Lung Cancer Second edition

Lung Cancer, Second Edition edited by Jack A. Roth, MD Professor and Chairman Department of Thoracic and Cardiovascul...

Author: Jack A. Roth | James D. Cox | Waun Ki Hong

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Lung Cancer, Second Edition

edited by

Jack A. Roth, MD Professor and Chairman Department of Thoracic and Cardiovascular Surgery Bud Johnson Clinical Chair Professor of Tumor Biology The University of Texas M.D. Anderson Cancer Center Houston, Texas

James D. Cox, MD Professor and Head Division of Radiation Oncology The University of Texas M.D. Anderson Cancer Center Houston, Texas

Waun Ki Hong, MD American Cancer Society Clinical Research Professor Charles A. LeMaistre Distinguished Chair in Thoracic Oncology Professor and Chairman Department of Thoracic/Head and Neck Medical Oncology The University of Texas M.D. Anderson Cancer Center Houston, Texas

b

Blackwell SCience

©1998 by Blackwell Science, Inc.

USA

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Canada

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Blackwell Science KK MG Kodenmacho Building 7-10 Kodenmacho Nihombashi Chuo-ku, Tokyo 104, Japan All rights reserved. No part of this book may be reproduced in any form or by any electronic or mechanical means, including information storage and retrieval systems, without permission in writing from the publisher, except by a reviewer who may quote brief passages in a review. First published 1998 Acquisitions: Christopher Davis Prod uction: Ellen Samia Manufacturing: Lisa Flanagan Typeset by Braun-Brumfield, Inc. Printed and bound by Braun-Brumfield, Inc. Printed in the United States of America 98 99 00 01 5 4 3 2 1 The Blackwell Science logo is a trade mark of Blackwell Science Ltd, registered at the United Kingdom Trade Marks Registry DISTRIBUTORS Marston Book Services Ltd PO Box 269 Abingdon, Oxon OX14 4YN England (Orders: Tel: 44-01235-465500 Fax: 44-01235-465555)

Australia Blackwell Science Pty Ltd 54 University Street Carlton, Victoria 3053 (Orders: Tel: 3-9347-0300 Fax: 3-9349-3016) Library of Congress Cataloging-in-Publication Data Lung cancer/edited by Jack A. Roth, James D. Cox, Waun Ki Hong.-2 nd ed. p. cm. Includes bibliographical references and index. ISBN 0-86542-573-6 1. Lungs-Cancer. 1. Roth, Jack A. II. Cox, James D. (James Daniel), 1938- . III. Hong, Waun Ki. [DNLM: 1. Lung Neoplasms-diagnosis. 2. Lung Neoplasms-therapy. WF 658 9604 1998] RC280.L8L765 1998 616.99'424 - dc21 DNLM/DLC for Library of Congress 97-37264 CIP

Contents Preface vii Contributors

ix

Markers of Susceptibility 1 Margaret R. Spitz, Xifeng Wu, and Scott M. Lippman 2 Biology of Preneoplastic Lesions

25

Jin Soo Lee, Li Mao, and Waun Ki Hong 3 Familial Predisposition To Lung Cancer

57

Thomas A. Sellers and Joan E. Bailey-Wilson 4 Preoperative Evaluation of the Patient with Lung Cancer 73

Rodol£o C. Morice 5 Lung-Sparing Operations for Cancer

87

Robert J. Ginsberg 6 Role of Thoracoscopy in the Diagnosis and Treatment of Lung Cancer 105

Joseph LoCicero III 7 Surgery for Small-Cell Lung Cancer

115

Thomas W. Shields and Karl Karrer 8 Extended Resections for Lung Cancer

135

Paolo Macchiarini and Philippe Dartevelle 9 High-Dose-Rate Remote Afterloading Endobronchial 163 Brachytherapy Ritsuko Komaki, Rodol£o C. Morice, and Garrett L. Walsh 10 Three-Dimensional Conformal Radiotherapy in Bronchogenic Carcinoma 181

Bahman Emami and Mary V. Graham 1 1 Combinations of Radiation Therapy and Chemotherapy for NSCLC 195

Ritsuko Komaki and James D. Cox

v

vi

Contents 12

Preoperative and Postoperative Adjunctive Therapy for Resectable NSCLC 207

John C. Ruckdeschel 217 Vincent A. Miller, Kenneth K. Ng, Stefan C. Grant, Ellen Early, and Mark G. Kris

13

New Chemotherapeutic Agents in NSCLC

14

Chemoprevention

235

Jonathan M. Kurie, Li Mao, Jin Soo Lee, Scott M. Lippman, Margaret R. Spitz, and Waun Ki Hong 15

Molecular Detection of Lung Cancer

253

David Sidransky 16

Fluorescence Detection

269

Stephen C. Lam and Branko Palcic 17

Photodynamic Therapy and Thoracic Malignancies Harvey I. Pass

18

Growth-Factor Receptors as a Target for Therapy

Roman Perez-Soler and John Mendelsohn 19

Immunologic and Biologic Approaches to Lung Cancer Therapy 343

David P. Carbone 20

Genetic Manipulations for the Treatment of Lung Cancer 369

JackA. Roth Index

383

287 309

Preface Five years ago, when the First Edition of Lung Cancer was published, the editors were optimistic that progress in reducing the mortality from this disease would result from insights in the biology of cancer and new treatment strategies. We remain optimistic and believe this book documents the considerable progress that has been made since the First Edition. The emphasis of many studies on the biology of lung cancer has shifted to studies of factors influencing predisposition and the development of preneoplastic lesions. Identification of the subset of smokers at high risk for developing lung cancer will be important for implementing chemoprevention strategies. Surgical studies have defined the appropriate minimal operation for lung cancer. Extended resections can now be done with acceptable risk and result in long-term survival for some patients. Technical advances in radiation therapy using threedimensional conformal techniques now permit higher doses of radiation to be delivered to tumors. Combined modality therapy and new chemotherapeutic agents are yielding higher response rates and improved survival. The final section of the book describes novel approaches that may emerge as important preventive, diagnostic, and therapeutic modalities in the near future. The editors hope that this book will provide a succinct and timely summary of recent advances and new research in the field of lung cancer. These advances are possible only in the context of broadly based multidisciplinary research and treatment programs. J.A.R. J.D.C. W.K.H.

vii

Contributors Joan E. Bailey-Wilson, PhD

National Center for Human Genome Research National Institutes of Health Bethesda, Maryland David P. Carbone, MD, PhD Vanderbilt Cancer Center Nashville, Tennessee James D. Cox, MD

Professor and Head Division of Radiation Oncology The University of Texas M.D. Anderson Cancer Center Houston, Texas Philippe Dartevelle, MD

Department of Thoracic and Vascular Surgery and Heart-Lung Transplantation Marie-Lannelongue Hospital (Paris-Sud University) Le Plessis Robinson France Ellen Early, MD

Memorial Sloan-Kettering Cancer Center New York, New York Bahman Emami, MD

Professor and Chairman Department of Radiotherapy Loyola University Medical Center Hines, Illinois Robert J. Ginsberg, MD, FRCS(C)

Professor of Surgery Cornell University Medical College Chief of Thoracic Service Department of Surgery Memorial Sloan-Kettering Cancer Center New York, New York ix

x

Contributors Mary V. Graham, MO

Assistant Professor of Radiology and Radiation Oncology Washington University School of Medicine St. Louis, Missouri Stefan C. Grant, MO

Assistant Professor of Medicine Cornell University Medical College Assistant Physician Memorial Sloan-Kettering Cancer Center New York, New York Waun Ki Hong, MO

American Cancer Society Clinical Research Professor Charles A. LeMaistre Distinguished Chair in Thoracic Oncology Professor and Chairman Department of Thoracic/Head and Neck Medical Oncology The University of Texas M.D. Anderson Cancer Center Houston, Texas Karl Karrer

Emeritus University Professor of Medicine University of Vienna Vienna Austria Ritsuko Komaki, MO

Department of Radiation Oncology The University of Texas M.D. Anderson Cancer Center Houston, Texas Mark G. Kris, MO

Cornell University Medical College Thoracic Oncology Service Division of Solid Tumor Oncology Memorial Sloan-Kettering Cancer Center New York, New York Jonathan M. Kurie, MO

Assistant Internist and Assistant Professor of Medicine Department of Thoracic/Head and Neck Medical Oncology The University of Texas M.D. Anderson Cancer Center Houston, Texas Stephen C. Lam, MO, FRCPfC)

Associate Professor Department of Medicine University of British Columbia Head, Bronchoscopy Program British Columbia Cancer Agency Vancouver, British Columbia Canada

Contributors

Jin

500

Lee, MD

Chief, Section of Thoracic Medicine Internist and Professor of Medicine Department of Thoracic/Head and Neck Medical Oncology The University of Texas M.D. Anderson Cancer Center Houston, Texas Scott M. Lippman, MD

Professor and Chairman Department of Clinical Cancer Prevention The University of Texas M.D. Anderson Cancer Center Houston, Texas Joseph LoCicero III, MD

Associate Professor Harvard Medical School Deaconess Surgical Associates Boston, Massachusetts Paolo Macchiarini, MD

Department of Thoracic and Vascular Surgery and Heart-Lung Transplantation Marie-Lannelongue Hospital (Paris-Sud University) Le Plessis Robinson France Li Mao, MD

Assistant Cell Biologist and Assistant Professor of Medicine Department of Thoracic/Head and Neck Medical Oncology The University of Texas M.D. Anderson Cancer Center Houston, Texas John Mendelsohn, MD

President Department of Clinical Investigation The University of Texas M.D. Anderson Cancer Center Houston, Texas Vincent A. Miller, MD

Instructor in Medicine Cornell University Medical College Clinical Assistant Physician Thoracic Oncology Service Department of Medicine Memorial Sloan-Kettering Cancer Center New York, New York Rodolfo C. Morice, MD

Associate Professor of Medicine Chief, Section of Pulmonary and Critical Care Medicine Department of Medical Specialties The University of Texas M.D. Anderson Cancer Center Houston, Texas

xi

xii

Contributors Kenneth K. Ng, MD

Cornell University Medical College Fellow in Medical Oncology Memorial Sloan-Kettering Cancer Center New York, New York Branko Paleie, PhD

Professor Department of Pathology University of British Columbia Head, Cancer Imaging Department of Physics British Columbia Cancer Agency Vancouver, British Columbia Canada Harvey I. Pass, MD

Professor of Surgery Wayne State University Chief, Thoracic Oncology Aerodigestive Program Director Karmanos Cancer Institute Detroit, Michigan Roman Perez-Soler, MD

Internist and Professor of Medicine Chief, Section of Experimental Therapeutics Deputy Chairman Department of Thoracic/Head and Neck Medical Oncology The University of Texas M.D. Anderson Cancer Center Houston, Texas Jaek A. Roth, MD

Professor and Chairman Department of Thoracic and Cardiovascular Surgery Bud Johnson Clinical Chair Professor of Tumor Biology The University of Texas M.D. Anderson Cancer Center Houston, Texas John C. Ruekdesehel, MD

Professor of Medicine and CEO H. Lee Moffitt Cancer Center and Research Institute University of South Florida Tampa, Florida Thomas A. Sellers, PhD, MPH

Division of Epidemiology School of Public Health University of Minnesota Minneapolis, Minnesota

Contributors

Thomas W. Shields, MO

Professor Emeritus of Surgery Northwestern University Medical School Chicago, Illinois David Sidransky, MO

Department of Otolaryngology-Head and Neck Surgery Division of Head and Neck Cancer Research Johns Hopkins University School of Medicine Baltimore, Maryland Margaret R. Spitz, MO, MPH

Professor and Chair Department of Epidemiology The University of Texas M.D. Anderson Cancer Center Houston, Texas Garrett l. Walsh, MO

Associate Professor and Associate Surgeon Department of Thoracic and Cardiovascular Surgery The University of Texas M.D. Anderson Cancer Center Houston, Texas Xifeng Wu, MO, PhD

Assistant Professor of Epidemiology The University of Texas M.D. Anderson Cancer Center Houston, Texas

xiii

xiv Notice: The indications and dosages of all drugs in this book have been recommended in the medical literature and conform to the practices of the general community. The medications described do not necessarily have specific approval by the Food and Drug Administration for use in the diseases and dosages for which they are recommended. The package insert for each drug should be consulted for use and dosage as approved by the FDA. Because standards for usage change, it is advisable to keep abreast of revised recommendations, particularly those concerning new drugs.

1 Markers of Susceptibility Margaret R. Spitz, Xifeng Wu, and Scott M. Lippman

Lung cancer accounts for 32% of all male cancer deaths and 25% of all female cancer deaths in the United States (1). It is estimated that in the United States in 1997 there will be 178,100 incident cases of lung cancer and 160,400 deaths (2). The prognosis for lung cancer remains dismal, with a 14% overallS-year relative survival rate for whites and 11% for blacks (2). The association between cigarette smoking and lung cancer risk has been one of the most intensely studied issues in cancer epidemiology. Traditional epidemiologic research relied on measures of "external exposure" (usually self-reported) in defining these associations (3). Classic epidemiology was limited in its capability to evaluate factors (beyond age, sex, and ethnicity) that affect host susceptibility to carcinogens. Once carcinogen-induced genetic and epigenetic events began to be elucidated, however, knowledge of the molecular etiology of lung cancer rapidly began to accumulate. There is an increasing interest in the use of biomarkers in cancer epidemiology to enhance exposure assessment, to gain insight into disease mechanisms, to understand acquired or inherited susceptibility, and to refine risk assessment (4). Vineis and Caporaso (5) have categorized molecular epidemiologic studies on mechanisms of tobacco carcinogenesis into three categories, which will be used in this chapter: 1. Markers of interindividual differences in susceptibility 2. Measures of internal exposures (including dose to the target tissue) 3. Early biological effects of exposure (e.g., cytogenetic damage, mutations) 1

2

Chapter 1

GENE-ENVIRONMENT INTERACTIONS Over 80% of lung cancers are attributed to tobacco exposure, and the relative risk for lung cancer in current smokers is twenty times greater than for those who have never smoked (6). However, only a fraction of smokers «20%) will develop lung cancer in their lifetimes. One of the challenges of quantitative human risk assessment of carcinogenic exposures and of molecular dosimetry lies in the need to account for interindividual variation in susceptibility. The existence of genetically determined factors that abrogate the effects of environmental carcinogens is one mechanism that may explain the wide range in susceptibility across populations. This biologic variability may occur at any phase of the multistage carcinogenic process. Evaluation of these differences is part of the broad field of ecogenetics, the study of genetically determined differences in response to environmental agents. Epidemiologic studies of familial aggregation of lung cancer provide indirect evidence for the role of genetic predisposition to lung cancer. Tokuhata and Lilienfeld (7) and Ooi et al. (8) found increased risks for lung cancer for both smoking and nonsmoking relatives of lung cancer patients. In men, but not in women, the effect from smoking appeared to be stronger than the familial effect. Sellers et al. (9) also documented an increased familial risk for lung cancer after controlling for the effects of age, sex, occupation, and smoking. They subsequently performed segregation analyses and reported results compatible with mendelian codominant inheritance of a rare major autosomal gene for lung cancer predisposition (10). Twin studies have also confirmed that monozygotic twins discordant for smoking behavior have different risks for lung cancer (11).

Metabolic POlymorphisms Any factor that influences carcinogen absorption, distribution, or accumulation in the target tissue will impact on cancer susceptibility. The dose of tobacco carcinogens to which lung tissue is exposed may be modulated by genetic polymorphisms in the enzymes responsible for activation and detoxification of these carcinogens. Genetic differences in these pathways are likely to be an important source of interindividual difference in susceptibility. These polymorphisms, although generally associated with low risks for cancer, are frequent in the population, and therefore the attributable risks are high. The cytochrome P450 super-multigene family of enzymes is involved in phase I metabolism. These oxidative processes may create products that are more reactive than the parent compounds and can be carcinogenic or mutagenic. The derivative compounds may covalently bind to DNA and form carcinogen-macromolecular adducts. Phase II metabolic processes generally inactivate these genotoxic compounds through

Markers of Susceptibility

3

conjugation (e.g., with glutathiones, glucuronides, sulfate esters), which promotes cellular excretion. Cytochrome P450 (CYP)lA1, the gene that codes for aryl-hydrocarbon hydroxylase (AHH), initiates a multienzyme pathway that activates polycyclic aromatic hydrocarbons including benzo-[a]-pyrene to highly electrophilic metabolites. The CYP1A1 gene is located on chromosome 15q22-qter. AHH activity is expressed in parenchymal lung and is inducible by interaction with an aryl-hydrocarbon receptorligand complex, with high affinity for polycyclic aromatic hydrocarbons. Binding of the receptor-ligand complex to the regulatory element leads to increased amounts of the CYP1A1 mRNA and protein. Activity has been measured in peripheral lymphocytes that have been activated by exposure to benzo-[a]-pyrene. Kellerman et al. (12), Kouri et al. (13), and Anttila et al. (14) showed that high CYP1A1 inducibility was associated with lung cancer risk, but these results were not confirmed in other studies (15). A statistically significant increase of AHH activity in neoplastic versus normal lung tissues was found in smokers (16), and lower crude mortality rates have been related to lower enzyme levels in surgically treated lung cancer patients (17). The sequencing of the CYP1A1 gene allowed investigation of the genetic background of the findings previously mentioned (18, 19). Three polymorphisms have been described. A restriction fragment length polymorphism (RFLP) downstream of the CYP1A1locus in the 3' noncoding region, after MspI digestion, was associated with tobaccorelated lung cancer risk in Japanese populations where the prevalence of the variant allele is about 30% (20). An A/G mutation on exon 7 determines an isoleucine/valine amino acid change (21). The activity of the CYP1A1 enzyme, as determined with the ethoxyresorufin assay, was elevated in individuals with such mutations of the coding region of the CYP1A1 gene (22). Another MspI RFLP also in the 3' noncoding region has been reported in individuals of African American descent (23). However, we (24) and London et al. (25) have previously reported that this novel African American-specific polymorphism was not associated with sizable risks for tobacco-induced lung cancer. MspI RFLP studies in Finnish, Norwegian, and US populations did not show clear associations between the CYP1A1 genotype and susceptibility to lung cancer (26-28). This may be explained by insufficient study sizes for detecting an association between cancer and a putative risk factor whose prevalence is between 0.05 (26) and 0.08 (29). A recent US study, however, did report an association between the MspI polymorphism and lung cancer risk (30). Individuals with the susceptible CYP1A1 genotype are reported to contract smoking-induced lung cancers at lower cigarette smoking levels than those with other CYP1A1 genotypes (31). The functional significance of these polymorphisms was further emphasized in a study in which CYP1A1 genotypes, gene expression levels, and enzymatic activity levels were measured in mitogen-stimu-

4

Chapter 1

lated lymphocytes. There was a threefold elevation in CYP1A1 enzymatic activity in exon-7 variant genotypes (32). When MspI and exon-7 genotypes were combined, there was an increased CYP1A1 inducibility and enzymatic activity in individuals with the exon-7 polymorphism and in those with both polymorphisms. The variation among populations at risk from having the MspI site and coinheritance of the alleles with CYP1A1 levels suggests that the restriction site does not directly affect lung cancer risk but that other nearby allelic variants, such as the isoleucine-to-valine mutation in the structural gene, may affect CYP1A1 expression (33). Cosma et al. (34) found that there was poor concordance between the MspI RFLP and the isoleucine-to-valine mutation in ex on 7. The exon-7 mutation is in tight linkage with the MspI RFLP in some Asian populations (35). This may explain the differences between the Japanese and the Norwegian studies; i.e., those mutations are part of racially distinct haplotypes. A CYP2D6 polymorphism, originally described by Ayesh et al. (36), has also been evaluated as a risk factor for lung cancer. CYP2D6 metabolizes a wide range of nitrogen-containing drugs, including neuroleptics, antidepressants, and beta-blockers. It also metabolizes the tobaccospecific nitrosamine, 4-(methylnitrosamino )-1-(3-pyridyl)-1-butanone (NNK), to mutagenic products (37). Unlike CYP1A1, CYP2D6 is not inducible, but individuals vary greatly in their CYP2D6 metabolic capacity, which is usually expressed as the metabolic ratio of debrisoquine metabolites to debrisoquine in urine. RFLP analysis with polymerase chain reaction (PCR) has made genotyping possible (38), and null alleles, slow metabolizing alleles, and a rare, ultrarapid allele due to amplification of the CYP2D6 have been described. Currently, about 95% of CYP2D6 variants can be detected by DNA analyses. Three common mutant forms have been related to poor metabolism of debrisoquine: CYP2D6A, CYP2D6B, and CYP2D6D. Rarely, deletion of a single lysine residue in CYP2D6C accounts for impaired metabolism. The poor metabolizer phenotype occurs in approximately 10% of the US population who have two nonfunctional CYP2D6 alleles. Heterozygotes for mutant alleles are intermediate metabolizers, and they cannot be well distinguished from homozygous wild-type individuals on the basis of debrisoquine phenotype. A summary analysis of seven independent studies yielded an aggregate odds ratio (OR) of 2.3 for lung cancer risk among extensive or intermediate metabolizers versus risk among poor metabolizers (39). Hirvonen (40) reported a significantly protective effect for being homozygous for variant alleles, but two larger studies in the United States found no such association (41,42). Several epidemiologic studies indicate that lung cancer risk is increased in extensive metabolizers (EMs) of debrisoquine (43,44). The extensive metabolizer genotype was overrepresented among Finnish lung cancer patients (27,40). However, inconsistent and negative results (42) have been reported, ascribable to


5

assay misclassification, noncorrespondence of phenotype/genotype, disease heterogeneity, exposure variation, and ethnic variation. Caporaso et al. (43) have presented the problem of misclassification of the phenotype using genotypic testing. The wild-type gene, characterized by a 29-kb allele, can also contain mutations that result in nonfunctional or absent proteins. These mutations, if present, result in the poor or intermediate metabolizer phenotype, despite the presence of the putative wild-type allele. To date, almost 13 polymorphisms have been identified; thus, misclassification by genotyping is problematic. Bouchardy et al. (45) recently reported a strong interaction in lung cancer risk between average daily consumption of tobacco and CYP2D6 activity. A dose-response effect of tobacco was detected only in smokers with the highest levels of CYP2D6 activity (46). The relationship between this polymorphism and nicotine addiction is also of interest, as CYP2D6 is involved in nicotine metabolism. Choler ton et al. (47) have suggested that extensive metabolizers might require a greater consumption of tobacco to maintain nicotine plasma levels that satisfy craving. The CYP2A genes are located as a cluster on the long arm of chromosome 19 (48). Polymorphisms also exist in CYP2A6, known to metabolize the tobacco-specific nitrosamine NNK (49), resulting in marked interindividual differences in gene expression in hepatic and extrahepatic tissues. Marked interindividual differences in phenotyping using coumarin have been detected, and a genotyping assay is available (50). Human CYP2E1 is inducible by ethanol and up to fiftyfold variability in enzyme activity is reported (51). CYP2E1 is involved in the metabolic activation of N-nitrosamines, butadiene, benzene, carbon tetrachloride, and other low-molecular-weight compounds. Induction of the enzyme results in a pronounced increase in the rate of microsomal lipid peroxidization (52), which is not seen after simultaneous administration of the CYP2E1 inhibitor, diallylsulfide. One RFLP located in intron 6, about 7000 base pairs downstream, revealed by Oral and identified as C (minor) alleles and 0 (common) alleles, has been associated with lung cancer in a Japanese case-control study (53). Two other RFLPs have been described, revealed by either Rsal or Pst! (54), that are in the transcription region of the gene. The Rsal polymorphism in the 5-flanking region of the gene affects transcriptional regulation of the gene but was not implicated in increased risk of lung cancer in a Japanese study that included 316 lung cancer patients (55) or in a Brazilian study (56). Substantial ethnic variations in allele frequencies have been reported (51). Kato et al. (57) found no association between the Oral RFLP and lung cancer risk in US whites or blacks, although the low prevalence of the C-minor allele (0.09) limits statistical power. Similarly, Hirvonen et al. (54) found no association between either the Oral or Rsal polymorphisms and lung cancer risk in their Finnish study. Persson et al. (58) concluded that the presence of a mutation in a putative binding site for

6

Chapter 1

hepatic transcription factor (HNF-l), which is thought to affect gene expression (c2 allele), might be associated with lower risk of lung cancer in Swedish populations. The glutathione S-transferases (GSTs) comprise five related gene families, of which four are cytosolic and one is microsomal (59). GSTs catalyze the conjugation of glutathione to several electrophilic compounds, including carcinogenic polycyclic aromatic hydrocarbons and cytotoxic drugs. Such conjugated xenobiotics are rendered harmless, and their excretion is enhanced. Affinity for activated carcinogens varies greatly among the classes, with GSTMI (also denoted GSTIl) having high affinity for trans-stilbene oxide and activated forms of benzo[a]-pyrene (60). The products of this gene cluster are detectable in various organ systems of the body. The presence or absence of the GSTMI gene constitutes the polymorphism, and the lack of GSTMI (the GSTMl null genotype) affects approximately 50% of the population (59, 61). A 98-100% correlation between phenotyping and genotyping for GSTMI is described (62). Several studies have suggested that the lack of GSTMI is a host determinant for lung cancer (especially squamous cell carcinoma) in smokers (59, 63-66), but contradictory phenotypic and genotypic studies have also been published (67, 68). The risk appears to be dependent on the extent of tobacco smoke exposure (31,69,70). Heckbert et al. (67) identified a reduced lung cancer risk for individuals (cases and controls) with intermediate or high GST activity and a history of 20 or more pack-years of smoking. NazarStewart et al. (66) noted that the protective effect of high GST activity was greater in heavy smokers (relative risk [RR] = 0.15; 95% confidence limits [CL] = 0.03-0.64) than in light smokers (RR = 0.61; CL = 0.14-2.6). The combined effects of GSTMI and CYPIAI polymorphisms have also been investigated (14,31, 71). The combination of the GSTMl-null genotype and the rare homozygous allele (G/G) of the CYPIAI gene resulted in a significantly higher relative risk for lung cancer than the risks calculated for either of these genotypes separately (71). A subsequent case-control study showed that individuals with the susceptible MspI or G/G genotype combined with the GSTMl-null genotype had significantly elevated odds ratios for lung cancer at a low-dose level of cigarette smoking (31). Nakachi et al. (31) reported that the GSTMl-null genotype interacts with the CYPIAI trait to produce very large risks for lung cancer (31). Individuals both homozygous for the valine CYPIAI allele and GSTMl-null had a 5.4-fold elevated risk, higher than the risk observed for either susceptible genotype alone. Similarly, a European study showed that expression of the GSTMI enzyme had a protective effect in patients with inducible CYPIAI (13). The ml / m2 and m2/ m2 genotypes of the MspI site and the isoleucine/valine genotype of the CYPIAI gene were slightly overrepresented in patients with squamous cell carcinoma of the lung in Sweden (72). A combined risk of lung cancer was evident for patients diagnosed before 66 years of age who car-


7

ried both the GSTMl-null genotype and the m2 alleles (OR = 3.0, 95% CL = 1.2-7.2). The glutathione S-transferase theta (GSTTl) has relatively high activity toward epoxy and peroxide compounds (73). GSTTI is important in the detoxification of naturally occurring monohalomethanes, as well as dichloromethane and arylepoxides such as benzo-[a]-pyrene found in tobacco (74). Approximately 60-70% of the human population are able to carry out this conjugative reaction (" conjugators"), whereas the remaining 30-40% are "nonconjugators." The conjugation is detoxifying with regard to monohalomethanes and ethylene oxide, but conjugation of dihalomethanes to formaldehyde yields a genotoxic intermediate (74), so that having the conjugator phenotype is not necessarily beneficial. The human GSTTI gene has recently been isolated, and the null ("nonconjugator") phenotype results from the absence of the GSTTI gene (74). There is no relationship with the GSTMI genetic defect. The loci for GSTMI and GSTTI are not linked. We have recently shown an interaction between the two null genotypes and lung cancer risk in blacks and Mexican Americans (75). The N-acetylation polymorphism segregates individuals into rapid, intermediate, and slow acetylator phenotypes via monogenic inheritance of the NAT2 locus. Approximately 40-70% of whites in Europe and North America are of the "slow acetylator" phenotype and are less efficient than "rapid acetylators" in the metabolism of agents containing primary aromatic amine or hydrazine groups (76). In addition to arylamines, these compounds include drugs (e.g., isoniazid, sulfonamides, caffeine), xenobiotics present in dyes, pesticides, and explosives (e.g., benzidine, 2-aminofluorene, and beta-naphthylamine), and several potential carcinogens generated during cooking. Rapid acetylation has been implicated as a risk factor for colon carcinoma. However, certain allelic variants associated with slow acetylator status were considered a secondary risk factor in lung cancer (77). Vineis et al. (78) have reviewed the evidence for an association between NAT2 polymorphisms and lung cancer risk. Epoxide hydrolase (EH) is another phase II enzyme, which catalyzes the conjugation of polycyclic aromatic hydrocarbons (PAH) epoxides. Although the products of hydrolysis are less reactive than the parent epoxide, the resultant diol is sometimes a precursor to a more carcinogenic form; thus, hydrolysis is not strictly a detoxification pathway (67). The activity of EH appears to be under genetic control of the Ah locus (67). There is both cytosolic (measured with trans-stilbene oxide as a substrate) and microsomal activity (measured with styrene oxide as a substrate). Cytosolic EH activity in lung tissue of recent smokers was significantly lower than in former smokers (79) and was correlated with number of days of cessation and inversely correlated with number of cigarettes smoked per day. These data suggest that inhibition of cytoso-

8

Chapter 1

lic EH activity by tobacco smoke may reduce the inactivation of carcinogenic epoxides and thereby increase susceptibility to cancer. Two DNA polymorphisms (which affect the activity of protein products) have been identified in exons 3 and 4 of the microsomal EH gene on the basis of an amino acid sequence variation tyrosine/histidine at position 113 (exon 3) and histidine/arginine at position 139 (exon 4), with prevalence of the rare alleles in 27% (histidine) and 21 % (arginine) of a largely white population (80). Substitution of the histidine at position 113 decreases the amount of the epoxide by approximately twofold, while a substitution of arginine at amino acid 139 increases the protein by twofold. These polymorphic sites could thus also playa role in the etiology of smoking-related cancers. In fact, in a small casecontrol study of smoking-related cancers, Heckbert et al. (67) reported a moderate protective effect of high or intermediate enzyme activity in the heaviest smokers. A common Aci I RFLP in codon 72 in the p53 gene has been reported and results in at least two forms of wild-type p53 protein (81). The polymorphism is the replacement of the amino acid arginine by proline, but the functional significance of the variant is as yet unknown. Three studies have evaluated the polymorphic variants as potential susceptible genotypes for lung cancer, with inconsistent results (82-84). Weston et al. (82), in 78 lung cancer patients and 72 controls with either chronic obstructive airways disease or other cancers, did not detect an association between the proline variant and lung cancer risk. However, in the larger Japanese study of 328 lung cancer cases and 347 healthy controls, the proline/proline genotype was found to be significantly implicated in risk (83). We genotyped 109 lung cancer patients and 114 controls for this polymorphism (84) and reported that the univariate risk estimate for the proline/proline genotype compared with the combined arginine/ arginine and arginine/proline genotype was 1.6 for blacks and 2.0 for Mexican Americans, but not statistically significant. Risk associated with the proline genotype was especially high for those whose lung cancer was diagnosed before 53 years of age (the 25th lower percentile of the study age distribution). When the pack-years were dichotomized at 30 (the median for the cases), risk associated with the susceptible genotype was elevated (over threefold) only for the lighter smokers. We also demonstrated that lung cancer patients with the proline/proline susceptible genotype had a significantly earlier age at diagnosis and a lower packyear consumption compared with the other genotypes (Table 1.1).

Mutagen Sensitivity Cytogenetic assays in peripheral blood lymphocytes have been extensively used to survey exposure and response of humans to genotoxic agents. The conceptual basis for this is the hypothesis that the extent of


Table 1.1.

9

Characteristics of lung cancer patients with three p53 genotypes Pack-years a

Age in years Genotype

No. of patients

Mean

(zSD)

Mean

(zSD)

Arginine / arginine Arginine / proline Proline / proline

34 54 21

65.4 62.9 56.4

10.7 9.8 9.4 a

55.5 55.7 49.0

29.3 35.0 28.9

aSignificantly lower than arginine/ arginine (P = 0.003) and arginine/proline (P

= 0.01).

genetic damage in the lymphocytes reflects critical events in carcinogenesis in the affected tissues (85). In a cohort study of 3182 workers occupationally exposed to mutagenic agents and studied for chromosomal aberrations at entry into the study, there was a statistically significant linear trend in cancer risk with increasing frequency of aberrations. The risk estimate was 2.1 in the highest stratum of aberrations (85). Studies such as this have confirmed the value of using chromosomal aberrations in peripheral lymphocytes as a marker of cancer risk. In order to test the hypothesis that within the population there are interindividual differences in susceptibility to carcinogenic agents, Hsu (86) developed the mutagen sensitivity assay in which the frequency of in vitro bleomycin-induced breaks is quantitated as a measure of mutagen sensitivity in the general population. In a case-control study of lung cancer, mutagen sensitivity (defined as ~1 break/cell) was significantly associated with lung cancer risk (87). Overall, 53.8% of lung cancer patients had mutagen-sensitivity scores greater than or equal to 1 break per cell, compared with 22.4% of the controls. The overall odds ratio for mutagen sensitivity after adjusting for ethnicity and smoking status was 3.6 (95% CL = 2.2-5.9). The risk estimate in current smokers was 2.5 (CL = 1.2-5.3). For former smokers, the odds ratio was even higher, 6.2 (CL = 2.7-16.1). Lighter smokers «1 pack/day) appeared to be at higher risk (5.7) than heavier smokers (3.2). The data were also dichotomized at 61 years of age (the median of the age distribution of the cases and controls). The risk estimate for younger patients was 4.9 (CL = 2.3-10.4) compared with 2.9 (CL = 1.5-5.6) for older patients. Highest risks associated with mutagen sensitivity were for squamous cell carcinoma (OR = 8.5). We also assessed the effect of cigarette smoking on the sensitivity profile of both cases and controls. There were no significant differences in mean breaks-per-cell values by current or former smoking status, stratified by pack-year history, although values for cases were consistently higher than those for controls. There was no trend for increasing mutagen sensitivity to be expressed with more extensive exposure history. When patients were categorized into quartiles of breaks-per-cell values, with 0.50 break per cell as the referent category, there was a

10

Chapter 1

dose-response relationship between lung cancer risk and mutagen sensitivity. The ORs stratified by quartile of induced breaks were 1.0, 1.4 (CL = 0.6-3.3), 2.5 (CL = 1.1-5.4), and 4.8 (CL = 2.3-10.0). The trend test by chi-squared analysis was significant (P < 0.001). We also performed stratified analysis using as the referent group nonsensitive, never-smoking subjects. The combined risk for mutagen sensitivity and ever smoking (OR = 28.1) was greater than the additive effects of smoking alone (OR = 8.1) and mutagen sensitivity (OR = 4.7). The risk for former smokers in the absence of mutagen sensitivity was 6.5; for mutagen sensitivity in nonsmokers, the estimate was 4.7. In the presence of both factors, the OR was 37.4. The respective relative risks for current smokers (OR = 9.3), mutagen sensitivity (OR = 4.7), and combined (OR = 23.3) followed the same pattern, although none of the interaction terms was statistically significant in the logistic model. We have also extended the bleomycin assay by substituting benzo-[a]pyrene to compare the frequency of in vitro chromosomal aberrations in lymphocytes of 57 lung cancer patients and 100 matched controls (88). The mean breaks-per-cell scores were 0.78 for the cases and 0.47 for the controls (P < 0.001), and the adjusted odds ratio was 8.9 (CL = 3.8-20.5) for lung cancer associated with increased frequency of these chromosomal aberrations. These findings suggest that the mutagen-sensitivity assay has the flexibility to be used with an array of test mutagens that require differing repair pathways and reflect different molecular mechanisms of carcinogenesis (89).

Cytogenetic Alterations Identifying consistent cytogenetic changes in lung cancer and evaluating the association between fragile sites and cancer breakpoints might provide insight into lung cancer pathogenesis and, in some cases, may aid in the positional cloning of causative genetic loci. The cytogenetic information on lung cancer is based largely on analysis of metastases, effusions, and established cell lines; few reports describe karyotypes of primary tumors, largely because of the technical difficulties involved. Furthermore, the karyotypes are frequently complex-with multiple genetic alterations-and precise assignation of breakpoints is difficult, especially when the quality of the preparations is suboptimal (90). Therefore, cytogenetic analysis of peripheral-blood cultures could provide a clue to the role of breakpoints in tumorigenesis. We have investigated whether the bleomycin-induced chromatid breaks are not randomly distributed but, rather, preferentially located at targeted hotspots along the genome, by studying the location of the chromatid breaks from primary blood cultures of 75 randomly selected cases of lung cancer and 78 controls matched on age, sex, and ethnicity to the cases (91). The frequency of induced chromatid breaks and the locations of the breaks were determined in Q-banded preparations.


I I

After adjustment for their length, the larger chromosomes had more breaks than smaller chromosomes in both cases and controls. The cases had significantly more breaks on chromosomes 4 and 5 than the controls did, with ORs of 4.9 (CL = 2.0-11.7) and 3.9 (CL = 1.6-9.3), respectively.

DNA Repair Capacity The ability to monitor and repair persistent DNA damage is another determinant of susceptibility to carcinogenesis. Generally, cellular responses to DNA damage fall into three major categories: direct reversal of damage (e.g., enzymatic photoreactivation), nucleotide excision repair, and postreplication repair (92). Benzo-[a]-pyrene diol-epoxide (BPDE)-DNA adducts formed are repaired by a nucleotide excision repair pathway that is responsible for the restoration of normal DNA structure (93). Evidence for DNA repair deficiency as an etiologic factor in cancer was initially elucidated in patients with xeroderma pigmentosum (XP) (94). These patients have a markedly increased risk of developing sunlight-induced skin cancers and a greatly increased frequency of developing cancers of internal organs associated with a defect in nucleotide excision repair. The paradigm of a link between decreased DNA repair and cancer susceptibility is now being extended to the general population. There is a range of diminished repair deficiencies both in XP patients, who represent the extreme end of the repair spectrum, and in the general population. The expression level of damaged reporter genes is the assay of choice (host-cell reactivation assay) for population studies, in which time, cost, and repeatability of measurements are major concerns. This assay uses undamaged cells, is relatively fast, and is an objective way of measuring repair (95). In the assay, a damaged nonreplicating recombinant plasmid (pCMV cat) harboring a chloramphenicol acetyltransferase (CAT) reporter gene is introduced by transfection into lymphocytes, and excision-repair of the damaged bacterial gene is monitored as a function of reactivated CAT enzyme activity (95). It has been demonstrated that both lymphocytes and skin fibroblasts from patients who have basal cell carcinoma but not XP have lower excision-repair rates than individuals without cancer (96-98). Therefore, the repair capacity of lymphocytes can be considered a reflection of the repair capacity of the donor. Wei et al. (89) have recently published the results of a pilot case-control study evaluating the role of DNA repair capacity (DRC) as a risk factor for lung cancer in 51 patients and 56 frequency-matched controls. The mean level of DRC following exposure to 75 !-lM BPDE in the cases (3.3%) was significantly lower than that in the controls (5.1 %) (P < 0.01). After adjustment for age, gender, ethnicity, and smoking status, the cases were five times more likely than controls to have reduced DRC (OR = 5.7; CL = 2.1-15.7).

12

Chapter 1

The mutagen-sensitivity assay measures aberrations or breaks in metaphase chromosomes; it does not detect subtle modifications of DNA such as interstrand or intra strand crosslinks (e.g., dimers) or point mutations. On the other hand, the host cell reactivation (HeR) assay cannot measure DNA-strand break repair, particularly double-strand break repairs. Wei et al. (89) have shown that DNA repair capacity in lymphoblastoid cell lines correlates with in vitro mutagen sensitivity to the same type of mutagens. Thus, these two assays may be complementary in measuring susceptibility at different levels, and using both assays should enhance our risk assessment.

MARKERS OF INTERNAL EXPOSURE: DNA-CARCINOGEN ADDUCTS The net effect of exogenous carcinogen exposure and inherited traits for absorption, metabolism, and DNA repair is the DNA-carcinogen adduct (99). This interrelationship between susceptibility markers and measurements of biologically effective dose, such as PAH-DNA adducts, is the focus of considerable interest and may be useful for estimating the risk for fixed mutations (99). DNA-carcinogen adducts are reaction products formed during the biotransformation of chemical carcinogens, by xenobiotic metabolizing enzymes, to reactive intermediates that are electrophilic and bind covalently to DNA. DNAadducts can be removed by DNA-repair processes or by cell death; however, chronic exposure often leads to steady-state accumulation in a target tissue. The biological potential of a given DNA adduct depends on many factors, including its mutagenic potential, repairability, location within a target gene, and, of course, the nature of the target gene. In general, however, the steady-state levels of specific DNA-carcinogen adducts in target tissues during chronic exposure are the net effect of exposure and absorption, metabolism and repair. The lungs of smokers have been shown to have BPDE-guanine adducts. These ad ducts in the guanine bases of DNA are in accordance with the main type of mutation (G to T transversions) found in the K-ras oncogene and p53 tumor suppressor gene (see p53 Mutations under Markers of Biologic Effects of Exposure). White blood cells are commonly used as a surrogate tissue to study the genetic damage in human populations exposed to environmental carcinogens. Tang et al. (100) reported that PAH-DNA adducts were significantly higher in cases with non-smaIl-cell lung cancer than in controls and in smokers and ex-smokers compared with nonsmokers. Moreover, in the cases, leukocyte ad ducts were correlated more strongly with adducts in tumor tissue than in non tumor lung tissue. Rothman et al. (101), however, has suggested that these adducts reflect dietary rather than inhalational exposures. In a study of 38 lung samples, Shields et al. (102)


13

reported that PAH-DNA adduct levels were positivelj associated with the CST-null genotype, but not with the CYP1A1 exon-7 mutation, nor with smoking or serum cotinine levels. They also reported no significant correlation with lung tissue adduct levels in a subset of 20 patients. Similarly, van Schooten et al. (103) studied white blood cell DNA adducts using both 32P post-labeling and ELISA techniques and found no correlation between adduct level, smoking history, age, or gender, and also no correlation between adduct levels in blood and lung. However, Wiencke et al. (104) compared the levels of aromatic DNA adducts in lung tissue from 31 lung cancer patients with adduct levels in peripheral mononuclear cells and reported good correlation. They concluded that the blood cells were a valid surrogate tissue for estimating the burden of DNA adducts in respiratory tissue. We have studied tobacco-related DNA ad ducts in the lymphocyte DNA of 46 lung cancer cases and 40 controls who were current smokers (105). For both cases and controls, there was an inverse association between age at smoking initiation and adduct level, with case levels consistently higher than control levels (although these differences were not statistically significant). In logistic regression analysis, the induced adduct level was an independent risk factor after adjustment for age, gender, ethnicity, and smoking (OR = 6.4; CL = 1.3-29.4). For both cases and controls, there was an inverse association between age at smoking initiation and adduct level (P < 0.05). In the cases (but not in the controls) there was a significant correlation (P < 0.02) between adduct level and bleomycin-induced breaks-per-cell score. There was a nonsignificant trend for higher adduct levels in CST-null cases compared with CST-positive cases. Li et al. (106) have reported the potential usefulness of an assay measuring in vitro BPDE-induced peripheral lymphocyte DNA adducts. In 21 lung cancer cases and 41 controls, they noted in logistic regression analysis that the level of induced adducts was an independent risk factor (OR = 4.7) for lung cancer. Because the assay utilizes the same dose of BPDE, the levels of induced adducts should reflect individual variation in response to the carcinogenic challenge. A summary of the relevant susceptibility markers, their prevalence, and associated risks with lung cancer is provided in Table 1.2.

MARKERS OF BIOLOGIC EFFECTS OF EXPOSURE Molecular Genetic Changes due to Cigarette Smoking p53 Mutations Mutations in the p53 tumor suppressor gene are the most common genetic alterations identified in human cancer and can be used as

14

Chapter 1

Table 1.2.

Markers of susceptibility of lung cancer risk Prevalence of variant allele or phenotype in controls

Ethnicity

Positive association Reference

0.30

Japanese

3

Kawajiri et al. (20)

0.37 0.15

Japanese Black

2.6 1.3

Hayashi et al. (21) London et al. (25)

0.09 0.15

Not specified Finnish

2.3 NA

Amos et al. (39) Fernandez-Salguero et al. (49)

0.24 0.48 0.17 0.03

English Japanese Taiwanese Black

NA NA NA NA

0.74 0.89 0.95 0.91 0.95 0.69 0.48 0.53 0.40-0.70

Japanese Finnish Swedish White Black Japanese Not specified Not specified

Yes No Yes No No No 3 No

Uematsu et al. (53) Hirvonen et al. (54) Persson et al. (58) Kato et al. (57) Kato et al. (57) Kato et al. (57) Nazar-Stewart et al. (66) Brockm611er et al. (68)

0.35 0.23 0.35 0.34 0.53 0.26

Not specified Not specified Japanese White Black MexicanAmerican

NA NA 1.7 No 1.6 2.0

Hassett et al. (80) Hassett et al. (80) Kawajiri et al. (83) Weston et al. (82) Jin et al. (84) Jin et al. (84)

Bleomycin sensitivity

0.22

4.6

Wu et al. (87)

BPDE sensitivity Chromosome 4 bleomycin sensi ti vi ty

0.22 0.23

8 4.9

Wu et al. (87) Wu et al. (91)

Chromosome 5 bleomycin sensi ti vi ty

0.23

3.9

Wu et al. (91)

DNA repair capability

0.50

MexicanAmerican and black White MexicanAmerican and black MexicanAmerican and black White

5.7

Wei et al. (89)

Markers Metabolic polymorphisms CYPIAI

MspI RFLP 3' noncoding region AIG mutation in exon 7 Another MspI RFLP 3' noncoding region CYP2D6 CYP2A6

CYP2El DraI polymorphism Pst! polymorphism

GSTMI GSTTI NAT2

EH Exon3 Exon4 p53 codon 72

Mutagen sensitivity

BPDE = benzo-[a]-pyrene diol-epoxide; RFLP = restriction fragment length polymorphism.


I5

"fingerprints" of specific exposures in molecular epidemiology (5). Distinct differences in the mutational frequency and type are found in different tumor types, and are attributed in part to the effects of specific mutagens. Most smoking-related mutations are transversions at GC base pairs, with about three-fourths being G:C -- T:A transversions (107). Experimental studies have indicated that bulky PAH compounds like benzo[a]-pyrene induce such transversions (108). Uranium miners and atomic bomb survivors with lung cancer, however, do not show G:T transversions (109, 110). More commonly, the latter group exhibited G:C -- A:T transitions. K-ras mutations have also been found to be associated with smoking (111), and these mutations also appear to be largely G -- T transversions (112). The distribution of mutations along the p53 gene is nonrandom and is characterized by several mutational hotspots, especially at codons 157,248, and 273, corresponding to amino acids within the DNA-binding domain of p53 (113). Codon 157 is a hotspot specific for lung cancer. Recently, Denissenko et al. (113) mapped the distribution of BPDE adducts along exons of the p53 gene in BPDE-treated HeLa cells and bronchial epithelial cells, and noted selective adduct formation at these three codons. These results provide the first direct etiologic link between a tobacco carcinogen and lung cancer mutations. The occurrence of p53 mutations has been associated with a history of heavy smoking (114, 115). p53 mutations have been reported to occur less frequently in those patients with lower levels of tobacco exposure (116). Wang et al. (117) reported a linear increase in the occurrence of G:T transversion mutations with increasing cumulative exposure to smoking. In 85 patients studied, 29% had mutations in the p53 gene. These patients were significantly older (75.1 vs. 59.8 years) and had smoked for more years (56.8 vs. 41.2) than those patients without such mutations. Kawajiri et al. (118) also found an increased frequency of mutations in heavy smokers compared with those who had never smoked, and Kondo et al. (119) noted an association between amount of smoking and frequency of p53 mutations. Brennan et al. (120) reported that, in patients with head and neck squamous cell carcinoma, exposure to tobacco and alcohol was associated with p53 mutations at nonendogenous mutation sites. On the other hand, nonexposed patients tended to exhibit mutations at sites containing cytidine phosphate guanosine dinucleotides (representing endogenous mutations). Mutation frequency in relation to specific risk genotypes has also been studied. Kawajiri et al. (118) reported that patients with CYP1A1 Msp and isoleucine-valine polymorphisms were 4.5- and 5.5-fold more likely, respectively, to have tumor-specific p53 mutations than those with nonsusceptible alleles. Furthermore, the risk was ninefold with a combination of either of the CYP polymorphisms and presence of the GSTM1-null genotype.

16

Chapter 1

Loss of Heterozygosity Loss of genetic material (loss of heterozygosity, or LOH) at several human chromosome loci containing candidate tumor suppressor genes has been identified in tobacco-related cancers and tobacco-exposed tissue in individuals without cancer. LOH at 3p14 (the region that includes the candidate tumor-suppressor fragile and histidine triad [FHIT] gene) and 9p21 (site of the p16 tumor suppressor gene) has recently been linked to tobacco-related carcinogenesis as relatively early and specific genetic alterations. Mao et al. (121) reported LOH at these sites in 45% (13/29) and 36% (12/33), respectively, of clinically and histologically defined oral premalignant lesions. Most recently, this group also studied bronchial epithelium in 54 smokers with minimal histologic abnormalities (122). Each smoker had bronchial biopsies from multiple lung sites. They observed LOH at 3p14 in 37% of 172 informative biopsies (75% of 36 informative cases). The respective figures for LOH at 9p21 were 59% and 24%. LOH at 17p13 (located within the p53 gene) occurred significantly less frequently than LOH at 3p14 and 9p21, suggesting that 17p13 LOH (and p53 alterations) is a relatively late genetic alteration in tobacco-related carcinogenesis. In addition to being the most frequent alteration, LOH at 3p14 was most tightly linked to smoking, occurring significantly more frequently in current than former smokers. These data provide the first molecular evidence that smoking cessation can reverse tobacco-related genetic alterations. The previous molecular findings indicate that LOH at 3p14 is the earliest specific genetic alteration in tobacco-related aerodigestive tract cancers. Related recent findings have identified FHIT located within the 3p14 region (3p14.2), and frequent abnormal transcripts have been observed in lung cancer (123). These data, together with other work showing that 3p14 is one of the most common fragile sites, suggest that FHIT may be a major target of carcinogens in cigarette smoke, and provide the first molecular evidence linking fragile site instability with human cancer.

SUMMARY Lung cancer risk is defined by the balance between metabolic activation and metabolic detoxification of xenobiotic compounds, as well as the efficiency of DNA repair. That lung cancer is caused by a single explanatory gene environmental interaction is unlikely; one marker may not have a strong effect but, in conjunction with other genes, may shift the risk profile in an unfavorable direction. Multiple susceptibility factors must therefore be accounted for to represent the true dimensions of gene environmental interactions. The number of cigarettes consumed


17

before onset of lung cancer might be lower in "susceptible" individuals than in "nonsusceptible" individuals (i.e., individuals with a susceptible genotype have a high risk for lung cancer even at a low cigarette dose level). The genetic component in risk tends to be lower at highdose levels, when the environmental influence may overpower genetic predisposi tion. The ability to identify smokers with the highest risks of developing cancer has substantial preventive implications. These subgroups could be targeted for the most intensive smoking cessation interventions, could be enrolled into chemoprevention trials, and might be suitable for screening programs not appropriate for the general population. Finally, studying susceptibility to common cancers and widely prevalent exposures may provide further insights into the basic mechanisms of carcinogenesis.

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21

locus: use of genotyping and phenotyping assays to assess association with lung cancer susceptibility. Carcinogenesis 1991;12:1533-1537. 65. Kihara M, Noda K, Okamoto N. Increased risk of lung cancer in Japanese smokers with class mu glutathione S-transferase gene deficiency. Cancer Lett 1993;71:151-155. 66. Nazar-Stewart V, Motulsky AG, Eaton DL, et a1. The glutathione Stransferase mu polymorphism as a marker for susceptibility to lung carcinoma. Cancer Res 1993;53:2313-2318. 67. Heckbert SR, Weiss NS, Hornung SK, Eaton DL, Motulsky AG. Glutathione S-transferase and epoxide hydrolase activity in human leukocytes in relation to risk of lung cancer and other smoking-related cancers. J Natl Cancer Inst 1992;84:414-422. 68. Brockmoller J, Kerb R, Drakoulis N, Nitz M, Roots I. Genotype and phenotype of glutathione S-transferase class mu isoenzymes mu and psi in lung cancer patients and controls. Cancer Res 1993;53:1004-1011. 69. Hirvonen A, Husgafvel-Pursiainen K, Anttila S, Vainio H. The GSTM1 null genotype as a potential risk modifier for squamous cell carcinoma of the lung. Carcinogenesis 1993;14:1479-1481. 70. Kihara M, Noda K. Lung cancer risk of GSTM1 null genotype is dependent on the extent of tobacco smoke exposure. Carcinogenesis 1994;15: 415-418. 71. Hayashi S, Watanabe J, Kawajiri K. High susceptibility to lung cancer analyzed in terms of combined genotypes of P450IA1 and Mu-class glutathione S-transferase genes. Jpn J Cancer Res 1992;83:866-870. 72. Alexandrie AK, Sundberg MI, Seidegard J, Tornling G, Rannug A. Genetic susceptibility to lung cancer with special emphasis on CYP1A1 and GSTM1: a study on host factors in relation to age at onset, gender and histological cancer types. Carcinogenesis 1994;15:1785-1790. 73. Meyer DJ, Coles B, Pemble SE, et a1. Theta, a new class of glutathione transferases purified from rat and man. Biochem J 1991;274:409-414. 74. Pemble S, Schroeder KR, Spencer SR, et a1. Human glutathione S-transferase theta (GSTT1): cDNA cloning and the characterization of a genetic polymorphism. Biochem J 1994;300:271-276. 75. Kelsey KT, Spitz MR, Zuo ZF, Wiencke JK. Deletion of glutathione Stransferase class mu and class theta genes interacts to enhance susceptibility to lung cancer in minority populations. Cancer Causes Control 1997;8 :554-559. 76. Blum M, Demierre A, Grant DM, Heim M, Meyer UA. Molecular mechanism of slow acetylation of drugs and carcinogens in humans. Proc Natl Acad Sci USA 1991;88:5237-5241. 77. Martinez C, Agundez JA, Olivera M, et a1. Lung cancer and mutations at the polymorphic NAT2 gene locus. Pharmacogenetics 1995;5:207214. 78. Vineis P, Landi MT, Caporaso N. Metabolic polymorphisms and the cancer risk: the evaluation of epidemiological studies. Med Lav 1992;83: 557-575. 79. Petruzzelli S, Franchi M, Gronchi L, et a1. Cigarette smoke inhibits cytosolic but not microsomal epoxide hydrolase of human lung. Hum Exp ToxicoI1992;11:99-103. 80. Hassett C, Aicher L, Sidhu JS, Omiecinski CJ. The human microsomal epoxide hydrolase gene (EPHX1): complete nucleotide sequence and structural characterization. Hum Molec Genet 1994;3:421-428. 81. de la Calle-Martin 0, Fabregat V, Romero M, et a1. AccII polymorphism of the p53 gene. Nucleic Acids Res 1990;18:4963.

22

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Weston A, Perrin LS, Forrester K, et al. Allelic frequency of a p53 polymorphism in human lung cancer. Cancer Epidemiol Biomarkers Prev 1992;1:481-483. Kawajiri K, Nakachi K, Imai K, Watanabe I, Hayashi S1. Germ line polymorphisms of p53 and CYP1A1 genes involved in human lung cancer. Carcinogenesis 1993;14: 1085-1 089. Jin X, Wu X, Roth JA, et al. Higher lung cancer risk for younger AfricanAmericans with the Pro/Pro p53 genotype. Carcinogenesis 1995;16: 2205-2208. Hagmar L, Brogger A, Hansteen IL, et al. Cancer risk in humans predicted by increased levels of chromosomal aberrations in lymphocytes: nordic study group on the health risk of chromosome damage. Cancer Res 1994;54:2919-2922. Hsu TC, Johnston DA, Cherry LM, et al. Sensitivity to genotoxic events of bleomycin in humans: possible relationship to environmental carcinogens. Int J Cancer 1989;43:403-409. Wu XF, Delclos GL, Annegers FI, et al. A case-control study of wooddust exposure, mutagen sensitivity, and lung-cancer risk. Cancer Epidemiol Biomarkers Pre v 1995;4:583-588. Wu X, Gu I, Hsu TC, et al. Differential sensitivity to benzo[a]pyrenediol-epoxide (BPDE) as a marker of lung cancer susceptibility. Proc Am Assoc Cancer Res 1997;38:618. Wei Q, Cheng L, Hong WK, Spitz MR. Reduced DNA repair capacity in lung cancer patients. Cancer Res Adv Brief 1996;56:4103-4107. Testa JR, Graziano SL. Molecular implications of recurrent cytogenetic alterations in human small cell lung cancer. Cancer Detect Preven 1993; 17:267-277. Wu X, Hsu TC, Annegers JF, et aI. A case-control study of nonrandom distribution of bleomycin-induced chromatid breaks in lymphocytes of lung cancer cases. Cancer Res 1995;55:557-56l. Legerski RJ. DNA repair capability and cancer risk. Cancer Bull 1994;46:228-232. Harris Cc. Interindividual variation among humans in carcinogen metabolism, DNA adduct formation and DNA repair. Carcinogenesis 1989;10:1563-1566. Cleaver JE, Kraemer K. Xeroderma pigmentosum. In: Scriver CR, et aI., eds. The Metabolic Basis of Inherited Diseases II. New York: McGrawHill, 1989:2949-2971. Athas WF, Hedayati M, Matanoski GM, Farmer ER, Grossman L. Development and field-test validation of an assay for DNA repair in circulating human lymphocytes. Cancer Res 1991;51:5786-5793. Alcalay I, Freeman SE, Goldberg LH, Wolf JE. Excision repair of pyrimidine dimers induced by simulated solar radiation in the skin of patients with basal cell carcinoma. J Invest DermatoI1990;95:506-509. Wei Q, Matanoski GM, Farmer ER, Hedayati MA, Grossman L. DNA repair related to multiple skin cancers and drug use. Cancer Res 1994; 54:437-440. Wei Q, Spitz MR, Gu I, et al. DNA repair capacity correlates with mutagen sensitivity in lymphoblastoid cell lines. Cancer Epidemiol Biomarkers Prev 1996;5:199-204. Shields PG. Pharmacogenetics: detecting sensitive populations. Environ Health Perspect 1994;102:81-87.

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Tang D, Santella RM, Blackwood AM, et al. A molecular epidemiological case-control study of lung cancer. Cancer Epidemiol Biomarkers Prev 1995;4:341-346. Rothman N, Poirier MC, Baser ME, et al. Formation of polycyclic aromatic hydrocarbon-DNA adducts in peripheral white blood cells during consumption of charcoal-broiled beef. Carcinogenesis 1990;11:12411243. Shields PG, Bowman ED, Harrington AM, Doan VT, Weston A. Polycyclic aromatic hydrocarbon-DNA adducts in human lung and cancer susceptibility genes. Cancer Res 1993;53:3486-3492. van Schooten FI, Hillebrand MI, van Leeuwen FE, et al. Polycyclic aromatic hydrocarbon-DNA adducts in white blood cells from lung cancer patients: no correlation with adduct levels in lung. Carcinogenesis 1992;13:987-993. Wieneke JK, Kelsey KT, Varkonyi A, et al. Correlation of DNA adducts in blood mononuclear cells with tobacco carcinogen-induced damage in human lung. Cancer Res 1995;55:4910-4914. Vulimiri SV, Baer-Dubowska W, Wu X, et al. Analysis of smokingrelated lymphocyte DNA adducts in a case-control study of lung cancer. Prec Am Assoc Cancer Res 1996;37:A1707. Li D, Wang M, Cheng L, et al. In vitro induction of benzo[a]pyrene diol epoxide-DNA adducts in peripheral lymphocytes as a susceptibility marker for human lung cancer. Cancer Res Adv Brief 1996;56:3638-3641. Hollstein M, Shomer B, Greenblatt M, et al. Somatic point mutations in the p53 gene of human tumors and cell lines: updated compilation. Nucleic Acids Res 1996;24:141-146. Carothers AM, Grunberger D. DNA base changes in benzo[a]pyrene diol epoxide-induced dihydrofolate reductase mutants of Chinese hamster ovary cells. Carcinogenesis (London) 1990;11:189-192. Vahakangas KH, Samet JM, Metcalf RA, et al. Mutations of p53 and ras genes in radon-associated lung cancer from uranium miners. Lancet 1992;339:576-580. Takeshima Y, Seyama T, Bennett WP, et al. p53 mutations in lung cancers from non-smoking atomic-bomb survivors. Lancet 1993;342:1520-1521. Slebos RJC, Hruban RH, Dalesio 0, et al. Relationship between K-ras oncogene activation and smoking in adenocarcinoma of the human lung. J Natl Cancer Inst 1991;83:1024-1027. Husgafvel-Pursiainen K, Kackman P, Ridanpaa M, et al. K-ras mutations in human adenocarcinoma of the lung: association with smoking and occupational exposure to asbestos. Int J Cancer 1993;53:250-256. Denissenko MF, Pao A, Tang M, Pfeifer GP. Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in p53. Science 1996;274:430-432. Ryberg D, Kure E, Lystad S, et al. p53 mutations in lung tumors: relationship to putative susceptibility markers for cancer. J Cancer Res 1994;54:1551-1555. Field JK, Spandidos DA, Malliri A, et al. Elevated p53 expression correlates with a history of heavy smoking in squamous cell carcinoma of the head and neck. Br J Cancer 1991;64:573-577. Koch WM, Patel H, Brennan J, Boyle JO, Sidransky D. Squamous cell carcinoma of the head and neck in the elderly. Arch Otolaryngol Head Neck Surg 1995;121:262-265.

24

Chapter 1 117. Wang X, Christi ani DC, Wiencke JK, et al. Mutations in the p53 gene in lung cancer are associated with cigarette smoking and asbestos exposure. Cancer Epidemiol Biomarkers Prev 1995;4:543-548. 118. Kawajiri K, Eguchi H, Nakachi K, Sekiya T, Yamamoto M. Association of CYP1A1 germ line polymorphisms with mutations of the p53 gene in lung cancer. Cancer Res 1996;56:72-76. 119. Kondo K, Tsuzuki H, Sasa M, et al. A dose-response relationship between the frequency of p53 mutations and tobacco consumption in lung cancer patients. J Surg OncoI1996;61:20-26. 120. Brennan JA, Boyle JO, Koch WM, et al. Association between cigarette smoking and mutation of the p53 gene in squamous-cell carcinoma of the head and neck. N Engl J Med 1995;332:712-717. 121. Mao L, Lee JS, Fan YH, et al. Frequent microsatellite alterations at chromosome 9p21 and 3p14 in oral premalignant lesions and their value in cancer risk assessment. Nature 1996;2:682-685. 122. Mao L, Lee JS, Kurie JM, et al. Frequent genetic alterations in bronchial epithelium of smokers. Proc Am Assoc Cancer Res 1997;38:246. 123. Sozzi G, Veronese ML, Negrini M, et al. The FHIT gene at 3p14.2 is abnormal in lung cancer. Cell 1996;85:17-26.

2 Biology of Preneoplastic lesions Jin Soo Lee, Li Mao, and Waun Ki Hong

Lung cancer, like other epithelial cancers, is believed to arise as a result of a multistep carcinogenic process (1). The possibility that this process can be blocked or reversed, thereby preventing the development of an invasive cancer (2, 3), has generated great enthusiasm for the study of the biology of premalignant lesions. Two fundamental concepts are widely used to describe the carcinogenesis of lung cancers. The first, field cancerization, was introduced in 1953 by Slaughter et al. (4) to describe the diffuse mucosal changes observed in patients with head and neck cancers, and hypothesizes that the entire field of bronchial trees is exposed to the carcinogenic insult (such as cigarette smoke) and is at increased risk of developing cancer. The second concept is the multistep carcinogenic process (5). The driving force behind this process is thought to be genetic damage caused by continuous exposure to carcinogens (e.g., cigarette smoke), as evidenced by 1) the linear relationship between DNA adduct in human lung and cigarette smoking (6-8), 2) the increased frequency of micronuclei in high-risk tissue and premalignant lesions (9), and 3) the accumulation of multiple genetic alterations in the bronchial epithelium of smokers (10-13). A recent study of benzo-[a]-pyrene adduct formation along the p53 gene exons provides further supporting evidence for an etiologic link between cigarette smoking and lung cancer (14). Hypothetically, the greater the degree of genetic damage in the premalignant lesions, the greater the risk of cancer development, as illustrated in patients with oral leukoplakia lesions (IS, 16). Moreover, these genetic alterations are believed to induce phenotypic alterations in the tissues, 25

26

Chapter 2

such as dysregulation of the proliferation and differentiation pathways. Therefore, a better understanding of the biology of premalignant lesions in the bronchial tree is expected to help us develop rational strategies for early detection and screening of lung cancer, as well as chemoprevention. In this chapter, the types of cells found in the bronchial epithelium and alveoli and their kinetics will be described as a reference for further discussion of potential premalignant lesions found in the lung. Then, the studies relevant to the concepts of field cancerization and multistep carcinogenic process will be discussed, with a major focus on genetic alterations, and the potential precursor lesions described in the literature will be reviewed.

BRONCHIAL EPITHELIUM The entire airway, from the trachea to the respiratory bronchioles, is covered with an epithelial lining (17, 18). In large airways, the epithelium is the ciliated pseudostraHfied columnar type. The thickness of the epithelial lining gradually decreases as the airways become smaller, so that in the terminal bronchioles, the epithelium consists of a single layer of ciliated cells that are more cuboidal than columnar; in the respiratory bronchioles, the cells are even flatter. The pseudostratified columnar epithelium of the human tracheobronchial tree is known to contain at least six major cell types, listed in Table 2.1 (19-22). Ciliated cells are dominant: They are present throughout the entire length of the air-conducting system extending into the respiratory bronchioles. The ciliated cells are believed to be terminally differentiated and unable to divide. Mucous cells comprise those with

Table 2.1.

Major cell types in adult human airway and alveolar epithelium

Airway epithelium Ciliated cells Mucous cells Small mucous granule cells Goblet cells Serous cells Clara cells Neuroendocrine (Ku1chitsky) cells Basal cells Alveolar epithelium Type I pneumocyte Type II pneumocyte Source: Data from Jeffrey PK and Reid L (19), McDowell EM et al. (20), Cruz E (21), and Jeffrey PK (22).

Biology of Preneoplastic Lesions

27

small mucous granules, which are characterized by electron-dense cytoplasm containing electron-lucent confluent granules (19), and goblet cells, which are distended to varying degrees by mucous secretion. The serous cells resemble the serous cells of submucosal glands, which are characterized by large, electron-dense granules, and are found in fetal lung but not in adult bronchial epithelium (23). Clara cells, a specialized type of nonciliated secretory cell located principally in small peripheral airways, are the progenitor cells of the bronchiolar epithelium (24). The neuroendocrine or Kulchitsky cells, containing dense core granules, occur as solitary cells distributed along the entire length of the tracheobronchial tree (25). The basal cells, characterized by the presence of cytoplasmic tonofilaments, are located at the base of the epithelium to form a continuous row along the basal lamina. The basal cells are somewhat stratified in the trachea and large bronchi but the basal cell layer becomes interrupted as the basal cell number diminishes with subsequent airway divisions. Other cell types not included in Table 2.1 but worth mentioning are intermediate cells and brush cells. The term intermediate cells, which was used to describe the polygonal cells occurring between the basal layer and the mucosal lining cells (26, 27), is a light-microscopic classification category rather than a specific cell type (22), and these cells are believed to represent a mixture of basal cells and small mucous granule cells (28). Brush cells are found in animal species but have not been convincingly shown in humans (18, 22). The alveoli are lined by two types of epithelial cells. Flattened type I cells are involved in gaseous exchange, and cuboidal type II cells, containing multilamellar inclusion bodies, are believed to be the source of alveolar surfactant.

CELL KINETICS OF EPITHELIAL CELLS IN THE A,RWAY Studies of animal airways have shown that airway surface epithelium is replaced very slowly. Normally, less than 0.5% of these cells are actively dividing at any given time (29-31). In the normal pathogen-free rat, the basal cells constitute about 70% of the dividing cell population (31). Although earlier studies showed that both basal cells and mucous cells were capable of division, many investigators concluded that the basal cell was the stem cell for the airway epithelium. Like the germinal (reserve) cells of the epidermis, the basal cell was thought to first divide to form new basal cells and "intermediate" cells, which were also capable of cell division, and then to differentiate into mucous cells and ciliated cells (32,33). However, a review of studies (34) showed that secretory cells (both Clara cells and mucous cells) are the primary progenitor cells and that the basal cell is not directly related to renewal of the airway epithelium

28

Chapter 2 after injury. Following injury to the bronchial epithelium by N02, for example, Evans et al. (35) demonstrated a fivefold increase in the labeling index of mucous cells, while the labeling index of basal cells was the same as that of the control animal. All the secretory cells (mucous, Clara, and serous cells) were labeled 1 hour after an injection of eH] thymidine deoxyribose (TdR), which highlights cells preparing to divide. If the basal cells were the progenitors of the columnar cells, the proliferation rate of basal cells would increase after injury; this did not occur. Other studies of airway injury and cell proliferation corroborate these observations (36-40). The primary progenitor cell in the upper airway is thus considered to be the mucous cell rather than the basal cell (34,40). This explanation of the mechanism of large airway epithelial renewal agrees with that in the terminal bronchiole, where Clara cells are capable of both cell division to form new secretory cells and differentiation into ciliated cells (24, 41). Because there are no basal cells in the terminal airways, these data strongly indicate that basal cells are not necessary for renewal of the columnar epithelium and the formation of new ciliated cells. However, this does not address the issue of stem cells versus repopulating cells. There might be different levels of progenitor cells, as is the case in the hematopoietic system (42). In the alveoli, type II cells are the immediate precursors of the nondividing type I cells (30, 43).

HISTOLOGIC CHANGES IN BRONCHIAL TREES OF SMOKERS In the 1950s and early 1960s, many investigators reported histologic changes in the bronchial epithelium associated with chronic smoking and lung cancer, including basal cell hyperplasia, stratification, squamous cell metaplasia, and carcinoma in situ (27, 44-49). Transitional metaplasia distinct from squamous metaplasia was also described (45, 47), but it should be regarded as an intermediate type of epithelial change preceding the devE.lopment of squamous metaplasia from mucous cells described by Trump et al. (50). Most notable is the work of Auerbach et al. (47-49t who carefully examined the serial sections of entire tracheobronchial trees and recorded three major types of epithelial changes (i.e., increase in number of cell rows, loss of cilia, and presence of atypical cells) (49). These observations were analyzed singly and in combination with other variables. There was a consistent direct correlation between the frequency of these epithelial changes and the amount of cigarette smoking, as shown in Table 2.2. The most striking finding was the frequency of carcinoma in situ, defined as a lesion composed entirely of atypical cells without cilia in an average thickness of five or more cell rows, which was observed in

29

Biology of Preneoplastic Lesions Table 2.2.

Histologic changes in bronchial epithelium according to smoking history compared with lung cancer patients Smokers Histologic changes

Never smoked

(n Lesions with cilia Hyperplasia (~3 rows) ~70% atypical cells ~90% atypical cells Lesions without cilia Overall 100% atypical cells

= 3324)

o

5

o~------~--------~--------~--------~------~~------~

o

50

Rest

100

150

200

250

Work rate rN)

Fig. 4.3. Measurement of oxygen consumption (Vo 2 ) during incremental work on a cycle ergometer. Maximal oxygen consumption (Vo 2 ) is identified by the plateau point where increasing work produces no further increase in Vo 2 . The peak V0 2 is reached when a flattening in the rate of V0 2 rise does not occur with increasing work rate, but the subject stops exercising because of exhaustion or dyspnea.

This point is called "maximal" \'02 (\'02maJ (Fig. 4.3). The presence of cardiopulmonary dysfunction in virtually all patients that undergo preoperative evaluation, however, prevents them from reaching this maximal state defined by a plateau in \'02' Thus, most often a symptomlimited "maximum" \'02 or peak \'02 is utilized in the data comparison of studies dealing with preoperative assessment. The maximum or peak \'02 represents the \'02 at which the patient stopped exercising because of exhaustion or dyspnea. The results of \'02 as a parameter of oxygen transport need to be interpreted in relation to the level of work performed, the individual's pulmonary function, corrections for body weight or body surface area, and comparisons to normal predicted values (38). Peak \102 values need careful interpretation, as a submaximal patient effort may be erroneously interpreted as maximal and lead to withholding of indicated surgery. Interpretation of the results of the exercise testing also take into account minute ventilation and respiratory rate, heart rate, hemodynamic parameters, electrocardiographic \'02'

Preoperative Evaluation of the Patient with Lung Cancer

81

evidence of myocardial ischemia or arrhythmias, and oxygen arterial saturation measured by pulse oximetry (39). In 1982, Eugene et al. (40) were the first to report the relationship between exercise performance and postoperative complications. These authors found a significant relationship between peak \'02 and postoperative mortality. No deaths occurred when the peak \'02 was greater than 1 L/min, while 75% mortality was reported among those patients with a peak \'02 of less than 1 L/min. Subsequent studies by Smith et al. (6) reported no significant relationship of cardiopulmonary complications after thoracotomy and preoperative pulmonary function in fourteen high-risk patients. However, all six patients with a peak \'02 of less than 15 mL/kg/min experienced postoperative complications, while only one of ten patients with a peak \'02 greater than 20 mL/kg/min had complications. Four of six patients with a peak \'02 between 15 and 20 mL/kg/min developed postoperative complications. Significant differences were found only between patients with a peak \'02 less than 15 mL/kg/min and those whose peak \'02 values were greater than 20 mL/kg/min. Similarly, Bechard et al. (41) reported that in a group of 50 patients who underwent pulmonary resection, there was a 29% mortality rate and a 43% morbidity rate in those patients with a peak \'02 of less than 10 mL/kg/min; 10.7% morbidity and no deaths in those with a peak \'02 between 10 and 20 mL/kg/min; and no complications in those with a peak \'02 of more than 20 mL/kg/min. Olsen et al. (42) found that, in 52 patients with chronic obstructive pulmonary disease and lung masses, \'02 values during submaximal exercise testing (20 and 40 watts) predicted intolerance to lung resection better than lung function calculated using quantitative lung scanning. We have evaluated the usefulness of exercise testing in the preoperative evaluation of patients with resectable lung lesions who were considered ineligible for surgery according to conventional pulmonary function criteria (5). In an initial study, exercise testing was performed on 37 patients with resectable lung lesions who were considered at very high risk because any of the following factors: 1) an FEV 1 of less than 40% of the normal predicted value; 2) a radionuclide calculated postlobectomy FEV 1 of less than 33% of the normal predicted value; and 3) a Pac02 of more than 45 mm Hg. The patients who reached a peak \'02 of 15 mL/kg/min or more were offered surgical treatment. Patients with a peak \'02 of less than 15 mL/kg/min were referred for nonsurgical therapy. In this study, no relationship was found between the patient's exercise performance and spirometric function. Only two of the surgically treated patients had postoperative complications consisting of pneumonia and atrial fibrillation, but no patient died as a result of surgery or complications. Subsequent studies performed by our group have reaffirmed the usefulness of exercise testing in evaluating high-risk patients for lung resection (4, 5). We have found no treatment-related deaths and a reasonable rate of postoperative complications (12%). In addition, there

82

Chapter 4

has been a striking survival benefit to surgical approach in these highrisk patients. The median duration of survival was 48 ± 4.3 months for the surgically treated patients, and 17 ± 2.7 months for those treated with radiation and/ or chemotherapy (P = 0.0014). While most studies have used an exercise peak Y02 of 15 mL/kg/min or more as a level for acceptable risk for operability, a lower cut-off criteria appears justifiable based on more recent data. For some patients, particularly the elderly and women, a peak Y02 of 15 mL/kg/min corresponds to 100% of maximal predicted. It would therefore be unreasonable to exclude them from surgery based on their inability to have an absolute normal level of exercise performance. Pate et al. (43) utilized a peak Y02 of 10 mL/kg/min or more as the acceptable value for operability in twelve high-risk patients. There were no postoperative deaths, and 9 complications occurred in 7 patients. Further analysis of our experience has shown that there are patients with peak Y02 less than 15 mL/kg/ min who survived surgical treatment with acceptable morbidity. All these patients had preoperative exercise peak Y02 values, that when adjusted for height, gender, and age were 50% or more of maximal predicted. A prospective study was conducted using the criteria of peak Y02 of 50% or more of maximal predicted, rather than an absolute value, as the cut-off value for acceptable surgical risk (3). This new standard allowed us to further improve the proportion of high-risk patients who can undergo lung resection by an additional 21 %. Not all studies have agreed in documenting the value of exercise testing for predicting postthoracotomy complications. Coleman et al. (44) found no significant relationship between peak Y0 2 and the development of postoperative complications in 47 patients who underwent lung resection. Their patients, however, experienced complications such as gastrointestinal bleeding, wound infection, and excess blood loss that are unlikely to be predicted by any single mode of preoperative testing. At present, however, most authors agree on the usefulness of exercise testing in the evaluation of the high-risk patient for lung resection (45). Exercise testing does not determine the functional contribution of the pulmonary parenchyma to be resected, nor does it help estimate the patient's postoperative pulmonary function. Thus, patients must be carefully assessed with tests of pulmonary function to ensure the adequacy of postresection lung function. In this framework, exercise testing is primarily useful in the preoperative selection of patients who have severe pulmonary dysfunction and pulmonary lesions amenable to limited surgical resection (Fig. 4.4). Because postoperative complications also occur in patients with normal pulmonary function and adequate exercise performance, a single test cannot predict all complications. Exercise testing should be considered an important complement to standard pulmonary and cardiovascular evaluation when selecting patients for pulmonary resection.

Preoperative Evaluation of the Patient with Lung Cancer

83

- - -

Resectable Lung Cancer

FEV 1> 700/0 or

FEV t < 700/0

Pco2 < 45

or Pco2 > 45

> 33%and Pco2 < 45

2: 50 0/0

Excercise Test pred. max.)

_ ..... < 50%

tVo2°/o

Fig. 4.4. Combined use of preoperative pulmonary function, radionuclide regional lung studies, and exercise testing in the evaluation of patients for pulmonary resection. (XRT = radiation; Chemo = chemotherapy.)

REFERENCES Cancer Statistics 1996: A Cancer Journal for Clinicians. 1996;46:26-27. Tisi eM. Preoperative evaluation of pulmonary function: validity, indications, and benefits. Am Rev Respir Dis 1979;119:293-310. 3. Morice RC, Walsh eL, Ali MK, Roth JA. Redefining the lowest exercise peak oxygen consumption acceptable for lung resection of high-risk patients. Chest 1996;110:4, 658. 4. Walsh eL, Morice RC, Putnam JB, et al. Resection of lung cancer is justified in high-risk patients selected by exercise oxygen consumption. Ann Thorac Surg 1994;58:704-711. 5. Morice RC, Peters EJ, Ryan MB, et al. Exercise testing in the evaluation of patients at high risk for complications from lung resection. Chest 1992; 101:361-365. 6. Smith TP, Kinasewitz GT, Tucker WY, Spillers WP, George RB. Exercise capacity as a predictor of post-thoracotomy morbidity. Am Rev Respir Dis 1984;129:730-734. 1. 2.

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8.

9.

10.

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15. 16.

17. 18.

19.

20.

21.

22.

23.

24.

25. 26.

Markos I, Mullan BF, Hillman DR, et al. Preoperative assessment as a predictor of mortality and morbidity after lung resection. Am Rev Respir Dis 1989;139:902-910. Sandvik MSc, Eriksen I, Thaulow E, et al. Physical fitness as an independent predictor of mortality among healthy, middle-aged, Norwegian men. N Eng J Med 1993;328:533-537. Gaensler EA, Cugell DW, Lindgren I, et al. The role of pulmonary insufficiency in mortality and invalidism following surgery for pulmonary tuberculosis. J Thorac Cardiovasc Surg 1955;29: 163-187. Miller JI, Grossman GD, Hatcher CR. Pulmonary function test criteria for operability and pulmonary resection. Surg Gynecol Obstet 1981;153:893895. Boysen PG, Block AI, Moulder PY. Relationship between preoperative pulmonary function tests and complications after thoracotomy. Surg Gynecol Obstet 1981;52:813-815. Lockwood P. Lung function tests results and the risks of postthoracotomy complications. Respiration 1973;30:529-542. Mittman C. Assessment of operative risk in thoracic surgery. Am Respir Dis 1961;84:197-207. Knudson RI, Slatin RC, Lebowitz MD, Burrows B. The maximal expiratory flow volume curve: normal standards, variability, and effects of age. Am Rev Respir Dis 1976;113:587-600. Milledge JS, Nunn JE Criteria of fitness for anesthesia in patients with chronic obstructive lung disease. Br Med J 1975;3:670-673. Neuhaus H, Cherniack NS. A bronchospirometric method of estimating the effect of pneumonectomy on the maximum breathing capacity. J Thorac Cardiovasc Surg 1968;55:144-148. Bergan E A simple method for determining the relative contribution of the right and left lung. Acta Chir Scand 1960;253(suppl):58. Kristersson S, Lindell S, Sranberg L. Prediction of pulmonary function loss due to pneumonectomy using 133Xe-radiospirometry. Chest 1972;62:694698. Olsen GN, Block AI, Tobias JA. Prediction of postpneumonectomy pulmonary function using quantitative macroaggregate lung scanning. Chest 1974;66:13-16. Wernly JA, De Meester TR, Kirchner PT, et al. Clinical value of quantitative ventilation-perfusion lung scans in the surgical management of bronchogenic carcinoma. J Thorac Cardiovasc Surg 1980;80:535-543. Ali MK, Mountain CF, Ewer MS, Johnston D, Haynie TP. Predicting loss of pulmonary function after pulmonary resection for bronchogenic carcinoma. Chest 1980;77:337-342. Ladurie ML, Ranson-Bitker B. Uncertainties in the expected value for forced expiratory volume in one second after surgery. Chest 1986;90: 218-225. Cooper JD, Trulock EP, Triantafillou AN, et al. Bilateral pneumonectomy (volume reduction) for chronic obstructive pulmonary disease. J Thorac Cardiovasc Surg 1995;109:106-119. Pigula FA, Keenan RI, Ferson PF, Landrenau RJ. Unsuspected lung cancer found in work-up for lung reduction operation. Ann Thorac Surg 1996;61: 174-176. McKenna Bolliger CT, Jordan P, Soler M, et al. Pulmonary function and exercise capacity after lung resection. Eur Respir J 1996;9:415-421.

Preoperative Evaluation of the Patient with Lung Cancer 27.

28.

29. 30.

31.

32.

33. 34. 35.

36. 37.

38. 39. 40.

41. 42.

43.

44.

45.

85

Adams WE, Perkins JF, Flores A, Chao P, Castellanos M. The significance of pulmonary hypertension as a cause of death following pulmonary resection. J Thorac Surg 1953;26:207-218. Harrison RW, Adams WE, Long ET, Burrows B, Reimann A. The clinical significance of cor pulmonale in the reduction of cardiopulmonary reserve following extensive pulmonary resection. J Thorac Surg 1958;36:352-358. Carlens E, Hanson HE, Nordenstrom B. Temporary unilateral occlusion of the pulmonary artery. J Thorac Surg 1951;22:527-536. Laros CO, Swierenga J. Temporary unilateral pulmonary artery occlusion in the preoperative evaluation of patients with bronchogenic carcinoma. Med Thorac 1967;24:269-283. Olsen GN, Block AJ, Swenson EW, Castle JR, Wynne JW. Pulmonary function evaluation of the lung resection candidate: a prospective study. Am Rev Respir Dis 1975;111:379-387. Fee JH, Holmes EC, Gewirtz HS, Remming KP, Alexander JM. Role of pulmonary vascular resistance measurements in preoperative evaluation of candidates for pulmonary resection. J Thorac Cardiovasc Surg 1975;75: 519-524. Mahler OA, Weinberg OH, Wells CK, Feinstein AR. The measurement of dyspnea. Chest 1984;85:751-758. Olsen GN. The evolving role of exercise testing prior to lung resection. Chest 1989;95:218-225. Lugo G, Arizpe 0, Dominguez G, Ramirez M, Tamariz O. Relationship between oxygen consumption and oxygen delivery during anesthesia in high-risk surgical patients. Crit Care Med 1993;21:64-69. Van Nostrand 0, Kjelsberg MO, Humphrey EW. Preresectional evaluation of risk from pneumonectomy. Surg Gynecol Obstet 1968;127:306-312. Olsen GN, Bolton RJW, Weiman OS, Hornung CA. Stair climbing as an exercise test to predict postoperative complications of lung resection. Chest 1991;99:587-590. Carlson OJ, Ries AL, Kaplan RM. Prediction of maximum exercise tolerance in patients with COPO. Chest 1991;100:307-311. Wassermann K, Hansen JE, Sue OY, Whipp BJ. Principles of Exercise Testing and Interpretation. Philadelphia, Lea & Febiger, 1987. Eugene J, Brown SE, Light RW, Milne SE, Stemmer EA. Maximum oxygen consumption: a physiologic guide to pulmonary resection. Surg Forum 1982;33:260-262. Bechard 0, Wetstein L. Assessment of exercise oxygen consumption: a preoperative criterion for lung resection. Ann Thorac Surg 1987;44:344-349. Olsen GN, Weiman OS, Bolton JWR, et al. Submaximal invasive exercise testing and quantitative lung scanning in the evaluation for tolerance of lung resection. Chest 1989;95:267-273. Pate P, Tenholder MF, Griffin JP, Eastridge CE, Weinman OS. Preoperative assessment of the high-risk patient for lung resection. Ann Thorac Surg 1996;61 :1949-1950. Colman NC, Schraufrasel DE, Rivington RN, Pardy RJ. Exercise testing in the evaluation of patients for lung resection. Am Rev Respir Dis 1982;125: 604-606. Gilbreth EM, Weisman 1M. Role of exercise testing in preoperative evaluation of patients for lung resection. Clin Chest Med 1994;15:389-403.

5 Lung-Sparing Operations for Cancer Robert J. Ginsberg

Following Evarts Graham's historic pneumonectomy in 1932, which initiated surgical management of lung cancer (1), total removal of the ipsilateral lung became the resection of choice in dealing with this disease and remained so for the next 20 years (2). Within a decade of Graham's groundbreaking work, however, Blades (3) and others described the individual ligation technique for lobectomy, and in 1939 Churchill and Belsey (4) reported in detail the anatomy of the lung segment and proposed the technique of segmentectomy. Initially, these procedures were reserved for benign conditions (5). Following World War II, PriceThomas (6) and Paulson and Shaw (7) independently developed and reported successful sleeve-resections of lobar bronchi for patients suffering from carcinoid tumors. Despite these technical advances, pneumonectomy remained the accepted treatment for removal of a malignant lung tumor, other than carcinoid tumor, until the early 1950s. It was then recognized that earlier stage lung cancers could be successfully treated by lobectomy if completely resected by this technique (8). During the past two decades, lung-conserving operations including sleeve-lobectomy, segmentectomy, wedge resection, and sleeve-pneumonectomy have been utilized as resections of choice in selected patients with lung cancer (8-12).

EVOLUTION OF LUNG-SPARING OPERATIONS By virtue of the underlying causative factors in their disease, patients suffering from lung cancer frequently have significant chronic obstruc87

/

88

Chapter 5 tive pulmonary disease (COPD). In many such patients, it was recognized that pneumonectomy would be impossible because of lack of appropriate pulmonary reserve. In order to maintain adequate lung function, sleeve-resection of an involved major bronchus, preserving uninvolved pulmonary tissue, was developed, initially as a compromise operation in patients with proximal tumors involving the mainstem bronchus where distal pulmonary tissue was uninvolved. Similarly, in small peripheral tumors, segmentectomy or wedge resection were performed when patients could not tolerate removal of even a lobe because of compromised pulmonary function. Retrospective analyses of these "compromise" lung-conserving procedures showed that, in early lung cancer, reasonable long-term survival could be expected, comparable to more extensive pulmonary resections. This was especially true when the disease had not involved regional lymph nodes (NO disease). Lobectomy and sleeve-lobectomy soon became procedures of choice in early stage disease, when these procedures allowed a complete resection. In 1973, Jensik (10) was the first to report the use of "limited pulmonary resection" (segmentectomy) as an intentional technique in peripheral early-stage (TI-2, NO) lung cancer. This seminal report of 73 cases suggested that survival rates were similar to those seen with lobectomy, the local recurrence rate was acceptable «10%), and the mortality rate was minimal «2%)-less than that seen with lobectomy. Another use of intentional limited pulmonary resection has been for T3 NO tumors with en bloc resection of chest wall, especially superior sulcus tumors. Many surgeons have employed wedge or segmental resection, combined with chest wall resection for small peripheral lesions locally invading the chest wall, as the procedure of choice when an adequate margin is obtained by this technique.

THEORETICAL CONSIDERATIONS Classically, an adequate resection for lung cancer includes removal of the primary tumor plus the draining local-regional lymph nodes. For early-stage lung cancer (Tl, T2, NO-I), the most minimal resection allowing the surgeon to totally encompass the regional lymphatic drainage in every instance is lobectomy (13) or bilobectomy in right lower tumors. Ishida et al. (14) demonstrated that for very small tumors (s 1 em) the incidence of lymphatic permeation and nodal-involvement tumors was zero. Tumors of 1.1 to 2 em had lymphatic spread in 17% of cases, and of those patients with tumors 2.1 to 3 em in diameter 38% had lymph channel or lymph node metastases. Ichinose et al. (15) have recently confirmed this result in a separate study. In a recent analysis by the National Cancer Hospital in Japan, of 305 patients with small «3 em)

Lung-Sparing Operations for Cancer

89

peripheral lung cancers, 68 patients were found to have lymphatic and lymph node involvement at pathologic staging (16). It is apparent from this data that tumors greater than 1 cm, which are resected by less than lobectomy, may well have incomplete resections performed, as not all regional lymphatic channels and lymph nodes are removed with such lesser resections. One must conclude that the larger the size of the primary tumor treated by less than lobectomy, the greater the chance of a local-regional recurrence by virtue of occult lymphatic spread and inadequate local-regional control of lymph nodes at the initial surgery. Theoretically, limited resection, if proven to be an adequate cancer operation, would provide distinct advantages in the management of patients with lung cancer. In most retrospective reviews, pneumonectomy mortality exceeds that of a sleeve procedure, and lobectomy carries a greater perioperative mortality than lesser resections (17). As welt patients surviving lung cancer frequently develop a second primary tumor that may be amenable to surgical resection (18). If such a patient had undergone a lung-conserving resection for the initial tumor, there would be a greater chance that another surgical resection could playa curative role if a second primary tumor developed. Moreover, with the introduction of thoracoscopic-assisted surgery, surgeons have developed a keen interest in applying limited resection to early-stage disease, utilizing video-assisted thoracoscopic techniques (19-22).

TYPES OF RESECTION The types of resection that conserve pulmonary tissue are listed in Table 5.1. Lung-conserving resections, by virtue of bronchoplastic procedures, are discussed elsewhere. This chapter focuses on procedures,less than lobectomy, utilized in the treatment of primary lung cancer. These fall into three broad categories: segmentectomy, wedge excision, and precision dissection (lumpectomy).

Table 5.1.

Lung-sparing procedures in thoracic surgery

Lobe-sparing procedures Segmentectomy Sleeve segmentectomy Wedge excision Precision dissection Lung-sparing procedures Bronchial sleeve resection Mainstem bronchus Intermediate bronchus Sleeve lobectomy Sleeve pneumonectomy

90

Chapter 5

s ........-Artery

Subpleural vein

\

\

\ \

Segmental vein

\ \ \ \ ..... \

'~

,

I ./

._------

-"

./

Fig. 5.1. The anatomy of a pulmonary segment. Note that the bronchus and artery cross through the segment together, whereas the segmental veins take an intersegmental course.

Segmentectomy The segmental anatomy of the lung is well described (Fig. 5.1). The lymphatic drainage of segments usually follows the segmental artery, typically without crossing to adjacent segments, although a small amount of crossover has been described by various authors (23). For this reason, segmentectomy theoretically would have advantages as a cancer operation because it can encompass most of the local lymphatic drainage. The classic segmentectomy includes isolation and division of the feeding segmental artery and bronchus prior to "stripping" the segment from its adjacent segments along the plane delineated by intersegmental veins (Fig. 5.2) (24). Those segments abutting a single other segment are most appropriate for segmentectomy by virtue of their technical simplicity, the classic segments including the lingula and the superior segment (Fig. 5.3). With the increasing use of mechanical staplers, surgeons frequently isolate the artery and lobar bronchus but ultimately separate the segment from the adjacent lung by using Inechanical staplers along a plane estimated to be the intersegmental area. This has some theoretical disadvantages, as infolding of the adjacent lung tissue occurs thereby decreasing the volume of the remaining functioning lung tissue at the


91

BronchuS

Bronchus Subpleural

vein Subpleural vein

-~--.....i6/cIll~lh-- Intersegmental veins

\

Segm.entaI

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vein ---i"~rtJn

\

\

\

-~)

--_ _---

_/

.......

Single unit (segment)

Anterior

segment

-'

---

.-

Compound unit (segment)

Intersegmental vein Apical

Upper lobe Middle lobe

Fig. 5.2.

The anatomic dissection required for a segmental resection.

Fig. 5.3.

Typical segmental resections used most frequently in the right lung. Sup = superior.)

(Post

= posterior;

completion of the procedure. As well, there is necessarily less accurate identification of the intersegmental plane, and appropriate lymphatic drainage areas may not be completely encompassed by the excision. On occasion, segmentectomy can be combined with sleeve resection of a larger bronchus, to treat segmental orifice tumors (e.g., superior segment) thus preserving the rest of the lobe.

92

Chapter 5 Peripheral tumors located at the margins of a segment or between segments have been treated by "double-segmentectomy." In addition, lingular-sparing upper lobectomies or superior segmental-sparing lower lobectomies have been utilized. Whether these "extended" limited resections are a worthwhile endeavor in order to preserve such limited functioning lung tissue has never been investigated.

Wedge Excision Small peripheral tumors can be totally removed by a large wedge excision, most frequently by employing mechanical staplers for division of parenchymal tissue, although the traditional "cut-and-sew" technique can be used (Fig. 5.4). In such resections, no attempt is made to encompass all lymphatic drainage areas; however, complete removal of the primary tumor is always the goal. It is not known how much adjacent normal lung tissue should be excised before an excision can be safely considered "adequate." It appears that at least 2 cm of uninvolved lung tissue surrounding the tumor should be removed, and frozen section control of margins is important.

Precision Dissection Perelman (25) and Cooper et al. (26) described the technique of "lumpectomy" as applied to pulmonary lesions. This procedure is applicable to more deeply seated lesions than those treated by wedge excision and is used when lobectomy cannot be considered because of poor pulmonary function and when segmentectomy is inappropriate because of tumor location. With this technique, cautery or laser dissection of pulmonary tissue is performed with the lung expanded, encompassing the tumor and a 2-cm margin of normal lung tissue, and utilizing the coagulative effects of cautery or laser to seal alveolar spaces. In the depths of the dissection, a feeding artery and bronchus are usually identified and ligated (Fig. 5.5). The raw pulmonary surfaces may be suture-closed or left exposed.

RESULTS OF LIMITED RESECTION FOR EARLy-STAGE LUNG CANCER

Segmentectomy Results of segmentectomy have been the most widely reported and fall broadly into two categories: 1) results for compromise operations,


93

E

Fig. 5.4. Techniques of wedge resection including the "cut-and-sew" method (A, B, C) and the use of mechanical staplers (0, E).

where pulmonary function allowed no greater resection, and 2) results for intentional resections of Tl-2 NO lung cancers, where the surgeon believed such a resection was adequate for cure and opted for this procedure rather than formal lobectomy (Tables 5.2, 5.3). In compromise situations where nothing more extensive can be considered, the results of such resections lead to a reasonable salvage rate with 5-year tumor-free survivals ranging from 15-30%. These series often include patients with N1 and N2 disease. The incidence of localregional recurrence, although not well documented, is higher than one would expect with NO tumors, and is somewhat higher than one would expect with lobectomy for the same stage of disease.

Fig. 5.5. A. A peripheral nodule situated such that a wedge resection or segmental resection cannot be used. B. With an expanded lung, precision dissection cautery begins by outlining the tissue to be excised. C. The small peripheral tumor nearly excised, together with normal surrounding lung. D. The primary tumor and surrounding normal lung have been removed. Note the bronchovascular unit that has been sutured and ligated at the base of the excision.


Fig. 5.5

(continued)

95

96

Chapter 5 Table 5.2.

Results of limited pulmonary resection performed as a compromise procedure for T 1 and T2 lesions

Author (ref), yr

# Pts

5-yr survival

Local recurrence

Bonfils-Roberts & Clagett (5), 1972 LeRoux (30), 1972 Shields & Higgins (31), 1974 Bennett & Smith (32), 1979 Hoffman & Rantsera (33), 1980 Kutschera (34), 1984 Errett et al. (11), 1985 McCaughan (27), 1991 Crabbe et al. (35), 1991 Pastorino et al. (36), 1991

18 17 29 44 33 57 100 64 24 61

39% 39% 30% 36% 26% 23% 69%* 62% 65% 55%

13% 5%

*Postoperative radiotherapy; 2-yr survival.

Table 5.3.

Results of limited pulmonary resection performed as an intentional procedure, mainly for T 1 NO tumors

Author (ref), yr

# Pts

Estimated 5-yr survival

OP mortality

Local recurrence

Jensik (37), 1986 Read et al. (38), 1990 Wain et al. (39), 1991 Warren & Faber (40), 1994 LCSe (41), 1995

296 113 164 68

52% 70% 0.8

IP = 0.039)

-~

·

:::J

en

65%

c: o.s

o

~ 0.4

8. 0.3 e 0.2

Cl.

101

~.

Ie

'-

~--4

~--

.·

-- __ --- _430% :

.-~--------------.

h._--------- __

4 __

0.1 O----------~~----------~~----------~----------~ 30 60 90 120

Time (Mas) Fig. 5.8. The survival of patients when comparing lobectomy plus en-bloc chest wall resection with comparable patients who underwent wedge resection plus en-bloc chest wall resection. (Reproduced by permission from Ginsberg RJ, Martini N, Zaman M et al. The influence of surgical resection and intraoperative brachytherapy in the management of superior sulcus tumor. Ann Thorac Surg 1994;57: J 440- J 445.)

(Fig. 5.8) (29). For this reason, we no longer advocate any procedure less than lobectomy in conjunction with the chest wall resection, even in small peripheral tumors invading the chest wall.

SUMMARY Limited resection is an effective tool as a compromise operation in those patients who cannot tolerate lobectomy in the management of lung cancer. In such cases, adjuvant radiotherapy should be considered. However, recent data from our own center and those performing volume reduction, question the need for less-than-Iobectomy resections even in the most severely compromised. Whenever possible, a complete resection should always be performed, as less-than-complete resection will ultimately lead to local-regional recurrence and failure of the operation to control the disease. Resection less than lobectomy as the standard operation for small peripheral tumors has, as yet, not been proven to be

102

Chapter 5

as effective as lobectomy and, therefore, lobectomy must be considered the minimum resection of choice.

REFERENCES 1. 2.

3. 4. 5. 6. 7. 8.

9.

10. 11.

12. 13.

14. 15.

16.

17.

Graham EA, Sedar JJ. Successful removal of the entire lung for carcinoma of the bronchus. JAMA 1993;101:1371-1374. Ochsner A, DeBakey M. Surgical considerations of primary carcinomas of the lung: review of the literature and report of 19 cases. Surgery 1940;8:992-1023. Blades B. Conservation of lung tissue by partial lobectomy. Ann Surg 1943;118:353. Churchill ED, Belsey R. Segmental pneumonectomy and bronchiectasis: lingular segment of the left upper lobe. Ann Surg 1939;109:481-499. Bonfils-Roberts EA, Clagett OT. Contemporary indications for pulmonary segmental resections. J Thorac Cardiovasc Surg 1972;63:433-438. Price-Thomas C. Conservative resection of the bronchial tree. J Arch ColI Surg Edinb 1956;1:169-171. Paulson DL, Shaw RR. Preservation of lung tissue by means of bronchoplastic procedures. Am J Surg 1955;89:347. Churchill ED, Sweet RH, Sutter L, Scannell JG. The surgical management of carcinoma of the lung: a study of cases treated at the Massachusetts General Hospital from 1930-50. J Thorac Cardiovasc Surg 1950;20:349365. DesLauriers J, Gaulin P, Beaulieu M. Long-term clinical and functional results of sleeve lobectomy for primary lung cancer. J Thorac Cardiovasc Surg 1986;91:871-879. Jensik RJ, Faber LD, Milloy FJ, Monson BO. Segmental resection for lung cancer: a 15-year experience. J Thorac Cardiovasc Surg 1973;66:563-572. Errett LE, Wilson J, Chiur C-J, Munro DO. Wedge resection as an alternative procedure for peripheral bronchogenic carcinoma in poor risk patients. J Thorac Cardiovasc Surg 1985;90:656. Deslauriers J, Bealie N, Benazera A, et al. Sleeve pneumonectomy for bronchogenic carcinoma. Ann Thorac Surg 1978;28:465-474. Iascone C, DeMeester TR, Albertussi M, et al. Local recurrence of resectable non-oat cell carcinoma of the lung: a warning against conservative treatment for NO and N1 disease. Cancer 1986;57:471. Ishida T, Yano T, Maeda K. Strategy for lymphadenectomy in lung cancer 3 cm or less in diameter. Ann Thorac Surg 1991;50:708-713. Ichinose Y, Yano T, Yokomaya H, et al. The correlation between tumor size and lymphatic vessel invasion in resected peripheral stage I non-smaIl-cell lung cancer-a potential risk of limited resection. J Thorac Cardiovasc Surg 1994;108:684-686. Asamura H, Nakayama H, Kondo H, et al. Lymph node involvement, recurrence and prognosis in resected small, peripheral, non-smaIl-cell lung carcinomas: are these carcinomas candidates for video-assisted lobectomy? J Thorac Cardiovasc Surg 1996;11 :1125-1134. Ginsberg RJ, Hill LD, Eagan RT, et al. Modern-day operative mortality for surgical resection in lung cancer. J Thorac Cardiovasc Surg 1983;86:654658.

Lung-Sparing Operations for Cancer 18.

19.

20.

21. 22.

23.

24.

25. 26. 27. 28.

29.

30. 31. 32.

33. 34. 35. 36. 37.

38. 39.

103

Pairolero PC, Williams DE, Bergstalh EJ, et al. Post surgical stage I bronchogenic carcinoma: morbid implications of recurrent disease. Ann Thorac Surg 1984;38:331-338. Mack M, Aronoff R, Acuff T, et al. The present role of thoracoscopy in the diagnosis and treatment of diseases of the chest. Ann Thorac Surg 1992;54: 403-408. Miller DL, Allen MS, Trastek VF, Deschamps e, Pairolero Pc. Video thoracoscopic wedge resections of the lung. Ann Thorac Surg 1992;54:410414. Landreneau RJ, Hazelrigg SR, Ferson PF, et al. Thoracoscopic resection of 52 pulmonary lesions. Ann Thorac Surg 1992;54:415-420. Lewis RJ, Caccavale RJ, Sisler GE, Mackenzie JW. Video-assisted thoracic surgical resection of malignant lung tumors. J Thoracic Cardiovasc Surg 1992;104:1679-1687. Riguet M, Hidden G, Oebesse B. Direct lymphatic drainage of lung segments to the mediastinal nodes: an anatomic study on 260 adults. J Thorac Cardiovasc Surg 1989;97:623-632. Ginsberg RJ, Vanecko RM. Pulmonary resections. In: Nora PF, ed. Operative Surgery: Principles and Technigues, 3rd ed. Philadelphia: WB Saunders, 1990:302-320. Perelman Ml. Lumpectomy for lung cancer. Chest 1986;89(suppl):335S336S. Cooper JO, Perelman M, Todd TRJ, Ginsberg RJ, et al. Precision cautery excision of pulmonary lesions. Ann Thorac Surg 1986;41:51-53. McCaughan B. Limited resection for stage I carcinoma of lung. Lung Cancer 1991 ;75:75. Abstract. Miller JI, Hatcher CR. Limited resection of bronchogenic carcinoma in patients with marked improvement of pulmonary function. Ann Thorac Surg 1984;44:240-243. Ginsberg RJ, Martini N, Zaman M, et al. The influence of surgical resection and intraoperative brachytherapy in the management of superior sulcus tumor. Ann Thorac Surg 1994;57:1440-1445. LeRoux BT. Management of bronchial carcinoma by segmental resection. Thorax 1972;27:70-74. Shields TW, Higgins GA Jr. Minimal pulmonary resection in treatment of carcinoma of the lung. Arch Surg 1974;108:420-423. Bennett WF, Smith RA. Segmental resection for bronchial carcinoma: a surgical alternative for the compromised patient. Ann Thorac Surg 1979; 27:170-172. Hoffman TH, Ranstera HT. Comparison of lobectomy and wedge resection for carcinoma of the lung. J Thorac Cardiovasc Surg 1980;79:211-217. Kutschera W. Segmental resection for lung cancer. J Thorac Cardiovasc Surg 1984;32:102-104. Crabbe NM, Patrissi GA, Fontenelle LJ. Limited resection for bronchogenic carcinoma. Chest 1991;99:1421-1424. Pastorino U, Valente N, Bedini V, et al. Limited resection for stage I lung cancer. J Surg Oncol1991;17:42-46. Jensik RJ. The extent of resection for localized lung cancer: segmental resection. In: Kittle CF, ed. Current Controversies in Thoracic Surgery. Philadelphia: WB Saunders, 1986:175-182. Read Re, Boop We, Schaeffer RC. Survival after conservative resection for T1 NO MO non-small cell lung cancer. Ann Thorac Surg 1990;49:391-400. Wain Je, Mathisen OJ, Hilgenberg AD, et al. Wedge and segmental resec-

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40.

41.

tion for primary lung carcinomas. Proc Am Assoc Thorac Surg May 1991. Abstract. Warren WH, Faber LP. Segmentectomy vs lobectomy in patients with stage I pulmonary carcinoma. J Thorac Cardiovasc Surg 1994;107:10881094. Ginsberg RJ, Rubinstein LV. Randomized trial of lobectomy versus limited resection for T1 NO non-small cell lung cancer. Lung Cancer Study Group. Ann Thorac Surg 1995;60:615-623.

6 Role of Thoracoscopy in the Diagnosis and Treatment of Lung Cancer Joseph LoCicero III

Minimally invasive access to the thorax has enjoyed periodic enthusiasm during the twentieth century. As recently as the late 1800s, open surgery of the chest was considered unsafe at the very least. But, with the development of the controlled artificial pneumothorax, thoracoscopy had an opportunity to develop (1). Shortly after the turn of the century, Jacobaeus used a cystoscope to view the inside of the chest (2). The technique flourished in Europe where it was used to produce a therapeutic pneumothorax in the treatment of tuberculosis, to diagnose and manage pleural problems such as effusions, empyemas, and primary and secondary malignancies of the pleura (3). Effective antituberculous chemotherapy, coupled with refinement in operative and anesthetic techniques, eventually overwhelmed the modest success of thoracoscopy, and by 1960 most institutions relegated the equipment to the back of the operating room closet or donated their devices to museums or private collections. However, some surgeons in the United States continued to practice the techniques of thoracoscopy and periodically wrote about its effectiveness for diagnosis of pleural diseases (4-6). In Europe, pulmonary physicians continued to perform thoracoscopy for pleural problems (7). Not until the 1990s was there an explosion in the use of thoracoscopy. Many factors made this possible: 1) video equipment became miniaturized, allowing easy attachment to rigid scopes, which produced highquality video images; 2) devices designed for use during laparoscopy 105

1 06

Chapter 6

and arthroscopy were adapted to the chest; and 3) single lung anesthesia was perfected, allowing undisturbed examination of the chest. The most striking improvement was the development of a thin in-line stapler (U.S. Surgical, Norwalk, CT) that could be used through a thoracoscopy port. Suddenly, it was possible to perform nearly any open thoracic procedure with the assistance of the video camera, a few instruments borrowed from many disciplines, and an endoscopic stapler. Most thoracic operations have now been tried with varying degrees of success. A few years have passed since the thoracoscopy counterrevolution. Time now allows for a more somber survey of the technique and its potential application to the diagnosis and management of lung cancer.

DIAGNOSIS Clinicians often guess at the diagnosis of newly discovered pulmonary nodules. Many textbooks of pulmonary medicine and thoracic surgery discuss radiologic factors that might lead one to strongly suspect the possibility of malignancy (8, 9). Several investigators have attempted to statistically determine the nature of pulmonary nodules based on age, gender, smoking history, and size of the lesion (10, 11). Using these criteria, along with a thorough history and physical, one has a reasonable chance at successfully divining the histopathology of the lesion in any specific instance. However, actual biopsy of the nodule and pathologic confirmation remain the only reliable methods of diagnosis. The best method of biopsy remains debatable. For central lesions or for patients with hemoptysis, bronchoscopy with visualization and biopsy is the easiest, safest, and. most reliable method of diagnosis. Peripheral lesions suspicious for cancer in patients with good pulmonary function may be removed at open thoracotomy by lobectomy or first, by wedge resection followed by lobectomy. This operation would be the most accepted procedure and is considered standard of care for early (Tl or T2) cancers. Lesions that are clearly benign by radiologic criteria, such as hamartomas or calcified granulomas in patients with appropriate history, need no biopsy, surgical or otherwise. However, for indeterminate nodules, video-assisted thoracic surgery has revolutionized management. A lesion as large as several centimeters may be removed using an endoscopic stapler and a small non-ribspreading incision (Fig. 6.1). A variety of series have shown the efficacy of such an approach. The most extensive survey to date was performed by the Video-Assisted Thoracic Surgery Study Group (12), which reported that during 1992, 865 nodules were resected out of a total of 1820 video-assisted thoracic surgical procedures. Cancer was discovered in 62% with minimal morbidity.

Role of Thoracoscopy in the Diagnosis and Treatment of Lung Cancer

107

Fig. 6.1. Solitary pulmonary nodule amenable to video-assisted technique. This J-cm nodule in the superior segment of the left lower lobe was easily removed via video-assisted wedge resection.

When considering video-assisted thoracic surgical procedures for indeterminate nodules, one must remember that additional equipment and operating time is necessary to perform the procedure. In the case of benign disease, the patient may be spared a rib-spreading thoracotomy, and the associated pain and morbidity. In the case of malignant disease, it adds time and expense to a planned lobectomy. However, in the situation of a patient with marginal pulmonary function where wedge resection may be considered the procedure of choice, morbidity, pain, and length-of-stay are markedly reduced. Thus, video-assisted thoracic surgery for nodule resection should be applied rationally. Table 6.1 offers such an approach. In the case of a Table 6.1. Application of video-assisted thoracic surgery for diagnosis of solitary pulmonary nodules

1. Preliminary data* are indeterminate. 2. Wedge resection is the primary planned therapy (i.e., for patients with poor pulmonary function) 3. Diagnosis of metastatic disease 4. High likelihood of benignancy *History, physical, review of old roentgenograms, and high-resolution CT.

1 08

Chapter 6

truly indeterminate nodule, a patient should be prepared for a thoracotomy and a lobectomy if the lesion is malignant. The procedures are best performed under the same anesthetic to minimize morbidity. Wedge resection may offer significant palliation in patients with poor pulmonary reserve as reported by Miller and Hatcher (13). When resection of a benign lesion is indicated, video-assisted thoracic surgery offers a significant advantage. In patients with probable metastatic disease from another site, a wedge resection may be diagnostic as well as therapeutic and easily performed by the video-assisted technique. Lobectomy by video-assisted thoracic surgery will be discussed separately (see under Treatment). Not all indeterminate nodules are best approached by a videoassisted technique (Table 6.2). It must be kept in mind that the procedure was conceived to decrease morbidity. Thus, the simplest procedure that will make the diagnosis and establish the cancer stage is best. In some patients, the most effective minimally invasive procedure may be bronchoscopy, mediastinoscopy, or, in special circumstances, radiologically guided transthoracic biopsy. Lesions that are deep in the parenchyma or centrally located in the hilus of the lung usually are not amenable to a video-assisted approach and carry a much higher complication rate (Fig. 6.2). Lesions that require rib-spreading for removal, such as those greater than 3 cm in diameter, defeat the purpose of the video-assisted procedure and thus should be avoided.

STAGING Nodal status as well as tumor size and location are important factors in staging of primary lung cancer. The purpose of preoperative staging is prognostication and consideration of initiation of preresection therapy or enrollment into anyone of a variety of national and international experimental neoadjuvant trials. In the latter instance, these trials are designed to answer such questions as which therapy or combination of therapies are the best modalities, and what is the role of surgery and/ or radiation in extensive local regional disease. A wide variety of techniques are available to determine tumor status (Table 6.3). Radiologic studies and bronchoscopic evaluation help to Table 6.2. Contraindications to video-assisted thoracic surgery wedge resection for diagnosis

Diagnosis and/or staging made by easier methods (e.g., bronchoscopy, mediastinoscopy, etc.) Central lesions deep within the lung substance Lesions larger than 3 em or requiring rib-spreading to remove


109

Fig. 6.2. Lesion not amenable to video-assisted wedge resection. This adenocarcinoma of the right lower lobe is centrally located directly over the inferior pulmonary vein. This lesion required lobectomy for removal. At the time of surgery, the lesion measured 4 x 5 cm and rib-spreading was required to remove the lobe and the tumor.

Table 6.3.

Minimally invasive approach to pretherapy evaluation of advanced local-regional disease*

Tumor status

Approach

CT MRI Bronchoscopy Thoracoscopy Echocardiogram EUS

Pleural metastasis

Endo bronchial

Chest wall

Vertebral body

Heart

Blood vessel

Esophagus

2° 3°

2°

2° 1°

2° 1°

3° 2°

3° 2°

3° 2°

3°

3°

4° 1°

4° 1°

4°

1° 1°

4°

1°

CT = computed tomography; EUS = esophageal ultrasound; MRI = magnetic resonance imaging. *Diagnostic modalities: 1° = primary; 2° = secondary; 3° = tertiary; 4° = quarternary.

define the tumor status for the size of airway obstruction, and the presence of lesions within 2 cm of the main carina. However, these studies fall short in defining chest wall invasion or invasion of mediastinal structures. Video-assisted techniques may playa role in the evaluation of such situations. Direct assessment of chest wall invasion is easily done by thoracoscopy. Nearly every reported surgical series on videoassisted thoracic surgery notes the ease of lysis of adhesions along the

110

Chapter 6

pleura of the chest wall and spine. The magnification provided by the scope and camera permits evaluation of invasion with minimal disturbance to the main tumor mass. In addition, once the thoracoscope is placed, washings of the pleural space are obtained easily for cytology in assessing the unusual situation of stage IV disease by virtue of occult pleural metastasis. Assessment of mediastinal invasion is more problematic. Most of the important structures of the mediastinum are blood containing such as the vena cava, the heart, and the aorta. The minimalist nature of the thoracoscopic approach permits only a cursory evaluation of invasion of these structures. More detailed evaluation without the ability to adequately control significant hemorrhage invites disaster. A better tool for assessment of such invasion may be the transesophageal echo. A great deal of experience has been gained by cardiologists, gastroenterologists, and surgeons from its use in the outpatient setting and in the cardiac surgical operating room. Although not 100% accurate, it is certainly a much less invasive assessment with minimal chance for morbidity. No large series exists to date for this technique because of the rarity of such T4 lesions. Evaluation of nodal status is very easily accomplished by mediastinoscopy for most levels. Table 6.4 presents the variety of techniques that are available for evaluation of nodal status. Both N2 and N3 nodes can and should be sampled by mediastinoscopy, which has become an outpatient procedure. With few exceptions, nodes in stations 1, 2, 3, 4, 5,6, and the anterior portion of 7 can be evaluated by using a combination of a supraclavicular incision and standard mediastinoscopy, mediastinotomy, or the extended mediastinoscopy as described by Ginsberg (14). Nodes easily evaluated by a video-assisted technique include stations 5, 6, the posterior portion of 7, and 8 and 9. The scope also can be used to assess some of the Nl nodes in groups 10 and 11 when nonanatomic resections are planned for patients with limited pulmonary function.

Table 6.4.

Minimally invasive approach to pretherapy evaluation of advanced local-regional disease Nodal stations

Approach

Supraclavicular Bx Mediastinoscopy Extended mediastinoscopy Thoracoscopy EUSw/Bx*

2

3

4

X

X X X

X

5

6

X X X

X X

A7

P7

8

9

10

11

X X

X X

X X

X

X

X

X

X X

X

x = acceptable biopsy method(s); A7 = anterior seven; P7 = posterior seven; Bx = biopsy; EUS = esophageal ultrasound. *Preliminary report only.


111

Assessment of nodes in groups 8 and 9 for lower-lobe lesions, with other N2 or N3 nodes negative, may shed light on a situation that previously could not be defined before resection. It might be possible to offer such patients neoadjuvant therapy to determine if their prognosis will be the same or better than other patients with mediastinal N2 disease. A recent report by Silvestri et al. (15) may make moot video-assisted evaluation of nodes in stations 5, 8, 9, and the posterior portion of 7. These investigators used a specially designed esophageal ultrasound probe with a biopsy needle to sample these nodes. This technique, when perfected, will offer an even less invasive method of evaluation of these nodal stations.

TREATMENT The development of endoscopic staplers and video-assisted technique raises the possibility of wedge resection as the primary therapy for lung cancer. This method has many attractive aspects, including parenchyma-sparing and minimal invasion of the chest. Arguments for its use have been advanced by several investigators. Miller and Hatcher (13) noted that patients with poor pulmonary function could tolerate wedge resections with a 35% 5-year survival rate. Before the new era of video-assisted thoracic surgery, Cooper et al. (16) proposed wedge resections with cautery and LoCicero (17) proposed resection with a laser. These techniques allowed for deep lesion removal without major pulmonary resection or anatomic distortion of the remaining lung caused by staple resections. Lewis (18), in advocating wedge resections for primary lung cancer, noted the arguments originally proposed against lobectomy instead of pneumonectomy for lung cancer. He pointed out that, ultimately, lobectomy was proven to be an ideal therapy for appropriate primary lung cancers. Although many surgeons were initially detractors of this technique, they later admitted the usefulness of lobectomy. In another report, Warren and Faber (19) showed that, for appropriate lesions located in a given segment of the lung, anatomic segmentectomy was equal to lobectomy in terms of survival. However, the Lung Cancer Study Group performed a randomized trial of various types of resection versus survival that was reported by Ginsberg (20). In this study, patients with Tl NO lesions were randomized to wedge resection or segmentectomy in one arm of the study and lobectomy in the other. This study showed that patients with a lobectomy had a 0.11% chance per year of recurrence versus a 0.22% chance per year for segmentectomy (not significant), and a 0.44% chance per year for wedge resection (P < 0.05). Neither pulmonary function performed at 6 months after surgery nor long-term (3 years) survival was noted to

11 2

Chapter 6

be different between groups. Thus, lobectomy remains the standard of care for primary lung cancer in any patient who can tolerate a lobectomy. Although a full discussion of the minimum pulmonary function criteria for lobectomy is beyond the scope of this chapter, it is important to note that the absolute floor has yet to be determined. For patients who do not meet the standard criteria for lobectomy, a phase II single-arm trial is under way in the Cancer and Leukemia Group B. Patients will receive a wedge resection followed by localized radiation. Data from this trial will probably not be available for at least another 3 years. Video-assisted minimally invasive lobectomy is presented as a potentially useful technique for appropriate patients. The major advantages of this approach are in relation to the minimally invasive technique itself. Smaller incisions without rib-spreading should produce less postoperative pain, less morbidity, better mobility, shorter length-of-stay, and an earlier return to normal activity or work for most patients. Some of these facts are beginning to surface in reports comparing the technique to historical controls. The only randomized limited center trial was performed by Kirby et al. (21), showing no difference between video-assisted lobectomy and standard lobectomy. Video-assisted lobectomy continues to be performed at several centers. No one technique of video-assisted lobectomy has emerged as the standard approach. Landrenau et al. (22) as well as Yim et al. (23) report series with minimal invasion and heavy reliance on endoscopic instruments. Lewis et al. (24) use a different approach with conventional staplers and a large non-rib-spreading incision assisted by the video thoracoscope. This group performs simultaneous stapling of hilar structures without an effort to individually ligate the vein, artery, or bronchus (24). Regardless of technique for video-assisted thoracic lobectomy, the major surgical principles must remain the same. An entire anatomic lobe completely containing the tumor must be removed. All of the lobar, interlobar, and mediastinal nodes should be removed, along with the specimen, to allow appropriate staging. The rib and skin incisions must be large enough to permit removal of the lobe without morcellation in order to allow anatomic pathological evaluation of the tumor and lung for appropriate staging.

TUMOR IMPLANTATION Recently another concern has arisen for all video-assisted thoracic procedures performed for cancer, namely, chest wall implantation with tumor. Fry et al. (25) reported this devastating complication that eventually resulted in a patient's death. Subsequently, Downey et al. (26), in association with the Video-Assisted Thoracic Surgery Study Group,


I 13

reported 21 cases of implantation from a variety of tumors, including primary lung cancer, metastatic disease, and mesothelioma. Although a great deal of discussion, both pro and con, has arisen from this report, it remains a serious concern. Surgeons must ensure that the chest wall is protected, using any method available, to prevent cancer implantation during extraction of specimens. Endoscopic bags, surgical gloves, and other devices have been used. Larger incisions may be necessary for larger lesions. Above all, the surgeon must not squeeze the tissue through the skin and subcutaneous tissue or through a port. A survey should be done of open cases to determine the magnitude of the problem compared to the standard approach. Such an evaluation has not yet been done.

REFERENCES 1. Webb WR. Thoracoscopy. Chest 1992;2:679-689. 2. Jacobaeus HC Uber die Moglichkeit der Zystoskopischen unter suchung Serosser hohlunger enzuwenden. Miinchen Med Wochenscher 1910;40: 2090. 3. Coba F. Atlas Thoracoscopicon. Heidelberg: Mailand, Springer, and Kupfer, 1928. 4. Kaiser LR. Diagnostic and therapeutic uses of pleurosocopy (thoracoscopy) in lung cancer. Surg Clin North Am 1987;67:1081-1086. 5. Rusch VW. Thorocascopy under regional anesthesia for diagnosis and management of pleural disease. Am J Surg 1987;154:274-278. 6. Miller JI, Hatcher CR. Thoracoscopy, a useful tool in the diagnosis of thoracic disease. Ann Thorac Surg 1978;26:68-72. 7. Boutin C, Viallat JR, Cargnino P, Farisse P. Thoracoscopy and malignant pleural effusions. Am Rev Respir Dis 1981;124:588-592. 8. Fishman AP, ed. Pulmonary Diseases and Disorders, 2nd ed. New York: McGraw-Hill,1988. 9. Shields TW, ed. General Thoracic Surgery. Baltimore: Williams & Wilkins, 1994. 10. Cummings SR, Lillington GA, Richard RJ. Managing solitary pulmonary nodules: the choice of strategy is a "close call." Am Rev Respir Dis 1986;184:453-460. 11. Edwards FH, Schaefer PS, Cohen AI, et al. Use of artificial intelligence for the preoperative diagnosis of pulmonary lesions. Ann Thorac Surg 1989 ;48:556-559. 12. Hazelrigg SR, Nunchuk SK, LoCicero I, Video-Assisted Thoracic Surgery Study Group. Video-assisted thoracic surgery study data. Ann Thorac Surg 1993;56:1039-1044. 13. Miller JI, Hatcher CR. Limited resection of bronchogenic carcinoma in patients with marked impairment of pulmonary function. Ann Thorac Surg 1987;44:340-343. 14. Ginsberg RI, Rice TW, Goldberg M, Waters PF, Schmocker BJ. Extended cervical mediastinoscopy: a single staging procedure for bronchogenic carcinoma of the left upper lobe. J Thorac Cardiovasc Surg 1987;94:673676.

11 4

Chapter 6 15. Silvestri GA, Hoffman BG, Bhutani MS, et al. Endoscopic ultrasound with fine needle aspiration in the diagnosis and staging of lung cancer. Ann Thorac Surg 1996;61:1441-1445. 16. Cooper JD, Perelman M, Todd TRJ, et al. Precision cautery excision of pulmonary lesions. Ann Thorac Surg 1986;41:51-53. 17. LoCicero J, Frederickson JW, Hartz RS, Michaelis LL. Laser assisted parenchyma sparing pulmonary resection. J Thorac Cardiovasc Surg 1989;97: 732-736. 18. Lewis RJ, Caccavale RJ, Sisler GE, MacKenzie JW. Video-assisted thoracic surgical resection of malignant tumors. J Thorac Cardiovasc Surg 1992;104:1679-1687. 19. Warren WH, Faber LP. Segmentectomy versus lobectomy in patients with stage I pulmonary carcinoma. Five-year survival and patterns of intrathoracic recurrence. J Thorac Cardiovasc Surg 1994;107:1087-1093. 20. Ginsberg RJ, Rubinstein LV. Randomized trial of lobectomy versus limited resection for T1 NO non-small cell lung cancer. Lung Cancer Study Group. Ann Thorac Surg 1995;60:615-622. 21. Kirby TJ, Mack MJ, Landrenau RJ, Rice TW. Lobectomy video-assisted thoracic surgery versus muscle sparing thoracotomy, a randomized trial. J Thorac Cardiovasc Surg 1995;109:997-1002. 22. Landrenau RJ, Mack MJ, Hazelrigg SR, et al. Video-Assisted Thoracic Surgery: A Minimally Invasive Approach to Thoracic Oncology. PPO Updates. Philadelphia: Lippincott, 1994. 23. Yim APC, Ko K, Chau W, et al. Video-assisted thoracoscopic anatomic lung resections: the initial Hong Kong experience. Chest 1996;109:13-17. 24. Lewis RJ, Sisler GE, Caccavale RJ. Imaged thoracic lobectomy: should it be done? Ann Thorac Surg 1992;54:80-83. 25. Fry WA, Siddikui A, Pensler JM, Mostofavi H. Thoracoscopic implantation of cancer with a fatal outcome. Ann Thorac Surg 1995;59:42-45. 26. Downey RJ, McCormack P, LoCicero J, Video-Assisted Thoracic Surgery Study Group. Dissemination of malignant tumors after video-assisted thoracic surgery: a report of 21 cases. J Thorac Cardiovasc Surg 1996;11: 954-960.

7 Surgery for Small-Cell Lung Cancer Thomas W. Shields and Karl Karrer

The role of surgical intervention in the multimodality management of small-cell lung cancer (SCLC) continues to be controversial. At most, only 5-8% of patients with the disease can be considered initially as potential surgical candidates. These are the patients who can be classified clinically as having stage I, II, or resectable stage IlIa disease, as defined by the International TNM system proposed in 1986 (1). This small group of patients comprises 15-25% of patients with limited disease. Actually, the number of potential patients is even smaller, if one excludes those patients with N2 disease.

SOURCES OF CONTROVERSY Many factors contribute to the continuing controversy and, at times, seem to be unresolvable. Some of the controversial aspects are the inability to establish the patient base with which to compare medical therapy alone versus the addition of surgical resection; the inaccuracy of clinical staging of the potential surgical candidates (2, 3); the histologic subtypes and heterogeneity of SCLC (intermediate vs. the lymphocytic [oat]-cell types [4-6]); the presence of a varying percentage of non-smaIl-cell tumor cell types in association with SCLC (7-9); and the occasional difficulty in differentiating a neuroendocrine typical or atypical carcinoid tumor from the more malignant neuroendocrine intermediate or small-cell tumor. The attempts to convert marginally resectable 115

11 6

Chapter 7

and nonresectable lesions into resectable ones by the use of neoadjuvant (initial) chemotherapy (10, 11) only further confuse the issue as to the proper role of surgical resection in this disease. Lastly, the lack of sufficient numbers of potential surgical candidates has prevented the conduct of satisfactory randomized clinical trials of medical therapy alone versus the addition of surgical resection to a satisfactory pre- or postoperative medical regimen to resolve the question. This latter circumstance has been the major stumbling block for many in the acceptance of any role of surgical resection in the management of SCLC. However, a review of some of these controversial factors may be helpful in placing the surgical data that are now available into a proper perspective.

Chemotherapy Alone in Potential Surgical Patients Current multimodality therapy with multiple drug regimens plus concurrent radiation therapy has resulted in 5-year survival rates as high as 20-40% in all SCLC patients with limited-stage disease (12, 13). In most of the studies, the patient population has not been broken down into TNM subsets. However, in one report from the Toronto Lung Oncology Group (14), a 25% 5-year survival rate was projected in 33 patients with very limited SCLC (clinical stages I and II, exclusive of patients with a peripheral nodule) who were treated by chemotherapy alone. In another report from the same group, in patients whose disease was more advanced but still localized and who were thought to be surgical candidates after receiving initial chemotherapy, but who did not undergo resection, a 5-year survival rate of only 13% was recorded (15). In a more recent report, these authors divided a group of limited disease patients into three clinical subgroups: 1) those with a radiologic negative (uninvolved) mediastinum, 2) those with a positive (involved mediastinum), and 3) those with supraclavicular involvement, associated pleural effusion, pneumonic consolidation, or atelectasis. With the use of an adequate chemotherapeutic regimen, they were only able to project as-year survival of 18% in group 1,6% in group 2, and 2% in group 3 (16). Similar results were reported from Hara et al. in Japan (17, 18). These authors reported a 25% long-term survival in patients with stage I or stage II disease and a 5.7% long-term survival in patients with stage IlIa disease who were treated with chemoradiation therapy alone. However, in the few patients with stage IlIa disease who had a complete remission following the medical regimen, the survival rate was increased to 15.4%.

Inaccuracy of Clinical Staging Despite the use of CT examination and, frequently, routine prethoracotomy mediastinal exploration in patients undergoing initial surgical

Surgery for Small-Cell Lung Cancer

I I 7

resection for SCLC, an error in the TNM clinical stage, as compared to the final TNM pathologic stage, is observed in one-third to two-thirds of patients (2,3). The error is most often clinical understaging; the opposite occurs only infrequently (3). Unsuspected N2 disease is encountered in almost 30% of resected patients. Of additional interest is that the incidence of error is observed more frequently in those patients with centrally located tumors, in whom the diagnosis of SCLC is not established preoperatively by the standard diagnostic procedures.

Histologic Subtypes and the Heterogeneity of SCLC It has been suggested that more favorable results of surgical resection have occurred in those patients with intermediate small-cell type histology (neuroendocrine carcinoma, intermediate cell type) (19) than in those with the lymphocytic (oat)-cell type (neuroendocrine carcinoma, small-cell type) (4-6), although the difference has not been reported to be statistically significant (6) (Fig. 7.1). Others have not accepted that the cell type occasions any difference in survival rate (20-22). The presence of non-smaIl-cell tumor cell types that theoretically are "chemoresistant" may unfavorably affect the results of chemotherapy alone in limited SCLC tumors. The incidence of such foci of non-smallcell tumor cells has been reported to be between 5 and 15% initially (9), 100 90 80 ~ ~

70

~ 60

.~

:J rI)

Q)

50

Cl

19 c 40 Q)

~ a.. 30 20 10 0

3

6

9

12 15 18 21 24

30

36

48

60

Months after operation

Fig. 7.1. SUNival of patients with resected small-cell carcinoma according to cell type. Difference in survival of intermediate Ie) and oat-cell (6.) types is not statistically significant. Oat-cell type, n = 49; intermediate-cell type, n = 110; intermediate vs. oat-cell type, P = 0.06432. (Reproduced by permission from Ohta M, Hara N, Nakada T. et al. Survival of patients with resected small cell carcinoma of the lung. Lung Cancer 1982;22:459-466 (in Japanese, English abstract).)

118

Chapter 7

and to be increased to as high as 25-35% after neoadjuvant chemotherapy (8, 15). However, with resection, the adverse effect of this tumor heterogeneity becomes nonoperative, and the clinical course of the disease is primarily correlated with the pathologic/postsurgical TNM (pTNM) stage of the disease (3, 15,23).

Differentiation of Typical and Atypical Carcinoids from the More Malignant (High-Grade) Neuroendocrine Tumors Many have maintained that typical and atypical carcinoids frequently have been mistakenly identified as small-cell tumors and that this accounts for some of the long-term survivors included in the surgically resected series of limited small-cell disease. This criticism has generally been countered by having the histopathology reviewed by referee pathologists, but, unfortunately, at times the differentiation of the various pulmonary neuroendocrine neoplasms cannot be absolute. The use of immunohistochemistry with specific monoclonal antibodies to more completely differentiate the various neuroendocrine tumors may put this particular issue to rest (24).

Use of Neoadjuvant Therapy to Convert Nonresectable Tumors into Resectable Disease The use of initial chemotherapy to convert borderline or frankly nonresectable localized SCLC into resectable tumors has tended only to obscure the role of surgical resection in SCLC. A basic premise in the selection of a patient for possible surgical resection should be that the limited disease is of such an extent that it is resectable by the standard criteria applied to non-small-cell tumors-that is, the disease can be reasonably classified as stage I, II, or resectable IlIa disease. Whether N2 disease is considered resectable remains unsettled in both SCLC and non-small-celliung cancer (NSCLC). The poor results obtained when this premise is not adhered to are well demonstrated by several pilot studies (10, 11), and by the prospective randomized trial conducted by the North American Lung Cancer Study Group (25,26).

Absence of Prospective Randomized Studies The small number of patients with potentially resectable SCLC, the inability to properly stage the patients clinically in one- to two-thirds of potential surgical candidates, and the observation that, in approxi-


I I9

mately one-third of patients who have the disease resected, the diagnosis is not made until thoracotomy, makes the likelihood of ever conducting a satisfactory randomized study a near impossibility. Therefore, the role of surgery can only be surmised from the interpretation of the data in the literature. Moreover, the total population base from which the surgical patients is derived is impossible to determine in most studies. Any comparison of survival with medically treated patients remains historical and is crude at best. The surgical series reported in the past 15 years (1982-1996) can conveniently be divided into four categories: 1. Resection alone, either without or with only inadequate systemic chemotherapy 2. Initial resection with adequate postoperative chemotherapy 3. Preoperative neoadjuvant chemotherapy followed by resection in the responders 4. Salvage resection after failure to respond to induction chemotherapy or relapse after initial response. The numbers of patients in these surgical series vary greatly, are generally small, and criteria for selection are far from being standardized. Comparisons among the various trials are difficult, and any conclusions must be tentative in nature.

Surgical Excision Only One of the major reviews of surgical resection in SCLC was from the Veterans Administration Surgical Oncology Group (VASOG) (27). In this report, the results of resection of patients with small-cell cancer entered into the Group's randomized prospective adjuvant chemotherapy trials were retrospectively analyzed. Of 148 patients, 132 survived for 30 days postoperatively and were available for follow-up study. In these 132 patients, an overall 5-year actuarial survival rate of 23% was recorded. A survival rate of 59.5% was observed in 26 patients, who were staged as having postsurgical Tl NO MO disease. Survival rates in the other surgical stages were lower (Fig. 7.2). Only one patient with N2 disease was observed among the long-term survivors. Most patients were salvaged by surgical resection of their SCLC, either without or with only the use of ineffective postoperative chemotherapy in their management. Earlier, Hayata et al. (28) reported similar results following the resection of peripheral lesions of the intermediate small-cell type of tumor. They reported an overa1l5-year survival rate of 28.1 % in those patients who had undergone complete resection of their local disease. Shore and Paneth (29) reported an overall 5-year survival rate of 25% after resection in 40 patients with SCLC. Hilar lymph node involvement was pres-

120

Chapter 7

100

- - T1 NO MO -T1NOMO ---- T2NO MO ---- T2N1 MO .......... T3 or N2

n == 26 n==16 n == 23 n == 39 n == 28

80

60

40

........ -- ....... 20

--.---.

°0~------~------~2--------~3--------~4------~

Follow-up year

Fig. 7.2. SUNival, computed by the life-table method, from postoperative day 30 (early trials) or from randomization (recent trials) by TNM classification for patients with undifferentiated small-cell carcinoma who had undergone a "curative" resection in the Veterans Administration Surgical Oncology Group lung trials. (Reproduced by permission from Shields TV!, Higgins GA Jr, Matthews MJ, Keehn RJ. Surgical resection in the management of small cell carcinoma of the lung. J Thorac Cardiovasc Surg 1982;84:481-488.)

ent in a majority of the resected patients, as well as in the long-term survivors. In the United Kingdom there continues to be support for resection only in patients with stage I or II disease. Prasad et al. (30) reported 5year survival rates of 35% and 23%, respectively, in these stage groups. In contrast, in the report by Sorensen et al. (31), only an 8.4% long-term survival rate was noted in 71 patients managed in a similar manner. However, Shah et al. (32) reported an overallS-year survival in 28 SCLC patients of 43.3% in 1992. There were 14 stage I, 5 stage II, and 11 stage III (10 were pT3 NO and 1 pT3 Nl) patients. The survival rates in each stage were 57.1 %,0%, and 55.5%, respectively. Smit et al. (33) reported similar results: 50% 5-year survival in stage I and II patients, and 20% 5year survival in stage III patients (these results are from a total of 20


121

patients, 16 of whom had complete resections). The explanation for the salutary results in these series remains elusive.

Initial Surgical Resection Followed by Chemotherapy In one of the YASOG trials, in which postoperative intermittent lomustine (CCNU) and hydroxyurea were used, the small number of patients who received this combination chemotherapy postoperatively had an improved long-term survival rate over those who did not (80.8% vs. 38.1 %, respectively). Meyer (34) subsequently reported an 80% and a 50% 5-year survival rate in a small number of highly selected SCLC patients with stage I and stage II disease, following resection and adequate standard postoperative chemotherapy. In Japan, Ohta et al. (35) reported a 50.8% 5-year survival rate in patients with stage I disease who were managed by initial surgical resection and followed by intensive postoperative chemotherapy. No longterm survivors with stage II disease were recorded, but there was a 14.8% survival rate for those with stage III disease; no patient with N2 disease survived. Other similar studies in the late 1980s, usually with small numbers of patients, have been reported with varying results, but all have shown the probable efficacy of postoperative chemotherapy after resection of stage I or II SCLC over surgical resection alone (36-38). Two of the early major studies relative to initial resection followed by chemotherapy are those of the University of Toronto Lung Oncology Group (TLOG) (2) and the International Society of Chemotherapy Lung Cancer Study Group (lSC) (3,39,40). Of note is that, in both studies, the diagnosis of SCLC was not established until thoracotomy or examination of the resected specimen in one-third to one-half of patients. Likewise, in both these studies, clinical understaging of the extent of the disease was common. In the TLOG study, the most frequent drug combination used postoperatively was cyclophosphamide, doxorubicin, and vincristine. Postoperative regional thoracic irradiation and prophylactic cranial irradiation (PCI) were used in many of the patients. In ISC I (preoperative diagnosis of SCLC known) and II studies (diagnosis of SCLC unknown until surgical resection), the patients were randomized to receive either eight courses of cyclophosphamide, doxorubicin, and vincristine (CAY), or two courses of three sequential drug combinations: cyclophosphamide, lomustine, and methotrexate; CAY; and ifosfamide and etoposide (YP-16). Local thoracic irradiation was given infrequently, and PCI was given to all patients free of disease at the completion of the postoperative chemotherapy. In the TLOG study, 63 patients underwent initial resection, and in 22 of these patients the diagnosis was not established until the examination of the surgical specimen. The tumors were pure small-cell lung can-

122

Chapter 7 Table 7. 1 . Postresection stage of initially resected small-cell lung cancer in 63 patients

Postresection stage

No. of patients

TINO T2NO Tl Nl T2Nl Tl N2 T2N2 T3NO T3Nl T3N2

8 10 6 18

5 9 3 1 3

Reproduced by permission from Shepherd FA, Evans WK, Feld R, et al. Adjuvant chemotherapy following resection for small-cell carcinoma of the lung. J Clin Oncol 1988;6:832838.

cer in 54, and a mixed small-cell and non-smaIl-cell cancer in nine. Six of the 63 patients had microscopic residual disease. The postoperative staging of these tumors is seen in Table 7.1. Of note is that the preoperative and postoperative stages were only the same for 35% of the patients, and unsuspected mediastinal node involvement was identified in 17 patients (27%). The projected 5-year survival rate for the entire group was 31 %; the survival rate for stage I was 48%; for stage II, 24.5%; and for stage IlIa, 24% (Fig. 7.3). In the ISC studies, 182 patients had been placed on study. The postoperative TNM staging showed more advanced disease than the preoperative clinical TNM staging in more than 25% of the patients and less advanced disease in 10%. The cell types of the tumors were oat-cell type in 34%, intermediate type in 57%, and SCLC combined with adenocarcinoma or squamous cell carcinoma in the remaining 9%. The resection was determined to be complete in 150 patients and incomplete in 32 (Table 7.2). The actuarial 4-year survival rate was 43% for the entire group, but was 47% and 23%, respectively, for the complete and incomplete resection groups (Table 7.3). The survival rates for the various pTNM stages are shown in Figure 7.4. In stage IlIa non-N2 disease, the projected 4-year survival rate was 71 %, and in stage IlIa N2 disease, it was 33%. As of May 1996, the actuarial survival curve for the patients with NO disease up to 80 months showed a 61 % survival rate. Of interest, the projected survival of patients with N1 and N2 disease is the same and is approximately that of the survival obtained by chemoradiation therapy alone. In the entire group of patients, 75% of the treatment failures occurred within the first 30 months due to progression of the disease. In 21 %, the cause of death was not directly tumor-related, and in 4% the cause was unknown.


123

CHAPTER 10

100

Stage 1 (19 cases) Stage 2 (24 cases) Stage 3 (20 cases)

,'' '

y=--,

80

:--1 L, , '--l

I'

I

I

L,

60

-----, '

L_____

~

I

L_

L,

, --------

L, I

'------------1

40

,

L,

L ____________ , IL __________ _

20

o o

2

4

3

5

Fig. 7.3. Survival by stage for patients treated with adjuvant chemotherapy after surgical resection for SCLC. Stage I vs. stage II, P= 0.134; stage I vs. stage III, P= 0.038. (Reproduced by permission from Shepherd FA, Evans WK, Feld R, et al. Adjuvant chemotherapy following resection for small-cell carcinoma of the lung. J Clin Oncol 1988;6:832-838.)

Table 7.2.

Complete and incomplete resections in ISC studies

Complete curative resection Incomplete resection Unknown*

NO

Nl

N2

Total

67 4

57 4

26

4

4

150 17 15

9 7

ISC = International Society of Chemotherapy. *Considered to be incomplete resections.

In both the ISC and TLOG studies, the results of resection of Nl disease were not dissimilar to those of N2 disease. The futility of an incomplete resection in stages II and IlIa N2 disease is evident from the ISC study, since this results in survival rates that are similar to those obtained when treating all limited-disease patients with chemoradiation therapy alone. In addition to the two aforementioned series, a number of other studies reporting satisfactory long-term survival in small numbers of patients undergoing surgical resection followed by chemotherapy have

124

Chapter 7

Table 7.3.

Actuarial 48-month survival after resection and postoperative adjuvant chemotherapy in the lSC lung cancer studies I and II

Complete resections

p stage I P stage II P stage III Non-N2 disease N2 disease All patients

Incomplete resections*

No. of patients

Survival (%J

No. of patients

Survival (%J

65 51 34 8 26 150

60 36

8 6 18 2 16 32

52 17

71 33 47

15 23

ISC = International Society of Chemotherapy; p = pathologic. *Includes 19 patients (10.4%) with known pathologic gross or microscopic residual disease, and 13 patients (8%) in whom the pathologic status of the resection was not recorded.

100 90 80

as> .~

:::J

en ~

0

- - pTNO - 69 Patients ---- pTN1 - 58 Patients -.. _..-.. - pTN2 - 36 Patients

70 60 50 40

31

30 20 10 0

17

ISC-Studies 1111 Per May 1996

0

10

20

30

40

50

60

70

80

Months After Surgery Fig. 7.4. Actuarial survival rate and numbers of patients at risk after surgery-forcure, followed by chemotherapy and prophylactic cranial irradiation for SCLC (pTNM: I, II, lIla) according to the N status. Six of the 163 patients excluded: 2 N2 M 1,2 T4 N2, and 2 T4 N 1.

appeared in the literature (17, 41-45). The major studies are recorded in Table 7.4. In the series by Macchiarini et al. (41), the long-term survival rates for patients with pT1 NO, pT2 NO, and pT3 NO disease were 65%, 43%, and 13%, respectively. From these data, it appears that complete resection of stage I disease followed by chemotherapy is quite satisfactory, whereas similar management of stage II and particularly stage IlIa N2 disease does not yield overly encouraging results.


125

Table 7.4. Projected 5-year sUNival of patients treated by initial surgical resection followed by chemotherapy with or without PCI Author (ref), yr

Stage I

Stage II

Stage III

Total

Hara et al. (17), 1991 Macchiarini et al. (41), 1991 Ulsperger et al. (42), 1991 a Kaiser et al. (43), 1992b Muller et al. (44), 1992 c Davis et al. (45), 1993

64 52 63 67 57 50

42

10.7 13 29

36 141

34 21

36

49 42 28 35

il4-year survival. b3-year survival. CS tage III contained no N2 disease.

Chemotherapy Followed by Adjuvant Surgical Resection The initial reports of preoperative (neoadjuvant) chemotherapy followed by resection in the responders suggested favorable results in small numbers of patients (46-49). However, the prospective studies reported by Prager et al. (10) and Meyer et al. (11) were disappointing. Only one-third to one-half of the patients entered into these studies were ultimately considered to be surgical candidates, and fewer underwent surgical resection. The survival rates were poor, and no patients with documented N2 disease had long-term survival. Improved selection of patients resulted in more favorable results, especially in patients with postsurgical stage I disease, although no differences could be identified in the three clinical stage groups (9, 15,49). In the TLOG neoadjuvant reports (15), 72 patients were identified prospectively as potential candidates for resection (excluding patients with peripheral lesions). All underwent intensive initial chemotherapy. The overall response rate to chemotherapy was 80% (complete response was 38%; partial response was 42%). After completion of the induction chemotherapy, 57 patients were believed to be candidates for resection, but for various reasons (protocol randomization and patient's refusal), only 38 patients underwent the operation. The postoperative pathologic stages and actuarial survival rates are shown in Table 7.5. The overall projected 5-year surgical rate was 36%. In an attempt to assess the contribution of resection, the survival of the resected patients was compared to that of patients who were eligible for operation but did not undergo it (Fig. 7.5). A significantly better survival rate (P = 0.049) of the surgical group was noted. Another observation in the Toronto study was the confirmation of the poor survival rate after the resection of persistent N2 disease. A reduction in local recurrence was, however, noted in this subset of patients. The Innsbruck Group reported the results of resection in 24 patients with stage IlIa N2 SCLC (50). An overall 24% projected 5-year survival

126

Chapter 7

Table 7.5.

Postoperative stage and long-term sUNival after initial chemotherapy and subsequent surgical resection No. of patients

Stage

I II

7 9

III

22

Survival rate (%J

71 38 18

Reproduced by permission from Shepherd FA, Ginsberg RJ, Patterson GA, Evans WK, Feld R, the University of Toronto Lung Oncology Group. A prospective study of adjuvant surgical resection after chemotherapy for limited small cell lung cancer. A University of Toronto Lung Oncology Group study. J Thorac Cardiovasc Surg 1989;97:177-186.

110

,-,

100 ~--.-.,. 90 (ij

>

.~.

~

(/J

o

~

J5 ~

e

0..

80

L.

L,

Surgery (n = 38) > p:: 0.049 No surgery (n:: 19) - - - -

~ I ..

.. I

.,I I

I

L,

l,

L____, I I

-,I

IL_________________________ _

2

4

Time in years Fig. 7.5. Comparison of sUNival in 38 patients receiving adjuvant surgical therapy, and 19 eligible patients who did not receive adjuvant surgical therapy for SCLC. (Reproduced by permission From Shepherd FA. Ginsberg RJ. Patterson GA. Evans WK. Feld R. the University of Toronto Lung Oncology Group. A prospective study of adjuvant surgical resection after chemotherapy for limited small cell lung cancer. A University of Toronto Lung Oncology Group study. J Thorac Cardiovasc Surg 1989;97: 177-186.)

rate was recorded; but, more importantly, in 13 patients who completed the comprehensive therapy protocol, there was a projected survival rate of 52%. This comprehensive multimodality approach consists of an initial cycle of chemotherapy (CAV plus cisplatin and etoposide), followed by radical excision when there is at least a 50% partial remission. The surgical resection is followed by a second cycle of chemotherapy; a split


127

course of local thoracic and supraclavicular irradiation; a third cycle of chemotherapy; and a second split course of local irradiation and simultaneous PCI. Although some have questioned the efficacy of the approach (51, 52), these encouraging results suggest that additional studies should be carried out in selected patients with limited N2 disease. The prospective randomized neoadjuvant trial conducted by the North American Lung Cancer Study Group (LCSG) has not confirmed the value of neoadjuvant chemotherapy followed by resection plus thoracic irradiation, compared to chemotherapy followed only with irradiation, in the management of limited SCLC (25, 26). In the LCSG study, 144 patients of 217 who had achieved an objective response following five courses of CAY chemotherapy were randomized to undergo either surgical resection of their disease followed by thoracic irradiation and PCI, or thoracic irradiation and PCI only. The other 73 patients were not believed to be candidates for resection because of the extent of the disease, the patient's general medical status, or their refusal of the surgical option. Sixty-eight patients were randomized to the surgical option and 76 to radiation therapy alone. Including the patients who underwent surgery off the study, 83% of the patients randomized to the resection group had the tumor resected (6.8% were incomplete resections). Analysis of the surgical specimens revealed persistent small-cell tumor in 73% of patients, no small-cell tumor in 27%, and non-smaIl-cell tumor in 11 %. In six patients, only non-smaIl-cell type tumor was present, and in two the non-smaIl-cell tumor was associated with persistent smallcell tumor. Survival was essentially the same at 24 months for both randomized groups (approximately 20%). Clinical and postsurgical stage did not identify any favorable group or groups of patients. Of interest was the observation that survival was least favorable in those patients with residual non-smaIl-cell tumor in the resected specimen. It was concluded that the addition of surgical resection was of no benefit in the management of localized SCLC over conventional treatment alone. It should be noted that only four patients in the surgical group were initially classified as having clinical stage I disease (5.7%). The number of patients with stage I disease in the radiation therapy alone group was not stated but may be assumed to be of the same approximate percentage. Thus, almost all patients in this study may be considered to have had more advanced initial disease, with either N1 or N2 involvement. In addition to this large randomized study, numerous reports of the use of neoadjuvant chemotherapy prior to surgical resection in smaller series of usually more highly selected patients have been reported. Some of these, such as the reports by Yamada et al. (53) and Zatopek et al. (54), revealed no benefit relative to long-term salvage by this approach. However, the report of Namikawa et al. (55) showed a 75% 2year survival in nine patients with stage I disease. Recently, Wada et al. (56) reported an 80% 5-year survival in stage I and stage II patients who received chemotherapy first in contrast to a 37.7% survival in those

1 28

Chapter 7

undergoing initial surgical resection; unfortunately, these data were not statistically significant. In stage III patients, the survival rates were 10% and 0%, respectively. These authors also noted that, again, the preoperative presence of N2 or N3 disease presaged a poor result.

Salvage Resection The most selective approach to the use of surgical therapy in SCLC patients is that of salvage resection in a few patients who have failed to respond or have relapsed after chemotherapy for limited disease. The TLOG (57) has reported 28 such operations. The indications were no response in five patients; partial response (residual ~3 cm) in 12; initial response followed by progression during chemotherapy in three; and relapse after complete response in eight. The clinical TNM (cTNM), postsurgical TNM (pTNM), and histopathology are shown in Table 7.6. The projected postsurgical resection 5-year survival rate was 23%. All patients with stage I disease were alive at the time of the report, com-

Table 7.6.

A comparison of preoperative clinical stage and postoperative pathologic stage and pathology

c Preoperative

Stage I Tl NO T2NO Stage II Tl Nl T2Nl Stage III Tl N2 T2N2 T3NO T3N1 T3N2 Pathology Small-cell only Small-cell plus adenocarcinoma Small-cell plus malignant carcinoid Small-cell, adenocarcinoma, squamous Adenocarcinoma only Squamous carcinoma only

p Postoperative

4

7

3 4

3 1 9

4 5

10 4 6

12 4 7 0 1 0

14 3 6 1 2 2

25 3

18 2 1 1 3 3

c = clinical; p = pathology. Reproduced by permission from Shepherd FA, Ginsberg R, Patterson GA, et al. Is there ever a role for salvage operation in limited small-cell lung cancer? J Thorac Cardiovasc Surg 1991;101:196-200.


129

pared with only one with stage II disease and two with stage III disease. Relapse or failure to respond to chemotherapy was thought to be due to the presence of an NSCLC or a mixed tumor in 10 of the 28 patients.

EVALUATION OF THE SURGICAL DATA A number of observations may be made from the aforementioned surgical reports. 1. Only a small number of SCLC patients are surgical candidates. These are the limited-disease patients, who can clinically be staged as having T1 NO MO or T2 NO MO (stage I) disease. Even with extensive staging, approximately one-fourth or more of patients will have more advanced disease (N1 or N2). The results of surgical excision with either pre- or postoperative chemotherapy (the policy of no chemotherapy in the ISC group is not recommended even in patients with pNO disease) are only slightly less than the results obtained in the resection of stage I NSCLC. The survival rate appears to be superior to that of all limited-disease patients treated with chemotherapy alone, with or without local irradiation. 2. Patients with clinical stage II (N1) disease are frequently upstaged to pathologic N2 (pN2) disease. The survival of pN1 disease patients is similar to that observed in patients with pN2 disease, regardless of the timing of the chemotherapy. The survival rate is approximately that of patients treated by chemotherapy (plus thoracic irradiation) alone. 3. Patients with stage IlIa disease are best separated into those without N1 or N2 disease, and those with N1 or N2 disease. Surgical resection may be considered in the favorable T3 lesions without lymph node involvement, as the survival rate in this small subgroup of patients is similar to the patients with stage I disease. The results of resection of N2 disease are poor and, as with Nl disease, are no better than those obtained by treatment with chemotherapy (with local irradiation) alone. 4. Postoperative chemotherapy is as satisfactory as neoadjuvant chemotherapy in patients with initially resectable stage I disease. Neoadjuvant therapy cannot convert marginal or nonsurgical lesions into potentially curative ones. Initial chemotherapy, regardless of the response of the tumor, cannot favorably influence the outcome of surgical resection in initially advanced stage II and stage III localized disease. This interpretation is supported by the Lcse and TLoe studies as well as the more recent studies recorded in the text (25,26,53,54). Its use confers no benefit in the management of patients with stage II or stage IlIa or I1Ib disease.

130

Chapter 7 5. Incomplete surgical resection is of no benefit in the multimodality treatment of SCLC patients. Resection should be abandoned if it appears that it would be incomplete. 6. Salvage surgical resection may be indicated in patients who fail to respond, or who have a recurrence after complete remission, when the disease can be clinically staged as stage I disease, because a number of such patients will have either an NSCLC or a mixed tumor, which is refractory to chemotherapy.

RECOMMENDED ROLE OF SURGICAL RESECTION IN SCLC Patients recommended for surgical management may be separated into two categories: those with a preoperative diagnosis of SCLC, and those in whom the diagnosis is not established until thoracotomy or later examination of the resected specimen. In either instance, a mandatory criterion is that the tumor be considered potentially completely resectable at thoracotomy.

Preoperative Diagnosis of SCLC In the patient with known SCLC, extrathoracic disease must be excluded by CT of the upper abdomen and brain and a radionuclide bone scan. The necessity of bone marrow biopsy has been questioned by several authors (58, 59), as the incidence of such involvement is minimal to absent in patients with very limited SCLC (stage I or II disease). The presence of an extra thoracic tumor (Ml disease) contraindicates any further surgical consideration. CT of the chest is done to detect any enlarged hilar or mediastinal nodes and the extent of any central disease. Regardless of the status of the mediastinal lymph nodes on CT examination, prethoracotomy mediastinal exploration is indicated for those patients who are to undergo initial surgical resection. The presence of N2 disease would exclude the patient from further surgical consideration, except under the conditions of an investigational trial, such as that being conducted by the Innsbruck Group (50). If it has been decided that neoadjuvant therapy is to be used prior to possible surgical resection, the mediastinal exploration should be withheld until completion of the chemotherapy. The presence of N2 disease at this time would contraindicate resection. Patients with peripheral stage I disease should undergo initial resection followed by chemotherapy. Those with clinical stage I disease in the central area may be managed by either the neoadjuvant approach or by initial surgery. The use of postoperative PCI, although almost routine in the past, is now questioned because of the low incidence of cerebral metastasis in


1 31

stage I patients (60) and its observed neurotoxicity. Further investigative trials, with lower total radiation dose and different fractionation, are indicated to settle this question. Patients with suspected stage II disease may not be candidates for surgical resection, except as investigational candidates. The value of surgical intervention in these patients is highly questionable. Patients with resectable stage IlIa non-N2 disease may be managed either by initial surgery or the neoadjuvant approach. Patients with stage IlIa N2 disease are not considered to be surgical candidates.

Diagnosis of SCLC Unknown Prior to Operation In patients with potentially resectable lung tumors (with the exception of those with a solitary peripheral nodule), in whom the tissue diagnosis remains unknown despite an adequate preoperative evaluation, CT of the chest and upper abdomen, as well as a mediastinal exploration, are indicated prior to thoracotomy. These are done in an attempt to obtain a tissue diagnosis, as well as to rule out any upper abdominal spread or N2 involvement. "Metastatic" evaluation of other asymptomatic organ systems for occult metastases also may be indicated in this subset of patients. When the diagnosis is established initially at thoracotomy, all stage I and stage II lesions should be resected, and appropriate mediastinal lymph node evaluation should be done. If the extent of the lesion is obviously a stage IlIa without N2 disease, resection is indicated only if a complete resection of all tumor can be done. Patients with N2 disease should undergo resection only if disease is minimal and a complete resection can be done. The resected patients should have a final pTNM classification. All patients should receive adequate intensive postoperative chemotherapy of six to eight cycles, depending on the selected chemotherapeutic regimen. The option of no chemotherapy in patients with stage I disease would appear to be inappropriate despite the results reported from England (32,33). Local thoracic irradiation should be considered for patients with Nl or occult N2 disease. PCI may be indicated in stage pIlla N2 disease, as there is a high relapse rate in the brain (>50 11'0) in these patients.

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Macchiarini P, Hardin M, Basolo F, et al. Surgery plus adjuvant chemotherapy for Tl-3 No small-cell lung cancer. Am J Clin OncoI1991;14:218-224. Ulsperger E, Karrer K, Denck H, ISC-Cancer Study Group. Multimodality treatment for small-cell bronchial carcinoma. Eur J Cardiothorac Surg 1991;5:306-310. Kaiser 0, Fritzshe A, Matthiessen W. Operation sindikation beim kleinzelligen bronchialkarzinom. Deutsche Midizen Wochenschrift 1992;117:103106. Muller LC, Salzer GM, Huber H, et al. Multimodal therapy of small cell lung cancer in TNM stages I through IlIa. Ann Thorac Surg 1992;54:493497. Davis S, Crino L, Tonato M, et al. A prospective analysis of chemotherapy following surgical resection of clinical stage I-II small-cell lung cancer. Am J Clin Oncol 1993;16:93-95. Hara N, Ohta M, Ichinose Y. Surgical adjuvant chemotherapy for lung cancer. Proc 14th Intern Congr Chemother, Kyoto 1985;155-157. Pazdur R, Rossof AH, Jensik R. Pulmonary resection following chemotherapy in small cell bronchogenic carcinoma. Proc Am Soc Clin Oncol, St. Louis 1982;1. Abstract C-561. Valdivieso M, McMurtrey M, Farha P, et al. Increasing importance of adjuvant surgery in the therapy of patients with small-cell lung cancer. Proc Am Soc Clin Oncol, St. Louis 1982;1. Abstract C-576. Shepherd FA, Ginsberg RJ, Feld R, Evans WR, Johansen E. Surgical treatment for limited small-cell lung cancer. The University of Toronto Lung Oncology Group experience. J Thorac Cardiovasc Surg 1991;101:385-393. Salzer GM, Muller LC, Huber H, et al. Operation for N2 small cell lung carcinoma. Ann Thorac Surg 1990;49:759-762. Bunn PA Jr. Operation for stage IlIa small cell lung cancer? Ann Thorac Surg 1990;49:691. Ginsberg RJ. Operation for small-cell lung cancer-where are we? Ann Thorac Surg 1990;49:692-693. Yamada K, Saijo N, Kojima A, et al. A retrospective analysis of patients receiving surgery after chemotherapy for small cell lung cancer. Jpn J Clin OncoI1991;21:39-45. Zatopek NK, Holoye PY, Ellerbrock NA, et al. Resectability of small-cell lung cancer following induction chemotherapy in patients with limited disease (stages II-IIIb). Am J Clin OncoI1991;14:427-432. Namikawa S, Den T, Kimura M, Kusagawa M. The role of surgical resection and the effects of neoadjuvant therapy in the management of small cell lung cancer. Surg Today 1994;24:342-346. Wada H, Yokomise H, Tanaka F, et al. Surgical treatment of small cell carcinoma of the lung: advantage of preoperative chemotherapy. Lung Cancer 1995;13:45-56. Shepherd FA, Ginsberg R, Patterson GA, et al. Is there ever a role for salvage operation in limited small-cell lung cancer? J Thorac Cardiovasc Surg 1991;101:196-200. Campling B, Quirt I, De Boer G, et al. Is bone marrow examination in small cell lung cancer really necessary? Ann Intern Med 1986;105:508-512. Manegold C, Krempien B, Bulzebruck P, Drings P. Value of bilateral iliac crest needle biopsy for pretherapeutic staging of bronchogenic carcinoma. Oncology 1989;46:226-229. Ichinose Y, Hara N, Ohta M, et al. Brain metastases in patients with limited small cell lung cancer achieving complete remission: correlation with TNM staging. Chest 1989;96:1332-1335.

8 Extended Resections for Lung Cancer Paolo Macchiarini and Philippe Dartevelle

More than half of all patients with non-smaIl-cell lung cancer (NSCLC) present with extra pulmonary or extra thoracic spread (stages III and IV) of their tumor at the time of diagnosis. Except for unusual circumstances in which a solitary metastatic focus manifests with a primary tumor, stage IV disease by virtue of extra thoracic metastasis is beyond the surgical domain (1) and is usually treated with palliative radiation or chemotherapy. By contrast, stage III disease is a disputatious issue when indications for extensive surgery are discussed. Tumors invading adjacent structures (T3) or mediastinal lymph nodes (N2) define stage IlIa disease, are considered potentially resectable since 5-year survival of 20-30% may be anticipated (2), and the likelihood for ultimate cure is higher for patients without N2 disease and in whom a complete resection can be accomplished. By contrast, tumors invading adjacent organs (T4) or unresectable lymph nodes (N3) define stage 11Th disease, and radiation, chemotherapy, or combinations of these modalities represent the gold standard of treatment. Despite the fact that these tumors usually indicate unresectability, growing evidence now exists that occasional individuals harboring T4 tumor without N3 lesions can obtain long-term survival and cure if completely resected.

CHEST WALL INVASION Less than 8% of patients undergoing resection for NSCLC have involvement of the chest wall (3). Tumors are usually peripheral in position, 135

136

Chapter 8

without hilar or mediastinal lymph node metastases, are less likely to extend beyond the parietal pleura to the soft tissue of the chest wall and ribs, and have reduced tendency for distant metastasis. Consequently, surgical resection has curative potential. Patients' complaints are the most reliable indications of chest wall involvement, as infiltration between the ribs may result in false negative bone or CT scans. Pulmonary function tests and quantitative perfusion lung scan are necessary to assess the patient's ability to withstand operation and whether the paradox motion of the residual wall requires stabilization. The goals of surgery are to completely resect the primary tumor with clear surgical margins and to maintain a normal respiratory physiology by restoring the rigidity of the chest wall and resected soft tissue. Knowledge of chest wall invasion preoperatively is important because entering the chest at a site remote from the chest wall invasion lessens the risk of tumor spillage, allows the surgeon to assess the extent of involvement, and avoids placement of the prosthetic material directly beneath the incision. As a general rule, all tumors except those invading the thoracic inlet or the anterior thoracic cage are approached through a standard posterolateral thoracotomy. Resection should include at least one segment of rib (with the related intercostal muscle) above and below the involved rib(s) and 3 to 5 cm laterally and medially. To prevent tumor spillage, the entire tumor-bearing area should be resected en bloc, and it is frequently easier to do the chest wall resection initially (small involvements) and then proceed with the pulmonary resection. For large involvements, it is easier to do a wedge excision of the tumorbearing area with a mechanical stapler and to resect the remainder of the collapsed lobe later. Frozen sections on the soft-tissue margins are mandatory to confirm completeness of the resection. The majority of the chest wall resections do not require prosthetic reconstruction. Resection of a portion of three or fewer ribs posteriorly rarely requires prosthetic replacement, as the scapula lessens the cosmetic and functional impact of the chest wall resection. Resection of larger defect, especially when located at the anterolateral aspects of the lower ribs, may require prosthetic replacement, yet the risks of infection should be balanced against the cosmetic and functional benefits of prosthetic replacement. Reconstruction can be accomplished using nonreinforced materials like Marlex mesh or Gore-tex patch. The advantage of Marlex mesh (Bard Inc.), over Gore-Tex patch (W.L. Gore and Assoc., Flagstaff), is that it allows the ingrowth of the surrounding tissue and remains rigid over time (4). For small defects, the Marlex mesh is doubled crosswise at a 90-degree angle for added strength, and tailored and sutured to the edges of the defect with nonabsorbable sutures. With larger and unsupported defects, the chest wall rigidity can be obtained by utilizing methylmethacrylate between two layers of Marlex mesh as described by Eschapasse et al. (5) and McCormack et al. (6).

Extended Resections for Lung Cancer

137

In recent years, we adapted a more anatomical reconstruction, which is less prone to infection. Following the chest wall resection, the Marlex mesh is anchored to the surrounding tissues so that it remains beneath the ribs; 28-Fr silicone chest wall tubes are then tailored so that they can be interposed between the healthy portion of the previously resected rib to cover the Marlex mesh. The methylmethacrylate is then spread into the chest tubes, and while it becomes settled, the tubes are shaped according to the course of the resected ribs. Once hard and cool enough, the tube edges are telescoped and fixed with nonabsorbable sutures to the edges of the corresponding ribs. Presently, chest wall reconstruction rarely requires the interposition of a myocutaneous flap. As with all other prosthetic substitutes, absolute sterility is required, and the amount of air leaks should be minimal. Five-year survival rates of 15-40% have been reported in the literature (Table 8.1). Long-term survival is chiefly influenced by the completeness of the resection, depth of chest wall invasion, and presence or absence of lymph node metastases (1). The importance of performing a complete resection is stressed by 5-year survival exceeding 50% in patients undergoing a complete excision (7) and the absence of 2.5-year survivors among incompletely resected (micro- or macroscopic residual disease) patients or among those in whom resection was not possible (7-13). Likewise, the depth of the chest wall invasion affects prognosis, as extension to the parietal pleura only is associated with a twofold increase of 5-year survival (62 vs. 35%) when compared to deeper involvements (7). Different opinions exist as to whether tumors confined to the parietal pleura can be resected by simple extrapleural mobilization, without resecting en bloc the adjacent soft and bony tissues, as long as the resection margins are negative. While McCaughan et al. (7) showed that

Table 8.1.

Results after complete resection of NSCLC invading the chest wall

S-yr survival Author (ref), yr

Patterson et al. (8), 1982 Piehler et al. (14), 1982 Paone et al. (10), 1982 McCaughan et al. (7), 1985* Ricci et al. (9), 1987 Allen et al. (13), 1991 Albertucci et al. (12), 1992 Shaw et al. (16), 1995 TOTAL

No. of cases

Mortality (%)

Overall

NO

Nl

N2

30 66 32 77 77 52 21 55

8.5 15 3.1 4 7.8 3.8 9.5 3.4

38 33 35 40 15 26 40 34

45 54 35 56 22 29 50 44.7

17 7 0 21 12 11 NS 38.4

0 7 NS 0 8

410

6.9 ± 4.1

32.6 ± 8.4

41.9 ± 12.1

15.2 ± 12.2

2.5

NS = not stated; - = no patients. Note: Values are data ± standard deviation. *The 5-year survival rate was 21 % for all N1 and N2 patients.

0 0

138

Chapter 8

extrapleural mobilization was sufficient for a significant number of patients whose tumors invaded the parietal pleura only, Piehler et al. (14) reported a high incidence of local recurrence after extrapleural dissection for tumors invading the parietal pleura. Although the subject remains arguable, the current trend favors en bloc chest wall resection for all symptomatic tumors invading the parietal pleura, the only exception being marginal patients with bulky tumors and compromised pulmonary function. Although the completeness of resection and depth of chest wall involvement are adverse prognostic factors, the survival of patients with this type of T3 tumor is predominantly influenced by the nodal status. As shown in Table B.1, most if not all series report no 5-year survivors with positive N2, compared to 5-year survival exceeding 50% for NO patients. In this sense, mediastinoscopy is advocated for patients with chest wall involvement and enlarged lymph nodes. There is a lack of uniformity of opinion regarding the value of radiation therapy either preoperatively or postoperatively (1). Proponents of preoperative radiation therapy argue that it limits the extent of resection required and reduces the incidence of local recurrence. In contrast, opponents argue that the amount of chest wall spared is negligible, damage to the unresected lung may complicate the postoperative course, and local control can be enhanced with postoperative radiation therapy as well. In the absence of randomized prospective trials, radiation therapy should be reserved for patients with positive surgical margins and/ or mediastinal nodes.

SUPERIOR SULCUS TUMORS Superior sulcus tumor (Pancoast tumor) refers to any primary bronchogenic tumor arising from the apex of either upper lobe and extending through the visceral pleura into the parietal pleura and the adjacent structures of the thoracic inlet (15). Patients usually present with pain in the shoulder and vertebral border of the scapula, and, as the tumor involves the brachial plexus, symptoms develop in the distribution of the T1 (ulnar distribution of the arm and elbow) and CB nerve roots (ulnar surface of the forearm and small and ring fingers). Direct extension to the stellate ganglion and sympathetic chain causes Horner's syndrome (true Pancoast-Tobias syndrome). Superior sulcus tumors are extremely difficult to diagnose at initial presentation. It is their primarily extrapulmonary location that is responsible for the characteristically clinical pattern associated with the Pancoast-Tobias syndrome (Fig. 8.1). Patients with tumors invading the anterior part of the thoracic inlet generally present with severe pain arising from and localized to the anterior chest wall (related to the invasion of the first intercostal nerve and first ribs). Less frequently, patients may


1 39

/\ n tC'rior

Scalenus \lusclp

Phrenic

Nerve r\lfiddle Scalenus Muscle

Subclavian Artery

Brachial ....- __IJI Plexus

Subclavian

Vein Fig. 8.1. The insertion of the anterior, middle, and posterior scalenus muscles on the first and second ribs, respectively, divides the thoracic inlet in three compartments: 1) an anterior compartment containing the platysma and sternocleidomastoid muscles, the external and anterior jugular veins, the inferior belly of the omohyoid muscle, the subclavian and internal jugular veins and their major branches, and the scalene fat pad; 2) a middle compartment containing the anterior scalenus muscle with the phrenic nerve lying on its anterior aspect the subclavian artery with its primary branches except the posterior scapular artery, the trunks of the brachial plexus, and middle scalenus muscle; and 3) a posterior compartment, lying posteriorly to the middle scalenus muscles and including the long thoracic and external branch of the accessorius spinalis nerves, posterior scapular artery, sympathetic chain and stellate ganglion, vertebral bodies, intervertebral foramina, and epiduritis.

present with phrenic nerve palsy and superior vena cava hemisyndrome; tumors usually do not invade the brachial plexus or other structures of the posterior thoracic inlet. Those tumors invading the middle path of the thoracic inlet present with signs and symptoms related to the compression or infiltration of the middle and lower trunks of the brachial plexus; patients may complain that this pain radiates to the shoulder and upper limb. Often, tumors spread into the middle scalenus muscle. Posterior tumors typically present with most of the signs and

1 40

Chapter 8

symptoms of the Pancoast-Tobias syndrome and are usually located in the costovertebral groove, invading the nerve roots of Tl, the posterior aspect of the subclavian and vertebral artery, sympathetic chain, stellate ganglion, and prevertebral muscles. The severity of this form is linked to the tumor's potential spread along the T1 and T2 nerve roots up to the spinal canal through the intervertebral foramina. Recent diagnostic advances have made it possible to establish the diagnosis of this entity early on, usually before the full-blown syndrome occurs. Diagnosis is established by history and physical examination, chest x-ray, bronchoscopy and sputum cytology, fine-needle transthoracic or transcutaneous biopsy, and aspiration and CT of the chest. Preoperative histologic diagnosis is imperative, as the Pancoast-Tobias syndrome may be triggered by diseases other than bronchogenic carcinomas. If there is preoperative evidence of mediastinal lymph node involvement, mediastinoscopy is mandatory because patients with clinical N2 disease are not suitable for operation. Neurologic examination, electromyography, and fluoroscopy delineate the tumor's extension to the brachial plexus, phrenic nerve, and epiduritis. Vascular invasion is studied with venous angiography, subclavian arteriography, and Doppler ultrasonography, and by MRI, especially for tumors approaching the intervertebral foramina to rule out invasion of the extradural space. Superior sulcus tumors continue to represent a major therapeutic challenge. Controversy still exists as to whether these tumors should be treated with a combined radiosurgical approach (16), high-dose curative primary radiotherapy (17-19), "sandwich" preoperative and postoperative radiotherapy (20), postoperative radiation therapy alone (21), or intraoperative brachytherapy combined with preoperative radiation therapy and operation (22, 23). The combined radio surgical approach was popularized by Chardock and MacCallum (24) and Shaw et al. (16), who reported 5-year survival in the range of 30% by using the combination of preoperative radiation therapy (30-45 Gy in 4 weeks, including the primary tumor, mediastinum, and supraclavicular region) and surgery; the majority of the surgical reports still include this radiosurgical option (Table 8.2). However, advocates of high-dose irradiation alone (25) claim that evidence supporting the assertion that long-term survival is better with combined radiosurgical therapy than with high-dose radiation therapy alone is lacking, although the latter treatment rarely sterilizes the tumor area without surgery (26) and results in a local tumor recurrence rate of up to 50% and dismal early (27) and long-term (18) survival. The initial encouraging local-regional control and survival data with "sandwich" preoperative and postoperative irradiation (20) have been neither confirmed nor denied in follow-up studies or by other authors. Recently, Ginsberg et al. (28), analyzing one of the largest series ever published, demonstrated no advantage for the use of intraoperative brachytherapy in completely or incompletely resected patients, and concluded that en bloc resection, including the chest wall, involved adjacent structures as

Extended Resections for Lung Cancer Table 8.2.

1 41

Results of patients treated surgically for superior sulcus tumors

Author (refJ, yr

No. of cases

Paulson (34), 1985 Anderson et al. (35), 1986 Devine et al. (36), 1986 Miller et al. (37), 1987 Wright et al. (26), 1987 Shahian et al. (20), 1987 McKneally et al. (33), 1987 Komaki et al. (38), 1990 Sartori et al. (29), 1992 Maggi et al. (31), 1994 Ginsberg et al. (28), 1994 Okubo et al. (30), 1995 Dartevelle (32), 1997

79 28 40 36 21 18 25 25 42 60 100 18 55

TOTAL

547

5-yr survival

Mortality

(%J

(%J

35 34 10 31 27 56 51 40 25 17.4 26 38.5 34 32.6 ± 12.5

3 7 8 NS 0 0 NS NS 2.3 5 4 5.6 0 3.4 ± 2.9

NS = not stated. Note: Values are data ± standard deviation.

well as lobectomy and must be considered the standard surgical approach for superior sulcus tumors combined with external radiation (preoperative, postoperative, or both). Although Horner's syndrome, N2 or N3 disease, and invasion of T4 are established adverse prognostic factors, the key factor determining cure of these tumors depends mainly on the completeness of surgical resection (28). We recently proposed a new surgical technique for resecting superior sulcus tumor (21). Through an L-shaped incision cervicotomy (Fig. 8.2), the sternal attachment of the sternocleidomastoid muscle is divided and the cleidomastoid muscle scraped from the clavicle; the resulting myocutaneous flap is then folded back to give a full exposure of the neck, thoracic inlet, and upper part of the anterolateral chest wall. Once the inferior belly of the omohyoid muscle is transected, the scalene fat pad and ipsilateral superior mediastinum are dissected and pathologically examined to exclude lymph node invasion. The tumor's extension to the thoracic inlet is then carefully assessed, and if the tumor is deemed completely resectable, the internal half of the clavicle is resected. Following resection of the subclavian vein, the scalenus anterior muscle if invaded is divided in tumor-free margins with or without preservation of the phrenic nerve. If there is an invasion of the subclavian artery, the vessel is resected and revascularized either with prosthetic grafts or end-to-end anastomosis. Following division of the middle scalenus muscle, the nerve roots of C8 and T1 are easily dissected until their confluence into the lower trunk of the brachial plexus. Thereafter, the prevertebral muscles are detached, along with the dorsal sympathetic chain and stellate ganglion, from the anterior surface of the C7-T1 vertebral bodies, per-

142

Chapter 8

~ '\

\

'\\

\ "

'II"'IL"

\............. ." _.. ~ .. _1//!IJr~J .I,~: /11 _ ~...• "" • " .• - ... - - - ) . • ..t~

,

/

/~:------- // ---_./

/

., ,' .

...,

Fig. 8.2. Right transcervical incision. The patient is placed in the supine position with the neck hyperextended and the head turned away from the involved side. An L-shaped skin incision is made from the angle of the mandible down to the sternal notch, is extended horizontally under the internal half of the clavicle, and prolonged into the deltopectoral groove or into the bed of the second or third intercostal space, as indicated by the extension of the lesion.

mitting visualization of the intervertebral foramina. The Tl nerve root is usually divided beyond visible tumor, just lateral to the Tl intervertebral foramen. Although tumor spread to the brachial plexus may be high, neurolysis is usually achieved without division of the nerve roots above T1. Operation is completed by performing the en bloc chest wall resection and the upper lobectomy rather than a simple wedge tumoral excision. Because of its fibrotic consequences and a reduced ability to deliver postoperative high-dose radiation, this operation should be performed without preoperative radiation. When indications are respected, this transcervical approach results in a complete resection rate of 100%, no postoperative mortality or major complications, 5-year and median survivals approaching 35% and 18 months, respectively, and local recurrence rates less than 1.8% (see Table 8.2) (29-38). With increasing experience, our patient population now includes tumors extending to the subclavian or vertebral vessels, scalenus muscles, phrenic nerve, and


143

LUNG COSTOTRANSVERSE FORAMEN

B

A

c Fig. 8.3. To completely resect bronchogenic tumors extending into the intervertebral foramina (A), a combined transcervical anterior and median posterior approach is used and an hemivertebrectomy made (B = line of transection) followed by spinal fixation (C).

vertebrae (32); significant survival differences among the different T4 tumor extensions have not been observed. More encouraging are our preliminary results with extensive resections for vertebral involvement (Fig. 8.3): Although a higher number of patients are required and a longer follow-up time as well, it seems that tumors extending into the intervertebral foramen without intraspinal extension may be resectable with promising long-term results. The lessons learned with superior sulcus tumors resected by our transcervical approach are that the absolute surgical contra indications are the presence of extrathoracic sites of metastasis and histologically confirmed N2 disease. Extensive involvement of the brachial plexus, subclavian vessels (35), and intervertebral foramina should not be considered a contraindication, provided a complete surgical resection may be anticipated.

CARINAL RESECTIONS Until recently, tumors invading the carina, either directly or by (sub)mucosal or extrabronchial spread, were considered inoperable and were treated primarily by radiation. As techniques of airway reconstruction have become more popularized, thoracic surgeons have reconsidered

144

Chapter 8

this challenging group of tumors. The usefulness of carinal surgery for NSCLC is still questioned because 1) the majority of the tumors are usually so extensive that complete resection is unrealizable, 2) the carinal involvement is most often secondary to diseased subcarinal or laterotracheal nodes, and 3) operative mortality exceeded or equalized the likelihood for long-term survival. There are, however, occasional tumors that are sufficiently localized to permit curative carinal, resection and reconstruction. On the right side, carinal resection should be considered in each patient whose tumor invades the first centimeter of the ipsilateral main bronchus, the lateral aspect of the lower trachea, carina, and contralateral main bronchus. This applies less frequently to the left side because the first centimeter of the left mainstem bronchus is rarely invaded by itself, and carinal invasion is more likely related to subcarinal extracapsular node spread. Considering the inherent risks of the operation, careful preoperative evaluation of each patient should encompass the patient's physiological and functional status and exact determination of the extent of the lesion. A precise delineation of airway and mediastinal node invasion should be obtained. Flexible and/or rigid bronchoscopic overview of the involved and remaining airway is obligatory to evaluate the overall length of the resection and whether a tension-free anastomosis is feasible. The maximal amount of airway that can be safely resected varies from patient to patient, and therefore the lower trachea, carina, and mainstem bronchi need to be carefully inspected in each patient. For tumors arising from the ventral segment of the right upper lobe, pulmonary angiography should be made, as amputation of the mediastinal artery supports invasion of the right pulmonary artery, phrenic nerve, and posterior aspect of the superior vena cava (SVC). Transesophageal echocardiography is actually the gold standard to evaluate a tumor's extension to the organs of the posterior mediastinum. Because invasion of the trachea beyond the lower 2 em and of the contralateral main bronchus beyond 1.5 em produces an excessive anastomotic tension, particular attention is made to assess the extent of the intraluminal invasion by performing biopsies on the mucosa and submucosa lying at least 2 em from the grossly tumoral surface. It is generally accepted that the safe limit for most carinal and lung resections is 4 em between the lower trachea and the contralateral main bronchus. When a carinal resection alone is planned, for instance for in situ carcinoma of the carina, the tracheobronchial resection may be somewhat more extensive because of the greater mobilization of the right lung unhindered by the aortic arch. Positive margins or recurrence on the postpneumonectomy stump are also indications for carinal resection. Mediastinal lymph nodes greater than 1.5 em in diameter require their histologic verification by cervical mediastinoscopy, as for any other resection of carcinoma of the lung. N3 disease and invasion of the ipsilateral mediastinal lymph nodes above the subcarinal space preclude resection, as there are no early «2 years) long-term survivors (32).


145

Although induction therapy may be advocated for such patients according to the surgeon's philosophy of mediastinal node invasion, we learned that this increases the technical difficulties of the operation and particularly the perioperative complication rate. By contrast, diseased subcarinal nodes may benefit from induction therapy, as occasional 5year survivors have been reported. We recommend that cervical mediastinoscopy be performed at the time of the planned operation to avoid fibrosis and limitation of the mobility of the airway. In the absence of N2 disease, invasion of the SVC,left atrium, and muscular wall of the esophagus should not be considered an absolute contraindication. The main difficulty during carinal surgery is the simultaneous control of the airway required to maintain satisfactory gas exchange and to ensure adequate surgical exposure of the trachea and main bronchi. Not surprisingly, distal ventilation during carinal resection and reconstruction has always been a major concern. Our preferred method of airway management parallels that described by Grillo and Mathisen (39-42). The patient is initially intubated with an extra-long oral endotracheal tube that can be advanced into the opposite bronchus if one-lung ventilation is desired. Once the carina has been resected, one main bronchus is intubated with a sterile, flexible tube whose connecting system is passed to the anesthesiologist. The tube can be safely and intermittently removed for brief periods of time to precisely place the sutures. Once the trachea and bronchi are ready to be approximated, the cross-field tube is withdrawn and the original oral tube advanced across the anastomosis into the main bronchus. This applies for right and left carinal pneumonectomy as well; in other types of carinal reconstruction, however, ventilation is best obtained with selective high-frequency ventilation catheters. Cardiopulmonary bypass is never required for carinal resection. Carinal resection without sacrifice of pulmonary parenchyma can be safely approached by a median sternotomy and transpericardial exposure of the tracheobronchial bifurcation between the SVC and ascending aorta and between the innominate vein and pulmonary artery superiorly and inferiorly. By contrast, carinal resection with sacrifice of the pulmonary parenchyma is best approached through an ipsilateral thoracotomy in the fifth (or fourth) intercostal space. On the right, the lower trachea and origin of both mainstem bronchi are easily exposed. On the left, exposure of the lower trachea and right main bronchus is hindered by the aortic arch. To gain exposure, the ligamentum arteriosum is divided and the aortic arch mobilized with (43) or without scarifying the first two intercostal vessels. Right carinal pneumonectomy is the most frequent type of resection and reconstruction for bronchogenic carcinoma (Fig. 8.4). No irrevocable step should be taken until resection is guaranteed. After proximal and distal division and suture of the arch of the azygos vein, the tracheobronchial bifurcation is gently mobilized and dissected. Mobilization of the lower trachea should be only to the extent needed and lim-

146

Chapter 8

A

B

c

D

Fig. 8.4. A. Right carinal pneumonectomy. B,e. Inverted technique of Barclay (46,47). Reconstruction may be made by an end-to-end anastomosis betvveen the left main bronchus and lower trachea and an end-to-side anastomosis betvveen the right and left main bronchi, if the stump of the right main bronchus is short (B) or betvveen the right main bronchus and lower trachea above the first anastomosis when a long residual right mainstem stump remains (C). D. Right upper lobectomy with resection of the carina, distal trachea with end-to-side anastomosis of the intermedius bronchus to the medial wall of the left main bronchus, 1 cm below the previously made tracheal to the left main bronchus end-to-end anastomosis.

ited to no more than 2 cm from the proposed lines of transection of the lower trachea and left main bronchus. Mobilization is obtained by flexing the neck, opening the pretracheal plane from the tracheobronchial bifurcation up to the cervical trachea either at the time of mediastinoscopy or thoracotomy, and by dividing the inferior pulmonary ligament. Further mobilization of the pulmonary hilum is achieved by performing aU-shaped intrapericardial incision that allows a 2-cm upward shift of the hilar structures, thus contributing to tension-free anastomosis. Laryngeal release is not necessary to gain further length. Dissection should be limited to the anterior surface of the lower trachea while preserving as much of the lateral tracheal blood supply as possible. The hilum and esophagus are then dissected. If there is no


1 47

SVC involvement, the pulmonary artery and vein(s) are stapled at their extrapericardial origin and the lung remains attached only by the main bronchus. Subsequently, the cross-field intubation system is installed on the operative field and the trachea and the contralateral main bronchus, respectively, are divided by sharp and straight transection lines. One should always keep in mind that to accomplish a tension-free anastomosis in this type of carinal resection, the safe length between the distal end of the lower trachea and contralateral main bronchus should not surpass 4 cm. Traction sutures are then placed on the lateral walls of the contralateral main bronchus, and frozen sections are obtained on the tracheal and bronchial stumps, as for other lung cancer resections. The decision to further resect or to leave residual tumor in the case of positive margins is chiefly influenced by the need to perform a tensionfree anastomosis and the assumption that residual tumor at the bronchial margin is not always indicative of a poor prognosis. Diseased subcarinal lymph nodes can be resected en bloc with the tracheobronchial bifurcation; otherwise, the soft tissue around the carina should be preserved as best as possible to salvage major branches of the bronchial artery(ies) to the contralateral lung. Before the cross-field intubation of the contralateral main bronchus, any blood spillage from the previously dissected area should be carefully suctioned to avoid blood spillage into the contralateral lung. After having hyperventilated the contralateral lung with 100% oxygen, reconstruction is initiated alternating period of total apnea to period of ventilation. The tracheobronchial anastomosis is then constructed (Fig. 8.5). After completing the anastomosis, the endotracheal tube is pulled back at a sufficient distance from the suture line to avoid injury from the tip of the tube. The tracheobronchial anastomosis is then covered by available autogenous vascularized tissue(s) that should separate the suture lines from nearby vascular structures. For left carinal pneumonectomy, the aortic arch remarkably hinders the performance of the anastomosis. However, a one-stage procedure with mobilization of the aortic arch as described previously should be preferred to a two-stage procedure where a left proximal pneumonectomy with positive margins is followed 2 to 3 weeks later by resection of the carina from the right side (44). Because of the limited space in the subaortic region and because it permits carinal reconstruction without interruption around the catheter, the high-frequency jet device may be preferred to the cross-field intubation system. If this device is not available in the operating room, the right main bronchus is intubated through the cross-field system and the right lung hyperventilated; this shifts the mediastinum toward the operating surgeons and reduces the depth of the operating field. Intraoperative discovery of SVC invasion or limited spread to the muscular wall of the esophagus should not be considered a contraindication to carinal resection because long-term results may be encouraging (45). If a truncular replacement of the SVC is planned, the vascular

148

Chapter 8

Suture technique for right carinal pneumonectomy. It starts with an internal, cartilage-to-cartilage running 4-0 polydiaxone (PDS) suture on the mediastinal walls of the tracheal and bronchial stumps (J -5). It is then tied and fixed with two independent PDS sutures whose knots are made outside the lumen. Thereafter. interrupted stitches of 3-0 PDS are placed on the remaining part of the anastomosis. Telescoping the smaller main bronchus into the lower trachea is acceptable and can prevent anastomotic disruption in patients requiring mechanical ventilation. Fig. 8.5.

procedure should be performed before division of the airway: The SVC is clamped proximally at the confluence of the brachiocephalic veins and distally at the cavoatrial junction and divided on each side of the tumor, thus facilitating exposure and stapling of the pulmonary artery in the interaorticocaval groove. The SVC is then reconstructed using a ringless, straight No. 18 or No. 20 polytetrafluoroethylene (PTFE) graft. Considering the risk of infection while opening the airways, the PTFE must be protected with an absorbed gauze of betadine. Next, the carinal pneumonectomy is performed. Finally, the graft is wrapped with a vascularized autologous flap. Carinal resection without pulmonary resection is usually limited to those rare lesions either implanted on the carina or at the origin of the main bronchi and carina. Ideally, the length of the resection between the lower trachea and the distal main bronchus should not surpass 2 cm. Depending on the extent of the invasion, different modes of reconstruction exist. For very small lesions implanted only on the carina, the carina as well as the first centimeter of either main bronchi can be safely resected and reconstruction afforded by approximating the medial walls


149

of the main bronchi to frame a neocarina with the trachea. The main difficulty with this archetype is that the left main bronchus cannot rise sufficiently to ensure an adequate mobility to the neocarina because it is retained in its original position by the aortic arch, even after dissection of its proximal 2 cm; the trachea, therefore, needs to be pulled down to the neocarina. Carinal reconstruction for bronchogenic carcinomas involving the lower trachea, carina, and proximal main bronchi to a greater extent may be accomplished in three ways: 1. The technique of Barclay et al. (46), where the right main bronchus is anastomosed end-to-end to the distal trachea and the left main bronchus implanted end-to-side across the mediastinum to the intermedius bronchus. This reconstruction is possible only if the right main bronchus is left sufficiently long. It is challenging, as access to the end-to-side anastomosis is difficult and requires hypoventilation of the right lung. 2. The "inverted" technique of Barclay described by Eschapasse et al. (47). The reconstruction is made by an end-to-end anastomosis between the left main bronchus and lower trachea and, depending on the length of the remaining right main bronchus, an end-to-side anastomosis between the right and left main bronchi (short right main bronchus) or between the right main bronchus and lower trachea above the first anastomosis (long right main bronchus). In our limited experience, however, the right main bronchus can be easily anastomosed to the lateral tracheal wall above the first anastomosis after the adequate releasing maneuvers, regardless of its residual length. 3. The technique of Grillo et al. (48), where the trachea is anastomosed to the right main bronchus in an end-to-end fashion and the left main bronchus to the trachea above the first anastomosis in an end-to-side fashion. Its indications are rare, and it is a very technical demanding procedure.

Occasionally, bronchogenic tumors arising from the right upper lobe can extend to the carina and lower trachea. The lower trachea and carina are resected and divided as for right carinal pneumonectomy, but this is done after ligation and division of the right upper lobe vessels. The transection line on the right bronchial tree is made at the origin of the intermedius truncus, after the right upper lobe takeoff. After intrapericardial U-shaped and pulmonary ligament mobilizations, the residual bilobe is elevated and anastomosed end-to-side to the left main bronchus, 1 cm below the previously made tracheal-to-medial wall of the left main bronchus. One can advocate the implantation of the intermedius bronchus on the lateral tracheal wall, but the tension on this anastomosis would be excessive, dictating either the previous modelage or sacrifice of the residual right lung. Results following carinal surgery for bronchogenic carcinoma derive

1 50

Chapter 8

primarily from the experience of few institutions worldwide. The six largest series reported over the last 15 years mainly reflect the results of carinal pneumonectomy and are listed in Table 8.3 (49-51). The overall operative mortality is 22 ± 12%. Jensik et al. (49) reported a 29% mortality rate, and most of the deaths followed bronchopleural fistulas, possibly related to the preoperative radiation delivered at doses of 32 to 50 Gy. Deslauriers et al. (44) reported a 29% mortality rate in 38 right-sided carinal pneumonectomies following respiratory failure caused by infection on the remaining lung. At Massachusetts General Hospital (41), all early deaths following right carinal pneumonectomy were related to a noncardiogenic pulmonary edema unresponsive to maximal treatment; late deaths were related to anastomotic complications caused by tension and ischemia. A comprehensive review of these mortality figures shows that the most common causes of early and late mortality are respiratory failure following noncardiogenic pulmonary edema or infection in the remaining lung and anastomotic complications, respectively. The fact that it could appear that mortality rates equal survival rates, but the technical pitfalls can be corrected by rigorous indications, meticulous attention to all surgical details, and careful anesthetic management. In our series of 60 carinal pneumonectomy patients who have been operated on for bronchogenic cancer since 1981, the operative mortality rate was 6.6% and the 5- and 10-year survival rates, including postoperative deaths, were 43.3% and 29%, respectively. Long-term survival was significantly influenced by the nodal status (NO-1 vs. N2, P = 0.02) and histology (squamous vs. nonsquamous, P = 0.03) in univariate analysis, and nodal status (P = 0.01) by multivariate analysis. Five-year survival was 42% for patients with either NO or N1 disease, but none of the patients with N2 disease survived beyond 46 months (52). The main challenge of carinal resection is to reduce the operative mortality by judicious indications, meticulous surgical details, and

Table 8.3.

Mortality and 5-year survival rates after carinal pneumonectomy for bronchogenic carcinoma Operative mortality (%)

Author (ref), yr

Jensik et al. (49), 1982 Deslauriers et al. (44), 1989 Tsuchiya et al. (50), 1990 Mathisen & Grillo (41), 1991 Roviaro et al. (51), 1994 Dartevelle & Macchiarini (52), 1996 TOTAL

Number of patients

Early

34 38 20 37 28 60

15 8

217

4.3 ± 6

3.3

Overall

S-yr survival (%)

29 29 40 18.9 4 6.6

15 13 59 (2-yr) 19 20 43.3

21.2 ± 14.1

22.1 ± 12

Note: Operative mortality includes early «30 days) and overall «30 days plus >30 days) deaths related to the procedure. Total mortality and 5-year survival are expressed as mean ± standard deviation. TotalS-year survival does not include the results of Tsuchiya et al. (50).


1 51

peri operative management. Indications should not include patients with preoperative diseased mediastinal nodes, an exception being those patients entering protocols to evaluate preoperative treatment. This last, however, increases the already remarkable morbidity and mortality rates. Diseased subcarinal nodes may benefit from surgery because they can be resected en bloc with tracheobronchial bifurcation; in our series, the only 2-year survivors among N2 patients were those whose subcarinal nodes were diseased. The long-term survival rates fully justify this procedure for NO or N1 disease, and we endorse the suggestion by Grillo and Mathisen (53) that these NO-1 patients be reclassified into stage lIlA. Because the major cause of failure is systemic relapse, adjuvant chemotherapy is recommended on an individual basis. The benefits of postoperative radiation therapy should be evaluated against its effects on the residual lung (e.g., subclinical pneumonitis) and mediastinum (e.g., lymphatic blockage).

SUPERIOR VENA CAVA INVASION Invasion of the SVC by right-sided bronchogenic carcinomas occurs in less than 1% of operable patients (4) and is usually regarded as an absolute surgical contraindication because of the dismal prognosis, absence of suitable graft material for reconstruction, and technical fear concerning the effects of SVC clamp age, graft thrombosis, and infection. However, recent anesthetic and surgical advances have lowered the surgical risks inherent to SVC clamp age and revascularization. Moreover, the feasibility of reconstructing the SVC has been ameliorated by the efficacy of the presently suitable graft materials. Among them, PTFE graft is the only material remaining patent as a venous substitute (54-61) and should be considered the material of choice for SVC reconstruction. Consequently, SVC replacement has increased its popularity and acceptance among thoracic surgeons. The SVC is usually subject to easy obstruction due to its anatomic site, thin wall, low hemodynamic pressure, and encirclement by chains of lymph nodes draining all of the right thoracic cavity. The carcinomas most commonly arise in the ventral segment of the right upper lobe and extend directly to the SVC and adjacent lymph nodes. However, surgical indications are restricted. SVC syndromes related to unresectable bronchogenic tumors should not be considered for SVC revascularization. Because the proximal anastomosis needs to be performed either at the origin of the SVC or at the level of one or both brachiocephalic veins, SVC revascularization can be made only if there is no invasion at the level of the cephalic venous bed. A preoperative workup evaluating the extension of the primary disease should be performed routinely. All patients should have a superior vena cavography (simultaneous injection through both upper limbs)

152

Chapter 8

before operation to delineate the site and extension of the venous obstruction, presence of possible proximal thrombosis, and to anticipate where the proximal graft anastomosis can be made. Echocardiography eliminates thrombosis extension into the right atrium and appreciates the patency of the jugular and axillary veins. Because a majority of patients with bronchogenic cancer may present with a clinically and radiologically silent SVC invasion, chest CT is the key diagnostic tool to anticipate SVC reconstruction. Brain CT scan should always be performed to eliminate brain metastasis or ischemic lesions that may increase brain edema during SVC clamping. The usual approach includes a right thoracotomy in the fifth intercostal space. For patients whose SVC is completely obstructed or tightly stenosed, intraoperative venous clamping results in a negligible hemodynamic compromise, as a functioning collateral venous network already exists and supplements the flow obstruction to the SVC. However, this has never been observed in our practice and is more common for mediastinal tumors or fibrosis. By contrast, in patients whose SVC invasion is unobstructed, an even, sharp venous clamping might trigger a hemodynamic cascade of events including decreased cardiac inflow and outflow, increased venous pressure of the cephalic territory, and alterations of the cerebral arteriovenous gradient leading to brain damage and intracranial bleeding. Several technical details may mitigate this hemodynamic instability in unobstructed SVC, and among them the most important are: 1. Pharmacologic agents and fluid implementation. They should increase

the venous blood return to the right atrium and maintain the physiologic arteriovenous gradient in the cerebral territory. The first target is achieved by an adequate compensation of all blood losses by blood components and macromolecules. Because the cranial venous pressure may rise up to 40 mm Hg during venous clamping, maintenance of the cerebral arteriovenous gradient requires fluid administration (average 15-20 mL/kg) to normalize the cardiac output and eventually vasoconstrictive agents to increase the mean arterial pressure. 2. Shortening the venous clamping time. To reduce the venous clamping time (which can be prolonged up to 45 min), an accurate surgical strategy should be defined. For right bronchogenic tumors with carinal or proximal pulmonary artery invasion, it is often easier to perform the vascular step first and then the airway procedure. During the latter, all attention should be directed to avoid prosthesis bacterial contamination. 3. Anticoagulation therapy. Intravenous sodium heparin (0.5 mg/kg) is given before clamping and is continued at a daily dose of 1 to 2 mg/kg thereafter. Warfarin agents are used at the time of hospital discharge and thereafter. Although intraluminal shunting of the blood from the brachiocephalic vein into the right atrium may reduce the hemodynamic consequences of venous clamping (61), they will likely throm-


153

bose and fill the operative field, making the performance of the distal anastomosis more difficult. When the circumference of the involved caval wall is less than 30%, a partial resection of the vein is possible. Its reconstruction can be made either directly with a running suture or indirectly with the interposition of a prosthetic or autologous pericardial or venous patch. Closure of up to 50% of the caval circumference can be made without hemodynamic imbalance. However, when there is a greater circumferential involvement, one should not hesitate to perform a total replacement of the SVC for both oncologic and hemodynamic reasons. Truncular replacement requires a tumor-free confluence of both brachiocephalic veins (Fig. 8.6); this procedure employs a straight, not ringed, PTFE graft. After proximal (brachiocephalic veins confluence) and distal (cavoatrial junction) clampage, the invaded segment of the vein is completely excised. The proximal anastomosis between the SVC stump and the prosthesis is then performed first using a continuous 5-0 polypropylene (Prolene, Ethicon, Inc., Somerville, NJ) suture started at the posterior aspect of the prosthesis in an inside to outside fashion. After completion of the prox-

A

B

c

Fig. 8.6. Extended right pneumonectomy with replacement of the superior vena cava (SVC). A. Control of the SVC on opening of the pericardium. B. Control of the right pulmonary artery in the interaorticocaval sinus. C. Resection of the SVc. D. SVC revascularization by a polytetrafluoroethylene graft following right pneumonectomy.

1 54

Chapter 8

imal anastomosis, the distal anastomosis is performed in the same way. Before tightening the stretches of the distal suture, the proximal clamp is released, and the prosthesis flashed with saline heparinized solution and de-aired. The distal clamp is then released and the knots tied. To avoid prosthesis kinking, the length of the graft should be adapted so that the distal anastomosis rests under tension. Revascularization from the left brachiocephalic vein requires a ringed PTFE graft (No. 12 or 13). Minimal dissection of the left brachiocephalic vein is also mandatory to avoid its rotation above the proximal anastomosis. The distal anastomosis can be performed either on the right atrium or appendage or the inferior stump of the SVC. Revascularization from the right brachiocephalic vein requires ringed grafts (No. 12 or 14) to maintain patency and to avoid compression by postoperative fibrosclerosis. Although the risks of kinking are minimal because the direction of the graft is almost vertical, the proximal anastomosis is not always easy to perform, as after its resection the right brachiocephalic vein is often short; thus, proximal anastomosis should be performed first. The distal anastomosis should be made on the SVC stump, and this architecture results in the straightest and shortest graft; however, it is rarely indicated for lung cancers. Thus far, our experience since 1980 includes fourteen patients whose NSCLC invaded the SVC extensively. There were eleven squamous cell lesions, and six patients received induction therapy. All patients but one were approached through a posterolateral thoracotomy. The majority of the patients required extended pneumonectomies, six of them being carinal pneumonectomies. Only once, an upper lobectomy was sufficient to radically resect the tumor burden. In the latter case alone, the confluence of the right brachiocephalic vein was also invaded, and revascularization was made between the left brachiocephalic vein and right atrium. There were six N2-diseased nodes and eight patients with NO-1 disease. All patients except one had a direct tumoral SVC invasion by cancer at the level of the pulmonary pedicle. Three major complications occurred: two bronchopleural fistulas (BPFs) and one extrapericardial cardiac herniation. In one of the two BPFs, the PTFE became infected and was removed through a Clagget operation; unfortunately, the patient died 1 month later from pneumonia of the remaining lung. Overall mortality rate was 7.1%. The 5-year survival rate, including postoperative death, was 31 %; five patients are still alive and diseasefree after 13 to 73 postoperative months.

INVASION OF THE LEFT ATRIUM, AORTA, AND MAIN PULMONARY TRUNK Complete resections in patients with tumors invading the left atrium, aorta, and main pulmonary trunk are often not possible and are associ-

Extended Resections for lung Cancer

1 55

ated with a high morbidity and mortality. In a series of 44 patients with surgically managed T4 tumors, Martini et al. (62) reported an 18% complete resection rate and an overall 5-year survival of 12% but no survival benefits for incomplete resections. Despite these disappointing results, en bloc resection of the primary tumor with part of the involved left atrium (59), aorta (63), and main pulmonary trunk (64) can sometimes result in complete resection and occasional cure. The invasion of the left atrium by NSCLC is typically discovered incidentally at thoracotomy in less than 4% of patients undergoing curative resection for NSCLC. The left atrium is usually invaded more frequently by direct extension rather than by tumor emboli protruding from the pulmonary veins. While a surgical benefit exists for occasional and selected patients whose left atriums are invaded directly (65), the role of surgery for patients whose left atrium is embolized is hotly debated (66), the major area of contention being whether the technical hazards of left atrial clamp age and risks of systemic tumor embolization (67) are worthwhile. We operated on 31 patients whose left atriums were either directly invaded (n = 17) or embolized (n = 14), with an overall survival rate of 21.6% (Macchiarini P, Dartevelle P, unpublished data, 1997). All patients whose left atriums were embolized relapsed in extrathoracic sites and died from their disease after a median diseasefree survival of 6 months. The only long-term survivors were those patients whose left atriums were directly invaded. These findings strongly suggest that when left atrium embolization is incidentally discovered at the time of thoracotomy, further resection should be discouraged. On the other hand, complete resection of tumors with partial invasion of the left atrial wall should not be denied because this procedure represents the only hope for cure, as suggested by the few reports in the literature (Table 8.4). Whether these resections should be done with (68, 69) or without cardiopulmonary bypass has yet to be determined. The results of Tsuchiya et al. (59) suggest that cardiopulmonary bypass may well increase the resectability for these tumors but results in no early and long-term survival benefit. The results of invasion of the aorta by bronchogenic tumors are limited to scattered reports, mainly because tumors are so locally extensive that resection is often impossible. Nakahara et al. (63) reported three patients with invasion of the aortic arch and left common or subclavian artery who were resected with or without femorofemoral bypass: Two died from systemic metastasis within 12 months from operation while the remaining patient was alive at 17 months postoperatively. However, Nakahara et al.'s results were neither further confirmed nor denied. Martini et al. (65) reported, among twenty patients with aortic involvement, only one 5-year survivor who underwent an incomplete resection combined with intraoperative brachytherapy. Finally, Tsuchiya et al. (59) operated on twenty-eight patients with either invasion of the aortic adventitia (n = 21) or of the aorta itself (n = 7) by tumors situated near Botallo's ligament or by metastatic sub aortic lymph nodes. Twelve

156

Chapter 8 Table 8.4. Results of patients treated surgically for bronchogenic tumors involving the left atrium

Author Iref), yr

No. of cases

5-yr survival 1%)

Mortality 1%)

Shirakusa & Kimura (68), 1991 Martini et a1. (65), 1994 Tsuchiya et a1. (59), 1994 Macchiarini & Dartevelle (unpublished data), 1997

12 8 44

NS 12.5 22

8.3 NS NS

31

21.6

3.2

TOTAL

95

18.7 ± 5.3

5.7 ± 3.6

NS = not stated. Note: Values are number ± standard deviation.

patients underwent a sub adventitial incomplete peeling of the tumor, and the aortic replacement was made on a temporary bypass between the left subclavian artery and the descending aorta in the remaining sixteen patients. There were only two (9.5%) patients who survived longer than 5 years, and it was concluded that peeling of the aortic adventitia is an inadequate procedure. Similarly, surgical reports dealing with tumors invading the pulmonary artery trunk are limited. Ricci et al. (64) reported pulmonary angioplasty under cardiopulmonary bypass in three patients whose NSCLC invaded the main pulmonary artery trunk; however, all patients died within 25 months following operation. Tsuchiya et al. (59) replaced the bifurcation of the pulmonary artery on cardiopulmonary bypass in six patients. Because all patients died within 30 months from operation, invasion of the pulmonary artery trunk was considered technically resectable but incurable biologically.

INVASION OF THE VERTEBRAL BODY Direct invasion of the vertebral body or the costovertebral angle by an NSCLC other than a superior sulcus tumor is rarely observed. Evidence of bone involvement is difficult to obtain preoperatively because of bone or CT scan's low accuracy in detecting it in less than massive invasion; hence, intraoperative diagnosis is limited by the fact that frozen sections cannot be performed on ossified tissue (70). Treatment options vary among radiation therapy alone, resection by shaving off the bone, and tangential (70) or hemivertebrectomy (32). The largest experience, reported by DeMeester and colleagues (70), provides evidence that for tumors with limited invasion of the periosteum below the third vertebral body, long-term survival (5-year survival, 42%) and cure for selected patients can be anticipated by combining a preoperative radia-


157

tion therapy (30 Gy) and an en bloc resection of the primary tumor and involved vertebral body. Resectability was based on the preoperative radiological absence of bony erosion and intraoperative absence of invasion into the costotransverse foramen. For tumors with more extensive invasion, McCormack (4) reported a 10% survival at 5 years by performing a total vertebrectomy and spinal stabilization; however, no conclusions were drawn as to its value.

INVASION OF THE MEDIASTINAL PLEURA, PERICARDIUM, AND DIAPHRAGM Anecdotal reports in the literature make it difficult to determine the value of surgery for patients with local invasion limited to the mediastinal pleura or fat, pericardium, and diaphragm. However, the results reported at the Memorial Sloan-Kettering Cancer Center (1) on 58 patients with invasion of the mediastinal pleura or fat, pericardium, diaphragm, vagus nerve, azygos vein, or pulmonary vessel justify complete resection in the absence of N2 disease, with a reported 5-year survival rate of 36% (median survival, 32 months).

MISCELLANEOUS Other intrathoracic structures are only rarely invaded by bronchogenic carcinomas. The esophagus is almost frequently invaded by subcarinal lymph nodes rather than by the primary tumor itself, and in this case, prognosis is dismal. In rare circumstances, the tumor fixes to the muscular esophageal wall, and survival can be expected by performing a limited muscular esophageal wall resection. Pleural effusion, whether positive for malignant cells, has not been associated with prolonged survival.

CONCLUSION Improved surgical techniques have increased the feasibility and radicality of extended operations for patients with potentially resectable but locally invasive NSCLC. Advances in the perioperative management and postoperative care, along with a careful patient selection, will likely make the operative mortality and morbidity less prohibitive. However, the thoracic medical and surgical community should promote all efforts

1 58

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to extend the surgical indications for locally advanced NSCLC, making these operations available whenever possible to patients in whom a cure can be achieved when none seems thinkable.

REFERENCES 1. Van Raemdonck DE, Schneider A, Ginsberg RJ. Surgical treatment for higher stage non-small cell lung cancer. Ann Thorac Surg 1992;54:9991013. 2. Naruke T, Goya T, Tsuchiya R, Suemasu K. Prognosis and survival in resected lung carcinoma based on the new international staging system. J Thorac Cardiovasc Surg 1988;96:440--447. 3. Grillo HC Pleural and chest wall involvement. Int Trends Gen Thorac Surg 1985;1:134-138. 4. McCormack PM. Extended pulmonary resections. In: Pearson FG, Deslauriers J, Ginsberg RJ, et aI., eds. Thoracic Surgery. New York: Churchill Livingstone, 1995:897-908. 5. Eschapasse H, Gaillard J, Henry F, et al. Repair of large chest wall defect. Ann Thorac Surg 1981;31:45-51. 6. McCormack PM, Bains M, Beattie EJ Jr, Martini N. New trends in skeletal reconstruction after resection of chest wall tumors. Ann Thorac Surg 1981;31:45-52. 7. McCaughan Be, Martini N, Bains MS, McCormack PM. Chest wall invasion in carcinoma of the lung. J Thorac Cardiovasc Surg 1985;89:836-841. 8. Patterson GA, Ilves R, Ginsberg RJ, et al. The value of adjuvant radiotherapy in pulmonary and chest wall resection for bronchogenic carcinoma. Ann Thorac Surg 1982;34:692-697. 9. Ricci C, Rendina EA, Venuta F. En bloc resection for T3 bronchogenic carcinoma with chest wall invasion. Eur J Cardiothorac Surg 1987;1:23-28. 10. Paone JF, Spees EK, Newton CG, et al. An appraisal of en bloc resection of peripheral bronchogenic carcinoma involving the thoracic wall. Chest 1982;81 :203-207. 11. Shah SS, Goldstraw P. Combined pulmonary and thoracic wall resection for stage III lung cancer. Thorax 1995;50:782-784. 12. Albertucci M, DeMeester TR, Rothberg M, et al. Surgery and the management of peripheral lung tumors adherent to the parietal pleura. J Thorac Cardiovasc Surg 1992;103:8-12. 13. Allen MS, Mathisen OJ, Grillo HC, et al. Bronchogenic carcinoma with chest wall involvement. Ann Thorac Surg 1991;51:948-951. 14. Piehler JM, Pairolero PC, Weiland LH, et al. Bronchogenic carcinoma with chest wall involvement: factors affecting survival following en bloc resection. Ann Thorac Surg 1982;34:684-692. 15. Pancoast HK. Importance of careful roentgen-ray investigations of apical chest tumors. JAMA 1924;83:1407-1411. 16. Shaw RR, Paulson DL, Kee JL Jr. Treatment of the superior sulcus tumor by irradiation followed by resection. Ann Surg 1961;154:29--40. 17. Komaki R, Roth], COX J. Superior sulcus tumors: results of irradiation of 36 patients. Cancer 1981;48:1563-1568. 18. Komaki R, Derus SB, Perez-Tamayo C, et al. Brain metastasis in patients with superior sulcus tumors. Cancer 1987;59:1649-1653.

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Mathisen DJ, Grillo HC Carinal resection for bronchogenic carcinoma. J Thorac Cardiovasc Surg 1991;102:16-23. Mathisen DJ, Grillo HC Carinal resection. In: Pearson FG, Deslauriers J, Ginsberg RJ, et a1. Thoracic Surgery. New York: Churchill Livingstone, 1995:345-354. Abbey-Smith R, Nigam BK. Resection of proximal left main bronchus carcinoma. Thorax 1979;34:616-620. Deslauriers J, Beaulieu M, McClish A. Tracheal-sleeve pneumonectomy. In: Shields TW, ed. General Thoracic Surgery, 3rd ed. Philadelphia: Lea & Febiger, 1989:382-387. Dartevelle P, Macchiarini P, Chapelier A. Tracheal-sleeve pneumonectomy. Ann Thorac Surg 1995;60:1854-1855. Barclay RS, McSwan N, Welsh TM. Tracheal reconstruction without the use of grafts. Thorax 1957;12:177-180. Eschapasse H, Vahdat F, Gaillard J, et a1. Reflexions sur la resection de la trachee inferieure et de la bifurcation bronchique. Ann Chir Thor Cardiovasc 1967;6:63-70. Grillo HC, Bendixen HH, Gephart T. Resection of the carina and lower trachea. Ann Surg 1963;158:889-893. Jensik RJ, Faber JP, Kittle CF, et a1. Survival in patients undergoing tracheal sleeve pneumonectomy for bronchogenic carcinoma. J Thorac Cardiovasc Surg 1982;84:489-496. Tsuchiya R, Goya T, Naruke T, et a1. Resection of tracheal carina for lung cancer. J Thorac Cardiovasc Surg 1990;99:779-787. Roviaro GC, Varoli F, Rebuffat C, et al. Tracheal sleeve pneumonectomy for bronchogenic carcinoma. J Thorac Cardiovasc Surg 1944;107:13-18. Dartevelle P, Macchiarini P. Carinal resection for bronchogenic carcinomas. Semin Thorac Cardiovasc Surg 1996;8:414-425. Grillo He, Mathisen DJ. Upper airway tumors: secondary tumors. In: Pearson FG, Deslauriers J, Ginsberg RJ, et a1., eds. Thoracic Surgery. New York: Churchill Livingstone, 1995:299-311. Chapelier A, Dartevelle P, Lenot B. Chirurgie de la veine cava superieure. Encycl Med Chir 1992;13:42185-42197. Dartevelle P, Levasseur P, Rojas A, et a1. Les remplacements de la veine cave superieure par protheses en PTFE. Chirurgie 1982;108:671-677. Dartevelle P, Chapelier A, Navajas M, et a1. Replacement of the superior vena cava with polytetrafluoroethylene grafts combined with resection of mediastinal-pulmonary malignant tumors: report of 13 cases. J Thorac Cardiovasc Surg 1987;94:361-366. Dartevelle P, Chapelier A, Pastorino U, et a1. Long-term follow-up after prosthetic replacement of the superior vena cava combined with resection of mediastinal-pulmonary malignant tumors. J Thorac Cardiovasc Surg 1991;102:259-265. Inoue H, Shohtsu A, Koide S, Ogawa J, Inoue H. Resection of the superior vena cava for primary lung cancer: 5 years' survival. Ann Thorac Surg 1990;50:661-666. Tsuchiya R, Asamura H, Kondo H, Goya T, Naruke T. Extended resection of the left atrium, great vessels, or both for lung cancer. Ann Thorac Surg 1994;57:960-965. Dartevelle P, Macchiarini P, Chapelier A. Superior vena cava resection and reconstruction. In: Faber LP, ed. Techniques of Pulmonary Resection. Philadelphia: WB Saunders, 1995.

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Piccione W, Faber LP, Warren WHo Superior vena caval reconstruction using autologous pericardium. Ann Thorac Surg 1994;50:417-422. Martini N, Yellin A, Ginsberg RJ, et al. Management of non-small cell lung cancer with direct mediastinal involvement. Ann Thorac Surg 1994;58: 1447-1451. Nakahara K, Ohno K, Mastumura A, Ogawa I, Inoue H. Extended operation for lung cancer invading the aortic arch and superior vena cava. J Thorae Cardiovasc Surg 1989;97:428-433. Ricci C, Rendina E, Venuta F, et al. Reconstruction of the pulmonary artery in patients with lung cancer. Ann Thorac Surg 1944;57:627-633. Martini N, Yellin A, Ginsberg RJ, et al. Management of non-small cell lung cancer with direct mediastinal involvement. Ann Thorac Surg 1994;58: 1447-1451. Whyte RI, Starkey TO, Orringer MB. Tumor emboli from lung neoplasms involving the pulmonary vein. J Thorac Cardiovasc Surg 1992;104:421425. Mansour KA, Malon CE, Craver JM. Left atrial tumor embolization during pulmonary resection: review of literature and report of two cases. Ann Thorac Surg 1988;46:455-456. Shirakusa T, Kimura M. Partial atrial resection in advanced lung carcinoma with and without cardiopulmonary bypass. Thorax 1991;46:484487. Piehler JM, Trastek VF, Pairolero PC, et al. Concomitant cardiac pulmonary operations. J Thorac Cardiovasc Surg 1985;90:662-667. DeMeester TR, Albertucci M, Dawson PI, Montner SM. Management of tumors adherent to the vertebral column. J Thorac Cardiovasc Surg 1989; 97:373-378.

9 H ig h-Dose-Rate Remote Afterload ing Endobronchial Brachytherapy Ritsuko Komaki, Rodolfo C. Morice, and Garrett L. Walsh

Non-small-celliung cancers (NSCLCs) are preferentially treated by surgical resection, whenever all known tumor can be encompassed and the patient can medically tolerate the resection. External radiation therapy is considered the treatment of choice for NSCLCs that are too advanced for resection or for patients who are medically inoperable but whose cancer has not spread beyond the regional lymph nodes. Among those who might be treated with curative intent for their un resectable locally advanced NSCLC, patients usually receive induction chemotherapy, because a few randomized studies (1-3) showed survival benefits for giving chemotherapy before definitive surgery or radiotherapy for patients with stage III NSCLC. However, patients presenting with postobstructive pneumonitis or severe hemoptysis are unable to receive neoadjuvant chemotherapy without an increased risk of neutropenic sepsis or fatal hemorrhage. High-dose-rate (HDR) endobronchial brachytherapy (EBBT) to quickly alleviate airway obstruction or hemorrhage, followed by definitive surgery or external beam radiation therapy with or without induction or concurrent chemotherapy, is becoming one of the important modalities in managing patients with NSCLC. HDR EBBT is also one important method for palliation to relieve cough, obstructive pneumonia, shortness of breath, or hemorrhage due to recurrent or metastatic endobronchial lesion after previous definitive thoracic irradiation. Although a palliative external radiation might be 20-40% effective in reso163

1 64

Chapter 9

lution of atelectasis, it will usually take a longer time to achieve aeration (4,5). Limitations of external beam radiotherapy are related to the toxicity of treatment, which is often significant. These include radiation esophagitis, pneumonitis, lung fibrosis, myelitis (rarely), and other surrounding tissue toxicities. Also, for patients who develop endobronchial metastasis from cancers other than lung cancer and who are unable to receive chemotherapy because of postobstructive pneumonia or hemorrhage, HDR EBBT offers a fairly rapid resolution of the local problem without causing the above-mentioned problems from external radiation therapy. This method can be combined with a palliative short course of external thoracic radiation therapy if cause of the symptoms is related to extrinsic compression as well as the endobronchial lesion. Brachytherapy to treat endobronchial lesions has been available for many years, but its use has increased substantially over the last decade due to development of equipment that allows for safe and reliable methods of delivering this treatment.

BAC.3 months) to benefit from palliation that will not occur immediately. History and physical examination including performance status; routine blood work including coagulation panels and complete blood count; and lung function tests and chest x-rays are necessary and must be reviewed by a radiation oncologist and a physician who will perform bronchoscopy to assess the patient's suitability for endobronchial or endotracheal therapy as well as tumor location and volume. Bronchoscopy is performed usually as an outpatient procedure under sedation and local anesthesia. Tumors that protrude into the lumen are considered suitable, as opposed to extrinsic tumors that compress the bronchus or the trachea. The location of the tumor, its length along the bronchus or trachea, and the percent occlusion of the lumen are recorded. The distance from the nostrils is noted, and the tumor is photographed for future comparisons.

RESULTS At the MD Anderson Cancer Center, 81 patients with lung cancer have undergone EBBT, with the procedure described previously between 1988 and 1993 (10). Fifty-four patients were male. Age ranged from 28to 77-years-old, with a median age of 59. Fifty-nine patients (73%) had Karnofsky performance status (KPS) of 70 or higher. The presenting symptoms showed 65 patients with shortness of breath, 53 patients with cough, 22 patients with hemoptysis, 31 patients with wheezing, and 11 with chest pain. Eight patients had severe, 13 had moderate, and 44 had mild shortness of breath. Sixteen (20%) did not have shortness of breath at presentation. The histology or cytology showed 37 patients with squamous, 14 with adenocarcinoma, 13 with NSCLC without specific cell types, 7 with adenoid cystic, 5 with small cell, 3 with large cell, and 2 with carcinoid tumor. Of the patients, 11% (9/81) had endobronchial treatment three times with a 2-week interval between the EBBT, and 67% (54/81) had treatment twice with 2-week intervals between the EBBT. Twenty-two percent (18/81) of the patients had only one application because they had excellent initial responses. About one-half had rapid progression of their disease and could not receive the second EBBT because of rapid deterioration. Ninety-three percent of the patients had 1500 cGy calculated at a distance of 6 mm from the center of sources for mainstem lesions, and the remaining 7% of the patients had the same dose calculated at a distance of 7.5 mm from the center of the sources for the tracheal lesions. Twenty-six patients (32%) had significant improvement, and 25 patients had moderate improvement. Seventeen patients had minimal

High-Dose-Rate Remote Afterloading Endobronchial Brachytherapy

1 71

improvement, and 11 patients had no change; 5 patients became slightly worse, and 2 patients became much worse after EBBT. Therefore, 87% (75/86) achieved some response, and of these 32% (22/69) had an excellent response objectively evaluated by bronchoscopy and chest x-ray (Figs. 9.4 and 9.5). The median duration of responses was 4.5 months, and the patients who had an excellent response had much longer survivals. Median survival was 5 months for all patients, ranging from 2 weeks to 43 months. There was no significant difference based on histology, although small-cell lung cancer (SCLC) patients appeared to be worse in terms of symptomatic relief and survival, as they were referred to MD Anderson at their terminal stage and they had a significant extrinsic component to their airways compressed by mediastinal nodal disease. The survival time was correlated to duration of palliation, especially for shortness of breath. The 16 patients who had excellent relief of shortness of breath after the treatment had a median survival of 13.3 months compared to 65 patients who had less response or no relief of the symptoms. Their median survival was 5.4 months (P = 0.0135). Other symptomatic relief was not significantly correlated with survival. Thirty-one patients had lesions on the left, 48 patients had lesions on the right (1 patient had bilateral lesions), and 2 patients had main lesions at the carina. The location of the tumor was correlated to the complication rate. The two patients who had lesions at the carina developed fatal complications due to 1) tracheal malacia-one patient was treated close to home with four applications of HDR EBBT after one attempt at our institution-and 2) a fistula-one patient received two applications of HDR EBBT and had a few biopsies and ultimately developed infection and the fistula. Three patients developed pneumothoraces that resolved after insertion of a chest tube without incident. However, the pneumothoraces did not cause fatal complications. One patient developed a severe complication due to tumor necrosis, although it did heal. Two patients developed stenosis of the trachea, and one patient developed hemorrhage. The last follow-up analysis in 1996 revealed six patients alive with disease and one patient alive without any evidence of disease at 43 months after completion of treatment. Seventy patients died of disease, three patients died with intercurrent disease, and one patient died of a second malignancy.

DISCUSSION Endobronchial or endotracheal lesions can cause life-threatening symptoms including shortness of breath, postobstructive pneumonitis, and hemoptysis. The majority of patients with lung cancer have either locally unresectable, medically inoperable, or metastatic disease.

, 72

Chapter 9

Fig. 9.4. A. This 68-year-old man presented with a 3-month history of increasing shortness of breath and hemoptysis, and the bronchoscopy revealed an exophytic lesion involving the carina, with 70% obstruction of right main bronchus and 50% obstruction of left main bronchus. The biopsy showed squamous cell carcinoma arising from the carina. Staging work-up indicated the patient stage to be stage Ills T4 N2 MO. B. Chest x-ray after two applications of HDR EBBT to give 3000 cGy at 6 mm to both main bronchi and at 7.5 mm to the distal trachea on 8/9/94 and 9128/94, with external radiation therapy 45 Gy in 15 fractions between 8/1 1 and 8/29/94. Complete resolution of endotracheal and endobronchial lesion was achieved with excellent symptomatic relief.

Fig. 9.5 A. This 72-year-old man has squamous cell carcinoma involving the right main bronchus (staged T3 N 1 MO) and was previously treated with external radiation therapy 64 Gy in 32 fractions outside of the MD Anderson Cancer Center; 1 month prior to consult to our clinic. He developed increasing shortness of breath, cough, and fever 2 weeks before this visir, and chest x-ray showed a total atelectasis on the right lung. He received 3000 cGy at 6 mm from the center of the sources to his right main stem on 2/1/95 and 2122/95. B. Chest x-ray obtained 1 month after completion of two EBBTs. Excellent symptomatic relief continued until the patients death due to distant metastasis.

174

Chapter 9

Radiation therapy is able to control local symptoms, although externalbeam radiotherapy requires a longer time to alleviate the symptoms compared to EBBT. To accomplish rapid alleviation of symptoms and improve functional status without causing esophagitis, pneumonitis, bone marrow suppression, or occasional myelitis, afterloading HDR brachytherapy is one of the optimal modalities for patients who need either short-term palliation or quick resolution of symptoms before receiving definitive treatment (such as chemotherapy and radiation therapy, with or without surgery). In theory, LDR brachytherapy offers the advantage of better tolerance in normal tissues; however, it has not shown a clinical benefit for endobronchial treatments when compared to HDR (Tables 9.1 and 9.2). LDR requires hospitalization, and displacement of the catheter for patients with respiratory distress and cough would be a major concern. There has been proof that rapidly proliferating tumors might be more beneficially treated by rapid radiation therapy to overcome proliferation of malignant cells (11, 12). Rates with LDR (8, 9, 13-17) were 53-92% in relieving symptoms, 17-91% improvement on the chest x-ray and 60-93% improvement bronchoscopically, compared to the results by HDR (IS, 18-28), which showed symptomatic relief between 50% and 99%, 36-11% improvement on the chest x-ray, and 74-100% improvement bronchoscopically. Fatal complications (hemoptysis) have been reported between 0 and 12% in LDR series and between 0 and 32% in HDR series (see Tables 9.1 and 9.2). Our results among responders showed 9 out of 81 patients Table 9.1.

Author (ref), yr

Schray et a1. (8), 1988 Locken et al. (B), 1990 Roach et a1. (9), 1990 Lo et a1. (14), 1992 Mehta et a1. (15), 1992 Paradelo et a1. (16), 1992 Raju et a1. (17), 1993 TOTAL LOR

Survey of LOR endobronchial reports

No of patients

65 18 17 87 66 32

39 324

No. of Rxs

Symptoms improved

X-ray improved

(%)

(%)

Bronchoscopy improved (%)

30 Gy at 5-10mm 16-39 Gy at 10mm 30 Gy at 5mm 25 Gy at 20mm 20-30 Gy at 20mm 20-56 Gy at 8-16 mm

1

NA

NA

60

11

1

83

NA

70

NA

1

53

17

60

0

1

59

NA

76

3

1

78

78

93

6

1

66

21

85

9

7-28 Gy at 10mm

1

79-92

91

89

0

53-92%

17-91%

60-93%

Dose

= low dose rate; Rxs = treatments; NA = not available.

Fatal hemoptysis

(%)

0-11%

High-Dose-Rate Remote After/oading Endobronchial Brachytherapy Table 9.2.

Survey of HDR endobronchial reports

Author (refJ. yr

Burt et al. (18), 1990 Miller & Phillips (19), 1990 Stout et al. (20), 1990 Aygun et al. (21), 1992 Bedwinek et al. (22), 1992 Mehta et al. (15), 1992 Sutedja et al. (23), 1992 Speiser & Spratling (24), 1993

No. of patients

50 88

100 62 38 31 31 144

151 Zajac et al. (25), 1993 Chang et al. (26), 1994 MDACC (27), 1995 Macha et al. (28), 1995 TOTAL

175

82 76 81 365 1299

HDR = high dose rate; RXs

Dose

15-20 Gy at lOmm 10 Gy at 10mm 15-20 Gy at 10mm 5Gyat 10mm 6Gyat lOmm 4Gyat 20mm 10 Gy at 10mm 10 Gy at 10mm 7-5 Gy at lOmm 10-47 Gy at 10mm 7Gyat 10mm 15 Gy at 6mm 5 Gyat 10mm

Symptoms X-ray No. of improved improved Rxs (%J (%J

Bronchoscopy improved

Fatal hemoptysis

(%J

(%J

1

50-86

46

88

1.0

3

NA

NA

80

o

1

50-86

46

NA

NA

3-5

NA

36

76

15.0

3

76

64

82

32.0

4+

88

71-100

85

3.0

3

82

NA

NA

3

85-99

NA

80

3

32.0 7.0

8.6

1-5

82

NA

74

o

3

79-95

NA

87

4.0

1-2

85

75

80

2.0

3-4

66

NA

NA

21.0

1-4

50-99%

36-100%

74-100%

0-32%

= treatments; NA = not available; MDACC = MD Anderson Cancer Center. (11 %) developed complications with 2 patients having fatal complications, although both of them had lesions at the carina extending to the main bronchus or trachea and were treated during an earlier period of this trial. We have not seen any fatal complications related to the treatment in the past several years. When the patients receive two or more catheters, the complication rate appears to be higher compared to onecatheter application. This might be related to the location of the tumor, its volume, and possible inhomogeneous dose distribution. It seemed that the patients who had lesions at the carina developed more serious complications compared to the rest of the lesions, although the number was too small to draw any definitive conclusions. All right-sided lesions had higher complications compared to those on the left side. We did not

176

Chapter 9

appreciate differences in the complication rate based on upper and lower lobe lesions, although Bedwinek et al. (22) reported higher complications in upper lobe lesions. A more recent one institutional study (29) showed that patients who were treated by HOR had more significant bronchoscopic improvement compared to the results from LOR (P = 0.05), although this was not a randomized study. No significant difference in complication rates was found and was low with both treatment approaches (29). Radiobiologic bases between LOR and pulsed HOR has not shown any major difference in the cervical carcinoma with respect to tumor control and late-normal tissue effect (11). Once fractionated HOR is used based on equivalent dose of LOR, the normal tissue and tumor effects will become similar (30). Subsequently, the choice of LOR and HDR will be dependent on the patient's convenience and economical aspects. Technical accuracy to avoid overdose to the normal mucosa is a critical aspect in HDR brachytherapy. Differential dose, depending on the size of the lumen, has been tried to reduce normal tissue complications and might also improve local tumor control by red ucing overdosage and underdosage, respectively (31). In addition, the use of radiochromic film to measure dose distributions might contribute to homogeneous dose distribution in the lumen (32). Survival was correlated to the improvement of symptoms, especially dyspnea. To prolong the duration of palliation as well as to hasten the degree of improvement, we are now trying to give Paclitaxel infusion for 24 hours followed by EBBT. Paclitaxel interacts and stabilizes microtubules to block cell cycle at the G2/M phase which is sensitive to radiation (33). Some investigators (34) treated patients with locally advanced lung cancer by using combined external and EBBT. The external radiation therapy was given with a dose of between 57 and 66 Gy, followed by two to four 192Ir endobronchial treatments, which delivered 5 to 15 Gy at a distance of 10 mm from the center of sources; this resulted in complete response in 77% of patients and partial response in 13% of patients. EBBT can be used as a boost technique for patients with prominent endobronchial lesions without distant metastases, to improve local control as well as to reduce complications to the surrounding normal tissue from external radiation therapy, especially in patients treated with combined chemotherapy and radiation therapy. Other modalities to eliminate endobronchial or endotracheal obstruction are mechanical removal of the tumor with combination ofYAG laser or diathermia (35), although the mechanical removal of the tumor might cause severe bleeding (36, 37). Electrocautery (38), cryotherapy (39), and photodynamic therapy (40) have been described elsewhere. As tumor regresses by application of HOR EBBT with or without external radiation therapy, stenosis at the site of the previous location of the tumor could be encountered, which can be dilated by stent insertion (41).

High-Dose-Rate Remote Afterloading Endobronchial Brachytherapy

177

CONCLUSIONS HDR afterloading endobronchial radiation is an effective palliative modality, relieving obstructive symptoms rapidly in a majority of treated patients. Prospective trials are needed to optimize total doses, dose rates, fraction sizes, number of fractions, and adequate dosimetry, and to investigate HDR EBBT combined with radiosensitizers or other modifiers to minimize complications and maximize prolongation of durable palliation or survival. Prolonging local control might increase the survival of patients with ominous locally advanced lung cancer if chemotherapy is added. The role of EBBT in the curative approach for lung cancer also needs to be investigated.

REFERENCES 1.

Dillman RO, Seagren SL, Propert KJ, et al. A randomized trial of induction chemotherapy plus high dose radiation versus radiation alone in stage III nonsmall cell lung cancer. N Engl J Med 1990;323:940-945. 2. Sause WT, Scott C, Taylor S, et a1. Radiation Therapy Oncology Group 88-08 and Eastern Cooperative Oncology Group 45-88: Preliminary results of a phase 1II trial in regionally advanced, unresectable nonsmall cell lung cancer. J Natl Cancer Inst 1995;87:198-205. 3. Roth JA, Fossella F, Komaki R, et a1. A randomized trial comparing perioperative chemotherapy and surgery with surgery alone in resectable stage IlIA nonsmall cel1lung cancer. J Natl Cancer Inst 1994;86:673-680. 4. Slawson RG, Scott RM. Radiation therapy in bronchogenic carcinoma. Ther RadioI1979;132:175-176. 5. Phillips TL, Miller RJ. Should asymptomatic patients with inoperable bronchogenic carcinoma receive immediate radiotherapy? Am Rev Respir Dis 1978;117:411-414. 6. Yankauer S. Two cases of lung tumor treated bronchoscopically. NY Med J 1922;115:741-742. 7. Moylan 0, Strubler K, Unal AB, et al: Transbronchial brachytherapy of recurrent bronchogenic carcinoma: new approach using flexible fiberoptic bronchoscope. Radiology 1983;147:253-254. 8. Schray MF, McDougall Jc, Martinez A, Cortese DA, Brutinel MW. Management of malignant airway compromise with laser and low dose rate brachytherapy. Chest 1988;93:264-269. 9. Roach M, Leidholdt EM, Tater BS, Joseph J. Endobronchial radiation therapy in the management of lung cancer. Int J Radiat Oncol BioI Phys 1990;18: 1449-1454. 10. Delclos ME, Komaki R, Garden A, et al. High dose rate remote afterloading endobronchial brachytherapy for recurrent endobronchial lesions. Radiology 1996;281 :279-282. 11. Brenner OJ, Hall EJ. Fractionated high dose rate versus low-dose-rate regimens for intracavitary brachytherapy of the cervix. I. General considerations based on radiobiology. Br J Radiol1991;64:133-141.

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13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

Begg AC, Hofland I, Moonen L, et al. The predictive value of cell kinetic measurements in a European trial of accelerated fractionation in advanced head and neck tumors: an interim report. Int J Radiat Oncol BioI Phys 1990;19:1449-1453. Locken P, Dillon M, Patel P, et al. Palliation of locally recurrent nonsmall cell lung cancer with low dose rate iridium-192 endobronchial implant combined with localized external beam irradiation. Endocurie Hypertherm OncoI1990;6:217-222. Lo TCM, Beamis JF Jr, Weinstein RS, et al. Intraluminal low-dose rate brachytherapy for malignant endobronchial obstruction. Radiother Oncol 1992;23: 16-20. Mehta MP, Peterent 0, Chosy L, et al. Sequential comparison of low dose rate and hyperfractionated high dose rate endobronchial radiation for malignant airway occlusion. Int J Radiat Oncol BioI Phys 1992;23:133-139. Paradelo Jc, Waxman MJ, Throne IJ, et al. Endobronchial irradiation with 192 Ir in the treatment of malignant endobronchial obstruction. Chest 1992;102:1072-1074. Raju PI, Roy T, Mcdonald RD, et al. Low dose rate endobronchial brachytherapy in the treatment of malignant airway obstruction. Int J Radiat Oncol BioI Phys 1993;27:667-680. Burt PA, O'Driscoll BR, Notley HM, Barber PV, Stout R. Intraluminal irradiation for the palliation of lung cancer with the high dose rate microselectyron. Thorax 1990;45:765-768. Miller JI, Phillips TW. Neodymium:YAG laser and brachytherapy in the management of inoperable bronchogenic carcinoma. Ann Thorac Surg 1990;50: 190-196. Stout R, Barber PY, Burt PA, O'Driscoll BR, Notley M. Intraluminal brachytherapy in bronchial carcinoma. Br J RadioI1990;63(Suppl):16. Abstract. Aygun C, Weiner S, Scariato A, Spearman 0, Stark L. Treatment of nonsmall cell lung cancer with external beam: radiotherapy and high dose rate brachytherapy. Int J Radiat Oncol BioI Phys 1992;23:127-132. Bedwinek J, Petty A, Bruton C, Sofield C, Lee L. The use of HDR endobronchial brachytherapy to palliate symptomatic endobronchial recurrence of previously irradiated bronchogenic carcinoma. Int J Radiat Oncology BioI Phys 1992;22:23-30. Sutedja G, Baris G, Schaake-Koning C, van Zandwijk N. High dose rate brachytherapy in patients with local recurrences after radiotherapy of nonsmall cell lung cancer. Int J Radiat Oncol BioI Phys 1992;24:551-553. Speiser B, Spratling L. Intermediate dose rate remote afterloading brachytherapy for intraluminal control of bronchogenic carcinoma. Int J Radiat Oncol BioI Phys 1990;18:1443-1448. Zajac AJ, Kohn ML, Heiser 0, Peters JW. High dose rate intraluminal brachytherapy in the treatment of endobronchial malignancy. Radiology 1993;187:571-575. Chang L-FL, Horvath J, Peyton W, Ling S-S. High dose rate afterloading brachytherapy in malignant airway obstruction of lung cancer. Int J Radiat Oncol BioI Phys 1992;28:211-212. Komaki R, Morice RC, Walsh GL, Garden AS, Advise M. High dose rate remote afterloading endobronchial brachytherapy. In: Aisner J, ed. Comprehensive Textbook of Thoracic Oncology. Baltimore: Williams & Wilkins (In press).

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38. 39. 40.

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Macha HN, Wahlers B, Reichle C, von Zwehl D. Endobronchial radiation therapy for obstructing malignancies: ten years' experience with iridium192 high-dose radiation brachytherapy afterloading technique in 365 patients. Lung 1995;173:271-280. Lo TC, Girshovich L, Healey GA, et al. Low dose rate versus high dose rate intraluminal brachytherapy for malignant endobronchial tumors. Radiother OncoI1995;35:193-197. Hall EJ, Brenner OJ. The dose-rate effect revisited: radiobiological considerations of importance in radiotherapy. Int J Radiat Oncol BioI Phys 1991; 21:1403-1414. Jones B, Bleasdale C, Tan LT, et al. The achievement of isoeffective bronchial mucosal dose during endobronchial brachytherapy. Int J Radiat Oncol BioI Phys 1995;33:195-199. Skwarchuk MW, Ochran T, Komaki R, Cundiff J, Travis EL. The use of radiochromic film to measure dose distributions resulting from high dose rate iridium-102 afterloading treatment. Int J Radiat Oncol BioI Phys 1996; 34: 173-181. Schiff PB, Fant J, Hurwitz SB. Promotion of microtubule assembly in vitro by taxol. Nature 1979;277:665-667. Cotter GW, Herbert DE, Ellingwood KE. Inoperable endobronchial obstructing lung carcinoma treated with combined endobronchial and external beam irradiation. South Med J 1991;84:562-565. Oumon JF, Shapsay S, Bourceraou J, et al. Principles for safety in application of neodymium-YAG laser in bronchology. Chest 1984;86:163-168. Mathisen OJ, Grillo HC Endoscopic relief of malignant airway obstruction. Ann Thorac Surg 1989;48:469-475. Mehta AC, Livingstone DR. Biopsy excision through a fiberoptic bronchoscope in the palliative management of airway obstruction. Chest 1987;91 :774-775. Hooper RG, Jackson FN. Endobronchial electrocautery. Chest 1985;87:712714. Walsh OA, Maiwand MO, Nath AR, et al. Bronchoscopic cryotherapy for advanced bronchial carcinoma. Thorax 1990;45:509-513. Smith SGT, Bedwell J, MacRobert AJ, et al. Experimental studies to assess the potential of photodynamic therapy for the treatment of bronchial carcinomas. Thorax 1993;48:474-480. Cortese OA, Edell ES. Role of phototherapy, laser therapy, brachytherapy and prosthetic stents in the managements of lung cancer. Mayo Clin Proc 1993;14:149-150.

10 Three-Dimensional Conformal Radiotherapy in Bronchogenic Carcinoma Bahman Emami and Mary V. Graham

Radiotherapy represents one of the primary treatment modalities for patients with carcinoma of the lung. Second to surgery, it remains the modality with the highest response rates and potential for cure. Nonetheless, the results of nonsurgical therapy for patients with non-smallcell lung cancer (NSCLC) have remained suboptimal. Local control with or without chemotherapy is 20-40%, and 5-year survival is less than 10-19(Yo (1-5). Still, radiotherapy represents one of the primary treatment modalities for patients with carcinoma of the lung (1, 2, 4, 6-8). With radiotherapy, local control is directly related to dose (4, 9) as well as the technical accuracy with which the dose is delivered to the target volume (4, 8, 10). The complex anatomy of the thorax, with the proximity of critical normal structures (i.e., the spinal cord and the lungs) to the primary tumor and unacceptable complications, has set a limit on the prescription dose to between 60 and 70 Gy (1, 2, 7, 11). In an effort to reduce the dose to the normal structures, a variety of "boost" techniques have evolved including oblique ports (4, 6), brachytherapy (12), high linear energy transfer (LET) particle beams (13), and intraoperative radiotherapy (14). There are, however, serious technicallimitations on clinical applications of each one of these technologies, which preclude their systematic use. Even with the addition of systemic therapy, local control and survival have remained poor (1, 2, 7). Use of chemoradiotherapy has resulted in modest improvement in survival, 181

182

Chapter 10

but local control remains poor-and a significant cause of treatment failure and resultant morbidity (3, 7, 15). In the last decade, technological advances in the areas of computers, faster CT scans, and graphics have given birth to three-dimensional conformal radiotherapy (3D-CRT) (16, 17). 3D-CRT has enabled radiation oncologists to delineate target volumes and normal tissue structures with superior accuracy. Dose calculation, with fully three-dimensional algorithms, has allowed clinicians to increase the dose to the tumor and decrease the dose to uninvolved normal structures. This has created a new opportunity for possible improved therapeutic outcome. The potential of this new technology and treatment of bronchogenic carcinoma are reviewed in this chapter. The specific goal of 3D-CRT is to provide a mechanism for increasing the tumor dose as a means of enhancing local tumor control (18). Laboratory and clinical reports indicate that there is a direct correlation between radiation dose and the probability of achieving local control in a variety of tumors (19-21). The maximum dose that can be delivered to the tumor has classically been restricted by the tolerance of normal tissues with the high-dose volume. The radiation dose-response relationships for tumor control and normal tissue injury are site specific and are influenced by a number of factors. The more important technical factors include 1) the precision of target volume definition and of dose delivery, 2) the dose given to this volume, and 3) the degree to which uninvolved normal tissues are excluded from the treatment volume (10, 22, 23). With 3D-CRT, it is often possible to design the spatial dose distribution to conform to the target volume while not increasing--or even reducing-the dose to normal tissues. This approach, therefore, has the potential to decrease the probability of normal-tissue toxicity and may permit dose escalation to the tumor to produce higher rates of local control (24-26).

DOSE-RESPONSE RELATIONSHIP IN CARCINOMA OF THE LUNG The Radiation Therapy Oncology Group (RTOG) conducted a series of dose escalation studies in the 1970s that established 60 Gy /30 fractions/6 weeks as the standard dose of radiation therapy for NSCLC (4). Although a tumor control rate of 50-60% was reported in trials with doses of 60 Gy (IS), later studies with 65 Gy using standard fractionation documented a tumor control level of only 10% at 2 years (7). The reason for the discrepancy is that in the former studies, assessment of local control was based on radiographic findings (chest x-ray), and in the latter studies it was based on biopsies 2 years after completion of course of treatment (7).

Three-Dimensional Conformal Radiotherapy in Bronchogenic Carcinoma

183

Based on principles advocated by Fletcher (21), the generally accepted dose for eradication of microscopic disease is 50 to 60 Gy and for gross disease of 1- to 3-cm dimension is approximately 75 Gy. Considering the advanced status of lung cancer patients when seen in the radiation therapy departments, it becomes evident why the modest dose of 60 Gy is inadequate for tumor control. From a biological point of view, the relationship between probability of tumor control and dose, above a certain threshold, is described as a sigmoidal curve. Theoretical tumor control probability (TCP) curves for three spherical tumors of I, 2, and 4 cm in diameter and one TCP curve that would incorporate all three tumor sizes in equal proportions are shown in Figure 10.1. From the report of LeChevalier et al. (7), there is a known data point of 10% TCP dose for lung cancer of 65 Gy. If one assumes the same slope for lung cancer as for the hypothetical curve shown in Figure 10.1, the TCP for bronchogenic carcinoma can be estimated for various tumor sizes. For example, tumor sizes larger than 4 cm can potentially have a 10% TCP dose of approximately 80 Gy. From this hypothetical curve, it is not unreasonable to assume that a 50-80% TCP for lung cancer would

-100 ~ o

1 em diameter = 107 clonogens eDo = 3.5 Gy

""-"

~

80

.£l ~

.0

a

L-

a.. a

L......

C

60

40

a

() L-

a E

::J

f-

20 0

52 56 60 64 68 72 76 80 84 88 92

Dose (Gy) Fig. 10.1.

Theoretical tumor control probability curves for three spherical tumors (thin solid lines) and one that would result from a study incorporating into the dose-response analysis all three tumors sizes in equal proportion (broken line). Hypothetical dose-response curve for lung cancer using similar shape and one data point of dose for 10% tumor control (5) are shown (thick broken line). Assuming similar slopes and considering the degree of shift to the right for lung cancer, hypothetical dose-response curves for various tumor sizes of lung cancer are also depicted (solid thick lines). (Reproduced with permission from Emami B. Three-dimensional conformal radiation therapy in bronchogenic carcinoma. Seminars in Radiation Oncology 1996;6:92-97.

1 84

Chapter 10

require doses of 100 Gy or more. Because with radiation therapy many factors influence local control probability, the slope of the curve and the dose for control of a given tumor may vary significantly.

INFLUENCE OF LOCAL CONTROL ON SURVIVAL Most clinical results available today have been obtained with doses ranging from 40 to 65 Gy. A total of 376 patients with stage Tl-3 NO-2 carcinoma of the lung entered the RTOG study (RTOG 73-01) to evaluate the effect of different doses of radiation (4, 15). The results indicated a better 3-year survival with 60 Gy compared to lower doses of radiation. Patients treated with 60 Gy had an overall intrathoracic failure rate of 33% at 3 years, compared to 42% of those treated with 50 Gy, 44% of patients receiving 40 Gy split-course, and 52% of those treated with 40Gy continuous course (15). Patients surviving 6 to 12 months exhibited a statistically significant increased survival when the intrathoracic tumor was controlled. Patients treated with 50 to 60 Gy showing tumor control had a 3-year survival rate of 22% versus 10% if they had intrathoracic failure (P = 0.05). In patients treated with 40 Gy (split or continuous), the respective survival rate was 25% in patients with thoracic tumor control versus 5% if the tumor was not controlled. This and additional studies have concluded that higher doses of radiation yield a greater proportion of complete response, higher intrathoracic tumor control, and better survival in NSCLC (4, 15,27,28). In RTOG 73-01, tumors less than 3 cm in diameter had a tumor control of 60% in contrast to only 40% for larger lesions (15). These observations support the need for higher doses of radiation to control larger tumors. However, this approach must be tempered by the effect of increasing the radiation dose to the surrounding normal tissues and the possibility of inducing life-threatening or fatal complications (29). In protocol RTOG 83-12, by using CT scans, computerized treatment planning, and dose optimization procedures, 75 Gy in 28 fractions was delivered to the gross tumor volume seen on CT scan, while the potentially involved lymph nodes in the hila and mediastinum received 50.4 Gy in 28 fractions (30). The results of this study revealed a median survival of 10 months with a 1-year survival of 41 % and a 3-year survival of 18% (31). These results were fairly comparable to a Cancer and Leukemia Group B (CALGB) protocol that analyzed only good risk patients and gave chemotherapy followed by 60 Gy of conventional fractionated radiotherapy (2). The importance of this study was that a significant proportion of patients in the RTOG 83-12 protocol would not have been eligible for the CALGB protocol based on exclusion criteria requiring a high performance status or minimal weight loss; unfortunately, these characteristics are common to lung cancer patients. Thus


185

the results of RTOG 83-12 suggested that accelerated fractionation concomitant boost radiotherapy alone could have comparable results to combined chemoradiotherapy regimens, possibly with considerably less toxicity. In an attempt to reduce late toxicity, the RTOG conducted a multiinstitutional prospective study on the effect of hyperfractionation on tumor control and survival of patients with inoperable NSCLC. Fractions of 1.2 Gy were administered twice a day (at 4- to 6-hour intervals). Patients were randomized to minimum total doses of 60.0 Gy, 64.8 Gy, 69.6 Gy, 74.4 Gy, and 79.2 Gy. Among 519 patients, 248 were favorable (Karnofsky performance status of 70 to 100 and weight loss of less than 5%) and 271 were unfavorable. No significant difference in disease-free survival was found in unfavorable patients among the five arms of the study. In favorable patients, there was a benefit in survival for tumor dose level of 69.6 Gy when compared with lower doses (9). It is not clear why the higher doses did not result in improved survival. There was increased pulmonary toxicity, and it is thought that this may have negatively interacted and produced inferior survival. It is important to note that patients in this protocol were treated with conventional twodimensional (20) radiotherapy technology.

EXPERIENCE WITH 2D-CONFORMAL RADIOTHERAPY Despite two decades of 2D-technology use, there are serious limitations to this technique, including: • Lack of realistic appreciation of target volumes. • Lack of appreciation of real volumes of normal tissue/organs irradiated to various doses. • Deficiencies in the algorithms of computing dose. • Failure to compute dose throughout the volume of interest. • Restriction of treatment to coplanar beams. • Failure to provide estimates of error. • Lack of tools to compare and judge rival plans. • Inadequate definition of geometric coverage of anatomic structures by external beams. • Failure to provide tools for specifying and verifying the accuracy of treatment delivery. With the current practice of radiotherapy (simulation film and possibly one slice of a CT scan), it is nearly impossible to accurately delineate target volumes that need to be radiated or to accurately estimate normal tissue volumes that need to be spared. Moreover, there is little information on the true volumes of normal tissues at risk, which are radiated to

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various doses of radiation therapy, and how these factors relate to clinical complication and later risks. Another important cause of deficiencies in the current 20-radiotherapy technique is the limited coplanar beam arrangement as well as its demonstration on limited slices of CT scan. By restricting beam arrangement and planning from one slice CT, the planner/radiotherapist assumes proper geometric volume coverage of anatomical structures by a given plan, which may not be a correct assumption. Finally, judgment of the merits of rival plans of treatment is difficult, secondary to the lack of information supplied by 20 plans. For these reasons, 3-D CRT has the potential to become a unique tool for appropriately increasing tumor dose and decreasing normal tissue complications (16, 32).

TECHNICAL RESULTS Three-dimensional treatment planning has led to a number of changes in the clinical approach to patient treatment. Steps for 3D treatment planning include: • • • • • • •

CT scanning in treatment position. Establishment of treatment and planning coordinate system. Target delineation. Normal organ delineation. Beam arrangement, number, aperture, and weight arrangement. Plan optimization. Plan evaluation, including normal organ, partial volume dose tolerances, and target coverage.

Conformal 3D treatment planning has resulted in an improved ability to delineate a target and to confine the dose to the intended target. It has also made it possible to delineate normal tissues and thus to quantitate normal tissue tolerance. Clinical experience has shown that there are significant differences in tumor coverage when blocking is designed using a 3D treatment planning system as opposed to a conventional simulated approach. In treating fourteen patients with NSCLC, Vijayakumar et al. (33) have shown the superiority of beam's eye view technique as opposed to 20 technique for tumor coverage and the sparing of normal tissues. Similar results have also been reported by Hodapp et al. (34). One of the driving forces spurring the development of 3D treatment planning is the need for treatment techniques that allow more conformation of the high-dose region to the target volume while minimizing normal tissue dose. Studies on 3D radiotherapy planning in lung cancer at the Radiation Oncology Center at Washington University, St. Louis,


187

was initiated in 1986 with participation in a National Cancer Institute (NCI) contract (35). In these planning experiments, the patients with advanced lung cancer were planned either with standard and/ or traditional 20 techniques as well as with 3D technology available at the institutions involved in the contract (30). Results of the investigation revealed that 3D treatment planning has significant potential for improving radiation treatment planning in lung cancer, both for tumor coverage and for sparing of normal tissue from high doses of radiation. It thus has the potential of becoming a significant step toward uncomplicated local-regional control of lung cancer. Graham et al. (36) compared four traditional beam arrangements (as routinely practiced in their clinic) and 3D-CRT treatment plans in ten patients with advanced bronchogenic carcinoma using full 3D technology. Evaluations were done using dose-volume histograms (DVH), dose statistics, dose surfaces, and an evaluation program developed through a contract with the NCI. Analysis confirmed an earlier observation that 3D technology produces better delineation of target volumes, better coverage of target volumes by the prescribed dose, and significantly improved protection of the critical structures from high doses of radiation therapy. It was found that beam arrangements commonly used for the treatment of NSCLC were often inadequate to safely deliver tumor doses of greater than 70 Gy. Three-dimensional conformal treatment plans with multiple-beam arrangements allowed dose to gross tumor to be escalated to at least 80 Gy while maintaining acceptable or improved doses to the normal surrounding tissues. The main dose-limiting structure for tumors of the lung is the surrounding lung tissue. While this tissue has a very low threshold dose for injury (approximately 20 Gy with conventional fractionation), the volume of normal lung tissue that can receive this dose, particularly in diseased lungs, has yet to be determined. Preliminary data were reported from Memorial Sloan-Kettering and published by Armstrong et al. (37) on 18 patients with NSCLC who were treated with 3D technology. In this small series, they experienced one grade III and one grade IV pulmonary toxicity. These higher grade pulmonary toxicities were correlated with 49% of the lung volume receiving more than 25 Gy. Martel et al. (29) from the University of Michigan have also reported on mean lung doses in 42 lung cancer patients who were divided into groups that did or did not have pulmonary complications. Patients who developed pneumonitis had received a higher dose to individual lungs (35 Gy and 18 Gy, respectively) than the patients who did not develop pneumonitis (29 Gy and 18 Gy, respectively). Martel et al. (29) also evaluated normal tissue complication probability (NTCP) model and DVH analysis in the design and implementation of dose escalation protocols for lung cancers. They were able to correlate the incidence of pneumonitis with NTCP calculations. This correlation allowed separation of patients at low and high risk of complications, but the exact NTCP score was not an exact percentage of the

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incidence of pneumonitis. The histogram reduction method of Kutcher et al. (38) allows one to generate a single-step DVH from the 3D dose distributions using the effective volume Ceffective) reduction scheme. Using this scheme, Ten Haken et al. (39) have developed and implemented a dose-escalation protocol, stratifying patients based on their effective volume, a parameter to assess risk of the development of pneumonitis after treatment. While this protocol has escalated patients up to at least 90 Gy (it is expected to go even higher), the development of pneumonitis has been low and acceptable. Oetzel et al. (40) also reported a retrospective analysis of 86 patients with lung and esophageal cancer, and correlated this mean ipsilateral lung dose with NTCP calculations. They concurred with Martel et al. (29), stating that there was a good correlation between the incidence of pneumonitis and a high NTCP calculation. Like the Martel study, the data was best fit when ipsilateral single-lung calculations were used. Graham et al. (41) reported a good fit between NTCP predictions and the incidence of pneumonitis when total-lung NTCP calculations were performed. In preparation for a dose-escalation study, an indication of patients at risk for development of pneumonitis, researchers at Washington University evaluated various parameters to predict the development of pneumonitis. Graham et al. (42) evaluated 70 patients who had been treated with 3D treatment planning. Patients were stratified for the development of pneumonitis by the volume of the gross tumor (GTV in mL), the mean ipsilateral lung dose (in Gy), the percentage of the ipsilateral lung receiving greater than 20 Gy, the percentage of the total lung volume receiving greater than 20 Gy, and the effective volume. The data for the development of grade II or higher and grade III or higher pneumonitis in the 70 patients is charted in Table 10.1. Review of this table

Table 10.1.

Ouartile ~Grade

1st 2nd 3rd 4th

Incidence of pneumonitis (%)

GlV ImL)

Mean dose IGy)

% of ipsilateral lung receiving >20 Gy

% of total lung receiving >20 Gy

Effective volume

20 21 25 29

7 10 38 42

8 23 29 33

0 23 25 45

7 0 21 25

8 0 19 27

0 5 14 36

" pneumonitis

32 12 27 27

>Grade //I pneumonitis 11 10 6 11

1st 2nd 3rd 4th

GTV

13 20

8 24

= gross tumor volume.


189

reveals that stratifying patients by GTV or mean dose to the ipsilateral lung fails to adequately stratify the patients for the development of pneumonitis. The percent volume of either the ipsilateral- or total-lung volumes and the effective volume appear to equally stratify the patients according to risk. The skill and technology used by various institutions in calculating an effective volume was expected to vary in this protocol. It was also anticipated that the easier parameter by which to stratify for pneumonitis risk would be the percentage of total lung receiving a threshold dose of 20 Gy. Thus, the currently ongoing RTOG protocol has been designed to stratify the patients for the dose escalation based on the percentage of the total lung receiving greater than 20 Gy. This will require that patients have completed their 3D plans prior to entering this study so that they may be put into the proper bin or level for dose escalation.

CLINICAL EXPERIENCE The potential advantages of 3D treatment planning for target volume coverage and normal tissue sparing over conventional techniques when applied to patients with lung cancer was initially identified by Emami et al. (30). DVHs and a variety of dose statistics (i.e., minimum dose, maximum dose, mean dose, percentage of a specified volume receiving the prescribed dose) were identified as useful tools in evaluating 3D plans. Three-dimensional target volumes and dose display were also important. Superiority of beam's eye view (BEV)-based radiation therapy in accurate delineation of treatment volumes and avoidance of geographic misses has been shown by Vijayakumar et al. (33). Graham et al. (43) applied the International Commission on Radiological Units (ICRU) No. 50 target-volume definitions and nomenclature to 3D treatment planning for lung cancer. The importance of using dual windows (i.e., "lung window" and "soft tissue window") CT settings for delineation of target volumes was clearly identified. Improved planning and dosimetric ability of 3D-CRT to increase doses to lung cancer targets have been shown by several authors (36, 39, 44). Langer et al. (45) reported on dosimetric studies of homogeneity in optimized 3D treatment plans of six patients with lung cancer. The authors noted that if an inhomogeneity limit of 20% within the tumor was allowed, a minimum tumor dose of over 80 Gy could be delivered, whereas if the inhomogeneity was restricted to 13-17%, the achievable minimum tumor dose fell to 44 to 64 Gy. Ha et al. (46), reporting on six patients planned with a computer-controlled system, concluded that the gain from computer-controlled radiation therapy strongly depended on the chosen lung tolerance limit. A 10- to 20-Gy gain in minimum target dose could be found for some patients, but the gain was significantly

1 90

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reduced for relatively small decreases in the amount of lung permitted to receive a dose of approximately 2 Gy. In a comparative dosimetric study of 3D-CRT and conventional technology in ten patients, Graham et al. (36) reported that doses of up to 80 Gy could be delivered to target volumes in all patients using 3D-CRT. With a planned target volume dose of 80 Gy for 3D-CRT technique and 70 Gy for conventional technique, the doses to the normal structures were similar or less with 3DCRT. Hazuka et al. (47) reported on 88 patients with NSCLC in whom BEVplanning was used. From their retrospective analysis, they concluded that it was feasible to deliver uncorrected tumor doses up to 74 Gy with standard fractionations using BEV display. Armstrong et al. (44), in a preliminary analysis of 3D-CRT in nine patients with unresectable NSCLC, suggested that 3D-CRT may provide superior delivery of high-dose irradiation with reduced risk to normal tissue. In their analysis, the effective volume and the percent volume of the total-lung volume exceeding 20 Gy (as a threshold dose) appeared to be the best parameters by which to stratify patients for risk of developing pneumonitis. Both parameters indicated the strong volume dependence of the lungs to irradiation. Further study and a parameterization of the NTCP formula were recommended. Graham et al. (43) reported results of 77 patients with bronchogenic carcinoma treated with 3D technology. The incidence of pneumonitis was correlated with the percent volume of the total lung exceeding 20 Gy as well as the effective volume for total lung (Graham M, personal communication, 1996). Table 10.2 is a tabulation of reported survivals for NSCLC patients treated with 3D-CRT. These results compare favorably with the results of chemoradiotherapy trials, though they have not been tested in a randomized fashion. Also, the results thus far reported have used modest

Table 10.2.

Survival rates for NSCLC patients treated with 3D-CRT (overall sUNival %)

Median survival Author (ref), yr

Hazuka et al. (47), 1993 (University of Michigan) Leibel et al. (48), 1996 (MSKCC) Graham et al. (43), 1995 (Washington University)

No of patients

88 33 70

Stage

HI (19) III-B (69) I-II (7) IIIA-B (28) All stages I-II (19) IIIA-B (54)

Dose (Gy)

(mo)

64-72

26 14 16.5

60-74

20

>60

1 yr

2 yr

62 36 33 56

44 90* 55*

3D-CRT = three-dimensional conformal radiotherapy; MSKCC = Memorial Sloan-Kettering Cancer Center. *Cause-specific survival.


191

doses (60-74 Gy). The results of dose-escalation trials are necessary to confirm the anticipated benefit of 3D-CRT for lung cancer.

REFERENCES 1.

Sause W, Scott C, Taylor S, et a1. 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-smaIl-cell lung cancer. I Natl Cancer lnst 1995;87:98-205. 2. Dillman R, Herndon J, Seagren S, Eaton W, Green M. Improved survival in stage III non-small cell lung cancer: seven year follow-up of Cancer and Leukemia Group B (CALGB) 8433 trial. I Natl Cancer lnst 1996;88:12101215. 3. Dillman RO, Seagren SL, Propert KJ, et a1. A randomized trial of induction chemotherapy plus high-dose radiation versus radiation alone in Stage III non-small cell lung cancer. N Engl I Med 1990;323:940-945. 4. Perez C, Pajak T, Rubin P. Long-term observations of the patterns of failure in patients with unresectable non-oat cell carcinoma of the lung treated with definitive radiotherapy: report by the Radiation Therapy Oncology Group. Cancer 1987;59:1874-1881. 5. Arriagada R, Le Chevalier T, Quolx E, et a1. Effect of chemotherapy on locally advanced non-small cell lung carcinoma: a randomized study of 353 patients. Int I Radiat On col BioI Phys 1991;20:1183-1190. 6. Emami B, Perez CA. Lung cancer. In: Perez CA, Brady LW, eds. Principles and Practice of Radiation Oncology, 2nd ed. Philadelphia: Lippincott, 1992:806-836. 7. LeChevalier T, Arriagada R, Tarayre M. Significant effect of adjuvant chemotherapy in survival in locally advanced non-small cell lung carcinoma. I Natl Cancer lnst 1992;84:58. 8. Emami B, Perez C. Carcinoma of the lung and esophagus. In: Levitt S, Khan F, Potish R, eds. Levitt and Tapley's Technological Basis of Radiation Therapy: Practical Clinical Applications. Philadelphia: Lea & Febiger, 1992:248-262. 9. COX ID, Azarnia N, Byhardt RW, et al. A randomized Phase IIII trial of hyperfractionated radiation therapy with total doses of 60.0 Gy to 79.2 Gy: possible survival benefit with;::: 69.6 Gy in favorable patients with Radiation Therapy Oncology Group Stage III non-small-cell lung carcinoma. Report of Radiation Therapy Oncology Group 83-11. I Clin Oncol 1990; 8:1543-1555. 10. Emami B, Melo A, Carter B. Value of computed tomography in radiotherapy of lung cancer. Am I RadioI1978;131:63-67. 11. COX I, Pajak T, Herskovic A, et a1. Five-year survival after hyperfractionated radiation therapy for non-small-cell carcinoma of the lung (NSCLC): results of RTOG protocol 81-08. Am I Clin OncoI1991;14:280-284. 12. Hilaris B, Martini N. The current state of intraoperative interstitial brachytherapy in lung cancer. lnt I Radiat Oncol BioI Phys 1988;15: 1347-1354. 13. Laramore G, Bauer M, Griffin T, et a1. Fast neutron and mixed beam radiotherapy for inoperable non-small cell lung carcinoma of the lung. Am I Clin Oncol 1986;9:233-243.

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Calvo FA, Ortiz de Urbina 0, Abuchaibe 0, et al. Intraoperative radiotherapy during lung cancer surgery: technical description and early clinical results. Int J Radiat Oncol BioI Phys 1990;19:103-109. Perez C, Bauer M, Edelstein S. Impact of tumor control on survival in carcinoma of the lung treated with irradiation. Int J Radiat Oncol BioI Phys 1986;12:539-547. Purdy JA, Emami B. Computed tomography and three-dimensional approaches to radiation therapy. In: Levitt S, ed. Technological Basis of Radiation Therapy: Practical Clinical Applications. Philadelphia: Lea & Febiger, 1992:56-66. Purdy J, Harms W, Matthews J, et al. Advances in 3-dimensional radiation treatment planning systems: room-view display with real time interactivity. Int J Radiat Oncol BioI Phys 1993;27:933-944. Purdy J. Three-dimensional treatment planning: a new era. In: Meyer J, Purdy J, eds. 3D Conformal Radiotherapy: A New Era in Irradiation of Cancer. Basal: Karger, 1996:146. Thames H, Peters L, Spanos WJ. Dose response of squamous cell carcinomas of the upper respiratory and digestive tracts. Br J Cancer 1980; 41(SuppI4):35-38. Mantravadi RVP, Gates JO, Crawford IN. Unresectable non-oat cell carcinoma of the lung: definitive radiation therapy. Radiology 1989;172:851855. Fletcher G. Clinical dose-response curves of human malignant epithelial tumors. Br J RadioI1973;46:1-12. Dutriex A. When and how can we improve precision in radiotherapy. Radiother OncoI1984;1:275-292. Perez CA, Stanley K, Grundy G, et al. Impact of irradiation technique and tumor extent in tumor control and survival of patients with unresectable non-oat cell carcinoma of the lung. Cancer 1982;50:1091-1099. Fuks Z, Leibel S, Rutcher G, Mohan R, Ling C. Three dimensional conformal treatment: a new frontier in radiation therapy. In: DeVita VI, Hellman S, Rosenberg S, eds. Important Advances in Oncology 1991:151-172. Goitein M, Abrams M, Rowell 0, Pallari H, Wiles J. Multi-dimensional treatment planning. II. Beam's eye-view, back projection and projection through CT sections. Int J Radiat Oncol BioI Phys 1983;9:789-797. McShan 0, Silverman A, Lanza 0, Reinstein L, Glicksman A. A computerized three-dimensional treatment planning system utilizing interactive color graphics. Br J RadioI1979;52:478-481. Chol NCH, Doucette JA. Improved survival of patients with unresectable non-small cell bronchogenic carcinoma by an innovated high-dose en-bloc radiotherapeutic approach. Cancer 1981;48:101-109. Ball 0, Matthews J, Woprotniuk V, Crennan E. Longer survival with higher doses of thoracic radiotherapy in patients with limited non-small cell lung cancer. Int J Radiat Oncol BioI Phys 1993;25:599-604. Martel MK, Ten Haken RK, Hazuka MB, et al. Dose-volume histogram and 3D treatment planning evaluation of patients with pneumonitis. Int J Radiat Oncol BioI Phys 1994;28:575-581. Emami B, Purdy JA, Manolis J, et al. Three-dimensional treatment planning for lung cancer. Int J Radiat Oncol BioI Phys 1991;21:217-227. Emami B, Perez CA, Herskovich A, Hederman MA. Phase lIlI study of treatment of locally advanced (T3T4) non-oat cell lung cancer with high dose radiotherapy (rapid fractionation): Radiation Therapy Oncology Group Study. Int J Radiat Oncol BioI Phys 1988;15:1021-1025. Graham M, Pajak T, Herskovic A, Emami B, Perez C. Phase lIlI study of


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treatment of locally advanced (T3/T4) non-oat cell lung cancer with concomitant boost radiotherapy by the Radiation Therapy Oncology Group (RTOG 83-12): Long-term results. Int J Radiat Oncol BioI Phys 1995;31: 819-825. 33. Vijayakumar S, Myrianthopoulos L, Rosenberg I, et al. Optimization of radical radiotherapy with beam's eye view techniques for non-small cell lung cancer. Int J Radiat Oncol BioI Phys 1991;21:779-788. 34. Hodapp N, Boesecke R, Schlegel W, Bruggmoser G, Wannenmacher M. Three-dimensional treatment planning for conformation therapy of a bronchial carcinoma. Radiother OncoI1991;20:245-249. 35. Photon Treatment Planning Collaborative Working Group. Evaluation of high energy photon external beam treatment planning: project summary. Int J Radiat Oncol BioI Phys 1991;21 :3-8. 36. Graham MY, Matthews JW, Harms WB, et al. Three-dimensional radiation treatment planning study for patients with carcinoma of the lung. Int J Radiat Oncol Bioi Phys 1994;29:1105-1117. 37. Armstrong J, Zelefsky M, Burt M, et al. Strategy for dose escalation using three-dimensional conformal radiation therapy for lung cancer. (In press) 38. Kutcher GJ, Burman C, Brewster L, Goltein M, Mohan R. Histogram reduction method for calculating complication probabilities for threedimensional treatment planning evaluation. Int J Radiat Oncol BioI Phys 1991;21:137-146. 39. Ten Haken R, Martel M, Kessler M, et al. Use of Yeff and iso-NTCP in the implementation of dose escalation protocols. Int J Radiat Oncol BioI Phys 1993;27:2689-2695. 40. Oetzel D, Schraube P, Hensley F, et al. Estimation of pneumonitis risk in three-dimensional treatment planning using dose-volume histogram analysis. Int J Radiat Oncol Bioi Phys 1995;33:455-460. 41. Graham MY, Drzymala RE, Jain NL, Purdy JA. Confirmation of dose-volume histograms and normal tissue complication probability calculations to predict pulmonary complications after radiotherapy for lung cancer. Int J Radiat Oncol BioI Phys 1994;30(Suppl1):198. 42. Graham M, Purdy J, Harms W, et al. Clinical results of three-dimensional radiation therapy for non-small cell lung cancer: The Washington University experience. Radiother Oncol 1997. 43. Graham MY, Purdy JA, Emami B, Matthews JW, Harms WB. Preliminary results of a prospective trial using three-dimensional radiotherapy for lung cancer. Int J Radiat Oncol Bioi Phys 1995;33:993-1000. 44. Armstrong JG, Burman C, Leibel SA, et al. Three-dimensional conformal radiation therapy may improve the therapeutic ratio of high dose radiation therapy for lung cancer. Int J Radiat Oncol BioI Phys 1993;26:685-689. 45. Langer M, Kijewski P, Brown R, Ha C. The effect on minimum tumor dose of restricting target-dose inhomogeneity in optimized three-dimensiona1 treatment of lung cancer. Radiother OncoI1991;21:245-256. 46. Ha CS, Kijewski PK, Langer MP. Gain in target dose from using computer controlled radiation therapy (CCRT) in the treatment of non-small cell lung cancer. Int J Radiat Oncol BioI Phys 1993;26:335-359. 47. Hazuka MB, Turrisi AT, Lutz ST, et al. Results of high-dose thoracic irradiation incorporating beam's eye view display in non-small cell lung cancer: a retrospective multivariate analysis. Int J Radiat Oncol Bioi Phys 1993;27:273-284. 48. Leibel S, Armstrong J, Kutcher G, et al. 3-D conformal radiation therapy for non-small cell lung carcinoma: clinical experience at the Memorial Sloan-Kettering Cancer Center. Front Radiat Ther OncoI1996;29:199-206.

Plate 1. Intrapleural photodynamic therapy after resection of mesothelioma. (aj Modified endotracheal tubes used to introduce laser fiber into the chest. (bJ Light delivery to the pleura.

Plate 2. Pretreatment (A and CJ and 30-day post-treatment (B and 0) bronchoscopic images of patients I fA and B) and 5 IC and OJ . A. Lesion is situated in the left mainstem bronchus on the division of the upper and lower lobes; biopsies showed squamous cell carcinoma (arrowheads) . B. Left mainstem bronchus 30 days following ITRp53A injection; five independent biopsies showed absence of viable tumor cells. C. Adenocarcinoma obstructing the right upper lobe orifice. D. Right upper lobe orifice (arrowheads) 30 days following treatment; biopsies in this region showed residual adenocarcinoma (29).

11 Combinations of Radiation Therapy and Chemotherapy for NSCLC Ritsuko Komaki and James D. Cox

The large proportion of patients with non-smaIl-cell lung cancer (NSCLC) who are unable to undergo resection with curative intent compels oncologists to reconsider alternative treatment strategies. For many of these patients, palliative radiation therapy or chemotherapy or enrollment in formal clinical trials may be appropriate management. For a select population, especially those with no evidence of distant metastasis and few symptoms (high performance status) plus minimal weight loss, it is appropriate to consider therapy with curative intent. What is the potential for cure in these cases? This is one of the most important questions for patients and physicians alike, and the answer is changing rapidly as a result of recent clinical trials combining radiation therapy and chemotherapy.

RATIONALE FOR COMBINING RADIATION THERAPY AND CHEMOTHERAPY Laboratory Investigations A thorough review of laboratory investigations of chemotherapy-radiation therapy interactions is beyond the scope of this chapter. However, excellent reviews are provided by Hill and Bellamy (1) and John et al. 195

1 96

Chapter 1 1

(2). The relevance of available laboratory data to clinical application is difficult to establish because of variations in dose-fractionation schedules (single vs. multiple), treatment sequence, timing, drug dosage, drug delivery method, and the duration of drug exposure in the clinical setting. A few generalizations can be made, however. Of the four possible ways of improving therapeutic effect put forth by Steel et al. (3)-toxicity independence, protection of normal tissues, spatial cooperation, and enhancement of tumor response-only the last two find consistent clinical expression in NSCLC. Spatial cooperation, that is, radiation therapy for the local-regional tumor and chemotherapy for metastasis, is obvious, but enhancement of tumor response is less clear. Terms such as radiation sensitization may have different meanings for different investigators. The terminology suggested by Steel and Peckham (4) (Fig. 11.1) is preferred. Although there are laboratory data indicating an increased resistance to certain drugs by tumor cells in vitro following exposure to

Protection

------l Sub-additive

« ~

o

OJ til

o

o

Supra-additive

Dose of 8 Fig. 11.1. An isob%gram is an isoeffect plot of the doses of two agents that together give a fixed biological effect. If dose-response curves are nonlinear, there is a region of uncertainty about the existence of "additivity." (Reproduced by permission from Steel GG, Peckham MJ. Exploitable mechanisms in combined radiotherapychemotherapy: the concept of additivity. Int J Radiat Oncol Bioi Phys 1979;5:85-91.)

Combinations of Radiation Therapy and Chemotherapy for NSCLC

197

ionizing radiations (5), the resistant phenotype does not have characteristics of multidrug resistance. Responses to other drugs may be unchanged or sensitivity may even be enhanced.

Clinical Rationale The empirical rationale for combining radiation therapy and chemotherapy is the all-too-frequent failure of either modality given alone. Further, they do not fail for the same reasons: Radiation therapy fails to address distant subclinical metastases, and chemotherapy fails to eradicate bulky unresectable tumors. It had been the hope of some that cytotoxic chemotherapy would eventually become so effective as to eliminate both types of disease, but in the far more sensitive small-cell lung cancer (SCLC) and even in strikingly chemosensitive diseases such as malignant lymphomas, bulky tumors require additional local treatment to achieve maximum control, so it is unlikely that NSCLC can be adequately treated with chemotherapy alone. The local tumor, whether untreated or inadequately treated, is the actual cause of death in most patients with unresectable NSCLC. Documentation of this comes from four independent sources. First, if radiation therapy alone is given, progression of tumor within the field of irradiation is associated with poorer survival than when the tumor is controlled (6-8). Second, causes of death in patients treated with palliative irradiation or single-agent chemotherapy are attributable more frequently to intrathoracic disease than to extra thoracic metastasis (9), especially for squamous cell carcinoma. Third, Saunders et al. (10) studied the causes of death among patients with localized but unresectable NSCLC treated with a few large fractions of irradiation (in order to investigate the hypoxic cell-sensitizing drug misonidazole); most of these patients were followed to necropsy. They demonstrated that 72% of patients so treated died of complications of the intrathoracic tumor and only 15% died of distant metastasis. Supporting data come from Perez et al. (11), who showed that improving local tumor control was associated with an increased incidence of distant metastasis. Finally, concurrent chemotherapy and radiation therapy improved local tumor control, which resulted in improved survival (12) (vide infra).

RESULTS OF COMBINING RADIATION THERAPY AND CHEMOTHERAPY Induction Chemotherapy Induction chemotherapy before radiotherapy has many attractive features. It permits the most immediate attack on all components of the

198

Chapter 1 1

tumor-both those evident clinically and those presumed to be present at subclinical levels. In addition, demonstration of response to systemic therapy provides justification for continuation of therapy during or following radiation therapy. Although several prospective randomized clinical trials have shown mixed survival results overall, three studies using cisplatin-based chemotherapy and continuous radiation therapy to total doses of 60 Gy or more produced favorable results. They represent a basis for planning future trials. The first positive trial was that conducted by the Cancer and Leukemia Group B (CALGB) (CALGB 8433) published by Dillman et al. (13). This trial compared induction therapy with cisplatin (100 mg/m2 on days 1 and 29) and vinblastine (5 mg/m2 weekly for 5 weeks) prior to radiation therapy (60 Gy at 2.0 Gy per fraction, 5 days per week) beginning on day 50, with the same radiation therapy alone beginning on day 1. This trial was closed before the planned accrual was reached because the improvement in survival on the induction chemotherapy arm met the early-stopping rules of the study. Long-term observations of these patients showed continued survival benefit (14). Detailed failure-pattern analyses of this trial were not published, but there was no indication that local tumor control was improved with this induction chemotherapy. The second positive trial was reported by Le Chevalier's group (15-17). This French cooperative group enrolled 353 patients in a study that compared induction chemotherapy with 3 monthly cycles of vinde sine (1.5 mg/m2 on days 1-2), cyclophosphamide (200 mg/m2 on days 2-4), cisplatin (100 mg/m2 on day 2), and lomustine (75 mg/m2 on day 3) followed by radiation therapy (65 Gy at 2.5 Gy per daily fraction, 4 days per week) beginning on day 75-80, with the same radiation therapy alone. They found an improvement in survival with induction chemotherapy, and they also showed a statistically significant reduction in the incidence of distant metastasis with induction chemotherapy but no improvement in local tumor control. In fact, due to their policy of systematic fiberoptic bronchoscopy and biopsy at the site of the original lesions 3 months after the start of treatment, they were able to demonstrate that there was no advantage in local control with induction chemotherapy and that failure rates were high (>80%) at the primary disease sites in both arms. The third positive trial was coordinated by the Radiation Therapy Oncology Group (RTOG 88-08) in conjunction with the Eastern Cooperative Oncology Group (ECOG 4588) and published by Sause et al. (18). It replicated the design and confirmed the benefit of the induction chemotherapy regimen used in the CALGB 8433 study. Komaki et al. (19) studied failure patterns among these patients and found improvement in control of distant metastasis, but only among patients with squamous cell carcinoma; there was no influence of chemotherapy on the local tumor. It is notable that most of the patients enrolled in the


199

French trial in which distant metastasis was decreased also had squamous cell carcinoma. From induction chemotherapy-radiotherapy studies to date, we can conclude that chemotherapy is important to the success of these regimens, especially cisplatin (20). Distant metastasis is decreased by the chemotherapy, perhaps selectively, among patients with squamous cell carcinoma. Control of the tumor within the field of irradiation is poorer than originally thought, and there is no benefit from induction chemotherapy on local control of that tumor.

Concurrent Single-Agent Chemotherapy The rationale for the addition of a single chemotherapeutic agent to radiation therapy is specifically to increase local tumor control. There are few if any advocates of single-agent chemotherapy who would suggest this as the best approach to control of distant metastasis. Schaake-Koning et al. (12) from the European Organization for Research and Treatment of Cancer (EORTC) conducted a very important trial comparing radiation therapy alone to the same therapy with cisplatin given once weekly (30 mg/m2) or daily (6 mg/m2). It is important to note that the same total dose of cisplatin was given in both arms of the study. The radiation therapy fractionation used in this trial would not be considered standard in the United States: 3.0 Gy was given for 10 fractions, 5 days per week for 2 weeks; followed by an interruption of 3 to 4 weeks; followed by 2.5 Gy per fraction for 10 fractions, 5 fractions per week for 2 weeks. The increase in overall time to deliver the total dose of irradiation might be a disadvantage based on studies from the RTOG (21). Nonetheless, there was improvement in local control with both methods of delivering cisplatin, and that improvement was reflected in a significant increase in overall survival (Fig. 11.2). Studies that have examined the question of concurrent single-agent chemotherapy, plus radiotherapy after induction combination chemotherapy, could be considered promising, but randomized comparisons with standard therapy have yet to be published. Sause et al. (22) presented a small series of patients treated with induction cisplatin/vinblastine, similar to CALGB 8433, followed by concurrent cisplatin (75 mg/m2) with standard radiation therapy (45 Gy at 1.8 Gy plus boost of 18 Gy at 2.0 Gy per fraction; total dose = 63 Gy). Komaki et al. (23) reported a somewhat larger experience with this regimen and found it both well tolerated and modestly encouraging (I-year survival rate of 65% and median survival duration of 15.5 months); it was less effective than concurrent combination cisplatin and etoposide plus radiotherapy in achieving local control but better tolerated (vide infra). CALGB investigators have conducted a randomized comparison of their standard induction cisplatin/vinblastine regimen followed by concurrent carbo-

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Fig. 11.2 A. SUNival without local recurrence. The time to local recurrence was significantly longer in the cisplatin groups (P= 0.015 overall). For the comparison of Group 2 with Group I, P = 0.15; Group 3 with Group I, P = 0.003; Group 2 with Group 3, P= 0.17; and Group I with Groups 2 and 3, P= 0.009. B. Overall survival in the treatment groups. The 4-year point denotes survival as of May 1991, for which P = 0.054 overall. Kaplan-Meier analysis showed that for the comparison of Group 2 with Group I, P = 0.36; Group 3 with Group I, P = 0.36; Group 3 with Group I, P = 0.009; Group 2 with Group 3, P = 0.20; and Group I with Groups 2 and 3, P= 0.04. (RT = radiotherapy.) (Reproduced by permission of the New England Journal of Medicine, from Schaake-Koning C van den Bogaert W, Dalesio 0, et al. Effects of concomitant cisplatin and radiotherapy on inoperable non-small-cell lung cancer. N Engl J Med 1992;326:524-530.)


201

platin rather than cisplatin during radiation therapy. This is an important comparison of a strategy that targets only distant metastasis versus one that attempts both reduction in distant metastasis and improvement in local tumor control.

Concurrent Combination Chemotherapy and Radiation Therapy The most aggressive approach to combining chemotherapy and radiation therapy is to initiate both modalities at the outset. It could be expected that this approach would carry the greatest risk of acute toxicity, and indeed this has been demonstrated in studies of SCLC. At present, it can justifiably be investigated only among patients with relatively little physiologic compromise from the malignant disease and few or no comorbid conditions. If considerable benefit were shown in comparative trials, it might be appropriate to consider this approach in less favorable patients. Oncologic investigators have great interest in the data that will emerge from clinical trials by 1999. Justification for the concurrent use of cisplatin-based chemotherapy regimens and radiation therapy comes from the prospective randomized comparative trial of the EORTC (12). In addition, a meta-analysis (20) of clinical trials comparing radiation therapy alone versus radiation therapy plus chemotherapy has suggested that only cisplatin-based regimens have been beneficial to date. The RTOG has conducted several pilot studies with dual endpoints, tolerance (hematologic and nonhematologic), and short-term survival (response rates have not been used in RTOG studies, as they do not predict survival in NSCLC and they are meaningless when concurrent treatments are administered). Survival varies widely, however, as pretreatment prognostic factors are such strong determinants. Recursive partitioning studies of the large RTOG database have assisted in identifying suitable groups of patients in which to compare outcomes across studies in order to select regimens for phase III comparative trials, which provide the only basis on which to justify changes in standard practice in treating this disease. The major prognostic variables found in RTOG studies are Karnofsky performance status (24), weight loss, and extent of nodal disease (N stage). The first RTOG trial of concurrent combination chemotherapy and radiation therapy was published by Byhardt et al. (25). It used the cisplatin/vinblastine regimen of CALGB 8433 plus hyperfractionated radiation therapy (HFX) (RTOG 90-15). HFX with a total dose of 69.6 Gy delivered as 1.2 Gy twice daily, 5 days per week, had been suggested as superior to 60 Gy at 2.0 Gy per fraction per day in a dose-seeking study published by Cox et al. (26). While RTOG 90-15 was being conducted, HFX was being compared to standard fractionation in RTOG 88-08 and ECOG 4588 studies. Although few patients in RTOG 90-15 had the most

202

Chapter 1 1

favorable prognostic factors, it is encouraging that the median survival among them was 12.2 months (25). A successor trial to RTOG 90-15, RTOG 91-06, employed the same design but advanced the hypothesis that oral etoposide given daily during much of the radiation therapy would be a more effective addition to cisplatin than vinblastine (27). Cisplatin, 75 mg/m2 IV on days 1 and 29 and etoposide 50 mg PO bid on days 1 to 14 and days 29 to 43 were given with HFX, also beginning on day 1. The toxicity of this regimen was considerable, especially esophageal toxicity. However, the median survival of 21 months and the 2-year survival rate of 42% for favorable patients were the best reported in such patients. Supporting data were reported by Reboul et al. (28) from Avignon, France using similar chemotherapy with more standard fractionation. To reduce the severity of acute effects, a modification of the RTOG 91-06 regimen was used in RTOG 92-04 (23): Cisplatin was given as in protocol 91-06, but instead of etoposide 50 mg PO days 1 to 14 and 29 to 43, the drug was omitted on the weekends when irradiation was not given, thus reducing the total number of days on which etoposide was administered from 28 to 20. This regimen was compared for toxicity and short-term survival in a randomized phase 1/11 study with the induction cisplatin/vinblastine followed by concurrent cisplatin and standard radiation therapy mentioned earlier. The results demonstrated a lower risk of nonhematologic toxicity with the induction regimen at the price of a higher in-field progression rate, but the survival for patients given either regimen was the same (median 15.5 and 14.1 months, and I-year survival rates of 65% and 58%, respectively). Unwilling to wait until the results of these several studies were available, the RTOG launched an ambitious three-arm randomized comparative trial (protocol 94-10) (Fig. 11.3) to address two questions. First, Was concurrent combination chemotherapy and standard radiation therapy better than induction chemotherapy and standard radiation therapy (60 Gy at 2.0 Gy per fraction, 5 days per week)? And second, Was a more intensive and more toxic chemotherapy /radiation therapy combination

RTOG 94-10: Vbl/P followed by Standard RT (RTOG 88-08) vs. Concurrent Vbl/P + RT (RTOG 90-15) vs. Concurrent E/P + HFX RT (RTOG 91-06) Fig. 11.3. Current phase III trial (RTOG 94-10) to test timing/sequencing chemotherapy and radiation therapy for unresectable NSCLC. As of 3/96, RTOG 94-10 had accrued 250/600 cases. (Vbl = vinblastine; P = cisplatin; RT = radion therapy; E = etoposide; HFX = hyperfractionation.)


203

superior to either or both arms? The latter regimen used cisplatin/ oral etoposide and HFX similar to RTOG 92-04. This protocol will be closed by April 1998 for accrual and the results will be available by April 1999.

Concurrent Combination Chemotherapy and Radiation Therapy Followed by Resection It is important to review this subject briefly because there is increasing interest in induction chemotherapy plus radiation therapy followed by surgical resection. An important intergroup study coordinated by the RTOG is being conducted for patients with marginally resectable tumors. The term marginally resectable necessarily includes a heterogeneous group of patients. There are patients whose disease might be considered resectable by one group of thoracic surgeons but unresectable by another group. Moreover, some patients would accept the risks of the operative procedure and others would not. The Southwest Oncology Group (SWOG) conducted a phase II study (8805) of the three-modality arm (29) in carefully selected patients with mediastinal lymph node metastasis documented by biopsy or percutaneous fine-needle aspiration. Combination chemotherapy with cisplatin (50 mg/m2 on days 1, 8, 29, 36) and intravenous etoposide (50 mg/m2 on days 1-5, 29-33) concurrent with radiation therapy (45 Gy at 1.8 Gy per fraction,S days per week) were administered for 5 weeks. After an interval of 2 to 4 weeks, thoracotomy and resection were attempted. In the 126 patients enrolled, the median survival was 15 months, and the 2-year survival rate was 40% (median period of observation was 2.4 years). This led to a randomized comparative trial to determine the role of surgery compared to chemotherapy and radiotherapy in this stage of disease (RTOG 93-09 and ECOG, SWOG 9336, INT 0139) (Fig. 11.4). Randomization is performed before patients are enrolled in the study, and all are treated with an induction chemotherapy /radiation therapy regimen similar to the SWOG study for 5 weeks. Half the patients proceed to surgical exploration after an interval of 2 to 4 weeks, and half the patients continue, without interruption, with chemotherapy and radiation therapy. The latter patients received a total dose of 61.0 Gy in 33 fractions. This is one of very few trials comparing surgical resection to radiation therapy since the United Kingdom's Medical Research Council (MRC) trial of the 1960s, which documented the superiority of resection (30). The addition of effective combination chemotherapy makes the question appropriate again.

CONCLUSION The successes to date from combining chemotherapy with radiation therapy for NSCLC, although quite limited, are sufficient to encourage

204

Chapter 11

RTOG 93-09 Int: Tl-3 N2 NSCLC R A

N D

ARM 1:

RT + Concurrent Induction Chemotherapy followed by Surgery plus Chemotherapy

ARM 2:

RT + Concurrent Induction Chemotherapy followed by RT Boost plus Chemotherapy

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Hypothesis: Local Control & Survival with RT/CT Equals that of RT/Cf Followed by Surgery Fig. 11.4. Intergroup study INT 0139 (RTOG 93-09). A phase III comparison between concurrent chemotherapy plus radiotherapy and concurrent chemotherapy plus radiotherapy followed by surgical resection for stage Ilia (Tl-3 N2) NSCLC. (RT = radiation therapy; CT = chemotherapy)

continuing this strategy. Major issues include the sequencing and timing of the two modalities and the contributing role of surgery in marginally resectable tumors. Newer and more effective drugs require phase I studies, which should be followed by phase III comparative trials. The slow pace of progress in NSCLC is in part the result of the initiation of a plethora of phase II trials once phase I studies were complete rather than early adoption of a comparative strategy once toxicity is shown to be tolerable. Results of ongoing phase III studies will provide the appropriate standards for comparison and for practice throughout the medical community.

REFERENCES 1. 2. 3. 4.

5.

Hill BT, Bellamy AS. Antitumor Drug-Radiation Interactions. Boca Raton, FL: CRC Press, 1990. John MJ, Flam MS, Legha SS, Phillips TL. Chemoradiation: An Integrated Approach to Cancer Treatment. Philadelphia: Lea & Febiger, 1993. Steel GG, Hill RP, Peckham MJ. Combined radiotherapy-chemotherapy of Lewis lung carcinoma. Int J Radiat Oncol BioI Phys 1978;4:49-52. Steel GG, Peckham MJ. Exploitable mechanisms in combined radiotherapy-chemotherapy: the concept of additivity. Int J Radiat Oncol BioI Phys 1979;5:85-91. Hill BT. In vitro drug-radiation interactions using fractionated X-irradiation regimens. In: Hill BR, Bellamy AS, eds. Antitumor Drug-Radiation Interactions. Boca Raton, FL: CRC Press, 1990:207-224.

Combinations of Radiation Therapy and Chemotherapy for NSCLC 6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

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Eisert DR, Cox JO, Komaki R. Irradiation for bronchial carcinoma: reasons for failure. I. Analysis as a function of dose-time-fractionation. Cancer 1976;37:2665-2670. Bauer M, Birch R, Pajak TF, Perez CA, Weiner JM. Prognostic factors in cancer of the lung. In: COX JO, ed. Lung Cancer: A Categorical Course in Radiation Therapy. Oak Brook, IL: Radiological Society of North America, 1985:87-112. Perez CA, Bauer M, Edelstein S, Gillepsie BW, Birch R. Impact of tumor control on survival in carcinoma of the lung treated with irradiation. Int J Radiat Oncol BioI Phys 1986;12:539-547. COX JO, Yesner R, Mietlowski W, Petrovich Z. Influence of cell type on failure pattern after irradiation for locally advanced carcinoma of the lung. From the Veterans Administration Lung Group. Cancer 1979;44:94-98. Saunders MI, Bennett MH, Oische S, Anderson PJ. Primary tumor control after radiotherapy for carcinoma of the bronchus. Int J Radiat Oncol BioI Phys 1984;10:499-501. Perez CA, Pajak TF, Rubin P, et al. Long-term observations of the patterns of failure in patients with unresectable non-oat cell carcinoma of the lung treated with definitive radiotherapy. Report by the Radiation Therapy Oncology Group. Cancer 1987;59:1874-1881. Schaake-Koning C, van den Bogaert W, Dalesio 0, et al. Effects of concomitant cisplatin and radiotherapy on inoperable non-smaIl-cell lung cancer. N Engl J Med 1992;326:524-530. Dillman RO, Seagren SL, Propert KJ, et al. A randomized trial of induction chemotherapy plus high-dose radiation versus radiation alone in stage III non-smaIl-cell lung cancer. N Engl J Med 1990;323:940-945. Dillman RO, Herndon J, Seagren SL, Eaton WL Jr, Green MR. Improved survival in stage III non-small-celliung cancer: seven-year follow-up of Cancer and Leukemia Group B (CALGB) 8433 trial. J Natl Cancer Inst 1996;88:1210-1215. Le Chevalier T, Arriagada R, Quoix E, et al. 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 1991;83:417-423. Arriagada R, Le Chevalier T, Quoix E, et al. ASTRO plenary: effect of chemotherapy on locally advanced non-small cell lung carcinoma: a randomized study of 353 patients. Int J Radiat Oncol BioI Phys 1991;20: 1183-1190. Le Chevalier T, Arriagada R, Tarayre M, et al. Significant effect of adjuvant chemotherapy on survival in locally advanced non-smaIl-cell lung carcinoma. J Natl Cancer Inst 1992;84:58. Sause WT, Scott CB, Taylor SG IV, et al. RTOG 88-08 and ECOG 4588: Preliminary results of a phase III trial in regionally advanced unresectable non-small cell lung cancer. J Natl Cancer Inst 1995;87:198-205. Komaki R, Scott CB, Sause WT, et al. Induction cisplatin/vinblastine decreased distant metastasis in unresectable squamous cell lung cancer (SCC): failure patterns in RTOG 88-08/ECOG 4588. Int J Radiat Oncol BioI Phys 1996;36S:206. Non-small Cell Lung Cancer Collaborative Group. Chemotherapy in nonsmall cell lung cancer: meta-analysis using updated data on individual patients from 52 randomised clinical trials. Br Med J 1995;311:899-909. COX JD, Pajak TF, Asbell S, et al. Interruptions of high-dose radiation therapy decrease long-term survival of favorable patients with unresectable

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22.

23.

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25.

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28.

29.

30.

non-small cell carcinoma of the lung: analysis of 1244 cases from 3 RTOG trials. Int J Radiat Oncol BioI Phys 1993;27:493-498. Sause WT, Scott C, Taylor S, et a1. Phase II trial of combination chemotherapy and irradiation in non-smaIl-cell lung cancer. Radiation Therapy Oncology Group 88-04. Am J Clin OncoI1992;15:163-167. Komaki R, Scott C, Ettinger D, et a1. Randomized phase II study of chemotherapy /radiation therapy combinations for favorable patients with locally advanced inoperable non-small cell cancer: Radiation Therapy Oncology Group 92-04. Int J Radiat Oncol BioI Phys 1977;38:149-155. Karnofsky DA, Burchenal JH. The clinical evaluation of chemotherapeutic agents in cancer. In: Macleod CM, ed. Evaluation of chemotherapeutic agents. New York: Columbia University, 1949:191-205. Byhardt RW, Scott CB, Ettinger DS, et al. Concurrent hyperfractionated irradiation and chemotherapy for unresectable non-small cell lung cancer. Cancer 1995;75:2337-2344. COX JD, Azarnia N, Byhardt RW, et a1. A randomized phase l/II trial of hyperfractionated radiation therapy with total doses of 60.0 Gy to 79.2 Gy: possible survival benefit with ~69.6 Gy in favorable patients with Radiation Therapy Oncology Group Stage III non-smaIl-cell lung carcinoma: Report of Radiation Therapy Oncology Group 83-11. J Clin OncoI1990;8: 1543-1555. Lee JS, Scott C, Komaki R, et a1. Concurrent chemoradiation therapy with oral etoposide and cisplatin for locally advanced inoperable non-smallcell lung cancer: radiation therapy oncology group protocol 91-06. J Clin OncoI1996;14:1055-1064. Reboul F, Yvelise B, Vincent P, et a1. Concurrent cisplatin, etoposide, and radiotherapy for unresectable stage III nonsmall cell lung cancer: a phase II study. Int J Radiat Oncol BioI Phys 1996;35:343-350. Albain K, Rusch VW, Crowley H, et a1. Concurrent cisplatin/etoposide plus chest radiotherapy followed by surgery for stages IIIA(N2) and IIIB non-smaIl-cell lung cancer: mature results of Southwest Oncology Group phase II study 8805. J Clin OncoI1995;13:1880-1892. Fox W, Scadding GH. Medical research council comparative time of surgery and radiotherapy for primary treatment of small-celled or oatcelled carcinoma of bronchus. Ten-year follow-up. Lancet 1973;2:63-65.

12 Preoperative and Postoperative Adjunctive Therapy for Resectable NSCLC John C. Ruckdeschel

Attempts to improve on the surgical outcome for non-smaIl-cell lung cancer (NSCLC) have continued unabated since our last review of this topic in 1992 (1). Although there have been several pivotal, randomized studies published, the overwhelming majority of reports have been nonrandomized phase II trials with highly variable efforts at pretreatment staging. In contrast to 1992, when most adjuvant therapies were postoperative, the more recent studies show a preponderance of preoperative interventions designed both to better control local disease and to simultaneously overcome clinically inapparent disseminated disease. The overwhelming problem slowing interpretation of clinical results in this arena is the continued failure to either perform or report adequate surgical staging data. Additionally, as it becomes more clear that various permutations of chemotherapy and irradiation significantly reduce tumor burden, the inadequacies of the current staging system (2) become more apparent.

CURRENT ISSUES RELATED TO THE STAGING SYSTEM Mountain's report of the MD Anderson and Lung Cancer Study Group (LCSG) surgical data in 1985 (2) greatly clarified the then existing stag207

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Chapter 12

ing system and added some degree of rationality to reporting of outcomes. Given the relative lack of efficacy of chemotherapy in the early 1980s, it is not surprising that Mountain was able to condense (lump) several subcategories. As the move to preoperative treatments has accelerated, the variability in response among patients with various subsets of stage IlIa and IIIb disease has become apparent. We have previously proposed a modification to the staging system that we feel better stratifies stage III disease for purposes of both prognostication and clinical research (3). Table 12.1 describes the differences between our system and Mountain's. In an attempt to lend some clarity to the clinical trials in stage III NSCLC, I will employ our proposed staging categories.

PROPOSED STAGING Stage 1111 NSCLC: T3 NO-1 These tumors comprise two separate clinical syndromes. The first is the traditional apical or Pancoast tumor that classically presents with shoulder pain, neurologic changes in the ipsilateral upper extremity, and evidence of autonomic dysfunction including Horner's syndrome. Paulson (4) reported on a surprisingly good outcome for patients with these tumors following preoperative radiation to 3000 cGy. Although widely used clinically for all patients with Pancoast tumors, the clinical benefit is seen only in patients who have no evidence of mediastinal adenopathy (4). There have been no controlled trials of therapy for Pancoast tumors due to their rarity. The LCSG could not identify sufficient

Table 12.1.

A more rationale proposal for stage III NSCLC

Clinical description *

Current stage

T3 lesions with no mediastinal nodes (NO or Nl); e.g., chest wall, Pancoast Tl-T3 lesions with microscopic or single station intra nodal metastases Tl-T3 lesions with multi station N2 disease but technically resectable Tl-T3 lesions with bulky N2 or N3 disease, or T4 disease TI-T4, NO-N3lesions with malignant pleural effusion

lIla

Proposed stage

lIla lIla IlIa (bulky N2) IIIb (all others) IIIb

IV

NSCLC = non-small-cell lung cancer.

*All T1 N descriptors are as described by Mountain (2). Reproduced by permission from Ruckdeschel IC, Wagner H, Robinson LA. Locally advanced lung cancer: controversies in management. Adv Oncol 1996;12:22-28.

Preoperative and Postoperative Adjunctive Therapy for Resectable NSCLC

209

numbers of these patients across North America to make a study feasible. The current Intergroup trial is a phase II exploration of combined chemoradiotherapy prior to surgical resection in patients who have a negative pretreatment mediastinal evaluation; results are pending. Nonapical chest wall tumors without N2 disease do surprisingly well (5-year survival 20-40% [2]) with surgery alone. An attempt by the LeSe to study the necessity for postoperative radiation did not accrue well and was halted, with most clinicians preferring to give postoperative irradiation.

Stage 1112 NSCLC: Minimal or Microscopic N2 Disease Although these patients have traditionally been included in clinical trials of patients with more extensive disease, the results of two very promising trials suggest they should be segregated clinically (5, 6). At our last report (1), the early results of the Rosell trial had just been reported and demonstrated an apparent benefit for preoperative chemotherapy. The maturation of the Spanish data (5) and the subsequent report by Roth et al. (6) clearly demonstrate a benefit for two to three cycles of preoperative chemotherapy with hazard ratios for survival improved five- to sevenfold compared to the surgical arms. A trial of preoperative paclitaxel-carboplatin in patients with T2-3, NO-1 disease is currently under way (7).

Stage 1113 NSCLC: Resectable N2 Disease, Multistation This group of patients has been the subject of numerous phase II and III trials of both pre- and postoperative therapy, although these studies have often included patients with stages 1112 and 1114 disease as well. Since the early postoperative trials of the LeSe (8-12), only a handful of randomized postoperative trials have been reported. Figlin and Piantadosi (13) described the final results of the last LeSe trial, comparing eAP (cyclophosphamide, doxorubicin, cisplatin) chemotherapy to no further therapy in fully resected N2 patients of all histologies and showed no benefit. Ayoub et al. (14) and Pisters et al. (15) have also reported on negative postoperative adjuvant trials. As noted earlier, the majority of trials for this subset of patients have explored preoperative chemotherapy or chemoradiotherapy. The details of these trials have been summarized elsewhere (16), but several patterns have emerged. The first is the lack of randomized trials. Fleck et al. (17) compared preoperative chemotherapy with MVP (mitomycin, vinblastine, cisplatin) (18,19) to the combined approach of concurrent 5 fluorouracil-cisplatin and irradiation popularized by the group at Rush-

210

Chapter J 2 Presbyterian (20). The combined modality arm was superior (17), but this report has only appeared in abstract form with no follow-up since 1993. Pass et al.'s report (21) suggested a benefit, but the numbers were far too small to be valid. Wagner et al. (22) described a randomized phase II LCSe trial of preoperative MVP compared to preoperative radiation with neither arm being superior and both demonstrating significant toxicity. The phase II trials have explored several different means of delivering preoperative therapy, with the most popular being concurrent chemoradiotherapy (23-42) (Table 12.2). Chemotherapy alone (43-51) and chemotherapy alternating with radiation (52, 53) have also been explored with similar results, although the initial resectability and tumor sterilization rates appear slightly inferior for the chemotherapyonly approaches. Several investigators have employed sequential combined therapy with the chemotherapy preceding the radiation (54) or sequential therapy followed by concurrent "sensitizing" doses of chemotherapy during the irradiation (55-58). No one approach appears superior. The current Intergroup trial explores the role of surgery in this setting, comparing etoposide-cisplatin and concurrent irradiation with or

Table 12.2.

Phase II trials of preoperative concurrent radiation and chemotherapy for stage lila NSCLC

Author (ref), yr

No. of patients

% Complete resection

Rx-related deaths (%)

No. pathologic complete response (%)

% Overall survival (yr)

Milstein et al. (23), 1996 Goldfarb et al. (24), 1995 Reboul et al. (25), 1995 Karp et al. (26), 1995 Belli et al. (27), 1995 Mathisen et al. (28), 1996 Rice et al. (29), 1995 Albain et al. (30), 1995 Wagner et al. (31), 1995 Katakami et al. (32), 1995 Weiden et al. (33), 1994 Maggi et al. (34), 1994 Choi et al. (35), 1994 Bedini et al. (36), 1993 Palazzi et al. (37), 1993 Yoneda et al. (38), 1993 Rovea et al. (39), 1993 Weitberg et al. (40), 1993 Rebischung et al. (41), 1994 Yashar (42), 1992 Fischer (51), 1994

36 16 24 17 25 40 42 126 25 42 85 18 35 38 43 25 15 53 73 36 60

56 44 NR 35 48 87 86 83 52 45 52 66 80 47 28 72 73 51 70 88 62

3 (8) NR NR NR 2 (8) 3 (8) 5 (12) 13 (10) 1 (4) NR 0 1 (5) 3 (9) NR 4 (9) 1 (4) NR NR 6 (8) NR NR

3 (8) NR NR 3 (18) 2 (8) 2 (5) 2 (5) NR 8 (32) 7 (17) 8 (9) 6 (33) NR NR 3 (7) 6 (24) 6 (40) NR 7 (10) 10 (28) NR

39 (2) NR NR NR NR NR 53 (2) 26 (3) NR NR NR NR 50 (2) 63 (2) NR 67 (3) NR NR NR 62 (3) NR

NSCLC = non-small-celliung cancer; Rx = treatment; NR = none reported.


211

without consolidative surgery. Accrual is modest to date but the study seeks to explore whether the surgical intervention is required. It has been demonstrated that the patients most likely to benefit are those with complete histologic clearing following the preoperative therapy (59,60). If this turns out to be true, the surgical resection may only serve as a prognostic procedure rather than adding therapeutic benefit.

Stage 1114 NSCLC: Bulky N2 or N3 Disease These patients are beyond the scope of this chapter, although Albain et al. (30) have suggested that some N3 patients (contralateral mediastinal nodes, not supraclavicular) do as well following preoperative therapy. What is clear, however, is that this subset of patients has served as a very effective stalking horse for therapeutic approaches that can be moved into the preoperative arena (61-69).

CONCLUSION Progress continues to be made against locally advanced NSCLC. Data is now quite conclusive that sequential chemotherapy radiation and/ or concurrent sensitizing doses of chemotherapy improve outcome in patients with truly unresectable NSCLC (stage 1114)' Despite numerous phase II trials, there is still no conclusive proof that concurrent therapy is superior. It is a mixed blessing that we now have newer and potentially superior chemotherapy regimens. It will require a reworking of new or existing trials to sort out their relative benefit. This will delay the answer to fundamental issues of drug-radiation sequencing and the need for surgical consolidation, as no one wants to be accused of using "inferior" chemotherapy.

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13 ·New Chemotherapeutic Agents in NSCLC Vincent A. Miller, Kenneth K. N g, Stefan C. Grant, Ellen Early, and Mark G. Kris

BACKGROUND Lung cancer continues to be the most lethal malignant tumor, annually killing more women than breast, ovary, and uterine cancer combined, and taking nearly four times as many lives as prostate cancer in men. In 1997, it is estimated that 178,100 cases of lung cancer will be diagnosed (1). Approximately 87% of these patients will eventually succumb to their illness. Although improvements in care have increased 5-year survival from 9 to 13%, translating into thousands of lives saved, better therapies are sorely needed. Chemotherapy has been studied largely in patients with stage IIIb (malignant pleural effusion) or stage IV non-smaIl-cell lung cancer (NSCLC). In this setting, combination chemotherapy composed solely of active agents, when given to patients with an adequate performance status, leads to an approximate doubling of median survival (from 4 to 8 months) and an enhanced quality of life (2). Earlier stage patients were treated with surgery or definitive radiation therapy. In the 1980s, the utility of chemotherapy preceding surgery or radiation in stage IlIa and IIIb (nonpleural effusion) NSCLC was demonstrated. The rationale for this included the following: 217

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1. The use of chemotherapy in earlier stages of disease was generally associated with higher response rates than when used in more advanced disease. 2. Chemotherapy was better tolerated in earlier stage patients. 3. Although technically feasible, single-modality (surgery or radiation) therapy was associated with dismal survival in patients with stage IlIa or I1Ib disease, largely due to metastatic spread. In multiple randomized trials, this multimodality approach was conclusively shown to improve the survival of patients with stage III NSCLC.

Five chemotherapeutic agents useful in NSCLC patients were identified in the 1970s and 1980s: vindesine, vinblastine, mitomycin, ifosfamide, and cisplatin (3). Trials of these drugs as two- and three-drug combinations have shown that the rate of response for combinationagent therapy appears to be higher than that of single agents, and the highest rates of response have been seen with the three-drug combinations, particularly in studies employing doses and schedules approaching those used in the single-agent phase II trials. Using cisplatin, 120 mg/m2 combined with two other active agents, major response rates of 40% and I-year survivals of 40% have been demonstrated in multicenter, randomized trials (4). Response rates have been shown to be higher for patients with stage III disease (5, 6). More recently, a number of agents (docetaxel, gemcitabine, irinotecan, paclitaxel, tirapazamine, topotecan, and vinorelbine) have been identified for the treatment of NSCLC. The availability of these drugs increase the chemotherapeutic options available for the treatment of metastatic disease. Furthermore, the encouraging results seen with older chemotherapy regimens in locally advanced disease suggest that these new drugs may be useful in that patient population as well as in individuals with high-risk stage I or II NSCLC. The encouraging response rates and survival data demonstrated have also spawned renewed interest in studying the role of new agents in the adjuvant therapy of completely resected NSCLC. This chapter summarizes the results of single-agent and combination regimens utilizing these new agents, and discusses ways to optimally define the role of these drugs in NSCLC.

DOCETAXEL Docetaxel (Taxotere) is a semisynthetic taxoid derived from the precursor 10-deacetylbaccatin III. Its mechanism of action, the promotion of microtubule assembly and inhibition of the depolymerization of tubulin, which stabilizes microtubules, is similar to that of paclitaxel. In five phase I studies using docetaxel formulated with the solubilizing agent


219

TWEEN 80 and employing various schedules, the dose-limiting toxicity was neutropenia. Nonhematologic side effects included mucositis, asthenia, rash, alopecia, infusion-related reactions, and peripheral neuropathy. Notably, longer infusions were associated with an increased incidence of mucositis. Antitumor responses were seen in a variety of solid tumors including NSCLC. Based on these trials, the recommended phase II dose was 100 mg/m2 given as a I-hour infusion every 3 weeks with dexamethasone premedication. Seven phase II trials of docetaxel as first-line therapy of NSCLC have been reported. A pooled major objective response rate of 26% in 280 patients was observed with a median survival of 10 months for this single agent (7-13). Five of the trials employed a dose of 100 mg/m2, while one trial used a dose of 75 mg/m2 (13) and another a dose of 60 mg/m2 (11). Although observed response rates were lower at the 60 and 75 mg/m2 doses, no difference in survival was apparent. Historically, patients with disease progression after initial chemotherapy for advanced NSCLC were either treated with best supportive care or referred for trials of phase I agents. This practice was well founded, as the median survivals reported with active agents used in the second-line setting were 5 months or less, and major objective response rates were routinely less than 10% (14). The lack of treatment alternatives for individuals with disease progression following cisplatin but with adequate performance status, combined with responses seen in "cisplatin failures" treated on phase I trials of docetaxel, engendered phase II trials of this agent in such patients. Two trials each enrolled 44 NSCLC patients with disease progression following cisplatin and employed a dose of 100 mg/m2 given as a I-hour infusion every 3 weeks (15, 16). Fossella et al. (15) reported a 21 % response rate (95% confidence interval [CL] = 9-33%) and a median survival of 42 weeks. Similarly encouraging results were observed in a parallel study at the University of Texas Health Science Center at San Antonio. The pooled response rate of 17%, 9-month median survival, and 38% I-year survival are unparalleled. The initial results were confirmed in a multicenter phase II study. Two large phase III studies comparing docetaxel to either best supportive care or another active chemotherapeutic agent (vinorelbine or ifosfamide) have nearly completed accrual and attempt to confirm these encouraging results.

GEMCITABINE Gemcitabine (Gemzar) (2'2'-difluorodeoxycytidine) is a fluorine-substituted cytarabine (ARA-C) analog. Like the parent drug, it is a prodrug requiring intracellular phosphorylation (17, 18). In contrast to cytarabine, gemcitabine has greater membrane permeability and enzyme

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affinity. The incorporation of, the phosphorylated gemcitabine into DNA appears to be the major mechanism by which gemcitabine causes cell death (19). Because of gemcitabine's inherent ability to inhibit DNA replication and repair, this drug is an attractive candidate for combination with drugs that damage DNA. Preclinical data have revealed gemcitabine to be active in human lung xenograft models (20-22). Combination studies with gemcitabine and cisplatin or paclitaxel in the human NSCLC xenograft CALU-6 demonstrate synergistic antitumor activity (22). Gemcitabine in combination with cisplatin is active against NSCLC in vitro (23, 24). The mechanism of synergy is unclear, although it is known that cytosine arabinoside and hydroxyurea can potentiate cisplatin effects by inhibiting interstrand crosslink repair. Gemcitabine also acts both as an inhibitor of ribonucleotide reductase and as a chain terminator (25, 26). Data from phase I trials have demonstrated important scheduledependent differences in gemcitabine toxicity (27,28). Studies employing frequent drug administration (daily for 5 days or twice weekly) led to a higher incidence of nonhematologic toxic effects including flulike symptoms (fever, malaise, headache) (29). Given this, the most commonly employed doses and schedule in NSCLC patients in phase II trials have been a weekly 30-minute infusion of 800 to 1250 mg/m2 for 3 weeks followed by a I-week rest period. The initial phase II trial reported by Abratt et al. (30) reported on 84 NSCLC patients who received either 1000 or 1250 mg/m2 once weekly for 3 consecutive weeks. A20% major objective response rate (95% CL = 12-31%) and 9.2-month median survival was seen, and hematologic toxicity was negligible. Nonhematologic toxicity such as edema, asthenia, and malaise were readily managed (30). In a second phase II trial conducted by the Manchester Lung Tumour Study Group in 82 NSCLC patients at a starting dose of 800 to 1000 mg/m2, 16 partial responses in 79 evaluable cases were observed (overall response rate = 20%; 95%CL= 12-31%), with a median response duration and median survival of 7 months (31). A European multicenter confirmatory trial treated 154 NSCLC patients at a dose of 1250 mg/m2 over 30 minutes for 3 consecutive weeks followed by 1 week of rest (32). A 17% major objective response rate (95% CL = 12-24%) was observed. Gemcitabine was very well tolerated; grade IV neutropenia was seen in only 1% of administered cycles, and thrombocytopenia was even less common. In Japan, similar results were obtained in phase II trials (33). Given this mild adverse effect profile, a recently completed phase I doseescalation study in chemotherapy-naive NSCLC patients sought to better define the phase II single-agent dose on the weekly schedule (34). This trial showed that a weekly dose of 2400 mg/m2 could be safely given. This trial reported a major objective response rate of 25% in 32 assessable patients, and a projected median survival of 49 weeks. Doselimiting toxicities were reversible transaminase elevation and myelosuppression. Whether use of this higher dose imparts a meaningful clinical benefit remains to be determined.


Table 13.1.

221

Phase" studies of gemcitabine with cisplatin in NSCLC patients

Author (ref), yr

Gemcitabine

Cisplatin

Sandler et al. (35), 1995 Steward et al. (36), 1995 Crino et al. (37), 1997 Shepherd et al. (38), 1996

1.0 g/m2 on days I, 8,15 1.0 g/m2 on days I, 8,15 1.0 g/m2 on days 1,8, 15 1.0-2.25 g/m2 on days I, 8, 15 1.0 g/m2 on days I, 8,15 1.2 g/m2 on days I, 8,15

100 mg/m2 on day 1 100 mg/m2 on day 15 100 mg/m2 on day 2 25-30 mg/m2 on days I, 8, 15 100 mg/m2 on day 15 100 mg/m2 on day 15

Abratt et al. (39), 1997 Anton et al. (40), 1996

No. of patients

RR% (95% Cl)

Median survival

l-yr survival

26

42 (23-63)

NR

37%

52

38 (28-52)

NR

40%

48

54 (40-68)

14mo

59%

50

30 (17-43)

5.5mo

NR

53

52 (37-63)

13mo

56%

40

47 (31-63)

NR

NR

RR = response rate; CL = confidence limit; NR = none reported.

Six phase II trials of gemcitabine and cisplatin have been conducted in patients with advanced NSCLC (35-40). These trials (Table 13.1) report a pooled response rate of 44% and equally promising median and I-year survival rates. Because of its mild adverse-effect profile, preclinical synergism with cisplatin, and ease of administration, gemcitabine merits further study in combination regimens, multimodality therapy, and possibly in conjunction with radiotherapy for locally advanced disease.

IRINOTECAN lrinotecan (Camptosar, CPT-l1) is a semisynthetic derivative of the plant product camptothecin, which inhibits the enzyme topoisomerase I. Phase I trials revealed the dose-limiting toxicities to be leukopenia and diarrhea, and the recommended phase II dose was 100 mg/m2 weekly (41). A phase II trial of irinotecan (42) treated 73 patients with inoperable or metastatic NSCLC with 100 mg/m2 weekly, with dose adjustment based on the leukocyte count on the day of retreatment. Twenty-three of 72 evaluable patients achieved a partial response (32%; 95% CL = 20-44%); no complete responses were observed. Grade III or IV leukopenia was seen in 25%, and grade III or IV diarrhea occurred in 21 %. The incidence and severity of diarrhea did not correlate with the total dose administered, and loperamide was not routinely employed. The median response duration was 15 weeks, and the median survival was 42 weeks. Irinotecan has also been explored in a phase I combina-

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tion trial with cisplatin (43). In this study, 27 untreated NSCLC patients received 3 consecutive weeks of irinotecan therapy, commencing at a dose of 30 mg/m2, and cisplatin 80 mg/m2, on the first day of each treatment cycle. The dose of irinotecan was successfully escalated to 60 mg/m2. Beyond this, leukopenia and diarrhea became dose-limiting. Major objective responses were seen in 14 patients (54%; 95% CL = 32-73%). These preliminary results are promising and justify additional confirmatory single-agent phase II studies, as well as testing in combination therapy.

PACLITAXEL Paclitaxel (Taxol) is a plant-derived antineoplastic agent initially isolated from the bark of the Pacific yew tree, Taxus brevifolia. Antitumor activity of the yew bark was first identified in the 1960s through a plantscreening program sponsored by the National Cancer Institute. In 1971, paclitaxel was isolated and characterized as the active component of the bark extract (44). Paclitaxel entered clinical trials in the early 1980s, and the dose-limiting toxicity was determined to be neutropenia. Other toxicities included infusion-related reactions, alopecia, peripheral neuropathy, mucositis, and myalgias/ arthralgias. The recommended dose of paclitaxel for phase II study ranged from 135 to 250 mg/ m 2, with infusion duration ranging from 3 to 24 hours every 3 weeks. At least 6 phase II trials of paclitaxel have been conducted in NSCLC patients previously untreated with chemotherapy (45-50). The results of these trials are summarized in Table 13.2. No definite conclusions regarding the optimal length of infusion are apparent. However, there is a suggestion that doses less than 200 mg/m2 may be associated with lower response rates. Neurotoxicity may be somewhat greater with shorter infusions, while neutropenia may be lessened. Infusion length beyond 3 hours does not affect the frequency of infusion-related reactions in appropriately preTable 13.2.

Single-agent phase" trials of paclitaxel in NSCLC patients untreated with chemotherapy

Author (ref), yr

Dose

Chang et a1. (45), 1993 Murphy et a1. (46), 1993 Hainsworth et a1. (47), 1995

250 mg/ m 2 x 24 hr 200 mg/ m 2 x 24 hr 135 mg/m 2 xl hr 200 mg/m2 xl hr 210 mg/m2 x 3 hr 175 mg/m2 x 3 hr 210 mg/m2 x 3 hr

Alberola et a1. (4S), 1995 Millward et a1. (49), 1996 Sekine et a1. (50), 1996 TOTAL

No. of patients

24 25 7 20 47 51 60 234


RR% (95% el)

Median survival

l-yr survival

21 24 2S 25 36 10 23

5.6mo 9.2mo Smo Smo NR 6.7mo 11.2 mo

40% NR 33% 33% 45% 40% 4S%

Smo

40%

23 (lS-2S)


Table 13.3.

223

Trials of paclitaxel in previously treated NSCLC patients

Author (ref), yr

Dose

No. of patients

RR% (95% CL)

Median survival

l-yr survival

Murphy et al. (52), 1994 Ruckdeschel et al. (51), 1994 Hainsworth et al. (47),1995

175-200 mg/m2 x 24 hr 200-250 m~/m2 x 24 hr 135 mg/m xl hr 200 mg/m2 xl hr

30 14 10 16

3 14 0 38

NR 4mo NR NR

NR NR NR NR

70

13 (7-23)

TOTAL RR

= response rate; CL = confidence limit; NR = none reported. medicated patients. Trials of this drug in second-line therapy have also been reported (Table 13.3) (47,51,52). Unlike docetaxel, there is less evidence for paclitaxel to be an active agent in second-line therapy; only one reported response has occurred at doses less than 200 mg/m2. Paclitaxel has been explored in combination with both cisplatin and carboplatin. Phase I trials of paclitaxel as a 24-hour infusion and cisplatin showed neutropenia to be dose-limiting and recommended a paclitaxel dose of 135 mg/m2 with cisplatin, 75 mg/m2. With routine use of granulocyte colony-stimulating factor (G-CSF), the paclitaxel dose could be increased to 250 mg/m2, and neurotoxicity became doselimiting (53, 54). On the basis of these results and with encouraging activity seen with the combination in phase I trials, the Eastern Cooperative Oncology Group (ECOG) undertook a large (n = 560) three-arm randomized trial comparing cisplatin and etoposide-a reference regimen from prior lung cancer trials-against cisplatin, 75 mg/m2, with either paclitaxel 135 mg/m2 or 250 mg/m2 as a 24-hour infusion with prophylactic filgrastim (55). The paclitaxel-based combinations both provided improved response rates (27 and 32%, respectively) versus the etoposide combination (12%) (P < 0.001). In addition, a trend for improved survival was seen with the paclitaxel-based combinations (10, P = 0.12 months and 10, P = 0.09 months vs. 8 months). No appreciable difference in efficacy was seen between the two paclitaxel dose levels. The EORTC also studied the combination of paclitaxel, 175 mg/m2 as a 3-hour infusion, with cisplatin, 80 mg/m2, versus teniposide and cisplatin. Again, the paclitaxel combination produced superior response rates. Median survival in both arms was 9 months (56). Current studies are evaluating the utility of a 96-hour paclitaxel infusion with cisplatin (57). Carboplatin was chosen to be combined with paclitaxel because of lessened neurotoxicity and ototoxicity as well as ease of administration (58). Phase II trials of this agent show an 11 % pooled response rate from 291 treated patients and a 6.5-month median survival (59). These results are inferior to those of single-agent cisplatin, the other conventional active agents, and the new agents described herein. However, the suggestion of a dose-response relationship and the capacity to now more

224

Chapter 13

reliably dose carboplatin using the Calvert formula fostered enthusiasm that the agent might meet criteria for activity. In the first reported trial of this combination, Langer et al. (60) administered paclitaxel as a 24-hour infusion, 135 to 215 mg/m2, on day I, followed by carboplatin with an area under the concentration-x time curve (AVC) of 7.5 to 53 advanced NSCLC patients. At a paclitaxel dose of 135 mg/m 2 neutropenia was dose-limiting without growth factor use. However, with prophylactic filgrastim, paclitaxel doses were successfully escalated to 215 mg/m2. In this trial, a 62% major response rate (95% CL = 48-78%) and I-year survival of 54% were observed. An alternative schedule using paclitaxel as a 3-hour infusion followed by carboplatin at an AVC of 6 has also been evaluated extensively (58). Paclitaxel doses were escalated in successive cohorts of patients from 150 to 250 mg/m2. At this highest dose level, neurotoxicity proved dose-limiting, and a paclitaxel dose of 225 mg/m2 has been recommended for further study. No prophylactic serotonin antagonist antiemetics or growth factors were routinely employed, and at this paclitaxel dose therapy was well tolerated. Major responses were reported in 20 of 32 (63%; 95% CL = 45-81%) patients, including two complete responses. A third trial employed an analogous schedule to that of Langer and used paclitaxel (135 or 175 mg/m2) and carboplatin (300 mg/m2 or AVC of 6), and treatment was given once every 4 weeks (61). Fourteen responses were seen in 51 patients (27%; 95% CL = 17-41%), with a median survival of 8.8 months and a I-year survival of 32%. A suggestion of higher response rates at the higher dose of paclitaxel was noted. One possible explanation for the results less favorable than those obtained by Langer include the reduced dose intensity achieved in this trial. Specifically, a mean lower carboplatin dose was given per cycle, and the retreatment interval of 4 weeks, rather than 3 weeks, in addition to the larger proportion of patients who received the lower dose of paclitaxel all may have contributed to a lower response rate and poorer overall survival. A four-arm randomized trial of ECOG is now under way and compares paclitaxel and cisplatin, paclitaxel and carboplatin, docetaxel and cisplatin, and gemcitabine and cisplatin.

TIRAPAZAMINE Tirapazamine (WIN 59075; SR 4233; 1,2,4-benzotriazin-3-amine 1,4dioxide) is the lead compound in a new class ofbioreductive anticancer drugs, the benzotriazine di-N-oxides, characterized by their preferential cytotoxicity for hypoxic cells. Tirapazamine is cytotoxic in drug concentrations capable of providing a differential large enough to kill hypoxic cells with minimal killing of well-oxygenated cells (62).


225

Tirapazamine damages hypoxic cells when it undergoes one-electron reduction by cytochrome P-450 reductase to a free radical, which induces extensive single- and double-strand breaks in DNA (63-65). This cytotoxic radical is oxidized to an inactive metabolite in well-oxygenated tissues. In preclinical studies, tirapazamine kills cancer cells when administered intraperitoneally to tumor-bearing mice and in fibrosarcoma xenografts (66). Under hypoxic conditions, tirapazamine reverses resistance to cisplatin in a human breast-cancer cell line (67) and demonstrates schedule-dependent synergism with cisplatin in a RIF-1 tumor (68). Lastly, the combination of tirapazamine and cisplatin produced additive cell-killing of human lung adenocarcinoma (A549) cells (69). The initial phase I trial of 28 patients with advanced solid tumors of single-agent tirapazamine defined the dose-limiting toxicity to be acute, reversible tinnitus and hearing loss at 450 mg/ m 2 (70). These patients were given a total of 50 courses of tirapazamine at doses ranging from 36 to 450 mg/m2 every 3 weeks. Reversible ototoxicity was observed in 1 (17%) of 6 patients treated at 330 mg/m2, 1 (25%) of 4 patients treated at 390 mg/m2, and 3 (100%) of 3 patients treated at 450 mg/m2. Muscle cramping and vomiting were also noted. Subsequently, Rodriguez et al. (71) conducted a phase 1/11 trial of the combination of tirapazamine and cisplatin in untreated patients with stage IIlb or stage IV NSCLC. In the phase I portion, 41 patients were treated with tirapazamine given at doses of 80 to 390 mg/m2, combined with cisplatin administered at doses of 75 or 100 mg/m2, every 3 weeks. A total of 151 courses were given. Observed toxicities of the combination included nausea and vomiting, transient diarrhea, rash, muscle cramping, and acute, reversible hearing loss. No dose-limiting myelosuppression was observed. The overall response rate in the phase I portion was 32%, with a median survival of 11 months. One-year survival was 43%, with a median follow-up of 18 months. One complete response and eleven partial responses were reported in 37 evaluable patients. A phase II dose of tirapazamine, 390 mg/m2, and cisplatin, 75 mg/m2, was selected because of higher incidences of nausea/vomiting and renal insufficiency observed with the cisplatin dose of 100 mg/m2. In the phase II portion, 27 of 33 patients were assessed with an overall response rate of 26%. Similarly, Treat et al. (72) reported a major response rate of 23% in a second phase II trial of 36 patients with stage IIIb or stage IV NSCLC, who previously did not receive chemotherapy. Tirapazamine was administered at a dose of 260 mg/m2 in 32 patients and 390 mg/m2 in 4 patients over 2 hours, followed 1 hour later by cisplatin at 75 mg/m2, every 21 days. Toxicities were similar to the previous trial by Rodriguez, except there was no otoxicity reported. In a third phase II trial of this combination employing the same dose and schedule as the phase II portion of the Rodriguez trial, Miller et al. (73) demonstrated a major objective response rate of 25% (95% CL =

226

Chapter 13

11-50%) in 20 chemotherapy-naive patients with stage IIIb or IV NSCLC. The median duration of response was 8 months, with a range of 6 to 11 months, and a projected I-year survival of 44%. No grade 4 toxicity was observed. These trials suggests that tirapazamine enhances the antitumor activity of cisplatin. In addition, the toxicity of the combination of tirapazamine and cisplatin is not substantially increased over cisplatin alone, and little myelosuppression occurs. A phase III multicenter trial comparing the combination to cisplatin alone has completed accrual. Because significant neutropenia rarely occurs with this combination, a phase 1/11 trial of tirapazamine and cisplatin plus paclitaxel has been initiated. Preclinical studies have also demonstrated that tirapazamine may enhance the cytotoxicity of radiation, suggesting the possibility of a role in combined modality therapy in NSCLC (74, 75).

TOPOTECAN Topotecan is a semisynthetic camptothecin derivative with broad antitumor activity in both human and animal tumors (76) and a favorable toxicity profile due to increased hydrophilicity and decreased protein binding (77). The agent was initially studied in a variety of schedules ranging from a 30-minute infusion every 3 weeks to a 120-hour infusion every 3 weeks, and neutropenia has generally been dose limiting. Based on data in human-mouse xenografts suggesting that prolonged exposure would maximize antitumor efficacy, Hochster et al. (78) evaluated topotecan as a 21-day continuous infusion. Dose-limiting hematologic toxicity has not been reached, with the calculated dose-intensity already exceeding that of the recommended phase II dose of 1.5 mg/m2 daily for 5 days. This seemingly favorable hematologic-toxicity profile, combined with the opportunity for prolonged drug exposure so as to affect dividing cells at the appropriate stage of the cell cycle, made this an appealing schedule for phase II investigation in NSCLC patients. A phase II trial of topotecan given as a 21-day continuous infusion using daily doses of 0.5 to 0.6 mg/m2 has been conducted by investigators in New York and London (79). One partial response in 26 patients was observed (4%; 95% CL = 2-18%), and a median survival of9 months and I-year survival of 39% were attained. Symptomatic benefit was also documented and was more evident in patients with stable or responding disease. A phase II trial of Lynch et al. (80) giving topotecan 2 mg/m2 daily for 5 days yielded no objective responses in a group of 20 NSCLC patients who had not received prior chemotherapy. Another phase II trial treated individuals with either topotecan, 1.5 mg/m2, as a 30-minute daily infusion for 5 days every 3 weeks (n = 38), or as a 3-day continu-

227


ous infusion at a dose of 1.3 mg/m2 per day every 4 weeks (n = 40) (81). Seven of 38 patients attained partial response on the former schedule (18%; 95% CL = 8-33%), whereas only three responses were seen on the latter schedule (8%; 95% CL = 2-22%); however, toxicity was comparable. The median survival on the daily 5-day schedule was 8.1 months compared to 5.9 months with continuous infusion therapy. An additional phase II trial at the MD Anderson Cancer Center administered topotecan at a dose of 1.5 mg/m2 daily for 5 consecutive days every 3 weeks (82). Six of 40 (15%; 95% CL = 6-30%) assessable patients attained a partial response with a median survival of 9 months and 1-year survival of 30%. Five of 6 responses were noted in patients with squamous cell carcinomas; an observation unconfirmed in other trials of this agent. These results are summarized in Table 13.4. Current trials are studying topotecan in combination with cisplatin, an agent with which it demonstrates preclinical synergism.

VINORELBINE Vinorelbine (Navelbine) is a semisynthetic vinca alkaloid approved in the United States as a single agent or in combination with cisplatin for the treatment of NSCLC. The compound was created by a modification in the catharanthine (rather than the vindoline) ring, which appears to be responsible for a favorable toxicity and activity profile. Phase II studies have generally employed a schedule of 30 mg/m2 weekly as a single 20-minute infusion. An initial phase II trial in NSCLC showed a response rate of 32% (95% CL = 23-41 %) in 97 patients (83). A subsequent phase II trial (84) reported a 29% response rate in 62 evaluable patients. Because of these promising results, randomized single-agent

Table 13.4.

Single-agent phase" trials of topotecan in NSCLC patients untreated with chemotherapy

Author (ref), yr

Dose

Lynch et a1. (80), 1994

2 mg/ m 2 for 30 min daily x 5 days 1.5 mg/m2 for 30 min daily x 5 days 1.3 mg/m 2 CVI x 3 days 1.5 mg/m2 for 30 min daily x 5 days 0.5-0.6 mg/m2 CVI x 21 days

Weitz et a1. (81), 1995

Perez-Soler et a1. (82), 1996 Kindler et a1. (79), 1997 TOTAL RR

No. of patients

RR% (95% el)

Median survival

l-yr survival

20

0

7.6mo

NR

38

18

8.1 mo

NR

40 40

8 15

5.9mo 9mo

NR 30%

26

4

9mo

39%

164

10(6-18)

8mo

= response rate; CL = confidence limit; NR = none reported; CVI = continuous venous infusion.

228

Chapter 13

and combination trials were undertaken. The first study (85) randomized 612 patients to receive vinorelbine in combination with cisplatin (120 mg/m2), vinorelbine alone, or vindesine with cisplatin. Response rates were 30%, 14%, and 19%, respectively. The median duration of survival was 9 months in the vinorelbine/cisplatin arm, and 7 months in both the vindesine/ cisplatin group and the single agent vinorelbine arm. Comparison of survival among the groups showed a statistically significant advantage for the vinorelbine/ cisplatin group (85). Neutropenia was significantly higher in the vinorelbine/ cisplatin-treated patients, but neurotoxicity occurred more frequently in the vindesine/ cisplatin cohort. A second trial randomized individuals to receive weekly vinorelbine or 5-fluorouracil (425 mg/m2/day for 5 days) with leucovorin (20 mg/ m 2/ day for 5 days). A 12% major response rate was observed in the vinorelbine arm (n = 143) compared to 3% for the 5-fluorouracil/leucovorin group-a result not attaining statistical significance (86). The median and I-year survival for the vinorelbine group (7 months and 25%) were prolonged compared to the 5-fluorouracil/leucovorin arm (5 months and 16%) (P = 0.03). On the basis of these two trials, vinorelbine was approved by the U.s. Food and Drug Administration (FDA) both as a single agent and in combination with cisplatin for advanced but ambulatory NSCLC patients. In a subsequent trial conducted by the Southwest Oncology Group, the combination of vinorelbine and cisplatin produced significant improvement in response rate and median and I-year survival versus single-agent cisplatin (87). Results of single-agent trials of vinorelbine are summarized in Table 13.5.

COMBINATION THERAPY AND FUTURE DIRECTIONS The identification of a number of new agents with activity in NSCLC affords the opportunity for improved results, but also mandates discretion in choosing which two- and three-drug combinations to pursue.

Table 13.5.

Single-agent trials of vinorelbine in NSCLC patients untreated with chemotherapy

Author (ref), yr

Dose

No. of patients

RR% (95% el)

Median survival

l-yr survival

Depierre et a1. (84), 1991 Yokoyama et a1. (83), 1992 Le Chevalier et a1. (85), 1994 Crawford et a1. (86), (1996)

30 mg/m2 25 mg/m 2 30 mg/m2 30 mg/m2

78 79 206 143

29 23 14 12

7.6mo 9.2mo 8mo 7mo

30% NR 30% 25%

506

17 (14-22)

8mo

28%

TOTAL



229

Historically, the addition of a third active agent to a combination adds to response rate but not survival in stage IV NSCLC patients. Often toxicity issues compromise dose intensity and offset theoretical gains in efficacy. Mathematically, more than 60 two-drug and several hundred three-drug combinations may now be generated without considering dose and schedules of the component agents. Thus, the development of successful combination chemotherapeutic regimens requires the continued identification of single agents with response rates reproducibly above 15%. The toxicities of component agents, together with their mechanisms of action, must be taken into account. Nonoverlapping toxicities and preclinically demonstrated synergism make for the most attractive combinations. Importantly, a component drug'S dose and schedule should be close to that used in single-agent phase II trials yielding the highest response rates. When optimization of dose or schedule requires supportive care measures such as combination antiemetics and cytokine growth factors, they should be used routinely. Secondary endpoints such as quality of life and cost must be assessed. The use of these newer agents either alone or in combination appears to have made a clear improvement in the survival of advanced NSCLC patients. Reproducible I-year survivals of 30-40% and median survivals of 9 to 10 months in cooperative group trials suggest that we have taken a real step forward in the treatment of advanced disease. It is likely the use of these agents will have an even greater impact in earlier stage patients as part of combined modality programs. In order to reach the next milestone in treating advanced disease, it will be important to be selective in choosing which two- and three-drug combinations to evaluate, while more rapidly testing agents of entirely different classes that can build on the successes of combination chemotherapy.

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Bonomi P. Carboplatin in non-small cell lung cancer: review of the Eastern Cooperative Oncology Group trial and comparison with other carboplatin trials. Semin Onco11991;18:2-7. 60. Langer CJ, Leighton JC, Comis RL, et al. Paclitaxel and carboplatin in combination in the treatment of advanced non-small-ce111ung cancer: a phase II toxicity, response, and survival analysis. J Clin Oncol 1995;13:18601870. 61. Johnson DH, Paul DM, Hande KR, et al. Paclitaxel plus carboplatin in advanced non-small-cell lung cancer: a phase II trial. J Clin Oncol 1996; 14:2054-2060. 62. Brown JM. SR 4233 (Tirapazamine): a new anticancer drug exploiting hypoxia in solid tumours. Br J Cancer 1993;67:1163-1170. 63. Baker MA, Zeman EM, Hirst VK, et al. Metabolism of SR 4233 by chinese hamster ovary cells: basis of selective hypoxic cytotoxicity. Cancer Res 1988;48:5947-5952. 64. Cahill A, White INH. Reductive metabolism of 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) and the induction of unscheduled DNA synthesis in rat and human derived cell lines. Carcinogenesis 1990;11:14071411. 65. Keohane A, Godden J, Stratford IJ, et al. The effects of three bioreductive drugs (mitomycin C, RSU-1069 and SR 4233) on cell lines selected for their sensitivity to mitomycin C or ionising radiation. Br J Cancer 1989;61: 722-726. 66. Holden SA, Teicher BA, Ara G, et al. Enhancement of alkylating agent activity by SR-4233 in the FSaIIC murine fibrosarcoma. J Natl Cancer Inst 1992;84:187-193. 67. Kusumoto T, Teicher BA, Holden SA, et al. Reversal of drug resistance to MCF-7/CDDP human breast carcinoma cells by SR-4233 or etanidazole (ETA)/hypoxia. Proc Annu Meet Am Assoc Cancer Res 1993;34:322. Abstract. 68. Dorie MJ, Brown JM. Tumor-specific, schedule-dependent interaction between tirapazamine (SR 4233) and cisplatin. Cancer Res 1993;53:46334636. 69. Dorie MJ, Brown JM. Potentiation of the anticancer effect of cisplatin by the hypoxic cytotoxin tirapazamine. In: Vaupel PW, Kelleher DK, Grunderoth M, eds. Tumor Oxygenation. New York: Gustav Fischer, 1995:125135. 70. Senan S, Rampling R, Graham MA, et al. Phase I and pharmacokinetic study of tirapazamine (SR 4233) administered every three weeks. Clin Cancer Res 1997;3:31-38. 71. Rodriguez GI, Valdivieso M, Von Hoff DD, et al. A phase 1/11 trial of the combination of tirapazamine and cisplatin in patients with non-small cell lung cancer. Proc Am Soc Clin Oncol 1996;15:382. Abstract. 72. Treat J, Greenberg R, Rodriguez R, et al. Tirapazamine and cisplatin: phase II trial in advanced stage non small cell cancer (NSCLC). Proc Am Soc Clin Oncol 1996;15:400. Abstract. 73. Miller VA, Pizzo B, Grant Sc, et al. Phase II study of tirapazamine, a unique bioreductive agent, with cisplatin in patients with advanced nonsmall cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 1996;15:403. Abstract. 74. Brown JM, Lemmon MJ. Tumor hypoxia can be exploited to preferentially sensitize tumors to fractionated irradiation. Int J Radiat Oncol BioI Phys 1991;20:457-461.

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Zeman EM, Hirst VK, Lemmon MJ, et al. Enhancement of radiationinduced tumor cell killing by the hypoxic cell toxin SR 4233. Radiother Oncol 1988;12:209-218. Burris H, Kuhn J, Wall J, et al. Early clinical trials of topotecan, a new topoisomerase I inhibitor (Meeting Abstract). Ann Oncol 1992;3(Suppl 1):118. Verweij J, Lund B, Beynen J, et al. Clinical studies with topotecan: the EORTC experience (Meeting Abstract). Ann OncoI1992;3(Suppl1):118. Hochster H, Liebes L, Speyer J, et al. Phase I trial of low-dose continuous topotecan infusion in patients with cancer: an active and well-tolerated regimen. J Clin OncoI1994;12:553-559. Kindler HL, Kris MG, Smith IE, et al. Continuous infusion topotecan as first-line therapy in patients with non-small cell lung cancer (NSCLC): a phase II study. Proc Am Soc Clin OncoI1997;16:472a. Lynch TJ, Kalish L, Strauss G, et al. Phase II study of topotecan in metastatic non-smaIl-cell lung cancer. J Clin OncoI1994;12:347-352. Weitz H, Jung S-H, Marschke RF Jr, et al. Randomized phase II trial of two schedules of topotecan for the treatment of advanced stage non-small cell lung carcinoma (NSCLC): a North Central Cancer Treatment Group (NCCTG) trial. Proc Am Soc Clin OncoI1995;14:348. Perez-Soler R, Fossella FV, Glisson BS, et al. Phase II study of topotecan in patients with advanced non-smaIl-cell lung cancer previously untreated with chemotherapy. J Clin OncoI1996;14:503-513. Yokoyama A, Furuse K, Niitani H. Multi-institutional phase II study of navelbine (vinorelbine) in non-small cell lung cancer. Proc Am Soc Clin OncoI1992;11:287. Depierre A, Lemaire E, Dabouis G, et al. A phase II study of navelbine (Vinorelbine) in the treatment of non-smaIl-cell lung cancer. Am J Clin OncoI1991;14:115-119. Le Chevalier T, Brisgand D, Douillard JY, et al. Randomized study of vinorelbine and cisplatin versus vindesine and cisplatin versus vinorelbine alone in advanced non-smaIl-cell lung cancer: results of a European multicenter trial including 612 patients. J Clin OncoI1994;12:360-367. Crawford J, O'Rourke M, Schiller JH, et al. Randomized trial of vinorelbine compared with fluorouracil plus leucovorin in patients with stage IV non-small cell lung cancer. J Clin OncoI1996;14:2774-2784. Wozniak AJ, Crowley H, Balcerzak GR, et al. Randomized phase III trial of cisplatin (CDDP) vs. CDDP plus navelbine (NVB) in treatment of advanced non-small cell lung cancer (NSCLC): report of a Southwest Oncology Group study (SWOG-9308). Proc Am Soc Clin Oncol 1996;15: 374. Abstract.

14 Chemoprevention Jonathan M. Kurie, Li Mao, Jin Soo Lee, Scott M. Lippman, Margaret R. Spitz, and Waun Ki Hong

Chemoprevention is an intervention made to prevent the development of invasive cancer in individuals or populations at increased risk, with the intention of reducing cancer incidence and mortality.

THE NEED FOR CHEMOPREVENTION Despite a variety of advances in surgical, radiotherapeutic, and chemotherapeutic treatments, non-small-celliung cancer (NSCLC) mortality in the United States has not changed during the last 30 years (1). Overall survival for NSCLC is approximately 10%, and it remains the number one cause of cancer-related death in the United States (1). Our failure to eradicate invasive disease has led investigators toward intervention at earlier disease stages, prior to the development of invasive NSCLC. This movement has stimulated an investigation of bronchial premalignancy, its associated molecular genetic changes, and mechanisms by which the neoplastic process might be interrupted. This chapter describes lung cancer chemoprevention in its present state, highlights the recent advances in our understanding of bronchial epithelial premalignancy, and discusses how these advances might be used to develop better lung cancer chemoprevention strategies in the future.

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CHEMOPREVENTION TRIAL DESIGN Chemoprevention trials are designed to prevent the development of cancer in cancer-naive patients (primary prevention) or in patients cured of a prior cancer (second primary prevention). Among smokers, the risk of developing lung cancer is less than 1% per year (2). Because of the low prevalence of lung cancer in this population, primary lung cancer chemoprevention trials require 20,000 to 30,000 participants followed for 5 to 10 years to reach statistical significance. This sample size can be reduced in second primary lung cancer prevention trials because the participants have a higher incidence of lung cancer than cancernaive patients. NSCLC patients who have been rendered free of disease develop second primary tumors at a rate of 2-4% per year (3). Second primary lung cancer prevention trials require the enrollment of approximately 1000 patients who must be followed for approximately 5 years. To reduce the cost and time commitment required for these studies, investigators have sought "biomarkers" that detect either the presence of increased cancer risk or a biologic response to chemopreventive treatment. Biomarkers of lung cancer risk can be divided into several categories, including those that reflect an inherited lung cancer susceptibility, damage associated with environmental exposure (cigarette smoke, asbestos, etc.), and the presence of premalignant cells. Using these biomarkers, the ultimate goal in chemoprevention research is to construct a risk model that can predict an individual's risk of developing lung cancer. In addition, sample size can be reduced through the use of biomarkers that reflect activation of signal transduction pathways associated with chemoprevention treatment. Because modulation of these biomarkers replaces cancer as an endpoint of the trial, these biomarkers are considered "intermediate endpoints" of chemoprevention trials. Depending on the characteristics of the biomarker, definitive biomarker trials can include approximately 100 participants treated for 6 to 12 months.

PRIOR LUNG CANCER CHEMOPREVENTION TRIALS Several large scale, primary lung cancer chemoprevention trials were recently completed. The Alpha-Tocopherol, Beta-Carotene (ATBC) Cancer Prevention Study involved a total of 29,133 male smokers in Finland who were randomized to treatment with alpha-tocopherol alone, betacarotene alone, a combination of alpha-tocopherol and beta-carotene, or placebo. This was based primarily on evidence that alpha-tocopherol (the most prevalent chemical form of vitamin E found in vegetable oils, seeds, grains, nuts, and other foods) and beta-carotene (a plant pigment found in many yellow, orange, and dark-green leafy vegetables and

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some fruit) might reduce the risk of lung cancer. Unexpectedly, lung cancer incidence and total mortality were higher in participants who received beta-carotene than among those who did not (4). In the BetaCarotene and Retinol Efficacy Trial (CARET), 14,254 heavy smokers and 4060 asbestos-exposed workers were randomized to treatment with either a combination of beta-carotene and retinol or placebo. Lung cancer incidence was 28% higher in participants receiving beta-carotene and retinol, leading to closure of the trial 21 months early (5). The Physician's Health Study, which involved 22,071 male doctors, randomized participants to aspirin, beta-carotene, both drugs, or placebo for a mean treatment duration of more than 12 years. Endpoints were overall mortality, cardiovascular mortality, and cancer incidence. This study failed to show a benefit in lung cancer incidence from beta-carotene (6). The results of the ATBC and CARET trials suggested that beta-carotene alone or beta-carotene plus vitamin A had no chemopreventive benefit and had excess lung cancer incidence and mortality. Several second primary lung cancer prevention trials are ongoing, examining the incidence of second primary tumors in lung cancer patients who have been rendered disease-free. A multicenter study sponsored by the National Cancer Institute is testing the efficacy of 13cis retinoic acid (13cRA) in this setting, and accrual to this study has neared completion. This trial is based on a randomized, placebo-controlled study demonstrating that 13cRA treatment reduced the incidence of second primary tumors in head and neck cancer patients rendered disease-free (7). A trial sponsored by the European Organization for Research and Therapy of Cancer (EORTC), coined the Euroscan trial, is investigating the efficacy of retinyl palmitate and N-acetylcysteine in the reduction of second primary tumors in completely resected NSCLC and head and neck cancer patients. This trial is based, in part, on a smaller study demonstrating that retinyl palmitate reduced the incidence of second primary tumors in NSCLC patients rendered disease-free (3).

BIOMARKERS Studies examining potential biomarkers of lung cancer risk have focused on changes in the bronchial epithelium, using random bronchial biopsies and sputum specimens. This approach is based on the "field carcinogenesis" hypothesis, which states that the process of lung carcinogenesis occurs over the entire field of bronchial epithelium exposed to carcinogens (8), suggesting that random biopsies and sputum specimens will reflect the state of airway epithelium throughout the lungs. Bronchial epithelial metaplasia, dysplasia, and sputum atypia are associated with NSCLC and a history of smoking (9-11). Sputum atypia was examined in a multicenter lung cancer screening trial, and atypia

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Chapter 14 did not positively predict lung cancer development (12). Interpretation of cytologic findings differed among the investigators in this trial, making conclusions difficult. Chemoprevention trials have investigated the effect of different agents on bronchial metaplasia and dysplasia and sputum atypia (Table 14.1).In participants withatleast a 15 pack-year smoking history, Mathe et al. (13) observed a reduction in bronchial metaplasia following treatment with etretinate, a synthetic retinoid. However, this trial was not randomized, and randomized trials have failed to show significant differences between the intervention and treatment groups (14-17). In smokers with at least a 15 pack-year history, Arnold et al. (14) examined the effect of 6 months of treatment with etretinate or placebo on sputum atypia and found no effect. In a population with a heavier smoking history and exposure to asbestos, McLarty et al. (15) observed no effect of beta-carotene and retinol on sputum atypia. In participants with at least a 20 pack-year smoking history, Lee et al. (17) found no effect of 13cRA on bronchial metaplasia, but bronchial metaplasia was reduced by smoking cessation, suggesting that, in active smokers, bronchial metaplasia is an acute reaction to cigarette smoke exposure. The previously mentioned trials have relied on white-light bronchoscopy for the detection of bronchial metaplasia and dysplasia. Anew technique utilizing a laser incorporated into a bronchoscope was reported to be 50% more sensitive in the detection of bronchial dysplasia than standard white-light bronchoscopy (18). This technique is based on the principle that light of specific wavelengths can stimulate intrinsic cellular fluoroflors such as flavins, riboflavins, nucleic acids, and proteins to fluoresce, emitting a spectral pattern of light typical of that particular tissue. Epithelial carcinogenesis is associated with altered levels of these fluoroflors; by using fluorescence spectroscopy, normal, dysplastic, and neoplastic tissues can be distinguished on the basis of their spectral patterns (19). This new bronchoscopic technique is under evaluation in chemoprevention trials as a method of enhancing the detection of bronchial premalignancy in smokers and former smokers. NSCLC and dysplastic bronchial epithelial cells contain genetic alterations that have been used as biomarkers of increased lung cancer risk. The p53 gene is mutated in approximately 50% of NSCLCs, and these mutations have also been found in bronchial dysplasia (20-25). However, p53 gene mutations occur over a large region, making p53 DNA

Table 14.1

Chemoprevention trials in active smokers with bronchial metaplasia

Author (ref), yr

Chemopreventive agent

Endpoint

Results

Mathe et a1. (13), 1982 Arnold et a1. (14), 1992 Lee et a1. (17), 1994 McLarty et a1. (IS), 1995 Heimberger et a1. (16), 1988

Etretinate Etretinate 13cRA Beta-carotene / retinol Folate/vitamin B12

Bronchial metaplasia Sputum atypia Bronchial metaplasia Sputum atypia Sputum atypia

Positive Negative Negative Negative Positive

13cRA = 13-cis retinoic acid.

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sequencing laborious. Although, in many cases, p53 mutations lead to a prolonged protein half-life and intracellular accumulation of p53 protein (20-25), the predictive value of p53 immunohistochemical staining for the presence of p53 mutations is variable, reducing the utility of p53 as a biomarker. The kirsten-ras (K-ras) gene is mutated in approximately 30°/c) of lung adenocarcinomas (26-28), which is too Iowa prevalence to be useful in NSCLC screening. Loss of heterozygosity (LOH) and genomic instability of chromosomes 3p, 9p, and 17p have been detected in NSCLCs and in bronchial dysplasia (29, 30) (Table 14.2). LOH is recognized as loss of one chromosomal allele, and genomic instability is defined as a loss or gain of genetic material within a chromosomal region. Histologically normal bronchial biopsies contain these genetic mutations, suggesting that these events occur early in the process of lung carcinogenesis. Up to 80% of smokers have evidence of LOH and genomic instability in bronchial tissue, demonstrating widespread damage associated with cigarette smoke exposure and a potentially sensitive marker of premalignant change. These changes persist following smoking cessation, suggesting that bronchial tissue does not return to normal with smoking cessation (30). Recent work has shown that mutant DNA can also be detected in blood plasma of lung cancer patients (31), which would facilitate the process of screening for individuals at increased risk. Use of these markers as intermediate endpoints in lung cancer chemoprevention trials has begun, and the results of these studies will require several years to complete. A number of biochemical markers that reflect the highly proliferative state of bronchial premalignancy and NSCLC cells are being tested as biomarkers of lung cancer risk and of response to chemopreventive intervention. This includes the expression of epidermal growth factor receptor (EGFR), proliferating cell nuclear antigen (PCNA), and Ki-67 (32-46). Cell culture models suggest that activation of the EGFR signaling pathway is mitogenic in normal human bronchial epithelial (HBE) cells (47). EGFR expression is increased in NSCLC cells and in bronchial metaplasia, and reversal of bronchial metaplasia is associated with decreased EGFR levels (48). While cell culture studies suggest that EGFR expression is modulated by retinoid treatment (49-52), EGFR expression in the bronchial epithelium did not detectably change in a randomized, placebo-controlled trial with 13cRA (48). PCNA is a cell cycle-associated protein, and PCNA expression is increased in actively

Table 14.2

Loss of heterozygosity (LOH) in bronchial epithelium of smokers*

Category

3p14

9p21

17p13

TOTAL

Per case Per biopsy

27/36 (75%) 64/172 (37%)

21/37 (57%) 41/168 (24%)

6/34 (18%) 12/161 (7%)

39/51 (76%) 103/238 (43%)

*Number with LOH/total number (percent).

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dividing cells (39-46). PCNA and Ki-67 expression are increased in NSCLC cells, and immunohistochemical studies of NSCLC biopsies suggest that increased PCNA levels may correlate with shortened survival (39-46). The signal transduction pathways activated by some chemopreventive agents have been defined, and these pathways are being investigated as biomarkers. For example, expression of the retinoid nuclear receptor RAR-f3 (retinoic acid receptor-beta) is under investigation as a marker of lung cancer risk and of response to retinoid treatment. Compared to its expression in normal bronchial mucosa, RAR-f3 expression is reduced in bronchial metaplasia, dysplasia, and NSCLC (53). Similarly, RAR-f3 expression is lower in oral leukoplakia biopsies than in normal oral mucosa (54). 13cRA treatment increases RAR-f3 expression in bronchial epithelium and in sites of oral leukoplakia, and this was associated with a clinical response in leukoplakia patients (54). Additional studies are necessary to examine the role of RAR-f3 expression in the bronchial epithelium as a marker of lung cancer risk and of response to retinoid treatment. While approximately 90% of lung cancers occur in smokers or former smokers, among smokers, the annual risk of developing lung cancer is less than 1%, suggesting the presence of constitutional factors that determine lung cancer susceptibility (55-63). Although lung cancer is the paradigm of an environmentally induced disease, host susceptibility is also an important determinant of risk. Risk assessment will be considerably enhanced by considering genetic predisposition to endogenous processes and exogenous exposures. That lung cancer is caused by a single explanatory gene-environmental interaction is unlikely; alone one marker may not have a strong effect, but in conjunction with other genes it may shift the risk profile in an unfavorable direction. Multiple susceptibility factors must therefore be accounted for to represent the true dimensions of gene-environmental interactions. The ability to identify current and former smokers with the highest risks of developing lung cancer has substantial implications for prevention and for the design of future intervention studies. These subgroups could be enrolled into chemoprevention trials and might be suitable for screening programs not appropriate for the general population. Finally, studying susceptibility to common cancers and widely prevalent exposures to carcinogens may provide further insights into the basic mechanisms of carcinogenesis.

FUTURE LUNG CANCER CHEMOPREVENTION STRATEGIES The outcome of previous lung cancer chemoprevention trials demonstrates that our current strategies have been ineffective in achieving our

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goals of reducing lung cancer mortality. Before proceeding with further chemoprevention trials, we should reassess every aspect of past trials, including target populations, chemopreventive treatments, and intermediate endpoints. Previous lung cancer chemoprevention trials have focused on people who are actively smoking. As a result of massive campaigns to educate the public about the hazards of cigarette smoking, there has been a substantial reduction in the percentage of adults who actively smoke in the United States. Since 1965, when the first National Health Interview Survey (NHIS) was initiated, the prevalence of smoking cessation has almost doubled (64-68). The most recent data available show that in 1991,29.9% of adult men and 19% of women were estimated to be former smokers, representing a 77% change since 1965. This translates into 43.5 million adult former smokers nationwide, of whom 15.3 million are aged 45 to 64 years and 10.9 million are older than 65 years. Altogether, 48.5% of "ever-smokers" are now former smokers. For several reasons, former smokers may be a better population than active smokers for lung cancer chemoprevention trials. First, ongoing exposure to carcinogens may negate the beneficial effect of chemoprevention. Second, bronchial metaplasia, which is under study as a biomarker of lung cancer risk, is increased acutely by exposure to cigarette smoke (17), suggesting that active smoking may obscure the analysis of biomarkers. Third, former smokers, defined as smokers who have quit smoking for at least 1 year, have overtaken active smokers in lung cancer incidence in the last decade, now accounting for over 50% of lung cancers (64-68). Previous studies examining bronchial metaplasia, dysplasia, and sputum atypia in smokers revealed several problems with their use as biomarkers. First, samplings of these abnormalities at different times revealed that they varied in presence and severity, suggesting that they are not stable changes. Related to this observation, levels of bronchial metaplasia decrease acutely with smoking cessation (17). Second, adenocarcinomas, which occur peripherally and are the most common NSCLC, are in many cases, not contiguous with dysplastic changes. This differs from head and neck cancer, in which dysplastic epithelial regions are frequently contiguous with squamous cell carcinomas (69). The lack of spatial association brings into question the value of metaplasia and dysplasia as predictors of lung cancer risk. Third, metaplasia and dysplasia were detected in approximately 60% of active smokers and in 30% of former smokers (17). For chemoprevention trials involving former smokers, a more sensitive biomarker of damage associated with cigarette smoking is needed. Recent studies provide evidence that genetic damage, in the form of LOH and genomic instability at chromosomes 3p, 9p, and 17p, is detectable in the bronchial epithelium of approximately 60% of former smokers, and these alterations appear to be more stable than bronchial

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metaplasia and dysplasia (30). Future trials should investigate these abnormalities as markers of lung cancer risk and of response to chemopreventive intervention. The agents chosen for previous lung cancer chemoprevention trials have been largely ineffective in reducing lung cancer incidence. While the reasons for this failure are probably multiple, the possibility that the agents chosen have limited biologic activity in this setting should be considered. Retinoids, which were used in the majority of these trials, have a defined mechanism of action. Recent studies illustrate that normal HBE cells are sensitive to the biologic effects of retinoids, but NSCLC cells are retinoid-resistant. These findings provide in vitro evidence that the chemopreventive effects of retinoids are limited to their actions on normal HBE cells. Strategies to augment the biologic effects of retinoids on dysplastic HBE cells and bronchial carcinoma in situ could enhance the chemopreventive effects of retinoids. The remainder of this chapter delineates the signaling pathways that mediate the biologic effects of retinoids on normal HBE cells, and alterations in retinoid signaling that have been found in NSCLC cells. Based on these findings, strategies to enhance the lung cancer chemopreventive effects of retinoids are discussed.

RETINOID SIGNALING IN HUMAN BRONCHIAL EPITHELIAL CELLS Retinoids, including retinol and retinoic acid stereoisomers, have pleiotropic effects on the growth and differentiation of normal HBE cells (Fig. 14.1). At low dosage (10- 10 M), all-trans retinoic acid (t-RA) stimulates the growth of normal HBE cells, and high doses (10- 6 M) are growth inhibitory (70, 71). Normal HBE cells undergo squamous differentiation in response to treatment with transforming growth factor-beta (TGF-~), interferon-gamma, phorbol esters, or serum, and retinoids inhibit this process (72-76). Grown in collagen gels, normal HBE cells undergo mucous differentiation in response to retinoid treatment (72). These findings demonstrate that retinoids control the growth and differentiation of normal HBE cells. Retinoids mediate their effects through the activation of retinoid nuclear receptors, which include the RAR and retinoid X receptor (RXR) gene families, each family containing three members (a, ~, y) (77-82). These receptors are transcription factors that function as RAR : RXR heterodimers or RXR homodimers (83-88). Ligand-binding transcriptionally activates the receptors. RARs bind to t-RA and 9-cis retinoic acid (9cRA), and RXRs bind to 9cRA (89, 90). In addition, retinoid receptor transcriptional activity is regulated by their association with accessory proteins that function as transcriptional coactivators or corepressors

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GO EGF Insulin Pituitary extract Hydrocortisone

Proliferation

TGF-p Serum TPA

Retinoids Growth Arrest

t •

Squamous Differentiation Fig. 14.1. Normal HBE cells proliferate in defined media, including the indicated constituents, and are induced to undergo terminal squamous differentiation following treatment with TGF-~, serum, or TPA. Squamous differentiation can be reversed or prevented by cotreatment with retinoids, and retinoid treatment alone can induce growth inhibition. The arrow with a cross-hatch indicates abrogation of this process by retinoid treatment. (HBE = human bronchial epithelial; EGF = epidermal growth factor; TGF-~ = transforming growth factor-~; TPA = phorbol esters.)

(91-96). Retinoid receptors regulate gene expression directly by binding to gene promoters and indirectly by binding to other transcription factors. RAR: RXR heterodimers and RXR homodimers bind to distinct DNA response elements, suggesting that they activate target genes involved in different signal transduction pathways (83-88). In normal HBE cells, several retinoid-responsive genes and transcription factors have been defined. t-RA increases the expression of TGF-f32 and insulin-like growth factor binding protein-3 (IGFBP-3) in normal HBE cells (97). TGF-f3 is a secreted protein that is a potent regulator of cellular growth and differentiation in a variety of cells (98, 99). IGFBP-3 is one of six IGFBPs, and these secreted proteins bind to insulin-like growth factors (IGFs), either enhancing or inhibiting their effects depending on the cell type (100-102). Further, evidence suggests that IGFBP-3 may also function by binding to a cell surface receptor

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(103, 104). RAR-a activation appears to be required for the increase in and IGFBP-3 induced by t-RA (97), implicating a specific retinoid receptor in the effects of t-RA. Further supporting its role in bronchial epithelial cells, RAR-a activation increases transglutaminase type II expression, which is associated with apoptotic cell death induced by t-RA in the rat bronchial epithelial cell line SPOC-1 (105). t-RA treatment of normal HBE cells also inhibits the transactivation potential of the activator protein-1 (AP-1) complex, which is a dimeric transcription factor containing fos and jun family members that has demonstrated a role in controlling cellular growth, differentiation, and transformation (106-109). In normal HBE cells, AP-1 inhibition contributes to the suppression of growth and squamous differentiation induced by t-RA (97, 110). The role of TGF-~2, and IGFBP-3, and AP-1 in retinoid signaling is the subject of ongoing research. In contrast to normal HBE cells, NSCLC cells are resistant to the growth-inhibitory effects of retinoids (Ill, 112), suggesting that, during the malignant transformation of HBE cells, retinoid signaling is altered. Supporting this hypothesis, in a subgroup of NSCLC cell lines, retinoid receptors are refractory to ligand-induced transcriptional activation. Retinoid receptor dysfunction in these NSCLC cells appears to be related to a loss of transcriptional coactivator function (113, 114). However, the majority of retinoid-resistant NSCLC cell lines express functional retinoid receptors, suggesting that other retinoid signaling components are altered in these cells. As one possibility, retinoid-resistance in cancer cells has been correlated with a retinoid signaling abnormality involving the AP-1 complex (115). These findings provide evidence that multiple retinoid signaling alterations contribute to the development of retinoid resistance in NSCLC cells (Fig. 14.2). Studies in retinoid signaling have implications for the use of retinoids in lung cancer prevention. If AP-1 inhibition and increased expression of TGF-~2 and IGFBP-3 contribute to the biologic effects of retinoids on normal HBE cells, then selective activation of these events may more effectively prevent lung cancer than activation of all retinoid-responsive pathways. RAR-a is important in activating the expression of TGF-~2, IGFBP-3, and transglutaminase type II in normal HBE cells. Recently, synthetic retinoids were developed that, unlike natural retinoids, activate specific retinoid receptors (116). RAR-a-specific retinoids should be considered in preclinical studies to examine their activity in lung cancer prevention. Similarly, synthetic retinoids have been developed that function specifically as AP-1 antagonists without activating retinoid receptors (117). These compounds may be useful in combination with RAR-a-selective retinoids in lung cancer chemoprevention trials. Because these retinoids activate a limited spectrum of signaling pathways, associated toxicities may be less than that of natural retinoids. Through this approach, the future of retinoid therapy in lung cancer chemoprevention will be based on our understanding of the retinoid signaling pathways that mediate the biologic effects of retinoids on HBE cells. TGF-~2

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RA

Retinoid Receptor

AP-1 Inhibition TGF-~ Expression IGFBP-3 Expression

t

t

Target Gene

Biologic Effect

Growth Suppression

Loss of Receptor Coactivator Function

Relief of AP-1 Inhibition

No Growth Suppression

Fig. 14.2. Retinoid signaling pathways in normal HBE cells and potential alterations of these pathways in NSCLC cells are simplistically illustrated. In normal HBE cells, all-trans retinoic acid binds as ligand to retinoid nuclear receptors (RARs and RXRs); retinoid receptors modulate the expression of target genes (increased TGF~ and IGFBP-3 levels) and the activity of other transcription factors (inhibition of AP-l activity); and these events potentially indirectly mediate the biologic effects of retinoid treatment (growth suppression). These processes are altered in NSCLC cells, potentially through loss of receptor coactivator function and relief of AP-l inhibition. (HBE = human bronchial epithelial; RA = all-trans retinoic acid; RAR = retinoic acid receptor; RXR = retinoid X receptor; AP-l = activator protein-I; TGF-~ = transforming growth factor-~; IGFBP-3 = insulin-like growth factor binding protein-3.)

REFERENCES Cancer Facts and Figures, 1994. Atlanta: American Cancer Society, 1994. Halpern MT, Gillespie BW, Warner KE. Patterns of absolute risk of lung cancer mortality in former smokers. J Natl Cancer Inst 1993;85:457-464. 3. 'Pastorino U, Infante M, Maioli M, et a1. Adjuvant treatment of stage I lung cancer with high dose vitamin A. J Clin OncoI1993;11:1216-1222. 4. The ATBC Cancer Prevention Study Group. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N Engl J Med 1994;330:1029-1035. 1. 2.

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15 Molecular Detection of Lung Cancer David Sidransky

The prognosis for patients with lung cancer is primarily dependent on the stage of the tumor at the time of clinical diagnosis. Currently, only 25-40% of all lung tumors are considered resectable at the time of initial assessment, and only 20% are found to have limited disease at the time of surgery. Because patients diagnosed early with stage I tumors have a 40-70% survival following surgical resection, the development of better early detection techniques is clearly a pressing issue (1-3). Current detection techniques can be broadly divided into three main categories: 1) blood analysis, 2) sputum analysis, and 3) staging. These separate approaches may be useful in different clinical situations or, under certain circumstances, complementary. An ideal screening test should be highly sensitive, very specific, and eventually must be available at very low cost. If scrutinized thoroughly, none of the current approaches is suitable for screening of lung cancer in the general population. Moreover, current approaches are clearly not ideal, even for high-risk populations such as heavy smokers. In the past few years, there has been a revolution in our understanding of the molecular genetic events that give rise to and lead to the progression of human cancer (4-6). Like other neoplasms, lung cancers are thought to progress through a series of specific histopathologic changes. These histopathologic changes are driven by the accumulation of various mutations in proto-oncogenes and tumor-suppressor genes (4). These oncogene mutations are believed to provide a specific growth advantage for affected cells, resulting in clonal outgrowth and tumor progression (5, 6). Thus, these genetic changes drive the progression of

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clinical cancers and have certain specific biologic consequences. We have demonstrated that these and other molecular genetic changes now provide rational new targets for the early detection of many neoplasms, including lung cancer (7, 8). Genetic-based approaches promise exquisite sensitivity because they are amenable to sensitive molecular techniques such as polymerase chain reaction (PCR). Moreover, they promise high specificity due to their intimate and perhaps unique association with the clonal progression of cancer. In this chapter, we provide some promising examples from translational studies that have used new molecular markers for the detection and staging of lung cancer. Although new imaging approaches are not discussed, their continued development as complementary tools for the localization of early tumors detected by novel molecular techniques is essential.

BLOOD ANALYSIS Theoretically, a simple blood test to detect the presence of early lung cancer would be optimal for general screening. It is conceivable that certain specific proteins (found in cancer but not normal tissue) could be excreted from early lesions and might be detectable after circulating in blood. In some ways, this would be analogous to the PSA test now used for screening of prostate cancer (9-11). Although numerous proteins expressed at high levels in primary lung cancer have been found circulating in blood, none has emerged as either sensitive or specific enough for lung cancer detection. While these markers have found a role in following patients with established disease for evidence of recurrence, they have not been found adequate for early detection (12, 13). Moreover, these markers are not necessarily associated with the progression of cancer, and in fact normal patients may also harbor low levels of these proteins. This alone is devastating for a marker considered for introduction into widespread screening for any low prevalence disease.

p53 Antibodies Another approach has emerged based on the ability to detect the presence of circulating antibodies against cancer-specific proteins. One particularly intriguing approach is the detection of p53 antibodies in patients with lung cancer. The p53 gene is the most commonly mutated gene in both non-small-cell and small-cell lung cancer (NSCLC, SCLC, respectively) (14, 15). Moreover, some p53 gene mutations lead to stabilization of p53 protein, resulting in an increased half-life, often associated with strong immunohistochemical staining. Several years ago, it


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was found that some patients with cancer harbor antibodies to this p53 protein (16,17). However, the number of cancer patients that harbor p53 antibodies is quite small. Studies in lung cancer and other tumor types have found that approximately 10-15% of affected patients harbor p53 antibodies (16-18). In most cases, patients with these antibodies harbor tumors with a p53 mutation (18). The mechanism by which patients make antibodies to the p53 protein is not well understood. Initial reports suggested that stabilization of the p53 protein and a subsequent prolongation of the half-life and/ or stabilization by heat-shock proteins might lead to an increased uptake by antigen-presenting cells, followed by eventual recognition by the immune system (19). There were also reports indicating increased immunogenicity due to conformational changes induced by specific mutations (18, 19). However, it has been suggested that class I and II human leukocyte antigens (HLAs) of the patient may be critical for presentation of a mutated p53 protein, leading to a break of tolerance and subsequent development of the antibodies (20). Recent work also suggests that, with more sensitive detection techniques, a higher percentage of patients may harbor antibodies (Couch M, Sidransky D, personal communication, 1997). Although most patients with lung cancer will not harbor these antibodies, the potential use of this type of screening approach has been highlighted in recent clinical reports (21, 22). In these anecdotal reports, high-risk smokers harbored circulating p53 antibodies up to 1 year before subsequently developing lung cancer. In these individual cases, tumors were found to have a p53 mutation and also stained for p53 protein. Although this assay is hindered by sensitivity, it highlights the potential usefulness of well-characterized protein markers intimately associated with cancer evolution.

DNA-Based Testing: Microsatellites Recently, progress has been made in a DNA-based test for the detection of cancer in the blood (23, 24). This analysis depends on PCRbased amplification of microsatellites, small repetitive elements spread throughout the genome. Because microsatellite loci are highly polymorphic, two separate alleles are usually identified in an individual's DNA (corresponding to paternal and maternal alleles), in which case the locus is termed heterozygous (25). In cancer, deletion of one of these heterozygous alleles detects deletion of a chromosomal segment and is termed loss of heterozygosity (LOH). Chromosomal deletions are characteristic of neoplastic tissue, and different tumor types harbor different chromosomal deletion patterns at specific chromosomal loci. Chromosomal deletions are thought to indicate the inactivation of a tumorsuppressor gene within the deleted segment (26).

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In addition to identifying chromosomal deletions, microsatellites can identify microsatellite instability or microsatellite alterations. In certain tumors, these microsatellite sequences have a striking tendency to undergo expansions or contractions in the repeated motif. In patients with the HNPCC syndrome, microsatellite instability results from a failure of the DNA-mismatch repair system due to mutations in specific genes (27-29). In many other tumors, however, specific microsatellites occasionally show new alleles or alterations (8,30). Although the mechanism that leads to these replication errors is unclear, they can serve as markers for the clonal detection of cancer. An affected progenitor cancer cell will pass this new genetic alteration to its daughter cells; the identification of a new allele (as a new band on a gel) is synonymous with clonal expansion (8). Historical evidence suggested that patients with cancer harbor increased amounts of circulating plasma or serum DNA (31,32). In fact, the amount of DNA was somewhat predictive of whether patients had cancer and, in some cases, whether they had metastatic cancer (31). Based on these studies, we sought to detect the microsatellite changes described previously (LOH or new alleles) in the serum of patients with cancer (23). We took normal lymphocytes as germ line control DNA and then compared it to DNA extracted from circulating plasma or serum. In head and neck cancer, we found that 6 (29%) out of 21 patients who had undergone surgery to remove the primary tumor harbored either microsatellite alterations or LOH in the serum (23). Importantly, all six patients with a positive test had advanced stage disease and the identical microsatellite alterations were found in the primary tumors. Three of these positive patients went on to develop metastatic disease. this is intriguing because metastatic disease is quite rare in head and neck cancer, a tumor type in which most patients fail from local-regional recurrence. In a complementary study, other investigators tested plasma DNA at similar microsatellite loci in patients with SCLC (24). In this study, investigators also found micro satellite alterations, again either new alleles or LOH, in the plasma of 16 (76%) out of 21 patients (24). While the clinical outcomes were not available for these patients, SCLC is a particularly aggressive tumor type with a high tendency for metastases. These studies suggest that patients with a high burden of disease and potential for metastases may be more susceptible to harboring these microsatellite alterations in serum or plasma. The mechanism of how mutated DNA from a primary tumor makes its way to the serum is not well understood. It is plausible that large necrotic tumors extravasate necrotic debris, including DNA and protein complexes, into the blood. Subsequently, sophisticated DNA tests can detect these abnormalities in serum. Alternatively, it is possible that actual viable tumor cells are circulating in the blood and in search of metastatic sites. The immune system may recognize these cells,leading to cell death and degradation with release of free DNA. Both of these


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hypotheses suggest that microsatellite alterations may be uncommon unless there is a high burden of disease. These studies confirm that naked tumor DNA is present in the blood of cancer patients and is accessible for analysis (23, 24). Moreover, they demonstrate that characteristic mutations, indicating the precise origin of this DNA from tumor cells, can be detected by PCR-based techniques. However, microsatellite analysis of serum or plasma has not been done in a large number of control patients or in large studies. Thus, these preliminary results await prospective, controlled studies to understand the significance of these alterations in blood. Still, even if the test is limited in use for early detection, it may be useful for prognosis. A positive microsatellite test in serum might be particularly useful for the treatment of tumors for which there is effective therapy but for which patient selection remains problematic. In cases with a positive test, where patients might be predisposed to developing metastatic disease, additional, more aggressive therapy might be warranted. The potential sensitivity of this test may permit selection of patients that would benefit most from systemic chemotherapy after surgery.

SPUTUM AND BRONCHOALVEOLAR FLUID ANALYSIS Cytologic diagnosis of lung cancer has been hindered by poor sensitivity and specificity. An attempt at lung cancer screening through the use of tri-annual sputum cytology and annual chest x-rays was found to be inadequate for the early detection of lung cancer in the Johns Hopkins Lung Project (JHLP) study (33, 34). Although additional early stage tumors were detected early by cytologic screening, there was no significant difference in mortality in more than 5000 participants screened by sputum cytology plus chest x-ray, compared to those screened by radiographs alone. This study dampened enthusiasm for large-scale screening trials for lung cancer and highlighted the difficulties of cytologic diagnosis alone. Armed with the understanding that tumors progress through a series of specific genetic changes in critical oncogenes, we now believe that novel molecular markers may augment cytologic diagnosis. Inactivation of the tumor suppression gene p53 is the most common genetic alteration in all lung cancers (14, 35), and activation of the proto-oncogene ras is common in adenocarcinomas (36). Based on preliminary results indicating that we could detect these oncogene mutations in urine and stool samples obtained from patients with bladder and colon cancer, respectively (37, 38), we proceeded to test the feasibility of this approach in sputum. The JHLP repository had stored cytologic samples from the previous studies (33, 34, 39) and provided the opportunity to

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Chapter 15 test sputum samples obtained before the clinical diagnosis of lung cancer was established (39).

peR-Based Assays to Detect Oncogene Mutations In the initial feasibility study (7), we chose patients from the trial with cytologic samples that were not diagnostic for cancer but who later developed lung cancer. Our goal was to determine whether molecular tests could augment conventional cytologic techniques. We extracted DNA from the sputum samples and paired tumor samples for diagnosis. Of the 15 patients in the trial, we found 10 (66%) who had either a ras or p53 mutation after sequence analysis of the tumor (7). Importantly, none of the patients we analyzed ever had a positive sputum cytology in the JHLP study. Sputum samples were analyzed by a PCRbased assay able to detect one mutant-containing cell among an excess background of 10,000 normal cells (37, 38). This assay is based on the amplification of sputum DNA, followed by cloning into a phage vector and transfer onto nylon membranes. Mutant-specific single-stranded molecules (oligonucleotide probes) are then synthesized and hybridized on these filters to identify the identical point mutations in either the K-ras or the p53 gene present in sputum. Using this assay, we detected neoplastic cells in previously negative cytologic sputum samples from 8 (80%) of the 10 patients who had tumors containing oncogene mutation (7). The ratio of cancer cells to normal cells was approximately 1:300 for these samples. Positive sputum samples harboring a clonal population of cancer cells were obtained from 1 to 13 months prior to clinical diagnosis. In at least two patients, we were not able to detect the mutation present in the tumor by analyzing the sputum sample. Patients with tumors that did not contain oncogene mutations and control patients without cancer were all negative by this assay. There was also no correlation between the site of the tumor and ability to identify mutations in sputum samples by this assay. Although quite promising, this approach is currently limited for many reasons. Although PCR amplification of either K-ras or the p53 gene is quite simple to perform, the cloning step is tedious and costly. Moreover, in these cases, we identified mutations in either the ras or p53 gene before we attempted to examine sputum. For general screening, we would have to identify most of the common p53 mutations or ras gene mutations that occur in the population. This would require the synthesis and testing of many probes. Given the variety of p53 mutations (14), we have calculated that we would need approximately 70 oligomers to detect 100 of the most common p53 mutations. To circumvent the cloning step, other methods are available that


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might be simpler and more economical for detection of rare mutant alleles among a predominance of wild-type alleles. There are methods that are based on the ligation of two oligomers specific for a given mutation before or after PCR (40). There are also amplification methods based on matched or mismatched primers that only amplify the mutant alleles (41). Most of these techniques have a similar sensitivity of approximately 1 abnormal cell among 2000 to 10,000 normal cells. Information gained from cytologic diagnosis in sputum through light microscopy also suggests that examination of two or three samples may improve the diagnostic yield significantly, and this may pertain to molecular diagnosis as well. Although this was a very small study, the ability to detect gene mutations 1 year prior to clinical diagnosis holds promise for screening of patients at high risk for this deadly disease. Other investigators have also used this approach, predominately studying ras gene mutations (42, 43). Utilizing approaches similar to those mentioned earlier, both groups used a method term called enriched peR, which allows sensitive detection of ras gene mutations. One group of investigators thought to use mutant ras as a biomarker for lung cancer and tested normal patients, smokers at high risk, and those with cancer (42). PCR identified K-ras codon-12 mutations in 10% of lung tissue obtained from patients with no lung disease, whereas the same mutation was detected in 60% of samples of normal-appearing lung tissue obtained from patients with NSCLC (42). Analysis of sputum samples of patients with NSCLC identified these mutations in 47% of patients, whereas only 12.5% of the controls diagnosed with nonneoplastic lung disease carried this mutation. Yakubovskaya et al. (42) concluded that at least some of these positive mutations reflected small clones of cells, with ras mutations in individuals exposed to high levels of cigarette smoke. This could be a direct result of the exogenous exposure, as tumors from patients who smoke have been clearly associated with a higher incidence of ras (44,45) and p53 mutations in numerous studies of different tumor types (46). Recently, the most common p53 gene mutations in lung cancer were associated with specific benzopyrene ad ducts at the same codons (47). Another group tested bronchoalveolar fluid in 52 patients with lung cancer and found a K-ras mutation in more than half of the patients with adenocarcinoma, and none in those with squamous cell carcinoma (43). Tissue samples from these patients always yielded the identical mutation, and no mutation was found in the fluid from 30 patients without cancer. Moreover, mutations were detected in four cases where cancer was suspected but not yet confirmed by histopathology. Both of these studies point to the need to perform additional molecular analysis in well-characterized smoking patients, normal controls, and cancer patients with paired sputum samples to assess the usefulness of these oncogene alterations as biomarkers of cancer development.

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Microsatellite Analysis Microsatellite analysis represents another potentially powerful approach for the detection of neoplastic cells in sputum. Although microsatellite alterations (new alleles) are very rare in NSCLC, they are much more common in SCLC (48). In fact, a small but persistent population of SCLCs display diffuse instability that rivals that seen in tumors from HNPCC patients. We have screened a large number of DNA nucleotide repeats, including trinucleotide and tetranucleotide repeat markers in primary lung cancers (8). These larger repeats have a much higher propensity for alterations in the germline and in somatic tissues such as cancer. Among these markers, we have found that specific repeats have been much more susceptible to alterations. In particular, the tetranucleotide repeat (AAAG)n appears to be a common target of these alterations in lung cancer. Data suggests that approximately 10 to 15 of these well-chosen tetranucleotide repeat markers may identify a specific new allele in up to 50% of NSCLCs. As noted earlier, these microsatellite alterations are useful as clonal markers for the detection of human cancer (8). In limited studies, we and others have found that microsatellite alterations can be detected in corresponding sputum samples from lung cancer patients (8, 49). Unfortunately, the detection of LOH and chromosomal deletions is not possible by microsatellite analysis in sputum (50). This is due to the large dilutional effect of normal epithelial cells that are shed and sloughed off into the upper aerodigestive tract. Recall that a typical sputum sample from a patient with lung cancer contains approximately 1 neoplastic cell among 300 normal (nonmutated) cells. Therefore, it will be necessary to combine a large number of these particularly susceptible microsatellite repeats in an effort to identify microsatellite alterations in sputum. Microsatellite analysis in bladder cancer (50) and other tumors suggests that this approach should be sufficiently sensitive for the detection of lung cancer. Analysis of a large number of paired tumor and sputum samples remains to be done.

Telomerase Another important marker of cancer progression is the reactivation of telomerase. Telomeres are specialized repeat structures at the end of chromosomes that allow faithful reconstitution of chromosomal length (51). Normal DNA replication occurs from the 5' to the 3' end, leaving the very end of the DNA strand of the telomere susceptible to continuous shortening through multiple rounds of replication. Eukaryotic cells have solved this problem with the formation of a telomerase complex that allows addition of specific telomere repeats through a ribonucleic protein complex, which includes an RNA component and several bound proteins. Investigators realized that telomerase activity could be impor-


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tant for the continued replication of cancer cells (52). Subsequent studies have found that many immortalized cells and primary tumors have reactivated telomerase, perhaps to prevent telomere shortening during multiple rounds of replication (53). In this regard, telomerase represents a potentially novel and a sensitive method for the detection of cancer. One method for the detection of telomerase activity is called the telomeric repeat amplification protocol (TRAP) assay (54). This assay allows the simple extraction of a protein and nucleotides from primary tumors or clinical samples. Extraction is followed by a short extension, where inherent telomerase activity initiates synthesis, followed by peR amplification for detection of a characteristic telomere repeat "ladder." These assays can be performed by radioactive or nonradioactive techniques. In addition, the TRAP assay is very sensitive with the potential to detect telomerase activity in 1 malignant cell among 10,000 normal tissue culture cells. Investigators have begun to assess the possibility of using telomerase for the detection of many types of cancers. Preliminary results have suggested that telomerase activity may be present in the urine of patients with bladder cancer and perhaps in the sputum of patients with lung cancer (Tarin 0, unpublished data, 1996). In recent reports of head and neck cancer, one-third of saliva samples were positive for telomerase activity in patients with primary head and neck cancer (55). However, the assay may be limited by the necessity to isolate intact RNA and protein enzymatic activity. Better sensitivity may be achieved by the collection of fresh samples and by the addition of RNA and proteinase inhibitors. False positives may also occur from contaminating normal lymphocytes in cytologic specimens that harbor low levels of endogenous telomerase activity. In the absence of larger published studies, feasibility studies have suggested that this approach is possible, but it is as yet untested for the detection of lung cancer.

STAGING The molecular markers that have been described are potentially useful not only for the detection of early lung cancer but also may be useful for assessing prognosis and improving staging in patients with lung cancer. Use of the p53 plaque assay described previously has suggested that occult neoplastic cells can be detected in the margins and lymph nodes of patients with head and neck cancer (56). In patients with positive tumor cells by molecular analysis, there was a high incidence of localregional recurrence and an overall decrease in survival. In patients with lung cancer, survival rates are significantly lower once local-regional lymph nodes contain metastatic disease. Patients with metastasis to peribronchial lymph nodes alone have an average 5-year survival of

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37-54%, whereas patients with ipsilateral nodal involvement have an average 5-year survival of 14-29% (57). Thus, the ability to detect micrometastatic nodal disease might identify patients at high risk of treatment failure who might benefit from more aggressive adjuvant therapy. Based on our work on head and neck cancer, we have initiated a prospective trial to analyze lymph nodes in patients with lung cancer by looking for occult neoplastic cells that harbor either a p53 or ras gene mutation previously identified in the primary tumor (58). p53 and ras oligonucleotide hybridization has detected occult cells, not identified by standard histopathology, in lymph nodes at all levels in some patients with stage I disease. These patients would have been considered as having a more advanced stage by molecular analysis, and clinical outcome is pending. These studies are also consistent with other immunohistochemical studies. Studies using monoclonal antibodies have found micrometastatic disease in more than half the lymph nodes of patients with stage I NSCLC (59). Several investigators have also demonstrated a decrease in disease survival in patients with bone marrow metastases identified by similar immunohistochemical techniques (60,61). Although immunohistochemistry is relatively rapid and simple to perform, the sensitivity and specificity of these new molecular assays should be more reliable. In patients with clinically negative disease by standard histopathologic examination, molecular analysis may be an important adjunct for accurate staging. Telomerase can also be considered as a tool to identify patients with occult metastatic disease to lymph nodes. However, our analysis suggests that many lymph nodes that are clearly negative by oligonucleotide plaque hybridization techniques appear to harbor telomerase activity by the TRAP assay (58). In fact, one-third of negative lymph nodes by molecular analysis remain telomerase positive despite 100fold dilution. This is consistent with reports indicating that normallymphocytes have low telomerase activity. Telomerase activity has also been reported in normal lymphocytes in lesions that are characterized by lymphocytic filtration. This activity probably plays a major role in the high false positive rate we observed in applying the TRAP assay to detect occult metastasis in lymph nodes. Because of this, telomerase assay may be limited in its specificity for the detection of occult neoplastic disease.

CONCLUSION An obvious problem in any type of new screening or detection approach using clinical samples is the need to detect an extremely small number of cancer cells among a large background of normal cells, espe-


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cially in bodily fluids such as sputum. Although the molecular strategies outlined in this chapter have solved these problems with molecular biology, the approaches still remain relatively challenging and require further development. These studies highlight the importance of basic science studies that demonstrated LOH in human cancer and continue with the development of molecular progression models for many types of neoplasms (62). Translational studies demonstrate that morphologic and cytologic analyses are not adequate for the detection of lung cancer. Molecular analysis promises to be more reliable, potentially detecting tumors at all grades and stages, including those that are often missed by cytology. In principle, these molecular approaches should be much more sensitive than standard histopathologic approaches. Moreover, because these new molecular strategies target genetic alterations that either drive or are a consequence of the neoplastic process, they should be much more specific. It is immediately apparent that these new technologic advances still need substantial development. In order to develop safe, effective screening tests for lung cancer, the tests will not only need to be reliable but will have to be highly cost effective. In this regard, it is encouraging that we have been able to automate some of these approaches such as the microsatellite analysis (63, 64). It is now possible to run 100 microsatellite markers by automated microcapillary analysis at a very reasonable cost. It is essential to develop these low-cost automated approaches and eventually to undergo carefully controlled trials. None of these approaches will gain entrance into standard clinical practice unless they undergo testing in prospective clinical trials. The development of reliable low-cost assays will allow us to begin to test, for the first time, the hypothesis that these molecular detection strategies can detect lung cancers early. Moreover, we have to demonstrate that these approaches will not only lead to an improvement in diagnosis but also to an improvement in overall survival. Our general approach has been to target patients at risk for recurrent disease for initial validation of these strategies, and this highlights the need to identify high-risk target populations. If we proceed with large-scale randomized trials, it will be very costly, and useful information will be scarce because of the low incidence of lung cancer in the general population. By selecting the appropriate high-risk population such as heavy smokers and those with chronic pulmonary disease, it may be possible to test the feasibility of these analyses in a much more thorough and rapid fashion. Selection of the appropriate high-risk population remains a challenge to better test and finally develop these assays for clinical use. Clonal genetic alterations appear to be promising as molecular diagnostic tools for lung cancer. Assays that detect microsatellite alterations or oncogene mutations will make this type of screening plausible, and efforts to automate and simplify these approaches will make them practical. The accumulation of paired patient samples to test these approaches in identification of high-risk populations is imperative to

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Chapter 15 begin this work. As technology improves, these approaches will need to be taken to the clinical setting for prospective trials with many patients. Although the effort and costs to run these trials may appear daunting, the promise of early detection and higher cure rates for lung cancer is clearly a worthwhile objective. Moreover, the identification of highrisk patients with occult spread of neoplastic cells to lymph nodes or into the circulation will greatly help to stratify patients for more aggressive therapy. Success will depend on close cooperation between basic scientists and clinicians as they continue to bring promising molecular approaches from the bench to the bedside.

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16 Fluorescence Detection Stephen C. Lam and Branko Palcic

Lung cancer is the most common cause of cancer death in North America. At present, only 15% of all patients diagnosed with lung cancer can be successfully treated as measured by 5-year survival rates. The primary reason for such a dismal cure rate is the fact that a great majority of all lung cancers are found at a very late invasive stage, and there is no curative treatment for advanced lung cancer. This is supported by the stage distribution at diagnosis in the general population (1) (Table 16.1). If, however, the stage distribution can be changed such that a higher proportion of the lung cancer patients can be diagnosed at an early stage, as in a recent study by Bechtel et al. (2) using sputum cytology examination, the survival rate can be dramatically improved from 1545%, even without any change in the treatment result for each stage (Table 16.2). Unfortunately, the yield of conventional sputum cytology examination is very poor. For example, in the Cooperative Early Lung Cancer Study sponsored by the National Cancer Institute, sputum cytology alone detected only 22% of the lung cancer cases in the prevalence study of smokers and 10% in subsequent screening of the same population (3).

This work was supported by the National Cancer Institute of Canada, the BC Health Care Research Foundation, the BC Science Council, and the BC Lung Association. The development of the light-imaging fluorescence endoscope (LIFE-LUNG) system was supported by Xillix Technologies Corp., Vancouver, BC, Canada. We would like to thank Jaclyn Hung, Ph.D., and Calum MacAulay, Ph.D., for their contribution to the fluorescence imaging studies.

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Table 16.1.

Stage distribution at diagnosis and 5--year survival rates

Stage

Stage distribution (%J

0 I II III IV

TOTAL

5-yr relative survival (%)

No. surviving 5 yr per 1000 cases diagnosed

0.6 26.2 6.9 29.4 36.9

90* 42 22 5 3

5 110 15 15 11

100.0

32

156

*The 5-year survival rate for those with stage 0 disease is from the report by Cortese et al. (21) in patients with roentgenographically occult lung cancer.

Table 16.2.

Stage

0 I II III IV

TOTAL

Stage distribution and predicted survival for population diagnosed by sputum cytology

Conventional stage distribution (%)

Stage distribution for cases diagnosed by sputum cytology (%)*

Predicted no. surviving 5-yr per 1 000 cases diagnosed by sputum cytology

0.6 26.2 6.9 29.4 36.9

14.0 74.0 2.0 B.O 2.0

126 311 4 4 1

100.0

100.0

446

*As projected from the study by Bechtel et al. (2).

However, recent advances in computer-assisted image analysis (4, 5) and development of monoclonal antibodies (6, 7) as well as molecular biology techniques (8) promise hope of a significant improvement in the sensitivity of sputum cytology examination to detect early lung cancer. The challenge will be how to localize small intraepithelial neoplastic lesions when atypical/malignant cells are found in the sputum while the chest x-ray is still normal.

BASIS FOR DETECTION AND TREATMENT OF INTRAEPITHELIAL NEOPLASIA There is precedence in other epithelial organs that suggests detecting and treating intraepithelial neoplastic lesions will lead to a reduction in the incidence and hence the mortality of invasive cancer. The first is the well-established effectiveness of the cervical cytology screening program coupled with treatment of preinvasive lesions with cryotherapy


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or the carbon-dioxide laser (9). The National Polyp Study in the United States suggests that removal of colonic polyps may lead to a reduction of invasive colon cancer (10). In a population at risk of developing esophageal cancer, a study in China showed that in patients found to have esophageal dysplasia by balloon cytology screening, chemopreventive intervention reduced the incidence of esophageal carcinoma by about 50% in those with severe dysplasia and by approximately 40% in those with mild dysplasia (11, 12). The success in these tumor sites offers hope that a similar strategy may also lead to a reduction in lung cancer mortality. There is general reluctance of chest physicians to adopt a similar strategy in lung cancer. Dogma from studies conducted in the 1970s dictates that there is no real gain in screening for early lung cancer (3, 13, 14). The apparent reversibility of intraepithelial neoplastic lesions (15, 16) and the long duration of the preinvasive phase (17) further augment the thinking that these lesions are benign and that there is no justification to detect or treat them. Recent advances in quantitative microscopy suggest that a lack of objective classification of intraepithelial neoplastic lesions is probably the cause of this misunderstanding (18). Although there is little disagreement in the diagnosis of normal tissue or invasive cancer, significant variation exists among pathologists in the interpretation of dysplasia or carcinoma in situ (19, 20). The problem with conventional subjective histopathologic or cytologic classification is illustrated by several studies. In a study by Cortese et al. (21), 13 patients with moderate or marked atypia in their sputum cytology were found to have lung cancer on bronchoscopy. Other studies had also found that approximately 10% of those with moderate or marked atypia in their sputum cytology actually had carcinoma in situ or invasive cancer (22). Current work in computer-assisted image analysis of cells and tissues as well as molecular genetic studies will lead to a more accurate definition of intraepithelial neoplasia (18, 23, 24). From a practical standpoint, when one considers that if only 40% of individuals with severe atypia and 10% of those with moderate atypia in their sputum cells will develop invasive lung cancer on follow-up (16), it would be prudent to identify individuals harboring these lesions and treat them, as the invasive phase of lung cancer is very short and the chance of survival is less than 15% at late invasive stages.

ISSUES IN DETECTION AND LOCALIZATION OF INTRAEPITHELIAL NEOPLASTIC LESIONS Carcinoma in situ and microinvasive cancers present a challenging diagnostic problem, even for experienced bronchoscopists. The reason for

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this is that these cancers are only a few cell layers thick (0.2-1.0 mm) and a few millimeters in surface diameter (25,26). Because of this, the lesions may not produce any visible abnormality on conventional, white-light bronchoscopy. In some cases, subtle changes exist, consisting of an increase in redness, granularity, or slight thickening of the mucosa only. Unfortunately, while some of these changes are seen in malignancy, they may also be seen in patients with chronic bronchitis. In a study by Woolner et al. (27), in situ carcinomas were visible bronchoscopically in less than 30% of cases. If no lesion is found on whitelight examination, a blind segmental brushing protocol, with or without biopsy of the bronchial spurs, has been used for localization (28-34). The problem with this approach is that multiple samples need to be taken. Although it can be done under local anesthesia, it is more commonly done under general anesthesia. The entire procedure generally takes 1 to 2 hours. Then there is the problem of cross contamination, especially if nondisposable brushes are used. Accurate interpretation of the numerous samples obtained from each patient requires meticulous examination of the samples by experienced cytotechnologists and cytopathologists who can make a diagnosis by assessing the number, staining, and appearance of atypical cells. Furthermore, if bronchial brushing shows malignant cells without a visible bronchoscopic abnormality, a repeat brushing of the same segment in a separate bronchoscopic procedure is required for confirmation before treatment can be undertaken. The lack of progress in the use of conventional white-light bronchoscopy for localization is illustrated by two studies conducted ten years apart. In a study reported by Cortese et al. (21) in 1983, 31.5% of the patients with roentgenographically occult lung cancer required more than one bronchoscopy for localization. In 1994, Bechtel et al. (2) reported in a similar group of patients that 39% required more than one bronchoscopy for localization. Clearly, the task would be much easier if the lesions could be made visible by other means for direct biopsy.

FLUORESCENCE BRONCHOSCOPY When the bronchial surface is illuminated by light, the light can be reflected, back-scattered, absorbed, or can induce tissue fluorescence. Conventional white-light examination makes use of the first three optical properties-a process that is known as reflectance imaging. The tissue autofluorescence is not visible to the unaided eye because the intensity is low and is overwhelmed by the reflected and back-scattered light. However, this autofluorescence can be demonstrated by illuminating the tissue surface with a shorter wavelength light, such as an ultravio-


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let light, and observing with a long-pass filter to remove the background excitation light. In 1933, Sutro and Burman (35) found that when surgically excised breast tissue was exposed to ultraviolet light from a Wood's lamp, normal breast tissue fluoresced green, while breast cancer tissue fluoresced purple. Unfortunately, because the difference between small thin tumors and the adjacent normal tissue was minimal and because the ultraviolet light was strongly absorbed by blood, this method was not found to be useful in delineating the resection margin at the time of surgery. This approach to tumor detection was later superseded by a surge of interest in the use of exogenous fluorescent drugs to enhance the contrast between normal and cancer tissues (36). The idea of using special fluorescing drugs that are preferentially retained in tumors for cancer detection has intrigued many investigators since 1924 (37). Many such compounds exist in nature, but to date most of the research has been concentrated on porphyrins, the beststudied of which is hematoporphyrin derivative (HpD), or its purified form porfimer sodium (Photofrin) (QLT Photo therapeutics, Vancouver, BC, Canada) (38-42). Fluorescence diagnosis of tumors using fluorescent compounds such as Photofrin is based on two principles: 1) that even at very low concentrations in tissue, Photofrin emits a red fluorescence, with peaks at 630 nm and 690 nm when excited by violet light (390-415 nm); and 2) that the concentration of Photofrin in malignant tissue is higher than in the adjacent nonmalignant tissues, from 3 hours to at least 72 hours after intravenous injection. Thus, tumors can be detected by the difference in Photofrin concentration and hence by fluorescence emission intensity. If a different drug is used, the principle of imaging remains similar, except that a different excitation wavelength and detection system may be required. There are several disadvantages to using an exogenous fluorescence drug such as Photofrin for tumor localization. The improvement in the signal-to-noise ratio in small thin preinvasive lesions is only 1.5 to 2 times at best (43). False positive fluorescence in areas of inflammation or metaplasia is on the order of 27-50%. There is the inconvenience of waiting between drug administration and the bronchoscopic examination. In addition, there is a potential for side effects such as skin photosensitivity, which lasts for 4 weeks or more (36). The cost of each bronchoscopy examination also increases several times if a fluorescent drug is added. To improve the signal-to-noise ratio, it was proposed that an endogenously produced fluorescent marker be used, an interesting concept. An example of this is a-aminolevulinic acid (ALA) (44, 45). ALA is a natural precursor of heme. Endogenous synthesis of ALA is the ratelimiting step in heme synthesis. When sufficient exogenous ALA is given, the feedback mechanism is disrupted, and this causes a buildup of endogenous protoporphyrin IX (PpIX) (46). ALA is not fluorescent, but PpIX is. Due to a difference in ferrochelatase activity between tumor

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and most normal tissues, there is a transient buildup of PpIX in tumor tissue that can be exploited for photedetection (47). The advantage of ALA is that the skin photosensitivity lasts for less than 24 hours versus 4 weeks or more after Photofrin. Kriegmair et al. (48) used intravesically administered ALA followed by fluorescence cystoscopy using violet light to detect early malignant or precancerous lesions in the bladder. The fluorescence cystoscopy had a sensitivity of 100% and a specificity of 68.5% for the detection of carcinoma in situ or dysplastic lesions. Huber et al. (49) used inhaled ALA in six patients and oral ALA in two patients for photodetection of lung cancer. They found that this method was highly sensitive for photodetection of malignant and premalignant lesions. However, the specificity was only 50%. A recent study by Awardh et al. (50) confirms the nonspecific accumulation and fluorescence of PpIX in areas of inflammation and metaplasia. The mechanism of increased accumulation of PpIX in inflammatory and metaplastic tissues is presumably due to heightened cellular metabolic activity in these tissues. Unfortunately, this limits the usefulness of ALA for photodetection of early lung cancer. In addition, with the exception of a higher tumor-to-normal tissue fluorescence and a shorter skin photosensitivity, the other previously discussed disadvantages associated with the use of exogenous fluorescence drugs remain. To overcome the problems associated with drug-enhanced photodetection of intraepithelial neoplastic lesions, the mechanism of tissue autofluorescence was reinvestigated by performing spectroscopy in vivo during bronchoscopic examinations, using a multichannel optical analyzer. The tissue autofluorescence spectra of dysplasia and carcinoma in situ when excited by violet (405 nm) and blue (442 nm) light (51), were found to be significantly different from those of normal bronchial tissues. An example of an autofluorescence spectrum from a patient with a severe dysplasia and a carcinoma in situ lesion is shown in Figure 16.1. When illuminated by a blue (442 nm) light from a helium-cadmium laser, normal bronchial mucosa is characterized by a strong signal in the green region (around 500 nm), with a much reduced intensity toward the red region. In contrast, premalignant and malignant bronchial tissues have a significantly reduced autofluorescence in the green region. In the far-red region of the spectrum, the fluorescence intensity of these tissues is similar. Using excised (in vitro) animal and human lung tissue, Alfano et al. (52, 53) observed subtle differences in spectral shape between normal and cancerous tissues; however, significant differences in fluorescence intensity were not observed. Hung (54) observed that the freezing and thawing of tissues at variable times after excision reduced the differences in fluorescence intensity between malignant and normal bronchial tissues, as well as altering the spectral shape of these tissues, yielding data similar to those reported by Alfano et al. We therefore conclude that the large differences in tissue autofluorescence


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Green chanoel 15

o 420

450

480

510

540

570

600

630

660

690

720

Fig. 16.1. In vivo autofluorescence spectra from a patient showing a significant decrease in fluorescence intensity in an area with severe dysplasia, and a separate area with carcinoma in situ. Excitation (442 nm) is by a helium-cadmium laser. The figure shows the spectral bands imaged by the lung-imaging fluorescence endoscope (LlFE)-Lung device. (CIS = carcinoma in situ.)

between normal and malignant sites are unique for the in vivo situation. The decrease in fluorescence intensity, especially in the blue-green region of the visible spectrum, is not unique for bronchial cancers and dysplasia; it was also observed in other epithelial tumors and dysplasia when spectroscopic measurements were made in vivo (55-57). The reason for the decrease in autofluorescence in intraepithelial neoplastic tissues has not been clearly defined. Most of the fluorescence comes from the connective tissue in the submucosa, while the epithelium contributes to a few percent of the overall fluorescence only (58, 59). However, thickening of the epithelial layer can reduce the fluorescence intensity by impeding transmission of fluorescence from the submucosa to the epithelial surface. Recently, it was observed that matrix metalloproteases such as stromelysin-3 and urokinase-type plasminogen activator, which can degrade the extracellular matrix, were found to be expressed in bronchial dysplasia and carcinoma in situ (60). It is likely that the drop in fluorescence intensity is due to both thickening of the epithelium and a loss of fluorophore concentration or efficiency. Preferential diffusion of longer wavelength (red) fluorescent light horizontally from the adjacent normal mucosa into the tumor area provides

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further opportunity to discriminate abnormal from normal tissue (36, 58,59). Recent advances in imaging technology make it possible to reexamine the use of tissue autofluorescence alone to detect early lung cancer.

Fluorescence Endoscopic Devices The fluorescence detection systems developed to date can be classified as imaging and nonimaging devices. The nonimaging systems detect signals that are related to fluorescence intensity, i.e., fluorescent light reaching the detector. In imaging systems, a picture of varying intensity of the fluorescence is obtained. Imaging devices are used to detect and locate tumors precisely, and to show their size and shape. Nonimaging instruments have been devised primarily to allow quantitative measurements of fluorescence or fluorescence ratios in different wavelength bands, but they only give an approximate location because of the signal-averaging effect. The most recent instrument permits quantitative measurements to be made in different spectral bands, by computer analysis of two- or three-color images, thus combining the two types of instruments into one. Most of these devices were developed for use in conjunction with exogenous or endogenous fluorescent drugs, but they illustrate some important concepts.

Nonimaging Methods Kinsey and Cortese developed a nonimaging device that could be used with a double-lumen fiberoptic bronchoscope (61, 62). A chopper technology with a filter wheel was used to illuminate the bronchial surface with violet light, alternating with white light at 30 Hz. The violet (405 nm) light for the excitation of Photofrin was obtained from a filtered 200 W mercury arc lamp. A fiberoptic detector guide, placed in one lumen of the bronchoscope, was used to transmit emitted fluorescence from the bronchial surface to a photomultiplier tube, to measure the red fluorescence characteristic of Photofrin after removing the reflected excitation light and some of the autofluorescence by a barrier filter. A lockin amplifier enhances the fluorescence signal, while the output from the amplifier generates a frequency-modulated audio signal, with the pitch of the signal being proportional to the intensity of the red fluorescence. The system allows the endoscopist to perform routine flexible fiberoptic bronchoscopy under white-light illumination (that is, without the 405 nm filter), while the detection system searches the Photofrin fluorescence. The audio signal alerts the endoscopist to areas of the tracheobronchial tree that are suspicious for cancer, so that biopsies for cytologic or pathologic confirmation can be obtained. Although it is a


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relatively simple technique, the device could not correct for changes in the intensity of the fluorescence signal due to variations in distance, angle, and intensity of the excitation light. Care and skill are required to position the endoscopic probe as the airway is scanned to locate a tumor. Another approach was made by Baumgartner et al. (63) and Huber et al. (64). A krypton iron laser, with a specially designed resonator to produce two excitation wavelengths at 405 nm and 470 nm, was used for illumination. The tissue autofluorescence spectra at these two excitation wavelengths are very similar, while the red Photofrin fluorescence viewed at 470 nm is much lower than that at 405 nm. Thus, if the fluorescence signals or images obtained from the two excitation wavelengths are captured and then subtracted, the net result is only the red fluorescence of Photofrin. A fluorescence photometer was developed by Potter and Mang (65) based on a similar principle, but using a much cheaper helium-neon laser. In this device, 612-nm and 638.2-nm light from two 5 mW heliumneon lasers was used for illumination. Two fiberoptic probes were available. A scanning probe composed of six receiving fibers and one illumination fiber was used to scan large areas of tissue. The second probe consisting of one illumination fiber maintained at a distance of 1 cm from the tissue and a second fiber placed in contact with the tissue to receive the 690-nm fluorescence signal that was used to pinpoint the areas of high fluorescence after scanning. This instrument was designed for the detection of nonpalpable metastatic breast cancers in the skin and micrometastases to regional lymph nodes (66-68). The detection of intraoperative metastases during mediastinoscopy, or the delineation of surgical margins during thoracotomy, are potential applications, but further research is required. Although such fluorescence detection probes are sensitive systems, the display signal is dependent on the distance and angle of the excitation light source and of the receiving light guide from the tissue surface. In an attempt to overcome the problem of variation of the fluorescence signal with angle and distance, a ratio fluorometer probe was devised by Profio et al. (69). On illumination by a violet light, normal tissues emit autofluorescence predominantly in the green region of the visible spectrum. In contrast, tissues containing Photofrin have significantly elevated red fluorescence. When the red fluorescence (690 nm) is ratioed against the green autofluorescence (560 nm), the red-green ratio is higher in the tumor area than in the adjacent normal area, because more Photofrin is retained in the tumor tissues. Since the red-green ratio is a dimensionless value, it is independent of the distance and angle between the illumination and the receiving light guides. The ratio fluorometer also has a built-in audio signal to herald red-green ratios that are above the normal background. Although subtraction and ratioing are important concepts, they have several disadvantages. First, whenever a positive signal is obtained, the

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exact source of the signal cannot be pinpointed, and the ability to precisely localize and delineate the extent of the cancer is important in making a decision regarding treatment. Secondly, as a single probe, it is essentially a one-pixel system. Small early cancers can be readily missed because of the field-averaging effect. Thirdly, a control (presumably normal) site is needed to calibrate these probes in vivo. If it were always possible to tell on conventional white-light examination whether a given site were normal, it would not be necessary to develop a fluorescencedetection device.

Imaging Methods The earliest fluorescence imaging system that can detect carcinoma in situ was developed by Profio and Doiron (70, 71). In this system, an image intensifier was attached to the eyepiece of a bronchoscope to magnify the fluorescence intensity by approximately 30,000 times. Although the sensitivity of the system was very high, with over 90% of the tumors showing positive fluorescence, the specificity was lowonly 50% (72, 73). The problem with the image-intensified system is that it involves a subjective judgment of the brightness contrast, whereas the human eye is more sensitive to color contrast. Although lesions with bright spots are readily detectable, areas with diffuse fluorescence and blurred margins, or areas with low contrast, are difficult to differentiate from the normal background. Another major problem is that the fluorescence intensity is dependent on the distance and angle of the bronchoscope from the bronchial wall, as well as on the intensity of the excitation light. Thus, when the tip of the bronchoscope is closer to the bronchial wall, or over cartilaginous areas, the fluorescence intensity is higher. This will often result in false positive signals. A system combining imaging and nonimaging detectors was designed by Kato et al. at Tokyo Medical College in Japan (74-78). The system consists of a pulse excimer laser and a spectral image analyzer. This allows the display of the fluorescence spectrum on the video monitor to distinguish between Photofrin fluorescence and tissue autofluorescence. Tissue autofluorescence subtraction from the Photofrin fluorescence can be made. The color and fluorescence images, as well as the fluorescence spectrum, can be observed simultaneously in real-time. However, the fluorescence intensity is still significantly influenced by the position of the bronchoscope in relation to the bronchial surface. Montan et al. (79) employed a very sophisticated approach using a Cassegranian telescope to collect the fluorescence light. A nitrogen laser was used for excitation. By using a spherical mirror divided into four individually adjustable sectors, four identical images can be sent to an image-intensified charge-coupled device (CCD) detector after passing through interference filters. Subtraction and ratioing of different images


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can be carried out by image processing to create pseudo-images delineating the tumor site. Approximately 5 seconds are required for image acquisition and processing. If properly developed into a practical clinical tool that can be used in real-time, this system may have some advantages over the other systems. The only imaging system that has received U.S. Food and Drug Administration (FDA) approval for the detection of early lung cancer is the light-induced fluorescence endoscope (LIFE)-Lung device that was originally developed at the British Columbia Cancer Research Center in collaboration with Xillix Technologies Corp. (Vancouver, BC). The device is also approved for clinical use in Canada, Germany, Japan, and France. LIFE-Lung is composed of a helium-cadmium laser as a light source (442 nm), two image-intensified CCD cameras with green and red filters, respectively, a computer with an imaging board, and a color video monitor (Fig. 16.2). A green and a red fluorescence image are cap-

Wum;,nation Console

Image Acquisition

RGB

Imaging Console

Display

Fluorescence Camera

White

light Source

8

= = o § Blue light

Blue

Fig. 16.2.

A schematic diagram of the lung-imaging fluorescence endoscope (LlFE)-Lung device. (Courtesy

of Xillix Technologies Corp., Richmond, Be. Canada)

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tured simultaneously in precise registration by the imaging board. The images are then combined to produce a color image on the video monitor such that normal tissue appears green and tumor tissue appears brown or brownish red. Bronchial dysplasia appears as a lighter brown color compared to carcinoma in situ. An abnormal area can be biopsied under direct vision for pathologic confirmation. Lesions as small as 1 mm in diameter and only a few cell layers thick can be localized by this method. It is important to note that the LIFE-Lung device makes use of tissue autofluorescence alone. It does not require exogenous fluorescent drugs such as Photofrin or endogenously generated fluorescent compounds such as ALA-induced PpIX.

CLINICAL RESULTS Fluorescence Detection with Porphyrin Drugs Clinical experience at the Mayo Clinic (78) using 2 mg/kg HpD and a nonimaging fluorescence detection system showed positive fluorescence in all five patients with roentgenographically occult, but bronchoscopically visible, cancers. Fluorescence examination was instrumental in localizing ten of eleven centrally located squamous cancers that were bronchoscopically invisible. There were three peripheral lung cancers that were not localized on fluorescence examination because they were beyond the reach of the relatively large, two-channel flexible fiberoptic bronchoscope. An area of marked dysplasia also showed weak fluorescence (78). Clinical testing of the image-intensifier fluorescence bronchoscope system (IFB) and the ratio fluorometer probe (RFP) showed that bronchial cancers of all histologic types had positive Photofrin fluorescence (70, 72, 73, 80-82). In seven patients with carcinoma in situ who had received 2 mg/kg Photofrin, the sensitivity of IFB and RFP was 93% and 70%, respectively. The specificity of IFB and RFP was 50% and 100%, respectively. When both methods were used together in the same patients, the sensitivity and specificity were 98% and 100%, respectively (82). Clinical experience with the other fluorescence detection devices suggests that they can all detect small radiographically occult lung cancers (64, 74, 78, 83, 84). However, the sensitivity and specificity of these devices cannot be evaluated from the data presented. In addition, most of these reports did not indicate what proportion of the in situ carcinomas detected by fluorescence examination was actually visible by conventional white-light bronchoscopy. At the present time, no fluorescence-enhancing drug has yet been approved by the FDA for detection of lung cancer.


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Fluorescence Bronchoscopy Using LIFE-Lung Clinical experience worldwide involving more than 600 patients showed that this device, in conjunction with white-light bronchoscopy, can improve the detection rate of intraepithelial neoplastic lesion and invasive cancer by more than two times compared to white-light examination alone (18, 36, 85, 86). The recently completed clinical trial in the United States and Canada showed that the detection rate for intraepi thelial lesions (moderate / severe dysplasia and carcinoma in situ) was improved by more than six times with the addition of the LIFELung device (87). The specificity of combined white-light and fluorescence bronchoscopy was 72% versus 83% with white-light bronchoscopy alone. However, correlation of false positive lesions with molecular studies of loss of heterozygosity (LOH) at one of five regions commonly deleted in lung cancers indicates that at least half of the "false positives" represent lesions damaged at the molecular level (18). Thus, there appears to be a correlation between abnormal fluorescence and molecular changes, irrespective of histopathologic change. The LIFE-Lung device was found to be useful in preoperative assessment of lung cancer patients to determine the extent of the endobronchial spread and to detect synchronous cancers. It was also useful in localizing roentgenographically occult, sputum cytology-positive, early lung cancers.

Research Application of Fluorescence Bronchoscopy The areas of the bronchial tree accessible to the fiberoptic bronchoscope have been carefully named. Using this nomenclature and by reviewing digital images captured by the LIFE device, the same area can be revisited and biopsied precisely in subsequent examinations. Thus, the outcome of preneoplastic lesions with or without chemopreventive intervention can be studied on a lesion-by-lesion basis using this method. The biopsy material also provides a valuable resource to study the effect of various chemopreventive agents and the pathogenesis of lung cancer (18).

SUMMARY Autofluorescence bronchoscopy has opened up a new and exciting possibility of detecting and localizing intraepithelial neoplastic lesions without fluorescent drugs. Fluorescence bronchoscopy can detect small lesions that are undetectable by conventional white-light bronchoscopy.

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It is useful in the preoperative assessment of lung cancer patients to

determine the extent of endobronchial spread and to detect synchronous in situ carcinomas that are invisible on conventional white-light examination. The ability to detect and biopsy intraepithelial neoplastic lesions will also allow us to have better management of lung cancer. In addition, it will allow us to study the natural history and molecular biology of lung cancer and to establish more precise intermediate endpoints to investigate the efficacy of chemopreventive drugs.

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Rava RP, Richards-Kortum R, Fitzmaurice M, et al. Early detection of dysplasia in colon and bladder tissue using laser-induced fluorescence. SPIE Proc 1991;1426:68-78. Cothren RM, Richards-Kortum R, Sivak MV, et al. Gastrointestinal tissue diagnosis by laser-induced fluorescence spectroscopy at endoscopy. Gastrointest Endosc 1990;36:105-111. Qu J, MacAulay C, Lam S, Palcic B. Optical properties of normal and carcinoma bronchial tissue. Appl Optics 1991;11:99-105. Qu J, MacAulay C, Lam S, Palcic B. Laser induced fluorescence spectroscopy at endoscopy: tissue optics; Monte Carlo modeling and in-vivo measurements. Optical Engineer 1995;34:3334-3343. Bolon I, Brambilla E, Vandenbunder B, et al. Changes in the expression of matrix proteases and of the transcription factor c-Ets-1 during progression of precancerous bronchial lesions. Lab Invest 1996;75:1-13. Cortese DA, Kinsey JH, Woolner LB, et al. Clinical application of a new endoscopic technique for detection of in situ bronchial carcinoma. Mayo Clin Proc 1979;54:636-641. Kinsey JH, Cortese DA. Endoscopic system for simultaneous visual examination and electronic detection of fluorescence. Rev Sci Instrum 1980;51:1403-1406. Baumgartner R, Fislinger H, Jocham 0, et al. A fluorescence imaging device for endoscopic detection of early stage cancer-instrumental and experimental studies. Photochem PhotobioI1987;46:759-763. Huber RM, Haubinger K, Krug M, Baumgartner R, Jocham D. Photodynamic diagnosis (POD) in lung cancer: report from a pilot study at Munich. Chest 1991;100:2S. Potter WR, Mang TS. Photofrin II levels by in vivo fluorescence photometry. In: Doiron DR, Gomer CJ, eds. Porphyrin Localization and Treatment of Tumors. New York: Alan R. Liss, 1984:177-186. Mang TS, McGinnis C, Castillio N, Potter WR. Fluorescence detection of occult nonpalpable metastases in lymph nodes following injection of low doses of Photofrin II. SPIE Proc 1988;997:118-125. Mang TS, McGinnis C, Khan S, Liebow C. Fiberoptic detection of low-level porphyrin concentrations in preclinical and clinical studies. SPIE Proc 1990;1201:390-399. Mang TS, McGinnis C, Crean DH, Khan S, Liebow C. Fluorescence detection of tumors: studies on the early diagnosis of microscopic lesions in preclinical and clinical studies. SPIE Proc 1991;1426:97-110. Profio AE, Doiron DR, Sarnaik J. Fluorometer for endoscopic diagnosis of tumors. Med Phys 1984;11:516-520. Profio AE, Doiron DR, King EG. Laser fluorescence bronchoscopy for localization of occult lung tumors. Med Phys 1979;6:532-535. King EG, Man G, LeRiche J, et al. Fluorescence bronchoscopy in the localization of bronchogenic carcinoma. Cancer 1982;49:777-782. Balchum OJ, Doiron DR, Profio AE, Huth Gc. Fluorescence bronchoscopy for localizing early bronchial cancer and carcinoma in situ. Recent Results in Cancer Research 1982:97-120. Balchum OJ, Profio AE, Razum N. Ratio fluorometer probe for localizing carcinoma in situ in bronchi of the lung. J Photochem Photobiol B 1987; 41:887-891. Kato H, Imaizumi T, Aizawa K, et al. Photodynamic diagnosis in respiratory tract malignancy using an excimer dye laser system. J Photo chern Photobiol B 1990;6:189-191.

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17 Photodynamic Therapy and Thoracic Malignancies Harvey 1. Pass

The use of light in the healing arts dates back to ancient Greece, when Herodotus first used sunlight to treat certain diseases (1). Raab (2) first described photodynamic therapy (PDT) in 1900 with experiments that showed the killing of paramecia with acridine and light (2). The treatment of skin cancer with eosin and light in 1903 constituted the first reported use of PDT in oncology (3). Since these initial reports, investigators have used various chemicals in efforts to optimize the cytotoxic effect of PDT. Hausman (4) found that porphyrins, naturally occurring iron-free or magnesium-free respiratory pigments present in the protoplasm of plant and animal cells, could promote photochemically induced cell death. Studies with hematoporphyrin derivative (HpD), a complex porphyrin mixture, showed that this sensitizer concentrates in tumors (5), projecting the use of HpD as an agent for tumor detection and cancer treatment. Lipson et al. (6) subsequently used multiple HpD injections and light exposures to treat a locally recurrent breast cancer, resulting in an objective tumor response, if not a cure. Ensuing studies by Dougherty and others (7-13) have explored the use of PDT in the treatment of a wide variety of malignancies. PDT is based on the science of photochemistry, the treatment with drugs that react to light. A basic photochemical reaction occurs when a sensitizer is exposed to light in the presence of oxygen. Absorbed light energy converts the sensitizer to an excited state that in turn interacts with molecular oxygen to form singlet oxygen or oxygen-free radicals. Singlet oxygen, the main product of a photochemical reaction, is a powerful oxidizing agent capable of damaging plasma membranes and 287

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other subcellular organelles. PDT is the use of the three components of a photochemical reaction, light, oxygen, and photosensitizer, to treat disease. In this chapter, we discuss the components of a photodynamic system, the mechanism by which PDT kills cells, and the potential applications of PDT in the treatment of lung and pleural malignancies.

COMPONENTS OF PHOTODYNAMIC THERAPY Light Light is high-velocity energy transmitted in electromagnetic waves. Although light possesses familiar waveform properties when interacting with prisms, mirrors, and lenses, it also displays particulate characteristics by delivering energy in discrete measurable units called photons. The absorption of photons by a photosensitizer is the first reaction of a photodynamic system, and photochemical reactions can occur only if light is absorbed (14). The action spectrum of a sensitizer defines the rate of a photochemical reaction when the sensitizer is stimulated by a given wavelength of light, and usually corresponds to the absorption spectrum of the sensitizer. Therefore, the absorption spectrum of a sensitizer determines the wavelength of light, measured in nanometers (nm), needed to achieve the maximum photodynamic effect. The absorption spectra of sensitizers generally fall within the visible light spectrum between ultraviolet (UV) and infrared (IR) light. Porphyrin sensitizers have an intense absorption band at the UV end of the spectrum called the Soret band (14). Light at the Soret band wavelength maximally excites a sensitizer (Fig. 17.1). Additionally, the energy of light is inversely related to its wavelength, with lower wavelengths being more powerful. For these two reasons, one might expect that lower wavelengths of light would be optimal for PDT. This is not the case, however. Because shorter wavelengths are attenuated by hemoglobin as they pass through tissue, the depth of tissue penetration by light is also inversely proportional to wavelength. Therefore, light at the IR end of the spectrum will penetrate much deeper into a tumor than UV light. Although stimulation of a porphyrin sensitizer with blue-violet light may result in more energy production, tissue penetration of less than 1 mm is inadequate to treat malignancy. Therefore, PDT with porphyrin-based sensitizers is usually performed with 630-nm red light, which gives tissue penetration up to 10 mm with acceptable production. The total amount of energy delivered depends on the duration of light delivery and the dose rate of light. The dose rate, or power density, of light is usually expressed in m W / cm 2 . In vitro, higher dose rate

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I 400

450

500

550

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Wavelength (nm) Fig. 17.1. Absorption spectrum of porphyrin sensitizers. Porphyrin sensitizers absorb light in the visible spectrum between ultraviolet (400 nm) and infrared (700 nm). The Soret band is represented by high absorption peak at 400 nm.

delivery to tumor cells at a given total energy results in greater PDT cytotoxicity (15). The relative inefficiency of PDT at lower dose rates may result from the ability of tumor cells to repair sublethal damage or to buffer toxic oxidative molecules. The importance of these potential protective capabilities remains questionable, as contradictory findings have been described in vivo. Using human mesothelioma allografts in nude mice, Foster demonstrated that decreasing the power density from 200 to 50 mW / cm 2 resulted in lower tumor regrowth rates (16). Low fluence rates may increase the level of singlet oxygen in areas of low capillary density, resulting in greater tumor cytotoxicity. The appropriate method of light delivery for PDT depends on the experimental or clinical situation. An apparatus as simple as a horizontally placed x-ray view box covered with a ruby red acetate filter can be used for in vitro PDT experiments (15). An argon pump-dye laser, exciting either Kiton red or rhodamine B and producing up to 5 W of red light, is a typical light source for PDT in the clinical environment. The laser is coupled to one or more fiberoptic cables used to convey light with minimal energy loss to the treatment field. The end of the cable is fitted with a tip designed to disperse light in a pattern appropriate for the clinical application. A cleaved tip projects light forward for treating flat surfaces, and a bulbous tip projects light in an isotropic spherical distribution for illuminating large cavities. A tip specially coated with

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Oxygen Although there are a few photodynamic systems that are oxygen independent, molecular oxygen is required to achieve cytotoxicity with porphyrin-based PDT(17, 18). PDT kills cells by selective destruction of essential molecules through photo-oxidative reactions (19). In type I photo-oxidation, an excited sensitizer transfers energy to a substrate compound, which in turn reacts with molecular oxygen to yield superoxide radical (0 2-), Type II photo-oxidative reactions involve direct interaction between excited sensitizer and oxygen to yield singlet oxygen, an extremely reactive oxidant. Probably of lesser importance, PDT may also activate the xanthine-oxidase pathway to produce superoxide (20). Because type I reactions are inefficient and dependent on high substrate concentrations, most sensitizers used today act through type II photo-oxidation. Several studies corroborate the importance of oxygen to photochemical systems. Using sodium dithionate to remove oxygen from the system, Lee et al. (18) showed that tumor cytotoxicity correlates directly with oxygen concentration. Mitchell et al. (17) used a customized glass Petri system to demonstrate that cells treated in hypoxic conditions are extremely resistant to PDT. Similar studies have been performed in vivo. Gomer and Razum (21) found that restricting the blood flow to the hind limb of albino mice inhibited the photosensitizing effects of porphyrin PDT. Although attempts to directly quantitate singlet oxygen in vivo have been unsuccessful (22, 23), investigators have used transcutaneous oxygen-detecting electrodes to measure microcirculatory damage after PDT (24, 25). This technology may one day be used to determine the clinical effectiveness of photochemical systems.

Photosensitizers Several properties determine the clinical effectiveness of a photosensitizer (Table 17.1). Because cancer is such a diverse disease, it is unlikely that a single sensitizer would effectively treat all malignancies. Likewise, the ability of an activated sensitizer to kill cancer cells must be balanced against that sensitizer's propensity for normal tissue toxicity. For these reasons, the perfect photosensitizer probably does not exist. The properties of optimal photosensitizers have been recently discussed by Moan (26). Selective uptake or retention of sensitizer by tumors increases tumor cytotoxicity while decreasing normal tissue toxicity. Most sensitizers presently used have tumor-to-tissue ratios of 2-5:1. This appears to be

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Properties of optimal sensitizers

Selectively concentrate in tumor tissue Efficiently generate singlet oxygen when exposed to light Absorb longer light wavelengths that penetrate deeply into tissue Photodeactivated in normal tissue to reduce side effects

an in vivo phenomenon, as in vitro studies using dihematoporphyrin ether (OHE), a purified form of HpO, fail to show differences in either sensitizer uptake or survival between normal and malignant cells. NIH3T3 cells transformed by the ras oncogene have similar sensitizer levels and PDT survival curves compared to the parent NIH3T3 cell line (27). Perry et al. (28) found no survival differences after PDT between normal lung fibroblasts and a variety of lung cancer cell lines. However, numerous in vitro studies have demonstrated differences in sensitizer retention between tumor and normal tissue. Levels of OHE in animal flank tumors remain elevated for 48 hours after intravenous injection, while skin and muscle exhibit significantly lower levels for 2 to 48 hours (27). The method of delivery appears to playa role in determining levels of sensitizer in tumor cells, depending on the type and location of tumor. In an ovarian cancer ascites model, Tocher et al. (29) showed that intraperitoneal injection resulted in maximum sensitizer levels. The direct administration of sensitizer to cancer cells without dependence on vascular delivery probability explains this phenomenon. The mode of presentation at the cell membrane can affect sensitizer uptake. The hydrophobicity and degree of aggregation of a sensitizer determine its internalization by the cell. When exposed to sensitizers in vitro, cells take up and retain liposomal-bound porphyrins much better than aqueous phase porphyrins (30). Cells sensitized with these liposomal-bound porphyrins sustain damage to the mitochondria and cytoplasm, while porphyrins in aqueous phase target the plasma membrane. In vitro, hydrophilic sensitizers localize to the extracellular tissue stroma. These sensitizers are not internalized by specific receptors and therefore deposit at the cell membrane (31). Lipophilic sensitizers such as OHE are transported to the cell membrane by low-density lipoproteins (LOL). LOL-bound fractions enter the lysosomal compartment by endocytosis, and on exposure to light, cause release of lysosomal hydrolases into the cytoplasm (32, 33). Studies of different tumor cell lines have revealed that tumors endocytose LOL at a faster rate than normal tissues, possibly accounting for the tumor-selective properties of LOLbound photosensitizers. In addition to uptake, hydrophobicity may contribute to sensitizer retention as well. Porphyrins become more water soluble as pH is decreased and would therefore be retained in acidotic tumor cells. Other theories regarding the selective uptake and retention of sensitizers involve tumor cell heterogeneity, tumor neovascularity, abnormal lymphatic drainage, and stromal cell binding.

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Lipophilic sensitizers that localize to the intracellular compartment also appear to generate more single oxygen when exposed to light than do hydrophilic sensitizers. These first-generation porphyrin sensitizers are not extremely efficient, however, for reasons discussed earlier. Maximal quantum yield of a photochemical system occurs with light wavelengths that correspond to the sensitizer's Soret band, which for porphyrin sensitizers is in the 400-nm region. Because light energy is also indirectly proportional to wavelength, the greatest quantum yield results from exposure to light just above the UV range. Light wavelengths in this range are impractical for PDT, however, because tissue penetration is directly proportional to wavelength. To balance maximal quantum yield with deepest tissue penetration, PDT is performed at higher wavelengths (>590 nm), corresponding to minor peaks further along the absorption spectrum of the sensitizer. Under these conditions, PDT sensitizers generate singlet oxygen with quantum yield of 0.2 to 0.6, with tissue penetration up to 10 mm. Investigators are developing second-generation sensitizers in efforts to increase quantum yield at longer wavelengths (34) (Table 17.2). Another goal in the search for better sensitizers relates to photolability, the deactivation of sensitizer by light. An ideal way to decrease the toxic side effects of PDT would be to use a sensitizer that could be deactivated ("photobleached") in normal tissue but that would remain cytotoxic in tumor tissue. A dose of sensitizer could be chosen that resulted in sensitizer concentrations high enough in the tumor to cause photodestruction of the tumor. Lower sensitizer concentrations in adjacent normal tissue would result in photodegradation of the sensitizer to nontoxic levels. Sensitizers in use today, particularly DHE, have photodegradation products that are also sensitizers and are therefore not photodegraded sufficiently to be deactivated (26).

EFFECTS OF PHOTODYNAMIC THERAPY IN VITRO CYTOTOXICITY A wide variety of cell lines show sensitivity to PDT (2, 28, 35-43). Comparison of different lung cancer histologies reveals few real differences Table 1 7.2.

Second-generation photosensitizers

Chlorins Chlorin E6 Purpurin Benzoporphyrin derivative Phthalocyanines Cationic sensitizers (rhodamine 123) Porphins (TPPS4)


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in the characteristics that influence PDT cytotoxicity (28). Intracellular sensitizer levels are relatively the same after correcting for total cellular protein, cell volume, and cell size. Likewise, the survival curves for a variety of thoracic malignancies, including adenocarcinoma, squamous cell carcinoma, large-cell carcinoma, small-cell carcinoma, and mesothelioma are quite similar (28) (Table 17.3). PDT generates singlet oxygen through the reaction of light with a photosensitizer. The resultant oxygen-free radicals mediate the toxic effects of PDT. The most impressive effect seen in vitro soon after cells are treated with PDT is damage to the plasma membrane (43,44). Membranes are the primary targets of PDT, partly because of the water-lipid partition coefficient of DHE. Porphyrins bind initially to the plasma membrane, followed by migration to intracellular regions. In only a few hours after PDT, normal cellular movement stops and large membrane blebs form on the cell surface (45). These large balloon-like structures, often as large as the cell itself, indicate severe membrane damage and are the first visible sign of photo-oxidation in the cell (46, 47). This disruption of the integrity of the plasma membrane results in leakage of intracellular contents out of the cell (48,49). After membrane blebbing occurs, cell division comes to a halt, and cell lysis begins. As alluded to earlier, PDT damages other membranes in addition to the plasma membrane. Because of these effects on cell membranes, PDT may injure the nucleus, lysosomes, Golgi apparatus, endoplasmic reticulum, and mitochondria. Mitochondrial damage after PDT may involve inhibition of oxidative phosphorylation and electron transport, with subsequent reduction in adenosine triphosphate (ATP) production (50, 51). One cellular target that appears to escape serious damage by PDT is nuclear DNA. While PDT can induce DNA strand breaks, this does not lead to cell death. PDT does not appear to be mutagenic (52). Resistance of cells to PDT may one day determine the clinical efficacy of PDT in cancer treatment. Several investigators have begun to examine the role of the multi drug resistance (MDR) phenotype in PDT resistance. In MDR associated with chemotherapy, a membrane pump actively transports drug out of the cell. Conceivably, this mechanism Table 17.3.

Examples of cell types sensitive to photodynamic therapy

Lung cancer Mesothelioma Pancreatic cancer Retinoblastoma Bladder cancer Ovarian cancer Leukemia Neuroblastoma Colon cancer

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could transport photosensitizer out of the cell, thereby reducing the photodynamic effect at treatment. This could become a particular problem when treating patients with combined chemotherapy-photodynamic therapy regimens. Similarly, patients who have previously failed chemotherapy might likewise develop cross-resistance to PDT. Studies comparing the sensitivities of cells that express MDR versus wild-type cells from the same cell line revealed no cross-resistance to PDT (53). Using cells made resistant to PDT by repeated PDT exposure, investigators have shown that this resistance is not due to decreased intracellular levels of sensitizer, the mechanism of action of classic MDR (54, 55). MDR may be associated with cross-resistance to PDT depending on the sensitizer used (56). A more plausible explanation is that most cases of PDT resistance involve the mechanism of action of PDT. Buffering of toxic oxygen radicals by free-radical scavengers could explain the induced resistance of cells to PDT. Studies by Ryter (57) have shown that PDT can enhance transcription and translation of oxidative stress genes. Many studies involving chemotherapy resistance have shown that free-radical buffering enzymes such as glutathione can protect cells against drugs that produce free radicals. While no one knows how cells become resistant to PDT, mechanisms other than MDR are probably involved.

In Vivo Cytotoxicity Flank tumors in mice respond rapidly to PDT with complete tumor regression by two days after treatment. These tumors undergo a marked coagulation necrosis, implicating the microvasculature as a target for PDT. Soon after PDT, blood flow stasis occurs in both arterioles and venules (58, 59). Vasoconstriction of arterioles, thrombosis of venules, and edema of perivascular tissues accompany the reduction in blood flow. Ben Hur (60) suggests that PDT induces the endothelium to release vasoconstrictors and clotting factors that cause local coagulation and stasis. This begins a cascade in which local blood flow stasis and endothelial damage retain red blood cells in the treatment field for a longer period of time, thereby exposing them to higher doses of PDT. Subsequent damage results in red cell agglutination, which further exacerbates the low flow state, and binding of neutrophils and platelets to damaged endothelium with release of prostaglandins and arichidonates that also act on the microvasculature. Other factors such as von Willebrand factor, platelet activating factor, and nitric oxide may also be involved. The photodynamic process itself may lead to alterations in tumor blood flow (61). A positive feedback cycle of oxygen depletion by the photo-oxidative system, acidosis, increased intra tumoral swelling with decreased blood flow, and worsening hypoxia could account for static blood flow followed by tumor necrosis. Nuclear magnetic resonance studies examining tumor energy substrate levels during and after


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PDT indicate that ATP levels are virtually undetectable by 2 to 4 hours (62-64) and appear to decline in a dose-dependent fashion (65). The antitumor effect of PDT may in part be due to modulation of the immune system. Various studies have documented the release of vasoactive agents such as prostaglandins, thromboxane, and histamine from murine peritoneal macrophages and mast cells (66-68). These mediators could contribute to the vascular and metabolic effects of PDT in mediating tumor necrosis. PDT also induces macrophages to release tumor necrosis factor (TNF) in a dose-dependent fashion (69). Moreover, Bellnier (70) has shown that intravenous TNF in combination with PDT resulted in an additive effect on tumor regression in a murine subcutaneous tumor model. The combination treatment resulted in essentially the same tumor response as doubling the PDT dose. PDT may exert a net inhibitory effect on the immune system, however. Contact hypersensitivity remains suppressed 2 weeks after PDT (71), and this effect is adoptively transferred by macrophages (72). PDT also inhibits the mitogen-stimulated division of human peripheral blood lymphocytes (73). Therefore, while PDT can induce nonspecific immune cells to produce substances such as TNF, prostaglandins, thromboxanes, and histamines that could either directly or indirectly mediate tumor necrosis, the overall effect of PDT on the immune system is unknown. The most efficacious use of the immune system in PDT may result from the treatment of tumors with antibody-sensitizer conjugates. A photosensitizing agent could be ligated with a monoclonal antibody with idiotypic specificity for a known tumor. Theoretically, this would increase the specificity of the sensitizer for the tumor, resulting in increased tumor cytotoxicity with reduced side effects in normal tissues. Mew (74, 75) demonstrated this concept by showing that monoclonal antibody-photosensitizer conjugates effectively mediate tumor regression in vivo and kill a variety of cell lines in vitro. PDT using monoclonal antibody-sensitizer conjugates appears to result in greater tumor specificity,longer remission, and less normal tissue toxicity in murine subcutaneous tumors (76). While similar constructs have demonstrated selective killing of human ovarian carcinoma and melanoma cells (77, 78), work in this area remains highly investigational.

CLINICAL ApPLICATIONS OF PHOTODYNAMIC THERAPY IN THORACIC CANCER (Table 17.41

Treatment of Early-Stage Lung Cancer PDT may also aid in the treatment of early-stage lung cancer. Several findings of the Mayo Lung Project indicate that a treatment modality

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Applications of photodynamic therapy in the treatment of thoracic cancer

Detection of lung cancer (experimental) Treatment of early or unresectable lung cancer (experimental) Palliation of bronchial (experimental) or esophageal (approved) obstruction Treatment of pleural malignancy (experimental)

such as PDT might be perfect for the treatment of this often difficult problem (79). In patients with positive sputum cytologies and negative chest films, most lesions are confined to the bronchial wall with no extrabronchial involvement. The depth of these lesions, approximately 5 mm, corresponds to the tissue penetration of red light. After an initial curative attempt, a large number of patients will recur with second primary lesions, indicating the failure of localized surgery to cure a more generalized disease. Moreover, these lesions tend to be centrally located squamous cell cancers. Because of their central location, major operations such as sleeve-resection or even pneumonectomy are required to manage early, noninvasive lesions. PDT has the ability to selectively treat large surface areas at risk, without the problems associated with major resections that may not be curative. Several clinical trials have examined the role of PDT in the treatment of early-stage lung bronchial cancer. Complete response rates have been as high as 30%; many of these patients also received radiation therapy, however, making interpretation of these findings difficult (80). PDT may provide long-term cures for selected patients with early-stage lung cancer (81, 82). At the Tokyo Medical College, 56 patients with 66 lesions have been treated with PDT for early-stage lung cancers. Tumor response to PDT was evaluated endoscopically, roentgenographically, cytologically, and histologically. A complete response rate (no tumor) was determined after PDT in 65% of patients and lesions, with an 8% subsequent recurrence rate in the patients having a complete remission. Most patients required multiple treatments to accomplish a complete remission. A similar experience with 70 patients has been reported at the Mayo Clinic, with a 65% complete response rate for mucosal limited squamous cell carcinomas that are 3 cm2 in surface area (83). Despite these encouraging results, surgery remains the best therapy for early-stage lung cancer. The choice of treatment should be based on consideration of factors such as the multicentricity of disease and the risk of lymph node involvement in patients who appear to have "early" disease (25%). Improved techniques for delivering sensitizer and light to endobronchial tissues, along with more sensitive diagnostic methods for the disease, are needed to counter these problems. Controlled randomized trials comparing PDT to more traditional therapies are needed to determine the overall safety and efficacy of PDT in the treatment of early-stage lung cancer.


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Palliation of Endobronchial Obstruction The most common use of PDT in the treatment of patients with thoracic malignancies has been in the palliation of endobronchial obstruction due to primary or metastatic lesions (28). Patients potentially benefiting from this treatment approach have complete or partial bronchial obstruction with resultant shortness of breath or segmental lung collapse with pneumonia. Palliation of acute bleeding from these lesions is not an indication for endobronchial PDT because this therapy does not provide immediate hemostasis. The treatment technique follows the general guidelines for other clinical applications of PDT (Fig. 17.2). The patient receives DHE, 2 mg/kg, intravenously 48 to 72 hours prior to therapy. This waiting period allows the tumor to develop high levels of sensitizer. The patient is then

Patient with tumor occluding bronchus. a. View of tumor through bronchoscope. b. Cylindrical laser fiber extending through the bronchoscope just before interstitial treatment. c. Residual tumor and open airway after clean-up bronchoscopy 72 hours later.

Fig. 17.2.

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treated with 630-nm red light from an argon pump-dye laser coupled to fiberoptic cables passed through a fiberoptic bronchoscope. A cylindrical fiber illuminator can be implanted into the tumor, or a cleavedtip fiber can illuminate the surface of the tumor. The light output at the end of the fiber is measured with a light meter prior to treatment. Based on the laser output, the duration of light exposure is calculated to achieve a desired total energy dose in joules, generally ranging from 5 to 900 J/ cm2 , depending on the site and method of treatment. This procedure is performed under general anesthesia with a single-lumen endotracheal tube. More than one lesion can be treated at a single bronchoscopy. Bronchoscopy is repeated 48 hours later to allow debridement of necrotic tissue and biopsies. By this time, the treated tumor tissue has undergone avascular necrosis and does not bleed. The tumor is debrided down to the bronchial wall until bleeding occurs, indicating that the level of treatment effect has been passed. Multiple phase II studies documenting the safety and efficacy of PDT for relieving endobronchial obstruction have been performed (84-92).

INTRAPLEURAL PHOTODYNAMIC THERAPY Due to the superficial nature of the treatment (5-10 mm) (dictated by the wavelength of light used for appropriate excitation of the sensitizer), it seemed logical that PDT might be of some use for intracavitary treatment if it could be applied to a surface that normally had a volume of 4 to 5 liters. The rationale behind such an approach was that, since it was known that the sensitizer is selectively retained by malignant tissue in vivo, one could deliver the sensitizer preoperatively, debulk the patient at the appropriate time down to some "critical thickness," and then deliver light to the entire cavity, possibly eradicating the residual disease. Such a hypothesis was a leap of faith because three things had to be proven prior to initiating any sort of human trial. 1. Thoracic malignancies are sensitive to PDT, specifically mesothelioma. We reported that thoracic malignancies, including mesothelioma, were sensitive to PDT (28). Keller et al. (35) also reported the ability of PDT to destroy human mesothelioma in vitro, which corroborated our findings. Feins et al. (93) investigated PDT in a nude mouse model of human malignant mesothelioma grown subcutaneously to a size of 0.2 to 0.4 cm3 . All PDT regimens were successful in destroying the tumor. The use of PDT for intrathoracic use in animal models has been limited. Pelton et al. (94), however, reported on intrathoracic organ injury associated with PDT, detailing the pathologic changes that occur within the lung, heart, trachea, and diaphragm of Sprague-Dawley rats after administration of tumoricidal PDT. Animals were injected with the photosensitizer Photofrin-II, 10 mg/kg, 24 hours before surface illumination of a portion of the target organ with gold vapor laser light (628


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nm) (124 J / cm 2). Twenty-four hours after treatment, the lung, heart, and trachea of rats subjected to PDT demonstrated parenchymal injury with alveolar and endothelial disruption, intra-alveolar hemorrhage, and fibrin deposition. Coagulation necrosis of myocardial fibers extending through the epicardium to involve up to 50% of myocardial thickness was observed. The National Cancer Institute (NCI) approach to animal studies (95) was more conservative, using larger animals that could possibly better represent 1) the human situation and 2) the mechanics of laser-light delivery. Twelve American foxhounds were subjected to right thoracotomy, 72 hours after receiving 6 mg/kg PIl, and red light was delivered to 1-cm circular areas on the pleural surface of the esophagus, heart, chest wall, and diaphragm, while the lungs were treated to 2-cm circular areas. Three other dogs had thoracotomy and light treatment, but did not receive preoperative sensitizer. Each of the above organs, in each dog, was illuminated with energy densities of 5, 10, 20, and 40 J/cm 2 • One dog (drug only-no light) and four fully treated animals were sacrificed at time points of 1 week, 1 month, and 6 months after treatment and studied for gross and microscopic injury. There were no differences between the PDT-treated and control animals. All dogs recovered from surgery without side effects and clinically had no complications at follow-up examinations. At autopsy, the amount and depth of injury was proportional to the dose of light in animals who received the drug. No injury was seen in the control animals. In PDTexposed animals, at 1 week, tissue in the light-field showed coagulation necrosis that progressed to fibrosis by 1 month. No tissue had damage deeper than 5 mm. The lung was the most sensitive organ, the chest wall the most resistant. The myocardium had superficial damage and coronary arteries showed no acute changes.

2. One could develop a technique to "uniformly" illuminate the entire chest cavity. Treating cells in a flask is vastly different from treating a complex geometric structure like the chest where there can be light-shadowing. In bladder PDT, in order to obtain more uniform light distribution, intralipid in concentrations between 0.1 and 0.5% had been used empirically. The effect of this intralipid on intracellular Photofrin-Il concentration or cytotoxicity, however, had not been explored. Our group treated Photofrin-Il-loaded cells with light while they were in various concentrations of intralipid and found that despite loss of sensitizer from the cells incubated in intralipid, there was significantly better PDT cytotoxicity with light-scattering media (96). It was possible, then, that the same strategy used to treat a relatively simple geometric space (an avoid sphere) like the bladder could be adapted to treat the pleural space. 3. One could actually record how much light is being delivered to the pleura. The ability to actually record the amount of light that the tissue is being subjected to during PDT would be critical in any trial of large cavity PDT treatment. As with any investigational therapy, accurate quantita-

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tion of the individual components of the PDT package would be necessary to make decisions regarding efficacy and toxicity. Therefore, in combination with the Biomedical Engineering and Instrumentation Program, a photodiode system was developed to record on-line light doses, and tested in a phantom model of the thoracic cavity. Initially using a four-photodiode system, light measurements were recorded in a phantom model of the chest, comparing buffered saline solution to intralipid as light-scattering media. Excellent light distribution from a single fiber placed in the center of the intralipid-filled model occurred, with uniform power densities noted (97). Maximum lightscattering occurred between 0.01 and 0.05% intralipid. The addition of blood to the intralipid, however, resulted in a 16-56% decrease in delivered light. This model allowed us to modify a photo diode system for use in the pleura-treated patients and also pointed out the difficulties in illuminating the anterior and posterior diaphragmatic gutters, as compared to the rest of the surface of the pleura. The Biomedical Engineering and Instrumentation Program modified the system to measure light intensity at a number of sites and to integrate the values in real-time to provide the light dose at each, all in a way that was convenient and safe to use in the operating room (98). This system evolved from the primitive one used for the Plexiglas thoracic model into the present system consisting of an amplifier box, isolation transformers, photodiodes, and a computer. A custom-designed amplifier box houses individual current to voltage converters for up to 12 photodiodes. Signals are transmitted to a Compaq Portable II computer with the appropriate software for photodiode calibration and location of each photodiode if recorded, as well as the real-time and cumulative J / cm2 . The system has proven to be very reliable and dispenses with the necessity of calculating the duration of PDT treatment to guide dosimetry. From these studies, a feasibility trial in eight patients was conducted, which demonstrated that mechanical delivery of light to the pleura using dye lasers could be accomplished safely up to a dose of 15 J / cm 2, 48 hours after sensitizer delivery (99). A phase I study of PDT in the management of pleural malignancies to define the maximal tolerated dose of PDT that could be delivered to the chest cavity after maximum cytoreductive surgery was then performed from June 1990 to April 1992 (100). The problem of uneven geometry for light distribution was overcome partially by using a light-scattering media that was simply poured into the chest in which the laser fiber was placed. The light-scattering media, low concentration intralipid, minimally absorbed the light and was well tolerated by tissues (Plate 1). Commercially available photodiodes, mounted on transparent lucite bases, gathered light to a custom-designed amplifier box, isolation transformers, and computer. Each photo diode gave instantaneous readings of light in J / cm2 as well as the cumulative dose seen. Such a system allowed treatment of a given area to a prescribed dose.


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Fifty-four patients with isolated hemithorax pleural malignancy were prospectively entered into the trial which, in cohorts of three patients, escalated the intraoperative light dose from 15 to 35 J/ cm 2 48 hours after intravenous delivery of Photofrin-I1, 2.0 mg/kg, and then escalated the light dose from 30 to 32.5 J/ cm 2 after a 24-hour sensitizeroperation interval. Twelve patients could not be debulked to the prerequisite 5-mm residual tumor thickness. The remaining 42 patients (31 mesothelioma,S adenocarcinoma, 4 sarcoma, 2 other) received 19 modified pleuropneumonectomies, 5Iobectomy-pleurectomies, and 18 pleurectomies. Intrapleural PDT was delivered using 630-nm light from two argon pump-dye lasers, and real-time as well as cumulative light dose were monitored using seven uniquely designed, computer-interfaced photodiodes. There was 1/54 (1.9%) less-than-30-day mortality from intraoperative hemorrhage. Operation duration was 6.5 ± 0.3 hours, median intensive-care-unit stay was 4.9 ± 0.3 days, and patients were discharged 12.4 ± 1.0 days after surgery. Arrhythmia (11 /42; 26%) was the most common complication, and 59% of patients were complication free. In the 48-hour sensitizer-operation group (n = 33), possible PDT-related complications included an empyema with late hemorrhage in one of three patients at 17.5 J/ cm 2, and a bronchopleural fistula at 35 J/ cm2 , At each of these light doses, three additional patients were treated without complication, Two patients with 24-hour sensitizer dosing and 32.5 J/ cm 2 developed esophageal perforations after pleuropneumonectomy at identical sites. These patients were salvaged with open thoracostomy, jejunostomy, and gastroesophageal junction stapling. Subsequent left colon esophagogastric interposition re-established normal swallowing in both. The maximal tolerated dose (MTO) was declared as 30 J/ cm2 light with a 24-hour dosing interval when none of the six patients (three original, three repeat) at that level developed toxicity. Consistent patient-to-patient light measurements were substantiated by analysis of the dosimetry system, which revealed a 0.99 correlation of the laser treatment time with patient body surface area, projected light dose, and available power density.

CONCLUSION At least five other centers have had an interest in the management of pleural mesothelioma with PDT (101-105). In reviewing the data, most of which is only presented in abstract form, it can be appreciated that the studies represent a tremendous amount of labor. Nevertheless, the PDT delivery is heterogeneous with regard to sensitizer used and dose of light used, and the staging of the patients treated is very unclear. It is far too early to draw conclusions regarding efficacy from these phase II trials, but they reinforce the efforts from the NCI group that the deliv-

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ery is feasible and can be performed safely. Moreover, the operative mortalities are low in all the studies. Phase III trials using this first generation of PDT mechanics are now being performed at this MTD in a homogeneous population of patients with pleural malignancies. Naturally in such patients, whose curative surgical options are close to zero with standard treatment, the advent of an intraoperative adjuvant that may obtain at least local control might be the first step toward improving survival.

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18 Growth-Factor Receptors as a Target for Therapy Roman Perez-Soler and John Mendelsohn

In neoplasia, a variable degree of cell de-differentiation is characteristically associated with uncontrolled proliferation, as a result of the loss of tumor-suppressor gene products that play an important role in cellcycle checkpoint control, the induction of abnormal oncoproteins that are directly involved in cell-proliferation signaling, or a combination of both. Neoplasia may be biologically similar to physiological tissue replacement in that stem cells proliferate in response to an external signal generally transmitted to the cell through the binding of one or several growth factors to specific cell-membrane receptors. However, only in physiological tissue replacement do consecutive cell generations progressively undergo differentiation and display downregulation of growth-factor receptors while losing their ability to proliferate. The rationale for attempting to block growth-factor function as an approach to cancer therapy is compelling (1). There is strong evidence that neoplastic cells gain a growth advantage by overexpression of a variety of growth-factor receptors and active secretion of growth factors in what constitutes an autocrine or paracrine stimulation of tumor growth. The overexpression and activation of these cell-signaling pathways is a poor prognostic factor in many malignancies. Finally, some of these pathways are involved in cancer cell sensitivity to cytotoxic agents, and their blockade or activation can be potentially exploited for the targeted chemosensitization of several human malignancies. There is a great heterogeneity among malignancies and individual tumors of a given histologic type in their dependence on different signaling path309

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ways for sustained growth. Therefore, the validation of growth factor-receptor blockade as an effective form of cancer therapy would be enhanced if the clinical trials took into consideration the prior identification of those signaling pathways that are predominantly involved in each individual tumor. Non-smaIl-cell and small-cell lung cancer (NSCLC and SCLC, respectively) are two diseases of related etiologies but distinct morphology, genetics, biology, and clinical behavior. There is definite evidence that NSCLC and SCLC differ in the growth-signaling pathways involved in maintaining their proliferation and possibly also their ability to metastasize. The epidermal growth-factor receptor (EGFR) and c-erb B-2 (HER-2) pathways are overexpressed in NSCLC tumors and not in SCLC tumors, whereas a variety of neuroendocrine pathways are overexpressed exclusively in SCLC tumors. The advances in the understanding of these pathways during the last few years have resulted in the development of different therapeutic strategies for lung cancer aimed at either specifically inhibiting lung cancer cell proliferation by growth factor-receptor-blocking agents or enzymatic inhibitors, sensitizing lung cancer cells to cytotoxic therapy, or targeting selectively cytotoxic agents to lung cancer tumors.

GROWTH-SIGNALING PATHWAYS AS A TARGET FOR THE TREATMENT OF NSCLC The EGFR pathway is thought to play an important role in the physiological turnover of the bronchial epithelium and in promoting its regeneration after damage. Numerous studies have shown that this pathway is overexpressed in many malignancies of epithelial origin, including most NSCLC tumors, and some studies suggest that overexpression tends to be associated with tumors that are more aggressive and have a worse prognosis, thus indicating this pathway's potential role in contributing to the sustained growth of these tumors. These findings have led to the conception and development of therapeutic strategies aimed at controlling cell proliferation through receptor blockade by monoclonal antibodies or specific receptor kinase inhibitors, and at targeting cytotoxics to tumors by using a variety of immunoconjugates or fusion proteins containing epidermal growth factor (EGF), transforming-growth factor-alpha (TGF-a), or anti-EGFR antibodies as targeting moieties. Furthermore, combinations of humanized monoclonal antibodies, which block EGFR function with high specificity, and cytotoxic agents have shown a striking synergism in preclinical studies and are under intensive clinical evaluation.


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The EGFR Pathway Physiology and Biochemistry EGF is a single-chain polypeptide of 53 amino acids that stimulates proliferation and differentiation of a variety of cell types through interaction with a specific cell-surface receptor (EGFR) (2). EGF is produced by a variety of human tissues, including the salivary glands, Brunner's glands of the duodenum, the kidney, the bronchial epithelium, and other endocrine organs (3). The EGF gene is located on human chromosome 4q25 and is initially synthesized as a large precursor of 1217 amino acids (4). TGF-a and amphiregulin (AR) are peptides structurally related to EGF that bind and also exert their biological effects exclusively through EGFR (5, 6). The human TGF-a and AR genes are located on chromosome 2p11-13 and 4q13-21, respectively (7, 8). TGFa is produced by a variety of adult tissues, apparently playing a role in regenerating the stem cells of epithelial surfaces, and is actively secreted in large amounts by a variety of malignant epithelial cell lines (9). EGF and TGF-a, in general, stimulate growth upon binding to EGFR. However, in some cancer cell lines with very high numbers of receptors-such as A431 cervix carcinoma cells, HSC-2 and NA squamous head and neck carcinoma (10), ES-4 esophageal carcinoma (11), NX002, CX140, and CX143 lung squamous carcinoma (12), and MDMB-468 breast adenocarcinoma cells (13, 14)-EGF and TGF-a can stimulate growth at low concentrations and also inhibit growth and, in some cases, induce apoptosis at high concentrations. AR has been found to be mostly a bifunctional growth modulator, able to both stimulate or inhibit growth depending on the experimental conditions and also the number of EGFR receptors present on the cells (15). EGFR (c-erb B-1) is a transmembrane glycoprotein of 170 kd that is expressed in many human tissues and neoplasms, including head and neck cancer, NSCLC, bladder cancer, glioblastoma, breast carcinoma, and ovarian carcinoma (2). EGFR is a member of a superfamily of structurally and functionally related membrane growth-factor receptors that have an associated tyrosine kinase activity (16). Other members of the family include c-erb B-2 (HER-2), c-erb B-3 (HER-3), and c-erb B-4 (HER-4). The EGFR gene is located on chromosome 7p13-p12 (17). The structure of EGFR consists of a 621-amino acid extracellular domain that is glycosylated and has two cysteine clusters, between which is located the ligand-binding region, a 23-amino acid hydrophobic transmembrane domain, and a 542-amino acid cytoplasmic domain that contains a highly conserved tyrosine kinase. Upon ligand-binding, the receptor can be autophosphorylated at five different tyrosine residues. This autophosphorylation triggers the proliferative signal. The receptor can also be a substrate for other tyrosine kinases, such as c-erb B-2, or serine-threonine kinases, such as protein kinase C. Upon ligand-bind-

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ing, there is receptor clustering and dimerization, which is followed by receptor internalization. Internalized EGFR can be recycled as in A431 cells (18) or metabolized and downregulated, which can desensitize the cells to growth-factor stimulation. Activation of EGFR by EGF or TGFa autoinduces the production and cleavage of pro-TGF-a (19). Activation of the EGFR pathway can occur as a result of EGF or TGF-a secreted by other cells in the same organ (paracrine activation), vicinal cells (juxtacrine activation), or the same cells that express EGFR (autocrine loop). In cells that secrete EGF or TGF-a and overexpress EGFR, cell growth is normally inhibited by antibodies that neutralize the growth factor, thus suggesting that the growth factor that binds to the receptor is secreted into the extracellular space. However, this is not always the case. This finding has led some to hypothesize that intracrine stimulation as a result of growth-factor binding to internalized receptor can occur. Such a mechanism has been demonstrated for platelet-derived growth factor (PDGF) (20).

Expression of EGFR, EGF, and TGF-a in Normal Lung In adult human lung, EGF immunoreactivity is observed in the secretory glanules and endoplasmic reticulum of serous acinar cells of bronchial glands, and EGFR immunoreactivity at the cell membrane of the basal cells and the bronchial surface of nonciliated cells, the nonciliated bronchiolar cells (Clara cells), and the type II pneumocytes in peripheral lung (21). Interestingly, the positivity is limited to the intercellular lateral membranes. These findings suggest that EGF is secreted into the bronchial surface fluid from the bronchial glands, where it stimulates the proliferation and differentiation of basal cells and nonciliated cells. Ciliated cells are well-differentiated cells that do not express EGFR and therefore cannot be stimulated by EGF. Access of EGF to the basal cells probably occurs when the ciliated cells exfoliate because of aging and physiological turnover or direct mucosal damage. In both cases, the intercellular junction of the basal cells is destroyed, thus allowing direct access of the EGF to the lateral basal cell membrane where EGFR is expressed. The access of EGF to the basal cells initiates the replacement of old or damaged ciliated cells by new, highly functional ciliated cells.

Expression of EGFR, EGF, and TGF-a in NSCLC Cell Lines, Premalignant Bronchial Lesions, and NSCLC Tumors NSCLC Cell Lines

The great majority of NSCLCs express EGFR and TGF-a. The levels of EGFR expression are often elevated. However, very high levels of EGFR expression, comparable to those of well-known cell lines such as A431 (100-fold normal levels), are rare. In a panel of eight NSCLC cell lines

Growth-Factor Receptors

as a Target for Therapy

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studied by Liu et a1. (22), expression of EGFR and TGF-a were assessed by Northern blot analysis. Only one of the NSCLC cell lines (MGH-7) (the only squamous carcinoma cell line) expressed higher EGFR mRNA than A431 cells. The level of expression in the other cell lines was two to ten times lower than in A431 cells. Higher expression has been observed in head and neck squamous carcinoma cell lines. Kamata et a1. (10) studied EGFR expression by binding of radiolabeled EGF to tumor cell membranes in a panel of six squamous cell carcinoma cell lines of the oral cavity, and found that two cell lines expressed about twice as many EGFR receptors compared with A431 cells, two others had a similar number of EGFR receptors, and the final two had about four to five times fewer EGFR receptors. Similarly, Cowley et a1. (23) found that head and neck cell lines had 5-50-fold higher EGFR expression than normal kera tinocytes. The secretion of TGF-a by many cell lines that express EGFR suggests the existence of an autocrine loop. Putnam et a1. (24) studied the production of TGF-a by a panel of NSCLC cell lines expressing EGFR. All cell lines expressed TGF-a by immunohistochemistry and mRNA. The growth of three cell lines could be stimulated with exogenous TGF-a, and inhibition by an anti-TGF-a antibody occurred in two cell lines but not in two others. Similar results have been observed with a panel of four colon carcinoma cell lines that overexpress EGFR and TGF-a, thus suggesting that other growth-factor pathways may be predominant (25). Cytoplasmic binding of receptor and ligand is also possible (intracrine activation) but has only been observed and described for the PDGF-receptor pathway. Notwithstanding the above, in most cell lines where an autocrine loop involving EGFR and EGF or TGF-a exists, EGFR blockade by monoclonal antibodies results in inhibition of EGFR function and reduced cell growth rate. Rabiasz et a1. (12) observed that both EGF and TGF-a inhibit the growth of NSCLC cell lines that produce TGF-a and have very high EGFR expression (100-fold higher than NSCLC tumors obtained from patients), a phenomenon observed with other cell lines that express extremely high levels of EGFR, where the level of inhibition has been shown to increase as the EGFR number increases. Interestingly, the growth-inhibitory effect of EGF and TGF-a in these cell lines was associated with arrest at G2/M, while growth inhibition by receptor blockade in cell lines with a lower number of EGFR receptors is associated with arrest at GO/1; also cell growth inhibition of high EGFR-expressing A431 cells by EGF has been associated with arrest at GO /1 (26). These observations suggest the potential use of EGFR agonistic agents for the treatment of the minority of NSCLC tumors with very high EGFR expression. However, recognition of these patients before therapy should be mandatory, as EGFR activation may enhance the growth of tumors with lower EGFR overexpression. Furthermore, experiments with xenografted cell lines indicate that cells inhibited in tissue culture may be stimulated by EGF in vivo (27).

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In the case of c-erb B-2, increased expression has been associated with chemoresistance in a large panel of NSCLC cell lines (see Premalignant Bronchial Lesions) (28). No similar studies correlating chemosensitivity and EGFR expression in an in vitro panel of cell lines have been reported. However, in pairs of parental cells and their variants resistant to cisplatin, acquired resistance has been associated with a lower cell proliferation and downregulation of EGFR receptors (29). This phenomenon has also been described for c-erb B-2 (30), but its clinical relevance remains unknown (9). Premalignant Bronchial Lesions

The recognition that lung cancer is a manifestation of a pathologic process that affects the whole bronchial epithelium has led some to investigate the frequency of genetic alterations in the normal, metaplastic, or dysplastic bronchial epithelium of patients with lung cancer. Pastorino et al. (31) analyzed 68 primary NSCLC tumors undergoing resection for early-stage carcinoma and the bronchial epithelium of the same patients. Overexpression of EGFR and c-erb B-2 and p53 mutations were found in 63%, 21 %, and 50% of tumors, respectively. Overexpression of c-erb B-2 was exclusively found in adenocarcinoma tumors. In the bronchial mucosa, EGFR overexpression was found in 39% of cases, c-erb B-2 overexpression in 14% of cases, and p53 mutations in 5% of cases. Sozzi et al. (32) studied the EGFR and HER-2 neu expression in the bronchial epithelium of patients with lung cancer by immunohistochemistry and found overexpression in 9 of 18 tumors and in 6 of 13 bronchial epithelium samples, respectively. Kurie et al. (33) studied EGFR expression in the bronchial mucosa of 69 chronic smokers and found EGFR overexpression in the areas of bronchial metaplasia compared to the areas of normal mucosa. All these studies suggest that early overexpression of these receptors in the bronchial epithelium may play a role in the pathogenesis of lung cancer and could be exploited as a target for early intervention or prevention strategies. NSCLC Tumors: Prognostic Implications of EGFR Overexpression

In bladder cancer and breast cancer, several studies have reported an association of EGFR overexpression and survival (2). Numerous studies have investigated the expression of EGFR, EGF, and TGF-a in NSCLC tumors using a variety of techniques, including immunohistochemistry with different monoclonal antibodies, membrane-binding of radiolabeled EGF, mRNA expression (Northern blot), and gene expression (Southern blot). These studies have consistently established that EGFR is overexpressed in about 60-70% of NSCLC, predominantly squamous carcinomas, and that the level of expression is about two- to threefold higher than in normal lung tissue. Less consistent are the findings regarding the impact of EGFR and/ or TGF-a expression on prog-


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nos is, although the majority of studies have reported significant correlations or trends between EGFR overexpression and either a shorter survival, a higher stage, or a lower degree of cell differentiation. No prospective studies aimed at defining the prognostic value of EGFR and EGF or TGF-a in NSCLC have been reported. Three studies analyzed EGFR expression by radiolabeled-EGF binding to tumor cell, fresh membrane preparations. Veale et al. (34) studied the correlation between EGFR and survival in 19 surgically treated NSCLC tumors. Seven patients survived for more than 5 years. There was no difference in stage between patients surviving more than 5 years and less than 5 years. Those who survived more than 5 years had a median EGFR concentration that was fourfold lower than that of the patients who survived less than 5 years. Although the number of patients studied is small, this is one of the few studies demonstrating a negative prognostic impact of EGFR overexpression assessed by a quantitative technique. No relationship between EGFR expression and stage was found. In a similar study, di Carlo et al. (35) analyzed the EGFR expression in fresh plasma membranes of 54 lung tumors (34 squamous and 20 adenocarcinomas). A moderate increase (2- to 5-fold) in the amount of radiolabeled EGF bound to the tumors in comparison with normal lung was found in most cases. Only 5% of patients showed a tenfold or greater increase in EGF-binding in comparison with normal lung. Radiolabeled EGF-binding correlated with poor cell differentiation but not with stage. Finally, Dittadi et al. (36) found similar results in a study of 51 NSCLC samples: a twofold increase in binding or radiolabeled EGF to fresh tumor membranes compared with normal lung. No relationship was found between EGFR expression and stage. However, there was a trend for a direct relation between EGFR expression and cellular grading. Most studies have used a qualitative assessment of EGFR expression by immunohistochemistry using different antibodies. Cerny et al. (37) reported EGFR expression in 80% of 48 NSCLC tumors. Using monoclonal antibody 528, Sobol et al. (38) observed immunoreactivity in 22 of 22 squamous cell carcinomas of the lung, 4 of 4 large cell carcinomas, 13 of 21 adenocarcinomas, and 0 of 9 SCLCs. Pave lie et al. (39) studied 63 primary NSCLC tumors and reported EGFR expression in 55% of patients. A positive correlation between EGFR overexpression and high metastatic rate, high rate of cell proliferation, and degree of dedifferentiation was observed. Gorgoulis et al. (40) studied the expression of EGFR and TGF-a by immunohistochemistry in 70 squamous cell lung carcinomas. EGFR expression alone was found in 0.7% of cases, EGFR and TGF-a in 3%, EGFR and EGF in 23%, and all three markers in 30% of cases. TGF-a alone was expressed in 4% of cases, EGF alone in 14% of cases, and both in 20% of cases. The frequency of lymph node metastases was significantly higher in tumors expressing EGFR and EGF, TGF-a and EGF, and all three markers, thus leading to the conclusion that the con-

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comitant overexpression of EGFR and EGF or TGF-a confers a more aggressive behavior. In a similar study, Tateishi et al. (41) studied the expression of EGFR and TGF-a in 131 patients with lung adenocarcinoma. The 55 patients with overexpression of EGFR and TGF-a or EGF had a more advanced stage and a shorter survival. The expression of either growth factor did not have any impact on survival in the absence of EGFR overexpression. The prognostic impact of the coexpression of EGFR and EGF or TGFa suggests a role for the autocrine loop in the spread of adenocarcinoma. Finally, in another study of 176 NSCLC tumors, Fontanini et al. (42) found that the EGFR immunoreactivity was higher in squamous cell tumors compared with adenocarcinoma tumors, and EGFR expression correlated with the presence of nodal metastases. However, no relationship between EGFR expression and T-status, grading, or proliferative activity (measured by Ki-67, premature condensation nuclear antigen (PCNA), and S-phase by flow cytometry) was observed, leading the authors to speculate that EGFR activation may influence the interaction of cells with matrix components or the cell-cell contact, both of which are important in determining tumor invasiveness. Two other studies have looked at the EGFR expression at the mRNA level. Rusch et al. (43) studied the expression of EGFR, TGF-a, EGF, and AR by Northern blot analysis in paired samples of primary tumors and uninvolved lung, in 57 consecutive patients who underwent resection and were followed prospectively. EGFR was expressed in 93% of specimens, TGF-a in 86%, and AR in 92%. EGF was not expressed in tumor or normal lung. Compared to normal lung, EGFR was overexpressed in 45% of cases, TGF-a in 61 %, and AR in 63%. Simultaneous overexpression of EGFR and TGF-a was observed in 38% of cases, and overexpression of EGFR and EGF in 21 % of tumors. In contrast with studies discussed earlier, there was no correlation with stage, cell type, or survival, although follow-up was short. Finally, Liu et al. (22) studied the expression of EGFR, c-erb B-2, and TGF-a in 29 primary tumors of NSCLC. The mean mRNA levels of TGF-a and EGFR were higher in carcinomas than in normal tissues (2.8- and 1.7-fold, respectively). However, only adenocarcinomas expressed a higher level of c-erb B-2 (2.2 times). When compared to normal lung, 55% of tumors had more than a twofold increase in expression of both TGF-a and EGFR, and 30% of tumors had more than twofold increase in expression of c-erb B-2. Mechanisms of Overexpression and Frequency of Gene Structural Alterations

Most studies indicate that EGFR overexpression in NSCLC is principally related to enhanced gene transcription or translation. However, a few studies have suggested that gene amplification may occur in a significant number of patients. Yamamoto et al. (44) studied 12 squamous cell carcinoma and 18 nonsquamous carcinoma cell lines by Southern blot hybridiza tion analysis and found amplifica tion of EGFR gene in 10 of the


31 7

12 squamous and in a of 18 nonsquamous. The degree of amplification was 2- to 50-fold compared with normal keratinocytes. These results must be taken with caution, as it is possible that EGFR gene amplification may be a mechanism of adaptation of squamous carcinoma cell lines to in vitro growth. However, some studies using human tumors have also found a relatively high incidence of gene amplification. Gorgoulis et al. (45) studied 18 squamous cell carcinomas of the lung and found EGFR overexpression by immunohistochemistry in 89% of cases, and gene amplification by Southern blot analysis in 27% of cases. Moreover, Testa et al. (46) and Spurr et al. (47) reported polysomy of all or part of the short arm of chromosome 7, where the EGFR is located, in 67% of NSCLC specimens, and Sozzi et al. (32) found alterations in chromosome 7 in four of the six normal bronchial epithelial samples from cancer patients. This suggests that EGFR overexpression secondary to gene amplification or alterations of chromosome 7 may not be as rare as thought by most investigators and, therefore, potentially exploitable for therapeutic purposes. Many studies have investigated the frequency of structural alterations in the EGFR gene in NSCLC. Grunberger et al. (48) studied the restriction fragment length polymorphisms (RFLPs) of the EGFR in 29 NSCLC tumor samples and in the adjacent normal lung, and could not detect significant differences in the specific polymorphic bands between tumor and paired nontumor lung tissues or between the different types of carcinoma. The authors concluded that they could not demonstrate amplification or rearrangement of the EGFR gene in lung cancer, and that the four polymorphic patterns identified did not appear to be involved in the pathogenesis of lung cancer. A few studies have reported the expression of truncated EGFR in a significant proportion of NSCLC tumors. A type III deletion mutant of the EGFR has been identified in glioblastomas. Garcia de Palazzo et al. (49) studied the prevalence of this mutation in 32 samples of NSCLC by immunohistochemistry. Native EGFR was detected in 12 of the 32 samples, and the type III deletion mutant in 5 (16%) of the specimens. The authors concluded that this type of EGFR may be a molecularly distinct tumorspecific antigen that can be exploited for the targeted therapy of a subset of NSCLC tumors. Specific monoclonal antibodies against EGFRvllI have been developed by Wikstrand et al. (50) and confirmed the expression of this truncated form of EGFR in a small subset of patients with NSCLC, breast carcinoma, and glioblastoma, but not in normal tissues. The authors conclude that development of these antibodies for the therapy of these subsets of tumors is warranted.

EGFR Blockade with Monoclonal Antibodies Because of the EGFR pathway's role in sustaining the growth of different human malignancies (including NSCLC) and conferring, in general,

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a more aggressive phenotype and worse prognosis, anti-EGFR monoclonal antibodies have been developed during the last two decades as therapeutic agents for these malignancies. The different antibodies developed vary in their affinity for EGFR, the epitope they recognize, and their ability to inhibit or stimulate EGFR tyrosine kinase activity. Monoclonal antibodies 225 and 528 are the best studied anti-EGFR antibodies (1). A human chimeric version of 225 (C225) is currently under extensive clinical investigation. Monoclonal antibodies 225 and 528 recognize specifically human EGFR, have an affinity for EGFR comparable to EGF, compete for EGF-binding, inhibit TGF-a autoinduction after EGFR activation (19), initiate EGFR internalization after binding, and inhibit EGFR tyrosine kinase activity (51-53). They specifically inhibit the in vitro proliferation and the in vivo growth of human malignant cells that depend on the presence of extracellular EGF or TGF-a for their baseline in vitro proliferation, or whose proliferation can be augmented by the addition of exogenous growth factor (54). In these cell lines, their effect is reversed by the addition of exogenous EGF (55). They do not affect the in vitro proliferation or the in vivo growth of the rare EGFR-expressing human cell lines that do not depend on the autocrine production of EGF or TGF-a for their growth. Their effect is cytostatic in most cell lines that overexpress EGFR, with the exception of rare cell lines like DiFi colon carcinoma, which are strictly dependent on the EGFR pathway for survival and in which the antibodies are cytotoxic and induce apoptosis (56-59). Intact monoclonal antibody 225 is as effective as its Fab fragments in inhibiting tyrosine kinase activity of EGFR on the cell surface, but it is much more effective in inhibiting cell growth because the intact antibody is able to dimerize EGFR, which results in receptor downregulation and removal from the cell surface (57-59). RG83852 or A108 is a murine IgG2a monoclonal antibody that binds to human EGFR and has a partial agonistic effect by stimulating receptor autophosphorylation but inhibits cell proliferation. A108 was found to inhibit the in vitro growth of several NSCLC cell lines overexpressing EGFR, and in vivo the growth of KB and NSCLC xenografts in nude mice (60, 61).

Biochemical and Cell Cycle Effects Secondary to EGFR Blockade As in the case of other biological regulatory pathways, activation of the EGFR pathway and other pathways of the same receptor superfamily normally, but not always, results in growth stimulation, whereas deprivation of growth factor or blockade of the pathway results in GO/1 blockade, decreased cell proliferation, and in some cases apoptosis. Depending on the cell line, the type and concentration of growth factor,


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and the number of EGFR receptors, stimulation of the EGFR pathway may also cause decreased cell proliferation and apoptosis. The first biochemical effect of EGFR activation is receptor autophosphorylation at one or more of five tyrosine residues. The downstream biochemical effects that link EGFR activation to cell proliferation or inhibition are under active investigation and are far from being fully elucidated. In both cases, such effects appear to be mediated by the induction of cell cycle-specific natural inhibitors. InA431 cells, cell growth is inhibited by EGFR activation by EGF and is associated with induction of p21 cip 1 /Wafl, a natural inhibitor of cyclin-dependent kinases, and arrest at GO/I. This effect can be blocked by EGFR tyrosine kinase inhibition by tyrphostins, thus suggesting that it is linked to tyrosine kinase activation (26), and augmented by TGF-B, which causes increased EGFR tyrosine phosphorylation (62). In DiFi colon carcinoma and DU145 prostate carcinoma, EGFR blockade by monoclonal antibody 225 causes cell growth inhibition and arrest at GO/I, as in most EGFR expressing cell lines. Arrest at GO/l has been found to be associated with induction of p27 kipl, a natural inhibitor of cdk2 and cyclin E, both at the mRNA and protein levels, thus suggesting that the antiproliferative effect of receptor blockade is mediated, at least in part, by the induction of a physiological cyclin inhibitor (63, 64).

Synergism Between EGFR Blockade and Cytotoxic Agents During the last few years, several studies have demonstrated a striking synergism between different anti-growth-factor antibodies and chemotherapeutic agents. Aboud-Pirak et al. (60) reported a remarkable synergism between the agonistic anti-EGFR antibody RG83852 and cisplatin in the treatment of KB xenografts in vivo. In the same context, Christen et al. (65) reported synergism between EGF itself and cisplatin in two cultured ovarian cell lines. Interestingly, in this latter study, synergism was only observed in parental cells but not in the variants with acquired resistance to cisplatin, despite the presence of functional EGFR. Baselga et al. (66) reported that the antagonistic anti-EGFR antibodies 528 and 225 also enhanced the antitumor effect of doxorubicin against A431 and MDA-468 breast cancer cells. The effect was only additive in cell culture. In contrast, the treatment of well-established xenografts with either agent alone temporarily inhibited tumor growth, whereas the combination of both agents resulted in a markedly enhanced antitumor activity. In the case of A431 xenografts, tumor eradication was commonly seen. A similar study using the same antibodies in combination with cisplatin gave very similar results (67). Well-established A431 xenografts could be eradicated with the combination, whereas either

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agent alone had little effect on tumor growth, and only an additive effect was observed in the in vitro experiments. The mechanisms of this in vivo synergism or targeted chemosensitization effect are under investigation and may vary depending on the chemotherapeutic agent and the status of regulator genes such as p53, Rb, Bcl-2, and BAX. Altered GI IS checkpoint control by EGFR functional blockade and cell dependence on the EGFR pathway for DNA repair andlor survival after damage have been suggested. Because the effect is more evident in vivo than in vitro, in vivo mechanisms such as enhanced access of the antibody to the tumor tissue as a result of an anti-angiogenesis effect of the cytotoxic agent and immunostimulation through the Fc receptor have also been postulated. In normal cells, cisplatin-induced DNA damage causes Gl arrest to allow for complete DNA repair before the cell engages in DNA synthesis. Cells that obey cell-cycle checkpoints may be less sensitive to cytotoxic damage as a result of more stringent checkpoint controls. Many tumor cells do not obey the GI/S checkpoint control as a result of molecular alterations (p53 mutations, Rb and pl6 deletions, etc.). In the recent studies that have shown that blockade of EGFR activation can cause arrest at a restriction point at GO/I through, at least in part, upregulation of p27 kipl (63,64), the arrest in tumor cells was never complete. However, when nonmalignant cells are treated with anti-EGFR monoclonal antibodies, complete growth arrest is observed (68,69). Violation of this checkpoint in the presence of an EGFR-blocking agent in a malignant cell whose DNA has been damaged by cisplatin may compound the effect of residual DNA damage and induce apoptosis. Other possibilities include dependence or increased dependence on the EGFR pathway for cell survival after damage or direct involvement of the EGFR pathway in DNA repair. Supporting the first hypothesis, doxorubicin has been shown to enhance the secretion of TGF-a by tumor cells, probably as a compensatory response to cell damage (66). Arrest at Gall secondary to EGFR blockade has been shown to be associated with protein-synthesis inhibition. Forced arrest of these cells at GO/I, a command they normally do not follow, may inhibit the synthesis of enzymes essential for proper repair of damage (57). As discussed below, Pietras et al. (70) and Arteaga et al. (71) have reported synergism in tumor cells overexpressing c-erb B-2 between the anti-c-erb-B2 monoclonal antibodies 405 and TAB250 and cisplatin, and in both cases the mechanism of synergism or sensitization appeared to be a reduced repair of platinum-DNA adducts.

Clinical Studies with Anti-EGFR Monoclonal Antibodies Different anti-EGFR monoclonal antibodies have been evaluated in clinical studies in the last few years. In all cases, no significant toxicities


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were observed at doses that resulted in tumor EGFR saturation. The best studied antibody is 225, whose clinical development is being actively pursued.

Phase I Study of 111'n-M22S

The initial clinical study of murine monoclonal antibody 225 was performed in patients with squamous cell carcinoma of the lung (72). The goals of this phase I study were to define the toxicity and pharmacokinetics, and tumor localization of l11In-225-IgGl. Groups of three patients received escalating doses of antibody intravenously over 1 hour. No toxicities were observed up to doses of 300 mg of antibody. The imaging studies revealed visualization of all primary tumors and metastases of larger than 1 cm detected by CT scan and chest x-ray at doses of 40 mg and higher. The serum concentration of ll1In-225 was more than 40 nM for more than 3 days at doses of 120 mg and higher, a concentration that if reached in tumors should saturate all EGFR receptors. Very high liver uptake was observed probably because of the expression of EGFR in normal liver cells and the phagocytic capacity of the liver. Antimouse antibodies were detected in all patients, thus preventing the repeated use of this antibody to block tumor EGFR for therapeutic purposes. This study suggests a potential use of antibody 225 as an imaging agent.

Phase I Studies with C22S Alone or in Combination

C225 is the human chimeric counterpart of 225. Both antibodies have the same effect on human EGFR. Two phase I studies, by single and four weekly intravenous infusions have been completed using doses from 5 to 100 mg/ m 2 in patients with EGFR-positive tumors for which no curative therapy exists (73). The only toxicity higher than grade 2 was a grade 3 aseptic meningitis in one instance. Side effects were limited to one grade 2 allergic reaction and asthenia, fever, flu-like symptoms, pain, and diarrea in a few patients. Because of the striking synergism between C225 with different chemotherapeutic agents in human xenografts, phase I combination studies of weekly C225 (100-400 mg/m2) with cisplatin (NSCLC and head and neck cancer), adriamycin (prostate cancer), and taxol (breast cancer) are in progress. The objective of these studies is to determine the toxicity of these combinations and the dose of C225 that results in saturation of EGFR receptors in tumor tissue, as determined indirectly by pharmacokinetic studies and directly by tumor EGFR saturation studies at different time points after C225 administration. The design of these studies is based on the hypothesis supported by the preclinical studies that optimum synergism requires continuous blockade of EGFR. Available preliminary pharmacokinetic studies performed in the context of these studies indicate that a dose of more than 20 nM can be achieved for 1

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week after the administration of a dose of 100 mg/m2 of C225. Repeated administration can keep concentrations at this level for periods of up to 3 months. This concentration can saturate EGFR in human cell lines in vitro. However, because of the poor vascularity and suboptimal access of monoclonal antibodies to solid tumors and because at this concentration the clearance of C225 has not achieved zero kinetics, it is possible that tumor EGFR saturation has not been achieved. The recommended dose for phase II studies will be that dose that achieves homogeneous tumor EGFR saturation for 7 days, which is the period between C225 infusions, and does not cause significant side effects (grade 3). A potential concern in these combination studies is the possibility of synergism in normal tissues, thus causing new or unexpected toxicities. However, EGFR is not expressed in hematopoietic cells, which are the main target of cytotoxic agents. In the case of cisplatin and taxol, increased nephrotoxicity or neurotoxicity is a remote possibility, as normal renal tubular cells and neurons have a very low EGFR expression (74), and the mechanisms of toxicity of cisplatin and taxol in these organs are probably different than the mechanisms of cytotoxicity. In the case of doxorubicin, no enhancement of cardiac toxicity should be expected because heart muscle cells do not express EGFR. Other organs that deserve to be monitored closely are the skin and the gut, which are rapidly dividing tissues and express EGFR. However, EGFR in gut epithelium may be poorly reached by monoclonal antibodies.

Phase I Study of Murine RG83852

fA 108)

A phase I toxicity and tumor-saturation study of Al08 was conducted in patients with NSCLC and head and neck cancer (75). No significant side effects were observed up to a dose of 600 mg/m2. Fresh tumor specimens were obtained 24 hours after therapy in ten patients (of whom five had a pretherapy sample taken). Tumor EGFR tyrosine kinase activity was determined in fresh tumor samples by autophosphorylation of EGFR isolated in immunocomplexes with Al08. Tumor EGFR saturation was assessed by comparing the EGFR tyrosine kinase activity in immunocomplexes of tumor specimens obtained after therapy with total EGFR tyrosine kinase activity assessed by exogenous addition of Al08 to tumor lysates. By this method, EGFR saturation was estimated to be close to 100% at doses of more than 400 mg/m2, both in tumor tissue and skin used as surrogate EGFR positive tissue. This was confirmed by immunohistochemistry studies. In five patients in whom pre- and post-therapy biopsies were taken, a three- to fourfold upregulation of EGFR tyrosine kinase activity in post-therapy specimens was observed in two patients. The conclusions of this study were that Al08 causes no toxic side effects at doses that result in high EGFR saturation, and it confirmed that Al08 has an agonistic effect on EGFR.


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Clinical Studies with Other Monoclonal Antibodies: A42S, R1, 9A, and ICR62

Monoclonal antibody A425 has many properties similar to 528 and has been used to image recurrent human gliomas and for therapy. No significant toxicities were observed (76). Monoclonal antibody R1 was one of the first produced. It does not block EGF binding and does not inhibit cell proliferation. It was explored as an imaging agent for gliomas and showed greater specificity than a control monoclonal antibody (77). More recently, a phase I trial of the rat monoclonal antibody ICR62 in head and neck and lung cancer patients has been reported with results similar to those reported for 225 and A108 (78). This antibody blocks the binding of EGF and TGF-a to EGFR, inhibits the in vitro growth of EGFR-expressing tumors, and eradicates xenografts in nude mice. Eleven patients were entered. No severe toxicities were reported at the higher dose, 100 mg/m2. Excellent tumor localization was demonstrated in tumor tissue at the higher dose.

The c-erb 8-2 Pathway

Physiology and Biochemistry Heregulin (HRG) is a peptide of the EGF family initially identified and purified from conditioned medium of MDA-MB-231 human breast cancer cells (79-81). It is a 44-kd membrane-associated protein with a long cytoplasmic tail. The gene is located on chromosome 8p12-p21. HRG can specifically bind to and stimulate tyrosine phosphorylation of p185 c-erb B-2 and p180 c-erb B-4, but not EGFR, in several human breast cancer cell lines and promotes cell proliferation as a result (75). In contrast, in some other malignant cell lines that overexpress p185 c-erb B-2, HRG does not stimulate cell growth (82). It has been suggested that binding of HRG to p185 c-erb B-2 may require an additional cellular component that has been identified as p180 c-erb B-4. The c-erb B-2 gene product is a 185-kd protein originally identified and cloned from a human breast carcinoma (16); c-erb B-2 belongs to the EGFR superfamily of receptors and is normally expressed at low levels in a variety of human secretory epithelial tissues (83). The gene is located on chromosome 17qll.2-q12. There is 82% homology with EGFR in the tyrosine kinase domain. The extracellular domains are more distantly related, and neither EGF nor TGF-a binds to p185 c-erb B-2. Heterodimerization between p185 c-erb B-2 and EGFR or p180 c-erb B-4 has been described and can lead to transphosphorylation of p185 c-erb B-2 in response to ligand-binding to the other receptors, suggesting the possibility of cross-talk between these various receptors and of developing synergistic therapeutic strategies targeting both types of

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receptors. Normal lung tissue does not significantly express HRG nor p185 c-erb B-2. Kern et al. (84) studied the expression of p185 c-erb B-2 in a panel of 14 cell lines of NSCLC-by Western, Southern, and Northern blot analysis. Human bronchiole epithelial cells were found to express p185 c-erb B-2 at low to undetectable levels by Western blot. In contrast, 13 of the cell lines expressed p185 c-erb B-2 and 8 of the 13 at levels at least twofold higher than that found in bronchiole epithelial cells. Overexpression was due to gene amplification in only 1 of the 8 cell lines with high p 185 c-erb B-2 expression.

Expression of c-erb 8-2 in NSCLC: Prognostic Implications The c-erb B-2 gene product is overexpressed in about 30% of hreast and ovarian carcinomas as a result of gene amplification. Overexpression of c-erb B-2 in breast and ovarian carcinoma has been found to be associated with a shorter survival (85). The c-erb B-2 gene product is also overexpressed in some NSCLC tumors, but gene amplification is rare; c-erb B-2 overexpression was found to be a poor prognostic factor in adenocarcinoma of the lung in several studies. Results in squamous carcinoma are discordant, some studies reporting overexpression in 35% of cases in comparison with normal lung and others no expression. Studies in adenocarcinoma have consistently reported overexpression in 30-35% of cases (86, 87). Weiner et al. (88) studied the expression of p185 c-erb B-2 by immunohistochemistry in NSCLC tumors and normal bronchial mucosa. Normal ciliated bronchial cells expressed p185 c-erb B-2 at low levels. Compared with the bronchial mucosa, p185 c-erb B-2 was overexpressed in 4 of 12 adenocarcinomas and in 3 of 5 squamous carcinomas. Kern et aL (89) reported thatp185 c-erb B-2 overexpression in patients with lung adenocarcinoma was associated with older age, more advanced stage, and a shortened survival. However, the effect on survival was independent of tumor stage. Tateishi et aL (90) studied the prognostic implications of the cooverexpression of EGFR and c-erb B-2 in NSCLC using 119 samples of human lung adenocarcinoma. Overexpression of both receptors was detected in 13% of cases and was associated with the presence of distant metastases when compared with tumors not expressing either EGFR or c-erb B-2. EGFR overexpression was not associated with a shorter survival, whereas c-erb B-2 overexpression had a significant negative impact on survival in patients who had tumors overexpressing EGFR. The same trend was observed in patients who had tumors without EGFR overexpression, but the difference did not reach statistical significance. Finally, Shi et aL (91) studied the expression of c-erb B-2 in 120 patients with lung cancer; c-erb 8-2 was expressed in 58.8% of NSCLC and not in SCLC. Thirty-three of 41 adenocarcinomas and 24 of 55 squamous carci-


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nomas overexpressed c-erb B-2 when compared with normal lung tissue; c-erb B-2 expression correlated with a more advanced stage. In other studies, overexpression of p185 c-erb B-2 was found to correlate with intrinsic resistance to different cytotoxic agents in a panel of 20 cell lines of NSCLC (27), and transfection of p185 c-erb B-2 was demonstrated to confer chemoresistance to H460 cells, which have a very low p185 c-erb B-2 expression (92). In contrast with these results suggesting a direct relationship between c-erb B-2 expression and chemosensitivity, other studies have demonstrated that a twelvefold acquired resistance to cisplatin in an ovarian carcinoma cell line was associated with a sevenfold downregulation of p185 c-erb B-2 and a slower cell proliferation (30). A similar phenomenon has been reported with a subvariant of cervical ME180 cells, whid'_ is threefold resistant to cisplatin and has a threefold lower expression of EGFR (29). Surprisingly, overexpression of c-erb B-2 has been associated with enhanced chemosensitivity to anthracyclines and taxanes in patients with breast cancer, despite the association of c-erb B-2 expression with poor prognosis (93).

c-erb B-2 Blockade with Monoclonal Antibodies Several murine monoclonal antibodies that bind to the extracellular domain of c-erb B-2 have been developed and characterized. Among them, antibody 4D5 has been humanized and is in clinical development for the treatment of breast cancer. No studies in NSCLC patients have been performed despite evidence that 25-30% of NSCLC tumors overexpress c-erb B-2 and that such overexpression is associated with a worse prognosis. Kern et al. (94) studied the effect of antibody 4D5 against a panel of human NSCLC cell lines and found that it inhibited the growth only of the p185 c-erb B-2--expressing cell lines in a dose-dependent manner. Antibody 4D5 had a weak agonistic effect on the tyrosine kinase function of p185 c-erb B-2, as shown by an increased p185 c-erb B-2 phosphorylation as a result of exposure to the antibody. Inhibition of 185 c-erb B-2 tyrosine kinase by genistein abrogated the growth-inhibitory effects of the antibody, thus suggesting that 4D5 inhibits the growth of p185 c-erb B-2-expressing NSCLC cell lines through an agonist effect on p185 c-erb B-2. Another anti-c-erb B-2 antibody, Tab 250, has also shown growth-inhibitory activity against p185 c-erb B-2-expressing cell lines and induction of c-erb B-2 autophosphorylation.

Synergism Between c-erb B-2 Blockade and Cytotoxic Agents As in the case of EGFR, marked synergism between cisplatin and the anti-c-erb B-2 agonistic antibodies 4D5 and Tab 250 has been described by different laboratories. However, in this case the synergism has not

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only been observed in vivo but also in vitro. Pietras et al. (70) studied the in vitro mechanism of synergism between the antibody 405 and cisplatin in breast and ovarian cancer cells that overexpress c-erb B-2. Treatment with cisplatin led to a marked, dose-dependent increase in unscheduled DNA synthesis that was reduced by combined treatment with 405. Therapy with 405 led to a 35-40% reduction in repair of cisplatin-ONA adducts after cisplatin exposure and, as a result, promoted drug-induced killing in target cells. Similar results were obtained by Arteaga et al. (71) with the antibody Tab 250 in SK Br-3 breast cancer cells. Tab 250 enhanced the cytotoxicity of cisplatin in this cell line. This effect was abrogated by the tyrosine kinase inhibitor tyrphostin 50864-2, thus suggesting that the synergism requires tyrosine kinase activation. The addition of Tab 250 did not alter the cellular uptake or efflux of cisplatin but increased cisplatin/ONA intrastrand adduct formation and delayed the rate of adduct decay. Taken together, these studies support a direct association between c-erb B-2 signal transduction and cisplatin-induced ONA repair. As in the case of EGFR, this synergism has not been observed in cell lines with acquired resistance to cisplatin, probably because these cell lines display downregulation of p185 c-erb B-2. Langton-Webster et al. (30) reported that a variant of SKOV-3 cells that is twelvefold resistant to cisplatin displays a sevenfold downregulation of p185 c-erb B-2. The growth-inhibitory effect of Tab 250 alone was similar in both cell lines, but synergism between cisplatin and Tab 250 was only observed in the parental cell line. These studies suggest that these combinations may be more effective in the treatment of tumors naturally resistant to cisplatin than in those that initially respond but eventually become refractory to the drug.

Clinical Studies with Anti-c-erb 8-2 Monoclonal Antibodies Phase I studies with humanized 405 showed lack of toxicity with good tumor localization. Two phase II clinical studies in patients with refractory breast cancer overexpressing c-erb B-2 have been reported: one study used 405 alone (95) and the other 405 at the same dose in combination with cisplatin (96). In both studies, 405 was given weekly (100 mg/m2) and cisplatin (75 mg/m2) on day 1 of each 4-week cycle in the second study. No toxicity from 405 was observed, and in the second study the toxicities observed were those expected for cisplatin. The response rates were 11.6 and 25%, respectively. The conclusions from the first study are that 405 alone has modest but significant antitumor activity in this poor prognosis population, demonstrating that receptor blockade can inhibit tumor growth in patients with cancer. The combination of 405 and cisplatin results in antitumor activity that is higher than that expected with cisplatin alone, as suggested by the preclinical studies.


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MDX-210 is a bispecific antibody recognizing c-erb B-2 and the Fc receptor of monocytes and macrophages. It targets in vitro the Fc receptor of immune effector cells to kill cells expressing c-erb B-2. It is currently being evaluated in clinical trials in patients with breast and ovarian carcinoma (97,98).

Specific Inhibitors of EGFR and c-erb 8-2 Tyrosine Kinase Because receptor tyrosine kinases participate in malignant cell proliferation, the development of small chemical entities with specific tyrosine kinase inhibitory activity is being extensively pursued (99). The compounds genistein, lavendustin A, ebstatin, and herbimycin A are natural and broad-spectrum tyrosine kinase inhibitors isolated from fungal extracts. They have served as a starting point in synthesizing analogs with a higher specificity. Several synthetic inhibitors with a high selectivity for the EGFR or c-erb B-2 tyrosine kinase have been identified and their biological activity reported. The tyrphostins RG13022 and RG-14620, and the 4,5-dianilinophthalimides have shown to inhibit selectively the growth of human malignant xenografts overexpressing EGFR. The quinazolines PD153035 and AG1478 have shown a high selectivity and potency in inhibiting EGFR tyrosine kinase. In particular, PD153035 is an extremely potent inhibitor of EGFR tyrosine kinase with a 5 pM inhibition constant, only inhibiting other purified tyrosine kinases at micromolar or higher concentrations (100). Specific inhibitors for c-erb B-2 tyrosine kinase have also been identified and include the tyrphostins AG825, AG1377, and AG879. Leading compounds are about to enter clinical trials. Potential advantages of these inhibitors compared with receptor blockade by monoclonal antibodies include the possibility of prolonged oral administration and enhanced tissue penetration because of their smaller size. Initial phase I clinical trials with two specific EGFR tyrosine kinase inhibitors are about to begin (CP 358774, Pfizer, and ZD 1839, Zeneca). Both compounds have acceptable oral bioavailability. No toxicity has been observed in dogs at doses that result in biologically active plasma concentrations. Both compounds have also shown remarkable in vivo antiproliferative activity in human xenografts with high EGFR expression (101, 102).

Targeting of Cytotoxic Therapy to EGFR or c-erb B-2-Expressing Tumors Anti-EGFR or c-erb B-2 monoclonal antibodies and EGF or TGF-a themselves have been used as carriers for the targeted delivery of cytotoxic

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agents, toxins, and radioisotopes to tumors. A general problem of this approach is toxicity to normal tissues that express these receptors. In the case of fusion proteins containing toxins, liver uptake is of particular concern, as these toxins are hepatotoxic. The good preclinical results obtained with human xenografts in nude mice can be misleading. Because these antibodies only recognize the human receptors, the observed antitumor activity in the xenografts cannot be evaluated in the context of the toxicities to be expected in humans as a result of uptake of the conjugate by normal tissues. The unexpected gastrointestinal toxicity observed with the conjugate BR96-Dox is an illustration of such limitation. With these caveats, several investigators have demonstrated that a certain degree of therapeutic selectivity can be accomplished by coupling drugs, toxins, or radioisotopes to anti-EGFR or anti-c-erb B-2 antibodies, as well as growth factors. Aboud-Pirak et al. (103) developed conjugates of doxorubicin and A108 and showed a markedly enhanced antitumor activity against KB xenografts in vivo when compared to free doxorubicin. Taetle et al. (104) reported a selective in vitro antitumor activity of an immunoconjugate of monoclonal antibody 528 and recombinant ricin A chain against cells with more than 5 x 104 EGFRs. Vollmar et al. (105) studied conjugates of 225 with ricin A chain and showed a selective cytotoxicity for EGFR-positive cells. More recently, singlechain antibody exotoxin A fusion proteins containing the heavy and light chains, with variable domain of 225, have been described and shown effective binding and growth-inhibitory activity in EGFRexpressing cells (106). Two of these constructs have reached clinical evaluation. Pai et al. (107) developed a TGF-a pseudomonas exotoxin fusion protein (TGF-aPE40) and demonstrated significant antitumor activity by the intrapleural route against two subcutaneous human xenografts. A phase I clinical study by intravesical administration has been conducted (108). DAB 389-EGF is an EGFR-specific fusion protein consisting of sequences of the diphtheria toxin and EGF (109). It is a potent, EGFR-specific cytotoxic agent that rapidly inhibits protein synthesis by a mechanism of action similar to that of diphtheria toxin itself. Preliminary results of a phase I clinical study have been recently reported (110). The maximum tolerated dose has not been reached, although significant renal and hepatic toxicity was observed. Bender et al. (111) reported that the antitumor activity of anti-EGFR monoclonal antibody 425 against human glioma xenografts is enhanced by conjugation with iodine-125 (1251). Epenetos et al. (112) reported a case of regression of a human glioma using the anti-EGFR monoclonal antibody-9A labeled with 131 1 and administered into the carotid artery, thus suggesting the potential use of radioconjugates by regional delivery to minimize systemic uptake. Other targeting strategies that are actively pursued are the use of immunoliposomes incorporating monoclonal antibodies or their frag-



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ments covalently coupled to phospholipids on the liposome surface. Park et al. (113) have recently reported such constructs with long-circulating liposomes (Stealth Sequus Pharmaceuticals Inc., Menlo Park, CA) coupled to the Fabs of 405 for the delivery of doxorubicin and shown an enhanced therapeutic index as a result of effective targeted drug delivery.

GROWTH-SIGNALING PATHWAYS AS A TARGET FOR THE TREATMENT OF SCLC A variety of neuropeptides, including bombesin/ gastrin-releasing peptide, neuromedin B, bradykinin, vasopressin, galanin, and gastrin, among others, are produced by the majority of SCLC cells and promote their clonal proliferation (114-116). Consequently, they have been proposed to act as autocrine/paracrine growth factors for these cells. The in vitro growth of many SCLC cell lines is stimulated by addition of these growth factors and inhibited by molecules that block their receptors. Different strategies to block these pathways have been developed, including the development of monoclonal antibodies to block the growth factors and the development of small neuropeptide antagonists to inhibit the receptor function. In addition to the neuroendocrine growth pathways, the insulin-like growth factor I and the transferrin-receptor pathways may also playa role in the growth of SCLC tumors (115, 116).

The GRP Pathway Physiology and Biochemistry Bombesin is a 14-amino acid neuropeptide originally isolated from the skin of frogs. Gastrin-releasing peptide (GRP) and neuromedin-B are the two mammalian homologues. Bombesin-like peptides (BLPs) can bind to specific cell-surface receptors. The bombesin/GRP receptor belongs to the G protein-coupled receptor family, has been cloned, and is localized in chromosome 18q21 (114-116). Binding ofbombesin/GRP to the receptors triggers an intricate series of intracellular steps, the end result being DNA synthesis and cellular proliferation. The initial biochemical step is phospholipase-C activation, which generates inositol 1,4,5-triphosphate and diacylglycerol and elevation of cytosolic calcium levels. About 70% of SCLC cell lines secrete bombesin in the culture medium and express high affinity receptors for BLPs. The addition of exogenous

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bombesin or GRP can stimulate their growth in serum-free conditions. These observations suggest that BLPs contribute to the proliferation of SCLC through an autocrine growth loop. In contrast with EGFR and c-erb B-2, the expression of BLPs and their receptors has not been reported to be of prognostic significance or to correlate with chemosensitivity in SCLC.

Blockade of Bombesin or GRP with Monoclonal Antibodies Blockade of bombesin itself rather than its receptor has been the main strategy explored. Several antibombesin monoclonal antibodies have been shown to effectively inhibit BLPs' related growth. Monoclonal antibody 2A11 binds to bombesin with high affinity and prevents its interaction with cellular receptors; it has also been shown to inhibit, both in vitro and in vivo in nude mice, the growth of SCLC cell lines that express bombesin receptors (115). In contrast with EGFR and c-erb B-2, no synergism with cytotoxic agents has been reported. A phase I study of 2A11 was performed in 14 patients with relapsed SCLC (117). No toxicities were observed at the highest dose administered, 250 mg/m2 three times per week for 4 weeks. The pharmacokinetic studies suggested that a dose of 200 mg is required to reduce GRP-receptor occupancy below 10% and to prevent GRP-mediated growth. A phase II study is being performed in the same patient population. Patients are treated at a dose of 250 mg/m2 three times per week for 4 weeks. Thirteen patients have been enrolled, and one partial response has been observed (115).

Small Neuropeptide Antagonists The design and synthesis of potent small-molecule inhibitors of bombesin and other neuroendocrine receptors is an active area of therapeutic research. A series of broad-spectrum peptide antagonists have been synthesized and found to be effective inhibitors of bombesin and other neuroendocrine growth factors in SCLC, both in vitro and in vivo, in xenografts (118, 119). Interestingly, all of these antagonists are derivatives of substance P, a polypeptide that is distinct from the BLPs. These broad-spectrum antagonists inhibit intracellular calcium mobilization stimulated by GRP, vasopressin, bradykinin, and galanin. One of these compounds is in a phase I clinical trial. Because many SCLCs probably depend for their growth on several of these mitogens, the broad-spectrum properties of these agents make them potentially more useful than monoclonal antibodies that target one single growth factor-receptor interaction. Somatostatin analogues have also been explored, and some


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of them have been found to be superior to bombesin/GRP antagonists (120, 121).

OTHER GROWTH FACTOR-RECEPTOR PATHWAYS

I nterleukin-4 Interleukin-4 (1L-4) was originally described as a B-cell growth factor. IL-4 may either suppress or enhance the growth of hematopoietic progenitor cells depending on the lineage and differentiation of target cells. A preliminary study using the MR6 anti-IL-4 receptor antibody showed that IL-4 receptors were present on a number of carcinomas including one case of lung cancer. A more recent study found expression of the IL-4 receptor in 10 of 29 squamous cell carcinomas and in 6 of 17 adenocarcinomas of the lung (122). None of the small cell carcinomas or carcinoid tumors stained. All but two of the IL-4 positive tumors also expressed EGFR. Positive staining was also seen in non-neoplastic bronchial epithelium and on lymphocytes and macrophages infiltrating the tumor stroma. Although the role of IL-4 receptors in lung cancer is unknown, the results of these studies suggest that they may have a role in differentiation or proliferation of squamous and adenocarcinomas and, therefore, they could be used as a therapeutic target.

Insulin-Like Growth Factor Insulin-like growth factor-1 (IGF-1) is a basic peptide of 70 amino acids, and is secreted by most SCLC cell lines where high affinity cellsurface receptors have been demonstrated. A quantitative study of IGF-1 also demonstrated a more than threefold overexpression in ten primary squamous carcinomas and adenocarcinomas of the lung, compared with normal lung tissue (123). The IGF-1 receptor is formed by two a (130 kd) and two ~ (90 kd) subunits and has an intrinsic tyrosine kinase activity. Activation results in autophosphorylation of the ~ subunits. Exogenous IGF-1 is mitogenic in many malignant cell lines, including SCLC cell lines, and its effect is inhibited at least in vitro by blockade of the receptor by monoclonal antibodies (114, 115).

Transferrin Transferrin is a plasma protein that serves as the principal iron transport protein (116). Some SCLC cells secrete transferrin and have cell-mem-

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Chapter J 8 brane transferrin receptors (114, 115). Monoclonal antibodies against transferrin or its receptor inhibit the growth of some SCLC cell lines by depriving them of iron, which is essential for cell proliferation (114, 115, 117). Clinical studies with the antitransferrin receptor monoclonal antibody 42/ 6-IgA have been reported (1).

SUMMARY AND FUTURE PROSPECTS There is a great deal of evidence indicating that growth factor-receptor pathways play an important role in the dysregulated growth of neoplasia. Studies during the last few years have identified specific pathways that are relevant to the different types of lung cancer: the EGFR pathway in squamous cell carcinoma, the c-erb B-2 pathway in adenocarcinoma, and multiple neuroendocrine pathways in SCLC. However, prospective and definitive studies of the prognostic implications of the different growth factor-receptor pathways in lung cancer, using standardized methods of receptor and growth-factor expression, have not been performed but would be extremely helpful in identifying the subsets of tumors in which these pathways are biologically relevant and therefore rational targets for specific therapy. Different strategies to inhibit cell growth by blocking or altering these pathways have been developed, including growth factor-receptor blockade by monoclonal antibodies or small synthetic molecules and peptides, and the use of targeting molecules as carriers of cytotoxic agents. Impressive preclinical activity has been observed with some of these agents, thus leading to their introduction and investigation in clinical trials. Although the ongoing clinical trials are undeniable testimony of the rapid progress that has occurred in this area of translational research during the last few years, validating this therapeutic approach in the clinical arena may not be a straightforward task. There is great redundancy in cellular signaling pathways and wide heterogeneity, even among tumors of the same histologic subtype, on the signaling pathway or pathways that play a predominant role in sustaining their uncontrolled growth. Blocking one signaling pathway may not be effective therapy for those tumors whose growth depends on more than one pathway or that can easily switch to alternative pathways as a result of external selection pressure. Combination blockade therapy may be the answer in these cases. However, overexpression of certain receptors suggests the possibility that in some cases the malignant cells may have become selectively dependent on that receptor's signaling, indicating the possibility of selectivity with single agents. Consequently, progress in our ability to identify, prior to therapy, which signaling pathways are


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relevant to each individual tumor will be essential to expedite the clinical development of these agents. Successful targeted therapy for solid tumors requires either 1) an agent or combination of agents that exclusively exert their single or combined effect on a tumor-specific biochemical target (antigen, receptor, enzyme, mutated protein, etc.), or 2) an agent that, although not exerting its effect on a tumor-specific biochemical target, has the ability to exclusively localize and homogeneously reach the tumor tissue. Monoclonal antibodies bind with high specificity to cell-surface receptors but have poor tumor penetration because of their high molecular weight and no preferential tropism for tumor sites. None of the growth factor-signaling pathways discussed in this chapter is tumor-specific, except for the truncated EGFR. However, all are overexpressed by severalfold in a significant proportion of lung cancers and other malignancies. Consequently, conjugates of cytotoxic agents with antireceptor monoclonal antibodies or growth factors only partially meet the criteria outlined above for successful targeted therapy, and their potential toxicity to normal tissues remains a logical concern. In contrast, there is significant evidence that anti-EGFR or c-erb B-2 monoclonal antibodies appear to synergize with cytotoxic agents preferentially or exclusively in tumor tissue (targeted synergism or chemosensitization) and, therefore, such combinations may constitute a much improved and most promising example of effective tumor targeting. An additional advantage of this targeted chemosensitization strategy is that it allows for dose escalation of the nontoxic antibody until the desired tumor level and homogeneity of tumor uptake are obtained, which is not the case with antibody-drug conjugates. Phase I combination studies of C225 and cisplatin, taxol, or doxorubicin are in progress in head and neck and NSCLC, breast, and prostate cancer, respectively. No potentiation of the expected toxicities of these agents has been observed so far, which is encouraging and strongly justifies pursuing the investigation of these combinations in phase II studies. The mechanisms of sensitization probably vary among tumors, depending on the integrity of the cell-cycle checkpoint controls and their dependence on the growth factor-receptor pathways. Their investigation remains a very important task to maximize the chances of success of these combinations in the clinical arena by guiding clinical trial design. The use of small synthetic organic molecules or pep tides to specifically block the enzymatic function of growth-factor receptors represents an alternative and very promising strategy. If the therapeutic goal is a prolonged cytostatic effect as a result of continuous blockade of the signaling pathway, these compounds have the advantage over monoclonal antibodies of potentially being amenable to oral chronic administration. However, in contrast with monoclonal antibodies, a high specificity in inhibiting enzymatic functions involved in signaling path-

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way has been demonstrated in vitro but not in vivo, and a high specificity is crucial to minimize the risk of toxicity to normal tissues. Given the great deal of effort by different academic and industrial groups in this area, it is reasonable to expect several of these compounds to reach clinical evaluation in the near future.

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19 Immunologic and Biologic Approaches to Lung Cancer Therapy David P. Carbone

Despite recent advances in treatment, approximately 90% of patients diagnosed with lung cancer eventually die of their disease. This is by far the worst overall cure rate of the common solid tumors in the United States (1) and contributes to the fact that, while it is not the most common type of cancer, lung cancer causes the largest number of cancer deaths in both men and women in the United States. In this clinical situation, the observation more than 25 years ago that postoperative empyema resulted in improved survival (2) provoked a decade of enthusiasm for effective adjuvant immunotherapy for this disease. Unfortunately, the tools available at that time were crude, and the studies were not convincingly positive. Interest ebbed in the mid- to late 1980s, and it was generally felt that immunotherapy had no future in the therapy of lung cancer. However, in just the same period, dramatic new discoveries were made about the mechanisms of antigen processing and presentation, the structure of class I major histocompatibility complex (MHC), and the cytokine networks involved in immune regulation. As a result of this research, the mechanisms of immune recognition are much better understood today, and the tools available for modulation of the immune response are dramatically more sophisticated. This has led to a renewed interest in the potential for the immunotherapy of solid tumors in general. It remains to be seen whether perceived sophistication translates to clinical utility, however, but the potential is real. In 343

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this chapter, the historical approaches to the immunotherapy of lung cancer are briefly reviewed, the molecular pathways now known to operate in the induction of iInmunity are outlined, and some of the current therapeutic approaches that may ultimately show clinical utility are described.

BeG AND BACTERIAL PRODUCTSIINONSPECIFIC" IMMUNOSTIMULANTS In the early 1970s, when the observation was made that empyema improved the outcome of resected lung cancer patients, little was known of the cytokine and cellular responses to inflammation. Therefore, in an attempt to reproduce the observed beneficial effects of empyema on lung cancer outcome without the dangerous side effects of virulent bacterial infection, a number of studies utilized either the vaccine strain of mycobacteria (bacille Calmette-Guerin, or BCG) or various bacterial cell products such as Nocardia rubra cell wall skeleton. McKneally et al. (3) conducted a randomized trial of intrapleural BCG versus no treatment in surgically resected stage I and II non-smaIl-cell lung cancer (NSCLC). This study showed a small but statistically significant prolongation of remission and survival for stage I patients only. A trial was conducted to confirm these findings by the Lung Cancer Study Group (4). In this study, 141 patients with resected stage II and III adenocarcinoma and large-cell carcinoma were randomized to receive either chemotherapy or intrapleural BCG. No untreated control arm was included. The chemotherapy arm was found to be statistically significant and superior to the BCG arm, and the BCG arm was not significantly different from historical controls. Interestingly, stage I patients, the only positive subgroup in the earlier trial, were not included in this trial, nor were squamous cell carcinomas. Also interesting is the fact that the positive finding of the trial (the benefit of adjuvant chemotherapy for resected NSCLC) is not generally accepted as true today. However, this trial is taken as proof of the lack of efficacy of BCG and, by many, as evidence that immunotherapy will not work for lung cancer. For small-cell lung cancer (SCLC), a randomized study was performed with the Southwest Oncology Group (SWaG) randomizing between two different chemotherapy regimens and either BCG (during chemotherapy) or no additional treatment (5). Of the 114 patients who survived more than 1 year after registration, there was a statistically significant improvement in survival in patients who received BCG, with a 35% versus 5% survival after an additional 2 years. The authors concluded that the magnitude of this benefit was not sufficient to warrant further investigation. In addition, immunotherapy during full-

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dose chemotherapy may not have been an optimal study design. However, these data may support the hypothesis that there is some actual benefit, though small, to adjuvant BCG in lung cancer and that more focused immune manipulation might result in improved therapeutic efficacy. Other studies have evaluated a bacterial cell wall component from Nocardia rubra (Nocardia cell wall skeleton, or N-CWS) administered intrapleurally or intradermally. In a randomized study of 87 patients with completely resected NSCLC treated with adjuvant chemotherapy or chemotherapy followed by N-CWS therapy, a significantly prolonged survival was noted in the immunotherapy group (6). This observation has not been seriously pursued. Levamisole was also studied as a supposed general immunostimulator. A randomized trial of only 74 patients was performed to evaluate the addition of levamisole to postoperative radiation in stage II and III NSCLC with positive nodes, and no advantage was observed for the addition of levamisole (7). This trial was of a very low statistical power, too low to detect a modest clinical benefit, and again excluded stage I patients.

ACTIVE IISPECIFIC" IMMUNOTHERAPY FOR LUNG CANCER The term specific immunotherapy applies to the use of tumor cells (either autologous or allogeneic) or purified tumor antigens in an attempt to boost antigen-specific responses to shared antigens (common between individual tumors) or unique tumor antigens present in each patient's tumor. Takita et al. (8) conducted a randomized trial of 86 resectable NSCLC patients who received either no postoperative treatment or two arms of treatment with different doses of tumor antigen in adjuvant. A significantly improved survival was observed in the subset of NO patients in the low-dose antigen group. Analysis of data from another three trials involving 234 stage I and II resected NSCLC patients found a 5-year survival benefit (P = 0.0002) in patients treated with specific tumor antigen extracts (9). No follow-up study has been reported. Souter et al. (10) have reported a negative randomized trial in which 95 patients were treated after resection for NSCLC with either a single intradermal injection of autologous irradiated tumor cells combined with Corynebacterium parvum 2 weeks after surgery or no additional treatment. No statistically significant differences were observed between the two arms. However, previous positive trials involved multiple injections continuing for several months after surgery, and recent data indicate that repeated antigen exposure is important for effective therapeutic immunity induction (11).

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TUMOR-INFILTRATING LYMPHOCYTES IN LUNG CANCER THERAPY Tumor-infiltrating lymphocytes, or TILs, are presumably enriched for those effector cells with tumor specificity (12). These cells are harvested from a tumor biopsy and expanded in vitro in the presence of cytokines and in the absence of potential tumor-derived immunosuppressive factors. Approximately 10 10 to 1011 of these cells are then reinfused into the patient from whom they were derived. Major efforts to test the efficacy of TIL have been made in the treatment of renal cell carcinoma and melanoma (with occasional clinical success), but little attention has been paid to lung cancer until recently. Tan et al. (13) harvested TIL from ten lung cancer patients with malignant pleural effusions, introduced the interleukin-2 (IL-2) gene into them with a recombinant retrovirus, and expanded them in vitro. The patients were then treated with 1 to 6 10 x 10 TIL intrapleurally. No reaccumulation was observed for at least 4 weeks in six of ten patients, and one patient had a lasting resolution of the effusion and an objective response of the primary tumor mass. Even more intriguing is a randomized study conducted by Ratto et al. in Italy (14). In this study, 113 patients with resected stage II, IlIa, and IIIb NSCLC were randomized to receive autologous TIL intravenously 6 to 8 weeks after surgery, with subcutaneous IL-2 for 3 months or control therapy. Stage II patients received either immunotherapy or no treatment, and stage III patients were randomized to receive immunotherapy plus radiation or chemotherapy plus radiation. This study demonstrated a statistically significant improvement in survival for the group as a whole, but, interestingly, the largest benefit was for immunotherapy plus radiation therapy in patients with stage IIIb disease. In this study, only T4 IIIb patients were eligible, and not patients with N3 disease. Also, perhaps unexpectedly, among these patients the benefit was almost entirely from reduction in local relapse rather than reduced development of metastatic disease. In sum, these trials suggest that there may be some benefit to adjuvant immunotherapy in lung cancer, but definitive trials are lacking. With the high incidence and mortality of lung cancer, even relatively small percentage differences can mean benefits to large numbers of people. The efficacy of adjuvant treatment of colon cancer was established in a randomized study of almost 1000 patients (15), and adjuvant treatment of breast cancer was studied in even larger numbers of patients, with relatively modest magnitudes of effects leading to widespread adoption in clinical practice. A major problem with historical immunotherapeutic approaches has been the lack of a clear and practical path to follow. Many of these treatments were laborious, specialized, difficult to standardize, and difficult to assess. These issues are being overcome in some of the newer approaches to immunotherapy.


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NEWER ApPROACHES In order to understand the next generation of studies, a little background information is needed. The immune cells responsible for tumor cell killing appear to be primarily MHC-restricted cytotoxic T lymphocytes (CTL). Such T cells detect target cells for killing by recognizing short peptide fragments of endogenous proteins, which are presented to them by class I MHC molecules on the surface of the target cell. Naturally presented peptides are found to be 8 or 9 residues long and are generated by endogenous antigen processing and transmembrane transport of both intracellular and membrane proteins (10-18). There are now specific examples in both patients and rodent models of tumor cell immunity generated by MHC-restricted CTLs detecting endogenous cytoplasmic peptide antigens (19-26). Thus, it is important to recognize that effective target antigens do not have to be found intact on the cell surface, but rather cytoplasmic and nuclear proteins can be very effective targets for CTL-mediated lysis. Also important to understand is that effective immunity induction requires cooperation between a number of cell types including antigen presenting cells, T helper cell subsets, and CTLs, mediated in large part by soluble factors called cytokines. It is also critical to understand that clinically evident tumors have clearly avoided an effective immune response. We now know a great deal about some of the potential mechanisms of immune escape. Among the identified or hypothesized explanations for lack of an observed response are: 1. Tumor-specific antigens do not exist or are structurally unable to induce an effective T-cell immune response (not within the available genetic repertoire or unable to bind to MHC). 2. Defects of antigen processing in the tumor cells, involving defects in the transport of peptides to the cell surface (e.g., defects in transporter associated with antigen processing (TAP) peptide transporter protein [27] or the \32 microglobulin light chain protein [28]) or loss of expression of the appropriate MHC molecule (29). 3. Inadequate "help" (cytokine environment or costimulatory interactions). 4. Tumor production of immune-suppressive factors (transforming growth factor-beta [TGF-\3], insulin-like growth factor-1 [IGF-1], IL-10). 5. A relative increase in the T helper-2 (Th2) cell subpopulation among CD4+ T cells. IL-10 secreted from the Th2 cell subpopulation is known to suppress the production of IL-2, interferon-gamma (IFN-y), and tumor necrosis factor-alpha (TNF-a) from the Th1 subpopulation, important for the development of antitumor cytotoxic T cells.

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6. Defects in signal transduction by the T-cell receptor (TCR) in T cells from cancer patients, resulting in an inability to respond to appropriate signals. If item 1 or 2 above is the case, no therapeutic maneuver is likely to result in an effective immune response. The newer approaches outlined in the next section, however, attempt to correct the other listed deficiencies of immune induction through modulation of the cytokine environment of the tumor cells or efficient presentation of an identified tumor antigen. Some of these approaches utilize gene therapy, and these will be discussed first.

GENE THERAPEUTIC ApPROACHES TO THE INDUCTION OF SPECIFIC IMMUNITY Gene therapy is the treatment of cancer using genetic material as a therapeutic agent. Gene therapeutic approaches aimed at gene replacement and those targeting growth-factor receptors in lung cancer are discussed in Chapter 18, but genetic therapies can also be designed to induce effective anticancer immunity. Gene therapy techniques can be used to deliver biologically active genes intended to alter the local immunologic microenvironment and increase the immunogenicity of cancer cells and the responsiveness of T cells, improve antigen presentation, or to provide missing paracrine factors. Because the induction of immunity is inherently systemic, and the targets are naturally arising tumor antigens, the resulting immune response has the potential to detect and kill non-gene-modified tumor cells. This characteristic has the greatest potential for improving outcome in lung cancer where distant metastases are a major clinical problem. A variety of genetic approaches have been demonstrated in model systems to assist in the induction of immunity. Allogeneic and syngeneic MHC class I and II genes, costimulatory molecule genes (e.g., B7), and cytokine genes have all been inserted into tumor cells to alter the immunologic environment and to overcome the defective induction of immunity in tumor-bearing hosts. These will be briefly reviewed.

MHC Molecules The MHC is a region of highly polymorphic genes whose products are expressed on the surface of most cells. MHC class I molecules bind endogenously synthesized peptide fragments and present them on cell surface for recognition by the TCR on CD8+ T cells. MHC class II molecules are primarily expressed on "professional antigen-presenting cells"


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such as macrophages and dendritic cells and are thought to be primarily responsible for binding peptide fragments derived from extracellular proteins and presenting them to T helper cells. This interaction ind uces the prod uction of cytokines necessary for the expansion of cytotoxic effectors. MHC molecules therefore playa key role in all phases of the immune response. Tumor cells usually have some level of expression of MHC class I molecules on their surface, but in many tumor cells, expression of MHC is low. Attempts to increase the immunogenicity of tumor cells by insertion of foreign (viral or allogeneic) genes was one of the first gene therapeutic strategies. Among foreign genes, allogeneic MHC molecules are a classic example. Allogeneic MHC class I genes introduced into tumor cells will induce allo-MHC-specific T cells. These allo-MHC-specific T cells presumably then assist in the generation of T cells specific for previously silent tumor-specific antigens. In a murine lung cancer system (3LL/3) from C57BL/6 mice (MHC type H-2 b), transfection of the allogeneic MHC molecule H-2L d caused a reduction in tumorigenicity and protection against unmodified 3LL/3 (30). Plautz et al. (31) showed that expression of a murine class I H-2Ks gene in CT26 mouse colon adenocarcinoma (H-2Kd) or MCA-106 fibrosarcoma (H-2Kd) induced a cytotoxic T-cell response to H-2Kd and, more importantly, to other antigens present on unmodified tumor cells which had not been recognized previously. Recently, allogeneic MHC transfection has been applied to humans with human leukocyte antigen-B7 (HLA-B7) gene transfer. Nabel et al. (32) reported the reduction of tumor size in a melanoma patient after the direct gene transfer of an HLA-B7 gene in a liposome complex. Clinical protocols of HLA-B7 gene transfer by lipofection in advanced cancers, including melanoma, renal cell cancer, and colon cancer, are under way. Therapeutic transfection of syngeneic MHC class I and II molecules has been attempted in several different tumor model systems. Transfection of syngeneic MHC class II (Ak) in sarcoma cells induced an immune response against both transfected tumor cells and nontransfected tumor cells (33). This finding suggests that transfected MHC class II molecules can induce T helper cells specific to nontransfected tumor cells as well. Low MHC class I molecule expression in some tumors may lead to decreased presentation of tumor-specific antigens on the cell surface and, as stated above, this could be one mechanism for their escape from immune surveillance. Transfection of syngeneic MHC class I molecules could induce or increase the presentation of endogenous peptide fragment derived from tumor-specific antigens, leading to the generation of CD8+ CTLs specific for parental tumor antigens. Once induced, this response can cause recognition and lysis of non-gene-modified cells with lower levels of MHC expression. Increased MHC class I expression on tumor cells may thus result in enhanced immunogenicity and decreased tumorigenicity of tumor cells. Plaskin et al. (34) showed that

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high expression of a transfected H-2Kb MHC gene in highly metastatic Lewis lung carcinoma cell (3LL) resulted in the conversion of a highmetastatic to a nonmetastatic or low metastatic phenotype and protection against the metastatic spread of 3LL.

Accessory Molecules (87)' Effective antitumor immunity is usually dependent on T-cell-mediated responses. Two kinds of signals are required for the activation of T cells. As described previously, the first signal is the antigen-specific binding of a peptide antigen-MHC complex on the surface of antigen-presenting cells (APCs) with antigen-specific T-cell receptors. This interaction is necessary but not sufficient to induce primary T-cell activation and production of essential cytokines such as IL-2. It is hypothesized that the presence of this first signal alone may induce a state of readiness to respond to the second signal known as costimulation. The second signal is transmitted by the antigen-independent binding of costimulatory molecules on APCs with their corresponding receptors on T cells. This signal is required to induce primary T-cell proliferation and other effector functions of the immune response. Without this second signal, the binding of antigen with TCR alone may cause prolonged unresponsiveness or even specific T-cell anergy. A number of molecules have been found to mediate this second signal, including the B7 family (unrelated to the HLA-B7 class I molecule described above), which interact with the CD28 receptor on T cells (35, 36). B7 is expressed on activated B cells, macrophages and dendritic cells, and is the ligand for CD28 and cytolytic T lymphocyte antigen (CTLA-4) on T cells (37, 38). Tumor cells may have tumor-specific antigens and MHC class I expression but only rarely express B7. The rationale for transfection of B7 into tumor cells is to provide or supplement the necessary costimulation for effective immune induction. B7-transfected tumor cells can directly stimulate CD8+ CTLs without the help of APC and CD4+ T helper cells. Townsend and Alison (39) demonstrated that B7 transfection into MHC class 1- and II-positive melanoma induced the rejection of tumors in vivo. This rejection was mediated by CD8+ T cells and did not require CD4+ T cells. Chen et al. (40) showed that B7 transfection in human papilloma virus (HPV)-16 E7 transfected tumors resulted in the loss of tumorigenicity and regression of E7+B7- tumors by the immune response mediated by CD8+ CTL. The costimulatory effect of B7 in antitumor immunity is dependent on tumor immunogenicity, as B7 transduction by a retroviral vector decreased the tumorigenicity and induced protective immunity only against immunogeneic tumors (41). B7 transduction via recombinant adenovirus into murine tumors expressing mutant p53 resulted in the induction of mutant p53-specific CTLs and loss of tumorigenicity as well as protective immunity against challenge by untransduced tumor (42). Similar induction of immunity was found


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for the PIA epitope in the P815 (murine plasmacytoma) tumor model. Another recent study demonstrated that B7 and CD28 interaction provided costimulatory signals not only for T cells but also for natural killer (NK) cells (43). Tumor-cell resistance to NK cells is mediated by expression of MHC class I molecules but the B7:CD28 interaction induced by B7 transfection can overcome the MHC class I-mediated inactivation of NK cells, increasing their tumoricidal activity. In contrast to defined antigen vaccine approaches, another important aspect of B7 gene therapy is the potential ability to increase the immunogenicity of all tumor-specific antigens, whether or not they have been identified. All of these studies provide the experimental groundwork for attempts to utilize B7 in cancer gene therapeutic approaches.

Cytokine Gene Therapy Rosenberg (44) was the first to report that systemic administration of IL-2 with or without in vitro expanded autologous lymphokine-activated killer cells (LAK) to advanced cancer patients was associated with some significant responses. The toxicities of systemic cytokine administration interfered with the popularization of this approach. To avoid systemic side effects and to better approximate normal physiologic conditions, gene therapy has been used to insert cytokine genes into tumor cells and to induce the production of cytokines in the vicinity of tumor. Local production of cytokines from tumors can modify the tumor's interactions with the host immune system. These altered immunologic microenvironments may provide favorable conditions for the host immune detection of tumor cells and previously unrecognized tumorspecific antigens. Effective recognition of these antigens and induction of a response can therefore allow detection and killing of non-gene-modified tumor cells with the same tumor-specific antigen, even though the tumor cells may be located far from the site of gene modification. Many cytokines have been tested for efficacy in animal models of cytokine gene therapy. IL-2, IL-4, IL-6, IL-7, IL-12, IFN-y, TNF-a, and granulocyte/macrophage colony-stimulating factor (GM-CSF), among others, have been investigated. IL-2 in particular has been investigated extensively. Fearon et al. (45) demonstrated that IL-2 eDNA transfected into murine colon carcinoma (CT26) resulted in the loss of tumorigenicity and bypassed deficient T-helper function in the generation of an antitumor response. Ley et al. (46) showed that IL-2-transfected murine mastocytoma (P815) cells induced P815-specific CTL, which led to regression of established tumors. Using a highly malignant and poorly immunogeneic Lewis lung carcinoma, IL-2 production by retrovirally transduced tumor cells induced antitumor CTL and eliminated the generation of lung metastasis (47). In natural immune responses, IL-2 is produced by CD4+ T cells activated by the binding of TCR and antigens presented by MHC class II molecules on APC. Secreted IL-2 will

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activate CD8+ T-cell precursors to cytotoxic T cells. IL-2 secretion by gene-modified tumor cells may activate CD8+ T cells directly, bypassing some of the T-helper cell requirement. IL-2 can also overcome defects in the signal transduction of T cells observed in tumor-bearing patients caused by the decreased levels of p56 lck and p59fyn (48). Clinical trials of IL-2 cytokine gene therapy in lung cancer are planned at several sites. IL-4 also shows antitumor effects in animal tumor models. IL-4 is produced by the Th2 subset of helper T cells and mast cells, and has many functions. It can induce LAK cells, stimulate B cell proliferation and maturation, and activate endothelial cells to express vascular cell adhesion molecules (49). Tepper et al. (50) demonstrated that IL-4 production from transfected tumor cells had antitumor effects in various tumor cell lines. This effect is blocked by anti-IL-4 antibody, related to the level of the production, and is evident in nude mice, implying that the effect is independent of T-cell responses. Infiltration of the transduced tumor site with macrophages and eosinophils suggested that inflammatory mechanisms were involved. IL-6 is the pleiotropic cytokine that can stimulate the differentiated functions of B cells and T cells and induce the production of mature myeloid cells and megakaryocytes (51). IL-6 has antitumor effects on murine models of lung cancer. Immunization with inactivated IL-6transfected Lewis lung carcinoma cells induced antitumor CTL and reduced the metastatic potential of these cells (52). IL-7 is a bone marrow stromal cell-derived cytokine that stimulates pre-B cell expansion. IL-7 also functions as a growth factor for thymocytes and CD4+ and CD8+ T cells. IL-7 can enhance antigen presentation indirectly by increasing expression of costimulatory adhesion molecules such as intercellular adhesion molecule (ICAM-1) and inhibit the production of immunosuppressive cytokines such as TGF-f3 (53). The production of IL-7 by retroviral transduction in fibrosarcomas can decrease tumorigenicity and induce protective immunity against unmodified tumor challenge, and IL-7 transduction of immunogenic tumors can cause the regression of established lung metastasis (54). A clinical trial of cytokine gene therapy with IL-7 is planned in lung cancer, but no preliminary results are available. IFN-y produced by activated T cells can modulate the immune response in a number of ways. IFN-y can induce MHC class I and II molecules, which will increase the presentation of antigen on the cell surface, and can induce the activation of macrophages (55). Low expression of MHC molecules on tumor cells is one postulated mechanism by which tumors escape from host immune surveillance. Increased expression of MHC molecules by IFN-y should increase the presentation of tumor-specific antigens and assist the induction of antigen-specific CTL. Transduction of a weakly immunogenic tumor (CMS-5) by retrovirus-IFN-y induced the abrogation of tumorigenicity and persistent and specific antitumor immunity against the unmodified CMS-5 challenge (56). The effect of IFN-y has also been demonstrated in the 3LL


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mouse lung cancer model. Retroviral IFN-y gene insertion into poorly immunogenic 3LL-D122 showed a significant decrease in tumorigenicity and metastatic potential, and induced tumor-specific CTLs when modified tumor cells were injected after irradiation (57). TNF has direct cytolytic effects on tumor cells, and attracts and augments the tumoricidal activity of macrophages (58). Systemic administration of TNF, however, was too toxic to achieve significant in vivo antitumor effects. Local production ofTNF-a by retroviral transduction into the TNF insensitive tumor cell line (J558L) drastically suppressed tumorigenicity in a syngeneic animal, even though it did not appreciably affect growth in vitro (59). Administration of an anti-type-3 complement-factor receptor to block the migration of inflammatory cells abolished the antitumor effects of TNF-a, which suggested the involvement of an inflammatory mechanism including the activation of macrophages (59). The antitumor effect of TNF-a was also proven in human lung cancer cell lines, even though most human tumor cells are relatively resistant to TNF-a. Insertion of human TNF-a cDNA into several human lung cancer cells resulted in decreased tumorigenicity in nude mice (60). Furthermore, injection of a mixture of 50% gene-modified and 50% parental cells also showed decreased tumor formation. These data suggest that local production of TNF-a can induce antitumor effects on human lung cancer cell growth, and that every tumor cell need not be gene-modified to produce local antitumor effects. GM-CSF appears to be one of the most active cytokines in the induction of antitumor immunity. In a comparison of the efficacy of a number of cytokines using retroviral vectors, GM-CSF demonstrated the most potent, specific, and long-lasting antitumor immunity (61). The antitumor immunity induction after gene therapy with GM-CSF was dependent on both CD4+ and CD8+ T cells. This activity may be related to GM-CSP's ability to promote the differentiation of hematopoietic precursors to dendritic cells and other professional antigen-presenting cells (62). Because culture and stable transduction of human tumors is problematic, "paracrine" GM-CSF release from gelatin-chondroitin microspheres mixed with irradiated tumor cells was tested and found to evoke antitumor immunity comparable to a GM-CSF-transduced tumor vaccine (63). We have designed and produced an adenovirusGM-CSF vector that also overcomes many of the limitations of in vitro culture of primary human tumors. Transduction of 3LL with this adenovirus-GM-CSF vector eliminated its tumorigenicity, induced tumorspecific CTLs, and the cure of established Lewis lung carcinoma tumors (64). Furthermore, we showed that this was associated with an increased number of dendritic cells in the tumor vaccine-injection site. IL-12 is a new cytokine with dramatic antitumor effects and relatively modest systemic toxicity in animal models. IL-12 has the ability to promote the differentiation of uncommitted T cells to Th1 cells, thought to be key to the production of effective antitumor immunity (65). IL-12 also activates the CD8+ T cells and NK cells and generates LAK cells (66).

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Tahara and Lotze (67) demonstrated that IL-12-retroviral transduction of murine sarcomas showed suppression of tumorigenicity, induction of protective antitumor immunity, and suppression of preinjected nontransduced tumors. The efficacy of IL-12 was confirmed when delivered via recombinant vaccinia virus (68). It also improves the efficacy of peptide immunization (69, 70). All of the data from cytokine gene-modified animal systems implies that locally released cytokines from tumor cells can alter the local immunologic microenvironment to help the host immune system detect previously unrecognized tumor-specific antigens and to induce the ability to kill the tumor cells. A number of human clinical trials utilizing cytokine gene-modified cells are under way, and the results of these are expected in the near future (71). To improve and facilitate the clinical application of cytokine gene therapy, several modifications of the published animal model tumor systems are being evaluated. Most animal studies, and some human trials, have used in vitro cultured autologous tumor cells as targets for gene transfer. In practice, this approach has serious limitations in a clinical setting, in that generating an autologous tumor cell line from each patient's tumor is difficult, expensive, and time-consuming, and not possible in many circumstances. However, the key to cytokine gene therapy appears to be the production of appropriate cytokines in the vicinity of tumors, and not necessarily from tumor cells themselves. Several types of cells have been used for gene transfer including fibroblasts, TILs, and endothelial cells (72). Transduction of fibroblasts has many advantages over transduction of autologous cancer cells, as they can be obtained from skin biopsies, grow easily in culture for many passages, and can be efficiently transduced with viral vectors in vitro. Fakhrai et al. (73) showed that immunization with a mixture of irradiated tumor cells and IL-2-transduced fibroblasts induced protective antitumor immunity and remission of established tumors. A similar result was reported for IL-12 (70). Human studies using transduced autologous fibroblasts are under way at the University of Pittsburgh. Genetic modification of immune effectors to increase cytotoxicity has also been explored. As discussed earlier, TILs are lymphocytes, which have homed to the tumor, within a tumor mass. Rosenberg et al. (74, 75) have utilized TNF gene-transduced autologous TILs in advanced cancer patients and demonstrated enhanced antitumor effects with less toxicity than systemic administration. IL-2 gene-modified TILs have shown safety and perhaps efficacy in lung cancer (73). Transduction with chimeric TCR and antibody has the potential to improve targeting and killing by these effectors as well (75). Another potential approach to improving the efficacy of immunemediated gene therapy is to combine two different immunogenes to obtain a synergistic effect. The modification of tumor cells by MHC molecules and the B7 co stimulatory molecule, or by combinations of different cytokines with different mechanisms of action, are possible


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approaches. Combination of B7-1 transduced tumors and concurrent administration of recombinant IL-12 can induce antitumor immunity synergistically (76). Combined gene therapy with B7 and IL-12 also showed an antitumor effect on lung metastases of the 3LL mouse lung carcinoma model (77). In this study, 3LL cells, transfected with the B7 plasmid and transduced with retrovirus-IL-12, induced 3LL-specific CTL and significantly reduced the number of metastases compared to parental 3LL (77). Combination of syngeneic MHC class I transfection and IL-2 transduction in the mouse melanoma cells also showed synergistic effects on the eradication of established lung metastasis by the combined effects of efficient CTL induction and NK/LAK activity compared with single gene-modified tumor cells (78).

THERAPY WITH DEFINED TUMOR ANTIGENS Many potential tumor-specific antigens have been postulated in tumor cells including viral antigens, fetal genes, and mutated oncogene, or tumor-suppressor gene products (79). Intensive studies of tumorspecific antigens during the last decade resulted in the identification of numerous antigens associated with different types of cancer, including melanoma, cervical cancer, colon cancer, and lung cancer (reviewed in [80]). During carcinogenesis, it is clear that multiple genetic and protein structural changes occur. The inactivation of tumor-suppressor genes and activation of oncogenes through mutation and/or dysregulated expression are examples of these genetic alterations. These alterations represent differences between cancer cells and normal cells. Most cancer cells are thus likely to have protein structural features or patterns of protein expression that could allow immune detection and elimination. Several groups (including ours) have clearly demonstrated the induction of the antitumor immune responses after immunization of control animals with appropriate antigens (reviewed in [81]). Carcinoembryonic antigen (CEA) is an oncofetal protein expressed by many adenocarcinomas, including those of lung origin. Recently, cotransduction of recombinant vaccinia virus-B7 (rV-B7) and rV-CEA resulted in CEA-specific T-cell responses and antitumor immunity against a murine carcinoma expressing CEA. These findings suggested that B7 increased the immunogenicity of CEA (82). Human immunotherapy studies evaluating the efficacy of CEA as an immunotherapeutic target are in progress. Mutations in the p53 tumor-suppressor gene are common in lung cancer. Mutations in p53 represent discrete differences between every tumor cell and its normal counterparts and therefore represent an attractive immunotherapeutic target. p53 is also relatively unusual among the tumor-suppressor genes in that most mutations are missense

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(causing a change in the protein coding sequence, rather than deletions or stop mutations), and tumor cells usually retain p53 protein production. In many cases, the mutant p53 protein product is also overexpressed (83). There are a few "hotspots" for mutations in p53 (such as codons 175,248, and 273), but these make up only a small fraction of the mutations observed. This complicates mutation-specific immunotargeting, as each patient is likely to have a different mutation. Mutant-specific epitope targeting may, however, not be necessary, as several authors have demonstrated efficient recognition of wild-type p53 sequences in murine models (84) or human cells in vitro (85). This occurs despite the fact that every normal cell in normal individuals expresses a low level of structurally identical p53 protein. The typical massive overexpression of the mutant protein may allow sufficient tumor-normal discrimination to be of clinical utility. The major practical advantage of targeting a wild-type epitope is that a single vaccine preparation can be used to target a wide variety of tumors producing different mutated p53 proteins, and these sequences can be selected to match the peptide-binding preferences of large classes of patients with common MHC antigens. Animal studies have demonstrated the induction of effective CTL responses against the mutant p53 protein. Using peptides that span mutation sites in mutant p53 to immunize animals, it is possible to show the development of p53-specific CTL. Some of these CTLs recognized the mutant peptide sequence and not the corresponding wild-type one (86). It is remarkable that a CTL response, which is specific for the presence of a single amino acid substitution in the p53 protein, can be generated in such animals. This underscores the specificity achievable when a finely tuned biologic process, such as the cellular immune system, is evoked, as opposed to the lack of specificity observed for standard chemotherapeutic agents. This CTL response provided tumor protection of control mice against subsequent challenge, with tumor cells bearing specific mutation (86-89). A recent study in animals demonstrated the enhancement of the immunogenicity of a mutant p53 epitope after transduction of the costimulatory molecule B7 via recombinant adenovirus (42). This underscores the fact that appropriate antigens may be present and not recognized without therapeutic manipulation.

ANTI-p53 IMMUNE RESPONSES IN CANCER PATIENTS Immune responses in cancer patients against p53 can occur in the absence of specific vaccination. Anti-p53 antibodies have been detected in the serum of human lung cancer patients and correlate with the presence of missense mutations in p53 (90). These antibodies are not thought to playa major role in antitumor responses, because p53 is not


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found intact on the surface of cells and therefore is not accessible to antibody binding. Interestingly, the antibodies that these cancer patients develop recognize sequences in either the amino-or carboxy-terminal regions of p53, and not in the central DNA-binding region of the molecule where most of the somatic mutations occur (91). As a consequence of this, these antibodies recognize both mutant and wild-type p53 in vitro despite the fact that individuals might be expected to be "tolerant" of these epitopes. Because these antibodies are found much less commonly, if at all, in patients without cancer or in cancer patients whose tumors do not have missense mutations in p53, it is possible that antip53 antibodies are induced due to the resulting overproduction of the mutant protein or a conformational change associated with the mutation. Cellular immune responses specific for p53 have also been observed in humans. Induction of the humoral responses described previously requires antigen presentation on APCs and helper T cells, so detection of these antibodies implies a T-cell response to the same antigen. CD4+ T cells (those which produce cytokines and proliferate in response to antigen) that respond to p53 pep tides have been reported in breast cancer patients (92). All of the patients from which helper T-cell responses could be detected also had antibodies against p53, demonstrating the existence of a combined cellular and humoral response in these patients. CTLs that are specific for either wild-type or mutant p53 epitopes have also been isolated. Melief and Kast (85) showed in vitro induction of human CTL to both normal and mutant p53 epitopes. Very low levels of CTL precursors recognizing wild-type p53 epitopes have even been identified in the peripheral blood of individuals with no apparent cancer (93). It is unclear what the clinical significance of these responses is and whether they are in vitro primary responses (artifacts) or true recall responses. In breast cancer patients, significant CTL responses specific for the mutant p53 in a cancer patient's tumor have been observed (94). In most cases, these CTLs recognized mutant but not the corresponding wild-type p53 sequences. Therefore, p53 protein can behave as an antigenic target for CTL during the natural process of tumorigenesis without external immunization or other immunotherapy. The role of these CTLs in affecting the clinical course of the cancer is unclear, but the majority of patients with these responses in this study were apparently cured of their disease with surgery alone.

INDUCTION OF EPITOPE-SPECIFIC IMMUNITY IN LUNG CANCER For defined target antigens, such as CEA and p53, induction of epitopespecific immunity in animals has been typically accomplished using synthetic pep tides (95,96). The use of peptides as immunogens is com-

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plicated, however, by their weak inherent immunogenicity and variable chemical and physical properties, so a variety of strategies have been employed to enhance the efficacy of peptide-based vaccines (97-100). The chemical and physical problems of protein- or peptide-based vaccines can be avoided by the use of genetic vaccines, purified plasmid DNA expression vectors encoding the entire cloned open reading frame of proteins introduced into living animals. These DNA vaccine vectors may generate substantial humoral and cellular immunity with little or no toxicity (101-105). We have shown the induction of T-cell epitopespecific (mutant p53) cellular immune responses and antitumor effects after introduction of a "genetic epitope" vaccine, consisting of an expression cassette containing only an oligonucleotide coding for the desired epitope (88). In this study, we utilized a particle gun that atraumatically delivers microscopic gold particles coated with the plasmid DNA into the shaved skin of living animals. A plasmid vector containing the adenovirus E3 protein leader sequence fused in frame with a short sequence of an epitome from p53 induced p53-specific was constructed, which facilitates transport of the mutant p53 epitope CTL and tumor protective immunity when introduced by the particle gun into the skin of animals. The use of epitope-minigene genetic vaccines may thus have significant potential for the induction of responses against identified T-cell epitopes in tumors. When the p53 gene was expressed in a recombinant vaccinia virus vector and used to immunize animals, protection from lethal tumor challenge was observed (84). Interestingly, the wild-type p53 was just as effective as the mutant p53 in this system. Studies with animals bearing pre-existing tumors (a more clinically relevant situation) have not yet been reported using recombinant viral immunization. At a number of sites including Vanderbilt University and the National Cancer Institute, human clinical trials are under way that aim to induce CTL in cancer patients using a custom p53- or ras-derived peptide corresponding to the mutant p53 or ras sequence in the particular patient's tumor (NCI T93-0148). In this trial, DNA from each patient's tumor is evaluated for mutations in p53 and ras, and if a mutation is found that alters the predicted protein sequence, a customized synthetic peptide is constructed and used to immunize the patient. Immunologic responses have been already observed. Future studies will undoubtedly use dendritic cells as antigen carriers and might be able to use peptides derived from normal p53 protein sequences or the intact p53 protein. A cocktail of p53 peptides is also possible, analogous to multivalent pneumococcal vaccines, particularly if wild-type sequences were used. With rapidly improving technology, inexpensive and rapid genetic analysis of tumors is a real possibility, but a vaccine composed of a collection of mutant peptides may be impractical given the huge number of different mutant sequences observed in most naturally occurring tumors.


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IMMUNE DEFECTS IN CANCER PATIENTS Demonstration of antitumor responses induced by peptide immunization in animals bearing pre-existing tumors has been much more difficult than demonstrating tumor protection, presumably due to tumorassociated immune dysfunction, which will be discussed in the next section. However, when repeated immunizations with p53 peptidepulsed dendritic cells were used in animals bearing palpable tumors, clinically significant antitumor effects were observed (11). The combination of this approach with the cytokine IL-12 yields further improvement in the antitumor effect (69). In these studies, repeated immunizations were important, and this stresses the difference between the common prophylactic immunization for infectious diseases and antigen-specific immunotherapy. The prior presence of the tumor appears to inhibit the ability of the individual to maintain a response, and repeated antigen exposure is essential. The development of clinically evident tumors implies a failure of the immune system to detect and reject cancer cells as foreign. As mentioned above, it is unclear whether this failure is entirely due to a property of the cancer cell or whether there are tumor associated alterations of the host that contribute to this evasion. In the 1970s, several groups studied whether patients with cancer could be effectively immunized against common antigens. Filber and Morton (106) have shown that only 60% of patients with localized potentially resectable neoplasms developed delayed cutaneous hypersensitivity (DCH) after sensitization with 2,4-dinitrochlorbenzene. More than 95% of healthy individuals and 100% of patients with benign tumors developed DCH (106). The same observations were reported by several other groups (107, 108). Stiver and Weinerman (109) immunized patients with localized cancer with influenza vaccine, and found that almost 93% of healthy individuals but only 36% of cancer patients developed fourfold or greater increases in antibody titers. During the last decade, various defects in the function of T lymphocytes were demonstrated in patients with cancer and tumor-bearing animals (reviewed in [111]). However, much less is known about the function of APCs in cancer. Several studies, however, have recently described defective functioning of professional APCs in tumor-bearing hosts (111-113). This will be briefly reviewed, along with its therapeutic implications.

Dendritic Cells as Inducers of Cellular Immune Responses Induction of CTL responses requires effective cooperation between at least three elements of the immune system: APCs (114, 115), T-helper

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cells, and cytotoxic T cells. The APC provides effective presentation of antigen and delivery of costimulatory signals to CD4+ and CD8+ cells, whereas the activated CD4+ cells together with monocytes/macrophages have to provide the cytokine support for antigen-specific CTL clonal outgrowth. Thus, effective responses to tumor-specific antigens may be possible if both CD4+ and CD8+ lymphocytes are engaged. The most potent APCs known are dendritic cells (DCs). They have several distinct features making them ideal candidates as vehicles for presenting tumor-specific antigens for immunotherapy. DCs express on their surface a high level of both MHC class I and II molecules, as well as a variety of adhesion and costimulatory molecules (116). DCs are extremely effective in the stimulation of secondary immune responses (100 times more potent than B cells or macrophages). DCs are the only cells capable of stimulating primary immune responses, including cytotoxic ones (116). They have a high potential to acquire, process, and present soluble antigens on their surface. Murine spleen dendritic cells and their cutaneous counterpartLangerhans' cells (LCs)-have been reported to induce specific cytotoxic immune response against tumor antigens (117, 118). Tumor protection has been described to be induced when DCs or LCs were used for induction of immune responses (117). DCs were about 100 times more effective in the induction of anti-influenza virus or anti-HIV-specific CTL responses than unseparated spleen cells (119, 120). DCs thus appear to be the ideal vehicle for cellular immunization. All of these studies, however, have been performed with DCs from normal animals. We have recently demonstrated a clear defect in DC function in patients with advanced cancer (121), but DCs from these patients function normally after growth from precursors in vitro, free from the influence of tumor-derived factors. There are also data from other labs to suggest that DC defects are present in human cancer patients (122). We have gone on to demonstrate that tumor-derived vascular endothelial growth factor (VEGP), a factor produced by most tumors and known to be important for tumor angiogenesis, is an important factor in the induction of this DC defect (123). Therapeutic approaches aimed at blocking the action of this factor to enhance immune induction as well as to inhibit angiogenesis are under way. A multimodality approach that utilizes optimal standard therapeutic modalities, followed by anti-angiogenic and immunotherapeutic strategies, may well ultimately prove to be superior to the traditional modalities of chemotherapy, radiation, and surgery alone.

SUMMARY When viewed objectively, even the early immunotherapy trials may have provided some evidence that lung cancer might be subject to


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immunotherapeutic approaches. The dramatic progress in understanding of the genetic and molecular basis of oncogenesis and the induction of immunity has led to a rejuvenation of efforts to apply this new knowledge to this common and refractory disease. The techniques being applied today are as different from the early attempts at immunotherapy as the first attempts to use nitrogen mustard or aminopterin are to the chemotherapy of today, and so historical"failures" of the immunotherapy of lung cancer should not discourage the investigation of future scientifically based therapeutics. Hopefully, real and convincing clinical efficacy will be demonstrated once some of these strategies are tested in carefully performed, randomized clinical trials with appropriate power to detect meaningful differences.

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20 Genetic Manipulations for the Treatment of Lung Cancer Jack A. Roth

The strategy for inactivating or replacing cancer-causing genes is analogous to the classic concept of gene therapy for replacing defective or nonfunctioning genes. Previous studies have shown that regional administration of viral vectors expressing wild-type p53 prevents the growth of tumors with mutant p53 and mediates regression of large established tumors. These data provide the rationale for clinical protocols recently approved by the National Institutes of Health (NIH) Recombinant DNA Advisory Committee and the U.S. Food and Drug Administration (FDA) to replace a defective p53 gene with a normal p53, expressed by either a recombinant retrovirus or recombinant adenovirus injected intratumorally. If these methods are efficacious, their lack of toxicity may provide a sufficiently high therapeutic index to allow their use as adjuvants to surgery in patients with earlier stages of cancer or as agents to prevent second primary cancers in individuals with genetically abnormal premalignant lesions. Although much research needs to be done, the possibility of specific gene targeting with a high therapeutic index makes gene activation or replacement a promising area for investigation. This study was partially supported by grants from the National Cancer Institute (NCI) and the National Institutes of Health (ROl-CA45187) [J.A.R.]; by an NCI Training Grant (CA09611) [J.A.R.]; by gifts to the Division of Surgery from Tenneco and Exxon for the Core Lab Facility; by the MD Anderson Cancer Center Support Core Grant (CA16672); by a grant from the Mathers Foundation; by a sponsored research agreement with Introgen Therapeutics, Inc.; and by the University of Texas Specialized Program of Research Excellence (SPORE) in lung cancer (P50-CA70907).

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Genetic abnormalities have been identified in the cancer cell that functionally contribute to the process of carcinogenesis. This provides the opportunity to target specific gene lesions and abnormal gene products with therapeutic interventions. The gene families implicated in carcinogenesis include dominant oncogenes and tumor-suppressor genes (t 2) (Table 20.1). Proto-oncogenes (the normal homologues of dominant oncogenes) participate in critical cell functions, including signal transduction and transcription, and only a single mutant allele is required to induce malignant transformation. Primary modifications in the dominant oncogenes that confer gain of transforming function include point mutation, amplification, translocation, and rearrangement. In comparison, tumor-suppressor genes require homozygous loss of function either by mutation, deletion, or both. Some tumor-suppressor gene products appear to playa role in governing proliferation by their regulation of transcription. Alterations in the expression of dominant oncogenes and tumor-suppressor genes may therefore influence certain characteristics of cells that contribute to the malignant phenotype. Therapies directed at abnormal gene products could be in the form of small molecules or could involve replacement of the defective gene with a functioning gene or a gene construct that inhibits production of the mutant gene product. Development of small molecule approaches is beyond the scope of this chapter; instead, the focus will be the use of gene constructs. Table 20.1 .

Oncogenes and tumor-suppressor genes altered in lung cancer

SClC

NSClC

Oncogenes

c-myc* L-myc N-myc c-raf c-myb c-erb B-1 (EGF-R) c-fms c-rlf

K-ras* N-ras H-ras c-myc c-raf c-fur c-fes c-erb B-1 (EGF-R) c-erb B-2 (Her2, neu) c-sis

Tumor-suppressor genes

p53*

bcl-1 p53*

RB*

RB

p16 3p

p16 3p

9p

9p

SCLC = small-cell lung cancer; NSCLC = non-small-celllung cancer. *Most frequently altered genes in tumors or cell lines evaluated. Source: Modified from Greenblatt MS, Reddel RR, Harris Cc. Carcinogenesis, cellular, and molecular biology of lung cancer. In: Roth JA, Ruckdeschel JC, Weisenberger TH, eds. Thoracic Oncology, 2nd ed. New York: Saunders.

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The basic concept in gene therapeutic approaches to monogenic diseases is to treat and potentially cure disease by inserting and expressing a normal copy of the mutant or deleted gene in host cells. In this way, monogenic diseases such as adenosine deaminase deficiency or Gaucher's disease could be treated and potentially cured. The identification of specific genes that contribute to the development of the cancer cell presents an opportunity to replace defective genes or inactivate genes that have gained transforming function and so treat and prevent cancer. However, this is a considerably more complex problem than in monogenic disease, as the development of cancer is associated with multiple genetic abnormalities. Multiple genetic abnormalities are present in cancer cell lines and fresh tumor samples (3-6). This is evident at the chromosomal level, where multiple chromosomal abnormalities have been identified. In addition, more and more oncogenes and tumor-suppressor genes are being identified. Thus, some have felt that gene replacement cancer therapy would not be possible because of the difficulties associated with correcting so many genetic abnormalities in one cell. However, several observations by us and others suggest otherwise. Several studies have shown that correction of a single genetic defect, such as eliminating expression of a dominant oncogene or adding a normal copy of a tumor-suppressor gene to a cell with deleted or mutated copies, can reduce or eliminate such critical characteristics of the malignant phenotype as tumorigenicity or anchorage-independent growth (7-9). Studies done in our laboratory support this concept, and their results have been applied to animal models and clinical protocols. Essential to the mediation of any therapeutic effect, however, is the efficient delivery of the therapeutic gene to the cancer cell. It was previously believed that most available vectors were very inefficient in transducing genes into cancer cells. However, studies in models of human tumors in vitro and in nu/nu mice show that uptake of a retroviral vector is efficient enough to mediate a therapeutic effect (10-13). Retroviruses in fact can be integrated into and express a transgene primarily in proliferating cells. Thus, this vector may be selective for cancer cells. Another viral vector derived from adenovirus can be taken up by both dividing and nondividing cells. Consequently, even a single infection in culture is sufficient to infect more than 90% of cancer cells (12). Furthermore, both of these viral vector types can penetrate three-dimensional tumor cell matrices (14, 15). Yet, despite the relatively high efficiencies of cancer cell transduction achieved by viral vectors, it is doubtful that all cancer cells in a tumor can be transduced. Fortuitously transduced cells can cause cell death in nontransduced cells by mediating a "bystander effect." This effect was first noted in brain tumor cells transduced with the herpes simplex thymidine kinase gene and then exposed to ganciclovir (16), and was thought to be mediated by passage of toxic metabolites of the ganciclovir through gap junctions, by phagocytosis of apoptotic vesicles, or by

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immune responses (17-19). Since then, the bystander effect has also been observed in lung cancer cells, containing endogenous mutant p53, that have been transduced with a retrovirus expressing the wild-type p53, although a different mechanism is probably involved (20). Together, these observations suggest that gene replacement in cancer has the potential for mediating therapeutic effects. Given the multiple genetic lesions in cancer cells, however, a critical question will be which genes to target.

DOMINANT ONCOGENES One possible group to target is dominant oncogenes and ras oncogenes, in particular, because members of the ras oncogene family are frequently mutated in many common malignancies (21). These genes, which are homologous to the rat sarcoma virus, code for a protein called p21 that is located on the inner surface of the plasma membrane, has guanosine triphosphatase (GTPase) activity, and may participate in signal transduction. The oncogenes themselves are activated by point nucleotide mutations that alter the amino acid sequence of p21. To test the idea of targeting one dominant oncogene for gene therapy, antisense technology was used to identify the effects of eliminating expression of a mutant K-ras oncogene in lung cancer cells (8). In brief, a homozygous mutation at codon 61 (i.e., substitution of a normal glutamine residue [CAA] by histidine [CAT]) was detected in a clone of the NCI-H460a large-cell undifferentiated human non-smaIl-cell lung cancer (NSCLC) cell line. An antisense K-ras RNA construct was then developed and transfected into H460a cells, which have, in addition to a K-ras mutation, five chromosomal deletions (specifically, chromosomes 1,2,9, 12, and 16). The construct selectively blocked the expression of mutant antisense K-ras RNA and reduced the growth rate of H460a tumors in nu/nu mice. Thus, although the cancer cells had multiple genetic abnormalities, the reversal of a single abnormality appeared to have profound effects on fundamental properties of the malignant phenotype, such as rapid proliferation and tumorigenicity. Having established that gene therapy targeted to a single gene in cancer cells is efficacious, the question becomes how to deliver the therapy. Retroviruses have been extensively studied as delivery vehicles in gene transfer protocols and, therefore, are a candidate vector for gene delivery in vivo (22). One approach was to create retroviral vectors that lack genes essential for replication. Such replication-defective vectors are capable of infecting cells and being integrated as proviruses, which can then express recombinant genes in the cells. Because gene constructs transduced by retroviruses are integrated preferentially in dividing cells, this technique gives proliferating cancer


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cells a selective advantage for expressing the gene construct. Moreover, retroviruses and cells modified by retroviral transduction have little acute toxicity, making multiple treatments with high-titer preparations feasible. Taking advantage of these traits, a retroviral vector system was developed for efficiently transducing a K-ras antisense construct into human cancer cells (10). Once the 1.8-Kb K-ras gene fragment DNA in antisense orientation to a j3-actin promoter was inserted into retroviral vector LNSX, and transduced into H460a cells expressing mutant K-ras, colony formation in soft agarose and tumorigenicity in an orthotopic nu/nu mouse model were dramatically decreased (11). We concluded then that an antisense K-ras construct can be expressed effectively in a retroviral vector, which can in turn efficiently transduce human cancer cells. Together, these studies in K-ras gene therapy show that retroviral supernatants expressing the appropriate therapeutic construct can mediate antitumor effects, that an antisense construct can mediate therapeutic tumor regression in vivo, and that therapeutic antitumor effects can occur following inhibition of oncogene expression.

TUMOR-SUPPRESSOR GENES Tumor-suppressor genes are a second potential target for therapy, with the p53 gene being a particularly good candidate. The p53 gene, which encodes a 393 amino acid phosphoprotein that can form complexes with viral proteins such as large T antigen and EIB, is the most commonly mutated gene identified to date in human cancers. Missense mutations are common in the p53 gene and in many cases will functionally impair the p53 gene product (23, 24). In addition, the mechanism of p53 transformation may vary depending on the type of p53 mutation. The p53 protein appears to be multifunctional, with major domains that can transactivate other genes, bind other proteins, bind sequencespecific DNA, and oligomerize with other p53 proteins. Abnormalities in one or more of these functions could eliminate or reduce the tumorsuppressor function of the p53 gene product. Failure of the mutant p53 protein to activate transcription of molecules essential for regulating the cell cycle and DNA repair or the untimely expression of molecules transcriptionally enhanced by the mutant p53 protein may make the cell more susceptible to genetic instability. Thus, the wild-type p53 gene may suppress genes that contribute to uncontrolled cell growth and proliferation or activate genes that suppress uncontrolled cell growth, so that the absence of the wild-type p53 protein or inactivation of the wild-type p53 protein may contribute to transformation. The p53 gene also regulates cell-cycle progression. If DNA damage occurs, the cell will arrest at the Gl checkpoint until the damage is

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repaired. Failure to repair DNA damage may then trigger apoptosis (programmed cell death). To study the effects of p53 gene therapy, a retroviral vector was created for efficient transduction of the wild-type p53 gene into human lung cancer cell lines H358a (deleted p53) and H322a (mutant p53) (20). In briet LNSX/p53 constructs incorporating p53 cDNA, driven by a 13actin promoter, mediated the stable integration of p53 into cancer cells. The consequent restoration of the wild-type p53 gene suppressed growth in cell lines with a mutated p53 but had no effect on transduced H460a tumor cells, which have an endogenous wild-type p53 gene. Furthermore, mixing experiments showed that transduced cells could reduce the growth rate of nontransduced cells, thus indicating a bystander effect. Because mutations in the p53 tumor-suppressor gene are especially common in human lung cancers and because the wild-type form of p53 is dominant over the mutant, restoration of wild-type p53 function in lung cancer cells may suppress their growth as tumors. Consequently, we also investigated the therapeutic efficacy of direct administration of a retroviral wild-type p53 expression vector (LNp53B) in a mouse model of human orthotopic lung cancer (13). H226Br cells, originally derived from a squamous lung cancer that metastasized to brain and having a point mutation (ATC to GTC) at ex on 7, codon 254, of the p53 gene, were used. Thirty days after inoculation with the H266Br cells, 63-80% of control mice showed macroscopic tumors of the right mainstem bronchus. However, subsequent treatment with LNp53B suppressed H226Br tumor formation in 62-100% of mice in dose-dependent fashion. Thus, direct administration of a retroviral vector expressing wild-type p53 may inhibit local growth in vivo of human lung cancer cells with abnormal p53 expression. Unlike retroviral vectors, adenovirus vectors can transduce both dividing and nondividing cells and may have a high affinity for infecting lung epithelium. Therefore, we also developed the adenovirus vector Adp53 for delivery of wild-type p53 (25, 26). Using this vector, high level of expression of exogenous p53 was achieved in H358 cells at a multiplicity of infection (MOl) of 30 plaque forming units (PFU)/ cell (H358 cells lack endogenous p53). In contrast, when H322 and H460 cells were infected at the same MOl, the level of expression of the exogenous p53 was three times higher than that of the endogenous mutated protein cells and 14 times higher than that of the endogenous wild-type protein in H460 cells. In another study, the time course of exogenous p53 expression after a single infection of 10 PFU / cell was studied in H358 cells. The protein expression peaked at postinfection day 3, sharply decreased after day 5, and lasted for at least 15 days. This is a critical point with respect to safety of the Adp53 vector because it shows that 1) transient p53 expression is sufficient for mediating apoptosis, but that 2) normal cells taking up the vector will express the exogenous p53 for only a short time. In short, Adp53 inhibited the proliferation of


375

lung cancer cells with mutated or deleted p53 but only minimally affected growth of cells with endogenous wild-type p53. To study the efficacy of Adp53 in inhibiting tumorigenicity, we again used the mouse model of orthotopic human lung cancer and H226Br cells (11). After 6 weeks, only 25% of Adp53-treated mice formed tumors, whereas in the vehicle or Ad5/RSV /GL2 control groups, 70-80% of treated mice formed tumors. Tumors in the Adp53 group were significantly smaller on average than those in the control groups. Together, these results indicate that Adp53 can prevent H226Br cells from forming tumors in the mouse model of orthotopic human lung cancer. We also examined whether Adp53 and cisplatin given in a sequential combination could induce synergistic tumor regression in vivo. Following direct intratumoral injection of Adp53 on 3 consecutive days, H358a tumors subcutaneously transplanted in nu/nu mice showed a modest slowing of growth; Adp53-injected tumors, however, regressed if cisplatin was first administered intraperitoneally on 3 consecutive days. Histologic examination revealed necrosis of tumor tissue in the area where Adp53 was injected in mice treated with cisplatin. In situ staining of tumor cells treated with Adp53 and then cisplatin showed extensive areas of apoptosis. In contrast, in situ staining of tumors treated with either cisplatin or Adp53 alone showed no apoptosis.

CLINICAL ApPLICATIONS The studies just described provide a rationale for clinical protocols involving intra tumoral injection of recombinant retroviruses and have been approved by the Recombinant DNA Advisory Committee (RAC) of the NIH and the FDA. The protocol aims to replace a defective p53 gene with a vector expressing normal p53 (27, 28). Patients with unresectable lung cancer obstructing a bronchus will have their tumors directly injected with the appropriate retroviral supernatant, while patients with unresectable local tumors or isolated metastases may undergo radiologically guided injection of the vector. The results of treatment of the first nine patients on the retroviral p53 protocol have been reported (29). All nine patients (median age, 68 years; range, 51-73) had a history of primary NSCLC. Of these nine, four patients had recurrent endobronchial lesions (three squamous cell carcinomas and one adenocarcinoma); these were treated with bronchoscopic injections of the retroviral p53 expression vector ITRp53A. Another four patients had chest wall lesions (two large cell carcinomas, one squamous cell carcinoma, and one adenocarcinoma); these were treated with percutaneous injections under computed tomographic (n = 3) or fluoroscopic guidance. The ninth patient had a left adrenal metastasis from a large-cell carcinoma; this was treated with percutaneous

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injection of the vector under CT guidance. All nine patients had recurrent or metastatic tumors that had progressed during prior treatment. Five of the nine patients had undergone surgical resection of either the primary lung cancer (n =3) or a brain metastasis (n =2). Five of the nine had received chemotherapy, and eight had received radiation therapy. Thus, all patients in the study had failed existing treatments and had cancers that were progressively growing. Polymerase chain reaction (PCR) analysis, using primers specific for the retroviral trans gene, was used to detect the presence of vector sequences in tumor biopsies taken before and after treatment. While PCR revealed no retroviral sequences in any of the pretreatment biopsies, it did reveal sequences in DNA extracted from post-treatment biopsies of two patients and from a postmortem tumor of one patient (Table 20.2). Moreover, in situ hybridization using the near probe showed nuclear localization of the vector in six patients (see Table 20.2). Thus, all patients, with the exception of one who did not complete the treatment protocol (patient 9), showed some evidence of gene transfer.

Table 20.2.

Assessment of gene transfer by ITRp53A retroviral vector a

In situ hybridization fscore)b

TUNEl

+

3 3

17.0 34.0

+ +

ND ND

Patient no.

DNA-peR

1 2 3 4

2 2 2 1

5 6 7

8 9

ND

ND

staining f%)C

ND 27.6 10.2 10.2

0.8 23.0

ND

NO = not done, because specimen unsatisfactory or unavailable (29). aResults shown are for indicated assays of post-treatment biopsy specimens. No pretreatment samples were positive for gene transfer in ONA-PCR and in situ hybridization assays. Examfles are shown in Fig. 20.1. All slides were coded and read by a single blinded observer without knowledge of the patient or collection date. The percentage of tumor cells with punctate nuclear staining was determined in 500 cells per slide (400x magnification). The slides were evaluated on the following scoring system: no staining, 0; 20% of cells stained, 3. Slides with a score ~1 were considered positive. The maximum score for each patient is given; significant increases in in situ hybridization were observed in 6 of 6 evaluable cases (P < 0.05, sign test). cCells with morphologic features of necrosis were not included among the TUNEL-positive cells. The percentage of cells with nuclear staining was determined in 500 cells per slide (400x magnification). The background level of TOT staining in pretreatment samples was

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