considered sub-optimal, and the modest survival benefit with TEC may have simply been compensation for the sub-optimal local-regional therapy.

Other Platinum Doublets A number of recent studies have attempted to improve on the outcomes with EP by substituting the etoposide in the platinum-doublet for an alternative, active agent. Encouraging results were initially seen in a Japanese trial using the topoisomerase-1 inhibitor, irinotecan, in lieu of etoposide (IP), but that promise was not upheld in two subsequent North American studies (Table 4) (39–42). It has been hypothesized that population-related differences in pharmacogenomics may explain the divergent results of JCOG 9511 and the two North American studies. Interestingly, comparison of pharmacogenomic analysis in JCOG 9511 and SWOG 0124 patients has been presented in abstract form, and these data suggest that polymorphisms in genes involved in irinotecan metabolism may explain toxicity differences between the Japanese and American patients (43). In a recent European study, the addition of irinotecan to carboplatin produced superior response rates and survival times relative to ECbp, but there are a number of concerns about the design of this study. The outcome of patients in this study on either arm are less than expected for ES-SCLC nowadays, and are inferior to both the IP and EP arms in any of the aforementioned three studies. These poor outcomes may at least be partly attributable to the mandatory dose reductions upfront in patients greater than 70 years of age. Furthermore, there were more elderly patients in the control arm. Therefore, etoposide–platinum doublets remain the gold standard. The substitution of etoposide for topotecan, another topoisomerase-I inhibitor, in a doublet with cisplatin also did not improve survival relative to EP in a phase III study (44).

Increased Dose Intensity or Density Having been recognized as one of the most chemosensitive common solid tumors of adulthood, there was rationale to determine if increased dose density or intensity would improve survival in SCLC. Although improved survival was demonstrated for dose-dense therapy in four randomized studies in SCLC reported between 1995 and 2000, the benefit is modest (45–48). The only study with a sizeable absolute survival benefit was conducted in Japan, used a non-platinum containing regimen, and had a small sample size of only 63 patients (47); thus, no meaningful conclusions can be drawn from this study. In a British study with 299 patients (both LS- and ES-SCLC) randomized to receive vincristine, ifosfamide, etoposide, and carboplatin every 4 weeks or every 3 weeks, survival was superior with the 3-week cycle (median survival 11.5 months vs. 14.2 months, 2-year survival rate 18% vs. 33%, P = 0.0014) (45). However, there were no

408

E.O. Hanrahan and B. Glisson

Table  4  Randomized Phase III trials comparing etoposide–platinum doublets with irinotecan– platinum doublets in extensive stage SCLC MST Study Treatment regimens N (months) Comments IP q4w (I 60 mg/m2 D1, Enrollment was terminated 77 12.8 Japanese early due to a statistically Clinical D8, D15; P 60 mg/ significant difference in Oncology m2 D1) survival at interim analysis Group 77 9.4 EP q3w (E 100 mg/m2 There were less toxicities (JCOG) P = 0.002 D1–3; P 80 mg/m2 D1) associated with IP that with trial 9511 EP in this Japanese study (39) Hanna et al. IP q3w (I 65 mg/m2 D1, 221 9.3 The regimens were modified (40) D8; P 30 mg/m2 D1) from the JCOG regimens in an attempt to improve EP q3w (E 120 mg/m2 110 10.2 delivery, reduce toxicity, D1–3, P 60 mg/m2 D1) P = 0.74 and be more consistent with the dosages and schedules generally used in United States Southwestern IP q4w (I 60 mg/m2 D1, 323 9.7 The regimens used were the Oncology same as those in the JCOG D8, D15; P 60 mg/ Group, trial m2 D1) SWOG 1-Year survival rates were 322 8.9 EP q3w (E 100 mg/m2 0124 (41) 39% (IP) and 33% (EP) D1–3; P 80 mg/m2 D1) Certain SNPs were associated with an increased risk of grade 3/4 diarrhea and neutropenia with IP IC q3w (I 175 mg/m2 D1; 105 8.5 Hermes et al. There were mandatory dose (42) reductions upfront in Cbp = AUC 4 D1) patients greater than 70 104 7.1 ECbp (E 125 mg/m2/ years of age. There were P = 0.02 day by mouth D1–5; more elderly patients on Cbp = AUC 4 D1)(Cbp the ECbp arm (41% vs. dose based on Chatelut 30%) formula)

substantial differences in toxicity between the arms, suggesting that administration of this regimen every 3 weeks should be considered “standard” rather than “dose-dense.” The same research group next evaluated the administration of cyclophosphamide, doxorubicin, and etoposide at 3- or 2-week intervals among 403 patients, with GCSF support in the dose-dense arm, but they did not find a clinically meaningful survival advantage in the dose-dense arm (median survival 10.9 months vs. 11.5 months, 2-year survival rate 8% vs. 13%, P = 0.04) (48). Two randomized trials have compared ifosfamide, carboplatin, and etoposide (ICE) every 4 weeks with dose-dense ICE administered every 2 weeks with filgrastim and autologous peripheral blood progenitor cell support. Although a small trial stopped at interim analysis (n = 83) found that survival and time to progression were significantly improved with the dose-dense regimen (49), a larger randomized

Small Cell Carcinoma of the Lung

409

phase III trial (n = 318) found that response rates, median time to progression, median survival (13.9 and 14.4 months in the standard and dose-dense arms respectively), and 1- or 2-year survival were not improved in the dose-dense arm (50). Although two randomized trials of high-dose compared to low-dose cyclophosphamide, methotrexate, and lomustine (CMC) were positive for survival impact on the high-dose arms in the 1970s, the results of multiple subsequent randomized trials have generally been negative as regards survival benefit (51–53). A metaanalysis in 1991 of 60 published studies in SCLC analyzed the relationship between intended chemotherapy dose intensity and response or median survival and showed that increasing relative dose intensity of CAV or EP does not correlate with improved outcome (54). Most relevant in the modern era are two subsequent randomized trials that compared high-dose with standard-dose etoposide–platinum doublet regimens among patients with ES-SCLC (55, 56). Neither of these two studies showed a survival advantage with high-dose regimens. To obtain greater tumor cell kill, several investigators have studied myeloablative doses of chemotherapy, with either autologous bone marrow or peripheral blood stem cell (PBSC) support. Overall, there have been no convincing data supporting the benefit of increased dose density or intensification of therapy with marrow or stem cell support in SCLC. Indeed, a recent comprehensive review published in 2007 documents the absence of consistent survival benefit for these approaches (57). Based on in vitro data suggesting that drug concentrations have to be increased at least threefold to obtain a similar level of cell lysis in resistant SCLC cell lines as in sensitive ones (58), a recent randomized study involving 140 patients with LS-SCLC or ES-SCLC evaluated the efficacy of a threefold increased dose intensity of ICE with autologous PBSC support (59). However, neither response nor survival rates were improved in the high-dose arm. Based on the above data, the use of chemotherapy as initial treatment for SCLC in doses that cause more than moderately severe myelotoxicity is not justified.

Prolonged Administration of Chemotherapy Generally randomized trials have not shown survival impact from administration of first-line chemotherapy beyond 4–6 cycles. Based on the SECSG trial discussed above, 4 cycles of EP is accepted as a minimum standard of care, though many clinical trials continue to include options for up to 6 cycles (31). However, after the completion of first-line therapy, virtually all patients with ES-SCLC and about 80% with LS-SCLC will develop relapse or progression. Therefore, the role of prolonged or maintenance therapy has been evaluated in randomized trials. However, in patients who did not progress during first-line etoposide–platinum-based chemotherapy, neither the administration of an additional 3 cycles of oral etoposide nor an additional 4 cycles of topotecan led to an improvement in overall survival (60, 61). Therefore, the administration of prolonged or maintenance chemotherapy in the front-line treatment of SCLC is not currently recommended.

410

E.O. Hanrahan and B. Glisson

First-Line Chemotherapy for Limited Stage SCLC SCLC is an aggressive disease with early systemic spread. A series published in 1973 showed that 63% of patients who underwent an attempt at curative resection and died within 30 days from post-operative complications had distant metastases at autopsy (62). Ninety percent of these patients had metastases to mediastinal lymph nodes. Indeed, substantial evidence documenting the futility of either surgery or radiotherapy alone as a treatment for early-stage SCLC was accumulated in the 1960s and 1970s. Therefore, SCLC is generally treated as a systemic disease, even when classified as limited-stage. Multiple trials in the 1970s and 1980s found that the use of chemotherapy as an adjunct to radiation improved survival relative to that with radiation alone in LS-SCLC (63). It is now well-accepted that the optimal treatment of LS-SCLC requires a combined modality approach (64), with chemotherapy being at the cornerstone of therapy. Alkylator and anthracycline-based regimens are associated with excessive pulmonary, cardiac, esophageal, and bone marrow toxicity when administered concurrently with thoracic radiation therapy. The chemotherapy regimen of choice is EP, both because of its favorable toxicity profile and improved survival rates when used in combination with thoracic radiation. Although cisplatin is more toxic than carboplatin, cisplatin is the preferred platinum agent in LS-SCLC because treatment is with curative intent and, as discussed earlier, cisplatin may be more efficacious than carboplatin (28). As in ES-SCLC, data do not support the use of dose dense, dose intense or maintenance chemotherapy, or the addition of other cytotoxins to the EP base in LS-SCLC. An apparent exception is a trial that randomized 105 patients with LS-SCLC to receive a 33% higher dose of cyclophosphamide and a 25% higher dose of cisplatin in only the first of 6 cycles of treatment with cyclophosphamide, cisplatin, doxorubicin, and etoposide. Patients receiving the higher doses of the two drugs in the first cycle survived significantly longer, with 2- and 5-year survival rates of 42% vs. 20% and 26% vs. 8%, respectively (P = 0.03) (65, 66). Thoracic radiation was administered alternating with chemotherapy after cycles 2, 3, and 4. It should be noted that the survival in the “high-dose” arm of this trial is essentially identical to what was obtained with standard dose EP and early accelerated hyperfractionated radiation in the INT-0096 study (67). Therefore, these data do not seem relevant in the context of modern chemoradiation regimens.

The Role of Surgery in Limited Stage SCLC Surgical resection is rarely appropriate for patients with SCLC. Only one randomized trial, conducted by the Lung Cancer Study Group, has formally assessed the role of surgery in LS-SCLC (68). This trial excluded patients with stage 1 (T1-2N0M0) disease. Patients received 5 cycles of CAV chemotherapy, and those who responded were randomized to TRT alone or surgery plus TRT. However, the addition of surgery did not affect survival or the pattern of relapse.

Small Cell Carcinoma of the Lung

411

Less than 5% of patients with SCLC present with stage 1 disease. In such rare cases, surgery may be considered for patients with peripheral tumors and with no evidence of locoregional nodal or distant metastatic disease. Prior to definitive surgery, patients should have pathologic confirmation that mediastinal nodes are not involved, either at mediastinoscopy or via endoscopic ultrasound-guided biopsy. Definitive surgery should include a lobectomy and dissection or comprehensive sampling of mediastinal nodes. Because of the high rate of micrometastatic disease in patients with SCLC, adjuvant EP chemotherapy should be administered (69, 70). If hilar or mediastinal nodes obtained at definitive surgery are found to have metastatic involvement, thoracic RT should be integrated into the adjuvant chemotherapy (71).

Thoracic Radiation in Limited Stage SCLC The integration of thoracic radiation therapy (TRT) with systemic chemotherapy is crucial in the management of LS-SCLC. This combined modality approach reduces the intrathoracic failure rates in LS-SCLC from 75–90% with combination chemotherapy alone to 30–60%, and has been shown to improve patient survival in two meta-analyses (72–76). The current standard of care for LS-SCLC has been defined by the INT-0096 study, in which the implementation of accelerated hyperfractionated TRT during cycle 1 of EP chemotherapy significantly improved survival relative to standard TRT. Nevertheless, some controversy still persists about the most effective radiotherapy dose and fractionation, and the optimal timing of chemotherapy in relation to TRT.

Thoracic Radiation Therapy Dose and Fractionation Traditionally, SCLC was treated with modest doses of radiation, ranging from 30 to 50  Gy, because it was considered to be a highly radiosensitive disease (77). However, intrathoracic recurrence rates are high when modest-dose, conventionally fractionated TRT is used (67, 78). Therefore, the alternative strategies of accelerated hyperfractionated TRT, TRT dose escalation and concomitant boost TRT are being evaluated. Standard or conventionally fractionated radiation schedules use once daily treatment fractions of 1.8–2.0  Gy, five times per week, continuously. The duration depends on the total dose of radiation administered, but is usually 6–7 weeks. Accelerated hyperfractionated radiation schedules deliver a course of radiation over a shorter treatment period (accelerated) and with a greater number of treatment fractions (hyperfractionation). Two major randomized phase III studies have evaluated whether accelerated hyperfractionated TRT may be superior to standard TRT, but their results are conflicting.

412

E.O. Hanrahan and B. Glisson

The North Central Cancer Treatment Group treated 310 patients with LS-SCLC with an initial 3 cycles of EP, and then randomized the 261 patients without progression to receive a further 2 cycles of EP with concomitant TRT, either once-daily (50.4 Gy in 28 fractions) or twice-daily (48 Gy in 32 fractions) (79, 80). Patients receiving twice-daily TRT were given a 2.5-week break from TRT midway through the course. Although there were no differences between the two TRT schedules in terms of rate of progression, site of disease recurrence or survival, it has been proposed that the delayed initiation of TRT and the treatment break in the twice-daily arm may have negated the benefits of hyperfractionated TRT. INT 0096 compared 45 Gy administered either twice daily (1.5 Gy per fraction) over 3 weeks or once daily (1.8 Gy per fraction) over 5 weeks in 417 patients with LS-SCLC. Patients were treated with 4 cycles of EP, and TRT was administered concomitantly, beginning with cycle one of chemotherapy (67). Survival was significantly improved within the twice-daily TRT arm (median survival 23 months vs. 19 months, 5-year survival rates 26% vs. 16%; P = 0.04). Grade 3 esophagitis was significantly more frequent with twice-daily TRT, occurring in 27% of patients (vs. 11%, P < 0.001). The accelerated hyperfractionated TRT approach in INT 0096 has not been widely adopted in North America. The Patterns of Care Study published in 2003 noted that less than 10% of patients with LS-SCLC received this regimen, while more than 80% were treated with once-daily TRT (81). Reasons for this include concerns about increased acute toxicity and the logistical issues involved with treating patients twice daily. However, it is generally accepted that the comparator arm of once-daily TRT to a dose of 45 Gy in INT 0096 is no longer an acceptable standard of care. Most patients on a once-daily TRT schedule now receive treatment to a total dose of at least 60 Gy, despite the fact that such an approach has not yet been rigorously evaluated in phase III studies. The Cancer and Leukemia Group B (CALGB) has studied high-dose, once-daily TRT for the treatment of LS-SCLC. In a phase I study, they determined that the maximum tolerated doses (MTD) of TRT in combination with EP chemotherapy were ³70 Gy in 35 fractions over 7 weeks with standard once-daily TRT and 45 Gy in 30 fractions over 3 weeks with accelerated twice-daily TRT (82). CALGB conducted a subsequent phase II study of TRT 70 Gy in 35 fractions concurrent with carboplatin and etoposide, administered after 2 cycles of induction paclitaxel and topotecan chemotherapy (83). The postchemotherapy tumor volume was used for TRT planning. This study confirmed that this high-dose, once-daily TRT schedule is safe and feasible. The overall toxic effects of therapy were comparable with other recent trials using more modest total doses of TRT, the incidence of severe esophagitis was lower than that reported with hyperfractionated accelerated TRT, and the outcomes were comparable to the hyperfractionated accelerated TRT arm of INT 0096. Concomitant boost radiation therapy has been shown to improve local tumor control in head and neck squamous cell carcinoma compared with standard radiation therapy (84). This approach allows acceleration of TRT but only uses hyperfractionated TRT near the end of the course of treatment, thereby avoiding twice-daily, large-field TRT. The Radiation Therapy Oncology Group (RTOG) has evaluated concomitant boost TRT in LS-SCLC. In a phase I trial, the MTD for concomitant

Small Cell Carcinoma of the Lung

413

boost TRT initiated with the cycle 1 of EP for LS-CLC (RTOG 9712) was found to be 61.2 Gy delivered in 34 fractions of 1.8 Gy per fraction over 5 weeks, with twicedaily TRT during the final 9 treatment days (85). This led to RTOG 0239, a phase II study of the 61.2 Gy concomitant boost regimen, commenced on day 1 of the first of 4 cycles of EP chemotherapy. The results of this trial have been recently presented in abstract form. There were 72 participants in the study, and the rates of esophageal and pulmonary toxicity were acceptable. The rate of grade 3–4 esophagitis was 17%, which compared favorably to the 27% in the twice-daily TRT (45 Gy in 3 weeks) arm in INT 0096 (86). At 2 years, the rates of local control and survival were 80 and 37%, respectively (87). This survival rate is inferior to the 60% rate that was targeted and to the 47% rate in the experimental arm of INT 0096. In comparison with the 45 Gy twice-daily schedule used in INT 0096, the regimens tested by the CALGB (70 Gy once-daily) and the RTOG (61.2 Gy in a concomitant boost approach) use considerably higher nominal radiation and yield substantially higher biologic effective doses (88). To determine whether such further intensification of TRT regimens may achieve greater improvements in local control and survival in LS-SCLC, a randomized phase III US Intergroup study was launched in March 2008 (89). This study compares the two experimental arms of 70 Gy oncedaily TRT and 61.2  Gy concomitant boost TRT with standard 45  Gy twice-daily TRT. Radiation therapy is started with the first cycle of EP chemotherapy. It is important to note that this study commenced before the 2-year outcome results of RTOG 0239 were available. An early interim analysis of both experimental treatment arms is planned to assess treatment-related toxicities, and the experimental arm with the highest rate of severe acute toxicities will be discontinued. It is unclear if the survival results of study RTOG 0239 will lead to any modifications in this plan. While the outcome of this phase III trial is awaited, however, 45 Gy twice-daily TRT should continue to be considered the standard of care in LS-SCLC.

Timing of Thoracic Radiation Therapy Relative to Chemotherapy Sequential, concurrent, and alternating approaches have been tried in integrating TRT with chemotherapy. Although an early meta-analysis suggested that the mode of TRT integration with chemotherapy did not significantly affect patient outcome, subsequent clinical trial data have shown that the use of concurrent chemoradiation is superior. In general, studies that failed to demonstrate the superiority of concurrent TRT used cyclophosphamide- or doxorubicin-based chemotherapy, where the toxicity of the combined-modality therapy required reduced dose intensity. In contrast, with the EP chemotherapy regimen that is better tolerated when administered with TRT, early concurrent integration of TRT is more feasible and effective. In an NCIC phase III trial of TRT (40 Gy in 15 fractions over 3 weeks) initiated with either the second or sixth cycle of alternating CAV/EP chemotherapy, progressionfree survival (P = 0.036) and overall survival (P = 0.008) were superior in the early

414

E.O. Hanrahan and B. Glisson

TRT arm (90). In a similar trial, Spiro et al. failed to detect a benefit for early vs. late TRT, but the results of this trial have been viewed skeptically because the overall median survival outcomes in both treatment arms were considerably less than in the NCIC trial (91). Jeremic and colleagues performed a randomized study of accelerated hyperfractionated TRT (1.5 Gy twice daily to 54 Gy) administered early (with weeks 1–4) or late (weeks 6–9) concurrently with ECbp chemotherapy (92). Survival was greater in the early TRT arm (34 months vs. 26 months). The Japanese Clinical Oncology Group reported a phase III trial comparing TRT (45 Gy:1.5 Gy twice daily for 3 weeks) administered either concurrently (commencing day 2 of the first cycle) or sequentially with EP (93). Survival was improved in the concurrent arm (Median 27.2 months vs. 19.7 months, HR 0.70, P = 0.02). At least four meta-analyses addressing the timing of TRT have recently been published, and are summarized in Table 5. Although not all individual trials have consistently shown a benefit for early TRT, the weight of the evidence suggests a modest survival benefit to the use of early rather than delayed TRT. Furthermore, the available data suggest that the benefit of early concurrent chemotherapy and TRT may be optimized by more intensified TRT delivered with full doses of chemotherapy.

Prophylactic Cranial Irradiation Approximately 10–14% of patients with SCLC have brain metastases (BM) at initial diagnosis (94). With improvements in treatment strategies leading to better systemic and locoregional control, BM as a site of failure in LS-SCLC is a major problem. In the absence of specific therapy to the CNS, approximately 60% of patients with LS-SCLC in complete remission develop BM by 3 years (94). With the hope that the CNS would be the only site of residual disease, prophylactic cranial irradiation (PCI) has been evaluated as part of the treatment of SCLC. Between 1977 and 1995, 11 prospective randomized trials of PCI were published (95). Although all but two trials reported a significant reduction in the incidence of brain metastasis, none demonstrated an improvement in survival with PCI. Auperin et al. conducted a meta-analysis of the individual patient data of 987 patients with SCLC in complete remission who took part in seven of these trials that compared PCI with no PCI (95). The majority of the patients in this meta-analysis had LS-SCLC. PCI reduced the incidence of BM by almost 50% and increased 3-year survival rates significantly from 15.3 to 20.7% (P = 0.01). PCI has traditionally been offered only to patients with LS-SCLC who have a complete or near-complete response to chemoradiation. Until recently, it was unclear whether or not patients with ES-SCLC should also receive PCI. A randomized trial by the EORTC Radiation Oncology and Lung Cancer group published in 2007 provides evidence that PCI reduces the incidence of symptomatic brain metastases and prolongs survival in ES-SCLC (96). Two hundred and eighty-six patients with ES-SCLC who responded to 4–6 cycles of chemotherapy were randomized to

Small Cell Carcinoma of the Lung

415

Table 5  Meta-analyses of early vs. late thoracic radiation therapy in limited stage SCLC Author Timeframe # Trials # Patients Definitions Results 2-Year survival with Fried et al. (201) 1987–2002 7 1,524 Early TRT: TRT early vs. late TRT, administered RR = 1.17 within 9 weeks of starting The benefit was chemotherapy greatest for hyperfractionated TRT (RR = 1.44) and platinumbased chemotherapy (RR = 1.3) No significant 1993–2006 7 1,244 Early TRT: TRT Pijlsdifferences in delivered Johannesma the 2- and 5-year within 30 days et al. and De survival with of starting Ruysscher early vs. late chemotherapy et al. (202– TRT (RR = 0.93), Short SER: time 204) but there was from the start of a significant any treatment survival until the end advantage with of radiotherapy short SER (SER) £30 days (RR for 5-year survival = 0.62) Spiro et al. (91) 1987–2006 8 1,849 No survival advantage with early vs. late TRT (HR = 0.96) There appeared to be a survival benefit with early TRT if all intended chemotherapy cycles were delivered (RR = 1.35) 2-Year survival with Huncharek et al. 1993–2002 8 1,575 Early TRT: early vs. late TRT, (205) radiation summary odds administered ratio (ORp) = 1.60 concurrently The 3-year survival with data with early chemotherapy vs. late TRT, Late TRT: ORp = 1.49 sequential or split-course TRT

receive PCI or no PCI. A complete response to chemotherapy was not required, and baseline brain imaging was not mandated in asymptomatic patients. A number of different PCI schedules were allowed, with total doses ranging from 20 to 30 Gy.

416

E.O. Hanrahan and B. Glisson

Patients in the PCI group had a lower risk of symptomatic BM (hazard ratio, 0.27; P < 0.001; 1-year cumulative incidence of symptomatic BM in PCI vs. control group 14.6% vs. 40.4%). In addition, there was a considerable improvement in survival in the PCI group (1-year survival rate 27.1% vs. 13.3%; median survival times 6.7 months vs. 5.4 months, P = 0.003). Although there was a statistically significant disease-free survival benefit with the use of PCI in this study, the absolute benefit was small (median DFS 14.7 weeks in the PCI group and 12 weeks in the control group) and seemed insufficient to account entirely for the overall survival benefit seen with PCI. The greater overall survival in the PCI group may also be due in part to a higher rate of second-line chemotherapy administration in the PCI group (68.0% of patients in the PCI group and 45.1% in the control group). On the basis of the meta-analysis by Auperin et al. and this recent EORTC study, PCI is now recommended for patients with either LS- or ES-SCLC who have a complete or near complete response to chemotherapy, and it should be considered in patients with a partial response (71). Despite the advantages offered by PCI, there have been concerns about treatment-related neurotoxicity in long-term survivors of SCLC. The EORTC ES-SCLC assessed long-term sequela and did not find any differences between those who did and did not receive PCI with respect to quality of life or cognitive function. However, these findings must be viewed cautiously because very few patients were alive and assessable at 1-year follow-up. The results of at least four other randomized trials using formal prospective standardized neuropsychological tests in the mid-1990s showed no difference in the rates of neurocognitive impairment between patients treated with PCI and those who did not receive PCI over follow-up periods ranging from 5 to 30 months (97–100). However, there may be an increased risk of neurotoxicity from PCI when it is given concurrently with or before chemotherapy, when radiation fractions are greater than 2.5 Gy, and when total radiation dose is more than 30 Gy (101). Commonly used PCI regimens involve 2.0- to 2.5-Gy daily fractions to a total dose of 25–30 Gy. However, the optimal total dose and fractionation schedule for PCI remain undefined, and are being examined in two randomized studies involving patients with LS-SCLC that completely responded to induction therapy: the international, phase III PCI-01-EULINT1 trial and a phase II/III trial by the RTOG. These studies are comparing a standard arm (25 Gy in 10 daily fractions) with two high-dose arms of 36 Gy in conventional fractions (2.0 Gy/fraction) or delivered as accelerated hyperfractionated radiation therapy (1.5 Gy twice daily).

Relapsed Disease Despite high response rates to first-line standard treatment, about 80% of LS-SCLC patients and virtually all patients with ES-SCLC will develop recurrent or progressive disease. Their prognosis is poor, and median survival is in the range of 4–6 months with second-line chemotherapy. Until relatively recently, there was limited

Small Cell Carcinoma of the Lung

417

evidence that second-line chemotherapy improves survival in recurrent SCLC (102). Response to additional therapy and survival from the time of relapse is influenced by response to initial chemotherapy and the progression-free interval following its completion (treatment-free interval, TFI). Administration of second-line chemotherapy is challenging because many of these patients have advanced age, comorbidities, poor performance status, and cumulative toxicities from their firstline therapy. Giaccone et al. were the first to observe response to first-line chemotherapy and TFI as important predictors of benefit from second-line therapy. They conducted a single-arm study of teniposide, a podophyllotoxin derivative, for the treatment of SCLC, and found that previously-treated patients had a significantly higher probability of response if they had TFIs of more than 2.6 months (103). A number of additional trials and retrospective analyses have had similar findings (104–107). Consequently, the classification of relapsed SCLC as sensitive or resistant evolved and, although this delegation has not been validated in prospective studies, it is widely accepted in oncology practice (108). Sensitive disease is that which responds to first-line therapy and has a treatment-free interval of at least 60–90 days. Resistant disease is that which exhibits no response to first-line treatment or relapses within 60–90 days of completing treatment. There is no generally accepted standard regimen for relapsed SCLC, and commonly employed approaches include reinduction/rechallenge with the regimen that was used first-line (usually an etoposide–platinum doublet) in sensitive disease, or the use of a non-cross resistant regimen, such as CAV or single-agent topotecan. Topotecan is the only drug with an FDA indication for treatment of relapsed SCLC. The evidence in favor of reinduction/rechallenge with the same chemotherapy used first-line is based on four, small retrospective series reported in the 1980s with a total of 70 patients (104–107). Patients with sensitive relapsed SCLC were treated with chemotherapy regimens that included some or all of the drugs in their initial treatment. Response rates in these analyses were high, ranging from 50 to 80%. It is unclear whether this high response rate is attributable to patient selection or to a true efficacy of this strategy in sensitive relapse. This reinduction approach has not been evaluated in a prospective randomized study. Nevertheless, it is widely accepted that patients with relapsed SCLC and a long TFI, typically 6 months or more, should be considered for re-treatment with the same drugs. Topotecan, a semisynthetic derivative of camptothecin, is the most extensively studied agent for relapsed SCLC, and it is the only agent currently approved by the FDA for the treatment of relapsed SCLC. Topotecan has shown significant antitumor activity and symptom palliation in both sensitive and resistant relapsed SCLC. The safety and efficacy of topotecan for relapsed SCLC was initially established in several phase II studies, with response rates ranging from 11 to 31% in sensitive disease and from 2 to 7% in resistant disease, and with median survival times of 25–36 and 16–21 weeks, respectively (109–112). Data from three subsequent randomized phase III studies led to the approval of topotecan in the United States and Europe, as outlined in Table 6 (102, 113, 114).

418

E.O. Hanrahan and B. Glisson

Table 6  Randomized, phase III studies of topotecan in relapsed SCLC Treatment RR MST Trial TFI N arms (%) (weeks) Comments 24.3 25 No difference in efficacy 107 Topotecan Von Pawel 60 days between the two treatment (1.5 mg/ et al. arms m2 IV (113) Greater symptom improvement D1–5 and tolerability with q3w) topotecan 18.3 24.7 104 CAV 90 days 153 Topotecan Eckardt 18.3 33 Similar efficacy outcomes with 2.3 mg/ et al. the more convenient oral m2/day (114) form Toxicity is altered, with the po D1–5 oral form producing less q3w 151 Topotecan neutropenia (47% vs. 64%) 21.9 35 but more diarrhea (36% vs. 1.5 mg/ 20%) than the IV form m2/day IV D1–5 q3w Refractory 71 Topotecan O’Brien 25.9 44% of patients on topotecan 7 2.3 mg/ and et al. arm had stable disease for m2/day sensitive (102) ³54 days relapse This trial provided evidence po D1–5 of the survival benefit of q3w 70 Best second-line chemotherapy 13.9 in SCLC supportive The impact of topotecan on care progression and survival was seen equally in patients with resistant or sensitive relapse Patients on topotecan also had slower QOL deterioration and improved symptom control

Additional options for recurrent SCLC include monotherapy with other cytotoxic agents. A number of single chemotherapy agents have demonstrated varying degrees of activity against recurrent SCLC in phase II studies. Irinotecan, while not studied nearly as extensively as topotecan in recurrent patients, appears to have similar benefits (115). Limited evaluation of the taxanes suggest a response rate in the range of 20–29% for paclitaxel and 25% for docetaxel (116–118). Response rates with single-agent vinorelbine or gemcitabine are less than 20%, and with pemetrexed monotherapy, the response rate is less than 5% (119). Palliative radiation is effective and is used commonly in drug-resistant and recurrent SCLC. The treatment of patients with recurrent SCLC is an ongoing area of clinical research, and patients, particularly those with resistant disease, should be considered for clinical trials of novel therapies.

Small Cell Carcinoma of the Lung

419

New Systemic Therapies for SCLC Cytotoxic Agents Amrubicin is a synthetic anthracycline, whose mechanisms of action include the inhibition of topoisomerase II. It has shown promising activity in SCLC in phase II studies. As monotherapy, response rates of 21–50, 52 and 76% have been reported in resistant, sensitive, and previously untreated SCLC, respectively (120–122). In a recently presented randomized phase II study, 76 patients with recurrent, sensitive ES-SCLC were randomized (2:1) to either amrubicin or topotecan. Response rates were significantly higher with amrubicin (36% vs. 8%, P < 0.012) (123). However, there was no significant difference in survival. In a Japanese phase II study, 36 elderly patients (70 years or older) with SCLC were treated first-line with amrubicin in combination with carboplatin. The response rate was 89%, with median PFS and OS of 5.8 and 18.6 months, respectively (124). A number of randomized, phase III studies are now evaluating amrubicin as monotherapy or in combination with cisplatin in the first-line setting, and as monotherapy in relapsed SCLC. Picoplatin is a sterically hindered platinum analogue designed to overcome platinum resistance. Picoplatin also appears to be less nephrotoxic and neurotoxic than cisplatin (125). Phase II studies suggest that single-agent picoplatin is active in relapsed SCLC (125, 126). The response rates with second-line picoplatin in platinum-resistant and platinum-sensitive patients have been reported to range from 4 to 15.4%, but with a disease control rate of 47% and median survival times of 27–36 weeks (126). A randomized phase III trial is ongoing to compare best supportive care with or without picoplatin in patients with refractory SCLC or disease progression within 6 months of completing first-line platinum-containing chemotherapy.

Molecular Biology and Novel Targeted Therapies Improving the survival rate of patients with SCLC requires a better understanding of tumor molecular biology and the subsequent identification of novel targets for the development of targeted therapeutic strategies. Tumor Suppressor Genes SCLC is characterized by chromosomal imbalances, with a high incidence of deletions on chromosomes 3p, 4q, 5q, 10q, 13q, and 17p, and a high incidence of gains at 3q, 5p, 6p, 8q, 17q, 19q, and 20q (127, 128). One of the most consistent chromosomal abnormalities in SCLC is deletion of much of the short arm of chromosome 3 (3p[14-25]), which is present in more than 95% of cases (128, 129). Allele loss involving 3p is one of the earliest genetic alterations found in SCLC and affects a number of putative tumor suppressor genes (TSG) that play important roles in cancer

420

E.O. Hanrahan and B. Glisson

pathogenesis, such as cell cycle control and the regulation of apoptosis. TSGs located on 3p include the fragile histidine triad gene (FHIT), which is downregulated with loss of protein in about 80% of SCLCs (130, 131), and SEMA3B (132–134), whose expression is commonly lost in SCLC through allele loss or promoter methylation. Other TSGs on 3p that are lost because of methylation of their promoter regions, rather than mutations, include RARb (the gene encoding the retinoic acid receptor-beta isoform) and Ras-association domain family 1 (RASSF1) (135), whose expressions are lost in 72 and 90% of SCLCs, respectively (136–138). The RB TSG is located on chromosome 13q14.11, and encodes for a nuclear protein that regulates the transition from G1-phase to S-phase of the cell cycle. It was initially determined to be abnormal in patients with retinoblastoma, but has since been found to be mutated in many other solid tumors. RB is deleted or mutated in about 90% of SCLCs (139). The TSG p53, located on chromosome 17p13.1, is frequently mutated in lung cancer, and p53 mutations have been shown to correlate with cigarette smoking (140). p53 is inactivated by mutations or deleted in approximately 90% of SCLCs (140). The TSG p19ARF encodes a protein that enhances p53, and its protein expression is lost in approximately 65% of SCLCs (141). Attempts to therapeutically exploit these p53 abnormalities in SCLC are being made. In a single-arm phase II trial, patients with ES-SCLC post-completion of first-line chemotherapy were immunized with a vaccine consisting of dendritic cells transduced with wild-type p53 (142). Despite induction of antigen-specific immunity, there were no objective responses. Interestingly, most vaccinated patients had clinical responses to second-line chemotherapy. An ongoing randomized phase II study in patients with ES-SCLC is evaluating if administering this vaccine therapy with or without tretinoin after completion of standard ECbp chemotherapy will improve response to second-line therapy upon relapse (143). Nonreceptor Proto-oncogenes Gene amplification and overexpression of members of the MYC gene family lead to oncogenic activity (144, 145). Amplification of a member of the MYC family occurs in 18–31% of SCLCs (146), and c-MYC amplification has been associated with a worse prognosis (147–150). BCL-2 is an antiapoptotic protein (151). Overexpression of BCL-2 is associated with resistance to chemotherapy. Although BCL-2 is expressed in about 75% of SCLCs, its expression does not correlate with disease progression (152). A randomized phase II study of a BCL-2 antisense oligonucleotide, oblimersen sodium, added to etoposide and carboplatin for the treatment of ES-SCLC did not improve recurrence free or overall survival, and newer small molecule inhibitors of BCL-2 are being studied (153). Growth Factor and Receptor Abnormalities Several receptor tyrosine kinases (RTKs) and/or their ligands are overexpressed in SCLC or associated stromal tissue, constituting functional autocrine and/or paracrine growth loops. Some of these are proto-oncogenes that play a key role in

Small Cell Carcinoma of the Lung

421

mediating the dysregulated cell growth, differentiation, survival and motility that is characteristic of malignant tumors. Up to 70% of SCLC express c-Kit and its ligand, stem cell factor (SCF), and the SCF/c-Kit pathway is functional in an autocrine or a paracrine fashion in SCLC (152). Despite this, clinical studies with imatinib, a c-Kit inhibitor, in SCLC failed to show anti-tumor activity in relapsed SCLC or as maintenance therapy after completion of first-line chemotherapy (154–156). c-Met is widely expressed in SCLC, but coexpression of its ligand, HGF, appears to be rare (157). However, serum HGF levels have been reported to be elevated in patients with SCLC (158, 159). Modulation of the c-Met/HGF pathway in SCLC in vitro has been shown to lead to changes in cell motility/migration and production of reactive oxygen species, providing evidence that this pathway is functional in SCLC and a potential therapeutic target (160). There are a number of inhibitors of the c-Met/HGF pathway in phase I and II clinical trials in solid tumors at present. An ongoing international, multicenter phase Ib/II clinical trial is evaluating AMG102, an anti-HGF antibody, in combination with platinum–etoposide chemotherapy as first-line treatment for ES-SCLC (143). The insulin-like growth factor-1 (IGF-1) receptor is expressed in most lung cancers, and its ligand, IGF-1, is present in more than 95% of SCLCs (161, 162). IGF-1/IGF-1R signaling plays an important role in the growth and survival of SCLC by potently activating the PI3K-Akt signal transduction pathway (163). Preclinical data support evaluating anti-IGF-1R therapy in SCLC (163, 164). An international, multicenter phase Ib/II study is currently evaluating AMG479, a monoclonal antibody to the ligand-binding domain of IGF-1R, in combination with platinum–etoposide chemotherapy for ES-SCLC. Chronic activation of the PI3K/Akt/mTOR pathway through a multitude of mechanisms, including inactivating mutations and loss of expression of PTEN, is common in SCLC (152). mTOR inhibitors, such as temsirolimus and everolimus, are being assessed in SCLC. In phase II studies, temsirolimus was evaluated as maintenance treatment after response to first-line chemotherapy in patients with ES-SCLC, and everolimus was evaluated as monotherapy in previously-treated patients (165). These agents were well-tolerated, but they did not have significant anti-tumor activity as monotherapy. Nevertheless, these mTOR inhibitors are now being assessed in combination with first-line platinum-based chemotherapy for SCLC (143). Angiogenesis Angiogenesis, the process of new blood vessel formation from existing vessels, is critical for tumor growth (166, 167). Vascular endothelial growth factor (VEGF) is a potent proangiogenic factor that is upregulated in tumors (168–170). Increased microvessel density and overexpression of VEGF in both small cell and non-small cell lung cancer significantly correlate with advanced disease stage and poor survival (171, 172). Angiogenesis and the VEGF-signaling pathway as therapeutic targets are an area of active research in SCLC. Results with this therapeutic strategy to date have been mixed.

422

E.O. Hanrahan and B. Glisson

Thalidomide has multiple antitumor effects, including angiogenesis inhibition through unknown mechanisms. Results were encouraging from the initial evaluation of thalidomide in combination with cisplatin-based chemotherapy in a small, randomized, phase III trial (173). However, a subsequent large (N = 724), randomized phase III trial of ECbp chemotherapy with or without thalidomide found no survival benefit with the use of thalidomide, and no further evaluation of this agent in SCLC is planned (174). Bevacizumab is a recombinant, humanized monoclonal antibody against VEGF, and it is approved for use in combination with chemotherapy in the treatment of advanced colorectal, breast, and non-small cell lung cancers. There are a number of recently completed and ongoing trials of bevacizumab in SCLC. Two phase II trials evaluating the combination of first-line chemotherapy with bevacizumab in patients with ES-SCLC have been reported in abstract form, with results suggesting a modest improvement in survival compared to historical controls (175, 176). A multicenter, randomized phase II study of first-line chemotherapy with or without bevacizumab for ES-SCLC (the SALUTE trial) is currently ongoing in the US and is expected to complete accrual in late 2009. Assuming the results of this trial are promising, phase III randomized trials will be required to confirm if bevacizumab improves outcome in ES-SCLC. The combination of bevacizumab with second-line therapy for SCLC is also being evaluated. A phase II study in the US of paclitaxel and bevacizumab in chemosensitive relapsed SCLC has been recently completed. Preliminary data have been presented in abstract form, but there did not appear to be any improvement in median PFS or survival above what can be achieved with standard second-line regimens (177). Other phase II studies that are ongoing or planned to commence in the near future are evaluating the combinations of docetaxel plus bevacizumab and oral topotecan plus bevacizumab for the second-line treatment of SCLC (143). The question of incorporating bevacizumab in sequence with or concomitantly with chemoradiation for LS-SCLC has also been addressed in phase II studies, with the latter approach appearing to improve response rates (178, 179). However, both sequential and concomitant administration of bevacizumab with chemoradiation were associated with an increased risk for tracheoesophageal fistula development. As a consequence, further studies of bevacizumab with chemoradiation in LS-SCLC have been abandoned. A number of orally-administered multitargeted receptor tyrosine kinase inhibitors (RTKIs) with activity against VEGF receptors have been or are being evaluated in SCLC. In a phase II study, sorafenib (RTKI of Raf-1, VEGFR-2, VEGFR-3, and PDGFR-a) administered to 82 patients with relapsed SCLC after one platinumbased regimen yielded survival outcomes similar to what would be expected with standard second-line chemotherapy, though response rates were only 5% in both sensitive and refractory relapse (180). An ongoing phase II study is evaluating the concomitant administration of sorafenib with EP for 4 cycles for the first-line treatment of ES-SCLC, with continuation of sorafenib for up to 1 year in the absence of disease progression or unacceptable toxicity (181). The NCIC-CTG conducted a phase II randomized trial of maintenance vandetanib (RTKI of EGFR, VEGFR and

Small Cell Carcinoma of the Lung

423

RET) or placebo in 107 patients with either LS- or ES-SCLC who had a confirmed response to chemotherapy with or without radiotherapy. Disappointingly, there was no difference in PFS (2.7 and 2.8 months) or median survival time (ZD6474 10.6 months vs. placebo 11.9 months) between the two groups (182). Cediranib (AZD2171; RTKI of VEGFR-1, -2 and -3, PDGFR-b and c-Kit) as a single agent had minimal anti-tumor activity in a phase II study among 25 patients with SCLC pretreated with only one previous regimen (183). Cediranib continues to be evaluated in SCLC, but only as part of combination regimens. A phase I study of cediranib in combination with etoposide and cisplatin for the first-line treatment of ES-SCLC is ongoing. Matrix Metalloproteinases Matrix metalloproteinases (MMPs) are a family of enzymes that play an important role in tumor development and progression through a number of mechanisms, including degradation and remodeling the extracellular matrix that facilitates endothelial and tumor cell migration. Indeed, increased expression of MMPs in SCLC has been identified as a negative predictor of survival (184). Despite this, two randomized phase III clinical trials of synthetic MMP inhibitors (marimastat and tanomastat) as maintenance therapy after completion of frontline chemotherapy in patients with SCLC failed to demonstrate a TTP or survival advantage (185, 186). CD56 Antigen CD56, also known as neural cell adhesion molecule, is a glycoprotein of the Ig superfamily that is expressed in almost all cases of SCLC. BB-10901 is a conjugate of a CD56-targeted antibody and the cytotoxin DM1, a microtubule-depolymerizing agent. BB-10901 allows targeted delivery of DM1 to CD56-expressing tumors. Following identification of the maximum tolerated dose in a phase I dose-escalating study in patients with SCLC and other CD56-positive solid tumors, a phase II extension of the trial to enroll 30 patients with relapsed SCLC or CD56-positive nonpulmonary small cell carcinoma is ongoing.

Conclusions Despite the high initial response rates of SCLC to chemotherapy and radiotherapy, most patients will develop recurrent disease within 2 years. The 5-year survival rate is only approximately 10–15% for patients with LS-SCLC and less than 1% for patients with ES-SCLC. Attempts to improve outcome through modifications of conventional chemotherapy have been unsuccessful to date. The greatest advances

424

E.O. Hanrahan and B. Glisson

in the first-line treatment of SCLC over the past 20 years have come from integration of full dose chemotherapy in the form of etoposide and cisplatin with concurrent thoracic radiation. The importance of the early integration of TRT into chemoradiation for LS-SCLC and of PCI in patients who respond to systemic chemotherapy, even those with ES-SCLC, have been established. Clinical trials continue to evaluate the optimal dosing and schedule of TRT and PCI, but any further advances will likely offer modest survival advantages at best. SCLC remains a therapeutic challenge. Amrubicin and picoplatin are new cytotoxic agents that are in phase III clinical trials for SCLC at present. However, perhaps the greatest hope for improving outcomes for patients with SCLC lies in our increasing understanding of the molecular biology of SCLC and the development of novel, molecularly targeted agents. Although a number of such novel agents have been frustratingly unsuccessful in phase II or III trials in SCLC to date, new potential therapeutic targets and agents continue to be identified and clinically evaluated. Whenever possible, patients should be considered for enrollment in clinical trials to evaluate these new therapies.

References 1. American Cancer Society. Cancer Facts and Figures 2009, Atlanta: American Cancer Society:2009 2. Govindan R, Page N, Morgensztern D et al (2006) Changing epidemiology of small-cell lung cancer in the United States over the last 30 years: analysis of the surveillance, epidemiologic, and end results database. J Clin Oncol 24:4539–4544 3. Ettinger DS, Aisner J (2006) Changing face of small-cell lung cancer: real and artifact. J Clin Oncol 24:4526–4527 4. De Stefani E, Boffetta P, Ronco AL et al (2005) Squamous and small cell carcinomas of the lung: similarities and differences concerning the role of tobacco smoking. Lung Cancer 47:1–8 5. Travis WD, Colby TV, Corrin B et al (1999) Histological typing of lung and pleural tumors, 3rd edn. Springer, Berlin 6. Brambilla E, Travis WD, Colby TV et al (2001) The new World Health Organization classification of lung tumours. Eur Respir J 18:1059–1068 7. Junker K, Wiethege T, Muller KM (2000) Pathology of small-cell lung cancer. J Cancer Res Clin Oncol 126:361–368 8. Micke P, Faldum A, Metz T et  al (2002) Staging small cell lung cancer: Veterans Administration Lung Study Group versus International Association for the Study of Lung Cancer – what limits limited disease? Lung Cancer 37:271–276 9. Green RA, Humphrey E, Close H, Patno ME (1969) Alkylating agents in bronchogenic carcinoma. Am J Med 46:516 10. Ignatius Ou SH, Zell JA (2009) The applicability of the proposed IASLC staging revisions to small cell lung cancer (SCLC) with comparison to the current UICC 6th TNM Edition. J Thorac Oncol 4:300–310 11. Vinjamuri M, Craig M, Campbell-Fontaine A et al (2008) Can positron emission tomography be used as a staging tool for small-cell lung cancer? Clin Lung Cancer 9:30–34 12. Brink I, Schumacher T, Mix M et al (2004) Impact of [18F]FDG-PET on the primary staging of small-cell lung cancer. Eur J Nucl Med Mol Imaging 31:1614–1620 13. van Loon J, Offermann C, Bosmans G et al (2008) 18FDG-PET based radiation planning of mediastinal lymph nodes in limited disease small cell lung cancer changes radiotherapy fields: a planning study. Radiother Oncol 87:49–54

Small Cell Carcinoma of the Lung

425

14. Onn A, Vaporciyan A. Chang J, Komaki R, Roth J, Herbst R. Cancer of the Lung. In DW. Kufe, RC. Bast Jr, WN. Hait, W Ki Hong, R. Pollock, RR. Weichselbaum, JF. Holland, Emil Frei III (eds). Cancer Medicine 1179–1224 15. Yip D, Harper PG (2000) Predictive and prognostic factors in small cell lung cancer: current status. Lung Cancer 28:173–185 16. Chute JP, Chen T, Feigal E et  al (1999) Twenty years of phase III trials for patients with extensive-stage small-cell lung cancer: perceptible progress. J Clin Oncol 17:1794–1801 17. Boral A, Lynch TJ (2000) Chemotherapy of small cell lung cancer. In: Skarin AT, Alexander E (eds) Multimodality treatment of lung cancer. Informa Health Care, New York, pp 381–404 18. Ihde DC (1992) Chemotherapy of lung cancer. N Engl J Med 327:1434–1441 19. Lowenbraun S, Bartolucci A, Smalley RV et al (1979) The superiority of combination chemotherapy over single agent chemotherapy in small cell lung carcinoma. Cancer 44: 406–413 20. Edmonson JH, Lagakos SW, Selawry OS et al (1976) Cyclophosphamide and CCNU in the treatment of inoperable small cell carcinoma and adenocarcinoma of the lung. Cancer Treat Rep 60:925–932 21. Bunn PA Jr, Cohen MH, Ihde DC et al (1977) Advances in small cell bronchogenic carcinoma. Cancer Treat Rep 61:333–342 22. Einhorn LH, Bond WH, Hornback N et al (1978) Long-term results in combined-modality treatment of small cell carcinoma of the lung. Semin Oncol 5:309–313 23. Evans WK, Osoba D, Feld R et al (1985) Etoposide (VP-16) and cisplatin: an effective treatment for relapse in small-cell lung cancer. J Clin Oncol 3:65–71 24. Evans WK, Shepherd FA, Feld R et  al (1985) VP-16 and cisplatin as first-line therapy for small-cell lung cancer. J Clin Oncol 3:1471–1477 25. Pujol JL, Carestia L, Daures JP (2000) Is there a case for cisplatin in the treatment of smallcell lung cancer? A meta-analysis of randomized trials of a cisplatin-containing regimen versus a regimen without this alkylating agent. Br J Cancer 83:8–15 26. Sundstrom S, Bremnes RM, Kaasa S et  al (2002) Cisplatin and etoposide regimen is superior to cyclophosphamide, epirubicin, and vincristine regimen in small-cell lung cancer: results from a randomized phase III trial with 5 years’ follow-up. J Clin Oncol 20:4665–4672 27. Skarlos DV, Samantas E, Kosmidis P et  al (1994) Randomized comparison of etoposidecisplatin vs. etoposide-carboplatin and irradiation in small-cell lung cancer. A Hellenic Co-operative Oncology Group study. Ann Oncol 5:601–607 28. Wolf M, Havemann K, Drings P, Hans K, Schroder M, Lenaz L et al (1989) Phase III study of an alternating chemotherapy with adriamycin/ifosfamide/vincristine and cisplatin/etoposide or carboplatin/etoposide in small cell lung cancer (Abstract). Proc Am Soc Clin Oncol 8:236 Abstract 919 29. Evans WK, Feld R, Murray N et  al (1987) Superiority of alternating non-cross-resistant chemotherapy in extensive small cell lung cancer. A multicenter, randomized clinical trial by the National Cancer Institute of Canada. Ann Intern Med 107:451–458 30. Fukuoka M, Furuse K, Saijo N et al (1991) Randomized trial of cyclophosphamide, doxorubicin, and vincristine versus cisplatin and etoposide versus alternation of these regimens in small-cell lung cancer. J Natl Cancer Inst 83:855–861 31. Roth BJ, Johnson DH, Einhorn LH et  al (1992) Randomized study of cyclophosphamide, doxorubicin, and vincristine versus etoposide and cisplatin versus alternation of these two regimens in extensive small-cell lung cancer: a phase III trial of the Southeastern Cancer Study Group. J Clin Oncol 10:282–291 32. Feld R, Evans WK, Coy P et  al (1987) Canadian multicenter randomized trial comparing sequential and alternating administration of two non-cross-resistant chemotherapy combinations in patients with limited small-cell carcinoma of the lung. J Clin Oncol 5:1401–1409 33. Goodman GE, Crowley JJ, Blasko JC et al (1990) Treatment of limited small-cell lung cancer with etoposide and cisplatin alternating with vincristine, doxorubicin, and cyclophosphamide versus concurrent etoposide, vincristine, doxorubicin, and cyclophosphamide and chest radiotherapy: a Southwest Oncology Group Study. J Clin Oncol 8:39–47

426

E.O. Hanrahan and B. Glisson

34. Loehrer PJ Sr, Ansari R, Gonin R et  al (1995) Cisplatin plus etoposide with and without ifosfamide in extensive small-cell lung cancer: a Hoosier Oncology Group study. J Clin Oncol 13:2594–2599 35. Hanna N, Ansari R, Fisher W et  al (2002) Etoposide, ifosfamide and cisplatin (VIP) plus concurrent radiation therapy for previously untreated limited small cell lung cancer (SCLC): a Hoosier Oncology Group (HOG) phase II study. Lung Cancer 35:293–297 36. Niell HB, Herndon JE II, Miller AA et  al (2005) Randomized phase III intergroup trial of etoposide and cisplatin with or without paclitaxel and granulocyte colony-stimulating factor in patients with extensive-stage small-cell lung cancer: Cancer and Leukemia Group B Trial 9732. J Clin Oncol 23:3752–3759 37. Mavroudis D, Papadakis E, Veslemes M et al (2001) A multicenter randomized clinical trial comparing paclitaxel-cisplatin-etoposide versus cisplatin-etoposide as first-line treatment in patients with small-cell lung cancer. Ann Oncol 12:463–470 38. Reck M, von Pawel J, Macha HN et al (2003) Randomized phase III trial of paclitaxel, etoposide, and carboplatin versus carboplatin, etoposide, and vincristine in patients with small-cell lung cancer. J Natl Cancer Inst 95:1118–1127 39. Noda K, Nishiwaki Y, Kawahara M et  al (2002) Irinotecan plus cisplatin compared with etoposide plus cisplatin for extensive small-cell lung cancer. N Engl J Med 346:85–91 40. Hanna N, Bunn PA Jr, Langer C et al (2006) Randomized phase III trial comparing irinotecan/ cisplatin with etoposide/cisplatin in patients with previously untreated extensive-stage disease small-cell lung cancer. J Clin Oncol 24:2038–2043 41. Natale RB, Lara PN, Chansky K, et al (2008) S0124: a randomized phase III trial comparing irinotecan/cisplatin (IP) with etoposide/cisplatin (EP) in patients (pts) with previously untreated extensive stage small cell lung cancer (E-SCLC). J Clin Oncol 26(May 20 suppl; abstr 7512) 42. Hermes A, Bergman B, Bremnes R et al (2008) Irinotecan plus carboplatin versus oral etoposide plus carboplatin in extensive small-cell lung cancer: a randomized phase III trial. J Clin Oncol 26:4261–4267 43. Lara P Jr, Redman M, Lenz H, et al (2007) Cisplatin (Cis)/etoposide (VP16) compared to cis/ irinotecan (CPT11) in extensive-stage small cell lung cancer (E-SCLC): pharmacogenomic (PG) and comparative toxicity analysis of JCOG 9511 and SWOG 0124. J Clin Oncol ASCO Annual Meeting Proceedings Part I 25(18S) (June 20 suppl; abstr 7524) 44. Eckardt JR, von Pawel J, Papai Z et al (2006) Open-label, multicenter, randomized, phase III study comparing oral topotecan/cisplatin versus etoposide/cisplatin as treatment for chemotherapy-naive patients with extensive-disease small-cell lung cancer. J Clin Oncol 24:2044–2051 45. Steward WP, von Pawel J, Gatzemeier U et  al (1998) Effects of granulocyte-macrophage colony-stimulating factor and dose intensification of V-ICE chemotherapy in small-cell lung cancer: a prospective randomized study of 300 patients. J Clin Oncol 16:642–650 46. Woll PJ, Hodgetts J, Lomax L et al (1995) Can cytotoxic dose-intensity be increased by using granulocyte colony-stimulating factor? A randomized controlled trial of lenograstim in smallcell lung cancer. J Clin Oncol 13:652–659 47. Fukuoka M, Masuda N, Negoro S et al (1997) CODE chemotherapy with and without granulocyte colony-stimulating factor in small-cell lung cancer. Br J Cancer 75:306–309 48. Thatcher N, Girling DJ, Hopwood P et al (2000) Improving survival without reducing quality of life in small-cell lung cancer patients by increasing the dose-intensity of chemotherapy with granulocyte colony-stimulating factor support: results of a British Medical Research Council Multicenter Randomized Trial. Medical Research Council Lung Cancer Working Party. J Clin Oncol 18:395–404 49. Buchholz E, Manegold C, Pilz L et al (2007) Standard versus dose-intensified chemotherapy with sequential reinfusion of hematopoietic progenitor cells in small cell lung cancer patients with favorable prognosis. J Thorac Oncol 2:51–58 50. Lorigan P, Woll PJ, O’Brien ME et al (2005) Randomized phase III trial of dose-dense chemotherapy supported by whole-blood hematopoietic progenitors in better-prognosis smallcell lung cancer. J Natl Cancer Inst 97:666–674

Small Cell Carcinoma of the Lung

427

51. Johnson DH, Einhorn LH, Birch R et al (1987) A randomized comparison of high-dose versus conventional-dose cyclophosphamide, doxorubicin, and vincristine for extensive-stage small-cell lung cancer: a phase III trial of the Southeastern Cancer Study Group. J Clin Oncol 5:1731–1738 52. Hong WK, Nicaise C, Lawson R et al (1989) Etoposide combined with cyclophosphamide plus vincristine compared with doxorubicin plus cyclophosphamide plus vincristine and with high-dose cyclophosphamide plus vincristine in the treatment of small-cell carcinoma of the lung: a randomized trial of the Bristol Lung Cancer Study Group. J Clin Oncol 7:450–456 53. Ardizzoni A, Tjan-Heijnen VC, Postmus PE et al (2002) Standard versus intensified chemotherapy with granulocyte colony-stimulating factor support in small-cell lung cancer: a prospective European Organization for Research and Treatment of Cancer-Lung Cancer Group Phase III Trial-08923. J Clin Oncol 20:3947–3955 54. Klasa RJ, Murray N, Coldman AJ (1991) Dose-intensity meta-analysis of chemotherapy regimens in small-cell carcinoma of the lung. J Clin Oncol 9:499–508 55. Ihde DC, Mulshine JL, Kramer BS et al (1994) Prospective randomized comparison of highdose and standard-dose etoposide and cisplatin chemotherapy in patients with extensive-stage small-cell lung cancer. J Clin Oncol 12:2022–2034 56. Heigener DF, Manegold C, Jager E et al (2009) Multicenter randomized open-label phase III study comparing efficacy, safety, and tolerability of conventional carboplatin plus etoposide versus dose-intensified carboplatin plus etoposide plus lenograstim in small-cell lung cancer in “extensive disease” stage. Am J Clin Oncol 32:61–64 57. Crivellari G, Monfardini S, Stragliotto S et al (2007) Increasing chemotherapy in small-cell lung cancer: from dose intensity and density to megadoses. Oncologist 12:79–89 58. Humblet Y, Feyens AM, Sekhavat M et al (1989) Immunological and pharmacological removal of small cell lung cancer cells from bone marrow autografts. Cancer Res 49:5058–5061 59. Leyvraz S, Pampallona S, Martinelli G et al (2008) A threefold dose intensity treatment with ifosfamide, carboplatin, and etoposide for patients with small cell lung cancer: a randomized trial. J Natl Cancer Inst 100:533–541 60. Hanna NH, Sandier AB, Loehrer PJ Sr et al (2002) Maintenance daily oral etoposide versus no further therapy following induction chemotherapy with etoposide plus ifosfamide plus cisplatin in extensive small-cell lung cancer: a Hoosier Oncology Group randomized study. Ann Oncol 13:95–102 61. Schiller JH, Adak S, Cella D et al (2001) Topotecan versus observation after cisplatin plus etoposide in extensive-stage small-cell lung cancer: E7593 – a phase III trial of the Eastern Cooperative Oncology Group. J Clin Oncol 19:2114–2122 62. Matthews MJ, Kanhouwa S, Pickren J et al (1973) Frequency of residual and metastatic tumor in patients undergoing curative surgical resection for lung cancer. Cancer Chemother Rep 3(4):63–67 63. Choy H, Pass H, Rosell R, Traynor A (2006) Lung cancer. In: Chang AE, Ganz PA, Hayes DF et al (eds) Oncology an evidence-based approach. Springer, Berlin, pp 545–621 64. Janne PA, Freidlin B, Saxman S et al (2002) Twenty-five years of clinical research for patients with limited-stage small cell lung carcinoma in North America. Cancer 95:1528–1538 65. Arriagada R, Le Chevalier T, Pignon JP et al (1993) Initial chemotherapeutic doses and survival in patients with limited small-cell lung cancer. N Engl J Med 329:1848–1852 66. Arriagada R, Pignon JP, Le Chevalier T (2001) Initial chemotherapeutic doses and long-term survival in limited small-cell lung cancer. N Engl J Med 345:1281–1282 67. Turrisi AT III, Kim K, Blum R et al (1999) Twice-daily compared with once-daily thoracic radiotherapy in limited small-cell lung cancer treated concurrently with cisplatin and etoposide. N Engl J Med 340:265–271 68. Lad T, Piantadosi S, Thomas P et al (1994) A prospective randomized trial to determine the benefit of surgical resection of residual disease following response of small cell lung cancer to combination chemotherapy. Chest 106:320S–323S 69. Tsuchiya R, Suzuki K, Ichinose Y et al (2005) Phase II trial of postoperative adjuvant cisplatin and etoposide in patients with completely resected stage I–IIIa small cell lung cancer: the Japan Clinical Oncology Lung Cancer Study Group Trial (JCOG9101). J Thorac Cardiovasc Surg 129:977–983

428

E.O. Hanrahan and B. Glisson

70. Brock MV, Hooker CM, Syphard JE et al (2005) Surgical resection of limited disease small cell lung cancer in the new era of platinum chemotherapy: its time has come. J Thorac Cardiovasc Surg 129:64–72 71. National Comprehensive Cancer Network (NCCN) Practice Guidelines in Oncology – Small Cell Lung Cancer. v2.2.2009 72. Warde P, Payne D (1992) Does thoracic irradiation improve survival and local control in limited-stage small-cell carcinoma of the lung? A meta-analysis. J Clin Oncol 10:890–895 73. Perez CA, Einhorn L, Oldham RK et al (1984) Randomized trial of radiotherapy to the thorax in limited small-cell carcinoma of the lung treated with multiagent chemotherapy and elective brain irradiation: a preliminary report. J Clin Oncol 2:1200–1208 74. Perry MC, Eaton WL, Propert KJ et al (1987) Chemotherapy with or without radiation therapy in limited small-cell carcinoma of the lung. N Engl J Med 316:912–918 75. Bunn PA Jr, Lichter AS, Makuch RW et al (1987) Chemotherapy alone or chemotherapy with chest radiation therapy in limited stage small cell lung cancer. A prospective, randomized trial. Ann Intern Med 106:655–662 76. Pignon JP, Arriagada R, Ihde DC et al (1992) A meta-analysis of thoracic radiotherapy for small-cell lung cancer. N Engl J Med 327:1618–1624 77. Carney DN, Mitchell JB, Kinsella TJ (1983) In vitro radiation and chemotherapy sensitivity of established cell lines of human small cell lung cancer and its large cell morphological variants. Cancer Res 43:2806–2811 78. Choi NC, Carey RW (1989) Importance of radiation dose in achieving improved loco-regional tumor control in limited stage small-cell lung carcinoma: an update. Int J Radiat Oncol Biol Phys 17:307–310 79. Schild SE, Bonner JA, Shanahan TG et al (2004) Long-term results of a phase III trial comparing once-daily radiotherapy with twice-daily radiotherapy in limited-stage small-cell lung cancer. Int J Radiat Oncol Biol Phys 59:943–951 80. Bonner JA, Sloan JA, Shanahan TG et  al (1999) Phase III comparison of twice-daily split course irradiation versus once-daily irradiation for patients with limited stage small-cell lung carcinoma. J Clin Oncol 17:2681–2691 81. Movsas B, Moughan J, Komaki R et al (2003) Radiotherapy patterns of care study in lung carcinoma. J Clin Oncol 21:4553–4559 82. Choi NC, Herndon JE II, Rosenman J et al (1998) Phase I study to determine the maximumtolerated dose of radiation in standard daily and hyperfractionated-accelerated twice-daily radiation schedules with concurrent chemotherapy for limited-stage small-cell lung cancer. J Clin Oncol 16:3528–3536 83. Bogart JA, Herndon JE II, Lyss AP et al (2004) 70 Gy Thoracic radiotherapy is feasible concurrent with chemotherapy for limited-stage small-cell lung cancer: analysis of Cancer and Leukemia Group B study 39808. Int J Radiat Oncol Biol Phys 59:460–468 84. Fu KK, Pajak TF, Trotti A et al (2000) 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 48:7–16 85. Komaki R, Swann RS, Ettinger DS et al (2005) Phase I study of thoracic radiation dose escalation with concurrent chemotherapy for patients with limited small-cell lung cancer: report of Radiation Therapy Oncology Group (RTOG) protocol 97-12. Int J Radiat Oncol Biol Phys 62:342–350 86. Komaki R, Moughan J, Ettinger D, et al (2007) Toxicities in a phase II study of accelerated high dose thoracic radiation therapy (TRT) with concurrent chemotherapy for limited small cell lung cancer (LSCLC) (RTOG 0239). J Clin Oncol 2007 ASCO Annual Meeting Proceedings Part I 25(abstr 7717) 87. Komaki R, Paulus R, Ettinger DS, et  al (2009) A phase II study of accelerated high-dose thoracic radiation therapy (AHTRT) with concurrent chemotherapy for limited small cell lung cancer: RTOG 0239. J Clin Oncol 27:15s(suppl; abstr 7527) 88. Socinski MA, Bogart JA (2007) Limited-stage small-cell lung cancer: the current status of combined-modality therapy. J Clin Oncol 25:4137–4145

Small Cell Carcinoma of the Lung

429

89. Three different radiation therapy regimens in treating patients with limited-stage small cell lung cancer receiving cisplatin and etoposide. http://www.clinicaltrials.gov. Accessed March 2009 90. Murray N, Coy P, Pater JL et al (1993) Importance of timing for thoracic irradiation in the combined modality treatment of limited-stage small-cell lung cancer. The National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 11:336–344 91. Spiro SG, James LE, Rudd RM et al (2006) Early compared with late radiotherapy in combined modality treatment for limited disease small-cell lung cancer: a London Lung Cancer Group multicenter randomized clinical trial and meta-analysis. J Clin Oncol 24:3823–3830 92. Jeremic B, Shibamoto Y, Acimovic L et al (1997) Initial versus delayed accelerated hyperfractionated radiation therapy and concurrent chemotherapy in limited small-cell lung cancer: a randomized study. J Clin Oncol 15:893–900 93. Takada M, Fukuoka M, Kawahara M et  al (2002) Phase III study of concurrent versus sequential thoracic radiotherapy in combination with cisplatin and etoposide for limitedstage small-cell lung cancer: results of the Japan Clinical Oncology Group Study 9104. J Clin Oncol 20:3054–3060 94. Pottgen C, Eberhardt W, Stuschke M (2004) Prophylactic cranial irradiation in lung cancer. Curr Treat Options Oncol 5:43–50 95. Auperin A, Arriagada R, Pignon JP et al (1999) Prophylactic cranial irradiation for patients with small-cell lung cancer in complete remission. Prophylactic Cranial Irradiation Overview Collaborative Group. N Engl J Med 341:476–484 96. Slotman B, Faivre-Finn C, Kramer G et al (2007) Prophylactic cranial irradiation in extensive small-cell lung cancer. N Engl J Med 357:664–672 97. Komaki R, Meyers CA, Shin DM et al (1995) Evaluation of cognitive function in patients with limited small cell lung cancer prior to and shortly following prophylactic cranial irradiation. Int J Radiat Oncol Biol Phys 33:179–182 98. Arriagada R, Le Chevalier T, Borie F et al (1995) Prophylactic cranial irradiation for patients with small-cell lung cancer in complete remission. J Natl Cancer Inst 87:183–190 99. van Oosterhout AG, Boon PJ, Houx PJ et al (1995) Follow-up of cognitive functioning in patients with small cell lung cancer. Int J Radiat Oncol Biol Phys 31:911–914 100. Gregor A, Cull A, Stephens RJ et al (1997) Prophylactic cranial irradiation is indicated following complete response to induction therapy in small cell lung cancer: results of a multicentre randomised trial. United Kingdom Coordinating Committee for Cancer Research (UKCCCR) and the European Organization for Research and Treatment of Cancer (EORTC). Eur J Cancer 33:1752–1758 101. Fonseca R, O’Neill BP, Foote RL et al (1999) Cerebral toxicity in patients treated for small cell carcinoma of the lung. Mayo Clin Proc 74:461–465 102. O’Brien ME, Ciuleanu TE, Tsekov H et al (2006) Phase III trial comparing supportive care alone with supportive care with oral topotecan in patients with relapsed small-cell lung cancer. J Clin Oncol 24:5441–5447 103. Giaccone G, Donadio M, Bonardi G et al (1988) Teniposide in the treatment of small-cell lung cancer: the influence of prior chemotherapy. J Clin Oncol 6:1264–1270 104. Batist G, Ihde DC, Zabell A et al (1983) Small-cell carcinoma of lung: reinduction therapy after late relapse. Ann Intern Med 98:472–474 105. Postmus PE, Berendsen HH, van Zandwijk N et al (1987) Retreatment with the induction regimen in small cell lung cancer relapsing after an initial response to short term chemotherapy. Eur J Cancer Clin Oncol 23:1409–1411 106. Giaccone G, Ferrati P, Donadio M et al (1987) Reinduction chemotherapy in small cell lung cancer. Eur J Cancer Clin Oncol 23:1697–1699 107. Vincent M, Evans B, Smith I (1988) First-line chemotherapy rechallenge after relapse in small cell lung cancer. Cancer Chemother Pharmacol 21:45–48 108. Huisman C, Postmus PE, Giaccone G et al (1999) Second-line chemotherapy and its evaluation in small cell lung cancer. Cancer Treat Rev 25:199–206 109. Ardizzoni A, Manegold C, Debruyne C et al (2003) European organization for research and treatment of cancer (EORTC) 08957 phase II study of topotecan in combination with cisplatin

430

E.O. Hanrahan and B. Glisson

as second-line treatment of refractory and sensitive small cell lung cancer. Clin Cancer Res 9:143–150 110. Eckardt J, Gralla R, Palmer MC, Gandara D, Laplante J, Sandler A, Fields SZ, Fitts D, Broom C (1996) Topotecan (T) as second-line therapy in patients (Pts) with small cell lung cancer (SCLC): a phase II study (abstract). Ann Oncol 7(suppl 5):107 (Abstract 513P) 111. Depierre A, von Pawel J, Hans K et  al (1997) Evaluation of topotecan (Hycamtin™) in relapsed small cell lung cancer (SCLC). A multicentre phase II study (abstract). Lung Cancer 18(suppl 1):35 112. von Pawel J, Gatzemeier U, Pujol JL et al (2001) Phase II comparator study of oral versus intravenous topotecan in patients with chemosensitive small-cell lung cancer. J Clin Oncol 19:1743–1749 113. von Pawel J, Schiller JH, Shepherd FA et  al (1999) Topotecan versus cyclophosphamide, doxorubicin, and vincristine for the treatment of recurrent small-cell lung cancer. J Clin Oncol 17:658–667 114. Eckardt JR, von Pawel J, Pujol JL et al (2007) Phase III study of oral compared with intravenous topotecan as second-line therapy in small-cell lung cancer. J Clin Oncol 25:2086–2092 115. Masuda N, Fukuoka M, Kusunoki Y et al (1992) CPT-11: a new derivative of camptothecin for the treatment of refractory or relapsed small-cell lung cancer. J Clin Oncol 10:1225–1229 116. Joos G, Schallier D, Pinson P et  al (2004) Paclitaxel (PTX) as second line treatment in patients (pts) with small cell lung cancer (SCLC) refractory to carboplatin-etoposide: a multicenter phase II study (abstract). Proc Am Soc Clin Oncol 22:7211 117. Smit EF, Fokkema E, Biesma B et al (1998) A phase II study of paclitaxel in heavily pretreated patients with small-cell lung cancer. Br J Cancer 77:347–351 118. Smyth JF, Smith IE, Sessa C et al (1994) Activity of docetaxel (Taxotere) in small cell lung cancer. The Early Clinical Trials Group of the EORTC. Eur J Cancer 30A:1058–1060 119. Tiseo M, Ardizzoni A (2007) Current status of second-line treatment and novel therapies for small cell lung cancer. J Thorac Oncol 2:764–772 120. Onoda S, Masuda N, Seto T et  al (2006) Phase II trial of amrubicin for treatment of refractory or relapsed small-cell lung cancer: Thoracic Oncology Research Group Study 0301. J Clin Oncol 24:5448–5453 121. Yana T, Negoro S, Takada M et al (2007) Phase II study of amrubicin in previously untreated patients with extensive-disease small cell lung cancer: West Japan Thoracic Oncology Group (WJTOG) study. Invest New Drugs 25:253–258 122. Ettinger DS, Jotte R, Lorigan P, et al (2009) Results of a phase II trial of single-agent amrubicin (AMR) in patients with extensive disease small cell lung cancer (ED-SCLC) refractory to firstline platinum-based chemotherapy: an update. J Clin Oncol 27:15s(suppl; abstr 8103) 123. Jotte R, Conkling P, Reynolds C, et al (2009) Results of a randomized phase II trial of amrubicin (AMR) versus topotecan (Topo) in patients with extensive-disease small cell lung cancer (ED-SCLC) sensitive to first-line platinum-based chemotherapy. J Clin Oncol 27:15s(suppl; abstr 8028) 124. Ishimoto O, Inoue A, Sugawara S, et al. Final result of phase II study of amrubicin (AMR) combined with carboplatin (CBDCA) for elderly patients with small cell lung cancer (SCLC). J Clin Oncol 27:15s, 2009 (suppl; abstr 8054) 125. Eckardt JR, Bentsion DL, Lipatov ON, et al (2009) Phase II study of picoplatin as secondline therapy for patients with small-cell lung cancer. J Clin Oncol 27(12):2046–2051 126. Treat J, Schiller J, Quoix E et al (2002) ZD0473 treatment in lung cancer: an overview of the clinical trial results. Eur J Cancer 38(suppl 8):S13–S18 127. Balsara BR, Testa JR (2002) Chromosomal imbalances in human lung cancer. Oncogene 21:6877–6883 128. Petersen I, Langreck H, Wolf G et al (1997) Small-cell lung cancer is characterized by a high incidence of deletions on chromosomes 3p, 4q, 5q, 10q, 13q and 17p. Br J Cancer 75:79–86 129. Graziano SL, Pfeifer AM, Testa JR et  al (1991) Involvement of the RAF1 locus, at band 3p25, in the 3p deletion of small-cell lung cancer. Genes Chromosomes Cancer 3:283–293

Small Cell Carcinoma of the Lung

431

130. Sozzi G, Pastorino U, Moiraghi L et  al (1998) Loss of FHIT function in lung cancer and preinvasive bronchial lesions. Cancer Res 58:5032–5037 131. Sozzi G, Veronese ML, Negrini M et al (1996) The FHIT gene 3p14.2 is abnormal in lung cancer. Cell 85:17–26 132. Nakamura F, Kalb RG, Strittmatter SM (2000) Molecular basis of semaphorin-mediated axon guidance. J Neurobiol 44:219–229 133. Castro-Rivera E, Ran S, Thorpe P et al (2004) Semaphorin 3B (SEMA3B) induces apoptosis in lung and breast cancer, whereas VEGF165 antagonizes this effect. Proc Natl Acad Sci U S A 101:11432–11437 134. Tomizawa Y, Sekido Y, Kondo M et  al (2001) Inhibition of lung cancer cell growth and induction of apoptosis after reexpression of 3p21.3 candidate tumor suppressor gene SEMA3B. Proc Natl Acad Sci U S A 98:13954–13959 135. Agathanggelou A, Cooper WN, Latif F (2005) Role of the Ras-association domain family 1 tumor suppressor gene in human cancers. Cancer Res 65:3497–3508 136. Virmani AK, Rathi A, Zochbauer-Muller S et al (2000) Promoter methylation and silencing of the retinoic acid receptor-beta gene in lung carcinomas. J Natl Cancer Inst 92:1303–1307 137. Burbee DG, Forgacs E, Zochbauer-Muller S et  al (2001) Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. J Natl Cancer Inst 93:691–699 138. Dammann R, Li C, Yoon JH et al (2000) Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat Genet 25:315–319 139. Shimizu E, Coxon A, Otterson GA et  al (1994) RB protein status and clinical correlation from 171 cell lines representing lung cancer, extrapulmonary small cell carcinoma, and mesothelioma. Oncogene 9:2441–2448 140. Hainaut P, Hernandez T, Robinson A et al (1998) IARC Database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualisation tools. Nucleic Acids Res 26:205–213 141. Gazzeri S, Della Valle V, Chaussade L et al (1998) The human p19ARF protein encoded by the beta transcript of the p16INK4a gene is frequently lost in small cell lung cancer. Cancer Res 58:3926–3931 142. Chiappori A, Sereno M, Gabrilovich D, et  al (2007) Phase II trial of patients (pts) with extensive stage small cell lung cancer (ES-SCLC) immunized with: immune sensitization to chemotherapy (CT). J Clin Oncol 2007 ASCO Annual Meeting Proceedings Part I. 25(18S) (June 20 suppl; abstr 3012) 143. Available at: http://www.clinicaltrials.gov. 144. Prins J, De Vries EG, Mulder NH (1993) The myc family of oncogenes and their presence and importance in small-cell lung carcinoma and other tumour types. Anticancer Res 13:1373–1385 145. Adhikary S, Eilers M (2005) Transcriptional regulation and transformation by Myc proteins. Nat Rev Mol Cell Biol 6:635–645 146. Richardson GE, Johnson BE (1993) The biology of lung cancer. Semin Oncol 20:105–127 147. Takahashi T, Obata Y, Sekido Y et al (1989) Expression and amplification of myc gene family in small cell lung cancer and its relation to biological characteristics. Cancer Res 49:2683–2688 148. Noguchi M, Hirohashi S, Hara F et  al (1990) Heterogenous amplification of myc family oncogenes in small cell lung carcinoma. Cancer 66:2053–2058 149. Funa K, Steinholtz L, Nou E et al (1987) Increased expression of N-myc in human small cell lung cancer biopsies predicts lack of response to chemotherapy and poor prognosis. Am J Clin Pathol 88:216–220 150. Brennan J, O’Connor T, Makuch RW et al (1991) myc Family DNA amplification in 107 tumors and tumor cell lines from patients with small cell lung cancer treated with different combination chemotherapy regimens. Cancer Res 51:1708–1712 151. Hale AJ, Smith CA, Sutherland LC et  al (1996) Apoptosis: molecular regulation of cell death. Eur J Biochem 236:1–26

432

E.O. Hanrahan and B. Glisson

152. Sattler M, Salgia R (2003) Molecular and cellular biology of small cell lung cancer. Semin Oncol 30:57–71 153. Rudin CM, Salgia R, Wang X et al (2008) Randomized phase II Study of carboplatin and etoposide with or without the bcl-2 antisense oligonucleotide oblimersen for extensive-stage small-cell lung cancer: CALGB 30103. J Clin Oncol 26:870–876 154. Dy GK, Miller AA, Mandrekar SJ et  al (2005) A phase II trial of imatinib (ST1571) in patients with c-kit expressing relapsed small-cell lung cancer: a CALGB and NCCTG study. Ann Oncol 16:1811–1816 155. Krug LM, Crapanzano JP, Azzoli CG et al (2005) Imatinib mesylate lacks activity in small cell lung carcinoma expressing c-kit protein: a phase II clinical trial. Cancer 103:2128–2131 156. Schneider B, Gadgeel S, Ramnath N, et  al (2006) Phase II trial of imatinib maintenance therapy after irinotecan and cisplatin in patients with c-kit positive extensive-stage small cell lung cancer (ES SCLC). J Clin Oncol 24:18S (suppl; abstr 17089) 157. Rygaard K, Nakamura T, Spang-Thomsen M (1993) Expression of the proto-oncogenes c-met and c-kit and their ligands, hepatocyte growth factor/scatter factor and stem cell factor, in SCLC cell lines and xenografts. Br J Cancer 67:37–46 158. Takigawa N, Segawa Y, Maeda Y et al (1997) Serum hepatocyte growth factor/scatter factor levels in small cell lung cancer patients. Lung Cancer 17:211–218 159. Bharti A, Ma PC, Maulik G et al (2004) Haptoglobin alpha-subunit and hepatocyte growth factor can potentially serve as serum tumor biomarkers in small cell lung cancer. Anticancer Res 24:1031–1038 160. Maulik G, Kijima T, Ma PC et al (2002) Modulation of the c-Met/hepatocyte growth factor pathway in small cell lung cancer. Clin Cancer Res 8:620–627 161. Reeve JG, Payne JA, Bleehen NM (1990) Production of immunoreactive insulin-like growth factor-I (IGF-I) and IGF-I binding proteins by human lung tumours. Br J Cancer 61:727–731 162. Minuto F, Del Monte P, Barreca A et al (1986) Evidence for an increased somatomedin-C/ insulin-like growth factor I content in primary human lung tumors. Cancer Res 46:985–988 163. Yeh J, Litz J, Hauck P et al (2008) Selective inhibition of SCLC growth by the A12 anti-IGF1R monoclonal antibody correlates with inhibition of Akt. Lung Cancer 60:166–174 164. Warshamana-Greene GS, Litz J, Buchdunger E et al (2005) The insulin-like growth factor-I receptor kinase inhibitor, NVP-ADW742, sensitizes small cell lung cancer cell lines to the effects of chemotherapy. Clin Cancer Res 11:1563–1571 165. Pandya KJ, Dahlberg S, Hidalgo M et al (2007) A randomized, phase II trial of two dose levels of temsirolimus (CCI-779) in patients with extensive-stage small-cell lung cancer who have responding or stable disease after induction chemotherapy: a trial of the Eastern Cooperative Oncology Group (E1500). A preliminary report. J Thorac Oncol 2:1036–1041 166. Folkman J (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med 285:1182–1186 167. Folkman J (1990) What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 82:4–6 168. Hicklin DJ, Ellis LM (2005) Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 23:1011–1027 169. Dvorak HF (2002) Vascular permeability factor/vascular endothelial growth factor: a critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy. J Clin Oncol 20:4368–4380 170. Nagy JA, Vasile E, Feng D et al (2002) Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis. J Exp Med 196:1497–1506 171. Bremnes RM, Camps C, Sirera R (2006) Angiogenesis in non-small cell lung cancer: the prognostic impact of neoangiogenesis and the cytokines VEGF and bFGF in tumours and blood. Lung Cancer 51:143–158 172. Lucchi M, Mussi A, Fontanini G et al (2002) Small cell lung carcinoma (SCLC): the angiogenic phenomenon. Eur J Cardiothorac Surg 21:1105–1110 173. Pujol JL, Breton JL, Gervais R et al (2007) Phase III double-blind, placebo-controlled study of thalidomide in extensive-disease small-cell lung cancer after response to chemotherapy: an intergroup study FNCLCC cleo04 IFCT 00-01. J Clin Oncol 25:3945–3951

Small Cell Carcinoma of the Lung

433

174. Lee SM, Woll PJ, James LE et al (2007) A phase III randomised double blind, placebo controlled trial of etoposide/carboplatin with or without thalidomide in advanced small cell lung cancer (SCLC). J Thorac Oncol 2(8 suppl):S306–S307 175. Ready N, Dudek A, Wang XF et al (2007) CALGB 30306: a phase II study of cisplatin (C), irinotecan (I), and bevacizumab (B) for untreated extensive stage small cell lung cancer (ES-SCLC). J Clin Oncol 25(18 suppl):400s (Abstract 7563) 176. Sandler A, Szwaric S, Dowlati A et al (2007) A phase II study of cisplatin (P) plus etoposide (E) plus bevacizumab (B) for previously untreated extensive stage small cell lung cancer (SCLC) (E3501): a trial of the Eastern Cooperative Oncology Group. J Clin Oncol 25(18 suppl):400s (Abstract 7564) 177. Jalal SI, Bhatia S, Einhorn LH, et al (2008) Paclitaxel (P) plus bevacizumab (B) in patients (pts) with chemosensitive relapsed small cell lung cancer (SCLC): a safety, feasibility and efficacy trial from the Hoosier Oncology Group. J Clin Oncol 26(abstr 19013) 178. Patton JF, Spigel DR, Greco FA, et al (2006) Irinotecan (I), carboplatin (C), and radiotherapy (RT) followed by maintenance bevacizumab (B) in the treatment (tx) of limited-stage small cell lung cancer (LS-SCLC): update of a phase II trial of the Minnie Pearl Cancer Research Network. J Clin Oncol 2006 ASCO Annual Meeting Proceedings 24(abstr 7085) 179. Spigel DR, Hainsworth JD, Farley C et al (2008) Tracheoesophageal (TE) fistula development in a phase II trial of concurrent chemoradiation (CRT) and bevacizumab (B) in limitedstage small-cell lung cancer (LS-SCLC). J Clin Oncol 26(15 suppl):410s (Abstract 7554) 180. Gitlitz BJ, Glisson BS, Moon J et al (2008) Sorafenib in patients with platinum (plat) treated extensive stage small cell lung cancer (E-SCLC): a SWOG (S0435) phase II trial. J Clin Oncol 26(15 suppl):433s (Abstract 8039) 181. http://www.clinicaltrials.gov 182. Arnold AM, Seymour L, Smylie M et al (2007) Phase II study of vandetanib or placebo in small-cell lung cancer patients after complete or partial response to induction chemotherapy with or without radiation therapy: National Cancer Institute of Canada Clinical Trials Group Study BR.20. J Clin Oncol 25:4278–4284 183. Ramalingam SS, Mack PC, Vokes EE et al (2008) Cediranib (AZD2171) for the treatment of recurrent small cell lung cancer (SCLC): a California Consortium phase II study (NCI#7097). J Clin Oncol 26(15 suppl):443s (Abstract 8078) 184. Michael M, Babic B, Khokha R et al (1999) Expression and prognostic significance of metalloproteinases and their tissue inhibitors in patients with small-cell lung cancer. J Clin Oncol 17:1802–1808 185. Rigas JR, Denham CA, Rinaldi D et al (2003) Adjuvant targeted therapy in unresectable lung cancer: the results of two randomized placebo-controlled trials of BAY 12-9566, a matrix metalloproteinase inhibitor (MMPI). Lung Cancer 41(2 suppl):S34 (Abstract O107) 186. Shepherd FA, Giaccone G, Seymour L et al (2002) Prospective, randomized, double-blind, placebo-controlled trial of marimastat after response to first-line chemotherapy in patients with smallcell lung cancer: a trial of the National Cancer Institute of Canada-Clinical Trials Group and the European Organization for Research and Treatment of Cancer. J Clin Oncol 20:4434–4439 187. List AF, Hainsworth JD, Davis BW et al (1986) The syndrome of inappropriate secretion of antidiuretic hormone (SIADH) in small-cell lung cancer. J Clin Oncol 4:1191–1198 188. Ilias I, Torpy DJ, Pacak K et  al (2005) Cushing’s syndrome due to ectopic corticotropin secretion: twenty years’ experience at the National Institutes of Health. J Clin Endocrinol Metab 90:4955–4962 189. Shepherd FA, Laskey J, Evans WK et al (1992) Cushing’s syndrome associated with ectopic corticotropin production and small-cell lung cancer. J Clin Oncol 10:21–27 190. Delisle L, Boyer MJ, Warr D et al (1993) Ectopic corticotropin syndrome and small-cell carcinoma of the lung. Clinical features, outcome, and complications. Arch Intern Med 153:746–752 191. Graus F, Keime-Guibert F, Rene R et  al (2001) Anti-Hu-associated paraneoplastic encephalomyelitis: analysis of 200 patients. Brain 124:1138–1148 192. Alamowitch S, Graus F, Uchuya M et  al (1997) Limbic encephalitis and small cell lung cancer. Clinical and immunological features. Brain 120(Pt 6):923–928

434

E.O. Hanrahan and B. Glisson

193. Abrams J, Doyle LA, Aisner J (1988) Staging, prognostic factors, and special considerations in small cell lung cancer. Semin Oncol 15:261–277 194. Ihde DC (1985) Staging evaluation and prognostic factors in small cell lung cancer. In: Aisner J (ed) Lung cancer. Churchill Livingstone, New York, pp 241–268 195. Urban T, Chastang C, Vaylet F et al (1998) Prognostic significance of supraclavicular lymph nodes in small cell lung cancer: a study from four consecutive clinical trials, including 1,370 patients. “Petites Cellules” Group. Chest 114:1538–1541 196. Ihde DC, Makuch RW, Carney DN et al (1981) Prognostic implications of stage of disease and sites of metastases in patients with small cell carcinoma of the lung treated with intensive combination chemotherapy. Am Rev Respir Dis 123:500–507 197. Levitan N, Byrne RE, Bromer RH et al (1985) The value of the bone scan and bone marrow biopsy staging small cell lung cancer. Cancer 56:652–654 198. Norlund JD, Byhardt R, Foley WD, Unger GF, Cox JD (1985) Computed tomography in the staging of small cell lung cancer: implications for combined modality therapy. Int J Radiat Oncol Biol Phys 11:1081–1084 199. Castrucci WA, Knisely JP (2008) An update on the treatment of CNS metastases in small cell lung cancer. Cancer J 14:138–146 200. Seute T, Leffers P, ten Velde GP et al (2008) Detection of brain metastases from small cell lung cancer: consequences of changing imaging techniques (CT versus MRI). Cancer 112:1827–1834 201. Fried DB, Morris DE, Poole C et al (2004) Systematic review evaluating the timing of thoracic radiation therapy in combined modality therapy for limited-stage small-cell lung cancer. J Clin Oncol 22:4837–4845 202. De Ruysscher D, Pijls-Johannesma M, Bentzen SM et al (2006) 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 24:1057–1063 203. Pijls-Johannesma MC, De Ruysscher D, Lambin P, et al (2005) Early versus late chest radiotherapy for limited stage small cell lung cancer. Cochrane Database Syst Rev Jan 25(1):CD004700 204. De Ruysscher D, Pijls-Johannesma M, Vansteenkiste J et al (2006) Systematic review and meta-analysis of randomised, controlled trials of the timing of chest radiotherapy in patients with limited-stage, small-cell lung cancer. Ann Oncol 17:543–552 205. Huncharek M, McGarry R (2004) A meta-analysis of the timing of chest irradiation in the combined modality treatment of limited-stage small cell lung cancer. Oncologist 9:665–672

Mesothelioma Mary Frances McAleer, Reza J. Mehran, and Anne Tsao

Abstract  Mesothelioma is a rare but devastating malignancy originating from mesothelial cells lining serosal surfaces of the pleura, peritoneum, pericardium, and tunica vaginalis. This chapter focuses on malignant pleural mesothelioma (MPM). In the United States, the incidence of MPM is declining; however, the worldwide incidence of MPM is expected to increase over the next decade, particularly in Europe, Asia, and Australia. Development of MPM is characterized by disease manifestation several decades following asbestos exposure, accounting for the lag between asbestos regulation and peak incidence of MPM in developed countries. In vitro, malignant transformation of normal mesothelial cells by asbestos fibers may be influenced by DNA damage following cell cycle perturbation caused by disruption of mitotic spindles and reactive oxygen species plus altered signal transduction leading to activation of proto-oncogene expression. MPM is predominantly a locoregional, diffuse, aggressive disease with median survival of 9–17 months regardless of the stage of the disease. Multimodality therapy is becoming the standard treatment for resectable MPM. Extrapleural pneumonectomy (EPP) and pleurectomy/decortication (P/D) have been used to manage localized disease. To date, radiotherapy is the only treatment modality that has been shown to reduce locoregional recurrence following resection of MPM. Radiotherapy is used to prevent tract seeding by the malignant mesothelial cells following pleural intervention, to improve locoregional disease control postoperatively, and to palliate symptoms in unresectable patients. Improvements in chemotherapy, especially use of cisplatin and pemetrexed, have yielded survival benefit in these patients. Combination of surgery, radiotherapy, and chemotherapy has resulted in median survival of 20–28 months in the most favorable patients. Given these discouraging results, there is clearly a need to improve therapeutic outcomes in MPM. Ongoing studies are investigating novel targeted biologic agents alone and in combination with standard treatments to increase MPM patient survival.

M.F. McAleer (*), R.J. Mehran, and A. Tsao The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA e-mail: [email protected]

D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_17, © Humana Press, a part of Springer Science+Business Media, LLC 2010

435

436

M.F. McAleer et al.

Keywords  Mesothelioma • Asbestos • Mesothelin • Extrapleural pneumonectomy • Intensity-modulated radiotherapy • Pemetrexed • Molecular targeted biologic agents

Epidemiology and Etiology Mesothelioma is a rare malignancy derived from mesothelial cells lining serosal surfaces of the pleura, peritoneum, pericardium, and tunica vaginalis (1). This chapter focuses on malignant pleural mesothelioma (MPM). Approximately 3,000 people are diagnosed with MPM each year in the United States (2, 3). The majority of MPM cases is the result of asbestos exposure and manifest several decades following asbestos exposure, accounting for the lag between asbestos regulation and peak incidence of MPM in developed countries (4, 5). Asbestos was banned in the United States in 1967; subsequently, the incidence of MPM in the United States was thought to have peaked in 2005 (3, 6, 7). However, some have speculated that MPM incidence may rise as a direct result of thousands of people inhaling asbestos and other particulate debris following the collapse of the World Trade Center towers on September 11, 2001 (8). Meanwhile, the worldwide incidence of MPM continues to rise and is not expected to peak for a decade (6, 9–12), a pattern that corresponds to that in the United States and other developed countries in which asbestos was used industrially and then stringently regulated. Despite the incontrovertible link between asbestos exposure and MPM incidence, asbestos continues to be used globally (5). There are three main cohorts of people who develop MPM after exposure to asbestos: (a) those who mined and milled asbestos; (b) those who used manufactured asbestos products, such as asbestos insulation, in their work; and (c) those who were incidentally exposed to asbestos particles released into the atmosphere (6). The incidence of MPM is two to ten times greater among men than women, likely because there have been more men than women in the industrial work force (3, 7, 11, 13). In the United States, approximately 80% of MPM patients are men. MPM is more prevalent in older populations, and less than 2% of these patients present with the disease before the age of 40 years (Fig. 1) (13). Since Wagner et  al. first reported an association between asbestos exposure and MPM in 1960 (14), more than 80% of these cases have been attributed to asbestos. The risk of developing MPM after asbestos exposure is related to the duration and extent of exposure as well as the type of asbestos (5, 11). Amphibole asbestos, which includes crocidolite and amosite, is associated with a 100- to 500-fold higher risk of inducing mesothelial carcinogenesis than serpentine chrysotile asbestos (5, 11). The physical properties of different types of asbestos fibers may influence the mechanism by which asbestos triggers MPM. While straight amphibole asbestos fibers have a long half-life in the lung, the more soluble and easily fragmented chrysotile asbestos fibers are more quickly eliminated from the lung (5).

Mesothelioma

437

Fig. 1  The projected incidence of mesothelioma in men and women from 1973 to 2000 in the United States estimated from Surveillance, Epidemiology, and End Results Program data, and U.S. asbestos consumption from 1973 to 1992. “Males 2000” and “Females 2000” data points correspond to estimated number of mesothelioma cases in males and females, respectively, over the period from 1973 to 2000; “Males 1992” and “Females 1992” data points correspond to estimated number of mesothelioma cases in males and females, respectively, over the period from 1973 to 1992. Reproduced with permission from Oxford University Press (7)

Inhaled asbestos fibers may lodge in the pleura and perturb mesothelial cells by injuring the cells directly or indirectly by inducing the release of inflammatory cytokines. Mesothelial carcinogenesis caused by asbestos fibers may also be induced by DNA damage following cell-cycle perturbation caused by the disruption of mitotic spindles and reactive oxygen species. The malignant transformation of mesothelial cells has additionally been attributed to altered signal transduction caused by the phosphorylation of mitogen-activated protein and extracellular signal-regulated kinases, leading to the activation of proto-oncogene expression (5). Although one would anticipate a higher environmental exposure risk following the increased industrial use of asbestos between 1930 and 1970, the incidence of mesothelioma among women has remained constant. This finding suggests that that there is a threshold of asbestos exposure associated with a negligible incremental risk of developing MPM as well as a background level of nonasbestos-related MPM in the general population (7). Potential nonasbestos-related etiologies of mesothelioma include erionite, a naturally occurring fibrous mineral found in some building materials; autosomaldominant genetic inheritance; simian virus 40 (SV40), a potent double-stranded

438

M.F. McAleer et al.

DNA oncogenic virus; thorium dioxide (Thorotrast), a radiologic contrast material; and prior thoracic radiotherapy. The connection between erionite exposure and mesothelioma was identified after Baris et al. found an abnormally high number of MPM cases among residents of the Cappadocia region in Turkey (15). An analysis of the soil and stucco in the area revealed the presence of erionite, which was believed to have caused MPM in this population via carcinogenic mechanisms similar to those of asbestos. Pedigree analysis of the Cappadocia population identified an autosomal-dominant inheritance pattern, suggesting that the Cappadocia population had a possible genetic predisposition to developing MPM (16). However, because MPM was not found in family members born outside Cappadocia, an interaction between genetic predisposition and erionite exposure could have caused the disease (17). Erionite deposits have also been found in parts of the United States, Japan, Europe, and New Zealand; however, excess MPM incidences in these areas have not been identified. In 1994, SV40-like DNA sequences were identified in 60% of human mesothelioma specimens tested by the U.S. National Institutes of Health (18), suggesting a link between SV40 and MPM. The source of the SV40 was determined to be contaminated Salk poliovirus vaccine used between 1955 and 1963. Since the initial report, numerous studies have evaluated mesothelioma specimens for SV40. However, a recent review of these studies’ data found that earlier studies showing a high prevalence of SV40 DNA in mesothelioma specimens were not reproducible and that analyses conducted since 2002 using newer polymerase chain reaction-based techniques have been predominantly negative for SV40 (19). Several registry studies have examined the incidence of mesothelioma following the administration of alpha-emitting Thorotrast, an imaging agent used from 1930 to 1955 that is retained mostly in the liver, spleen, and lymph nodes for life, resulting in the constant irradiation of adjacent mesothelial surfaces. A Danish study suggested that higher Thorotrast exposure increases the incidence of mesothelioma (20), and a German study found nine cases of mesothelioma, including two peritoneal tumors adjacent to the spleen, in patients who received Thorotrast and no instances of mesothelioma in the study’s control cohort (21). In contrast, a Japanese study failed to demonstrate an excess of mesothelioma cases in patients who had received Thorotrast (22). Some studies have indicated that MPM may be caused by thoracic radiotherapy. Deutsch et al. found mesothelioma in three of 9,342 breast cancer patients who had received postoperative radiotherapy as part of breast-conserving therapy (23). None of the patients had a history of asbestos exposure, and MPM occurred on the same side as the breast cancer in each patient. Similarly, the incidence of MPM has been found to be higher in Hodgkin lymphoma and non-Hodgkin lymphoma survivors who received thoracic radiotherapy (24). Although this increase was significant only for male Hodgkin lymphoma survivors, the absolute number of case was very small: only 26 cases of mesothelioma were identified from approximately 22,000 Hodgkin lymphoma and 101,000 non-Hodgkin lymphoma patients.

Mesothelioma

439

Thus, while the data supporting the role of these nonasbestos agents as putative etiologies of MPM are conflicting, they may contribute to the background level of this rare malignancy (7).

Histologic Subtypes Mesothelioma results from the malignant transformation of mesothelial cells, which form a monolayer lining of the serosal surfaces of the pleura, peritoneum, pericardium, and tunica vaginalis. Mesothelioma has three histologic subtypes: epithelioid, the most common form of mesothelioma, comprising at least 50% of cases; sarcomatoid, comprising 15–20% of cases; and biphasic, comprising about 30% of mesothelioma cases (Fig. 2) (25). Historically, the histologic diagnosis of pleural mesothelioma has been difficult because of the tumor’s morphologic similarity to various benign (reactive) and malignant processes and the frequent inadequacy of tissue samples obtained for analysis. Epithelioid mesotheliomas are subclassified into tubulopapillary, deciduoid, storiform-like, fascicular-like, multicystic, papillary, microcystic, and granular tumors (26). Epithelioid mesothelioma and adenocarcinoma share many morphological features. Like adenocarcinoma, epithelioid mesothelioma stains positive for pan-cytokeratin, cytokeratin 7, and occasionally cytokeratin 20. Therefore, a panel of other immunohistochemical stains must be used to distinguish between adenocarcinoma and epithelioid mesothelioma because no marker that is specific to epithelioid mesothelioma but not adenocarcinoma (or vice-versa) has been identified (27). The use of a panel of markers increases the sensitivity and specificity in establishing the true diagnosis. Immunohistochemical stains supporting a diagnosis of epithelioid mesothelioma include calretinin, cytokeratins 5/6, and Wilms’ tumor gene (WT-1). Two recently identified markers, podoplanin and D2-40, stain virtually all epithelioid mesotheliomas and no adenocarcinomas and can thus be used to discriminate between these diagnoses (28). Mesothelin, previously used exclusively as a mesothelioma marker, has recently been shown to stain a large proportion of lung, ovarian, peritoneal, and pancreatic adenocarcinomas, thus rendering the stain

Fig. 2  Representative histology of (a) epithelioid, (b) biphasic, and (c) sarcomatoid mesothelioma stained with hematoxylin and eosin

440 Table  1  Immunohistochemical staining profile of from Beasley (27)) Histology Pancytokeratin Cytokeratin 5/6 Mesothelioma Epithelioid + + Biphasic + Epithelioid + Sarcomatoid + Rare Carcinoma Adenocarcinoma + − Sarcomatoid Weak −

M.F. McAleer et al. mesothelioma versus carcinoma (modified Calreticulin

WT-1

D2-40

+ + + Epithelioid + Epithelioid + Epithelioid + +/− Rare + − −

− −

− −

less effective in discriminating between epithelioid mesotheliomas and adenocarcinomas. However, a negative mesothelin stain can still be used to rule out epithelioid mesothelioma (Table 1) (29). Sarcomatoid, or mesenchymal, mesothelioma is subclassified according to whether the predominant connective tissue component is fibroblastic, musclelike, cartilaginous/osseous, angiomatoid, or fibrous (25). Sarcomatoid mesotheliomas mimic sarcomas and pulmonary sarcomatoid carcinomas. Combining the cytokeratin markers CAM5.2 and AE1/AE3 with WT-1 staining has been shown to have a 92.3% sensitivity and 95.3% specificity for identifying sarcomatoid mesothelioma (30). Biphasic, or “mixed,” mesothelioma has the morphologic and immunohistochemical staining features of both epithelioid and sarcomatoid mesothelioma (25, 27). Numerous multivariate analyses, including the two studies that established the commonly used prognostic scoring systems for mesothelioma patients, have shown that histology is a significant prognostic factor for survival. Specifically, a multivariate stepwise model used by the Cancer and Leukemia Group B (CALGB) to identify factors predictive of survival in mesothelioma patients revealed that a nonepithelioid histology is associated with a 33% increased risk of early death (31). Similarly, the European Organization for Research and Treatment of Cancer (EORTC) showed that patients with sarcomatoid mesothelioma have a 2.7-fold higher relative risk of death than patients with epithelioid mesothelioma. No significant difference between biphasic and epithelioid histologies was observed in this analysis (32).

Diagnosis and Staging The treatment of MPM patients largely depends on the stage of the tumor at diagnosis. The work-up of patients suspected to have MPM includes chest X-rays and computed tomography (CT) scans. The majority of MPM patients have a pleural-based mass and pleural effusion identified on these scans. Magnetic resonance imaging (MRI)

Mesothelioma

441

Fig. 3  Representative (a) coronal and (b) axial images of a fused positron emission tomography/ computed tomography scan from a patient with right-sided malignant pleural mesothelioma. The yellow signals correspond to high standardized uptake values. Note the involvement of pleural surfaces on the right with relative sparing of the underlying lung parenchyma

of the chest can be used to identify local invasion of the tumor into the ribs and/ or diaphragm; this information is important in determining whether the tumor is resectable. The role of positron emission tomography (PET) remains undefined in mesothelioma; however, because of mesothelioma’s increased metabolic activity, PET may be useful in identifying MPM patients who are not candidates for surgery because of contralateral nodal or distant metastatic disease (Fig. 3) (33, 34). In one study, multivariate analysis of PET scans from 65 MPM patients revealed that tumors with a high standard uptake value (SUV) were associated with a 3.3-fold higher risk of death than tumors with low SUVs (35), suggesting that PET scans may be prognostic indicators in MPM. Although their use in detecting mesothelioma has not yet been approved by the U.S. Food and Drug Administration, serum mesothelin-related peptide (SMRP) and serum osteopontin levels have been found to be associated with mesothelioma. SMRP level has been shown to have a sensitivity of 84% and a specificity of 100% for identifying mesothelioma and may be useful for monitoring tumor bulk and treatment response after surgical resection (36, 37). The mean SMRP level has been found to be significantly higher in MPM patients than in individuals with known asbestos exposure or lung cancer (37). SMRP levels have also been directly correlated with MPM stage: patients with stage 2–4 disease were found to have significantly higher SMRP levels than patients with stage 1 disease (37). Osteopontin, a glycoprotein involved in cell–matrix interactions, is overexpressed in many human tumors, including mesothelioma. High serum osteopontin levels in individuals exposed to asbestos have been found to be correlated with the presence of MPM, with a sensitivity of 77.6% and a specificity of 85.5% (38). Serum osteopontin levels have also been found to be independent prognostic factors for survival in MPM patients, with low serum levels corresponding to improved survival (39).

442

M.F. McAleer et al.

A definitive mesothelioma diagnosis requires histologic confirmation. The preferred method of obtaining a histologic specimen for pathologic evaluation is with a direct thoracoscopic approach. However, a combination of cytomorphologic analysis and an immunohistochemical staining panel that includes calretinin, epithelial membrane antigen, human milk fat globule membrane antigen-2, and carcinoembryonic antigen has been found to have a more than 90% sensitivity and specificity for diagnosing mesothelioma from pleural effusions (40). Following histologic confirmation of MPM, the disease is classified as localized or advanced using American Joint Committee on Cancer (AJCC) staging guidelines (41), which are based on criteria specified by the International Mesothelioma Interest Group (Table  2) (42). On the basis of a recent study of surgical nodal sampling, Flores et al. suggested updating the staging system for MPM based on the findings that patients with N1 disease have overall survival comparable to N0 disease, whereas those with multiple N2 nodal site involvement have significantly worse survival compared with single station N2 disease (43). MPM is clinically and pathologically staged. As with other malignancies, disease stage directs management and is prognostic of outcome (44). The role of locoregional therapies, namely surgery and radiotherapy, is determined principally by the extent of the primary tumor and lymph node involvement. Primary tumors are considered resectable if they involve only the ipsilateral pleura and no extrapleural structures except the endothoracic fascia, mediastinal fat, a solitary area of soft tissue on the chest wall, and/or nontransmural pericardium. Despite the complex nodal drainage patterns of the pleura, which are distinct from lung parenchymal lymphatic drainage, the AJCC’s guidelines for nodal staging mirror those used for primary bronchogenic tumors. MPM commonly metastasizes to the peritoneum, contralateral pleura, and contralateral lung, but it can also metastasize to other uncommon sites, including the thyroid and prostate (41). Given the morbidity and mortality associated with multimodality therapy for MPM, using laparoscopy and mediastinoscopy to accurately ascertain disease stage and identify candidates for aggressive treatment – specifically, those without stage IV disease – is essential (45).

Therapy for Locoregional Disease Surgery Surgical treatment options for MPM patients include video-assisted thoracotomy surgery (VATS), pleurectomy/decortication (P/D), and extrapleural pneumonectomy (EPP). VATS is used mainly for diagnostic and palliative purposes, while P/D and EPP are used with curative intent. EPP is generally considered the only surgical treatment that results in long-term, disease-free survival in MPM patients. EPP is an extensive procedure, consisting of en bloc removal of the parietal and visceral pleura, involved lung, mediastinal

Mesothelioma

443

Table 2  International Mesothelioma Interest Group Staging for pleural mesothelioma (modified from Zucali and Giaccone (156)) Identifier Definition Primary tumor (T) TX Not assessable T0 No evidence of primary tumor T1a Primary tumor involves ipsilateral parietal pleura, including mediastinal and diaphragmatic pleura, without visceral pleural involvement T1b Primary tumor involves ipsilateral parietal pleura, including mediastinal and diaphragmatic pleura, with focal visceral pleural involvement T2 Primary tumor involves ipsilateral parietal pleura and at least one of the following – Diaphragmatic muscle – Confluent visceral pleura (including fissure) – Extension into lung parenchyma T3 Locally advanced but potentially resectable tumor of ipsilateral pleural surface with involvement of at least one of the following – Endothoracic fascia – Mediastinal fat – Chest wall soft tissues (solitary focus) – Pericardium (nontransmural) T4 Locally advanced but technically unresectable tumor of ipsilateral pleural surface with at least one of the following – Diffuse or multifocal chest wall involvement – Rib destruction – Transdiaphragmatic extension into peritoneum – Direct extension into contralateral pleural – Direct extension into mediastinal organs – Invasion of spine – Extension to internal surface of pericardium – Pericardial effusion with positive cytology – Myocardial invasion – Brachial plexus invasion Regional lymph nodes (N) NX Not assessable N0 No nodal metastases N1 Metastases to ipsilateral bronchopulmonary and/or hilar lymph nodes N2 Metastases to subcarinal and/or ipsilateral mediastinal or internal mammary lymph nodes N3 Metastases to contralateral hilar, mediastinal, or internal mammary lymph nodes and/or ipsilateral or contralateral supraclavicular lymph nodes Distant metastases (M) MX Not assessable M0 No distant metastases M1 Distant metastases Stage TNM grouping IA T1a N0 M0 IB T1b N0 M0 II T2 N0 M0 III Any T3 Any N1–N2 M0 IV Any T4 Any N3 M1

444

M.F. McAleer et al.

lymph nodes, diaphragm, and pericardium. The procedure is usually reserved for patients who have had an extensive surgical staging and are found to not have mediastinal adenopathy or transdiaphragmatic spread of the disease. The 2-year overall survival rate for MPM patients who undergo only EPP ranges from 10 to 37%; however, survival rates may increase when EPP is performed in conjunction with other therapies (46–51). In recent series undertaken at major cancer centers, the mortality rate within 30 days of EPP ranged from 3.4 to 8% (52, 53). However, the morbidity rate for EPP is high, with frequent cardiac and pulmonary complications (50, 53, 54). Because of the high morbidity risk and the mortality risk associated with EPP, careful selection of patients for this procedure is warranted (48, 53, 55). P/D involves an open thoracotomy and the removal of the parietal and visceral pleura. Because P/D is a cytoreductive surgery, it should not be used alone if the goal is cure (56). Patients who undergo P/D alone often have disease recurrence, typically at the site of the primary tumor. However, P/D as part of a multimodality treatment approach may yield clinical benefits for MPM patients that are superior to those achieved with EPP (47). In a study by Flores et al., among 663 patients who underwent multimodality therapy with either P/D or EPP, patients who underwent EPP had a lower overall survival rate than those who underwent P/D (hazard ratio = 1.4, P < 0.001) (47). However, this analysis was retrospective and had potentially confounding factors, including a higher percentage of patients with earlystage disease in the P/D cohort. As expected, disease recurred predominantly at distant sites in patients who underwent EPP and adjuvant therapy, particularly highdose hemithoracic radiotherapy, but tended to recur locally in the ipsilateral hemithorax in patients who underwent P/D. Ongoing clinical trials such as the Mesothelioma Video-Assisted Thoracoscopic Cytoreductive Pleurectomy and Talc Pleurodesis (MesoVATS) trial and the Mesothelioma and Radical Surgery (MARS) trial may help clarify the optimal surgical therapy for MPM patients (11). The MesoVATS trial is a randomized phase III study aimed at determining whether surgical pleurectomy or palliative pleurodesis is more effective at preventing pleural fluid recurrence in patients with a malignant pleural effusion. The MARS trial is a phase III study in which surgically resectable MPM patients receiving neoadjuvant chemotherapy and radiotherapy are randomized to undergo EPP or no EPP. The MesoVATS trial results are anxiously awaited while the MARS trial is still recruiting.

Radiotherapy Radiotherapy has played several roles in the treatment of pleural mesothelioma patients, mainly as part of multimodality definitive treatment to improve locoregional control following en bloc resection of early-stage disease; as a prophylactic to reduce the incidence of painful recurrences in instrumentation tract sites; and as palliative therapy to relieve pain, dyspnea, and/or superior vena cava syndrome in patients with advanced disease. However, given the low incidence of mesothelioma,

Mesothelioma

445

few prospective data have been collected and few randomized control trials have investigated the efficacy of using radiotherapy to treat mesothelioma. Definitive Therapy Using radiotherapy alone as a potentially curative treatment for mesothelioma is neither practical nor recommended. Irradiation of the entire target tumor volume would expose the normal underlying lung tissue to an excessive radiation dose. When a therapeutic dose of 60 Gy is delivered to the pleura, >50% of the ipsilateral lung is likely to receive >20 Gy of radiation (57). Concerns about the high toxicity of this treatment are supported by a retrospective study in which 12 pleural mesothelioma patients were treated definitively with up to 50 Gy of radiation alone but no improvement in survival and a >16% treatment-related mortality rate was reported (58). Owing to the diffuse nature of the disease, which makes achieving clear surgical margins exceedingly difficult, local recurrence rates as high as 60% following aggressive surgical resection of early-stage pleural mesothelioma have been reported (59, 60). Not unexpectedly, the rate of locoregional recurrence observed after P/D, a less cytoreductive procedure than EPP, has been shown to exceed 90% (60). Various radiotherapy techniques have thus been used to reduce mesothelioma’s high recurrence rate following surgery. In a retrospective analysis of adjuvant radiotherapy in 123 patients treated nonpalliatively with P/D, 54 patients received intraoperative brachytherapy with permanent 125I seed implants (160 Gy matched peripheral dose), temporary 192Ir implants (30 Gy to 1-cm plane from implant), and/or 32P instillation (10–15 mCi in 0.5 L for an estimated 30 Gy to the pleural surface) to gross residual tumor (61). All patients were also treated with ipsilateral hemithoracic external beam radiotherapy (median dose, 42.5 Gy). The median overall survival was 13.5 months, and the 2- and 5-year actuarial overall survival rates were 23 and 5%, respectively. Although brachytherapy was associated with significantly shorter survival (11 months with brachytherapy vs. 18 months without brachytherapy, P = 0.006), this may have been due to the ineffectiveness of radiotherapy at eradicating gross residual disease in these patients. External-beam radiation doses ³40 Gy were associated with improved survival; however, selection bias, with healthier patients receiving higher doses of radiation, may have contributed to this outcome. The 1-year local control rate was 42%, with two thirds of patients eventually having a documented recurrence. Patients receiving <40 Gy and patients receiving ³40 Gy had the same time to locoregional recurrence. Four patients experienced grade 3 or 4 dyspnea and pneumonitis, and 10% of patients experienced severe chronic pulmonary symptoms that lasted for at least 3 months after completing radiotherapy. One patient died of pneumonitis after receiving 39.6 Gy but no brachytherapy. While postoperative radiotherapy following P/D significantly improved local disease control, the delivery of tumoricidal radiation doses was limited by the dose constraints of the remaining normal lung tissue.

446

M.F. McAleer et al.

Radiotherapy has also been used to improve local control and long-term outcomes following EPP. One of the earliest reports of adjuvant radiotherapy following EPP described the outcome of 120 consecutive pleural mesothelioma patients treated between 1980 and 1995 (62). In this series, all patients underwent en bloc resection of the ipsilateral lung, pleura, pericardium, and diaphragm; most patients then received four to six cycles of doxorubicin (50–60 mg/m2) and cyclophosphamide (600 mg/m2) with or without cisplatin (70 mg/m2) beginning 4–6 weeks after surgery; and then 30 Gy of external-beam radiotherapy to the ipsilateral hemithorax plus a 20- to 25-Gy boost to any regions at high risk for tumor recurrence. The median overall survival was 21 months, which was longer than the median survival of patients who underwent P/D and adjuvant radiotherapy (61). A subsequent subset analysis of 46 patients from the cohort treated with EPP and adjuvant therapy revealed that the most common site of first recurrence was the ipsilateral hemithorax (35% of this patient subset and 67% of all recurrences) (59). While only 35 of these patients received adjuvant radiotherapy, a comparison of local recurrence rates in patients who received radiotherapy and patients who did not receive radiotherapy revealed a nonsignificant trend towards improved local control following radiotherapy (local-only recurrence in 9% with vs. 27% without radiation). To improve the delivery of therapeutic doses of radiation – >40 Gy (63) to the at-risk surgical cavity – Rusch et al. conducted a phase II trial to test the feasibility of postoperatively treating the ipsilateral hemithorax with 54 Gy of radiation in 30 fractions using anterior and posterior fields with 6-MV or higher photons with electron supplementation in areas that required shielding of normal tissues (64). Of the 55 patients who underwent EPP followed by radiotherapy, seven patients (13%) had locoregional recurrence. Five patients experienced grade 4 toxicities, and two patients had life-threatening or fatal complications as a result of the treatment protocol. In a follow-up study, disease recurred locoregionally in 13 (37%) of 35 patients; in nine of these patients, disease recurred within the inferior border of the treatment field, which extended from the T12 to L3 vertebral bodies (65). To improve the therapeutic ratio of postoperative radiotherapy while sparing numerous adjacent normal structures, including the contralateral lung, spinal cord, heart, kidneys, liver, and esophagus, several groups have investigated the feasibility and tolerability of intensity-modulated radiotherapy (IMRT) (Fig.  4). In a retrospective analysis of 13 mesothelioma patients treated with adjuvant IMRT (54 Gy) following EPP and chemotherapy, Allen et  al. reported a 46% incidence of fatal pneumonitis with a median onset of 30 days from the completion of radiotherapy (66). Although all patients received chemotherapy, including 11 patients who received 225  mg/m2 heated cisplatin intrapleurally at the time of surgery, Allen et al. suggested that the high incidence of pneumonitis was related to the radiation dose delivered to the contralateral lung. In the patients who died of pulmonary toxicity, the volume of normal lung receiving ³20 Gy ranged from 15.3 to 22.3%; the volume of normal lung receiving ³5 Gy ranged from 81 to 100%; and the mean lung dose ranged from 13.3 to 17 Gy. The local tumor control rate with this regimen was not reported.

Mesothelioma

447

Fig. 4  Example of an intensity-modulated radiotherapy treatment plan for a patient with rightsided mesothelioma following extrapleural pneumonectomy. Representative (a) axial, (b) sagittal, and (c) coronal images are shown with the corresponding (d) dose-volume histogram and (e) regions of interest (ROI) statistics. cGy centigray; Bst boost; PTV planning target volume; CTV clinical target volume; lt or LT left; rt or RT right; Iso isodose

Ahamad et al. reported 100% local control in the contoured target volume after a median follow-up of 9 months (range, 5–27 months) among 28 pleural mesothelioma patients who received IMRT after EPP (49). Patients received 45–50 Gy, with focal boosted areas receiving 60 Gy. No grade 4 or higher toxicity was reported, and the most common side effects were grade 2 or 3 nausea or vomiting (65% of patients) and fatigue (62% of patients). The outcome of 61 patients receiving IMRT after EPP was reviewed. Only 3 patients (5%) had recurrent disease within the radiation portal, 8 patients (13%) had locoregional recurrences, and 33 patients (54%) had distant recurrences (52). The incidence of fatal pulmonary events, which occurred in only 10% of patients receiving IMRT after EPP (67), was markedly less than that reported by Allen et al (66). Fewer fatal pulmonary outcomes occurred in patients receiving mean lung doses £8.5 Gy, and the only predictive factor for pulmonary-related death was the volume of normal lung receiving ³20 Gy. Although this approach was tolerable and improved locoregional disease control, the median overall survival of patients who received IMRT after EPP was only 14.2 months (52). Although adjuvant radiotherapy following aggressive resection of MPM has been shown to improve locoregional disease control in certain patients, there have

448

M.F. McAleer et al.

been no randomized controlled trials supporting the use of radiotherapy either alone or as part of a multimodality treatment approach (68). Prophylactic Therapy The seeding of mesothelial tumor cells along instrumentation tract sites is a common complication following diagnostic and therapeutic pleural intervention in MPM patients, and occurs in 15–20% of cases (69, 70). Tract metastases are often painful and difficult to manage. Three prospective randomized trials have explored, using radiotherapy, ways to prevent malignant mesothelioma seeding along tract sites. Boutin et al. compared the incidence of tract metastasis in 20 pleural mesothelioma patients who received radiotherapy to sites of needle biopsy, thoracoscopy, or chest tube intervention to that in 20 patients who did not receive radiotherapy (69). Beginning 10–15 days after pleural intervention, 21 Gy in three fractions was delivered over 3 days using en face electrons (12.5–15 MeV) with a 1-cm paraffin bolus treated to the 90% isodose line. None of the patients who received radiotherapy developed subcutaneous nodules along the intervention sites, whereas eight patients (40%) who did not receive radiotherapy developed metastatic nodules (P < 0.001); of these, five patients had recurrences in thoracoscopy trocar or chest tube drain sites, and seven patients had nodules occurring along cytology or needle biopsy tracts. The mean time to tract recurrence in patients in the no-radiotherapy arm was 6 months (range, 1–13 months). Bydder et al. randomized 28 MPM patients who had undergone drain placement/ thoracoscopy, fine needle aspiration, or Abrams needle biopsy to receive a single fraction of 10 Gy to the procedure site with 9-MeV electrons prescribed at 100% (without a bolus) within 15 days of the thoracic intervention (71). The incidence of procedure site metastasis was then compared to that in 30 mesothelioma patients randomized to receive no radiotherapy. Regardless of the type of thoracic intervention, no difference in the rate of tract metastasis was found between the control arm (total of three metastases; 10%) and the radiotherapy arm (total of two metastases; 7%). O’Rourke et al. randomized 61 mesothelioma patients who underwent invasive pleural procedures to receive best supportive care only or 21 Gy in three consecutive fractions with 250-kV photons or 9- to 12-MeV electrons prescribed to 100% within 21 days (72). Tract metastases were identified in four patients receiving radiotherapy and in three patients receiving best supportive care only, with an estimated hazard ratio for developing tract metastasis in the two groups of 1.28 (95% confidence interval, 0.29–5.73). The median time to tract recurrence following pleural intervention was 2.4 months for the radiotherapy arm and 6.4 months for the best supportive care arm (P = 0.801). On the basis of these three studies’ findings, it is difficult to conclude whether prophylactic radiotherapy following chest wall intervention in MPM patients prevents disease recurrence at tract sites. First, all three trials included very few patients. Second, different radiation techniques were employed in each trial, specifically

Mesothelioma

449

with regard to dose, fractionation, and bolus use. Finally, the timing of radiation delivery varied among the trials.

Chemotherapy and Biological Therapy Although currently investigational, chemotherapy can be delivered in a neoadjuvant, intrapleural, or adjuvant setting in patients with resectable MPM. Previously, resectable mesothelioma was treated with surgery and radiotherapy only. Chemotherapy was not included because few agents had significant efficacy; however, this has changed with the advent of third-generation chemotherapeutics and new targeted agents. Ongoing clinical trials of drug treatments may ultimately lead to a change in standard practice. Neoadjuvant Chemotherapy To date, few trials of neoadjuvant chemotherapy have been completed, largely because of the small numbers of patients who are candidates for an aggressive regimen of chemotherapy followed by resection and subsequent radiotherapy. However, there are several advantages to using neoadjuvant chemotherapy in MPM. First, the chemotherapy may reduce the tumor size and facilitate surgical resection. Second, neoadjuvant treatment provides prognostic information: patients with chemosensitive tumors have a better prognosis than patients with tumors that do not respond to chemotherapy. Third, in patients with chemoresistant disease that continues to grow despite chemotherapy, futile surgical procedures that have significant associated morbidity and mortality can be avoided. The major disadvantage of neoadjuvant treatment is that some patients with resectable disease may develop toxicity that delays or prevents surgery. Five major prospective trials have explored the use of neoadjuvant chemotherapy, EPP, and adjuvant radiotherapy to treat mesothelioma (Table 3) (46, 73–76). These trials used platinum-doublets with either gemcitabine or pemetrexed and demonstrated median survival times ranging from 16.6 to 25.5 months. Patients who had a complete or partial response to the cisplatin–pemetrexed regimen had a greater overall survival than patients with stable or progressive disease (13.9 months vs. 9.1 months; P = 0.076) (46). Compliance with therapy varied, with completion rates of EPP after neoadjuvant chemotherapy ranging from 42 to 84%. Additional studies are needed to elucidate the benefit of neoadjuvant systemic therapy in MPM patients. However, the studies’ results indicate that with better systemic treatment options, it is possible to improve survival in MPM patients and potentially cure them with multimodality therapy. The addition of targeted therapies to neoadjuvant treatment provides a new avenue for clinical research. At The University of Texas M. D. Anderson Cancer Center, a trial of induction therapy with dasatinib, an oral Src kinase inhibitor, has

Table 3  Trials of neoadjuvant chemotherapy for resectable malignant pleural mesothelioma Adjuvant pN2, Median no. No. EPP, no. Response XRT, no. Study, no. patients PFS mos. patients Regimen cycles patients rate patients Histology, no. patients 34 Cisplatin– 4 50 29.3% 42 13.1 Krug et al. 60 epithelioid2 pemetrexed (46), 75 biphasic 1 sarcomatoid 12 other Weder et al. 14 epithelioid3 biphasic 0 Cisplatin– 3 16 32% 13 16.5 (73), 19 2 sarcomatoid gemcitabine Flores et al. 14 epithelioid4 biphasic 7 Cisplatin– 4 8 26% 8 NR (75), 21a 1 sarcomatoid gemcitabine 14 Cisplatin– 3 37 NR 36 13.5 Weder et al. 42 epithelioid14 gemcitabine (74), 61 biphasic 3 sarcomatoid 2 unknown Rea et al. (76), 20 epithelioid1 biphasic 5 Cisplatin– 4 17 33% 15 NR 21 gemcitabine pN2 pathologic N2; EPP extrapleural pneumonectomy; XRT radiotherapy; PFS progression-free survival; OS overall survival; NR not reported a Histology was reported on the 19 patients who underwent neoadjuvant chemotherapy

25.5

19.8

19.0

23.0

Median OS, mos. 16.6

450 M.F. McAleer et al.

Mesothelioma

451

been undertaken after preclinical studies showed that MPM tumors overexpress Src kinase and that dasatinib has antitumor efficacy against MPM cell lines (77). In this study, patients with resectable disease by EPP or P/D are scheduled to receive neoadjuvant dasatinib followed by surgical resection, adjuvant radiotherapy, and possibly additional adjuvant chemotherapy. Resected tumors are examined for modulation of the active Src kinase marker Tyr419; if positive for modulation, patients are eligible to receive dasatinib as maintenance therapy for two additional years. Intrapleural Chemotherapy Several trials have investigated delivering intrapleural chemotherapy after surgical resection of MPM. The rationale for intrapleural delivery is that the chemotherapy would be in direct contact with MPM cells because the tumor grows as a pleural rind around the lung. Most of these trials have used intrapleural cisplatin, but some have investigated novel agents such as liposome-entrapped cis-bisneodecanoatotrans-R-R-1,2-diaminocyclohexane platinum (II) (78–84). Other studies have investigated administering hyperthermic (42°C) intrapleural chemotherapy to increase chemotherapy uptake into the pleural space (82, 85–90). In general, these studies found that while hyperthermic intrapleural chemotherapy is feasible, the therapy causes additional toxicities following EPP or P/D – commonly atrial fibrillation and renal insufficiency (82, 86). Although these intrapleural therapies (normothermic or hyperthermic) were theoretically scientifically sound, they have not shown a survival benefit in clinical studies. Therefore, intrapleural chemotherapy for MPM patients should be limited to clinical trials (82, 86, 89, 90). Intrapleural Gene Therapy and Immunotherapy Intrapleural gene therapy involves injecting transgene-containing adenoviral vectors directly into the pleural space. Sterman et al. conducted a phase I trial in which 21 untreated MPM patients received intrapleural adenoviral vectors containing the herpes virus thymidine kinase suicide gene (Ad-HSVtk) and then underwent 2 weeks of ganciclovir therapy (91, 92). When ganciclovir is given after Ad-HSVtk has been incorporated into the tumor cells, thymidine kinase intracellularly metabolizes ganciclovir into ganciclovir triphosphate, a cytotoxin (93). The adenoviral vector also induces a host inflammatory response to tumor cells containing the transgene (91, 92, 94, 95). Ultimately, 11 patients had successful transfer of the HSVtk gene into the superficial layers of their tumors, and two patients survived long-term – over 6.5 years (92). However, further evaluation revealed that the antitumor effect was likely due to the host inflammatory effect rather than to a direct anticancer effect of the Ad-HSVtk. Subsequently, Sterman et  al. conducted a phase I trial in which an adenoviral vector containing interferon-b, an immune stimulant, was injected into the pleural space in ten patients (seven of whom had mesothelioma) (95). Seven patients had

452 Table 4  Intrapleural immunotherapy Agent Interferon-g (40 × 106 U) twice weekly for 2 months (98) Interleukin-2 (3 × 104 IU − 36 × 106 IU) (99) Interleukin-2 (21 × 106 IU/m2/day for 5 days) (97)

M.F. McAleer et al.

No. patients 19

No. responses (%) 5 (26)

21 22

4 (19) 12 (55)

successful gene transfer, and three of the mesothelioma patients had stable disease at 60 days. The main toxicities were transient hypoxia and reversible elevations of liver enzyme levels, but none of the side effects compromised the intrapleural delivery of adenoviral interferon-b. Several phase II trials have investigated intrapleural immunotherapy in mesothelioma patients. Although the therapies have shown some antitumor activity, it has not translated into a survival benefit (Table 4) (96–99). Among the regimens used have been interferon-g and interleukin-2 (IL-2). Adverse events have included fever, leucopenia, pleural empyema, catheter infection, and flu-like symptoms (97–99). Patients with epithelioid mesothelioma have had better outcomes than patients with sarcomatoid or biphasic mesothelioma (97). Another trial has studied the combination of intrapleural chemotherapy and adjuvant systemic chemotherapy. In 49 MPM patients, Lucchi et al. administered neoadjuvant intrapleural IL-2 (18 × 106 IU/day for 3 days) followed by P/D, postoperative intrapleural epirubicin (25 mg/m2 for 3 days), postoperative systemic IL-2 (18 × 106 IU/day for 3 days), adjuvant radiotherapy (30 Gy), adjuvant systemic chemotherapy (cisplatin (80 mg/m2) on day 1 and gemcitabine (1,250 mg/m2) on days 1–8 for six cycles), and maintenance therapy with subcutaneously administered IL-2 (3 × 106 IU/day, three times per week) (96). Patients’ median overall survival was 26 months, and the median duration of compliance for IL-2 maintenance therapy was 10 months. Novel intrapleural therapies with systemic chemotherapy, immunotherapy, or gene therapy have not yet shown a survival benefit for MPM patients, and whether the additional toxicity is warranted is not yet known. Currently, performing intrapleural therapy outside a clinical trial is not recommended.

Therapy for Advanced Disease Radiotherapy Radiotherapy has long been used to palliate pain, dyspnea, superior vena cava obstruction, Pancoast’s syndrome, dysphagia, neurological deficits secondary to brain metastases, and other symptoms associated with pleural mesothelioma. In an early retrospective review of symptomatic pleural mesothelioma patients, effective

Mesothelioma

453

palliation was associated with radiation doses ³40 Gy (63). In another retrospective analysis, 70% of patients undergoing palliative radiotherapy reported overall symptom improvement, with radiotherapy providing better control of pain and neurological deficits secondary to brain metastases than of superior vena cava obstruction or Pancoast’s syndrome (58). No clear dose effect was reported, with regimens of 20 Gy in five fractions producing symptom relief comparable to courses of 30–40 Gy in 10–15 fractions. Bissett et al. assessed the effectiveness of radiotherapy for palliation of chest wall pain using patient and physician reports of pain control and records of analgesic use before and following radiotherapy delivery (100). Although 13 of 19 evaluable patients initially reported reduced pain after radiotherapy, nine of 12 patients reported worsening pain by 3 months and six of seven patients reported worsening pain by 5 months. De Graaf-Strukowski et al. reported similar rates and pain relief duration based on self-reporting and analgesic use measurements. In that retrospective analysis, 91 patients treated with a median dose of 36 Gy delivered in 4-Gy fractions (including three patients who were also treated with hyperthermia) had 50% improvement of their chest wall pain, while only 39% of 73 patients treated with a median dose of 30 Gy delivered in smaller fractions (including 18 patients who were treated with hyperthermia) had 50% improvement of their pain (101). The median duration of pain relief was 69 days for patients receiving larger fractions and 98 days for patients receiving smaller fractions. However, the number of patients in all these studies was small, the radiotherapy techniques used in the studies were outdated, and the measure of symptom control was not standardized (102). Thus, a valid assessment of the efficacy of radiotherapy in the palliation of pleural mesothelioma symptoms is currently unavailable.

Chemotherapy Before 2003, the majority of agents used to treat advanced MPM-produced response rates below 20% (103). A 2006 meta-analysis of 119 trials for advanced MPM reported that combination chemotherapy yielded higher response rates than single-agent therapy, with cisplatin-containing regimens having the best results (104). When platinum agents were combined with gemcitabine or irinotecan, the response rate was 26.1%; platinum–anthracyclines combinations produced a response rate of 32.4%. However, an ongoing phase III trial comparing active supportive care to two chemotherapy regimens (mitomycin–vinblastine–cisplatin and weekly single-agent vinorelbine) has not so far shown that either chemotherapy regimen improves survival compared to active supportive care (105). Despite the unsatisfactory results of many prior chemotherapy studies for advanced MPM, the results of a phase III trial using cisplatin and pemetrexed demonstrate that advances in chemotherapy can be made (106). Pemetrexed, a multitargeted antifolate, inhibits purine and pyrimidine synthesis via dihydrofolate reductase, thymidylate synthase, and glycinamide ribonucleotide formyltransferase (107, 108). The trial was conducted in 456 chemo-naïve patients with advanced

454

M.F. McAleer et al.

MPM and compared cisplatin (75 mg/m2) and pemetrexed (500 mg/m2) administered every 3 weeks with cisplatin alone (75 mg/m2 IV every 3 weeks) (106). Patients treated with cisplatin and pemetrexed had a 41.3% response rate, a median time to progression of 5.7 months, and a median overall survival of 12.1 months. A qualityof-life analysis showed that symptoms improved by week 15 of therapy with the combination regimen (109). In the United States, this regimen is now the accepted standard of care for chemo-naïve patients with advanced MPM. Other studies have investigated combinations of platinum agents and antifolates such as raltitrexed, which has been investigated in Europe but not in the United States. Raltitrexed in combination with cisplatin yielded a median survival of 11.4 months but no improvement in quality of life (110). Studies of combined raltitrexed and carboplatin therapy yielded response rates ranging from 6 to 22%, a median time to progression of 6.5–7 months, and a median overall survival of 9.3–12.7 months (111–113). Although an antifolate is now the standard frontline therapy for unresectable MPM, other agents such as gemcitabine, vinorelbine, vinflunine, and irinotecan have also been investigated. Gemcitabine in combination with cisplatin has yielded response rates ranging from 16 to 48% and a median overall survival of 9.4–11.2 months (114–116). As single-agent therapies, gemcitabine and cisplatin yield more modest response rates ranging from 0 to 31% (117). Treatment with vinorelbine alone has yielded a response rate of 24% and a median overall survival of 10.6 months (118). Patients treated with newer vinca alkaloid agents such as vinflunine have had a 13.8% response rate and a median overall survival of 10.8 months (119). Irinotecan has been investigated in Japan and by the CALGB in the United States. However, while Japanese phase II trials of irinotecan in combination with other agents such as methotrexate or doxorubicin reported a response rate of 24%, the CALGB irinotecan trial showed a 0% response rate and substantial toxicity (120). On the basis of these results, irinotecan is no longer being studied in the United States. There is currently no standard regimen for mesothelioma that has failed to respond to frontline chemotherapy or that has recurred. As general guidelines, if pemetrexed was not given as frontline therapy, it should be given as salvage therapy alone or in combination with platinum-based therapy (121, 122). In patients who previously received pemetrexed, salvage therapy options include gemcitabine or vinorelbine given alone or in combination. In a phase II trial, the combination of gemcitabine (1,000  mg/m2) and vinorelbine (25  mg/m2), given on days 1 and 8 every 3 weeks for up to six cycles, was found to have activity in patients for whom pemetrexed-based therapy had failed (123). These patients had a response rate of 7.4%, with stabilization of disease in an additional 37% of patients, and a median time to progression of 2.8 months.

Biologic Therapy The future of MPM therapy rests on novel therapeutics. Several agents have been investigated in MPM, with variable results. Despite their success in treating other

Mesothelioma

455

solid and hematologic tumors, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) inhibitors have not shown any clinical benefit as single-agent therapies for MPM. Agents that have been studied include the EGFR inhibitors gefitinib and erlotinib (124, 125) and the PDGFR inhibitor imatinib mesylate (126). Although its activity as a single agent is limited, imatinib mesylate in combination with chemotherapy remains under investigation in clinical trials. Several other classes of agents are also being investigated. Anti-angiogenic Agents Bevacizumab, a monoclonal antibody that targets vascular endothelial growth factor (VEGF), has been shown to increase the overall survival duration in patients with colorectal, breast, and non-small-cell lung cancers. Although a phase II trial of 115 chemo-naïve MPM patients randomized to receive cisplatin and gemcitabine or cisplatin, gemcitabine, and bevacizumab demonstrated no difference in survival (127), subsequent analyses of plasma VEGF showed that patients with baseline plasma VEGF levels below the median had longer, progression-free and overall survival when treated with bevacizumab (P = 0.02 and 0.0066, respectively). Several oral multikinase inhibitors that target angiogenesis are being investigated for the treatment of advanced MPM. In a phase II trial, patients receiving vatalanib alone (targeting VEGF receptor (VEGFR)-1, -2, and -3, PDGFR, and c-Kit) had a response rate of 11%, a stable-disease rate of 66%, a median progression-free survival of 4.1 months, and a median overall survival of 10 months (128). A CALGB trial of sorafenib (targeting VEGFR-2, PDGFR, and Raf) given at 400 mg twice daily found a response rate of 4.4%, a median failure-free survival of 4.1 months, and a median overall survival of 10.4 months among MPM patients who were chemo-naïve or previously treated with pemetrexed (129, 130). Paradoxically, chemo-naïve patients had poorer outcomes than patients who had already received chemotherapy. Semaxanib (targeting Flk-1/ kinase domain insert receptor) and thalidomide have both shown some clinical activity in MPM patients (131, 132). Thalidomide alone also may stabilize MPM (133). Ongoing trials of other anti-angiogenic agents include trials of vandetanib (targeting VEGFR, EGFR, and RET) with pemetrexed in chemo-naïve patients (sponsored by CALGB); AZD2171 (targeting kinase domain insert receptor, Flt-1 and -4, and PDGFR) in previously treated patients (Southwest Oncology Group) and in chemo-naïve patients in combination with cisplatin–pemetrexed (Southwest Oncology Group 0905); sunitinib (targeting VEGFR-2, PDGFR-b, c-Kit, and Flt-3) in both frontline and salvage therapy settings (National Cancer Institute of Canada); and dasatinib (targeting Src, PDGFR, ephrin kinase, c-Kit, and bcr-abl) in the neoadjuvant (M. D. Anderson Cancer Center) and salvage therapy setting (CALGB). Thus, anti-angiogenic therapy may provide a benefit for certain MPM patients.

456

M.F. McAleer et al.

Histone Deacetylase Inhibitors Histone deacetylation prevents transcription factors from accessing genomic DNA and thereby leads to cell cycle progression and growth. Histone deacetylase inhibitors prevent deacetylation and establish control over the cell cycle. In MPM, the precise mechanism of action is unknown, although preclinical models suggest a direct antitumor effect with apoptosis. Studies have also suggested that caspase and bcl-xL activation may be involved in the antitumor effect (134, 135). Vorinostat, (suberoylanilide hydroxamic acid), a histone deacetylase inhibitor, showed some efficacy against advanced MPM in a phase I trial. MPM patients, most of whom had undergone previous therapy, received 300 or 400 mg of vorinostat orally twice daily for three consecutive days per week, and two partial responses were achieved (136). Side effects of treatment with histone deacetylase inhibitors include fatigue, anorexia, dehydration, nausea/vomiting, and diarrhea. On the basis of the phase I trial results, a phase III trial is investigating vorinostat as salvage therapy in MPM patients. Trials investigating vorinostat plus cisplatin and pemetrexed are also ongoing (137). Ribonuclease Inhibitors Ranpirnase controls carcinogenesis by targeting tumor-cell tRNA and inhibiting protein synthesis, which leads to cell-cycle arrest in the G1 phase. A phase III trial of 105 MPM patients receiving ranpirnase (480 mg/m2 weekly) or doxorubicin (60  mg/m2 every 3 weeks) did not reveal a difference in overall survival. However, in a subgroup analysis, the patients categorized as CALGB prognostic criteria groups 1–4 and EORTC low-risk criteria had a 2-month survival benefit when treated with ranpirnase rather than doxorubicin (138–141). The most common toxicities of ribonuclease inhibitors include drug hypersensitivity, renal toxicity (e.g., proteinuria and azotemia), fatigue, and peripheral edema. A large, international phase III trial comparing the efficacy of doxorubicin to that of doxorubicin and ranpirnase is ongoing. Proteasome Inhibitors Proteasome inhibitors block nuclear factor-kB and can cause apoptosis. Normal proteasomes form complexes with ubiquinated proteins to promote protein degradation. Murine xenograft studies have shown that proteasome inhibitors have some antitumor effect in MPM (142, 143). Currently, two European trials are investigating the proteosome inhibitor bortezomib alone and in combination with cisplatin for the treatment of MPM (142). Other Targets and Agents The c-Met tyrosine kinase oncogene is overexpressed and activated in MPM cell lines and tumor tissue (144, 145). c-Met mutations, primarily in the juxtamembrane

Mesothelioma

457

region, have also been reported to occur in the tumors of some MPM patients (144), although some reports suggest that this is a rare occurrence (146). Both c-Met siRNA and SU11274, a small-molecule tyrosine kinase inhibitor, exert an antitumor effect on mesothelioma cell lines by inhibiting cell migration and mobility. PHA665752, another MET inhibitor, has been used in preclinical models and is thought to have activity against MPM independent of MET mutation (146). The MET inhibitor 17-Allylamino-demethoxygeldanamycin targets the adenosine triphosphatebinding region of HSP90 and has preclinical antitumor activity against MPM (147). Another MET inhibitor, pegylated arginine deaminase, is being compared to best supportive care in an ongoing phase II trial for chemonaïve MPM patients (148). Additional new targets in mesothelioma include the insulin-like growth factor pathway, methyl ethyl ketone pathway, and the phosphatidylinositol-3-kinase/protein kinase B pathways (149–151). Antimesothelin monoclonal antibodies, such as SS1P, have been labeled with toxic molecules and are under investigation for MPM therapy (152, 153). Other possible targets for future intervention include protein kinase Cb2 (154), and methylated p15INK4B, p16INK4A, RASSF1A, and NORE1A (155), which are associated with poor prognosis in patients with MPM.

Conclusions For MPM patients with resectable tumors, surgery and adjuvant radiotherapy remain the standard practice in the United States. Some controversy remains over which type of surgical resection most benefits patients, but the MARS and MesoVATS trials should help elucidate this issue. Although a growing body of evidence suggests that systemic therapy may enhance the benefits of surgery and radiotherapy, this remains to be established in clinical trials. Unfortunately, the optimal multimodality management of MPM patients remains unclear. The use of systemic chemotherapy remains experimental, but further investigation of systemic treatment in clinical trials is warranted. For chemo-naïve MPM patients with unresectable tumors, treatment with cisplatin and pemetrexed is the standard approach in the United States. Although there is no standard salvage therapy for advanced MPM, single-agent gemcitabine or vinorelbine is commonly used. Further investigation of novel therapies is warranted, and enrolling advanced MPM patients in clinical trials is highly encouraged.

References 1. Hassan R, Alexander R (2005) Nonpleural mesotheliomas: mesothelioma of the peritoneum, tunica vaginalis, and pericardium. Hematol Oncol Clin North Am 19:1067–1087 2. Price B (1997) Analysis of current trends in United States mesothelioma incidence. Am J Epidemiol 145:211–218 3. Weill H, Hughes JM, Churg AM (2004) Changing trends in US mesothelioma incidence. Occup Environ Med 61:438–441

458

M.F. McAleer et al.

4. Lanphear BP, Buncher CR (1992) Latent period for malignant mesothelioma of occupational origin. J Occup Med 34:718–721 5. West SD, Lee YCG (2006) Management of malignant pleural mesothelioma. Clin Chest Med 27:335–354 6. Robinson BW, Lake RA (2005) Advances in malignant mesothelioma. N Engl J Med 353:1591–1603 7. Price B, Ware A (2004) Mesothelioma trends in the United States: an update based on surveillance, epidemiology, and end results program data for 1973 through 2003. Am J Epidemiol 159:107–112 8. Landrigan P, Lioy PJ, Thurston G et al (2004) NIEHS World Trade Center Working Group. Health and environmental consequences of the world trade center disaster. Environ Health Perspect 112:731–739 9. Lin R-T, Takahashi K, Karjalainen A et al (2007) Ecological association between asbestosrelated diseases and historical asbestos consumption: an international analysis. Lancet 369:844–849 10. Peto JHJ, Matthews FE, Jones JR (1995) Continuing increase in mesothelioma mortality in Britain. Lancet 345:535–539 11. Treasure T, Sedrakyan A (2004) Pleural mesothelioma: little evidence, still time to do trials. Lancet 364:1183–1185 12. Murayama TTK, Natori Y, Kurumatani N (2006) Estimation of future mortality from pleural malignant mesothelioma in Japan based on an age-cohort model. Am J Ind Med 49:1–7 13. Larson T, Melnikova N, Davis SI, Jamison P (2007) Incidence and descriptive epidemiology of mesothelioma in the United States, 1999–2002. Int J Occup Environ Health 13:398–403 14. Wagner J, Sleggs CA, Marchand P (1960) Diffuse pleural mesothelioma and asbestos exposure in the North Western Cape Province. Br J Ind Med 17:260–271 15. Baris YISA, Ozesmi M, Kerse I et al (1978) An outbreak of pleural mesothelioma and chronic fibrosing pleurisy in the village of Karain/Urgüp in Anatolia. Thorax 33:181–192 16. Carbone M, Emri S, Dogan AU et al (2007) A mesothelioma epidemic in Cappadocia: scientific developments and unexpected social outcomes. Nat Rev Cancer 7:147–154 17. Dikensoy O (2008) Mesothelioma due to environmental exposure to erionite in Turkey. Curr Opin Pulm Med 14:322–325 18. Carbone M, Pass HI, Rizzo P et  al (1994) Simian virus 40-like DNA sequences in human pleural mesothelioma. Oncogene 9:1781–1790 19. Shah K (2006) SV40 and human cancer: a review of recent data. Int J Cancer 120:215–223 20. Andersson M, Wallin H, Jönsson M et al (1995) Lung carcinoma and malignant mesothelioma in patients exposed to Thorotrast: incidence, histology and p53 status. Int J Cancer 63:330–336 21. van Kaick G, Dalheimer A, Hornik S et al (1999) The German thorotrast study: recent results and assessment of risks. Radiat Res 152:S64–S71 22. Ishikawa YMT, Machinami R (1995) Lack of apparent excess of malignant mesothelioma but increased overall malignancies of peritoneal cavity in Japanese autopsies with Thorotrast injection into blood vessels. J Cancer Res Clin Oncol 121:567–570 23. Deutsch M, Land SR, Begovic M, Cecchini R, Wolmark N (2007) An association between postoperative radiotherapy for primary breast cancer in 11 National Surgical Adjuvant Breast and Bowel Project (NSABP) studies and the subsequent appearance of pleural mesothelioma. Am J Clin Oncol 30:294–296 24. Teta M, Lau E, Sceurman BK, Wagner ME (2007) Therapeutic radiation for lymphoma: risk of malignant mesothelioma. Cancer 109:1432–1438 25. Attanoos R, Gibbs AR (1997) Pathology of malignant mesothelioma. Histopathology 30:403–418 26. Cerruto C, Brun EA, Chang D, Sugarbaker PH (2006) Prognostic significance of histomorphologic parameters in diffuse malignant peritoneal mesothelioma. Arch Pathol Lab Med 130:1654–1661 27. Beasley M (2008) Immunohistochemistry of pulmonary and pleural neoplasia. Arch Pathol Lab Med 132:1062–1072

Mesothelioma

459

28. Ordóñez NG (2007) What are the current best immunohistochemical markers for the diagnosis of epithelioid mesothelioma? A review and update. Hum Pathol 38:1–16 29. Ordóñez N (2003) The immunohistochemical diagnosis of mesothelioma: a comparative study of epithelioid mesothelioma and lung adenocarcinoma. Am J Surg Pathol 27:1031–1051 30. Kushitani K, Takeshima Y, Amatya VJ, Furonaka O, Sakatani A, Inai K (2008) Differential diagnosis of sarcomatoid mesothelioma from true sarcoma and sarcomatoid carcinoma using immunohistochemistry. Pathol Int 58:75–83 31. Herndon JE, Green MR, Chahinian AP, Corson JM, Suzuki Y, Vogelzang NJ (1998) Factors predictive of survival among 337 patients with mesothelioma treated between 1984 and 1994 by the Cancer and Leukemia Group B. Chest 113:723–731 32. Curran D, Sahmoud T, Therasse P, van Meerbeeck J, Postmus PE, Giaccone G (1998) Prognostic factors in patients with pleural mesothelioma: the European Organization for Research and Treatment of Cancer experience. J Clin Oncol 16:145–152 33. Benamore RE, O’Doherty MJ, Entwisle JJ (2005) Use of imaging in the management of malignant pleural mesothelioma. Clin Radiol 60:1237–1247 34. Truong MT, Marom EM, Erasmus JJ (2008) Preoperative evaluation of patients with malignant pleural mesothelioma: role of integrated CT-PET imaging. J Thorac Imaging 21:146–153 35. Flores RM (2005) The role of PET in the surgical management of malignant pleural mesothelioma. Lung Cancer 49:S27–S32 36. Robinson BW, Creaney J, Lake R et al (2005) Soluble mesothelin-related protein – a blood test for mesothelioma. Lung Cancer 49(Suppl 1):S109–S111 37. Pass HI, Wali A, Tang N et  al (2008) Soluble mesothelin-related peptide level elevation in mesothelioma serum and pleural effusions. Ann Thorac Surg 85:265–272 38. Pass HI, Lott D, Lonardo F et al (2005) Asbestos exposure, pleural mesothelioma, and serum osteopontin levels. N Engl J Med 353:1564–1573 39. Cappia S, Righi L, Mirabelli D et  al (2008) Prognostic role of osteopontin expression in malignant pleural mesothelioma. Am J Clin Pathol 130:58–64 40. Grefte J, de Wilde PC, Salet-van de Pol MR, Tomassen M, Raaymakers-van Geloof WL, Bulten J (2008) Improved identification of malignant cells in serous effusions using a small, robust panel of antibodies on paraffin-embedded cell suspensions. Acta Cytol 52:35–44 41. Greene FL, Balch CM, Page DL, Haller DG, Fleming ID, Morrow M, Fritz AG (eds) (2002) AJCC cancer staging manual, 6th edn. Springer, New York 42. Rusch VW (1995) A proposed new international TNM staging system for malignant pleural mesothelioma. From the International Mesothelioma Interest Group. Chest 108:1122–1128 43. Flores RM, Routledge T, Seshan VE et al (2008) The impact of lymph node station on survival in 348 patients with surgically resected malignant pleural mesothelioma: implications for revision of the American Joint Committee on Cancer staging system. J Thorac Cardiovasc Surg 136:605–610 44. Sugarbaker DJ, Strauss GM, Lynch TJ et al (1993) Node status has prognostic significance in the multimodality therapy of diffuse, malignant mesothelioma. J Clin Oncol 11:1172–1178 45. Rice DC, Erasmus JJ, Stevens CW et  al (2005) Extended surgical staging for potentially resectable malignant pleural mesothelioma. Ann Thorac Surg 80:1988–1992 discussion 92–93 46. Krug LM, Pass HI, Rusch V et al (2007) A multicenter phase II trial of neo-adjuvant pemetrexed plus cisplatin (PC) followed by extrapleural pneumonectomy (EPP) and hemithoracic radiation for stage I-III malignant pleural mesothelioma (MPM). Proc Am Soc Clin Oncol (abstr 7561) 47. Flores RM, Pass HI, Seshan VE et  al (2008) Extrapleural pneumonectomy versus pleurectomy/decortication in the surgical management of malignant pleural mesothelioma: results in 663 patients. J Thorac Cardiovasc Surg 135:620–626, 6e1–6e3 48. Rusch VW, Piantadosi S, Holmes EC (1991) The role of extrapleural pneumonectomy in malignant pleural mesothelioma. A Lung Cancer Study Group trial. J Thorac Cardiovasc Surg 102:1–9

460

M.F. McAleer et al.

49. Ahamad A, Stevens CW, Smythe WR et al (2003) Promising early local control of malignant pleural mesothelioma following postoperative intensity modulated radiotherapy (IMRT) to the chest. Cancer J 9:476–484 50. Martino D, Pass H (2004) Integration of Multimodality Approaches in the management of malignant pleural mesothelioma. Clin Lung Cancer 5:290–298 51. Jaklitsch MT, Grondin SC, Sugarbaker DJ (2001) Treatment of malignant mesothelioma. World J Surg 25:210–217 52. Rice DC, Stevens CW, Correa AM et al (2007) Outcomes after extrapleural pneumonectomy and intensity-modulated radiation therapy for malignant pleural mesothelioma. Ann Thorac Surg 84:1685–1692 discussion 92–93 53. Sugarbaker DJ, Jaklitsch MT, Bueno R et al (2004) Prevention, early detection, and management of complications after 328 consecutive extrapleural pneumonectomies. J Thorac Cardiovasc Surg 128:138–146 54. Chang MY, Sugarbaker DJ (2004) Extrapleural pneumonectomy for diffuse malignant pleural mesothelioma: techniques and complications. Thorac Surg Clin 14:523–530 55. Sugarbaker DJ, Flores RM, Jaklitsch MT et al (1999) Resection margins, extrapleural nodal status, and cell type determine postoperative long-term survival in trimodality therapy of malignant pleural mesothelioma: results in 183 patients. J Thorac Cardiovasc Surg 117:54–63 discussion 63–65 56. Rusch VW (1997) Pleurectomy/decortication in the setting of multimodality treatment for diffuse malignant pleural mesothelioma. Semin Thorac Cardiovasc Surg 9:367–372 57. Stevens CW, Forster KM, Smythe WR, Rice D (2005) Radiotherapy for Mesothelioma. Hematol Oncol Clin North Am 19:1099–1115 58. Ball D, Cruickshank DG (1990) The treatment of malignant mesothelioma of the pleura: review of a 5-year experience, with special reference to radiotherapy. Am J Clin Oncol 13:4–9 59. Baldini EH, Recht A, Strauss GM et al (1997) Patterns of failure after trimodality therapy for malignant pleural mesothelioma. Ann Thorac Surg 63:334–338 60. Pass HI, Temeck BK, Kranda K et  al (1997) Phase III randomized trial of surgery with or without intraoperative photodynamic therapy and postoperative immunochemotherapy for malignant pleural mesothelioma. Ann Surg Oncol 4:628–633 61. Gupta V, Mychalczak B, Krug L et al (2005) Hemithoracic radiation therapy after pleurectomy/ decortication for malignant pleural mesothelioma. Int J Radiat Oncol Biol Phys 63:1045–1052 62. Sugarbaker DJ, Garcia JP, Richards WG et  al (1996) Extrapleural pneumonectomy in the multimodality therapy of malignant pleural mesothelioma. Results in 120 consecutive patients. Ann Surg 224:288–294 discussion 94–96 63. Gordon WJ, Antman KH, Greenberger JS, Weichselbaum RR, Chaffey JT (1982) Radiation therapy in the management of patients with mesothelioma. Int J Radiat Oncol Biol Phys 8:19–25 64. Rusch VW, Rosenzweig K, Venkatraman E et al (2001) A phase II trial of surgical resection and adjuvant high-dose hemithoracic radiation for malignant pleural mesothelioma. J Thorac Cardiovasc Surg 122:788–795 65. Yajnik S, Rosenzweig KE, Mychalczak B et al (2003) Hemithoracic radiation after extrapleural pneumonectomy for malignant pleural mesothelioma. Int J Radiat Oncol Biol Phys 56:1319–1326 66. Allen AM, Czerminska M, Janne PA et al (2006) Fatal pneumonitis associated with intensitymodulated radiation therapy for mesothelioma. Int J Radiat Oncol Biol Phys 65:640–645 67. Rice DC, Smythe WR, Liao Z et al (2007) Dose-dependent pulmonary toxicity after postoperative intensity-modulated radiotherapy for malignant pleural mesothelioma. Int J Radiat Oncol Biol Phys 69:350–357 68. Chapman E, Berenstein EG, Diéguez M, Ortiz Z (2006) Radiotherapy for malignant pleural mesothelioma. Cochrane Database Syst Rev 3:CD003880 69. Boutin C, Rey F, Viallat JR (1995) Prevention of malignant seeding after invasive diagnostic procedures in patients with pleural mesothelioma. A randomized trial of local radiotherapy. Chest 108:754–758

Mesothelioma

461

70. Metintas M, Ak G, Parspour S et al (2008) Local recurrence of tumor at sites of intervention in malignant pleural mesothelioma. Lung Cancer 61:255–261 71. Bydder S, Phillips M, Joseph DJ et  al (2004) A randomised trial of single-dose radiotherapy to prevent procedure tract metastasis by malignant mesothelioma. Br J Cancer 91:9–10 72. O’Rourke N, Garcia JC, Paul J, Lawless C, McMenemin R, Hill J (2007) A randomised controlled trial of intervention site radiotherapy in malignant pleural mesothelioma. Radiother Oncol 84:18–22 73. Weder W, Kestenholz P, Taverna C et al (2004) Neoadjuvant chemotherapy followed by extrapleural pneumonectomy in malignant pleural mesothelioma. J Clin Oncol 22:3451–3457 74. Weder W, Stahel R, Bernhard J et al (2007) Multicenter trial of neo-adjuvant chemotherapy followed by extrapleural pneumonectomy in malignant pleural mesothelioma. Ann Oncol 18(7):1196–1202 75. Flores RM, Krug LM, Rosenzweig KE et  al (2006) Induction chemotherapy, extrapleural pneumonectomy, and postoperative high-dose radiotherapy for locally advanced malignant pleural mesothelioma: a phase II trial. J Thorac Oncol 1:289–295 76. Rea F, Marulli G, Bortolotti L et al (2007) Induction chemotherapy, extrapleural pneumonectomy (EPP) and adjuvant hemi-thoracic radiation in malignant pleural mesothelioma (MPM): feasibility and results. Lung Cancer 57:89–95 77. Tsao AS, He D, Saigal B et al (2007) Inhibition of c-Src expression and activation in malignant pleural mesothelioma tissues leads to apoptosis, cell cycle arrest, and decreased migration and invasion. Mol Cancer Ther 6:1962–1972 78. Rusch V, Saltz L, Venkatraman E et al (1994) A phase II trial of pleurectomy/decortication followed by intrapleural and systemic chemotherapy for malignant pleural mesothelioma. J Clin Oncol 12:1156–1163 79. Rusch VW (1994) Trials in malignant mesothelioma. LCSG 851 and 882. Chest 106(Suppl):359S–362S 80. Colleoni M, Sartori F, Calabro F et al (1996) Surgery followed by intracavitary plus systemic chemotherapy in malignant pleural mesothelioma. Tumori 82:53–56 81. Lee JD, Perez S, Wang HJ, Figlin RA, Holmes EC (1995) Intrapleural chemotherapy for patients with incompletely resected malignant mesothelioma: the UCLA experience. J Surg Oncol 60:262–267 82. Chang MY, Sugarbaker DJ (2004) Innovative therapies: intraoperative intracavitary chemotherapy. Thorac Surg Clin 14:549–556 83. Rice TW, Adelstein DJ, Kirby TJ et al (1994) Aggressive multimodality therapy for malignant pleural mesothelioma. Ann Thorac Surg 58:24–29 84. Lu C, Perez-Soler R, Piperdi B et al (2005) Phase II study of a liposome-entrapped cisplatin analog (L-NDDP) administered intrapleurally and pathologic response rates in patients with malignant pleural mesothelioma. J Clin Oncol 23:3495–3501 85. Zellos LS, Sugarbaker DJ (2002) Multimodality treatment of diffuse malignant pleural mesothelioma. Semin Oncol 29:41–50 86. Richards WG, Zellos L, Bueno R et al (2006) Phase I to II study of pleurectomy/decortication and intraoperative intracavitary hyperthermic cisplatin lavage for mesothelioma. J Clin Oncol 24:1561–1567 87. Yellin A, Simansky DA, Paley M, Refaely Y (2001) Hyperthermic pleural perfusion with cisplatin: early clinical experience. Cancer 92:2197–2203 88. Carry PY, Brachet A, Gilly FN et al (1993) A new device for the treatment of pleural malignancies: intrapleural chemohyperthermia preliminary report. Oncology 50:348–352 89. Van Ruth S, Baas P, Haas RL, Rutgers EJ, Verwaal VJ, Zoetmulder FA (2003) Cytoreductive surgery combined with intraoperative hyperthermic intrathoracic chemotherapy for stage I malignant pleural mesothelioma. Ann Surg Oncol 10:176–182 90. Ratto GB, Civalleri D, Esposito M et al (1999) Pleural space perfusion with cisplatin in the multimodality treatment of malignant mesothelioma: a feasibility and pharmacokinetic study. J Thorac Cardiovasc Surg 117:759–765

462

M.F. McAleer et al.

  91. Sterman DH, Treat J, Litzky LA et  al (1998) Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with localized malignancy: results of a phase I clinical trial in malignant mesothelioma. Hum Gene Ther 9:1083–1092   92. Sterman DH, Recio A, Vachani A et al (2005) Long-term follow-up of patients with malignant pleural mesothelioma receiving high-dose adenovirus herpes simplex thymidine kinase/ ganciclovir suicide gene therapy. Clin Cancer Res 11:7444–7453   93. Molnar-Kimber KL, Sterman DH, Chang M et al (1998) Impact of preexisting and induced humoral and cellular immune responses in an adenovirus-based gene therapy phase I clinical trial for localized mesothelioma. Hum Gene Ther 9:2121–2133   94. Sterman DH, Kaiser LR, Albelda SM (1998) Gene therapy for malignant pleural mesothelioma. Hematol Oncol Clin North Am 12:553–568   95. Sterman DH, Recio A, Carroll RG et al (2007) A phase I clinical trial of single-dose intrapleural IFN-beta gene transfer for malignant pleural mesothelioma and metastatic pleural effusions: high rate of antitumor immune responses. Clin Cancer Res 13:4456–4466   96. Lucchi M, Chella A, Melfi F et al (2007) A phase II study of intrapleural immuno-chemotherapy, pleurectomy/decortication, radiotherapy, systemic chemotherapy and long-term sub-cutaneous IL-2 in stage II-III malignant pleural mesothelioma. Eur J Cardiothorac Surg 31:529–533 discussion 33–34   97. Astoul P, Picat-Joossen D, Viallat JR, Boutin C (1998) Intrapleural administration of interleukin-2 for the treatment of patients with malignant pleural mesothelioma: a Phase II study. Cancer 83:2099–2104   98. Boutin C, Viallat JR, Van Zandwijk N et  al (1991) Activity of intrapleural recombinant gamma-interferon in malignant mesothelioma. Cancer 67:2033–2037   99. Goey SH, Eggermont AM, Punt CJ et al (1995) Intrapleural administration of interleukin 2 in pleural mesothelioma: a phase I-II study. Br J Cancer 72:1283–1288 100. Bissett D, Macbeth FR, Cram I (1991) The role of palliative radiotherapy in malignant mesothelioma. Clin Oncol (R Coll Radiol) 3:315–317 101. de Graaf-Strukowska L, van der Zee J, van Putten W, Senan S (1999) Factors influencing the outcome of radiotherapy in malignant mesothelioma of the pleura – a single-institution experience with 189 patients. Int J Radiat Oncol Biol Phys 43:511–516 102. Waite K, Gilligan D (2007) The role of radiotherapy in the treatment of malignant pleural mesothelioma. Clin Oncol 19:182–187 103. Ong ST, Vogelzang NJ (1996) Chemotherapy in malignant pleural mesothelioma. A review. J Clin Oncol 14:1007–1017 104. Ellis P, Davies AM, Evans WK, Haynes AE, Lloyd NS (2006) The use of chemotherapy in patients with advanced malignant pleural mesothelioma: a systematic review and practice guideline. J Thorac Oncol 1:591–601 105. Muers M, Fisher P, Snee M, et al (2007) A randomized phase III trial of active symptom control (ASC) with or without chemotherapy in the treatment of patients with malignant pleural mesothelioma: first results of the British Thoracic Society (BTS)/Medical Research Council (MRC) MS01 trial. Proc Am Soc Clin Oncol (abstr LBA7525) 106. Vogelzang NJ, Rusthoven JJ, Symanowski J et al (2003) Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol 21:2636–2644 107. Hazarika M, White RM, Booth BP et al (2005) Pemetrexed in malignant pleural mesothelioma. Clin Cancer Res 11:982–992 108. Hazarika M, White RM, Johnson JR, Pazdur R (2004) FDA drug approval summaries: pemetrexed (Alimta). Oncologist 9:482–488 109. Gralla R, Hollen P, Liepa A et al (2003) Improving quality of life in patients with malignant pleural mesothelioma: results of the randomized pemetrexed + cisplatin vs cisplatin trial using the LCSS-meso instrument. Proc Am Soc Clin Oncol 22:621 abstr 2496 110. Van Meerbeeck JP, Gaafar R, Manegold C et al (2005) Randomized phase III study of cisplatin with or without raltitrexed in patients with malignant pleural mesothelioma: an intergroup

Mesothelioma

463

study of the European Organisation for Research and Treatment of Cancer Lung Cancer Group and the National Cancer Institute of Canada. J Clin Oncol 23:6881–6889 111. Ceresoli GL, Zucali PA, Favaretto AG et al (2006) Phase II study of pemetrexed plus carboplatin in malignant pleural mesothelioma. J Clin Oncol 24:1443–1448 112. Bruno C, Luca C, Manlio M et al (2006) Pemetrexed (MTA) and carboplatin (CBDCA) in advanced pleural mesothelioma (MPM): evaluation of the activity and toxicity in a series of 178 chemonaive patients. Lung Cancer 54:S48 113. Papi M, Genestreti G, Tassinari D et al (2006) Pemetrexed in combination with carboplatin for the treatment of patients with malignant mesothelioma. Lung Cancer 54:S49 114. Byrne MJ, Davidson JA, Musk AW et  al (1999) Cisplatin and gemcitabine treatment for malignant mesothelioma: a phase II study. J Clin Oncol 17:25–30 115. Nowak AK, Byrne MJ, Williamson R et al (2002) A multicentre phase II study of cisplatin and gemcitabine for malignant mesothelioma. Br J Cancer 87:491–496 116. Van Haarst JM, Baas P, Manegold C et al (2002) Multicentre phase II study of gemcitabine and cisplatin in malignant pleural mesothelioma. Br J Cancer 86:342–345 117. Janne PA (2003) Chemotherapy for malignant pleural mesothelioma. Clin Lung Cancer 5:98–106 118. Steele JP, Shamash J, Evans MT, Gower NH, Tischkowitz MD, Rudd RM (2000) Phase II study of vinorelbine in patients with malignant pleural mesothelioma. J Clin Oncol 18:3912–3917 119. Margery J, Dabouis G, Dark G et al (2006) Vinflunine (VFL) in first line treatment of malignant pleural mesothelioma (MPM): final results of a European phase II study. Lung Cancer 54(Suppl):S47 120. Kindler H, Herndon J, Zhang C, Green M (2005) Irinotecan for malignant mesothelioma: a Phase II trial by the Cancer and Leukemia Group B. Lung Cancer 48:423–428 121. Sorensen J, Sundstrom S, Perell K, Thielsen A (2006) Pemetrexed second-line treatment in malignant pleural mesothelioma following platinum-based first-line treatment. Lung Cancer 54(Suppl):S46 122. Janne PA, Wozniak AJ, Belani CP et  al (2006) Pemetrexed alone or in combination with cisplatin in previously treated malignant pleural mesothelioma: outcomes from a phase IIIB expanded access program. J Thorac Oncol 1:506–512 123. Zucali P, Garassino I, Ceresoli G et al (2006) Treatment with gemcitabine and vinorelbine (GEMVIN) as second-line chemotherapy in pemetrexed-pretreated patients with malignant pleural mesothelioma. Lung Cancer 54(Suppl):S48 124. Govindan R, Kratzke RA, Herndon JE II et al (2005) Gefitinib in patients with malignant mesothelioma: a phase II study by the Cancer and Leukemia Group B. Clin Cancer Res 11:2300–2304 125. Garland LL, Rankin C, Gandara DR et al (2007) Phase II study of erlotinib in patients with malignant pleural mesothelioma: a Southwest Oncology Group Study. J Clin Oncol 25:2406–2413 126. Mathy A, Baas P, Dalesio O, van Zandwijk N (2005) Limited efficacy of imatinib mesylate in malignant mesothelioma: a phase II trial. Lung Cancer 50:83–86 127. Karrison T, Kindler H, Gandara D et al (2007) Final analysis of a multi-center, double-blind, placebo-controlled, randomized phase II trial of gemcitabine/cisplatin (GC) plus bevacizumab (B) or placebo (P) in patients with malignant mesothelioma. Proc Am Soc Clin Oncol (abstr 7526) 128. Jahan T, Wang L, Kratzke R et al (2006) Vatalanib (V) for patients with previously untreated advanced malignant mesothelioma (MM): a phase II study by the Cancer and Leukemia Group B (CALGB 30107). J Clin Oncol 24:7081 129. Janne P, Wang X, Krug L, Hodgson L, Vokes E, Kindler H (2006) Phase II trial of sorafenib (BAY) 43-9006) in malignant mesothelioma: CALGB 30307. Lung Cancer 54(Suppl):S51 130. Janne P, Wang X, Krug L, Hodgson E, Vokes E, Kindler H (2007) Sorafenib in malignant mesothelioma: a phase II trial of the Cancer and Leukemia Group (CALGB 30307). Proc Am Soc Clin Oncol 25(18S):435S abstr 7707

464

M.F. McAleer et al.

131. Kindler H, Vogelzang J, Chien K et al (2001) SU5416 in malignant mesothelioma: a University of Chicago Phase II Consortium Study. Proc Am Soc Clin Oncol 20:1359 abstr 1359 132. Pavlakis N, Williams G, Harvey R et al (2002) Thalidomide alone and in combination with cisplatin/gemcitabine chemotherapy for malignant mesothelioma (MM): preliminary results from two phase II studies. J Clin Oncol 21:19b 133. Baas P, Boogerd W, Dalesio O, Haringhuizen A, Custers F, Zandwijk NV (2005) Thalidomide in patients with malignant pleural mesothelioma. Lung Cancer 48:291–296 134. Cao XX, Mohuiddin I, Ece F, McConkey DJ, Smythe WR (2001) Histone deacetylase inhibitor downregulation of bcl-xl gene expression leads to apoptotic cell death in mesothelioma. Am J Respir Cell Mol Biol 25:562–568 135. Sonnemann J, Hartwig M, Plath A, Saravana Kumar K, Muller C, Beck JF (2006) Histone deacetylase inhibitors require caspase activity to induce apoptosis in lung and prostate carcinoma cells. Cancer Lett 232:148–160 136. Krug LM, Curley T, Schwartz L et al (2006) Potential role of histone deacetylase inhibitors in mesothelioma: clinical experience with suberoylanilide hydroxamic acid. Clin Lung Cancer 7:257–261 137. Sharma S (2006) Histone deacetylase inhibitors. Lung Cancer 54(suppl):S51 138. Vogelzang N, Taub R, Shin D et al (2000) Phase III randomized trial of onconase (ONC) vs. doxorubicin (DOX) in patients (Pts) with unresectable malignant mesothelioma (UMM): analysis of survival. Proc Am Soc Clin Oncol 19:2274 abstr 2274 139. Pavlakis N, Vogelzang NJ (2006) Ranpirnase – an antitumour ribonuclease: its potential role in malignant mesothelioma. Expert Opin Biol Ther 6:391–399 140. Fennell DA, Parmar A, Shamash J et al (2005) Statistical validation of the EORTC prognostic model for malignant pleural mesothelioma based on three consecutive phase II trials. J Clin Oncol 23:184–189 141. Von Pawel J, Costanzi J, Hardiman S (2006) A comparison of CALGB and EORTC paradigms in the assessment of ONCONASE for the treatment of unresectable malignant mesothelioma (UMM). Lung Cancer 54(Suppl):S51 142. Fennell D, Gaudino G, Porta C, Mutti L (2006) Proteasome inhibitors for treatment of malignant mesothelioma. Lung Cancer 54(Suppl):S50 143. Gordon G, Mani M, Maulik G, Mukhopadhyay L, Bueno R (2006) Preclincial studies of the protesome inhibitor bortezomib (velcade) in malignant pleural mesothelioma (MPM). Lung Cancer 54(Suppl):S50 144. Jagadeeswaran R, Ma PC, Seiwert TY et al (2006) Functional analysis of c-Met/hepatocyte growth factor pathway in malignant pleural mesothelioma. Cancer Res 66:352–361 145. Ramasamy J, Zumba O, Kindler H, Salgia R (2006) The role of c-Met in mesothelioma. Lung Cancer 54(Suppl):S53 146. Shimizu S, Krug L, Rushch V, Ladanyi M (2006) MET in mesothelioma: mutational screening, phosphorylation status, and sensitivity to the MET inhibitor PHA-665752. Lung Cancer 54(Suppl):S55 147. Korfee S, Gauler T, Nishio K et al (2006) 17-AAG, an inhibitor of HSP90, suppresses cellular growth of mesothelioma cell lines in vitro. Lung Cancer 54(Suppl):S54 148. Szlosarek P, Fennell D, Rudd R et al (2006) A randomized phase II trial of pegylated arginine deaminase (ADI-PEG 20) in patients with malignant pleural mesothelioma. Lung Cancer 54(Suppl):S54 149. Kratzke R (2006) IGF pathway activation in mesothelioma. Lung Cancer 54(Suppl):S54 150. Miraki-Mound F, Martinelli C, Camacho-Hubner C (2006) Effects of insulin-like growth factor binding protein-3 on human malignant pleural mesothelioma cell growth and apoptosis. Lung Cancer 54(Suppl):S53 151. Jacobson B, Patel M, De A et al (2006) Activation of 4E-BP1 represses IGF-1 mediated capdependent translation in malignant pleural mesothelioma. Lung Cancer 54(Suppl):S55 152. Hassan R, Bullock S, Premkumar A et al (2007) Phase I study of SS1P, a recombinant antimesothelin immunotoxin for targeted therapy of mesothelin expressing mesotheliomas, ovarian, and pancreatic cancer. Proc Am Soc Clin Oncol (abstr 3553)

Mesothelioma

465

153. Hassan R, Bera T, Pastan I (2004) Mesothelin: a new target for immunotherapy. Clin Cancer Res 10:3937–3942 154. Faoro L, Loganathan S, Westerhoff M et al (2008) Protein kinase C beta in malignant pleural mesothelioma. Anticancer Drugs 19:841–848 155. Destro A, Ceresoli GL, Baryshnikova E et al (2008) Gene methylation in pleural mesothelioma: correlations with clinico-pathological features and patient’s follow-up. Lung Cancer 159:369–376 156. Zucali PA, Giaccone G (2006) Biology and management of malignant pleural mesothelioma. Eur J Cancer 42:2706–2714

Advances in Oncology Clinical Research: Statistical and Study Design Methodologies B. Nebiyou Bekele

Abstract  In recent years, investigators have increasingly recognized the inadequacy of the current phase I, II, and III clinical trial framework. As a response, new designs using innovative statistical techniques have been developed to add increased flexibility to the drug development process. The primary focus of this chapter is to provide an overview of increasingly popular adaptive clinical trial designs and outline the advantages and limitations of such methods. In this chapter, we review advantages of adaptive designs over more traditional group sequential designs. Major topics to be covered include adaptive methods for dose finding in oncology, outcome adaptive clinical trial designs, randomized discontinuation designs, advances in the statistics of personalized medicine, the role of Bayesian methods within the adaptive design arena, and sample size re-estimation methods. Keywords  Adaptive designs • Innovative clinical trial designs

Introduction In recent years, investigators have increasingly recognized the inadequacy of the current phase I, II, and III clinical trial framework. As a response, new designs using innovative statistical techniques have been developed to add increased flexibility to the drug development process. The focus of this chapter is to discuss current trends in innovative clinical trial design and outline the advantages and limitations of such methods. The most common type of design used in clinical trials are fixed sample designs in which design input parameters, such as sample size, target population, and how patients are assigned to treatment, are determined during the design phase of the study. B.N. Bekele (*) Department of Biostatistics, Division of Quantitative Sciences, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA e-mail: [email protected] D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_18, © Humana Press, a part of Springer Science+Business Media, LLC 2010

467

468

B.N. Bekele

Consequently, if the assumptions that lead to the choice of a particular set of design input parameters are incorrectly specified, the trial is at increased risk of being negative, thus wasting the time and resources of those involved in the clinical trial process including patients, investigators, and the pharmaceutical sponsor. To mitigate the possible negative consequences of design input parameter misspecification, designs in which the input parameters are assessed during trial conduct are gaining increased popularity. One of the earliest advances in clinical trial design and conduct has been the development of group sequential methods for randomized clinical trials (1). In these designs, patients are randomized to treatment and the accumulating data is sequentially evaluated at prespecified points in the trial (usually after a certain number of patients have been evaluated for the primary endpoint or after a certain number of events have been observed). The goal of a group sequential design is to stop a trial early without increasing the type I error rate (i.e., the probability of declaring that an ineffective drug is effective at some point in the trial) and without decreasing the power (that is, the probability that a clinically beneficial drug is declared effective at some point in the trial). Early stopping may be for both superior efficacy, for futility (stopping an arm early because there is no treatment benefit from receiving that therapy) or for safety. The main characteristics of these designs include sequential monitoring of trial endpoints and early stopping based on interim accumulating evidence. An alternative to the group sequential framework described above is known as an adaptive clinical trial design. The basic idea behind an adaptive design strategy is to use data collected during the trial to impact some aspect of the trial design (e.g., sample size, randomization probabilities, target population, etc.). For example, later in this chapter, we will discuss adaptive methods that allow an investigator to assess, mid-course, if the sample size calculated at the start of the trial is truly adequate given the observed treatment effects, event rates, or other factors impacting the sample size calculation. Adaptive designs can also be used to target effective treatments by allocating more patients to those treatments, which appear most effective or be used to target patient subsets that are more likely to respond to a new therapy. The main advantage of adaptive designs is that they allow the investigator an increased flexibility, which is not possible with standard fixed sample or group sequential designs. Even with the added flexibility of adaptive designs there has been some resistance to their implementation. For example, Metha and Tsaitis provide a theoretical rationale for preferring group sequential designs over adaptive methods (2). They argue that well-designed group sequential methods are more efficient than adaptive designs. Yet, these theoretical arguments do not convincingly counter-balance four key arguments in favor or increased flexibility. First, the group sequential framework does not address all clinical trial design needs. Many times, endpoints, monitoring strategies, and scientific questions do not easily fall within the group sequential framework. This is because the group sequential framework provides stopping rules for early termination, but do not provide other types of guidance related to the clinical trial design. For example, phase I oncology clinical trials utilize interim decision rules, which go beyond early stopping (e.g., dose escalation/de-escalation).

Advances in Oncology Clinical Research: Statistical and Study Design Methodologies

469

Second, at an interim time-point the investigator may observe a smaller clinically meaningful effect than what was originally proposed during the design stage of the trial. This point is important because when sample sizes are calculated, they are based on some estimate of a compound’s treatment effect. Specifically, most traditional clinical trials typically do not target the smallest clinically meaningful treatment effect. This is due to the inverse relationship between treatment effect assumed and required sample size; the smaller the treatment effect, the larger the sample size required to adequately power the study. A consequence of the inverse relationship between the sample size and treatment effect is that investigators often choose an effect based on the resources the team is willing to commit to the trial. If the investigators do not have the financial wherewithal and clinical trial infrastructure for a large trial, then they target treatment effects that are larger than the smallest clinically meaningful effect so as to limit the total trial sample size. Yet, once some patients have been enrolled and their outcomes observed, the investigators may find that the treatment effects are smaller than originally hypothesized. In the situation described above, the adaptive clinical trial framework could be used to potentially adjust sample size upward based on the new estimate of the treatment effect. The third argument in favor of adaptive designs is that investigators may not wish to make an upfront monetary commitment for a large clinical trial in which the underlying assumptions are based on hypothesized treatment effects. Thus from a monetary perspective, one would commit some resources initially, wait and see how the trial progresses and then, if the results warrant, commit more resources. Lastly, adaptive designs allow for other types of mid-course corrections which would otherwise cause a trial to terminate or require protocol amendments, which must assure institutional review boards that the proposed changes do not call into question the study’s design integrity. Examples of mid-course corrections using adaptive methods include possibly changing the primary endpoint, or dropping a treatment arm or dose (3, 4). In the rest of the chapter, we discuss adaptive methods for dose finding in oncology, outcome adaptive clinical trial designs, randomized discontinuation designs, advances in personalized medicine, other adaptive methods, the role of Bayesian methods within the adaptive design arena, and sample size re-estimation methods. We then conclude the chapter with a discussion of factors impacting the future implementation of innovative design methods.

Adaptive Methods for Dose Finding in Oncology Traditionally, drugs developed in oncology have had the potential to produce severe toxicities. To ensure that large numbers of patients did not experience toxicity, phase I oncology trial designs have followed a different path than most other therapeutic areas. While in most therapeutic areas phase I clinical trials are usually carried out in healthy volunteers, in oncology, phase I trials of new compounds are carried out on cancer patients. In addition while the main purpose of phase I trials outside

470

B.N. Bekele

of oncology is to characterize the pharmacokinetic and pharmacodynamic profile of a compound, in oncology the main purpose of a phase I clinical trial is to determine the highest dose with acceptable toxicity. Because one of the most important constraints on the conduct of these trials is the need to limit the number of patients who experience severe toxicity, these studies are conducted in a sequential manner. Therefore, patients are enrolled in small cohorts, such that once a given cohort of patients has been treated and their outcomes assessed, the toxicity outcomes observed from these patients are used to select the dose for the next cohort. The purpose of this sequential approach is to decrease the chance that large numbers of patients are treated at doses that are too toxic. Those new to oncology drug development may wonder how dose-finding based on toxicity informs the researcher regarding which dose is best with respect to efficacy. The answer lies in the basic assumption that both toxicity and response increase with dose. Thus, phase I oncology trials attempt to determine the dose that has the highest acceptable toxicity level since by assumption this dose will also result in the highest efficacy. The highest dose with acceptable toxicity is called the maximum tolerated dose (MTD). While there are dozens of designs available to estimate the MTD, the two main design types used in practice are either algorithmic in nature (e.g., 3 + 3 design) or adaptive model-based designs (i.e., designs based on a statistical model). While the 3 + 3 design is the most commonly used design in phase I oncology settings, the purpose of this design is not to produce accurate estimates of the probability of toxicity at a given dose but to quickly identify a dose level that does not exhibit too much toxicity. Alternatively, adaptive model-based designs are more amenable to the goal of precisely estimating (i.e., estimating with more certainty) the MTD. An in-depth discussion of adaptive model-based designs for phase I oncology designs can be found in Chevret (5). Using these adaptive model-based designs requires that the investigator explicitly specify a target probability of toxicity. The target probability of toxicity represents the rate of toxicity acceptable to the investigator (the 3 + 3 design has an implicit target rate of toxicity of 17%). For compounds associated with very severe lifethreatening toxicities, the target probability may be set by the investigator at 0.10 (i.e., 10%), while for other compounds with more mild toxicities it may be acceptable to set the target probability of toxicity at 0.35. The decision to escalate, stay, or de-escalate from the current dose based on the most current data is made on the basis of which dose has expected probability of toxicity closest to the target toxicity. An important advantage of model-based phase I designs is that they combine information from patients treated at different dose levels, that is, to “borrow strength,” in order to more reliably predict the toxicity risk for future patients. A second advantage is the ability to adjust the target probability of toxicity to match the characteristics of the compound under investigation. For example, an investigator assessing the toxicity profile of a compound in a drug-class with the most common type of toxicity being 3 and 4 fatigue may be willing to accept a 40% toxicity rate, while an investigator assessing a new gene therapy may accept no more than a 10% toxicity rate. A third advantage of model-based methods is, unlike the 3 + 3 design, that the cohort size is not limited to three patients and more importantly a variable cohort

Advances in Oncology Clinical Research: Statistical and Study Design Methodologies

471

size may be used. Although one could argue that algorithmic designs can also use alternative cohort sizes, the complication associated with changing the cohort size when using X + X algorithmic approaches (i.e., 2 + 2, 4 + 4, 5 + 5, etc.) is that the implicit targeted rate toxicity changes with the size of the cohort. We should note that there are other algorithmic designs, which do not tie the implicit target toxicity rate to the cohort size but these methods are very rarely used and tend to place too many patients on doses that are too toxic (6). Although model-based designs have been available since the early 1990s, these methods have not gained as wide an audience as biostatisticians would like. One explanation for this is that it may be difficult to explain these adaptive methods to non-statisticians because the statistical models are complex and viewed as black boxes by some clinicians (7). In addition, the complex nature of the models requires the availability of computer code. While these are real concerns, we believe that the cost of increased complexity is more than outweighed by the added flexibility these designs provide. Below we review some of the innovative designs used in early phase oncology trials, to give the reader a sense of the types of adaptive trial designs developed for dose finding in oncology. One interesting innovation models time to toxicity in phase I oncology trials. Using this strategy, each cohort of patients is not required to be completely followed before the next patient or group is assigned a dose (unlike traditional designs for phase I clinical trials). This design has most appeal in cases where one wishes to evaluate the safety of a new compound over a long period of time (i.e., 3 months or longer). Using a traditional approach would result in trials that are excessively long. A new method, called the time-to-event continual reassessment method, allows patients to be entered into a trial before all patients currently enrolled have been completely observed (8–10). A trial enrolling 24 patients in cohorts of size 3 and utilizing a toxicity assessment window of 90 days would take 3 years to complete using a traditional method. Alternatively, the time required using the time-totoxicity method could be reduced to 15 months. There has been some discussion in the literature regarding how to ensure that the time-to-toxicity method is safe. Care should be taken in implementing the time-to-toxicity method when toxicities occur late in the assessment window and accrual is relatively rapid. Some authors propose an alternative approach, which allows the trial to suspend accrual if there is uncertainty regarding toxicity at a given dose (8). Another innovation in phase I trial designs focuses on determining the optimal number of cycles to treat a patient (11). Unlike typical phase I clinical trials which are designed to determine a maximum-tolerated dose, an investigator may be interested in determining how often (i.e., how many cycles of administration) an agent could be safely administered to determine the long-term toxicity due to cumulative toxicity effects. The framework accounts for a patient’s entire sequence of administrations. This work has been extended to jointly find the MTD and to determine the maximum tolerated number of cycles (12). As noted previously, the underlying assumption of oncology dose-finding trials is that both toxicity and response increase with dose. If this assumption is not tenable (for example, after a certain dose, increased treatment does not result in increased benefit), then using the typical phase I approach may not work.

472

B.N. Bekele

To address these issues, adaptive methods for dose finding in phase I/II clinical trials based on trade-offs between the probabilities of treatment efficacy and toxicity have been developed (6, 13–16). An investigator may take this approach because the underlying nature of the compound under investigation indicates that traditional assumptions of oncology dose-finding studies such as a monotone relationship between dose and toxicity does not exist. These methods are most effective when toxicity and efficacy are not correlated through dose or only correlated through dose for a subset of doses. The above ideas have been extended using an approach in which dose finding is based on jointly modeling a toxicity outcome and biomarker expression levels (17). In this work, the authors applied their method to a clinical trial of a new gene therapy for bladder cancer patients. In the design of this bladder cancer trial, biomarker expression was an indication that the new therapy was biologically active. For ethical reasons, the trial was designed such that patients would be enrolled sequentially, with the dose for each successive patient chosen using both toxicity and biomarker expression data from patients previously treated in the trial. The modeling framework incorporated correlation between the toxicity and biomarker expression. The authors showed that the design reliably chooses the preferred dose using both toxicity and expression outcomes under various clinical scenarios. While most phase I dose finding focuses on modeling toxicity as a binary outcome (dose-limiting toxicity is, for purposes of the statistical model, recorded as either being observed or it is not for a given patient), there has been some work in modeling toxicity by its severity. In this context, toxicity is modeled as a set of ordinal toxicity outcomes (18, 19). The work by Bekele and Thall was successfully used to model the relationship between toxicity and dose in a phase I trial of gemcitabine for soft tissue sarcoma. This approach was taken because in phase I oncology trials of new cytotoxic agents, patients typically are at risk of several qualitatively different toxicities, each occurring at several possible severity levels. The oncologists planning the trial desired to account for differences in importance among several toxicities, and they wanted the dose finding method to utilize the information that a low-grade toxicity observed at a given dose is a warning that a higher grade of that toxicity is likely to occur at a higher dose. Because conventional methods do not address these issues, they developed a method for dose finding based on a set of correlated, ordinal-valued toxicities with severity levels that vary with dose. They also developed a method for eliciting the set of weights quantifying the clinical importance of each level of each type of toxicity. Dose/schedule finding trials are a new type of oncology trials in which investigators aim to find a combination of dose and treatment schedule with a large probability of efficacy yet a relatively small probability of toxicity (20). A major difference between traditional dose finding and dose/schedule finding is that while the risk of toxicity is assumed to increase with dose in dose-finding trials, in dose/schedule finding trials the risk of toxicity adheres to a partial order. In this framework, the authors use a sensible dose–schedule allocation scheme for finding the dose and schedule most likely to result in acceptable levels of toxicity and high levels of response.

Advances in Oncology Clinical Research: Statistical and Study Design Methodologies

473

Outcome Adaptive Clinical Trials Randomized Outcome Adaptive Clinical Trials Once an acceptable dose has been selected during the dose-finding phase of a compound’s development, a whole host of methods are available for assessing the activity of a new treatment. In recent years, there has been an increased realization that some of the standard approaches to assessing the activity of a drug may be inadequate. For example, a new compound may not result in significant numbers of partial or complete responses in the general population of patients with the disease but may be active for a subset of patients. Thus using a standard approach like a Simon-two stage design targeting response for all patients would, in all likelihood, be a waste of time and effort. In this section, we focus on some innovative trial designs that could possibly be used to address the above situation concentrating on adaptive randomization and enrichment strategies. Randomized outcome adaptive clinical trials change the randomization probabilities in light of the accruing data (21–23). These studies start by randomizing patients to the different treatments with equal probability. These trials usually begin by initially enrolling some minimum number of patients using equal randomization. After this equal randomization phase, the randomization probabilities adapt in favor of the better performing treatments. The basic idea behind this method is to reduce the number of patients who receive inferior treatment while still accruing convincing evidence within the clinical trial. The following is an example of an application of adaptive randomization techniques. In this trial, the investigators evaluated the effectiveness of a combination of three drugs (an immunosuppressive agent, a purine analog anti-metabolite, and an anti-folate) to prevent graft-versushost disease (GVHD) after transplantation. The study used adaptive randomization and was designed to enroll a maximum of 150 patients where the first 30 patients enrolled into the trial were equally randomized to the three treatment arms. A success was defined in this study as “alive with successful engraftment, without relapse, and without GVHD 100 days after the transplant.” The design called for comparing each treatment to the control arm (i.e., the combination treatment with the immunosuppressive agent and anti-folate) in terms of the probability of success in the following manner. As information accrued about the treatments, the investigators altered the randomization probabilities from equal randomization to biased randomization based on the probability that each treatment-specific success probability exceeded that of the control arm success probability. That is, the randomization would adapt to favor treatments associated with success probabilities that were greater than that of the control. The use of an outcome adaptive strategy as outlined above typically impacts important parameters related to the trial design. These parameters include the total sample size, type I error rate, and power (and are generally known as the design’s operating characteristics). The only way to assess the impact of the proposed adaptive decision rules on the design’s operating characteristics is via computer simulation.

474

B.N. Bekele

Consequently, computer simulation has become an important tool in assessing and implementing adaptive methods. Additionally, one of the biggest challenges to utilizing adaptive methods is the availability of software to assess the designs. This software summarizes the average behavior of the proposed method under a wide variety of situations (called scenarios). In addition to the power, type I error rate, etc., other factors summarized as part of the operating characteristics include the number of patients assigned to each treatment, the probability of selecting a treatment as most efficacious, and the probability of stopping a trial if all treatments are ineffective. The statistician should consider a wide variety of possible scenarios ranging from very pessimistic, such as the case when no treatment provides any benefit, to optimistic cases in which treatment benefits are assumed to be large. Above we discussed the need for computer code for computer simulations for adaptive designs. Computer simulations are not only needed for outcome adaptive designs to assess the activity of a drug, but are also used in the other contexts such as phase I studies.

Real-Time Updating of Adaptive Clinical Trials In addition to having computer code for simulations, many adaptive methods (including model-based dose-finding designs and adaptive randomization designs require software for real-time updating of the statistical model developed for the clinical trial. Real-time updating is an important aspect of adaptive trial designs because many of these designs incorporate early stopping rules allowing the investigator to stop for lack of efficacy, superiority, excessive toxicity, or they incorporate data-dependent decision rules. For example, in a single arm phase II trial which compares the progression-free survival rate of a new compound against a historical control, an investigator may desire to stop the trial early if there is evidence that the new compound results in worse outcomes relative to the historical standard. A common stopping rule under this setup is to stop the trial if at any point there is high probability that the risk of progression in the experimental arm is lower than the historical risk of progression. This probability is computed each time a new patient is slated to enter the trial or when a patient already enrolled experiences disease progression. Typically, calculation of this probability is achieved computationally and requires development of statistical software to monitor the stopping rules and assess if the stopping boundaries have been crossed. Note that software can mean anything from a simple macro written solely for use by the collaborating statistician to a stand-alone desk-top computer program written for use by other statisticians, to a web-based application intended for use by nonstatistician research staff. The kind of application that is developed is a function of who the end-user will be and how often the design will be implemented. For example, for a single-institution trial designed to evaluate outcomes for a rare disease with slow accrual in which the statistical model must be updated every 4 to 6 weeks or so, all that may be required is a simple macro which will be used by

Advances in Oncology Clinical Research: Statistical and Study Design Methodologies

475

the collaborating statistician. In contrast, a rapidly accruing multi-center, multi-arm study may require that real-time updating be performed via a web-based application or a telephone voice-response system. Part of the work involved in implementing adaptive methods is in determining the exact software needs of a particular trial design. In addition, the investigators should include someone knowledgeable about the components needed to carry out an adaptive design. These components include: (1) the statistical engine; (2) the database; and (3) the user interface. The statistical engine performs all of the statistical calculations. The user typically does not see this part of the program. The database stores all of the patient level data used for statistical calculation by the statistical engine. The user interface is used to input data (which is then stored in the database), or obtain output such as the treatment randomization of the next patient enrolled into a clinical trial implementing an adaptive randomization scheme. The level of programming sophistication used in a clinical trial is a function of the type of trial, the type of target user, the resources the investigator is willing to invest into the trial, and how quickly the trial needs to be up and running. The more sophisticated the design, the more seamless the interaction between the statistical engine, database, and interface.

Randomized Discontinuation Trials An alternative adaptive strategy is based on a group of clinical trial methods known as enrichment designs. An example of an enrichment design is the randomized discontinuation design (24). This approach is used for testing new agents in heterogeneous populations. In this design, all enrolling patients are initially treated with an experimental treatment and subsequently followed for some fixed period of time (this period is disease-specific and should be sufficient to assess whether the patient has experienced response, stable disease, or progression). After the patients are evaluated for response, those patients who respond to treatment continue receiving treatment with the new drug, while those patients who have experienced disease progression are moved off the drug/study. Lastly, patients who have experienced stable disease are randomly assigned between continued administration of the drug or standard of care and then followed for another fixed period of time. The rationale for randomizing patients in stable disease stems from the need to determine whether those patients are stable due to the treatment or due to the indolent nature of the disease. The main purpose of the design is to provide an enrichment strategy to focus on those patients most likely to benefit from treatment (yet for whom benefit is unclear). For an enrichment strategy to work, the patients selected for the randomized discontinuation phase of the study should be relatively homogeneous. If this is not the case, then the strategy is more likely to fail. For example, restricting the randomized discontinuation phase of the trial to patients with stable disease may not result in a homogeneous set of patients. It could be that some patients have slow growing

476

B.N. Bekele

tumors while others are responsive to treatment. Essentially, this design assumes that the classification of stable disease is more important than the varying clinical, biological, and demographic differences. This assumption may not be tenable in all situations.

Personalized Medicine Recently, the pharmaceutical industry has developed medications based on known treatment mechanisms of action. In addition, recent advances in understanding the genetic nature of many different types of cancer has lead to personalized medicine strategies. While there are similarities to traditional medicine, these approaches involve an extension of traditional clinical medicine taking advantage of the cutting edge of genetics research. The approaches taken in designing personalized medicine trials include designs similar to adaptive randomization strategies or to enrichment strategies used in randomized discontinuation designs. The advent of these designs has been driven by a desire to increase the success rate associated with drug development. The aim of personalized medicine is to use genetic information as a guide in individualizing a patient’s medical care, which will lead to fewer adverse events, more positive trials targeting populations that can benefit from a treatment. Primarily, this information would be used to select the treatments most likely to be efficacious for that patient, or identify patients for whom preventative measures could possibly reduce the patient’s future cancer risk. Examples of personalized medicine have been used in oncology. Zhou et al. (2008) use an outcome adaptive randomization framework to modify the treatment assignment probabilities according to the performance of each treatment during the trial. As the trial progresses, more patients can be treated with more promising regimens based on the updated data. The method by Zhou et al. uses an adaptive randomization technique to not only find the most effective treatment, but also find the patient subgroups most likely to respond to treatment (25). There are many advantages to this approach in that one can model, in real time, which treatments are most effective and change focus if the data so indicate. We should note that there are some practical disadvantages to these approaches also. One disadvantage is that such an approach requires that patient genetic characteristics must be assessed in real-time. If the lab performing the tests is not Clinical Laboratory Improvement Amendments (CLIA) certified, then the lack of CLIA certification indicates that the lab performing the assessments does not meet certain Federal requirements, potentially resulting in regulatory concerns regarding the validity of the trial outcomes. An alternative approach to the adaptive randomization framework for personalized medicine trials would be to use a two-stage enrichment strategy in which during the first stage the investigator enrolls a large heterogeneous cohort of patients and follows them for a fixed length of time. After this cohort has been followed and evaluated for a host of factors including response and correlative biomarkers,

Advances in Oncology Clinical Research: Statistical and Study Design Methodologies

477

the investigator identifies the target population and/or drug using advanced statistical correlative techniques. Then during the second stage, the targeted population and/ or treatment is validated prospectively (26). The main disadvantage of this approach is that if the targeted population was incorrectly selected during the first stage, then the trial will end in failure.

Other Adaptive Methods Other examples of successful applications of adaptive designs include singletreatment phase II studies that jointly consider efficacy and toxicity, with stopping rules based on both endpoints (3, 27–30). Another interesting innovation is the seamless phase II–III design (4, 31). With this design, randomization begins within the context of a small phase II study that collects survival information but has an intermediate endpoint (such as tumor response) as the primary outcome. Based on early results with respect to this intermediate endpoint, the study may expand to a large randomized phase III study using a definitive endpoint such as overall survival. Berry et al. discuss a design that simultaneously seeks the best dose of a drug in an adaptive way and maintained a randomized comparison with placebo (3). Other examples exist in the statistical literature (32).

Bayesian Designs While some adaptive designs are Bayesian and some Bayesian designs are adaptive, we take some time here to sketch a brief introduction to Bayesian methods. Before we can start discussing the Bayesian paradigm, the reader should be aware that there are two camps on how to perform statistical inference. One camp utilizes Frequentist inferential procedures while the other camp utilizes Bayesian procedures. The Frequentist camp (which give birth to the p-value and confidence interval) is much more established in the medical literature than the Bayesian camp. The lack of exposure of Bayesian methods in medical applications is mostly driven by the fact that Bayesian methods are computationally expensive, thus they have been practical only for the last 15–20 years. The basic difference between the two approaches is that under the Frequentist paradigm inferences are made on statistics (quantities that characterize the distribution of observed outcomes) conditional on unknown fixed parameters (quantities that characterize the distribution of the population from which observed outcomes are drawn). In the Bayesian paradigm, inferences are made on parameters conditional on observed data. In other words, parameters are treated as random quantities in which the uncertainty regarding the value of the parameter can be expressed via probability. Because parameters are treated as random (unlike the Frequentist paradigm which fixes the parameters), a Bayesian analysis can say

478

B.N. Bekele

something like: “the probability that the median PFS is greater than 12 months is 0.84.” Such a statement is not possible under the Frequentist framework. The importance of being able to make direct probability statements on parameters has also propelled the Bayesian paradigm into the forefront of adaptive trial design. Bayesian methods are ideal for decision-making (i.e., minimizing risk or maximizing utility) and for prediction. Additionally, Bayesian methods are ideal for combining disparate sources of information. Thus, an investigator can construct a probability model to combine the information from the current study with historical data and with any information available from ongoing studies using the Bayesian framework. For a general discussion of the Bayesian framework see Berry (2006) (33). Increasingly clinical trials are incorporating Bayesian ideas, particularly in situations where there is the desire to include adaptive randomization. Such clinical trials change the randomization probabilities in light of the accruing data. The study may start randomizing patients to the different treatments with equal probability. Then, perhaps after enrolling some minimum number of patients, the randomization probabilities adapt to favor the better performing treatments. Bayesian methodology may enter the study by way of using posterior probability calculations to influence the randomization probabilities (23). The ethical idea is to reduce the number of patients who receive inferior treatment while still accruing convincing evidence within the clinical trial. To implement Bayesian adaptive methods, the statistician and clinical investigators must decide on the decision rules, which will lead the investigator to stop, such as basing decisions on the probability that the treatment’s response rate exceeds a threshold value. Next, the statistician carries out a large number of simulations under various scenarios. The statistician and principal investigator review the simulation results with the clinical investigators, allowing them to decide on trial conduct rules that yield the best operating characteristics. Simulations under various scenarios also help reveal sensitivity of the operating characteristics to prior assumptions.

Sample Size Reassessment Methods There are a wide variety of sample size adaptive methods. The aim of sample size re-estimation is to appropriately size a trial such that there is adequate chance of rejecting the null hypothesis given the observed data. The need for sample size reestimation stems from the fact that during the trial design stage, the size of a treatment’s effect cannot be predicted with certainty. Optimally, these methods should be prespecified and be designed in a way that the integrity of the study results (control of the type I and type II error rates) is not put into jeopardy. Sample size reviews, also known as internal pilot studies, are exemplified by the work of Wittes and Brittain (34, 35). These methods only review sample size via an assessment of some nuisance parameter (such as the variance) compared to what was expected at the start of the study. Implementation of these methods do

Advances in Oncology Clinical Research: Statistical and Study Design Methodologies

479

not result in early stopping for either efficacy or safety. These methods are the easiest to implement and are widely accepted. A second type of adjustment characterized by data re-weighting has gained increased attention in the statistical literature. Typically, these methods increase sample size in a reasonable re-estimation scheme while strictly controlling the type I error rate and power by giving relatively more “weight” to data collected after the sample size adjustment and relatively less weight to data collected after the sample size adjustment (36, 37). Lastly, a common approach taken is to use a conditional power (CP) approach to perform sample size re-estimation. CP is defined as the probability of rejecting the null hypothesis at the end of the trial, given the data observed so far. This concept differs for the standard power in that CP is calculated at an interim time-point after some patients have enrolled into the study but before the maximum sample size has been observed. In contrast, the standard power calculation is calculated before any patients have been enrolled. Thus, based on the data in hand, the CP can be used to characterize probability of success for a trial, given the data observed to date and the planned additional future patients. The typical use of the CP approach is to provide the investigator with a measure of futility (in other words, low CP for rejecting the null hypothesis implies that a treatment should be terminated). Because the CP is a function of the number of planned patients who have not yet enrolled to a trial, this method can be used to modify sample sizes also. Examples of this approach can be found in various works (38–40).

Discussion In this chapter, we discussed current trends in clinical trial design, and outlined the advantages and possible limitations of these designs. Major topics covered included adaptive methods for dose finding in oncology, outcome adaptive clinical trial designs, randomized discontinuation designs, advances in personalized medicine, the role of Bayesian methods within the adaptive design arena, and sample size re-estimation methods. Development of these new methods has been driven by the high cost of clinical trial development, desire for more flexibility in clinical trials and the growing realization that even early phase clinical trials should provide the investigator with useful information. The lack of useful early phase clinical trial information partially stems from the use of low (or inappropriate) efficacy thresholds (especially in early phase oncology clinical trials). This has led to a situation in which most trials conducted are nonrandomized and use endpoints such as response which, for a given compound, may not correlate with later phase endpoints such as overall survival or progression-free survival. For example, an antiangiogenic compound may not result in significant numbers of partial or complete responses, yet it may delay progression. Thus, using a standard Simon-two stage design targeting response could be a massive waste of effort. One can posit a guess

480

B.N. Bekele

as to why things are as they are. It could be due to sheer inertia, or a hope that “something will fall out,” or an unwillingness to commit resources to compounds which are not proven. The promise of adaptive methods is that they are applicable during the earliest phases of clinical trial development and their flexibility allows the investigator to ask better questions or discover which questions should be asked.

References 1. Jennison C, Turnbull BW (1999) Group sequential methods with applications to clinical trials. Chapman & Hall/CRC, Boca Raton 2. Tsiatis AA, Mehta C (2003) On the inefficiency of the adaptive design for monitoring clinical trials. Biometrika 90:367–378 3. Berry DA, Müller P, Grieve AP et al (2001) Adaptive Bayesian designs for dose-ranging drug trials. In: Gatsonis C, Carlin B, Carriquiry A (eds) Case studies in Bayesian statistics V. Springer Verlag, New York, pp 99–181 4. Thall PF (2008) A review of phase 2–3 clinical trial designs. Lifetime Data Anal 14:37–53 5. Chevret S (2006) Statistical methods for dose-finding experiments. Wiley, West Sussex 6. Ivanova A, Montazer-Haghighi A, Mohanty G, Durham SD (2003) Improved up-and-down designs for phase I trials. Stat Med 22:69–82 7. Rosenberger WF, Haines LM (2002) Competing designs for phase I clinical trials: a review. Stat Med 21:2757–2770 8. Bekele BN, Ji Y, Shen Y, Thall PF (2008) Monitoring late-onset toxicities in phase I trials using predicted risks. Biostatistics 9:442–457 9. Cheung YK, Chappell R (2000) Sequential designs for phase I clinical trials with late-onset toxicities. Biometrics 56:1177–1182 10. Normolle D, Lawrence T (2006) Designing dose-escalation trials with late-onset toxicities using the time-to-event continual reassessment method. J Clin Oncol 24:4426–4433 11. Braun TM, Yuan Z, Thall PF (2005) Determining a maximum-tolerated schedule of a cytotoxic agent. Biometrics 61:335–343 12. Braun TM, Thall PF, Nguyen H, de Lima M (2007) Simultaneously optimizing dose and schedule of a new cytotoxic agent. Clin Trials 4:113–124 13. O’Quigley J, Hughes MD, Fenton T (2001) Dose-finding designs for HIV studies. Biometrics 57:1018–1029 14. Ivanova A (2003) A new dose-finding design for bivariate outcomes. Biometrics 59:1001–1007 15. Thall PF, Cook JD (2004) Dose-finding based on efficacy-toxicity trade-offs. Biometrics 60:684–693 16. Ji Y, Li Y, Yin G (2007) Bayesian dose finding in phase I clinical trials based on a new statistical framework. Stat Sin 17:531–547 17. Bekele BN, Shen Y (2005) A Bayesian approach to jointly modeling toxicity and biomarker expression in a phase I/II dose-finding trial. Biometrics 61:344–354 18. Bekele BN, Thall PF (2004) Dose-finding based on multiple toxicities in a soft tissue sarcoma trial. J Am Stat Assoc 99:26–35 19. Yuan Z, Chappell R, Bailey H (2007) The continual reassessment method for multiple toxicity grades: a Bayesian quasi-likelihood approach. Biometrics 63:173–179 20. Li Y, Bekele BN, Ji Y (2008) Dose-schedule finding in phase III clinical trials using a Bayesian isotonic transformation. Stat Med 27:4895–4913 21. Bartlett RH, Roloff DW, Cornell RG, Andrews AF, Dillon PW, Zwischenberger JB (1985) Extracorporeal circulation in neonatal respiratory failure: a prospective randomized study. Pediatrics 76:479–487

Advances in Oncology Clinical Research: Statistical and Study Design Methodologies

481

22. Berry DA, Wolff MC, Sack D (1994) Decision making during a phase III randomized controlled trial. Control Clin Trials 15:360–378 23. Thall PF, Wathen JK (2007) Practical Bayesian adaptive randomisation in clinical trials. Eur J Cancer 43:859–866 24. Rosner GL, Stadler W, Ratain MJ (2002) Randomized discontinuation design: application to cytostatic antineoplastic agents. J Clin Oncol 20:4478–4484 25. Zhou X, Liu SY, Kim ES, Herbst RS, Lee JL (2008) Bayesian adaptive design for targeted therapy development in lung cancer – a step toward personalized medicine. Clin Trials 5:181–193 26. Temple RJ (2005) Enrichment designs: efficiency in development of cancer treatments. J Clin Oncol 23:4838–4839 27. Thall PF, Russell KE (1998) A strategy for dose-finding and safety monitoring based on efficacy and adverse outcomes in phase I/II clinical trials. Biometrics 54:251–264 28. Thall PF, Simon R (1990) Incorporating historical control data in planning phase II clinical trials. Stat Med 9:215–228 29. Thall PF, Simon RM, Estey EH (1996) New statistical strategy for monitoring safety and efficacy in single-arm clinical trials. J Clin Oncol 14:296–303 30. Thall PF, Sung HG (1998) Some extensions and applications of a Bayesian strategy for monitoring multiple outcomes in clinical trials. Stat Med 17:1563–1580 31. Inoue LYT, Thall PF, Berry DA (2002) Seamlessly expanding a randomized phase II trial to phase III. Biometrics 58:823–831 32. Spiegelhalter DJ, Abrams KR, Myles JP (2004) Bayesian approaches to clinical trials and health-care evaluation. John Wiley & Sons, Ltd, Chichester, UK 33. Berry DA (2006) Bayesian clinical trials. Nat Rev Drug Discov 5:27–36 34. Wittes J, Brittain E (1990) The role of internal pilot studies in increasing the efficiency of clinical trials. Stat Med 9:65–71; discussion 2 35. Wittes J, Schabenberger O, Zucker D, Brittain E, Proschan M (1999) Internal pilot studies I: type I error rate of the naive t-test. Stat Med 18:3481–3491 36. Cui L, Hung HMJ, Wang SJ (1999) Modification of sample size in group sequential clinical trials. Biometrics 55:853–857 37. Hung HMJ, Cui L, Wang SJ, Lawrence J (2005) Adaptive statistical analysis following sample size modification based on interim review of effect size. J Biopharm Stat 15:693–706 38. Li G, Shih WJ, Wang Y (2005) Two-stage adaptive design for clinical trials with survival data. J Biopharm Stat 15:707–718 39. Li G, Shih WJ, Xie T, Lu J (2002) A sample size adjustment procedure for clinical trials based on conditional power. Biostatistics 3:277–287 40. Proschan MA, Hunsberger SA (1995) Designed extension of studies based on conditional power. Biometrics 51:1315–1324

Palliative Care for Patients with Lung Cancer David Hui and Eduardo Bruera

Abstract  Lung cancer is associated with significant mortality and morbidity. Palliative care plays an important role along the disease continuum for patients with advanced lung cancer. Mastery of core palliative care clinical skills, such as symptom management, prognostication, communication, decision making, and transition of care, would allow oncologists to better serve patients and families. Common symptoms in lung cancer patients, such as dyspnea, pain, fatigue, cough, hemoptysis, anorexia, cachexia, depression, and anxiety, can usually be managed effectively. For patients with severe symptoms or psychosocial distress, early involvement of the palliative care team is essential. Keywords  Lung cancer • Palliative care • Prognosis • Communication skills • Transition of care

Introduction Lung cancer is the second most common cancer in North America, and remains the leading cause of cancer death (1). Up to 70% of non-small cell lung cancer (NSCLC) patients have locally advanced or metastatic disease at the time of diagnosis, with a median survival of 10–12 months. Among those with resectable disease, approximately 60% relapse within 5 years. The median survival for patients with small cell lung cancer (SCLC) is similar, being only 16–24 months for limited stage disease and 6–12 months for extensive stage disease. Furthermore, patients with advanced lung cancer tend to have a high symptom burden. NSCLC patients report an average of 14 symptoms while SCLC patients experience an average of

D. Hui and E. Bruera () Department of Symptomatic Center and Palliative Care, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA e-mail: [email protected] D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_19, © Humana Press, a part of Springer Science+Business Media, LLC 2010

483

484

D. Hui and E. Bruera

17 symptoms (2). The most common symptoms include pain, dyspnea, cough, fatigue, anorexia, cachexia, and depression (3), resulting in substantial physical and psychological suffering for patients, along with distress for their families. Given the significant mortality and morbidity associated with lung cancer and its treatments, palliative care plays an essential role along the disease continuum for the great majority of patients. Oncologists serving lung cancer patients should be competent in performing regular symptom assessments and in providing appropriate supportive measures. They should also be highly skilled at discussing prognosis and addressing transition of care and end-of-life issues. For patients with high symptom burden or complex psychosocial issues, consultation of a palliative care team early in the disease trajectory would be beneficial. In this chapter, we address a number of core palliative care skills and concepts essential for oncologists working with lung cancer patients. Specifically, we discuss issues related to palliative care access, prognostication, and explore the decisionmaking process for patients with advanced cancer. We also review the management of common symptoms seen in patients with advanced lung cancer.

Role of Palliative Care in Patients with Advanced Lung Cancer Patient Access to Palliative Care Palliative care aims to improve the quality of life of cancer patients and their families through early identification, assessment and treatment of symptoms, and to minimize suffering through effective management of psychosocial and spiritual concerns (4). Good symptom management not only improves patients’ quality of life, but also supports patients through intensive cancer therapies. Advanced cancer patients are living longer as a result of advances in cancer therapy, leading to a greater demand for palliative care. Indeed, the World Health Organization (WHO) recognizes palliative care as one of the four pillars of modern oncology (5). Despite the general recognition that palliative care plays a crucial role for advanced cancer patients, the availability and degree of integration of palliative care is highly variable among hospitals and cancer centers across the United States (6, 7). The quality of palliative care services is also heterogeneous. In its 2001 report, the Institute of Medicine identified eight key barriers to provision of effective palliative care, including the separation of palliative and hospice care from potentially lifeprolonging treatment within the health-care system, limited reimbursement for supportive care services, inadequate training of healthcare personnel in symptom management and other palliative care skills, and limited high quality research (8). A recent survey of health care professionals showed that the name “Supportive Care” is preferred over “Palliative Care,” a term that is frequently associated with hospice and end of life (9). This interesting finding reflects, to a certain extent,

Palliative Care for Patients with Lung Cancer

485

the stigma regarding death and dying. To maximize the availability and utilization of palliative care, we need to overcome a number of financial, infrastructural, professional, social, and educational barriers (10). For instance, timely access to effective symptom management for cancer patients can potentially be improved by increasing the palliative care training for oncologists. While palliative care is commonly associated with end-of-life care, it is most effective when incorporated early in cancer management (4, 8, 11). Indeed, timely incorporation of palliative care strategies in patients with advanced cancer can help to optimize symptom management (12), improve psychosocial interventions, enhance coordination of care, and facilitate patients’ explicit transition from curative to palliative intent (13, 14). This understanding led to the development of the comprehensive cancer care model, which integrates supportive care along with anticancer therapy from the time of diagnosis (Fig.  1). Lung cancer represents a prototype for comprehensive cancer care because of the natural history of this disease, with the majority of patients diagnosed with incurable cancer.

Survival Estimation Survival estimation is of utmost importance to patients, families, and oncologists, particularly in the advanced cancer setting. Accurate prognostication is essential for oncologists in formulating their recommendations. Patients’ predicted survival can have a profound impact on important decisions such as palliative care and hospice referral, initiation of specific medications, and avoidance of aggressive therapies. a Active treatment

b

Palliation

Active treatment Palliation

c Comprehensive Cancer Care

Fig. 1  Evolving models of cancer care. The transition from curative to palliative care changes overtime. (a) Currently, palliative care is often introduced too close to the end of life, which significantly limits its effectiveness. (b) Early integration of palliative care would allow optimization of symptom control, and allow the palliative care team to build a strong therapeutic relationship with patients as they move along the cancer journey. (c) Comprehensive cancer care in which active cancer treatment and palliation are coordinated in a harmonious fashion, with both services available to patients throughout the disease continuum, depending on patients’ specific needs at any given time

486

D. Hui and E. Bruera

As cancer patients progress over the course of their illness, knowing what to expect could provide them with a sense of control and facilitate the process of advance care planning (15). Clinicians have consistently been found to overestimate survival (16). Several prognostic factors have been identified for patients with advanced lung cancer, which not only help enhance the accuracy of survival predictions, but may also contribute to clinical decision making. Poor performance status, weight loss, male gender, and the presence of metastatic disease are associated with shorter survival in patients with advanced NSCLC and SCLC (17–19). There is also emerging evidence that patient reported outcomes (PROs) carry prognostic significance in patients with NSCLC (20). With far advanced disease (i.e., expected survival <3 months), tumor histology tends to lose its prognostic significance while other patient-related factors such as performance status, anorexia–cachexia, delirium, and dyspnea predominate (21). A number of laboratory abnormalities, such as leukocytosis, lymphocytopenia, hypoalbuminemia, hypercalcemia, elevated lactate dehydrogenase, and C-reactive protein (22), have also been shown to confer a poor prognosis. Prediction models incorporating multiple factors have been developed for the palliative population (23–26), with the Palliative Prognostic Score being the most validated in various settings (27–30). Even with the most sophisticated prognostic model, it is important to recognize that there will always be uncertainty in survival predictions due to the inherent nature of cancer deaths, mediated by acute complications such as infections and thromboembolism. Thus, it is imperative for oncologists not only to polish the science of prognostication, but also to further the art of communication, gently guiding patients and families through times of uncertainty (31). A recent review suggests that physicians tend to underestimate patients’ need for information, yet at the same time overestimate patients’ appreciation of their prognosis (32). While each patient–physician interaction should be individualized, a number of important strategies may be helpful for clinicians when discussing prognosis (Table 1). The truth may be difficult, but the sharing of it should not be.

Decision Making at the End of Life Lung cancer patients are bombarded with numerous decisions involving cancer treatments, philosophy of care, and end-of-life planning from the time of diagnosis until death. Many of these decisions are highly complex and emotionally charged. One of the key roles of an oncologist is to help guide patients through the maze of difficult choices by providing individualized recommendations, taking into account the patient’s preferences, disease state, treatment options, and resources. It is important to note that clinical decision making is a continuous process with the content of discussion ever evolving to reflect the changing goals and health status of the patient. In order to make sound decisions, patients need to have a good understanding of the natural history of lung cancer. Yet, the literature suggests significant gaps in

Palliative Care for Patients with Lung Cancer

487

Table 1  General strategies for discussing prognosis Key element Comments Context Ensure a quiet, comfortable, and safe environment Sit down and speak to patient at eye level Ask the patient if he/she wants to be accompanied by family/ friend during the discussion How much does the Explore patient’s understanding of his/her illness and expectations patient know? A clear understanding of patient’s reasons to explore prognosis can How would learning help clinicians to shape their discussion that addresses patient’s about prognosis help goals and to provide a personalized management plan the patient? Deliver information Emphasize the uncertainties in prognostication Discuss prognosis in terms of days, weeks, months, rather than specific numbers or median survival Information should be related such that it is measured to the perceptions of the patient’s intellectual comprehension and emotional resilience Empathic response Simple remarks such as “this is a difficult time for you.” can be helpful Empowering Take this opportunity to help patients define achievable goals. Initiate discussions about important advance care planning issues such as philosophy of care, living will, and code status Provide follow-up and Reassure patients regarding continuity of care support resources Introduce other members of the interprofessional team, such as chaplain and social worker, who will be able to provide further counseling Content partly based on the following refs. (31, 81, 82)

communication between patients and oncologists. For instance, a number of studies have demonstrated that one-third of patients with metastatic lung cancer believe that their cancer is curable (33, 34). While denial likely plays an important role, these studies highlight the need for enhanced communication, regular assessments of patient’s understanding and decision-making aids. Patients’ preferences regarding their role in clinical decision making vary greatly. While the majority of patients prefer to make decisions in conjunction with their oncologists, approximately 5–15% would rather make the decision on their own, and another 10–30% would favor their oncologists making healthcare decisions on their behalf. Importantly, studies have shown that physicians are not able to consistently predict the decision-making preferences of their patients (35–37), highlighting the need to specifically explore this important issue with patients. With the increasing availability of effective cancer therapies, advanced cancer patients are faced with more choices than ever. In addition to the potential for improved survival, antineoplastic treatments may offer symptom relief through tumor shrinkage or stabilization (38) (Fig.  2). However, the symptom benefits tend to be incomplete and short term, and would need to be balanced against potential harm, including adverse treatment side effects, frequent clinic visits, and financial burden. For advanced NSCLC patients, randomized controlled trials comparing gemcitabine (39) and docetaxel (40) to best supportive care have reported quality of life

488

D. Hui and E. Bruera Cancer ↑ size ↑ metabolism

Cancer treatments Chemotherapy Targeted therapy Radiation Surgery

Θ

↑ mass effect Altered cellular function

⊕

Tissue damage Cytokine/hormone release Afferent signals

Θ

Perception of symptoms

Supportive measures

Θ

Psychosocial interventions

Fig. 2  Pathophysiology of cancer-related symptoms and potential modifiers. As cancer progresses, patients develop worsening symptoms due to increasing tumor mass or activity, causing compression, obstruction, inflammation, and altered metabolism. Strategies to manage cancer-related symptoms include anti-neoplastic therapies that decrease tumor size and/or metabolism, symptomatic measures for palliation, and psychosocial interventions. Since many symptoms in advanced cancer patients are multifactorial in nature, a multitude of these approaches are required for optimal symptom control

improvement in the chemotherapy arms. However, these are not placebo control blinded studies, and thus subjected to reporting bias, particularly with subjective endpoints such as symptom intensity. Erlotinib has also been shown to provide symptom control through disease stabilization in the second- or third-line palliative therapy setting for patients with metastatic NSCLC (41). Recently, a predictive and prognostic model including ten factors has been developed for NSCLC patients receiving erlotinib (42), which may help clinicians identify patients who would not benefit from this therapy and thus spare them from unnecessary side effects. Continuation of active cancer therapy should not prevent patients from engaging in advance care planning. However, patients and clinicians have the tendency to avoid discussing end-of-life issues until it is too late, as it is easier to just focus on cancer treatments (43). When death approaches, many of the complex issues surrounding goals of care remain unresolved, which could lead to overly aggressive management such as endotracheal intubation, and significant distress in patients and families. One of the key reasons for delaying end-of-life discussions is the worry about destroying hope. Yet, it is important to understand that authentic hope is based on a good understanding and acceptance of reality. To facilitate a smooth transition, patients and their families should be provided with accurate clinical information and realistic expectations early on in the disease process, with frequent and regular assessments to check their understanding and goals as their cancer progresses. Timely discussions of advance directives, code status, and arrangements after death can help bring peace of mind to patients and families, knowing that their wishes will be respected.

Palliative Care for Patients with Lung Cancer

489

Referral to other members of the interprofessional team, such as palliative care, social work, and pastoral care, will facilitate provision of supportive care under a holistic environment, preparing patients and families for the journey ahead.

Symptom Management Dyspnea Dyspnea is a subjective awareness of difficulty in breathing, which may be associated with the distressing sensation of suffocation. It is the most common and most feared symptom among lung cancer patients, occurring in 40–60% of patients from the time of diagnosis (3), and increases up to 90% closer to the end of life. Dyspnea is an important prognostic factor for patients with advanced lung cancer (44). Causes of dyspnea can be classified as cancer-related, treatment-related, and psychological factors. Progressive disease may result in parenchymal metastasis, lymphangitic carcinomatosis, airway obstruction, atelectasis, pleural effusion, and/ or tense ascites causing difficulty breathing. Complications of cancer, including thromboembolism, pneumonia, sepsis, and anemia of chronic disease, may also contribute to the sensation of breathlessness. Lung cancer patients also tend to have poor respiratory reserve because of chronic obstructive pulmonary disease (COPD) and/or prior lung resections, predisposing them to the development of respiratory symptoms. In advanced cancer patients with significant cachexia, shortness of breath may also be related to loss of respiratory muscles, an under-recognized etiology. In a small proportion of patients, no identifiable etiologic factors may be found. Assessment of dyspnea includes a focused history and physical to determine the severity of shortness of breath, assess associated symptoms, and identify correctable causes. Oximetry can determine oxygen saturation level, although hypoxemia is not necessary for patients to experience dyspnea. Chest imaging can provide further information regarding intrathoracic pathologies. Management of dyspnea consists of reversal of underlying causes, such as pulmonary embolism and pneumonia, along with symptomatic relief. A good disease response from systemic agents or radiation may prove useful to improve dyspnea. Patients with large pleural effusion would benefit from thoracentesis, and insertion of a PleureX cathether may be considered for those with recurrent effusion. Pleurodesis can be performed using talc, tetracycline, doxycycline, etoposide, or bleomycin, with a success rate between 40 and 90% (45). For patients with severe dyspnea or far advanced cancer, early initiation of supportive measures is essential. Bronchodilators should be given to patients with COPD, or if bronchospasm is suspected. Supplemental oxygen is beneficial for patients with hypoxemia, although its role in dyspneic patients who are not hypoxemic remains unresolved (46). A prospective, double-blind crossover trial included 14 advanced cancer patients, and found significant improvements in dyspnea comparing oxygen to air by mask (47). However, a larger study with 33 patients did not confirm this benefit (48).

490

D. Hui and E. Bruera

Non-invasive mechanical ventilation, such as bilevel positive airway pressure (BIPAP) and continuous positive air pressure (CPAP) devices, may represent options for selected patients after detailed discussions of risk and benefits, and review of goals of care. Opioids have proven palliative benefit for dyspnea. In a crossover study with ten cancer patients, Bruera et al. showed that subcutaneous morphine was more effective at relieving dyspnea compared to placebo 60  min after drug administration (49). A systemic review suggested that oral and parenteral but not nebulized opioids are effective in managing dyspnea (50). Initiation and titration of opioids for dyspnea is similar to that for pain control (Table 2). Corticosteroids are used empirically for dyspneic patients, particularly those with lymphangitic carcinomatosis and superior vena cava syndrome, although evidence is lacking. Benzodiazepines have been compared in a number of randomized controlled trials with no demonstratable benefits, and should be limited to patients with anxiety disorders. For patients with refractory dyspnea, palliative sedation with midazolam or propofol may be considered as a last resort to relieve suffering, but only after detailed discussions with patients, family members, and the healthcare team. A number of non-pharmacologic interventions have been proposed for management of dyspnea. Bronchoscopic procedures, such as resection, electrocautery, cryotherapy, laser, balloon dilatation, and brachytherapy, may provide palliation of symptoms caused by endobronchial tumor for selected patients. For further information, the interested reader is referred to a recently published evidence-based clinical practice guideline by the American College of Chest Physicians on palliative care in lung cancer (51). A recent Cochrane systemic review, based mostly on studies conducted in COPD patients, examined a variety of non-pharmacologic interventions for relief of dyspnea (52). This showed strong evidence for neuro-electrical muscle stimulation and chest wall vibration. There was moderate strength evidence to support the use of walking aids and breathing training, but low or insufficient evidence to support the use of acupuncture/acupressure, music, relaxation, fan, counseling and support, case management, and psychotherapy. Further studies are required to determine the feasibility and efficacy of these measures, particularly in patients with advanced cancer.

Pain Pain is ranked as the second most distressing symptom after dyspnea among patients with inoperable lung cancer, with its severity increasing as disease progresses (3). In a recent population-based study, 60% of lung cancer patients reported having pain, and half of them described the pain as moderate to severe (53). Uncontrolled pain can result in reduced sleep, decreased function, altered mood, and significantly compromised quality of life. Pain in lung cancer patients can be related to multiple etiologies. Pleuritic pain may suggest local disease invasion of pleura or chest wall, pneumonia, or pulmonary embolism. Persistent achy pain may be due to bone metastasis, while back

Initial scheduled dosec Short-acting: 5–10 mg PO every 6 h long acting: N/A

Available dose formulations Short-acting: 5, 7.5, 10 mg tablets; 2.5 mg/5 ml liquid, in combination with acetaminophen long-acting: N/A Codeine PO/IV/ 67 mg PO 30–60 mg PO Short-acting: 30–60 mg every 6 h long-acting: Short-acting: 15, 30, 60 mg tablets SC N/A Long-acting: N/A Short-acting: 15, 30 mg tablets; 10 mg/5 ml, Morphine PO/ 10 mg PO 5–15 mg PO Short-acting: PO: 5–10 mg every 4 h; IV: 20 mg/5 ml, 20 mg/ml liquid IV/SC 2–3 mg IV/SC 2–4 mg every 4 h Long-acting: 15 mg every 12 h, or 20 or 30 mg Long-acting: 15, 30, 60, 100 mg as every 12 h presentation once daily Oxycodone PO/ 6.7 mg PO 5–10 mg PON/A Short-acting: 5 mg PO every 4 h Short-acting: 5, 15, 30 mg tablets; 5 mg/5 ml, IV/SC Long-acting: 10 mg PO every 12 h 20 mg/ml liquid Long-acting: 10, 20, 40, 80 mg tablets as 12 h preparations 3.3 mg PO 5–10 mg PO Short-acting: 5 mg PO every 4 h Short-acting: 5,10 mg tablets Oxymorphone PO/IV/SC 0.5 mg IV/SC Long-acting: 5 mg PO every 12 h Long-acting: 5, 10, 20, 40 mg as 12 h preparation Short-acting: PO 2 mg every 4 h. IV/SC: Short-acting: 2, 4, 8 mg tablets; 1 mg/ml liquid 1–3 mg PO Hydromorphone 2 mg PO 0.5–1 mg every 4 h Long acting: N/A PO/IV/SC 0.5–1.5 mg IV/SC Fentanyl IV/TD 40 mcg IVd Patch strength: 12 (delivers 12.5), 25, 50, 75, N/A Consider switching to fentanyl patch after 100 mcg/h patient is on stable dose of opioid. Choose appropriate patch strength based on the total daily morphine dose Methadone See belowe N/A 5 mg every 12 h 5, 10, 40 mg tablets; 5 mg/5 ml, 10 mg/5 ml liquid Table adapted and modified from MD Anderson Cancer Pain Guidelines (83) a Dosages equivalent to 10 mg of oral morphine are listed unless otherwise specified b In opioid-naïve patients c In opioid-naïve patients using more than three rescue doses/day d 100 mcg fentanyl patch releases the equivalent of 200–400 mg of morphine per day e To convert morphine-equivalent daily dose (MEDD) to methadone, divide MEDD by 5 if MEDD <500 mg, 10 if MEDD 500–1,000 mg, and 20 if MEDD >1,000 mg

Table 2  Common opioids for pain control Equi-analgesic Initial rescue doseb Opioid/routes dosesa Hydrocodone 67 mg PO 5–10 mg PO PO/IV/SC

Palliative Care for Patients with Lung Cancer 491

492

D. Hui and E. Bruera

pain should alert the clinician to the possibility of spinal cord compression. Furthermore, procedures such as thoracotomy and pleurodesis can also be associated with significant pain. Given the high prevalence of pain in lung cancer patients and the availability of effective treatments, it is important for clinicians to perform regular assessment of pain and to search for potential causes. A simple pain scale from 0 to 10 or visual analog scale is reliable in assessment changes in pain intensity, and may help to follow the effect of treatment. Recently, a staging system has been proposed to standardize reporting of cancer pain. This system takes into account the mechanism of pain and predictive factors for pain control (54), including incident pain, psychological distress, addictive behavior and cognitive function. Early and regular pain assessments, along with patient education and effective treatments, can help to optimize pain control for most cancer patients. Mild pain may be treated with acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs), and/or weak opioids (Table  2). For moderate to severe cancer pain, strong opioids such as morphine, hydromorphone, oxycodone, fentanyl, and methadone are recommended (Table 2). Opioid rotation should be considered if and when patients develop signs of opioid neurotoxicities, including significant sedation, myoclonus, and hallucinations. Immediate release and controlled release formulations of morphine provide similar pain relief (55). Patients should be educated in the appropriate use of breakthrough medications, and counseled regarding the side effects of constipation and nausea, with laxatives and metoclopramide prescribed for prophylaxis. The fear of addiction should also be explored. For neuropathic pain, opioids (56), gabapentin (57), tricyclic antidepressants, and venlafaxine (58) have all demonstrated efficacy, with number needed to treat (NNT, defined as the number of patients who would need to be treated to derive one clinical benefit) of 2.6, 4.3, 3.6, and 3.1, respectively. The role of other selective serotonin reuptake inhibitors for neuropathic pain remains to be defined. Treatment of the underlying cause where possible can provide pain alleviation and reduce use of analgesia. For instance, palliative radiation may be useful for bone metastasis, and chemotherapy may improve quality of life. Steroids can be effective for decreasing swelling around tumor and relief of pain, although its effects tend to be temporary, and they should be avoided long term because of potential significant side effects. Bisphosphonates may also be useful for management of pain related to bone metastases in cancer patients.

Fatigue Cancer-related fatigue (CRF) is “a distressing, persistent, and subjective sense of tiredness or exhaustion related to cancer or cancer treatment that interferers with usual functioning” (NCCN). CRF can be distinguished from typical tiredness as it does not correspond to the patient’s level of exertion, and is not usually relieved by sleep or rest. The diagnosis of lung cancer is a particularly strong risk factor for developing CRF (59).

Palliative Care for Patients with Lung Cancer Altered physiology • Cytokine dysregulation • Serotonin neurotransmitter dysregulation • HPA axis dysfunction ? • Circadian rhythm disruption • Vagal afferent activation • Alterations in muscle ATP metabolism Direct cancer burden and cancer treatments Co-morbidities • Anemia • Sleep disorders • Pain • Psychiatric issues • Electrolytes • Nutritional issues • Inactivity • Co-morbidities

493

Physical weakness

CANCER RELATED FATIGUE

↓ function Psychological distress

↓ QOL Economic impact Family problems

Mental changes

Fig. 3  Pathophysiology of cancer-related fatigue. Cancer and its treatments may result in altered physiology in neurologic and endocrine function. They may also indirectly lead to a number of co-morbid conditions, which may contribute to CRF further, compromising patients’ quality of life and function. Effective management of CRF includes treatment of any reversible contributing factors, exercise, pharmacotherapy, and multi-disciplinary intervention

At diagnosis, about 40% of cancer patients reported CRF. This increases to 80–90% during cancer therapy, with fatigue typically worst at the end of treatment for patients receiving radiation, and coinciding with the nadir of blood counts for those on chemotherapy. As patients approach the end of life, almost all experience significant CRF. CRF has a significant impact on quality of life, daily function, employment, and household financial status. CRF is believed to be a multifactorial process (Fig. 3). Cancer and its treatments may result in altered physiology, leading to cytokine dysregulation, serotonin neurotransmitter dysregulation, hypothalamic–pituitary axis changes, circadian rhythm disruption, vagal afferent activation, and alterations in muscle ATP metabolism. Other contributors associated with cancer and its treatments that may play a role include anemia, sleep disturbance, depression, anorexia, inactivity, organ dysfunction, and hormonal deficiency. Identification of these potentially reversible factors is one of the keys to successful management of CRF. CRF is common, pervasive, and debilitating, but under-reported by patients and under-recognized and under-treated by clinicians. The National Comprehensive Cancer Network (NCCN) recommends regular screening of patients using a 0- to 10-point scale, with scores of 1–3, 4–6, and 7–10 indicating mild, moderate, and severe fatigue, respectively (60). In addition to regular assessments, all patients should be educated on the nature of CRF and common strategies to combat CRF, such as energy conservation strategies and distraction (games, music, reading, socializing). For patients with a fatigue score ³4 out of 10, it is important to search for and treat any potential contributors (Table  3), and consider further measures for symptom relief.

494

D. Hui and E. Bruera

Table 3  Contributing factors to CRF (mnemonic ASTHENIC) Assessment Interventions Anemia CBC Transfusions as needed Sleep disturbances History, sleep study Improve sleep hygiene Sleep medication Throbbing pain History and physical Pain management Head (e.g., depression, anxiety) History Depression management Electrolytes   Hyponatremia Electrolytes, Ca, Mg Correction of electrolyte abnormalities   Hypokalemia   Hypomagnesemia   Hypocalcemia   Hypercalcemia Nutritional failure History, weight, Nutritional counseling,   Anorexia–cachexia albumin corticosteroids (short term) Inactivity History Exercise Comorbidities   Cardiac/pulmonary failure History, vitals Treat underlying cause   Hepatic/renal failure Cr, LFTs   Endocrine failure    Hypothyroidism TSH, fT4    Hypogonadism Testosterone level    Adrenal insufficiency AM cortisol   Infections History, cultures

Of all interventions for CRF, exercise is associated with the strongest evidence. A Cochrane metaanalysis that included 22 randomized controlled trials with 1,172 patients concluded that exercise was more effective than the control intervention, with a standardized mean difference of −0.23 (95% confidence interval −0.33 to −0.13) (61). However, the optimal type, intensity, frequency, and duration of exercise intervention remain to be determined. A reasonable regimen may consist of 30–60 min of walking daily. Pharmacologic treatments may also help to combat CRF and to improve patients’ quality of life. A recent metaanalysis that combined two randomized controlled trials comparing methylphenidate to placebo (62, 63) found a statistically significant effect in the management of CRF (64). Modafenil was associated with improvement of fatigue in a subgroup analysis of patients with fatigue score of at least 6 out of 10, and warrants further investigation (65). A recent Phase II randomized trial of American Ginseng also revealed some interesting activity, although further confirmatory studies are required (66). The role of corticosteroids for CRF remains inconclusive, although short-term use of less than 2 weeks is considered generally acceptable for palliative care patients. Other pharmacologic agents have so far failed to demonstrate significant clinical benefit for CRF, and include dextroamphetamine, donezepil, bupropion, paroxetine, sertraline, and megestrol. Erythropoiesis-stimulating agents, such as epoetin alfa and darbepoetin, should not be used for treatment of anemia in patients with advanced cancer except those on

Palliative Care for Patients with Lung Cancer

495

chemotherapy because of the risk of thromboembolism, and a trend towards increased mortality, which is thought to be related to stimulation of tumor growth (67). Rather, transfusions may be considered on an as-needed basis.

Cough and Hemoptysis Cough is present in over 65% of patients at the time of diagnosis of lung cancer (68). Chronic cough, defined as a duration of greater than 2 months, can be a distressing symptom. Complications of cough include exhaustion, insomnia, anxiety, headaches, dizziness, hoarseness, musculoskeletal pain, and incontinence. While chronic cough in lung cancer patients is most likely related to tumorcausing airway irritation, it is important to rule out other pulmonary etiologies, such as pneumonia, aspiration, thromboembolism, and pleural effusion, particularly if there has been a change in severity. Other conditions, including COPD, asthma, bronchiectasis, interstitial lung disease, congestive heart failure, gastroesophageal reflux disease and medications, such as ACE-inhibitors, may also contribute to cough. A focused history and physical examination should assess the severity of the cough and look for any reversible etiologies. It is important to characterize the duration and frequency of the cough and any associated symptoms. If the cough is productive or if hemoptysis is present, the amount of sputum or blood should be quantified. In addition to the treatment of any reversible processes, cough suppressants should be considered for persistent cough. Non-opioid antitussive agents such as dextromethorphan, benzonatate, and inhaled sodium cromoglycate (69) may be tried, while opioids remain the mainstay of treatment. Codeine and hydrocodone are used most commonly for cough relief, although strong opioids are also effective. One trial included 27 non-cancer patients with persistent cough randomized to slow released morphine 5 mg twice daily or placebo. Morphine was found to provide rapid and highly significant reduction of daily cough scores by 40% (70). For cough related to bronchospasms, bronchilators may be useful. Levodropropizine has been demonstrated to be equivalent to hydrocodone for cough suppression, although it is not available in the United States. Hemoptysis is often alarming to patients, regardless of amount of expectorated blood. Approximately 20% of lung cancer patients experience hemoptysis during the course of disease. It is usually related to tumor invasion of blood vessels lining the airways, but could be caused by other pathologies such as massive pulmonary infarction. Massive hemoptysis, defined as greater than 200 ml of blood in 24 h, is associated with mortality rate as high as 59–100%. Urgent diagnostic and therapeutic bronchoscopy is warranted, and in some cases bronchial artery embolization may be required. Endotracheal intubation may be necessary to maintain airway and optimal oxygenation, highlighting the need to address patient’s goals of care early on in the disease process rather than waiting until the catastrophic event.

496

D. Hui and E. Bruera

Tranexamic acid, an anti-fibrinolytic agent, may have a role in management of persistent bleeding in the palliative care setting, although its effectiveness for management of hemoptysis in cancer patients remains to be confirmed (71).

Anorexia and Cachexia Anorexia happens in 16% of lung cancer patients at diagnosis, and increases to 56% for patients with far advanced disease (3). Weight loss, commonly associated with anorexia, is a well-established poor prognostic factor in lung cancer. Cachexia is defined as involuntary, accelerated loss of skeletal muscle in the context of a chronic inflammatory response. The resulting weight loss cannot be adequately treated with aggressive feeding. This is in contrast to starvation, which is characterized by a loss of mostly adipose tissue, and can potentially be reversed with appropriate feeding. Cachexia represents a paraneoplastic process, mediated by release of cytokines such as TNF, IL-1, IL-6, and lipid-mobolizing factor (72). The resulting metabolic imbalance leads to increased basal energy expenditure, decreased appetite, weight loss, and may be associated with xerostomia, dysphagia, fatigue, and autonomic dysfunction. Moreover, cancer and its treatments can cause nausea, pain, taste changes, odynophagia, early satiety, bowel obstruction and depression, further contributing to anorexia and cachexia. Identification and treatment of any reversible causes of anorexia and cachexia can be helpful (73). Examples include metoclopromide for early satiety and/or nausea, opioids for mucositis, nystatin for oropharyngeal or esophageal candidiasis, and antidepressants for depression. Patients with far advanced cancer should be encouraged to eat for enjoyment, without worrying too much about caloric intake. Aggressive measures, such as parenteral or enteral feeding, have limited impact on survival for this population, and may significantly compromise quality of life. Thus, supplemental nutrition should generally be limited to patients for whom starvation is a major component of weight loss such as those with severe esophagitis after chemoradiation. Dietician referral for nutritional support and counseling can be helpful. For patients in whom severe anorexia and cachexia significantly impact their quality of life, appetite stimulants such as megestrol acetate and steroids represent potential therapeutic options. A metaanalysis concluded that megestrol acetate improves appetite and weight (74). However, it is also associated with thromboembolism, adrenal insufficiency, and potentially androgen deficiency in men, and should be used with caution. Dronabinol, a cannabinoid derivative, has been shown to be less effective for anorexia palliation than megestrol acetate in a randomized trial (75). Corticosteroids may also boost appetite in the short term, although they are associated with significant side effects, and their effects tend to wane off within a few weeks. Thus, corticosterioids are generally recommended for patients with prognosis less than 6 weeks, while megestrol acetate may be used for selected

Palliative Care for Patients with Lung Cancer

497

individuals for longer durations. Other pharmacologic interventions, such as melatonin, l-carnitine, mirtazapine, and olanzapine, have no proven benefit.

Depression and Anxiety Patients with advanced cancer are at risk for developing depression or anxiety disorders, given the life-threatening nature of their illness. Clinicians working with lung cancer patients should monitor them for the development of these disorders, with prompt psychiatric referral and treatment when indicated. The diagnosis of depression is particularly challenging in patients with advanced cancer, partly because depressed mood may represent a natural reaction to the multiple stressors while living with a life-limiting illness. Patients not only face an impending loss of their lives, but also experience significant decline in function over time. Moreover, many of the symptoms of depression, such as anorexia, weight loss, insomnia, and fatigue, are also common at the end of life. However, when grief, sense of hopelessness, feelings of worthlessness or guilt significantly interfere with daily function and patients’ quality of life, active treatment for depression should be sought. Hypoactive delirium frequently mimics depression, and should be ruled out along with other organic etiologies such as hypothyroidism and brain metastases prior to diagnosis of depression. Social history, previous history of psychiatric disorders, alcohol use, and substance use should also be explored. All patients with suspected depression should be assessed for suicide risk. Risk factors for suicide in cancer patients include advanced age, poor prognosis, lack of social support, sense of hopelessness, and delirium (76). Depression can be effectively managed with a combination of non-pharmacologic and pharmacologic interventions. Antidepressants such as selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants can be useful (77). The choice of medication depends on patient’s symptoms. Patients with psychomotor agitation may benefit from sedating medications such as mirtazapine and nortriptyline, while those with psychomotor retardation may be prescribed fluoxetine. The effect of SSRIs may not be noticeable until 4–6 weeks; thus, patients with a poor prognosis may benefit from psychostimulants instead. Methylphenidate, dexamphetamine, methylamphetamine, and pemoline are more immediate acting, and have demonstrated efficacy for the treatment of depression in far advanced cancer patients (78). Compared to treatment as usual, psychotherapy is associated with significant decrease in depression score in advanced cancer patients who have not yet been diagnosed with depression (79). Different types of psychotherapy include supportive psychotherapy, cognitive behavioral therapy, and problem-solving therapy. However, psychotherapy has not been studied in detail in advanced cancer patients with depression. Patients with significant depression should be referred to psychiatry for further management.

498

D. Hui and E. Bruera

Physical, psychosocial, and existential stressors may all contribute to anxiety in advanced cancer patients. Thus, management of anxiety requires multifactorial intervention addressing various factors. Effective symptom management, supportive psychotherapy and spiritual counseling are all crucial. Relaxation techniques including guided imagery and meditation may be useful for some patients. Although a Cochrane review suggested there is not enough evidence to recommend any specific pharmacotherapy for anxiety (80), benzodiazepines have been most commonly prescribed for anxiety. Other potential medications include antidepressants, buspirone, chlorpromazine, haloperidol, olanzapine, risperidone, hydroxyzine, methotrimeprazine, and thioridazine. Further research is required to determine the role of these agents for patients with advanced cancer.

Conclusion Given the significant morbidity and mortality associated with lung cancer, palliative care plays a crucial role in optimizing patient care and minimizing suffering along the disease continuum. Many of the symptoms in lung cancer patients, such as dyspnea, pain, fatigue, cough, hemoptysis, anorexia, cachexia, depression, and anxiety, can be managed by pharmacologic and non-pharmacologic interventions. Oncologists should be skilled at communicating prognosis, discussing goals of care, facilitating transition of care, and helping patients and families navigate through tough times and uncertainties. For patients with significant physical, psychosocial, and/or existential distress, early involvement of palliative care and the interprofessional team is essential.

References 1. Jemal A, Siegel R, Ward E et al (2008) Cancer statistics, 2008. CA Cancer J Clin 58:71–96 2. Hopwood P, Stephens RJ (1995) Symptoms at presentation for treatment in patients with lung cancer: implications for the evaluation of palliative treatment. The Medical Research Council (MRC) Lung Cancer Working Party. Br J Cancer 71:633–636 3. Tishelman C, Petersson LM, Degner LF, Sprangers MA (2007) Symptom prevalence, intensity, and distress in patients with inoperable lung cancer in relation to time of death. J Clin Oncol 25:5381–5389 4. Pain Relief and Palliative Care (2002) In: National cancer control programmes: policies and managerial guidelines, 2nd ed. World Health Organization, Geneva, pp 83–91 5. Palliative Care (1995) In: National cancer control programmes: policies and managerial guidelines. World Health Organization, Geneva, pp 82–86 6. Coluzzi PH, Grant M, Doroshow JH, Rhiner M, Ferrell B, Rivera L (1995) Survey of the provision of supportive care services at National Cancer Institute-designated cancer centers. J Clin Oncol 13:756–764 7. Goldsmith B, Dietrich J, Du Q, Morrison RS (2008) Variability in access to hospital palliative care in the United States. J Palliat Med 11:1–9

Palliative Care for Patients with Lung Cancer

499

8. IOM National Cancer Policy Board (2001) Improving palliative care for cancer. Institute of Medicine, Washington, DC 9. Fadul N, Elsayem A, Palmer JL et al (2009) Supportive vs. palliative care: what’s in a name? A survey of medical oncologists and mid-level providers at a comprehensive cancer center. Cancer 115(9):2013–2021 10. Miyashita M, Sanjo M, Morita T et al (2007) Barriers to providing palliative care and priorities for future actions to advance palliative care in Japan: a nationwide expert opinion survey. J Palliat Med 10:390–399 11. Levy MH, Back A, Benedetti C et al (2009) NCCN clinical practice guidelines in oncology: palliative care. J Natl Compr Canc Netw 7(4):436–473 12. Rabow MW, Dibble SL, Pantilat SZ, McPhee SJ (2004) The comprehensive care team: a controlled trial of outpatient palliative medicine consultation. Arch Intern Med 164:83–91 13. Meyers FJ, Linder J, Beckett L, Christensen S, Blais J, Gandara DR (2004) Simultaneous care: a model approach to the perceived conflict between investigational therapy and palliative care. J Pain Symptom Manage 28:548–556 14. Meyers FJ, Linder J (2003) Simultaneous care: disease treatment and palliative care throughout illness. J Clin Oncol 21:1412–1415 15. Lamont EB, Christakis NA (2003) Complexities in prognostication in advanced cancer: “to help them live their lives the way they want to”. JAMA 290:98–104 16. Christakis NA, Lamont EB (2000) Extent and determinants of error in doctors’ prognoses in terminally ill patients: prospective cohort study. BMJ 320:469–472 17. Paesmans M, Sculier JP, Lecomte J et al (2000) Prognostic factors for patients with small cell lung carcinoma: analysis of a series of 763 patients included in 4 consecutive prospective trials with a minimum follow-up of 5 years. Cancer 89:523–533 18. Paesmans M, Sculier JP, Libert P et al (1995) Prognostic factors for survival in advanced nonsmall-cell lung cancer: univariate and multivariate analyses including recursive partitioning and amalgamation algorithms in 1, 052 patients. The European Lung Cancer Working Party. J Clin Oncol 13:1221–1230 19. Thorogood J, Bulman AS, Collins T, Ash D (1992) The use of discriminant analysis to guide palliative treatment for lung cancer patients. Clin Oncol (R Coll Radiol) 4:22–26 20. Maione P, Perrone F, Gallo C et al (2005) Pretreatment quality of life and functional status assessment significantly predict survival of elderly patients with advanced non-small-cell lung cancer receiving chemotherapy: a prognostic analysis of the multicenter Italian lung cancer in the elderly study. J Clin Oncol 23:6865–6872 21. Maltoni M, Caraceni A, Brunelli C et  al (2005) Prognostic factors in advanced cancer patients: evidence-based clinical recommendations – a study by the Steering Committee of the European Association for Palliative Care. J Clin Oncol 23:6240–6248 22. Stone PC, Lund S (2007) Predicting prognosis in patients with advanced cancer. Ann Oncol 18:971–976 23. Bruera E, Miller MJ, Kuehn N, MacEachern T, Hanson J (1992) Estimate of survival of patients admitted to a palliative care unit: a prospective study. J Pain Symptom Manage 7:82–86 24. Chuang RB, Hu WY, Chiu TY, Chen CY (2004) Prediction of survival in terminal cancer patients in Taiwan: constructing a prognostic scale. J Pain Symptom Manage 28:115–122 25. Morita T, Tsunoda J, Inoue S, Chihara S (1999) The Palliative Prognostic Index: a scoring system for survival prediction of terminally ill cancer patients. Support Care Cancer 7:128–133 26. Yun YH, Heo DS, Heo BY, Yoo TW, Bae JM, Ahn SH (2001) Development of terminal cancer prognostic score as an index in terminally ill cancer patients. Oncol Rep 8:795–800 27. Maltoni M, Nanni O, Pirovano M et al (1999) Successful validation of the palliative prognostic score in terminally ill cancer patients. Italian Multicenter Study Group on Palliative Care. J Pain Symptom Manage 17:240–247 28. Pirovano M, Maltoni M, Nanni O et al (1999) A new palliative prognostic score: a first step for the staging of terminally ill cancer patients. Italian Multicenter and Study Group on Palliative Care. J Pain Symptom Manage 17:231–239

500

D. Hui and E. Bruera

29. Glare PA, Eychmueller S, McMahon P (2004) Diagnostic accuracy of the palliative prognostic score in hospitalized patients with advanced cancer. J Clin Oncol 22:4823–4828 30. Glare P, Virik K (2001) Independent prospective validation of the PaP score in terminally ill patients referred to a hospital-based palliative medicine consultation service. J Pain Symptom Manage 22:891–898 31. Loprinzi CL, Johnson ME, Steer G (2000) Doc, how much time do I have? J Clin Oncol 18:699–701 32. Hancock K, Clayton JM, Parker SM et  al (2007) Discrepant perceptions about end-of-life communication: a systematic review. J Pain Symptom Manage 34:190–200 33. Chow E, Harth T, Hruby G, Finkelstein J, Wu J, Danjoux C (2001) How accurate are physicians’ clinical predictions of survival and the available prognostic tools in estimating survival times in terminally ill cancer patients? A systematic review. Clin Oncol (R Coll Radiol) 13:209–218 34. Mackillop WJ, Stewart WE, Ginsburg AD, Stewart SS (1988) Cancer patients’ perceptions of their disease and its treatment. Br J Cancer 58:355–358 35. Bruera E, Sweeney C, Calder K, Palmer L, Benisch-Tolley S (2001) Patient preferences versus physician perceptions of treatment decisions in cancer care. J Clin Oncol 19:2883–2885 36. Bruera E, Willey JS, Palmer JL, Rosales M (2002) Treatment decisions for breast carcinoma: patient preferences and physician perceptions. Cancer 94:2076–2080 37. Grunfeld EA, Maher EJ, Browne S et al (2006) Advanced breast cancer patients’ perceptions of decision making for palliative chemotherapy. J Clin Oncol 24:1090–1098 38. Stinnett S, Williams L, Johnson DH (2007) Role of chemotherapy for palliation in the lung cancer patient. J Support Oncol 5:19–24 39. Anderson H, Hopwood P, Stephens RJ et  al (2000) Gemcitabine plus best supportive care (BSC) vs BSC in inoperable non-small cell lung cancer–a randomized trial with quality of life as the primary outcome. UK NSCLC Gemcitabine Group. Non-Small Cell Lung Cancer. Br J Cancer 83:447–453 40. Shepherd FA, Dancey J, Ramlau R et  al (2000) Prospective randomized trial of docetaxel versus best supportive care in patients with non-small-cell lung cancer previously treated with platinum-based chemotherapy. J Clin Oncol 18:2095–2103 41. Bezjak A, Tu D, Seymour L et  al (2006) Symptom improvement in lung cancer patients treated with erlotinib: quality of life analysis of the National Cancer Institute of Canada Clinical Trials Group Study BR.21. J Clin Oncol 24:3831–3837 42. Florescu M, Hasan B, Seymour L, Ding K, Shepherd FA (2008) National Cancer Institute of Canada Clinical Trials Group. A clinical prognostic index for patients treated with erlotinib in National Cancer Institute of Canada Clinical Trials Group study BR.21. J Thorac Oncol 3:590–598 43. Temel JS, McCannon J, Greer JA et al (2008) Aggressiveness of care in a prospective cohort of patients with advanced NSCLC. Cancer 113:826–833 44. Werner-Wasik M, Scott C, Cox JD et  al (2000) Recursive partitioning analysis of 1999 Radiation Therapy Oncology Group (RTOG) patients with locally-advanced non-small-cell lung cancer (LA-NSCLC): identification of five groups with different survival. Int J Radiat Oncol Biol Phys 48:1475–1482 45. Walker-Renard PB, Vaughan LM, Sahn SA (1994) Chemical pleurodesis for malignant pleural effusions. Ann Intern Med 120:56–64 46. Cranston JM, Crockett A, Currow D (2008) Oxygen therapy for dyspnoea in adults. Cochrane Database Syst Rev (3):CD004769 47. Bruera E, de Stoutz N, Velasco-Leiva A, Schoeller T, Hanson J (1993) Effects of oxygen on dyspnoea in hypoxaemic terminal-cancer patients. Lancet 342:13–14 48. Bruera E, Sweeney C, Willey J et al (2003) A randomized controlled trial of supplemental oxygen versus air in cancer patients with dyspnea. Palliat Med 17:659–663 49. Bruera E, MacEachern T, Ripamonti C, Hanson J (1993) Subcutaneous morphine for dyspnea in cancer patients. Ann Intern Med 119:906–907 50. Jennings AL, Davies AN, Higgins JP, Gibbs JS, Broadley KE (2002) A systematic review of the use of opioids in the management of dyspnoea. Thorax 57:939–944

Palliative Care for Patients with Lung Cancer

501

51. Kvale PA, Selecky PA, Prakash UB, American College of Chest Physicians (2007) Palliative care in lung cancer: ACCP evidence-based clinical practice guidelines (2nd edition). Chest 132:368S–403S 52. Bausewein C, Booth S, Gysels M, Higginson I (2008) Non-pharmacological interventions for breathlessness in advanced stages of malignant and non-malignant diseases. Cochrane Database Syst Rev (2):CD005623 53. van den Beuken-van Everdingen MH, de Rijke JM, Kessels AG, Schouten HC, van Kleef M, Patijn J (2007) High prevalence of pain in patients with cancer in a large population-based study in The Netherlands. Pain 132:312–320 54. Fainsinger RL, Nekolaichuk CL (2008) A “TNM” classification system for cancer pain: the Edmonton Classification System for Cancer Pain (ECS-CP). Support Care Cancer 16:547–555 55. Wiffen PJ, McQuay HJ (2007) Oral morphine for cancer pain. Cochrane Database Syst Rev (4):CD003868 56. Eisenberg E, McNicol E, Carr DB (2006) Opioids for neuropathic pain. Cochrane Database Syst Rev 3:CD006146 57. Wiffen PJ, McQuay HJ, Edwards JE, Moore RA (2005) Gabapentin for acute and chronic pain. Cochrane Database Syst Rev (3):CD005452 58. Saarto T, Wiffen PJ (2007) Antidepressants for neuropathic pain. Cochrane Database Syst Rev (4):CD005454 59. Forlenza MJ, Hall P, Lichtenstein P, Evengard B, Sullivan PF (2005) Epidemiology of cancerrelated fatigue in the Swedish twin registry. Cancer 104:2022–2031 60. Mock V, Atkinson A, Barsevick A et al (2000) NCCN Practice Guidelines for Cancer-Related Fatigue. Oncology (Williston Park) 14:151–161 61. Cramp F, Daniel J (2008) Exercise for the management of cancer-related fatigue in adults. Cochrane Database Syst Rev (2):CD006145 62. Bruera E, Valero V, Driver L et  al (2006) Patient-controlled methylphenidate for cancer fatigue: a double-blind, randomized, placebo-controlled trial. J Clin Oncol 24:2073–2078 63. Fleishman S, Lower E, Zeldis J, Faleck H, Manning D (2005) A phase II, randomized, placebo-controlled trial of the safety and efficacy of dexmethylphenidate (d-MPH) as a treatment for fatigue and “chemobrain” in adult cancer patients. Breast Cancer Res Treat 94:S214 64. Minton O, Stone P, Richardson A, Sharpe M, Hotopf M (2008) Drug therapy for the management of cancer related fatigue. Cochrane Database Syst Rev (1):CD006704 65. Morrow GR, Jean-Pierre P, Roscoe JA et  al (2008) A phase III randomized, placebo-controlled, double-blind trial of a eugeroic agent in 642 cancer patients reporting fatigue during chemotherapy: a URCC CCOP Study. J Clin Oncol 26:504s 66. Barton DL, Soori GS, Bauer B et al (2007) A pilot, multi-dose, placebo-controlled evaluation of American ginseng (panax quinquefolius) to improve cancer-related fatigue: NCCTG trial N03CA. J Clin Oncol 25:493s 67. Bennett CL, Silver SM, Djulbegovic B et al (2008) Venous thromboembolism and mortality associated with recombinant erythropoietin and darbepoetin administration for the treatment of cancer-associated anemia. JAMA 299:914–924 68. Kvale PA (2006) Chronic cough due to lung tumors: ACCP evidence-based clinical practice guidelines. Chest 129:147S–153S 69. Moroni M, Porta C, Gualtieri G, Nastasi G, Tinelli C (1996) Inhaled sodium cromoglycate to treat cough in advanced lung cancer patients. Br J Cancer 74:309–311 70. Morice AH, Menon MS, Mulrennan SA et al (2007) Opiate therapy in chronic cough. Am J Respir Crit Care Med 175:312–315 71. Tscheikuna J, Chvaychoo B, Naruman C, Maranetra N (2002) Tranexamic acid in patients with hemoptysis. J Med Assoc Thai 85:399–404 72. MacDonald N (2007) Cancer cachexia and targeting chronic inflammation: a unified approach to cancer treatment and palliative/supportive care. J Support Oncol 5:157–162 discussion 164–166, 183 73. Del Fabbro E, Dalal S, Bruera E (2006) Symptom control in palliative care – Part II: cachexia/ anorexia and fatigue. J Palliat Med 9:409–421

502

D. Hui and E. Bruera

74. Berenstein EG, Ortiz Z (2005) Megestrol acetate for the treatment of anorexia-cachexia syndrome. Cochrane Database Syst Rev (2):CD004310 75. Jatoi A, Windschitl HE, Loprinzi CL et al (2002) Dronabinol versus megestrol acetate versus combination therapy for cancer-associated anorexia: a North Central Cancer Treatment Group study. J Clin Oncol 20:567–573 76. Chochinov HM (2001) Depression in cancer patients. Lancet Oncol 2:499–505 77. Fulcher CD, Badger T, Gunter AK, Marrs JA, Reese JM (2008) Putting evidence into practice: interventions for depression. Clin J Oncol Nurs 12:131–140 78. Candy M, Jones L, Williams R, Tookman A, King M (2008) Psychostimulants for depression. Cochrane Database Syst Rev (2):CD006722 79. Akechi T, Okuyama T, Onishi J, Morita T, Furukawa TA (2008) Psychotherapy for depression among incurable cancer patients. Cochrane Database Syst Rev (2):CD005537 80. Jackson KC, Lipman AG (2004) Drug therapy for anxiety in palliative care. Cochrane Database Syst Rev (1):CD004596 81. Baile WF, Buckman R, Lenzi R, Glober G, Beale EA, Kudelka AP (2000) SPIKES-A six-step protocol for delivering bad news: application to the patient with cancer. Oncologist 5:302–311 82. Clayton JM, Hancock KM, Butow PN, et al (2007) Clinical practice guidelines for communicating prognosis and end-of-life issues with adults in the advanced stages of a life-limiting illness, and their caregivers. Med J Aust 186:S77, S79, S83–S108 83. M.D. Anderson Cancer Pain Guideline for Adults (2008) http://utm-ext01a.mdacc.tmc.edu/ mda/cm/cwtguide.nsf/LuHTML/SideBar1

The Future of Lung Cancer Sophie Sun, Joan H. Schiller, Monica Spinola, and John D. Minna

Abstract  Current treatments for lung cancer have limited effectiveness and unfortunately, therefore, prognosis remains poor. Major insights into the molecular pathogenesis of lung cancers have led to new and exciting approaches to improve prevention, detection, staging and treatment of lung cancer. This chapter reviews recent advances and discusses the potential clinical applications of these novel strategies, in the future. Keywords  Lung cancer • Molecularly targeted therapy • Personalized medicine

Past, Present, and Beyond From an historical perspective, lung cancer was a rare disease until the early 1900s, when the incidence of malignant lung tumors began to rise, initially among men and then among women in the 1960s. Landmark epidemiologic studies from the 1950s established the causal link between cigarette smoking and lung cancer (1). Surgical treatments for lung cancer emerged in the 1930s, with the first successful pneumonectomy reported in 1933 (2). The prognostic significance of nodal metastases was subsequently recognized, and surgical mediastinal lymph node sampling became a key component of staging. Current surgical approaches use lobectomy or, if necessary, pneumonectomy for curative treatment. Improvements in preoperative evaluation and postoperative care have lowered operative mortality rates to less than 5%. In the 1950s, radical radiotherapy was introduced for potential cure of lung cancer, and significant technologic developments in radiation therapy planning and delivery have improved such that approximately 15% of patients with early-

S. Sun, J.H. Schiller, M. Spinola, and J.D. Minna (*) Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX, 75390-8593, USA e-mail: [email protected] Grant Support: NCI Lung Cancer SPORE P50CA70907, DOD PROSPECT W81XWH-07-1-0306, an IASLC Fellowship (Spinola). D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_20, © Humana Press, a part of Springer Science+Business Media, LLC 2010

503

504

S. Sun et al.

stage disease can be cured with radiotherapy alone. In the late 1970s and early 1980s, drug combinations using cisplatin with a vinca alkaloid or etoposide were identified as being active against lung cancers. Over the last few decades, numerous randomized trials have evaluated various chemotherapy regimens, demonstrating that platinum-based doublet chemotherapy provides modest survival benefit when compared with no chemotherapy (3, 4).

Newer Strategies for Diagnosis and Treatment Despite modest improvements in conventional cytotoxic therapies and combined modality treatments for lung cancer, the prognosis for patients remains poor. Intensive research efforts over the last decade have resulted in significant advances in our understanding of lung cancer biology. Based on this knowledge, several new molecular strategies to detect and treat lung cancer have emerged. A major breakthrough in translational oncology has been the discovery of key oncogenic signals involved in pathogenesis of lung cancer and the development of therapies targeting these pathways. Drugs targeting cell proliferation pathways and angiogenesis are now approved for use in the clinic, and clinical trials of several other targeted agents are underway. The rapid progress made in recent years offers new and exciting strategies to improve our ability to prevent, diagnose, and treat this challenging disease.

Molecular Targeted Therapies Studies in the molecular and cellular biology of lung cancer have revealed the “circuit diagram” of molecules and pathways driving lung cancer pathogenesis. Genetic and epigenetic alterations of specific molecules resulting in the activation of pathways important in carcinogenesis have been identified. In searching for targeted therapies, special attention is paid to identifying key genes that lung cancer cells absolutely require for their malignant phenotype and survival, frequently thought of as “oncogene addictions” (5). To date, several novel drugs targeting these key pathways have been developed (Table 1), and clinical trials thus far have yielded encouraging results (see chapters by Fossella, Wheatley-Price and Shepherd, Horn and Sandler, and Besse and Soria).

Towards Individualized Therapy At present, lung cancer patients are treated based on their stage of disease, tumor location, histology, and clinical features such as performance status and co-morbidities. The benefit of such an approach is modest, and the therapy is often associated with significant side effects. Thus, the appropriate choice of treatment remains a challenge for lung cancer oncologists. The goals of “personalized” medicine will be to develop

The Future of Lung Cancer

505

Table 1  Select targeted agents in clinical development in lung cancer Target Drug Trade name EGFR pathway inhibitors EGFR Gefitinib Iressa

EGFR

Erlotinib

Tarceva

EGFR Cetuximab EGFR Matuzumab EGFR Panitumumab EGFR, HER2 Lapatinib EGFR, HER2 HKI-272 EGFR, HER2 BIBW2992 EGFR, HER2, ERB4 CI-1033 VEGF/VEGFR pathway inhibitors VEGF-A Bevacizumab

Erbitux

VEGFR-2, EGFR ZD6474; vandetanib VEGFR-1-3 AZD2171 VEGFR-1-3, PDGFR, SU11248; sunitinib c-KIT, FLT-3 PTK787; vatalanib VEGFR-1-3, PDGFRbeta, c-KIT, c-fms VEGFR-1-3, PDGFR, AG-013736; axitinib c-KIT AMG 706 VEGFR-1-3, PDGFR, c-KIT Ras/Raf/MEK pathway inhibitors Ras Tipifarnib (FTI) Ras Lonafarnib (FTI) Raf-1, VEGFR-2,3, BAY 43-9006; PDGFR, c-KIT sorafenib MEK CI-1040 MEK PD-0325901 MEK AZD6244 IGF1/IGF1-R pathway inhibitors IGF1-R CP751,871 IGF1-R AMG 479 IGF1-R MK-0646 IGF1-R OSI-906 PI3K/Akt/PTEN pathway inhibitors PI3K Ly294002 mTOR Rapamycin; sirolimus mTOR CCI-779; temsirolimus mTOR RAD001; everolimus mTOR AP23573

Zactima Recentin Sutent

Vectibix Tykerb TOVOK

Avastin

Stage of development Approved with advanced NSCLCa Approved for advanced NSCLC Phase III Phase I Phase II Phase II Phase II Phase III Phase II Approved for advanced NSCLC Phase III Phase II/III Phase II Phase II

Champix

Phase II Phase I

Zarnestra Sarasar Nexavar

Phase III Phase III Phase III Phase II Phase I/II Phase I Phase II/III Phase III Phase II/III Phase I

Rapamune

Phase I Phase I Phase I/II Phase I/II Phase I (continued)

506

S. Sun et al.

Table 1  (continued) Target

Drug

Trade name

Stage of development

Tumor suppressor gene therapies p53 p53 retrovirus Phase I p53 p53 adenovirus Advexin Phase I (Ad5CMV-p53) FUS1 FUS1 nanoparticle Phase I Proteosome inhibitors Proteasomes Bortezomib Velcade Phase II Histone deacetylase inhibitors Zolinza Phase II Histone deacetylase Suberoylanilide hydroxamic acid (SAHA); Vorinostat Histone deacetylase Depsipeptide Phase I Polo-like kinase (PLK) inhibitors PLK-1 BI 6727 Phase II PLK-1 GSK-461364 Phase I Telomerase inhibitors Telomerase GRN163L Phase I Hedgehog inhibitors Hedgehog GDC-0449 Phase I/II a Limited to patients who have previously taken gefitinib and who are benefiting from or have benefited from treatment. FTI farnesyl transferase inhibitor. Adapted from Sun S, Schiller JH, Spinola M, Minna JD (2007) New molecularly targeted therapies for lung cancer. J Clin Invest 117:2740–2750

tools to sample a patient’s tissue (e.g., tumor, blood, and/or sputum) for molecular analyses, then integrate clinical information with molecular data to facilitate diagnosis, refine prognosis, predict response to treatment, and determine susceptibility to toxic side effects, in order to identify the best treatment plan for each individual patient (Fig.  1). Tailoring patients’ treatments to their tumors will likely improve response to therapy, prolong survival, minimize exposure to the side effects of unnecessary treatment, and ultimately change treatment paradigms for the better. Several approaches for the molecular analysis of lung tumors have recently been developed, providing powerful tools to compile extensive molecular information from individual tumors. These include analyses of tumor tissue obtained from biopsies, lymph node sampling, and/or resection, as well as non-invasive methods using biomarkers detectable in peripheral blood, sputum or skin.

Tumor Molecular Profiling To date, molecular studies of lung tumors have ranged from characterizing single or small numbers of biomarkers, to large scale genome- and proteome-wide analyses using mRNA, microRNA, and protein expression profiling, and DNA copy number

The Future of Lung Cancer

507 Clinical staging and diagnosis: CT,PET imaging, bronschoscopy, mediastinoscopy

Blood sample

Tumor sample

Germline DNA polymorphisms, circulating tumor cells, serum proteomics

Molecular profiles: mRNA, microRNA, protein expression, DNA copy number, mutations

Integrated disease prognosis

Bad prognosis

Good prognosis, early stage (cured with surgery or radiotherapy)

Preparation of molecular signatures to predict response to chemotherapy and targeted drugs; assess efficacy and toxicity using germline pharmacogenomic and radiogenomic profiles

No further treatment

Best treatment plan for patient’s tumor

Delivery of treatment (chemotherapy, targeted therapy, radiation) – with feedback of results to refine molecular predictors

Fig. 1  Algorithm for developing personalized medicine for lung cancer

and mutation analyses. Due to tumor heterogeneity, conventional classification and staging schemes for lung cancer have limited power to predict prognosis, as suggested by the wide range in clinical outcomes for patients with the same histology and stage of disease. Gene expression profiling of lung tumors has identified distinct molecular subtypes that correlate with clinical outcome, suggesting this approach may complement current methods (6–9). Several groups have used genomic profiling to develop prognostic molecular signatures that predict prognosis (10–12). But which of the many prognostic expression signatures should we use? Preliminary collective analyses of large microarray datasets have identified common gene signatures in the early stage of NSCLC that, in combination with standard clinical data, predict survival better than clinical variables alone (12, 13). Further collaborative studies integrating tumor molecular data from different array datasets, with testing on different patient datasets, will help identify reproducible signatures to be further tested in clinical trials. While tempting, comparison of different approaches and signatures will need to be performed with great caution. The integration and development of clinically validated tumor prognostic signatures (or drug sensitivity signatures) is complex and will require attention to the biostatistical methods used, adequate sample size, development of prediction rules using a priori selected classification methods, validation in completely independent data sets, as well as proof that the signatures significantly add to known clinical factors (14).

508

S. Sun et al.

Researchers have also described sets of genes in lung cancers, the expression levels of which can predict response to specific anti-tumor drugs (15). Genomewide expression profiling has also been used to develop signatures of drug sensitivity to individual anti-tumor drugs (16, 17). These studies provide rationale for the use of molecular predictors of drug sensitivity to optimize and individualize therapy. However, multiple predictive gene sets will need to be integrated into common drug sensitivity signatures for individual therapies and further evaluated in clinical trials. The same biostatistical concerns exist for predicting response to drugs as for predicting a prognosis.

Non-invasive Molecular Testing and Monitoring Biopsies of lung tumors are particularly difficult as they are frequently associated with complications, including pain and risk of pneumothorax. Thus, for diagnosis, fine needle aspirations are often performed, yielding sparse quantities of tissue insufficient for molecular characterization. The difficulty in obtaining adequate tissue for molecular testing of lung tumors has been a major hurdle in translating laboratory findings to the clinic. New technologies that permit non-invasive molecular analysis of readily accessible tissues, such as peripheral blood, sputum or skin, have been developed and will likely be valuable diagnostic and predictive tools. For example, a highly sensitive and specific strategy to identify and isolate viable tumor-derived epithelial cells (circulating tumor cells) using a unique microchip-based device has recently been developed (18). In a pilot study of patients with metastatic non-small cell lung cancer, investigators were able to isolate an adequate number of circulating tumor cells of sufficient purity using this technique to perform serial molecular analyses, such as EGFR mutation analysis, providing proof of principle that this approach may be useful to detect genetic markers that guide therapy and to perform serial tumor genotyping during therapy to determine if drugs are reaching the predicted molecular targets (19). The use of serum proteomic profiling may also enhance our ability to diagnose, and predict survival and drug sensitivity. Initial studies have identified proteomic patterns which may help distinguish different lung cancer histologic subtypes, which may differentiate primary lung tumors from metastases, and which may classify nodal involvement and predict prognosis (20). Identification of specific serum protein peptides by mass spectrometry may also be useful for early detection and predicting response to therapy (21). The field of pharmacogenomics has attracted wide interest, and studies have identified germline DNA polymorphisms in genes involved in drug metabolism and DNA repair that may predict response and toxicity to therapy (see chapter “Pharmacogenetics of Lung Cancer”). Numerous genetic and epigenetic alterations found in lung tumors are also detectable in sputum (22–25) and may augment current approaches for screening, diagnosis, and monitoring on therapy.

The Future of Lung Cancer

509

Molecular imaging permits non-invasive visualization of key molecules, cellular processes, and pathways and may be useful to select patients who are more likely to respond to a specific targeted therapy, to assess response to treatment, and to confirm that therapies are “hitting” the predicted target(s). A large number of molecular probes have now been developed, targeting key oncogenic pathways involved in angiogenesis, proliferation, hypoxia, apoptosis, receptor expression, and metastasis (26). Positron emission tomography (PET) imaging probes for EGFR, and markers of angiogenesis are in early stages of clinical development in lung cancer (27, 28). Future progress in this field will likely add to the armamentarium of molecular tools available for individualizing cancer therapy.

Exploring the Tumor Microenvironment The tumor microenvironment may provide additional clues to predict prognosis and response to treatment, and uncover novel therapeutic opportunities (see chapter “Tumor Microenvironment”). Unique proteins and gene expression signatures originating from the lung tumor microenvironment have recently been identified (29, 30). Future studies will characterize the multiple components of the tumor microenvironment, including molecular pathways involved in tumor angiogenesis, paracrine growth regulation and immune response, and will lead to the identification of therapeutic approaches aimed at exploiting the tumor microenvironment.

Preclinical Models for Studying New Therapies A series of immortalized human bronchial epithelial cell lines (HBEC) has recently been developed, providing a valuable in vitro system to assess the oncogenic potential of the multiple genetic changes found in lung cancer (31). These cell lines can be genetically manipulated, do not form soft agar colonies or tumors in nude mice, and appear histologically similar to normal human bronchial epithelium in three-dimensional culture. HBECs carrying mutant KRAS, p53 knockdown, or mutant EGFR alone or in different combinations have been generated; they acquire the ability to grow in soft agar and invade in three-dimensional culture, but do not induce tumor formation in nude mice, suggesting that additional genetic changes are needed to confer a full malignant phenotype (32). Recent genome-scale studies of lung cancer have revealed countless genetic alterations in lung cancers and future research will undoubtedly uncover additional genes and pathways. Thus, the HBEC system is a powerful tool to systematically validate important genes and molecular pathways as potential therapeutic targets. A synthetic lethal screen is a novel method to identify new genes mediating response to a treatment. In a recent study, using a genome-wide paclitaxel synthetic lethal screen of a lung cancer cell line, 87 candidate paclitaxel-sensitizing loci were identified, and several of these targets were able to sensitize lung cancer cells to

510

S. Sun et al.

paclitaxel concentrations 1,000-fold lower than otherwise required (33). Further studies using this novel approach are expected and will likely lead to the discovery of therapeutic targets to complement existing therapies and identify new combinatorial treatment strategies. Although in vitro studies are useful, they cannot recapitulate the carcinogenic process of human lung cancer. To this end, several transgenic mouse models for lung cancer have been developed using innovative strategies (34). In particular, novel mouse models using tetracycline-inducible or Cre/loxP recombination gene expression systems have enabled the evaluation of different mutations in oncogene and tumor suppressor genes contributing to lung tumorigenesis. For example, two groups have engineered mouse models with conditional KRAS alleles, demonstrating that somatic activation of KRAS induces lung adenocarcinoma (35, 36). A small-cell lung cancer mouse model has also been developed by inactivating both Rb and p53 (37). Mouse modeling will serve as a robust tool to further elucidate and validate molecular targets for therapy.

Targeting Cancer Stem Cells Recent evidence suggests that most cancers may arise from “cancer stem cells,” a rare subpopulation of stem-cell like tumor cells with the ability to self-renew, proliferate and differentiate into the variety of cells which make up a tumor (38). Cytotoxic treatments, such as chemotherapy and radiation, target rapidly proliferating cells that, according to the cancer stem cell model, represent the bulk of the tumor, but are incapable of self-renewal. Cancer stem cells may be inherently resistant to conventional therapies due to their low proliferative rate, drug transporter expression and increased capacity for DNA repair. Thus, the development of therapy targeting cancer stem cells represents a potentially effective strategy to completely eradicate tumors, even in advanced stage disease. The identification of cancer stem cell markers may also be useful for early detection and provide predictive and prognostic information (39, 40). To date, cancer stem cells have been identified in several malignancies, including acute myelogenous leukemia, brain, breast, prostate, and colon cancers (41–46). Attempts to characterize and isolate lung cancer stem cells are ongoing, and results from preliminary studies are encouraging (47, 48). Potential strategies to develop cancer stem cell-targeted therapies can be derived from our knowledge of the signals required by normal stem cells to survive and function. Stem cell self-renewal is a tightly regulated process and developmental pathways such as Wnt, Notch, and hedgehog, are critical in the fate determination of normal stem cells (49–52). It has been postulated that deregulation and reactivation of these signaling pathways can lead to stem cell expansion and malignant transformation. In support of this, alterations in Wnt, Notch, and hedgehog pathways have been reported in several tumor types, including lung cancer (53–56). Recent preclinical studies indicate that pharmacologically targeting cancer stem cell pathways are feasible, and further development and potential clinical testing of

The Future of Lung Cancer

511

inhibitors of these pathways are forthcoming (Fig.  2). Other candidates for cancer stem cell-targeted therapy include inhibitors of key stem cell survival pathways, modulators of the cancer stem cell microenvironment or “vascular niche” (57), factors inducing stem cell differentiation (58), inhibitors of DNA repair, or immunotherapies against cancer stem cell antigens (59). A. Self-renewal pathways Wnt Hedgehog

anti-Wnt, anti - Fzdantibodies

anti-Hh antibody

Cyclopamine Smo antagonists BMPs

Sm o

E. Differentiation pathways

L I G A N ND O T C H

Inhibitors of protein complexes (e.g. b −Cat-Tcf or Wnt-Fzd)

HH

WNT FZD

PTCH

Notch

B. Cancer stem cell antigens Cancer stem cell vaccine

TACE/ ADAM10

g -secretase inhibitor g -secretase

Cancer stem cell antigen

DSH

Smo

GSK3β CSK1α

b-C at P

ubiquitination and degradation

AXIN APC

N I C D

b-C at

nucleus GLI

ACT

GLI

REP

b-Cat TCF

D. Cell survival

Telomerase inhibitors (GRN163L)

Telomerase

Checkpoint kinase inhibitors

N I C CSL D Chk1, Chk2

C. DNA repair

Fig. 2  Cancer stem cell-specific therapeutic approaches. Hedgehog (HH), Notch, and Wnt signaling are key stem cell self-renewal pathways that are deregulated in lung cancer, and thus represent potential therapeutic targets (a). Agents inhibiting the hedgehog pathway include monoclonal antibodies against HH ligand and cyclopamine, which is a small molecule inhibitor of smoothened (SMO). Monoclonal antibodies against Wnt ligand and frizzled (FZD) receptor and inhibitors of protein complexes mediating Wnt signaling, such as Wnt–FZD or b-catenin-transcription factor (b-Cat-TCF), are examples of ways of targeting the Wnt pathway. Strategies of blocking or silencing Notch signaling can be either selective, such as the targeting of individual Notch receptors with antisense or monoclonal antibodies, or nonselective, such as the use of soluble receptor decoys that sequester Notch ligands or g-secretase inhibitors. Solid and dashed arrows represent multiple components of these pathways that, for simplicity, are not detailed here. These components also represent potential therapeutic targets. Other methods of targeting cancer stem cells include immunotherapy-based approaches against antigens present on cancer stem cells (b); targeting cancer stem cell mechanisms of resistance to cytotoxic therapy by inhibiting DNA repair enzymes such as the checkpoint kinases (Chk1, Chk2) (c); targeting stem cell-specific survival mechanisms with telomerase inhibitors (GRN163L) (d); and inducing stem cell differentiation with soluble factors such as bone morphogenetic proteins (BMPs) (e). GLI glioma-associated oncogene; GLIACT active form of GLI; GLIREP repressor form of GLI; NICD Notch intracellular domain; CSL, CBF1 suppressor of hairless, Lag-1; TACE TNF-a-converting enzyme; ADAM10 a disintegrin and metalloprotease domain 10; PTCH patched homolog; GSK3b glycogen synthase kinase 3b; CSK1a cyclin-suppressing kinase 1; DSH disheveled. Adapted with permission from Sun S, Schiller JH, Spinola M, Minna JD (2007) New molecularly-targeted therapies for lung cancer. J Clin Invest 117:2740–2750

512

S. Sun et al.

Summary Our understanding of the biology and molecular basis of lung cancers has increased considerably over the last decade. Consequently, promising strategies to improve screening, early detection, prevention, staging, and treatment of lung cancer have been developed. However, the future clinical application of these novel approaches will rely on further development in several areas. First, a priority will be to develop early detection methods that will likely involve spiral CT imaging in combination with genetic epidemiology, serum proteomics, and sputum and blood biomarkers. Second, more individualized treatment strategies will need to be developed. The recent discovery of the correlation between EGFR mutations and response to EGFR tyrosine kinase inhibitor therapy has demonstrated that molecular typing to guide therapy selection is possible. Prospective trials will be needed to prospectively validate feasibility and efficacy of selecting therapy based on patient and tumor molecular profiles. Several molecularly-targeted approaches are being developed, and molecular and clinical tools will be needed to guide the development and use of these agents. For example, this may include biomarkers that indicate whether drugs are reaching target and resulting in predicted changes by identifying molecular alterations in circulating tumor cells in blood or by molecular imaging. Last, ongoing study of cancer biology – tumor microenvironment, synthetic lethal screens to identify chemosensitizing targets, cancer stem cells and development of therapies targeting these new pathways will have profound implications for the potential cure of this disease.

References 1. Doll R, Hill AB (1954) The mortality of doctors in relation to their smoking habits; a preliminary report. Br Med J 1:1451–1455 2. Graham EA, Singer JJ (1984) Landmark article Oct 28, 1933. Successful removal of an entire lung for carcinoma of the bronchus. JAMA 251:257–260 3. Johnson DH (2000) Evolution of cisplatin-based chemotherapy in non-small cell lung cancer: a historical perspective and the eastern cooperative oncology group experience. Chest 117:133S–137S 4. Schiller JH, Harrington D, Belani CP et al (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 346:92–98 5. Weinstein IB, Joe AK (2006) Mechanisms of disease: oncogene addiction – a rationale for molecular targeting in cancer therapy. Nat Clin Pract 3:448–457 6. Inamura K, Fujiwara T, Hoshida Y et al (2005) Two subclasses of lung squamous cell carcinoma with different gene expression profiles and prognosis identified by hierarchical clustering and non-negative matrix factorization. Oncogene 24:7105–7113 7. Beer DG, Kardia SL, Huang CC et  al (2002) Gene-expression profiles predict survival of patients with lung adenocarcinoma. Nat Med 8:816–824 8. Ramaswamy S, Ross KN, Lander ES, Golub TR (2003) A molecular signature of metastasis in primary solid tumors. Nat Genet 33:49–54 9. Moriya Y, Iyoda A, Kasai Y et al (2009) Prediction of lymph node metastasis by gene expression profiling in patients with primary resected lung cancer. Lung Cancer 64(1):86–91 10. Potti A, Mukherjee S, Petersen R et  al (2006) A genomic strategy to refine prognosis in early-stage non-small-cell lung cancer. N Engl J Med 355:570–580

The Future of Lung Cancer

513

11. Chen HY, Yu SL, Chen CH et  al (2007) A five-gene signature and clinical outcome in non-small-cell lung cancer. N Engl J Med 356:11–20 12. Shedden K, Taylor JM, Enkemann SA et al (2008) Gene expression-based survival prediction in lung adenocarcinoma: a multi-site, blinded validation study. Nat Med 14:822–827 13. Lu Y, Lemon W, Liu PY et al (2006) A gene expression signature predicts survival of patients with stage I non-small cell lung cancer. PLoS Med 3:e467 14. Michiels S, Koscielny S, Hill C (2007) Interpretation of microarray data in cancer. Br J Cancer 96:1155–1158 15. Kikuchi T, Daigo Y, Katagiri T et al (2003) Expression profiles of non-small cell lung cancers on cDNA microarrays: identification of genes for prediction of lymph-node metastasis and sensitivity to anti-cancer drugs. Oncogene 22:2192–2205 16. Bild AH, Yao G, Chang JT et al (2006) Oncogenic pathway signatures in human cancers as a guide to targeted therapies. Nature 439:353–357 17. Hsu DS, Balakumaran BS, Acharya CR et al (2007) Pharmacogenomic strategies provide a rational approach to the treatment of cisplatin-resistant patients with advanced cancer. J Clin Oncol 25:4350–4357 18. Nagrath S, Sequist LV, Maheswaran S et al (2007) Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 450:1235–1239 19. Maheswaran S, Sequist LV, Nagrath S et  al (2008) Detection of mutations in EGFR in circulating lung-cancer cells. N Engl J Med 359:366–377 20. Yanagisawa K, Shyr Y, Xu BJ et al (2003) Proteomic patterns of tumour subsets in non-small-cell lung cancer. Lancet 362:433–439 21. Solomon B, Gregorc V, Taguchi F et al (2006) Prediction of clinical outcome in non-small cell lung cancer (NSCLC) patients treated with gefitinib using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) of serum. In: ASCO annual meeting proceedings part I. June 20, 2006. J Clin Oncol 24(Suppl 18S):7004 22. Wistuba II, Behrens C, Virmani AK et al (2000) High resolution chromosome 3p allelotyping of human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple, discontinuous sites of 3p allele loss and three regions of frequent breakpoints. Cancer Res 60:1949–1960 23. Palmisano WA, Divine KK, Saccomanno G et al (2000) Predicting lung cancer by detecting aberrant promoter methylation in sputum. Cancer Res 60:5954–5958 24. Belinsky SA, Liechty KC, Gentry FD et al (2006) Promoter hypermethylation of multiple genes in sputum precedes lung cancer incidence in a high-risk cohort. Cancer Res 66:3338–3344 25. Li R, Todd NW, Qiu Q et al (2007) Genetic deletions in sputum as diagnostic markers for early detection of stage I non-small cell lung cancer. Clin Cancer Res 13:482–487 26. Wester HJ (2007) Nuclear imaging probes: from bench to bedside. Clin Cancer Res 13:3470–3481 27. Pal A, Glekas A, Doubrovin M et al (2006) Molecular imaging of EGFR kinase activity in tumors with 124I-labeled small molecular tracer and positron emission tomography. Mol Imaging Biol 8:262–277 28. Beer AJ, Lorenzen S, Metz S et al (2008) Comparison of integrin alphaVbeta3 expression and glucose metabolism in primary and metastatic lesions in cancer patients: a PET study using 18F-galacto-RGD and 18F-FDG. J Nucl Med 49:22–29 29. Stearman RS, Dwyer-Nield L, Grady MC, Malkinson AM, Geraci MW (2008) A macrophage gene expression signature defines a field effect in the lung tumor microenvironment. Cancer Res 68:34–43 30. Zhong L, Roybal J, Chaerkady R et al (2008) Identification of secreted proteins that mediate cell-cell interactions in an in vitro model of the lung cancer microenvironment. Cancer Res 68:7237–7245 31. Ramirez RD, Sheridan S, Girard L et al (2004) Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res 64:9027–9034 32. Sato M, Vaughan MB, Girard L et al (2006) Multiple oncogenic changes (K-RAS(V12), p53 knockdown, mutant EGFRs, p16 bypass, telomerase) are not sufficient to confer a full malignant phenotype on human bronchial epithelial cells. Cancer Res 66:2116–2128 33. Whitehurst AW, Bodemann BO, Cardenas J et al (2007) Synthetic lethal screen identification of chemosensitizer loci in cancer cells. Nature 446:815–819

514

S. Sun et al.

3 4. Dutt A, Wong KK (2006) Mouse models of lung cancer. Clin Cancer Res 12:4396s–4402s 35. Jackson EL, Willis N, Mercer K et al (2001) Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev 15:3243–3248 36. Guerra C, Mijimolle N, Dhawahir A et al (2003) Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. Cancer Cell 4:111–120 37. Meuwissen R, Linn SC, Linnoila RI, Zevenhoven J, Mooi WJ, Berns A (2003) Induction of small cell lung cancer by somatic inactivation of both Trp53 and Rb1 in a conditional mouse model. Cancer Cell 4:181–189 38. Jordan CT, Guzman ML, Noble M (2006) Cancer stem cells. N Engl J Med 355:1253–1261 39. Glinsky GV, Berezovska O, Glinskii AB (2005) Microarray analysis identifies a deathfrom-cancer signature predicting therapy failure in patients with multiple types of cancer. J Clin Invest 115:1503–1521 40. Liu R, Wang X, Chen GY et al (2007) The prognostic role of a gene signature from tumorigenic breast-cancer cells. N Engl J Med 356:217–226 41. Bonnet D, Dick JE (1997) Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3:730–737 42. Lapidot T, Sirard C, Vormoor J et al (1994) A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367:645–648 43. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100:3983–3988 44. Singh SK, Hawkins C, Clarke ID et al (2004) Identification of human brain tumour initiating cells. Nature 432:396–401 45. Patrawala L, Calhoun T, Schneider-Broussard R et al (2006) Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells. Oncogene 25:1696–1708 46. Ricci-Vitiani L, Lombardi DG, Pilozzi E et al (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445:111–115 47. Ho MM, Ng AV, Lam S, Hung JY (2007) Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res 67:4827–4833 48. Kim CF, Jackson EL, Woolfenden AE et  al (2005) Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121:823–835 49. Liu S, Dontu G, Wicha MS (2005) Mammary stem cells, self-renewal pathways, and carcinogenesis. Breast Cancer Res 7:86–95 50. Dontu G, Jackson KW, McNicholas E, Kawamura MJ, Abdallah WM, Wicha MS (2004) Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res 6:R605–R615 51. Taipale J, Beachy PA (2001) The Hedgehog and Wnt signalling pathways in cancer. Nature 411:349–354 52. Liu S, Dontu G, Mantle ID et al (2006) Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res 66:6063–6071 53. Reya T, Clevers H (2005) Wnt signalling in stem cells and cancer. Nature 434:843–850 54. Olsen CL, Hsu PP, Glienke J, Rubanyi GM, Brooks AR (2004) Hedgehog-interacting protein is highly expressed in endothelial cells but down-regulated during angiogenesis and in several human tumors. BMC Cancer 4:43 55. Nickoloff BJ, Osborne BA, Miele L (2003) Notch signaling as a therapeutic target in cancer: a new approach to the development of cell fate modifying agents. Oncogene 22:6598–6608 56. Daniel VC, Peacock CD, Watkins DN (2006) Developmental signalling pathways in lung cancer. Respirology 11:234–240 57. Yang ZJ, Wechsler-Reya RJ (2007) Hit ‘em where they live: targeting the cancer stem cell niche. Cancer Cell 11:3–5 58. Piccirillo SG, Reynolds BA, Zanetti N et al (2006) Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature 444:761–765 59. Bao S, Wu Q, McLendon RE et  al (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444:756–760

Index

A a5b1, 255, 267. See also Integrins ABT-263, 268 Accelerated repopulation, 163, 338 Adaptive designs, oncology clinical research dose finding maximum tolerated dose (MTD), 470 model-based designs, 470–471 phase I trial, 471–472 personalized medicine, 476–477 phase I oncology trials, 471 randomized discontinuation trials, 475–476 randomized outcome, 473–474 real time updating, 474–475 seamless phase II-III design, 477 Adenocarcinoma precursor lesions, 17 Adenocarcinoma subtypes, 3. See also Bronchioloalveolar carcinoma Adjuvant and neoadjuvant therapy clinical trials chemotherapy arm, 142–143 chemotherapy regimens, 148 cisplatin-based, 143 disease-free survival rates, 144 meta-analysis, 146–147 and observation groups, 145 pathological stages, 143 platin-based chemotherapy, 142 postoperative radiotherapy, 143 radiation, 140–141 randomization, 143 current standard of practice, 141, 148 induction chemotherapy, 149–155 (see also Induction chemotherapy) meta-analyses hazard ratio, 146 survival benefit, 147 molecular markers, 149

UFT-based trials, 147–148 AE-941, 245 AEE788, 209, 215 African American. See Ethnicity/race Akt angiogenesis, role, 257 attachment/adhesion, cell, 357, 359 integrins, 359 IGF-1R, 365, 421 inhibition/targeting, 126–127, 129, 130, 257, 265, 347, 357, 360, 505 mutation, 7 p-AKT, 340 pathway, 126, 256, 257, 359–360, 421 preneoplasia, 126, 257 resistance, role in chemotherapy, 337, 340, 359–360 EGFR TKIs, and MET, 263 radiotherapy, 167 trastuzumab, 271 signaling, 126 AMG102, 54, 255, 263, 421 AMG951, 255, 269 Amrubicin, SCLC, 419 Anethole dithiolethione (ADT) and budesonide, 118 Angiogenesis accelerated repopulation, role in, 338 angiogenesis pathways/mechanisms COX-2, 45, 120 ELR+CXC Chemokines CXCR2/CXCL8, 56 CXCL5, 120 EGFR family, 206 fibroblast growth factor (FGF) 2/basic FGF/FGF receptors, 8 HGF/c-Met, 53 HIF-1a, 56, 338 12-LOX, 123

515

516 Angiogenesis (cont.) matrix metalloproteinases (MMPs), 39 NF-kB, 56 PDGF/PDGFR, 338 PI3K/AKT, 257 stromal cell co-culture/macrophages, 29, 32 IL-1b, 57 VEGF/VEGFR, 8, 55, 120, 233–234, 338, 421 brain metastases, 12 drug delivery to tumor, 55, 56, 230, 338 inhibitors AE-941 (neovastat), 245 bevacizumab, 55–57, 235–239, 422 with EGFR inhibition, 237 tracheoesophageal fistula, with chemoradiation, 422 clinical trials/systemic therapies, 55–57, 231–245, 421–423, 455 cediranib, 244–245, 423 COX-2 inhibition, 39 dasatinib, 455 gene therapy, bystander killing, 308–309 integrin inhibition, 266 matrix metalloproteinase (MMP) inhibition, 43, 245, 423 PDGFR inhibition, 242–244 protein kinase C inhibition, 264–265 RNAi, 319 semaxanib, 455 sorafenib, 239–240, 422, 455 sunitinib, 242–243, 455 thalidomide, 243–244, 422, 455 toxicity profiles, chemotherapy, 229–230 tumor development, 228, 230 vandetanib, 215, 241–242, 422–423, 455 vatalanib, 455 VEGF/VEGFR inhibition, 55–57, 234–245, 422 intracellular adhesion molecule (ICAM)-1, 57 lung cancer development/progression, 8, 29, 54–55, 120, 228, 230 microvessel density, 9, 234, 421 NSCLC, 8 PET imaging, 509 prognosis, impact on, 234, 421 tumor microenvironment, 54–57 Anorexia and cachexia, 496–497 Anti-EGFR therapy, front-line treatment, 187–189

Index Antisense oligonucleotides (AS ODNs) Bcl-2, oblimersen, 317 clusterin, OGX-011, 315–316 C-Raf-1, ISIS 5132, 316–317 H-ras, ISIS 2503, 316 protein kinase C-a , ISIS 3521, 314–315 survivin, LY2181308, 317–318 Anti-tublin agents, 165–166 Anti-VEGF monoclonal antibodies, 186–187, 235–239 Anxiety, 497–498 AP23573, 505 Apoptosis Bcl-2 targeting, 267–268 death receptors, 269–270 inhibitors (see Chemotherapy resistance) proteasome inhibitors, 268–269 APC, tumor suppressor gene, mutations, deletions, methylation, 7, 13–14 stem cell self-renewal pathways, 511 Aprinocarsen (ISIS 3521), 255, 265 Arachidonic acid (AA) metabolism and carcinogenesis, 119–120, 125 chemoprevention, 119–120 cytosolic phospholipase A2 (cPLA2), 119–120, 130 eicosanoids, 47, 119 ARQ 197, 54, 255, 264 Asian. See also Ethnicity/race EGFR mutations, 206, 217, 218 EGFR TKI efficacy, 100, 211, 213, 217 Her-2/neu mutations, 6 Asbestos, MPM, 436 AS ODNs. See Antisense oligonucleotides ATM, tumor suppressor gene, mutation, deletion, 7 Atypical adenomatous hyperplasia (AAH, atypical alveolar hyperplasia). See Chemoprevention aVb1, 255, 266. See also Integrins Axitinib, 230, 236, 245 AZD6244, 255, 262, 505 B BAC. See Bronchioloalveolar carcinoma Basic fibroblast growth factor (bFGF, FGF2). See also Fibroblast growth factor receptors angiogenesis, 9 Chemoprevention, 129–130 Chemotherapy resistance, 337, 363, 365

Index expression, 9 integrins, 266 mast cells, 32 pathway activation, 9 serum levels, 57 Bayesian designs, 477–478 Bcl-2, Bcl-xL, and related factors chemotherapy resistance, 337, 340, 362–363, 420 histone deacetylase inhibitors, 456 IHC, NSCLC vs. SCLC, 4 AS ODNs, oblimersen, 314, 317, 362, 420 NF-kB, 368 P53, 307 protein kinase C, 361 RNAi, 319 targeting, apoptosis, 267–268 BEC2 antibody, 290–292 Belagenpumatucel, 280–281 Bevacizumab efficacy and serum VEGF, bFGF, E-selectin, ICAM-1, 57, 455 in patients with brain metastases, anti­ coagulants, hypertension, 238 mesothelioma, 455 NSCLC, 55–57, 186–187, 208, 210, 211, 235–238 proteomic profile of response, 237 SCLC, 238–239, 422 toxicity, 186, 229 with erlotinib, 211, 237 bFGF. See Basic fibroblast growth factor BIBF 1120, 230, 236, 245 BIBW-2992, 209, 215, 505 Bombesin/gastrin-releasing peptide, SCLC, 10 Bone morphogenetic proteins, 511 Bortezomib, 506. See also Proteasome mesothelioma, 456 NF-kB, 52, 268 NSCLC, 52–53, 255, 268–269 SCLC, 53 B-raf activation, 206 chemotherapy resistance, 361, 365 mutations, 5, 7, 262 pathway, 259, 262, 316 targeting MEK inhibitors, 262, 505 sorafenib, 236, 239, 422, 455, 505 Brain metastases bevacizumab, 57, 238 molecular predictors, 11–12 prophylactic cranial radiation, 414–415 SCLC, incidence, 402

517 early vs. delayed thoracic radiotherapy, 163 Breast cancer 1 (BRCA1), chemotherapy resistance, 336, 338–340, 351 Breast cancer resistance protein (BCRP), chemotherapy resistance, 339, 343–344 Bronchial dysplasia, 118, 126 Bronchioloalveolar carcinoma (BAC), 3, 16–18, 114, 211 B7.1 (CD80+) vaccine, 285 C Cachexia and exia, 496–497 Cancer cell growth pathways IGF-1R, 255–258 MAPK/ERK kinase (MEK), 263–264 mTOR, 257–259 Ras pathway FTIs, 261–262 RAF/MAPK, 262 Cancer related fatigue (CRF), 492–495 Cancer stem cells targeting, 510–511 Carcinogen metabolism genes, 89–90 Carcinoma in situ (CIS), 5, 15–16, 108–109, 114 Caspases Bcl-2, 267 chemotherapy resistance, 336, 354, 355 NF-kB, 357 RasGAP, 360 IAP-1, 364 P53, 307 histone deacetylase inhibitors, 456 b-catenin, 511 Caveolin-1, and chemotherapy resistance, 336, 339, 356 MDR1, 342 CD56 antigen, 423 CDH13 methylation, 7, 13 prognosis, 14 CDK (CDK4) mutation, amplification, 7, 13 CDKN2A. See p16Ink4 Cediranib (AZD2171) mesothelioma, 455 NSCLC, 232, 236, 244 SCLC, 233, 244–245, 423 targets, 236, 244, 423, 455 toxicity, 229, 244 VEGF levels and circulating endothelial cells, 245 Celecoxib. See COX-2 inhibitors Cell cycle checkpoint arrest, 163

518 Cell cycling chemotherapy resistance, 332, 337, 339, 365–368 radiation resistance, 163 Cetuximab (Erbitux, IMC-C225). See also Epidermal growth factor receptor efficacy in NSCLC and EGFR expression, 217 NSCLC, 187, 189, 209, 216 radiosensitization, 167, 169 ChChemoprevention biomarkers for risk of tumor progression, 110–112 categories of studies (Primary, Secondary, Tertiary), 109–110 clinical trials anethiole dithiolethione, 115, 118 budesonide, 115 broccoli sprout extract, 123, COX inhibitors, celecoxib, 39, 47, 49, 108, 115, 121, 123, 129 curcumin, 123 green tea extract, 123 iloprost, 115, 122 N-acetylcysteine, 108, 110 retinoids, Vitamin A, b-carotene, 108, 110, 114–117, 125–126, 129 selenium, 115, 118 thiazolidinediones (PPARg agonists), 49, 127 vitamin E (alpha-tocoherol), 108, 115, 117 combinatorial agents, 128 cyclin D1, 124 definition of chemoprevention, 108 EGFR inhibitors, 124–125 epigenetic targets, 127–128 high-risk groups, 109–111 histone deacetylase inhibitors, 124, 128, 130 insulin-like growth factor (IGF) axis, 126 methylation, promoter CDKN2A, 128 E-cadherins, 128 IGFBP-3, 128 laminins, 128 MGMT, 128 p16, 128 RAR-b, 128 methyltransferase inhibitors, 125, 128, 130 selenium, 128 PI3K/Akt, 126–127 polyunsaturated fatty acid synthesis pathways

Index arachidonic acid (AA) metabolism, 119–120 15-prostaglandin dehydrogenase (15-PGDH), 122 prostaglandin E2 suppression, 108, 123 prostaglandin I2, antineoplastic effects. 121–122 lipoxygenase (LOX)/Leukotriene/ Linoleic acid pathway 5-LOX and leukotriene modifiers, 122–123 15-LOXs, 123–124 PPARg, 127 preneoplasia/Progression from hyperplasia to invasive cancer, 108–109, 114 atypical adenomatous hyperplasia (AAH, atypical alveolar hyperplasia), 5, 15–18, 110, 114, 118 mutations and promoter methylation in, 17, 18, 114 intraepithelial neoplasia (IEN), 114, 116, 121–122, 124, 129–130 retinoids and rexinoids, 125–126, 128–129 second primaries, 110 smoking cessation, 112–113 age of cessation, and risk, 112 pharmacologic agents bupropion, 113 nicotine replacement, 113 varenicline, 113 reduction in smoking, 112 Chemoradiotherapy clinical impact of distant metastasis, 167 induction vs. concurrent chemotherapy, 168–169 impact of, 163 systemic therapy, mechanisms anti-tublin agents, 165–166 EGFR inhibitors, 167 gemcitabine, 166–167 platinum-based agents, 164–165 Cisplatin vs. carboplatin, 165 topoisomerase inhibitors, 166 techniques 3-dimensional conformal radiation therapy (3D CRT), 169–170 intensity-modulated radiation therapy (IMRT), 169–170 proton beam therapy (PBT), 170 treatment, biological basis for, 162–163 hypoxia, 162

Index Chemosensitivity signatures, 11 Chemotherapy. See Individual drugs Chemotherapy as targeted therapy, 334 Chemotherapy resistance apoptosis inhibitors annexin IV, 337, 365 antiapoptotic factors, 365 attachment, 336, 356–357 Bcl-2, 337, 340, 362–363, 368 Bcl-xL, 337, 340, 361, 363, 365, 368 caveolin-1/caveolae organelles, 336, 339, 342, 356 cell attachment, 356–357 clusterin, 336, 365 CXCL12, 336, 357 cyclooxygenase-2 (COX-2), 336, 339, 355–356 EGFR, 336, 340, 343, 351, 357–358 ERK/MEK pathway, 337, 339–340, 351, 358–359, 361 bFGF, 337, 363, 365 gangliosides, altered membrane, 337, 365 growth hormone releasing hormone, 337, 360, 365 heat shock proteins (HSPs), 337, 356 hepatocyte growth factor (HGF), 337, 365 HER-2/neu, 337, 340, 358 hyaluronan (CD44), 337, 365 IAPs, 337, 364, 368 IGF-1R, 337, 365 c-kit, 339, 355 K-ras and RasGTPase regulator RasGAP, 339, 340, 360–361 livin, 337, 364 c-myc, 337, 365 MKP1, 337, 361 myeloid cell leukemia-1 (Mcl-1) protein, 337, 363 Nrf2/heme oxygenase-1, 337, 364 PKC, 337, 340, 344, 346, 361, 365 PPARg splice variant, 336, 365 PTEN/PI3K/Akt/mTOR pathway, 340, 357, 359–360, 365 P21WAF1/CIP1, 337, 339, 353, 357, 364–365, 367 P27Kip1, 149, 339, 357, 366–367 P70S6K and S6 phosphorylation, 337, 360 survivin, 337, 339, 364 telomeres, hTERT, telomerase, 336, 356 STAT3, 337, 365

519 Stromal-cell-derived factor 1 (see CXCL12) TRAIL decoy receptors, 337, 365 XIAP, 337, 354, 361, 364–365 Apoptotic response reduction apoptosis signal transduction, 336, 355 Bad and Bid, 340, 357, 363 Bak and Bax, 336, 340, 363 big-h3, 336, 357 caspases, 336, 354–355, 360 connexin 32, 336 DNA mismatch repair deficiency, 94–95, 167, 336, 339, 352 FUS1, 336, 355 GML protein, 336, 339, 355 p53-binding protein 2, 336, 353 p53 mutation/overexpression, 333, 336, 339–340, 350, 352–355, 358, 366 proapoptotic factors, 355 SAPK/c-Jun N-terminal kinase (JNK) and c-Jun, 336, 354 cell cycling cell cycle phase, 337, 365–366 CHK2, 337, 367, 511 Cyclin D1, 333, 367–368 E2F1 and E2F4, 337, 367 Eg5, 339, 368 mitotic slippage/aneuploidy, 337, 366 mitotic spindle checkpoint (MSC), 337, 367 14-3-3 regulatory proteins, 337, 339, 367 retinoblastoma protein (RB), 337, 340, 366 S-phase kinase-associated protein 2 (SKP2) and p27Kip1, 337, 366–367 cell lines/xenografts, 335–338 classification, 332–333 damage tolerance, 352 deoxycytidine kinase, 335, 347 DNA repair breast cancer 1 (BRCA1), 336, 338–340, 351 dihydropyrimidine dehydrogenase, 336, 352 DNA-dependent protein kinase, 336, 352 DNA mismatch repair (MMR), 352 ERCC1, 9, 94, 97, 333, 336, 339, 340, 349–351 FANCD2, 340, 351 fragile histidine triad gene (FHIT), 336, 352

520 Chemotherapy resistance (cont.) homologous recombination repair, 164, 166, 336, 340, 349, 351–352 Hus1, 336, 352 high mobility group box 2 (HMG2), 336, 352 NER pathway components, 349–350 Nonhologous end joining repair, 164, 352 Nucleotide excision repair (NER), 90, 94–95, 97–99, 336, 339–340, 349, 358 Rad51, 336, 340, 351 ribonucleotide reductase M1 (RRM1), 336, 339, 340, 350–351 topoisomerase IIa, 336 thymidylate synthase, 336, 352 xeroderma pigmentosum A, 90, 336, 350 xeroderma pigmentosum C, D, G, 90, 97, 99, 333, 350 XRCC1, 333, 350 dose-response curve flattening, 168, 332 drug and oxygen delivery/blood flow, 334–335, 338, 340 flow-limited vs. membrane limited drugs, 334 HIF-1a, 335, 338, 340, 342, 348 drug detoxification cytotoxicity bypass, 346 deoxycytidine deaminase, 333, 335, 346 dihydrodiol dehydrogenase (DDH), 335, 346 folate pools, 335, 346 glutathione (GSH), 93, 95, 335, 344–345, 369 glutathione-cysteine ligase, 95, 335, 345 glutathione peroxidase, 95, 335, 345, 362 glutathione reductase, 95, 335 glutathione-S-transferase-pi (GST p ), 93, 95, 333, 335, 340, 345 metallothioneins, 335, 345–346 NQ01, 333 peroxiredoxin V (PrxV), 335, 346 thymidine, 335 drug efflux breast cancer resistance protein (BCRP), 339, 343–344 lung resistance protein (LRP), 335, 339, 340, 344 glutathione-X-conjugate pump (GS-X) (see Multidrug resistance protein) MDR1/p-glycoprotein (P-gp), 93, 334–335, 339, 342–343

Index multidrug resistance protein (MRP), 93–94, 333, 335, 339, 342–344, 347 P-type adenosine triphosphatase (ATP 7B), 93–94, 335, 344 Ral-interacting protein (RLIP76/ RALBP1), 335, 344 drug uptake, 340–341 cell membrane characteristics, 335 rigidity/fluidity, 335, 341 phospholipids/sphingomyelin, 335, 341 cholesterol, 335, 341 fatty acids, 335, 341 CTR1, 335, 341 human equilibrative nucleoside transporter 1 (hENT1), 335, 339, 342, 347 membrane transporters, 335, 340–341 Na+, K+ ATPase, 335, 339, 341 extracellular pH, 335, 338 factors for, 339–340 host genetic polymorphisms, 90–99, 333–334, 346, 350–351, 368–369 intracellular pH, 335, 347, 369 platinums, 347 polo-like kinase, 348 Target increase/decrease/alteration: folate pathway, 335, 347 thymidylate synthase, 183, 347, 357 fragile histidine triad gene, 335 stathmin (oncoprotein 18), 335, 339, 347 tubulin (Class IIIb tubulin), 335, 339, 348, 353 topoisomerase (topo) II-a, 335, 336, 339, 348–349 transcription factors, 368–369 activating transcription factor 4 (ATF4), 338, 369 clock, 338, 369 HIV-1 Tat interacting protein 60 (Tip60), 338, 369 NF-kB , 338, 357, 364, 368 SNAIL, 338, 368 TWIST, 338, 368 Stem cells, 510 Chromosomal LOH/deletions/abnormalities 3p LOH, deletions, 4, 8, 15, 16, 109, 114, 420 miRNA-128b, 12 tumor suppressor genes, 312, 419–420 FHIT, 420

Index SEMA3B, 420 RARb, 420 RASSF1, 420 8p21-23, LOH, 4, 5, 15 9p21 LOH, 5, 16, 110, 114, 115 p16Ink4, 16 10p15 LOH, 4 11p15.5, and RRM1, 9 17p, 114 1q23, 4 9q22-32, 4 13q, 4, 15, 419–420 16q, 114 detection, aneusomy, sputum, 112 GWAS, 92 gemcitabine and response to radiation, 166 mitotic spindle checkpoint, 367 rearrangements, 3 SCLC, 398, 419–420 tumor development, 3, 13, 111 CI-1033, 209, 215, 505 CI1040, 255, 262, 505 Cilengitide, 255, 266 Cisplatin and carboplatin NSCLC, 9, 32 adjuvant/neoadjuvant, 142–154, 270 chemoradiotherapy, 162–165, 168–169 advanced disease, 48, 178–191, 207, 209, 211, 213–216, 228, 231–233, 235–236, 238–244, 256–260, 265, 267–270, 310, 314–315, 317 SCLC, 403–410, 415, 417 mesothelioma, 446, 449–456 radiosensitization, 164–165 resistance, 9, 32, 48, 88, 309, 333–369 pharmacogenetics, 92–99 c-Jun N-terminal kinase (JNK) and c-Jun, 354 CL-3877785, 209 Clinical trials regulation, cost per life-year saved, and life-years lost vs. saved, ix Clusterin chemotherapy resistance, 336, 365 OGX-011, 315–316 C-raf, antisense oligonucleotides (ISIS 5132, LErafAON), 314, 316–317 Cough and hemoptysis, 495–496 COX-2. See Cyclooxygenase-2 CP-751,871, 255, 256, 258 CRF. See Cancer related fatigue CTNNB1, mutation, 7 Cyclin D1 carcinogenesis, 109

521 chemotherapy resistance, 333, 368 overexpression in preneoplastic lesions, 17, 124 proteasomal degradation by rexinoids, 126 suppression by EGFR TKIs, 124–125 PPARg ligands plus HDAC inhibitors, 127 rexinoids (with proteasomal degradation), 126 upregulation by attachment, 357 EGFR, 124 NF-kB, and smoking, 121 Cyclophosphamide and EGF vaccine, 287 mesothelioma, 446 NSCLC, 150 resistance, 339–340, 343–344, 346, 353–354, 357 SCLC, 233, 244, 401, 404–406, 408–410, 413, 417–418 Cyclooxygenase (COX)-2. See also Arachidonic acid metabolism angiogenesis, 44, 120 constitutive expression/upregulation, 44–45, 108, 121 chemotherapy resistance, 336, 339, 355 EGFR inhibitor resistance, 45 EMT, 52 E-cadherin, ZEB1, Snail, 52 genotype and chemotherapy resistance, 355 growth hormone releasing hormone, 365 IGFBP-3 suppression, with PI3K/AKT upregulation, 126 induction by EGFR, ErbB2 and ErbB3, 124 induction by IGF-1, 126 inhibition of immune response, 39, 44, 120 intraepithelial neoplasia (IEN), expression in, 121 invasion, 44, 120 macrophages, expression in, 44 metastasis, 44 polymorphism and lung cancer risk, 45, 121 and prostaglandin E2 (PGE2), 44–47, 120–121 PPARg interactions, 49, 127 prognosis, 45, 49, 108, 116, 121, 355 regulation by NF-kB, 121 resistance to apoptosis, 44, 120–121 role, prostanoid synthesis/arachidonic acid metabolism, 44, 108, 119–121 tumorigenesis, 120–121

522 Cyclooxygenase (COX)-2 Inhibitors apoptosis induction, 121 cardiovascular events, 46–47, 49, 121 chemoprevention, 45, 108, 121, 130 combination with EGFR inhibition, 45–46, 51, 124 combination with chemotherapy, 46–47, 121, 340, 355–356 E-cadherin expression, 51 IGF-1R down-regulation, 126 impact of COX-2 expression on efficacy, 46–47, 121, 356 impact on immune function, 39 inhibition of tumor cell growth, 355 Ki67 modulation, 45, 121 lung cancer risk, 45, 109 PPARg agonists, 47, 127 RAR-b, 116–117 selenium, 118 survivin modulation, 45, 121 Cyclophilin (Cyp)-B, 289–290 Cytotoxic agents, 179–180, 419 D Dasatinib mesothelioma, 449, 451, 455 targets, 449, 455 Death receptors, 255, 269–270 Dendritic cells and ectopic lymph nodes, 33–37 vaccines, 280, 282, 284, 288, 289, 311, 420 Depression and anxiety, 497–498 Dexosome vaccine, 288 Diffuse idiopathic pulmonary neuroendocrine hyperplasia, 15 Dihydrodiol dehydrogenase, chemotherapy resistance, 335, 336, 346 DMBT1 abnormal expression, SCLC vs. NSCLC, 4 DNA methyltransferase, 118, 314 Inhibition, 128 Inhibitors, 125, 128, 130 DNA repair chemotherapy resistance, 349–352 genes, 90–91 Docetaxel NSCLC, 52–53, 97, 179, 181–184, 186, 195–199, 208–211, 215–216, 231–232, 237, 241, 243–244, 259–260, 267–268, 270, 315, 336, 339, 340, 343 vs. pemetrexed, 201 TAX 317, 196–197

Index TAX 320, 197–199 SCLC, 418 resistance, 350–351, 356, 362–363, 365, 367 (see also Taxanes) Doxorubicin. See also Topoisomerase (topo) II-a inhibitors mesothelioma, 446, 454, 456 resistance, 335–338, 343–344, 346, 348, 353–354, 356–366, 368 SCLC, 396, 404–406, 408, 410, 413 Drug detoxification, chemotherapy resistance, 344–346 Drug efflux, chemotherapy resistance, 341–344 DUSP6, gene signatures and prognosis, 11 Dysplasia, 5, 15–16, 109–110, 114–116, 118, 122, 126, 234 Dyspnea, 489–490 E E-cadherin EMT, 9, 51 COX-2, PGE2, ZEB1, and Snail, 51 inactivation by hypermethylation, 128 PPARg, 127 EGFR. See Epidermal growth factor receptor EKB-569, 209, 215 Elderly patients, 190 amrubicin plus carboplatin, SCLC, 419 bevacizumab, 55, 236, 238 erlotinib, 211 EML-ALK (Echinoderm microtubuleassociated protein-like 4 gene/anaplastic lymphoma kinase gene) fusion, 3, 5 EMT. See Epithelial-mesenchymal transition Enzastaurin, targets, assessment in NSCLC, and impact on VEGF levels, 255, 265 Ephrin receptor (EPH3, EPH10, EphA, EphB), mutations, 7 ephrin kinase receptor targeting dasatinib, 455 XL647, 236 Epidermal growth factor receptor (EGFR) chemoprevention, 124–125 chemoradiotherapy, 167 chemotherapy resistance, 336, 340, 343, 351, 357–358 EGFR gene alterations EGFR amplification, polysomy, gene copy number, 6, 7, 16, 18

Index EGFR mutations, 5, 7, 16, 100, 206, 213 and gene copy number, 18 in atypical adenomatous hyperplasia (AAH), 17 in bronchiolar epithelium, 18 EGFR IHC expression, 8, 202, 206, 358 in carcinoma in situ, 109 and prognosis, 206, 358 EGFR Inhibitors, 505 clinical trials clinical trials ongoing, 210 combined with chemotherapy, 187–188, 207, 209, 211, 214, 216, 358 COX-2 inhibitors, 45–46, 51 VEGF/VEGFR inhibitors, 57, 237 front-line therapy, NSCLC, 188–189, 207, 209, 211, 213–216, 358 IPASS, 188–198, 207, 213 elderly patients, 211, 213 previously-treated NSCLC, 201–202, 208 BR.21, 201–202, 211, 212 ISEL, 214 randomized trials cetuximab, 209 erlotinib and gefitinib, 207–208 TKIs in development, 209, 215 erlotinib, 5, 46, 51–54, 100–101, 167, 187–189, 201–203, 206–215, 217–219, 231, 237, 243–244, 257–262, 264, 268, 270–271, 366, 455, 488, 505 gefitinib, viii, 5, 46, 48, 100–101, 167, 187–189, 201, 206–210, 213–215, 217–219, 231–232, 239–241, 259–260, 271, 343, 363, 455, 505–506 markers of resistance COX-2 expression, 45 EMT (loss of E-cadherin), 6, 9, 51 IGF-1R activation, 6 mi-RNA-128b, 12 mutations & alterations of EGFR gene T790M (exon 20), 6, 219 D761Y (exon 19), 100 D770-N771 inversion (exon 20), 100 EGFR gene CA dinucleotide repeats, 100

523 mutations or alterations of other genes K-ras mutations, 213, 219, 261 c-Met amplification, 6, 54, 263 proteomic signature, 14 markers of sensitivity activating mutations, 5, 213, 217, 218 clinical factors associated with activating mutations, 5 E-cadherin expression, 51 EGFR protein expression, 217, 218 EGFR gene copy number 217, 218 clinical features associated with sensitivity, 202, 211, 217 monoclonal antibodies, 216 other tumor types BAC, 212–213 mesotheliomas, 455 RNAi, 320 histology and EGFR expression/mutation SCLC vs. NSCLC, 4 adenocarcinoma vs. Squamous cell carcinoma, 5, 8 interactions of EGFR COX-2/PGE2, 124 HSP90, 356 PPARg agonists/rosiglitazone, 48 RAR-b/RXR signaling, 117, 126 pharmacogenetics, 100 radiosensitization, 167, 169 signaling pathway, 206 Epidermal growth factor vaccine, 286–287 Epigenetic, 2, 13, 29, 108, 116–117, 125, 127, 129, 163, 332–333, 504, 508 Epigenetic methylation profiling, 13–14 Epithelial-mesenchymal transition (EMT) and inflammation/COX-2, 49–51 and co-culture with stromal cells, 29 and stem cell properties, 51 and zinc finger transcription factors (TWIST, SNAIL), 51, 368 molecular pathology, 9 resistance to chemotherapy, 368–369 to EGFR TKIs, 6, 9 stem cell properties, 51 ErbB2. See Her-2/neu ErbB3, gene signatures and prognosis, 11 ErbB4, mutations, 7 ERK. See Extracellular signal-regulated kinase Erlotinib. See Epidermal growth factor receptor (EGFR) Etaracizumab (MEDI-522, Abegrin), 255, 266

524

Index

Ethnicity/race, and lung cancer age at presentation, 78 histology, 77–78 lung cancer development/mortality risk, 71–77 stage at presentation, 78–79 survival, 79–82 treatment, 79 Etoposide. See also Topoisomerase (topo) II-a inhibitors NSCLC, 142, 143, 147, 148, 150, 151, 154, 181, 182, 233, 238, 243–244, 305, 318 pharmacogenetics, 333 pleurodesis, 489 radiosensitization, 166 resistance, 333, 335–346, 348–369 SCLC, 403–410, 412, 417, 420, 421, 423–424 Everolimus (RAD001), 255, 257, 259–261, 271, 421, 505 Excision repair cross-complementation group 1 protein (ERCC1) prognosis, 9 resistance to chemotherapy, 9, 94, 97, 333, 336, 339, 340, 349–351 to radiotherapy, 166 polymorphisms, 97, 149, 333 Extracellular matrix metalloproteinase inducer (EMMPRIN), 41–42 Extracellular signal-regulated kinase (ERK) chemotherapy resistance, 337, 339–340, 351, 358–359, 361 EGFR, 206 inhibitors AZD6244, 262 CI 1040, 262 p-ERK/ERK phosphorylation, 262, 339–340, 359 hypoxia, 359 c-MET, 263 pathway, 43, 206, 256, 259, 262

hepatocyte growth factor production, 263 tumor microenvironment/stroma, 28–29 Fibroblast growth factor receptors 1 and 2. See also Basic fibroblast growth factor angiogenesis, 8 expression, 8 targeting (BIBF 1120), 236 Fibroblast growth factor receptor 4 (FGFR4), mutations, 7 Flk-1. See Vascular endothelial growth factor receptor Flt-1. See Vascular endothelial growth factor receptor Fragile histidine triad (FHIT) gene chemotherapy resistance, 335, 336, 352 inactivation or loss in tumorigenesis, 16, 109, 420 Frizzled, 511 Front-line treatment, NSCLC anti-EGFR therapy cetuximab, 189 gefitinib and erlotinib, 187–188 INTACT-2 trial, 188 TRIBUTE and TALENT trial, 188 anti-VEGF therapy, 186–187 carboplatin vs. cisplatin, 183–184 chemotherapy effects, 178–179 cytotoxic agents, 179–180 duration of, 185–186 elderly patients, 190 non-platinum combinations, 184–185 platinum-based doublets vs. cisplatin, 180 vs. non-platinum single agents, 181 randomized study of, 183 trials of, 182 poor performance status patients,190–191 FTIs. See Farnesyl transferase inhibitors Fucosyl GM-1 (Fuc-GM1), 290–291 FUS1 Chemotherapy resistance, 336, 355 Gene therapy, nanoparticles, 312, 506

F Farnesyl transferase inhibitors, and ras inhibition, NSCLC 126, 130, 261–262, 361, 505 IGFBP-3, and PI3K/AKT, 126 Fatigue, 492–495 First-line chemotherapy, SCLC, 403–405 Fenretinide. See Retinoids Fibroblasts

G a (1,3)-Galactosyltransferase, 289 GDC-0449, 506 Gefitinib. See Epidermal growth factor receptor (EGFR) Gemcitabine mesothelioma, 449–450, 452–455 mobilization of circulating endothelial cells, 56

Index NSCLC, 97, 150, 151, 153, 178–191, 209, 216, 231–232, 236–237, 240, 243–244, 265, 268, 270, 314–315, 487 pharmacogenetics, 333 radiosensitization, 166–167 resistance, 9–10, 333–343, 346–347, 349–352, 354–359, 361–368 SCLC, 418 Gene-based therapies AS ODNs Bcl-2, oblimersen, 317 clusterin, OGX-011, 315–316 C-Raf-1, ISIS 5132, 316–317 H-ras, ISIS 2503, 316 protein kinase C-a , ISIS 3521, 314–315 survivin, LY2181308, 317–318 p53 gene therapy chemotherapy, 310 clinical trials, 309 DNA damaging agents, NSCLC, 309 expression pathway, 308 preclinical studies, 308–309 radiation therapy, 310–311 systemic metastases, 311–312 tumor suppressor genes, 307 RNAi antitumor effects, 319 ASO and RBZ nucleotides, 318 liposomal-based technologies, 320 siRNA cancer therapeutics, 320–321 Glioma-associated oncogene (GLI), 511 Glutathione chemotherapy resistance, 93, 95, 335, 344–345, 369 depletion by taxanes, and radioresistance, 165 glutamate-cysteine ligase/gammaglutamylcysteine synthase, 95, 335, 345 Glutathione peroxidase and glutathione reductase, and chemotherapy resistance, 95, 335, 345, 362 Glutathione-S-conjugate pump, and chemotherapy resistance, 342, 344 Glutathione-S-transferase anethole dithiolethione, 118 chemotherapy resistance, 93, 95, 333, 335, 340, 345 glutathione-S-transferase-p, 94, 95, 333, 335, 340, 345 pharmacogenetics, 90, 95 roles, 89

525 Glycogen synthase kinase (GSK) 3b mutation, 7 stem cell self-renewal pathways, 511 GRN163L, 506, 511 Ground glass opacities, and atypical adenomatous hyperplasia, 17, 114 GSH. See Glutathione GSK-461364, 506 GST. See Glutathione-S-transferase GVAX vaccine, 281–284 H Heat shock proteins (HSPs) chemotherapy resistance, 356 mesothelioma, 457 Hedgehog, self renewal/stem cell pathway, 506, 510, 511 Hepatocyte growth factor (HGF, scatter factor). See also c-MET chemotherapy resistance, 337, 365 signaling and role in tumor development, 53–54, 263 SCLC, 421 targeting, AMG102, 255, 263, 421 Her-2/neu, ErbB2, 206, 209, 216, 505. See also p185; Trastuzumab chemotherapy resistance, 337, 340, 358 in preneoplasia and lung cancer development, 4–6, 17, 109, 124 mutations and gene copy number/gene amplification, NSCLC, 4–6, 17, 216 vaccines IDM-2101, 284 dendritic cell, 289 XL647, 236 HETEs. See Hydroxyeicosatetraenoic acids HGF. See Hepatocyte growth factor HIF-1a. See Hypoxia-inducible factor-1a Histological types of lung cancer, 2–4 Histone deacetylation, 116, 127–128 Histone deacetylase inhibitors, 123, 130, 356, 456, 505 Hispanic. See Ethnicity/race HKI-272, 209, 215, 219, 505 HODEs. See Hydroxyoctadecadienoic acids H-Ras, 259, 261, 319. See also K-ras ISIS 2503, 316 hTERT. See Human telomerase reverse transcriptase Human bronchial epithelial cell lines (HBEC), 509

526 Human telomerase reverse transcriptase (hTERT) and telomerase chemotherapy resistance, 336, 356 inhibitors (GRN163L), 506 pharmacogenetics, 91 and RAR-b expression, 117 RNAi, 319 self-renewal pathways, stem cells, 511 tumorigenesis, 16, 91 vaccine therapy (GV1001, HR2822), 282, 288 Hydroxyeicosatetraenoic acids (HETEs), and carcinogenesis, 119, 122–124, 127 Hydroxyoctadecadienoic acids (HODEs), and carcinogenesis, 119, 124, 127 Hydroxyl radicals, 162, 164 Hyperplasia, 15–16, 108–110, 114, 118 Hypoxia inducible factor (HIF)-1a. See also Angiogenesis angiogenesis, 8, 56 chemotherapy resistance, 335, 338, 340, 342, 348 I IAPs. See Inhibitor of apoptosis proteins IDM-2101 vaccine, 284–285 Induction chemotherapy, NSCLC clinical trials chemotherapy arm, 150–151 gemcitabine/cisplatin, 153 pathologic complete responses, 151 phase III randomized trials of, 151 platin-based regimens, 150 postoperative mediastinal radiation therapy, 150 radiographic response and complete resection, 150 randomization, 151 current standard of practice, 155 meta-analyses, 153–154 surgical morbidity and mortality, 154 Inflammation carcinogenesis, 28–29, 49–51, 57–58 EMT, 49–51 radiation sensitivity, 100 Inflammatory cytokines and carcinogenesis, 28, 50, 123, 437 “inflammasome”, 50 Ifosfamide NSCLC, 144, 150–151, 197–198 resistance, 350, 353–354, 360, 362 SCLC, 405–408

Index IgA, peripheral blood, and chemosensitivity, 11 IGF-1R. See Insulin-like growth factor-1 receptor IGFBP-3. See Insulin-like growth factor binding protein-3 Iloprost (PGI2 agonist) chemoprevention, 122, 127 PPARg activity, increase, 127 Inhibitor of apoptosis proteins (IAPs), chemotherapy resistance, 338, 364, 368 Innovative clinical trials. See also Adaptive designs, oncology clinical research Bayesian designs, 477–478 fixed sample designs, 467 group sequential, 468 sample size reassessment methods, 478–479 Insulin-like growth factor (IGF-1) chemotherapy resistance, 359 COX-2 interactions in carcinogenesis, 126 expression in SCLC, 421 serum, and lung cancer risk, 126 Insulin-like growth factor binding protein-3 (IGFBP-3) chemoprevention, 130 COX-2, suppression by, 126 induction of apoptosis, 126 methylation, 128 serum levels, decreased, and lung cancer risk, 126 prognosis, 126 Ras, and resistance to IGFBP-3, 126 farnesyl transferase inhibition, 126 retinoids, 125–126 Insulin-like growth factor-1 receptor (IGF-1R) chemotherapy resistance, 337, 365 COX-2 inhibitor, down-regulation by, 126 EGFR mutant vs. wild type and IGF-1R kinase phosphorylation, 257 EGFR TKI resistance, 6 expression in lung cancer, 256, 421 pathway, 255–256, 359, 421 targeting, 256, 457 AMG 479, 256, 258, 421, 505 AMG 655, 258 AVE1642, 256 CP-751,871, 255–256, 258, 505 squamous cell carcinoma, 256 MK-0646, 505 NVP-ADW742, 256 OSI-906, 505 RI507, 256, 258

Index INSR mutation, 7 Integrins activation of signaling pathways, 266, 359 angiogenesis, 264, 266 chemotherapy resistance, 357, 359 EMT, 9 inhibition, 266 inhibitors cilengitide, 255, 266–267 etaracizumab (Abegrin, MEDI-522), 255, 266 peptidomimetics (S247), 266 volociximab (Vitaxin, MEDI-523), 266–267 ligands, 266 PPARg-induced upregulation, 127 roles, 266 Interleukin-1a, peripheral blood, and chemosensitivity, 11 Intraepithelial neoplasia (IEN). See Chemoprevention Irinotecan gene expression profiles and outcome, 11 mesothelioma, 453–454 NSCLC, 179 pharmacogenetics, 333, 334 radiosensitization, 166 resistance, 333, 338, 342–344, 349–351, 353–354, 356, 362 SCLC, 407–408, 418 K KDR. See Vascular endothelial growth factor receptor K-ras targeting farnesyl transferase inhibitors, 261, 361, 505 RNAi, 319 gene copy number, 13 geranylgeranyltransferase, 261 and growth hormone resleasing hormone, 365 mouse models, 50, 56, 509, 510 mutations, 4–8, 10, 16–18, 29, 49, 109, 114, 145, 149, 206, 257, 260–262, 316 prognosis, 360 resistance to chemotherapy, 339–340, 360–361 resistance to EGFR inhibitors 213, 218–219, 361

527 pathway, 43, 126, 206, 256, 259, 261–262, 266, 308 B-Raf/MAPK, 262, 316 L Lapatinib, 209, 215, 505 Latinos. See Ethnicity/race L-BLP-25 vaccine, 284 LCK, gene signatures and prognosis, 11 Lipoxygenase (LOX)/Linoleic acid/ Leukotriene pathway, and carcinogenesis, 118–119, 122–124, 127, 130 LKB1 mutation, deletion, or inactivation, 4, 5, 8 Locoregional disease therapy, mesothelioma chemotherapy and biological therapy gene therapy and immunotherapy, 451–452 intrapleural, 451 neoadjuvant chemotherapy, 449–451 radiotherapy brachytherapy, 445 intensity-modulated radiotherapy treatment plan, 447 prophylactic therapy, 448–449 therapeutic doses, 446 surgery clinical trials, 444 extrapleural pneumonectomy (EPP), 442 pleurectomy/decortication (P/D), 444 treatment options, 442 Lonafarnib, 261, 361, 505 Loss of heterozygosity (LOH), 4–5, 10, 13, 16, 114 LOX. See Lipoxygenase L523S vaccine, 286 Lung cancer mortality and survival rates, vii, 106, 141 Lung resistance protein (LRP), chemotherapy resistance, 335, 339, 340, 344 Ly294002, 505 M Macrophages COX-2, 44, 123 dendritic cells, 280 IL-1b and angiogenesis, 56 and Mast cells, 30–33 prognosis, 30–33 T reg cell populations, 38 tumor development and progression, 29–30

528 Malignant pleural mesotheliomas (MPM). See Mesothelioma Mammalian target of rapamycin (mTOR) apoptosis inhibition, 359–360 cancer cell growth pathways, 257, 266, 359–360, 396 resistance to other agents, 271, 359–360 inhibitors in clinical development, 255, 257, 259–260, 421, 505 prevention, 127, 129, 130 Mapatumumab (TRM-1, HGS-ETR1), 255, 270 MAPK. See Mitogen activated protein kinase MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) chemotherapy resistance, 358–359 inhibitors (CI 1040, AZD6244, XL518, RDEA119, AZD8330, GSK1120212, RO5126766), 255, 262, 505 inhibition of radiation-induced MEK phosphorylation by EGFR TKIs, 167 pathway, 259 Matrix metalloproteinases (MMPs) COX-2, 120 epithelial-to-mesenchymal transition, 9 inhibitors, clinical trials, 40, 43–44, 245, 423 integrins, 266 NF-kB, 121 NSAIDs, 43 PPARg, 127 prognosis, NSCLC, 41–43 RECK, 43 SCLC, 423 tumor development, 40, 120 Matuzumab, 209, 216, 505 Mcl-1. See Myeloid cell leukemia-1 protein MDM2 mRNA levels with p53 gene therapy, 311 mutation, amplification, 7, 13 inhibition by p14ARF, 308 P53 inhibition, 308 Melanoma-associated antigen E-3 (MAGE-3), 287 c-MET. See also Hepatocyte growth factor EGFR TKI resistance, 54, 263 inhibitors (PF-02341066, ARQ197, XL880), 54, 264, 421 mesothelioma, 456–457 mutation, amplification, overexpression, 5, 54, 263 signaling and role in tumor development, 53–54, 263 SCLC, 421

Index Mesothelioma advanced disease, therapy anti-angiogenic agents, 455 chemotherapy, 453–454 c-Met tyrosine kinase oncogene, 456–457 histone deacetylase inhibitors, 456 proteasome inhibitors, 456 radiotherapy, 452–453 ribonuclease inhibitors, 456 diagnosis and staging coronal and axial images, 441 locoregional therapy role, 442 MRI, 440 PET, 441 pleural mesothelioma, 443 SMRP level, 440 epidemiology and etiology amphibole asbestos, 436 MPM, 436 nonasbestos agents, 438, 439 projected incidence of, 437 SV40, 438 histologic subtypes biphasic, 440 vs. carcinoma, 440 epithelioid, 439 sarcomatoid, 440 locoregional disease, therapy chemotherapy and biological therapy, 449–452 intrapleural therapy: chemotherapy, 451 gene therapy and immunotherapy, 451–452 radiotherapy, 444–449 surgery, 442, 444 Meta-analyses mesothelioma combination vs single agent chemotherapy, 454 NSCLC adjuvant chemotherapy, 142, 146–148 UFT-based, 147–148 induction chemotherapy, 153–155 advanced NSCLC chemotherapy vs. best supportive care, 178 cisplatin vs. carboplatin, 184 maintenance therapy, 186 number of agents, 179–180 platinums vs. non-platinums, 184–185 radiotherapy

Index cisplatin regimens plus radiotherapy, 162 postoperative radiotherapy, 141 risk of cancer gene polymorphisms, 89–90 selenium, 118 rofecoxib, myocardial infarction, 46 SCLC chemotherapy dose-intensity, 409 platinums vs. non-platinums, 405 prophylactic cranial irradiation, 414, 416 thoracic radiation concurrent vs. sequential with chemo, 413 early vs. delayed, 395, 414–415 yes vs. no, 411 symptom control anorexia, 496 cancer-related fatigue, 494 Metallothioneins, chemotherapy resistance, 95, 335, 339, 345–346 Methylation, promoter, 3–4, 7, 13–16, 91, 112, 114, 116–118, 127–128, 332, 359, 367, 420 methylation profiles, 10, 13–14, 111 MicroRNAs (miRNAs), roles, normal and in tumor development, 12, 321 prognosis, 12, 28, 93, 506–507 Microvessel density. See Angiogenesis Mitogen-activated protein kinase (MAPK), 266 activation by COX-2 and EGFR TKI resistance, 45 chemotherapy resistance, 337, 358, 361, 364 mesothelioma development, 437 mutations, 7 pathways, 123, 125, 127, 206, 256, 262, 266, 316 Mitogen-activated protein kinase (MAPK) phosphatase-1, chemotherapy resistance, 337, 361 Mitomycin-C mesothelioma, 453 NSCLC, 142, 144, 149–151, 185 resistance, 335, 343, 345, 350, 353–354, 357, 360, 362 Mitoxantrone, 360 MMD, gene signatures and prognosis, 11 MMP. See Matrix metalloproteinase Molecular pathology, lung cancer characteristics, 5 differences, NSCLC and SCLC, 4 molecular profiling studies

529 DNA copy number profiles, 13 epigenetic methylation profiling, 13–14 integrative approaches, 14–15 microRNA (miRNA) profiles, 12 proteomic signatures, 14 RNA signatures, 11–12 NSCLC angiogenesis, 8 chemotherapy associated markers, 9–10 EMT, 9 genetic abnormalities, 4–8 pathology, 3–4 pathogenesis adenocarcinoma precursor lesions, 17 non-smoking-related adenocarcinoma precursors, 17–18 pathways, 16 profiling DNA analysis, adenocarcinoma, 7 squamous cell carcinoma preneoplastic lesions, 15–16 recessive oncogenes, 3 SCLC molecular abnormalities, 10 pathology, 10 Molecular targeted agents, 254–255 antiangiogenic-related agents integrins, 266–267 PKC, 265–266 apoptosis Bcl-2 targeting, 267–268 death receptors, 269–270 proteasome inhibitors, 268–269 biochemotherapy combination, 270–271 cancer cell growth pathways IGF-1R, 255–258 MAPK/ERK kinase (MEK), 263–264 mTOR, 257, 259, 260 Ras pathway, 259, 261–262 Molecular targeted therapy, 504–506 Motesanib (AMG 706), 54, 230, 236, 245, 505 MPM. See Mesothelioma mTOR. See Mammalian target of rapamycin Mucin (MUC)-1, 284 Multidrug resistance protein (MRP), 342 c-Myc (MYC) chemotherapy resistance, 337, 365 gene amplification, overexpression, 4, 10, 13, 420 prognosis, 365 regulation by 26S proteasome, 268 Myeloid cell leukemia-1 (Mcl-1) protein, chemotherapy resistance, 337, 363

530 N Native American. See Ethnicity/race Neoadjuvant therapy. See Adjuvant and neoadjuvant therapy N-cadherin, and EMT, 9 Never-smokers, lung cancer in, 108 NF1, tumor suppressor gene, mutations, 7 NF-kB. See Nuclear factor-kB Nicotine addiction genes, 91 Nimotuzumab, 209, 216 NKX2-1. See TITF-1 Non-invasive molecular testing and monitoring, 508–509 Nonsteroidal anti-inflammatory drugs (NSAIDs). See COX-2 inhibitors Notch, self renewal/stem cell pathway, 510, 511 NTRK, mutation, 7 Nuclear factor-kB (NF-kB) activation by cigarette smoke, 121 chemotherapy resistance, 338, 357, 364, 368 inhibition by IkB, 52 IkB kinase phosphorylation and proteasomal degradation of IkB, 52 inhibitors bortezomib, 52–53 CHS828, SU6668, 52 12-LOX, 123 proteosome inhibitors, 52–53, 268, 456 tumor microenvironment, 52–53, 58 O Obatoclax (GX15-070), 267 Oncology clinical research and designmethodology. See Adaptive designs, oncology clinical research Opioids, 491 Oxaliplatin, NSCLC, 231, 270, 369 P p14ARF induction by ras and myc, 308 silencing by CDKN2A methylation or deletion, 127–128, 308 increased MDM2 levels/p53 inactivation 308 p16Ink4 benefit from adjuvant chemotherapy, lack of correlation with p16 expression, 149, 367

Index inhibition of cyclin D1/cyclin-dependent kinase-4,-6 complexes, 124 mutations, LOH, inactivation, deletion, methylation, 4, 5, 7, 14, 15, 128 methylation by smoking, 128 reexpression by inhibition of histone deacetylase or DNA methyltransferase, 128 prognosis, 14 SCLC, 10 p21CIP1/WAF1: cell cycle. 308 chemotherapy resistance, 357, 367 p53, 308 senescence, 308 p27KIP1, chemotherapy resistance, 339, 357, 366–367 P53 chemotherapy resistance, 333, 336, 339–340, 350, 352–355, 358, 366 gene therapy, 307–313, 506 molecular pathology (including mutations, deletions & LOH), 4–5, 7–8, 10, 15–17, 92, 109, 113, 420 murine models, 509–510 radiopotentiation by chemotherapy, 165–166 RNAi, 319–320 tumor suppression mechanisms and apoptosis, 307–308 vaccine therapy (IDM-2101), 284, 291 p-185. See HER-2/neu Pacific Islander. See Ethnicity/race Paclitaxel. See also Taxanes gene expression profiles and outcome, 11 impact on subsequent docetaxel efficacy, 199, NSCLC, 48, 55–56, 142, 145, 151–152, 169, 179–191, 209–210, 216, 231–232, 235–238, 240–242, 245, 256–261, 265, 267, 269, 310, 315, 317 resistance, 48, 335–336, 338, 342–343, 346, 349, 353, 356–361, 363–366, 368 SCLC, 406, 412, 418, 422 synthetic lethal screen, 509–510 T reg cells & cytokines, 38 Pain, 490–492 Palliative care models, 485 pathophysiology of, 488 role of decision making, 486–489 patient access, 484–485 prognosis discussion, 487

Index survival estimation, 485–486 symptom management anorexia and cachexia, 496–497 cough and hemoptysis, 495–496 depression and anxiety, 497–498 dyspnea, 489–490 fatigue, 492–495 pain, 490–492 Panitumumab, 209, 216, 230, 505 Paraneoplastic syndromes, SCLC, 398–400 Pazopanib (GW786034), 230, 236, 245, 270 PD-0325901, 505 PDGFR. See Platelet derived growth factor receptor Pemetrexed mesothelioma, 449–450, 453–457 NSCLC, 52–53, 182–183, 186, 199–201, 208, 210–211, 215, 231, 237, 244. 262, 265, 268 resistance, 335–336, 346–347, 357, 366 SCLC, 418 targets, 199–200 Performance status, 178–179, 190–191, 196, 199, 202, 210, 215, 244, 403, 417, 486 Peroxisome proliferator-activated receptorgamma (PPAR-g) agonists (thiazolidinediones ciglitazone, rosiglitazone, pioglitazone, troglitazone), 47–49, 127 and 15-prostaglandin dehydrogenase (15-PGDH), 47–49, 122, 129 and 15-LOX-2, 124 chemoprevention, 127, 130 prognosis, reduced expression, 49 splice variant, 48–49 and chemotherapy resistance, 336, 365 15-PDGH. See 15-Prostaglandin dehydrogenase Personalized medicine, 476–477, 506, 507 PF-00299804, 209, 215 PGE2/PGI2. See Prostaglandin E2 and I2 Pharmacodynamics, 96–97 Pharmacogenetics components of, 93 EGFR-target therapy, 100 genetic predisposing factors apoptosis genes, 91–92 carcinogen metabolism genes, 89–90 DNA repair genes, 90–91 genetic variants, 91–92 nicotine addiction, 91 platinum drug therapy, 92–99 multiple variants, cumulative effect of, 97–99

531 pharmacodynamics, 96–97 pharmacokinetics, 95 radiogenetics, 99–100 Pharmacokinetics, 95 Phosphatase and tensin homolog (PTEN) chemotherapy resistance, 359 LOH, 4 mutation/deletion/hypermethylation, 7, 257, 271, 359, 421 pathway, 257, 359, 505 PPARg, rosiglitazone, 48 SCLC, 421 trastuzumab resistance, 271 Phosphoinositide 3-kinase (PI3K) activation by COX-2, 126, 129 EGFR, 124, 206 Her-2/neu, 206 IGF/IGF-1R, 126, 256, 365, 421 integrins, 266, 357 c-MET and HGF, with gefitinib resistance, 263 RasGAP, 360 chemoprevention, 129–130 chemotherapy resistance, 337, 357, 359–360, 365 inhibitors deguelin and myo-inositol, 126 enzastaurin, 265 Ly294001, 505 mutation/amplification, 7 pathway, 126–127, 257 SCLC, 421 PI3K. See Phosphoinositide 3-kinas) PKC. See Protein kinase C PKI-166, 209, 215 Platelet derived growth factor receptor (PDGFR) angiogenesis, 8, 338 chemotherapy resistance, 338, 365 HIF-1a, 338 inhibitors AMG706, 505 axitinib, 236, 505 BIBF 1120, 236 cediranib (AZD2171), 244, 423, 455 dasatinib, 455 imatinib mesylate (Gleevec), 455 motesanib, 236 pazopanib, 236 sorafenib, 236, 239, 422, 505 sunitinib, 236, 243, 455, 505 vatalanib, 455, 505 mutations, 7

532 Platinums doublets, 407, 408 drug binding, 347 etoposide, triplet regimens, 406–407 multiple variants, cumulative effect of, 97–99 pharmacodynamics, 96–97 pharmacokinetics, 95 radiotherapy, 164–165 Polo-like kinase: Chemotherapy resistance, 348 inhibitors, 506 Polysialic acid (polySA), 291–292 Polyunsaturated fatty acid metabolic pathways, chemoprevention arachidonic acid (AA) metabolism, 119–120 COX-2, PGE 2 , and PGI 2 , 120–121 12-LOX, 123 5-LOX and leukotriene modifiers, 122–123 15-LOXs, 123–124 15-PGDH, 122 PGI 2 , 121–122 Postoperative radiotherapy (PORT), NSCLC, 141 PPARg. See Peroxisome proliferator-activated receptor gamma (PPARg) Prevention. See Chemoprevention Prognosis/survival, vii, 106 Prognostic factors, lung cancer Bcl-2, association with favorable prognosis, 267, 363 COX-2 expression, 45, 49, 108, 116, 121, 355 C-reactive protein, 486 dendritic cells, 35, 36, 58 dihydrodiol dehydrogenase, 346 dyspnea, 489 EGFR gene copy number and protein expression, 124, 206, 217 EGFR mutations, 218 EMMPRIN, 42 EMT, 9 ERCC-1, 9, 97, 149 ethnicity/race, 73–74, 79–82 IGFBP-3, serum, 126 gender, 486 gene expression profiles, 11–12, 28–29, 149, 507 tumor microenvironment cytokine gene signature, 29 genetic/single nucleotide polymorphisms, 88 Her-2/neu (ErbB2), 124, 358 K-ras, 149, 261, 316, 360–361 LDH, 403, 486

Index leucocytosis, 486 macrophages and mast cells, 30–33, 58 matrix metalloproteinases (MMPs), 40–43 c-MET, 263 metagene model, 149 methylation, RASSF1A, RUNX3, CDH13, p16Ink4, APC, 14 microvessel density, 8, 234 c-Myc, 365, 420 p27, 149 performance status, 190, 403, 486 PPARg reduction, 49 proteomic profiles, 14, 508 RAR-b, 116 RECK, 43 S100 family of inflammation-related proteins, 50 smoking, 88 stage, 141, 401, 403, 486, 507 stem cell markers, 510 survivin, 317, 318 TGF-b2, 280 tissue inhibitors of MMPs (TIMPs), 40, 43 T reg cell recruitment, 38, 58 TTF-1 (TITF-1), 369 tumor-induced bronchus-associated lymphoid tissue (Ti-BALT), 36 VEGF expression, 8, 55, 234 weight loss, 486, 496 Prognostic factors, mesothelioma, 440–442, 457 15-Prostaglandin dehydrogenase (15-PGDH) chemoprevention, 122, 129 inhibition of PGE2, 47, 122, 125, 129 inhibition by EGFR, 125 induction by EGFR TKI, 122, 129 NSAIDs, 122 PPARg, 122, 127, 129 tumor suppressor function, 122 Prostaglandin E2 (PGE2) cell growth regulation, 45 EGFR activation, 124 EGFR TKI resistance, 124 EMT, 49–51 E-cadherin, Zeb1, Snail, Slug, 51 G protein-coupled receptors, 45 EGFR pathway activation, 45 immune regulation and inhibition, 39, 44, 49–50 inactivation by 15-PGDH, 47, 122, 125 inhibition, 45 PPARg agonists (Rosiglitazone, pioglitazone), 47, 49 invasion, 45

Index PGE2 receptor type 3, and tumorigenesis, 121 production, arachidonic acid metabolism, 119–120, 125 Src signaling, 121 Prostaglandin I2, protection against lung cancer, 121–122 Proteasome cell cycle regulation and apoptosis, 268 inhibitors, 255, 268–269, 456, 506 (see also Bortezomib) and NF-kB, 52–53, 268, 456 Protein kinase B. See AKT Protein kinase C (PKC), 255, 457 angiogenesis, 264–265 chemotherapy resistance, 337, 340, 344, 346, 361, 365, inhibitors aprinocarsen (ISIS 3521, ISI 641A), 265, 314–315 enzastaurin, 255, 265 mesothelioma, 457 NF-kB and 12-LOX, 123 tumor development, 265, 314 Protein phosphatase, peripheral blood, and chemosensitivity, 11 Proteomic signatures, 14, 508 Proton beam therapy (PBT), 170 PTEN. See Phosphatase and tensin homolog PTPRD, mutations, 7 Q Quality of life, and Systemic Therapy mesothelioma, 454 NSCLC, 179, 181–182, 185, 189–190, 199, 211, 240, 242 SCLC, 416 symptoms and palliative care, 484, 487, 490, 492–494, 496–497 R R1507, 255, 256, 258 Racial and ethnic diversity histology and stage, 77–79 immigration and acculturation, 82 incidence, 72–73 mortality, 73–74 risk factors family history, 76 genetic variations and metabolic pathways role, 77

533 occupational and environmental exposures, 76–77 race and diet, 76 smoking and tobacco consumption, 74–76 survival, 79–82 treatment, 79 Radiation treatment. See Chemoradiotherapy Radiogenetics, 99–100 Radiosensitization by systemic agents, 163–167 Randomized clinical trials (RCTs), chemoprevention, 115 alpha-tocopherol (AT), 117–118 anethole dithiolethione (ADT) and budesonide, 118 retinoid, 114–117 selenium, 118 Raf. See B-raf Rapamycin, 257, 505. See also Sirolimus RARb. See Retinoids Ras. See K-ras RasGAP, 360 Ras-associated domain family 1 (RASSF1) gene hypermethylation molecular pathology, 4 prognosis, 14 SCLC, 420 mesothelioma, 457 RASSF1. See Ras-associated domain family 1 RB. See Retinoblastoma protein Recessive oncogenes, 2–3 Retinoblastoma protein (RB) and phosphorylated RB (p-RB) chemotherapy resistance, 337, 340, 366 cyclin D1/cyclin-dependent kinase-4,-6, phosphorylation by, 124 mouse model, 510 mutation/inactivation/LOH, 4, 7–8, 10, 15, 420 regulation of cell proliferation, 307, 366 Retinoic acid signaling. See Retinoids Retinoids association with COX-2 and telomerase, 116, 117 chemoprevention, 108, 110, 114–117, 125–126, 130 chicken ovalbumin upstream promotertranscription factors (COUP-TFs), 117 EGFR expression, 124 fenretinide (N-(4-hydroxyphenyl) retinamide, 4-HPR), 128, 129 IGFBP-3 expression, 125

534 Retinoids (cont.) prognosis, 116 RARb alternative splicing and dominantnegative variants, 117 RARb promoter methylation/epigenetic silencing, 4, 7, 13, 109, 117, 125, 128, 420 retinoids combined with COX-2 inibitors, 129, 355 RXRs and rexinoids, 125–126 proteasomal degradation of cyclin D1, 126 suppression by tobacco carcinogens, 116 tretinoin plus dendritic cell/p53 vaccine, SCLC, 420 tumor development, 4, 7, 13, 109, 116 Retinoid randomized clinical trials (RCTs), 114–117 Rexinoids. See Retinoids Ribonucleotide reductase M1 (RRM1), 166 chemotherapy resistance, 10, 336, 339, 340, 350–351 gemcitabine target, 9 prognosis, 9 radiosensitization by gemcitabine, 166 Ribozymes (RBZ), 318–319 Risk factors for lung cancer development, 45, 49, 71–77 asbestos exposure, 111 “at risk” population, 108 biomarkers and high-risk groups, 111–112 COPD, 49–50, 111 diet, ethnicity and lung cancer risk, 76 ethnicity/race, 71–77 ethnicity/race interaction with family history of lung cancer, 76 immigration/acculturation, 82 metabolic pathways, 77 occupational and environmental exposures, 76–77 smoking, tobacco consumption, 74–76 family history, 76, 88, 111 gender, 72–73, 78, 111 genetic/single nucleotide polymorphisms, 88–89 apoptosis genes, 91–92 carcinogen metabolism genes, 89–90 CLPTM1L, 91 DNA repair genes, 90–92 hTERT, 91 nicotine addiction genes, 91 high risk groups, 110 IGF-1, IGFBP-3, serum, 126 inflammation, 49–50, 122

Index endoplasmic reticulum stress-induced unfolded protein response, 50 preneoplastic lesions, 109, 111, 114 reduction in risk COX-2 inhibitors/NSAIDs/aspirin, 109, 121 glucocorticoids, 118 retinoids, 114, 116 rosiglitazone, 49 selenium (and effect on COX-2 and 5-LOX), 118 smoking/smoking cessation, 74–76, 91, 108, 111–113 vitamin A deficiency, 116 Risk factors for lung cancer mortality ethnicity/race, 73–74, 79–82 gender, 73 smoking/smoking cessation, 112 Risk index. See Prognosis/survival; Prognostic factors RNA interference (RNAi) antitumor effects, 319 ASO and RBZ nucleotides, 318 liposomal-based technologies, 320 siRNA cancer therapeutics, 320–321 RNA interfering silencing complex (RISC), 318 RNA signatures, 11–12 Rofecoxib, 46, 47. See also COX-2 inhibitors RUNX3 methylation and prognosis, 14 RXR. See Retinoids S Second primary lung cancers, in patients with a prior cancer, 110 g-Secretase, 511 Selenium randomized clinical trials (RCTs), 118 Self-renewal pathways, 511. See also Stem cells Serum mesothelin-related peptide (SMRP), 441 Serum proteomic profiling, 508 Sirolimus, 257, 505 Small cell lung cancer (SCLC), 10 classification, 397 epidemiology and etiology, 396–397 histology, 397 history and prognosis, 403 initial management alternating/sequential regimens, 405–406 etoposide-platinum-based triplet regimens, 406–407 first-line chemotherapy, 403–405, 410 increased dose intensity/density, 407–409 platinum doublets, 407, 408

Index prolonged administration, 409 metastatic sites, 402 molecular pathology, 398 paraneoplastic syndromes, 398–400 prophylactic cranial irradiation, 414–416 relapsed disease, 416–418 staging, 401–403 surgical resection, 410–411 systemic therapies angiogenesis, 421–423 CD56 antigen, 423 cytotoxic agents, 419 growth factor and receptor abnormalities, 420–421 matrix metalloproteinases (MMPs), 423 nonreceptor proto-oncogenes, 420 tumor suppressor genes, 419–420 Thoracic Radiotherapy (TRT) dose and fractionation, 411–413 early vs. late meta-analyses, 415 timing, 413–415 Smoke, from wood, fuels, and cooking, 112 Smoking cessation, 112–113 and tobacco consumption age-related racial differences, 75 mentholated cigarettes, 75 non Hispanic Whites, 74 prevalence rates, 75–76 Smoothened, 511 SN-38. See Irinotecan Sorafenib, 505 mesothelioma, 455 NSCLC, 231, 239–240 SCLC, 233, 236, 240, 422 targets, 236, 239, 422, 455 toxicity, 229, 262 S-phase kinase associated protein 2 (SKP2) and p27Kip1, 366–367 chemotherapy resistance, 337, 366–367 Squamous cell carcinoma preneoplastic lesions, 15–16 Squamous dysplasia, and lung cancer pathogenesis, 5, 15 Src inhibitors BIBF 1120, 236 dasatinib, 449, 451, 455 mesothelioma, 449, 451, 455 PGE2-dependent cell growth, 121 Stage, 141, 401, 403, 443, 486, 507 STAT1, gene signatures and prognosis, 11 Stem cell factor PI3K/Akt pathway activation, 359

535 SCLC, 421 Stem cells EMT, 51 pathways, Wnt, Notch, Hedgehog pathways, 510–511 properties, 510 targeting, 510 vascular niche, 511 Stump recurrence, 110 Sunitiinb, 505 mesothelioma, 455 NSCLC, 232, 236, 242–243 targets, 236, 242, 455 toxicity, 229, 271 T Taxanes. See also Docetaxel; Paclitaxel NSCLC, 150, 154, 181, 184, 186, 198, 216 radiopotentiation, 165 resistance, 335–340, 342–344, 348–351, 353–355, 357, 359–362, 366–368 SCLC, 404, 418 Telomeres/telomerase. See Human telomerase reverse transcriptase Tegafur/UFT, 147–148 Temsirolimus (CCI-779), 257, 259–260, 422, 505 TGFa and b. See Transforming growth factor a and b Thalidomide antiangiogenesis, targets, 236, 244 mesothelioma, 455 NSCLC, 232, 236, 243 SCLC, 233, 243–244, 422 toxicity, 229 Thoracic radiation therapy (TRT), SCLC dose and fractionation, 411–413 early vs. late meta-analyses, 415 timing, 413–415 Three-dimensional conformal radiation therapy (3D CRT), 169 Thromboxane A2 carinogenesis, 120 chemotherapy resistance, 335, 341 Tipifarnib, 261–262, 505 TITF1 (NKX2-1, TTF-1, thyroid transcription factor-1) adenocarcinoma vs. squamous cell carcinoma, 6 amplification, gene, 4, 6, 7, 13 EGFR mutation, 6 role, 6

536 TKIs. See Tyrosine kinase inhibitors (TKIs) Tobacco and smoking consumption, 74–76 Topoisomerase, as a therapeutic target, 334 Topoisomerase (topo) II-a inhibitors and chemotherapy resistance, 335–336, 339, 348–349, 352, 366, inhibitors, 166, 349 (see also Amrubicin; Doxorubicin; Etoposide; Mitoxantrone) Topotecan resistance, 335–338, 354, 366 SCLC, 407, 409, 412, 417–419, 422 TP53. See p53 TRAIL. See Tumor necrosis factor-related apoptosis-inducing ligand receptor Transcriptase catalytic subunit antigen, 288 Transcription factors, chemotherapy resistance, 368–369 Transforming growth factor a, 189, 206 Transforming growth factor b, 50, 99, 100, 122, 280, 293 antisense oligonucleotides (Belagenpumatucel), 280–282 Trastuzumab (Herceptin) NSCLC, 209, 216 PTEN loss, AKT/mTOR activation, and resistance, 271, 359 radiopotentiation, 167 TSG101 abnormal transcripts, SCLC vs. NSCLC, 4 TTF-1. See TITF-1 Tuberous sclerosis complex (TSC) 1/2 AKT and mTOR, 257 mutations, 7, 257 Tubulin as a chemotherapy target, 334 chemotherapy resistance, 335, 339, 347–348, 353 Tumor microenvironment, 509 angiogenesis anti-angiogenic therapies, 55–57 chemotherapy regimen, 56 pro-angiogenic and angiostatic peptides, 56 pro-angiogenic and anti-angiogenic factors, 55 VEGF, 55–56 cyclooxygenase (COX)-2 and prostaglandin E2 cytoplasmic staining, 44 gefitinib and celecoxib, 46 G protein-coupled receptors (GPCR), 45 isoforms, 44

Index rofecoxib, 46 dendritic cells and ectopic lymph nodes adenoviral vectors (AdV), 34 B cells, 36 CCL21, 34 cytokines and chemokines, 34 cytotoxic T lymphocytes (CTL), 34 MHC antigens, 33 hepatocyte growth factor (HGF) and c-Met, 53–54 inflammation and epithelial–mesenchymal transition carcinogenesis, 49 COPD, 50 E-cadherin expression, 51 inflammasome, 50 macrophages and mast cells cytotoxic, 32 gastric carcinoma, 30 immune effector cells, 30 islet macrophage density, 31 platinum-based combination chemotherapy, 32 stromal macrophage density, 31 matrix metalloproteinases (MMPs) ECM and non-ECM molecules, 40 extracellular matrix metalloproteinase inducer (EMMPRIN), 41–42 MMP inhibitors (MMPIs), 40, 43, 44 musculoskeletal toxicity, 40, 44 RECK expression, 43 NF-k B bortezomib, 52, 53 I k B kinase (IKK), 52 PPARg and 15-prostaglandin dehydrogenase chronic rosiglitazone/pioglitazone treatment, 49 splice variant, 48–49 thiazolidinediones (TZDs), 47, 49 troglitazone treatment, 47, 48 translational study, 29 T regulatory cells ovarian cancer, 38 paclitaxel, 38 prostaglandin E2 (PGE2), 39 role of, 39 tumor-infiltrating lymphocytes (TIL), 37, 38 Tumor molecular profiling, 506–508 Tumor necrosis factor receptor superfamily, 269 Tumor necrosis factor-related apoptosisinducing ligand receptor (TRAIL), 255, 270, 368

Index decoy receptors 1 and 2, chemotherapy resistance, 337, 365 Tumor suppressor genes, 419–420 Tyrosine kinase inhibitors (TKIs), 187–188 in development, 215 erlotinib, 211–213 gefitinib, 213–215 V Vaccine therapy NSCLC, development of B7.1 (CD80 + ), 285 belagenpumatucel, 280–281 CypB, 289–290 cancer stem cells, 511 demographics and trials results, 282–283 dendritic cells, 289 dexosome, 288 epidermal growth factor, 286–287 a (1,3)-galactosyltransferase, 289 GVAX, 281–284 IDM-2101, 284–285 L-BLP-25, 284 L523S, 286 melanoma-associated antigen E-3 vaccine, 287 transcriptase catalytic subunit antigen, 288 SCLC, development of BEC2, 290–292 demographics and trials results, 291 Fuc-GM1, 290 polySA, 291–292 WT1, 292–293 Vandetanib impact of VEGF levels on efficacy, 242 mesothelioma, 455 NSCLC, 210, 215, 231–232, 236, 241–242 SCLC, 233, 242, 422–423 targets, 209, 215, 236, 241, 422–423, 455, 505 toxicity, 229 Vascular endothelial growth factor (VEGF). See also Angiogenesis; Bevacizumab angiogenesis, 8, 120, 233–234, 338, 421 COX-2, 120 chemoprevention, 130 chemotherapy resistance, 338, 340 inhibitors (see Angiogenesis; Bevacizumab) isoforms, 233–234 microvessel density, 234, 421

537 prognosis, 8, 55, 234, 421 radiation upregulation, 165 resistance to EGFR inhibition, 237 signaling pathway, 237 targeting, 234–235, 421, 455, 505 Vascular endothelial growth factor receptor (VEGFR). See also Angiogenesis; Bevacizumab expression on cancer cells, 235 inhibition, effect on blood vessel formation and regression, 234 inhibitors (see Angiogenesis; Bevacizumab) mutations and amplification, 7 VEGFR-1,-2,-3, 234, 236 VEGF. See Vascular endothelial growth factor VEGFR. See Vascular endothelial growth factor receptor Vimentin, EMT, 9 Vinca alkaloid. See also Vinorelbine mesothelioma, 454 NSCLC, 142, 147, 149, 154 radiosensitization, 165–166 resistance, 335–338, 340, 342–345, 347–351, 353–354, 358, 362, 366–367 SCLC, 404 Vinblastine. See also Vinca alkaloid mesothelioma, 453 NSCLC, 142–144, 148–149, 162, 168 Vincristine. See also Vinca alkaloid resistance, 339, 340, 343, 344, 346, 354, 355, 361. 363, 365, 369 SCLC, 404–407, 409–410, 413, 417–418 Vindesine. See also Vinca alkaloid NSCLC, 142–144, 148–149 resistance, 339, 342–343, 353 Vinflunine, mesothelioma, 454. See also Vinca alkaloids Vinorelbine. See also Vinca alkaloid mesothelioma, 453–454 NSCLC, 142–144, 146–149, 179–185, 189–190, 197–198, 209, 213, 216, 239, 258, 310 resistance, 333–337, 339–340, 342–345, 347–349, 352–353, 355–358, 360, 362–363, 367 SCLC, 418 Volociximab (Vitaxin), 255, 266–267 W Wilm’s tumor gene (WT1), 292–293 Wnt, self renewal/stem cell pathway, 42, 510–511

538 X XL185, 264 XL647, 209, 215, 219, 230, 232, 236, 245 XL880, 264 X-linked inhibitor of apoptosis protein (XIAP), chemotherapy resistance, 337, 354, 361, 364, 365

Index Xq22.1 LOH, 4 XRCC 1 Chemotherapy resistance, 333, 350 Polymorphisms lung cancer risk, 91 radiation toxicity, 100 XRCC5, 6, 7, 99