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

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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

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subpopulation of patients, as happened with gefitinib. The other side of the problem is that with p40% 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

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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

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

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

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

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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

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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

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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

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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

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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

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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

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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

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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 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

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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).


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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

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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).


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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).

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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


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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

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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.


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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).

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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.


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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

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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).


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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).

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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.


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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.

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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

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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

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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

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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

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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

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