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~i-~arkly staining nuclei e a proliferation focus in the aid of imrnunohistochemis- (B) after microdissection to | odissected cells purified by L MOLECULAR EVENTS IN LUNG CARCINOGENESIS 225 MOLECULAR ANALYSIS OF PREMALIGNANT LESIONS Dysplastic squamous epithelium consists of only a few layers of epithelial cells. Biopsies typically contain not only regions of dysplastic epithelium but also histologically normal epithelium and supporting connective tissue. Therefore, analysis of biopsies by techniques that do not either physically separate dysplastic epithelium from the remainder of the specimen or detect mutant gene products from within a mixture of mutant and wild type alleles is fruitless. Microdissection of dysplastic epithelium has been useful in providing purified tissue for analysis (Fig. 3). The resulting DNA can be analyzed by polymerase chain reaction (PCR)-based methods. Several classes of genetic alteration can be detected in this material. Point mutations affecting specific target genes, such as ras or p53, can be documented by a variety of techniques, including direct sequence analy- sis of PCR products (Fig. 4) or single-strand conformational polymor- phism analysis of PCR products followed by sequence analysis. Genes in which mutations occur at one or a few codonsmfor example, activat- ing ras mutations--are more easily analyzed than genes with many possible mutation sites, such as p53. Chromoso~nal deletions are detected at a molecular level by loss of heterozygosity for polymorphic markers in dysplastic tissue (Fig. 5). The extent of deletion can be assessed by analyzing several allelic markers at different locations on the chromo- some of interest. To conserve dysplastic material, genomic DNA is best initially analyzed on each individual to determine which markers are informative. Fluorescence in situ hybridization (FISH) can provide simi- lar information on individual cells (Figure 6). Unstable short nucteotide repeats may undergo expansion in malignancy. These are detected by the presence of multiple PCR products of varying size when tumor or dysplastic DNA is amplified using primers that flank the region of repeats, a phenomenon referred to as microsatellite instability.~4 DNA methylation, one mechanism of gene inactivation, can be detected by a technique involving chemical modification followed by PCR.3 Rabbitts and colleagues~3 were among the first to apply molecular methods to the analysis of dysplastic respiratory epithelium. They de- tected loss of heterozygosity for chromosome 3p markers in dysplastic tissue found in proximity to lung cancer resection specimens. More recently, Hung and coworkers34 have performed an analysis in six resec- tion specimens. Dysplastic lesions exhibited loss of heterozygosity for chromosome 3p markers, always losing the same allele as for the adjacent tumor. These two seminal studies have supported the concept that lung cancers arise from a field of abnormal epithelium harboring one or more mutations. Similar findings have been reported for loss of heterozygosity of chromosome 9p, another site of known or suspected tumor suppressor genes important in lung cancer.4° p53 point mutations have been inferred by immunohistochemistry and confirmed bv molecu- lar means in dysplasias adjacent to tumors.~, ~-~ Recent studi~s suggest

226 G A T C G C Figure 4. Point mutation can be detected by single strand polymorphism (SSCP) analysis. A, PCR products obtained by amplification with primers encompassing p53 exons 5 and 6 are separated by denaturing polyacrylamide gel electrophoresis. One mutant sequence amplified from tumor (NSCLC) has a markedly retarded mobility in comparison to strands obtained by amplification of wild type DNA from uninvolved lung (Lung). DNA can be extracted from cells microdissected from either H&E stained (He) or immunostained (IH) sections. Mutation is confirmed by direct sequencing indicated in (B) by substitution of T for G (arrow) in codon 158 in this case. PCR Analysis of Mi CA02) CAoo) --~'CACACACACACA( GTGTGTGTGTGT( ~ CACACACACA~ , GTGTGTGTGT' Patterns of All Case I T N r~ ' Probe 1 Probe 2 Norm~ Figure 5. Allelic loss is t':=-,.~.~ dimeric, trimeric or tetrarve-.= Polymorphism at these sites in DNA from both target ce ~ =-- (top frame) for decimeric =-~--- separated by non-denat~---~_~ lengths have differing moc alleles (Case 1), the prese,-c=- -- tive result, Case 2), loss c-" (homozygous deletion, C=~--e-- - contaminate DNA from ~K---: - intensity of control allele=-, of allelic loss in flanking~'~-~-_= fbr quality and quantity c.= C',- site of potential deletion.

Lung ~ T-C I norphism (SSCP) analysis• )assing p53 exons 5 and 6 ;is. One mutant sequence t in comparison to strands !ung (Lung). DNA can be -le) or immunostained (IH) in (B) by substitution of T ! i i i i I l- t t MOLECULAR EVENTS IN LUNG CARCINOGENESIS 227 PCR Analysis of Microsatellite Dinucleotide Repeats CA02) Repeat Formula -~CACACACACACACACACACACACA GTGTGTGTGTGT~T~T~T~TGT~ Electrophoretic Pattern CA0o) --~CACACACACACACACACACA GTGTGTGTGTGTGTGTGTG~ Patterns of Allelic Loss in Lung Carcinoma Case 1 Case 2 Case 3 Case 4 Case 5 T N T N T N T N T N Normal Uninformative LOH Homozygous • Deletion Figure 5. Allelic loss is typically determined by amplification of polymorphic nucleotide dimedc, trimeric or tetrameric repeat sites which exist throughout the human genome. Polymorphism at these sites consists of variability in the length of the repeats. Repeat sites in DNA from both target cells and normal control cells are amplified by PCR as diagrammed (top frame) for decimeric and dodecimeric CA repeats• Strands from both alleles are separated by non-denaturing polyacryiamide get electrophoresis. Strands of differing lengths have differing mobilities. Possible results (bottom frame) include retention of both alleles (Case 1), the presence of only one allele in both normal and target DNA (unihforma- tive result, Case 2), loss of one allele (loss of heterozygosity, Case 3), loss of both alleles (homozygous deletion, Case 4). In some cases, a small amount of normal DNA may contaminate DNA from target cells in which case a weak band in both alleles but normal intensity of control alleles may indicate homozygous deletion, particularly in the presence of allelic loss in flanking regions (Case 5). Amplification may be multiplexed as a control for quality and quantity of DNA in each amplification reaction. (Bottom frame, probe 1 is site of potential deletion, probe 2 is a non-deleted control region.) Probe Probe 2

228 MILLER & FRANKLIN Figure 6. Fluorescent in situ hybridization for chromosome 9 markers performed on touch preparation of squamous carcinoma of lung. The centromeric region is labeled with a red probe while unique sequences at 9p21 are labeled with a green probe. Of the three interphase nuclei shown, the normal nucleus (upper/eft) has two copies of chromosome 9 indicated by red signals (centromeric) and two copies of unique sequence at chromosome 9p21 indicated by green signals. The two remaining nuclei have lost one copy of chromo- some 9 as indicated by the presence of only one red signal. Homozygous loss of sequences at chromosome 9p21 is indicated by complete absence of green signal. (Courtesy of Dr. Marileila Varella-Garcia and Kalpana Rao) that chromosome 3p loss of heterozygosity precedes p53 mutation.I°, 1~ Ras mutations, which occur in a subset of NSCLC, are not frequently found in adjacent dysplasias and are therefore thought to be later events23 Aneuploidy has also been reported in preinvasive airway epi- thelial lesions.~ Finally, increased cytosine DNA methyltransferase ex- pression has been demonstrated in an animal model of lung carcinogene- sis and may lead to the inactivation of growth regulatory genes.3 MECHANISMS OF FIELD CARCINOGENESIS The term field carcinogenesis was originally used to describe the occurrence of multiple carcinomas arising in patients with oral squa- mous cell carcinomaZ7 The lower respiratory epithelium is also suscepti- ble to the development of multiple primary carcinomas. The mechanism of field carcinogenesis is currently not clear. One possibility is that the massive exposure of the respiratory epithelium to the carcinogens pres- ent in tobacco smoke results in the independent initiation of multiple epithelial cells. Sozzi and colleagues~° have recently reported the analysis of several pairs of independent primary lung cancers. Different carcino- mas from the same individual did not share p53 point mutations or loss |' of the same 3p allele, although carcinomas and adjacent dysplasias did 1 I I ] ] :] share mutations. This fi that carcinomas arise fr~ not show shared mutati~ We have recently st dysplasia but no overt c identified at multiple sit~ mutation or PCR artifac airway epithelial cell ck widespread areas of the in the bladder, with m~.~ mutation, but this mech prior to the developme: currently thought to be this case likely represent: mechanism (i.e., expansi~ genesis. When the most are defined, it wil lial clones with SUMMARY AND CLINK Many critical issues analysis of premalignani (1) What is the usual Because premalignant d some of the mutations f in the dysplasia and the events are "late" events The technical problems i simultaneously for man order of mutational eve~ analyzed per lesion for this information can non investigation. (2) What are the eaz vast majority of genetic. to lesions that were mors. These mutations sense as mutations in a F in high-risk individuals cede the development rate either as yet to be d tions in sputum or s~ epithelium. (3) What Some early experience s~ for epitheiium to harbor

MOLECULAR EVENTS IN LUNG CARCINOGENESIS 229 9 markers performed on touch ,~ric region is labeled with a red ~l~len probe. Of the three ~s~opies of chromosome 9 ique sequence at chromosome have lost one copy of chromo- qomozygous loss of sequences green signal. (Courtesy of Dr. :cedes p53 mutation.I°. 1~ CLC, are not frequently ~re thought to be later preinvasive airway epi- A methyltransferase ex- ~del of lung carcinogene- :egulatory genes2 y used to describe the >atients with oral squa- ithelium is also suscepti- inomas. The mechanism ~e possibility is that the to the carcinogens pres- nt initiation of multiple tty reported the analysis nc,~,l~. Different carcino- . ~ mutations or loss acrJ~ent dysplasias did share mutations. This finding, in a small sample, supports the theory that carcinomas arise from an area of mutated epithelium, but it does not show shared mutation by different tumors. We have recently studied a patient with extensive airway epithelial dysplasia but no overt carcinoma.~ Identical p53 point mutations were identified at multiple sites in both lungs. The presence of a germline p53 mutation or PCR artifact was ruled out. This case demonstrates that an airway epithelial cell clone with a mutation can expand and populate widespread areas of the lungs. A precedent for this has been reported in the bladder, with multiple primary lesions sharing an identical p53 mutation, but this mechanism has not been previously shown to occur prior to the development of carcinoma.73 Because p53 mutation is not currently thought to be a common early event in lung carcinogenesis, this case likely represents an unusual target gene, but perhaps a frequent mechanism (i.e., expansion of a mutated epithelial clone) of field carcino- genesis. When the most common early mutations in lung carcinogenesis are defined, it will be possible to determine with what frequency epithe- lial clones with mutation expand. SUMMARY AND CLINICAL IMPLICATIONS Many critical issues remain to be resolved in terms of the molecular analysis of premalignant lesions: (1) What is the usual order of mutational events in hmg carcinogenesis? Because premalignant dysplasias adjacent to tumors frequently harbor some of the mutations found in the tumor, a comparison of mutations in the dysplasia and the tumor should at least be able to define which events are "late" events, that is, found in tumor and not in dysplasia. The technical problems inherent in analyzing small amounts of material simultaneously for many mutations has delayed the elucidation of the order of mutational events in lung carcinogenesis. Up to 25 loci may be analyzed per lesion for loss of heterozygosity using multiplex PCI~, so this information can now be obtained. This is currently an area of intense investigation. (2) What are the early mutational events in hmg carcinogenesis? The vast majority of genetic analysis of dysplastic lesions has been restricted to lesions that were discovered in resection specimens containing tu- mors. These mutations cannot be considered to be "early" in the same sense as mutations in a premalignant colonic polyp. Longitudinal studies in high-risk individuals are needed to determine which mutations pre- cede the development of lung cancer. These studies will need to incorpo- rate either as yet to be developed improved methods of detecting muta- tions in sputum or serial bronchoscopy and biopsy of abnormal epithelium. (3) What are the correlates between histologic dysplasia and mutation? Some early experience suggests that histotogic dysplasia is not necessary for epithelium to harbor mutated clones.-~-~

230 " MILLER & FRANKLIN (4) What is the biologic consequence of the presence of mutated airway epithelial clones? Several groups have now found frequent mutations shared by numbers of airway epithelial cells. These studies have been performed largely in subjects with lung cancer, chronic obstructive pul- monary disease, or abnormal sputum cytology. Thus, it is unclear if these changes are associated with smoking alone or with the development of smoking-induced lung disease. It is likely that our knowledge of the different molecular events involved in lung carcinogenesis, their usual sequence, their biologic consequences, and their prognostic meaning will advance significantly in the near future. This will allow the better definition of extremely high-risk individuals, as well as new and earlier endpoints for treatment. Other than smoking cessation, we have no validated interventions for premalignancy in the lung. Promising new approaches to the treatment of bronchial premalignancy, including local ablative measures, dietary modification, and chemopreventive agents, need to be developed and validated. References 1. Aguayo SM, Miller YE, Waldron JA Jr, et al: Brief report: Idiopathic diffuse hyperplasia of pulmonary neuroendocrine cells and airways disease. N Engl ] Med 327:1285, 1992 2. Alexandrie AK, Sundberg MI, Seidegard J, et al: Genetic susceptibility to lung cancer with special e~mphasis on CYPIA1 and GSTMI: A study on host factors in relation to age at onset, gender and histological cancer types. Carcinogenesis 15:1785, 1994 3. Belinsky SA, Nikula KJ, Baylin SB, Issa JP: Increased cytosine DNA-methyltransferase activity is target-cell-specific and an early event in lung cancer. Proc Natl Acad Sci USA 93:4045, 1996 4. Bosken CH, Hards J, Garter K, Hogg JC: Characterization of the inflammatory reaction in the peripheral airways of cigarette smokers using immunocytochemistry. Am Rev Respir Dis 145:911, 1992 5. Brauch H, Johnson B, Hovis J, et al: Molecular analysis of the short.arm of chromosome 3 in small-cell and non-small-cell carcinoma of the lung. N Engl J Med 317:1109, 1987 6. Bunn PA, Brenner DG, Helfrich B, et al: Effects of recombinant neutral endopeptidase (NEP, EC 3.4.24.11) on the growth of lung cancer cell lines in vitro and in vitro. Submitted 7. Burrows B, Knudson RJ, Camilli AE, et al: The "horse-racing effect" and predicting decline in forced expiratory volume in one second from screening spirometry. Am Rev Respir Dis 135:788, 1987 8. Caporaso N, DeBaun MR, Rothman N: Lung cancer and CYP2D6 (the debrisoquine polymorphism): Sources of heterogeneity in the proposed association. [review]. Phar- macogenetics 5(spec no)S129-134, 1995 9. Caporaso NE, Land[ MT: Molecular epidemiology: A new perspective for the study of toxic exposures in man. A consideration of the influence of genetic susceptibility factors on risk in different lung cancer histologies. Med Lay 85:68, 1994 10. Chung GT, Sundaresan V, Hasleton P, et al: Sequential molecular genetic changes in lung cancer development. Oncogene 11:2591, 1995 11. Chung GT, Sundaresan V, Hasleton P, et al: Clonal evolution of lung tumors. Cancer Res 56:1609, 1996 12. Cohen AJ, Bunn PA, Franklin W, et al: Neutral endopeptidase: Variable expression in human lung, inactivation in lung cancer, and modulation of peptide-induced calcium flux. Cancer Res 56:831, 1996 13. Cohen AJ, Skidgel R, Bunn I Chest, in press 14. Cohen BH, Diamond EL, G cancer and chronic obstruct[ 15. Cook RM; Burke BJ, Buchha and expression analysis of cancer. ] Bio[ Chem 268:170~ 16. Cook RM, Miller YE, Bunn features, staging, and treatrc 17. Cook RM, Moore M, Johnso in human small cell Lung ca 18. Crofts F, Taioli E, Trachman genotypes. Carcinogenesis 1 19. Daly MC, Douglas Jg, Bleeh arm of chromosome 3 h3 a t 20. Daly MC, Xiang RH, Buchl- in a small cell lung cance~ activity. Oncogene 8:1721, 1 21. DrabkJn HA, Mendez MJ, I deletion in the small-cell lu Cancer 5:67, 1992 22. Falco ]P, Baylin associa~d growth factors n Invest 85:1-740, 1990 23. Fontana RS, Sanderson program. J Occup Med 28:7 24. Franklin WA: The biology Med 1-7:309, 1_996 25. Franklin WA, LaRosa F, epithelium as a mechanism 26. Franklin WA, Todd S, Ge'~ immunophenotypic, and Chest 109(3 supp1):26S, 19 . 27. Frost JK, Ball WC Jr, Levin (prevalence) radiologic and Respir Dis 130:549, 1984 28. Ganju RK, Sunday M, Tsar" mas. Relationship to cellul; 29. Giaccone G, Battey J, Gaze- lines. Cancer Res 52(9 sup~ 30. Gray DA, Inazawa J, Gupt at 3p21.3, in human lung t 31. Harbour JW, Lai SL, Wha, ot ti~e human retinoblasto[ 32. Hensel CH, Hsieh CL, Gaz retinoblastoma susceptibili 33. Hosoe S, Shigedo Y, Uen cbromosome 3 in small c{ 10:297, 1994 34. Hung J, Kishimoto Y, Sug an early stage in the path, 35. [siam SS, Schottenfeld D: smokers: A 25-year prosp," Cancer Epidemiol ]~liilk'na~ "~o. Johnson BE, Saka~ phism studies sho¢~lSnsi patients" tumors. J Clin h

sence of mutated airway ~d frequent mutations ~ese studies have been hronic obstructive pul- us, it is unclear if these th the development of rent molecular events quence, their biologic advance significantlv efinition of extremelj, ~dpoints for treatment. ated interventions for ~ches to the treatment ive measures, dietary to be developed an~l ~pathic diffuse hyperplasia Engl j" Med 327:1285, 19q2 sceptibility to lung cancer host factors in relation to enesis 15:1785, 1994 . le DNA-methyltrans ferase ~ncer. Proc Natl Acad Sci .~he inflammatory reaction ~ocytochemistry. Am Rev short arm of chromosome ~ngl J Med 317:1109, Iq87 nt neutral endopeptidase -'s in vitro and in vitro. ~g effect" and predictin,~ reening spirometD.. Am ~'P2D6 (the debrisoquine ;ociation. ~reviewI. l'har- - 'spective for the study of of genetic susceptibility .5:68, 1994 cular genetic changes in of lung tumors. Cancer ,: Variable expression in ,eptide-induced calcium i i i i i ! t t MOLECULAR EVENTS IN LUNG CARCINOGENESIS 231 13. Cohen AJ, Skidgel R, Bunn PA, et al: Carboxypeptidase M inactivation in lung cancer. Chest, in press 14. Cohen BH, Diamond EL, Graves CG, et al: A common familial component in lung cancer and chronic obstructive pulmonary disease. Lancet 2:523, 1977 15. Cook RM, Burke BJ, Buchhagen DL, et al: Human aminoacylase-1. Cloning, sequence, and expression analysis of a chromosome 3p21 gene inactivated in small cell lung cancer. J Biol Chem 268:17010, 1993 16. Cook RM, Miller YE, Bunn PA Jr: Small cell lung cancer: Etiology, biology, clinical features, staging, and treatment. Curt Probl Cancer 17:69, I993 17. Cook R/vl, Moore M, Johnson B, et al: A compound mutation affecting the ACY1 gene in human small cell lung cancer. Submitted 18. Crofts F, Taioli E, Trachman J, et al: Functional significance of different human CYP1A1 genotypes. Carcinogenesis 15:2961, 1994 19. Daly MC, Douglas ]B, Bleehen NM, et ah An unusually proximal deletion on the short arm of chromosome 3 in a patient with small cell lung cancer. Genomics 9:113, 1991 20. Daly MC, Xiang RH, Buchhagen D, et al: A homozygous deletion on chromosome 3 in a small cell lung cancer cell line correlates with a region of tumor suppressor activity. Oncogene 8:1721, .1993 21. Drabkin HA, Mendez MJ, Rabbitts PH, et al: Characterization of the submicroscopic deletion in the small-cell lung carcinoma (SCLC) cell line U2020. Genes Chromosom Cancer 5:67, 1992 22. Falco JP, Baylin SB, Lupu R, et al: v-rasH induces non-small cell phenotype, with associated growth factors and receptors, in a small cell lung cancer cell line. J Clin Invest 85:1740, 1990 23. Fontana RS, Sanderson DR, Woolner LB, et al: Lung cancer screening: The Mayo program. J Occup Med 28:746, 1986 24. Franklin WA: The biology of bronchial premalignancy. Semin Respir Critical Care Med 17:309, 1996 25. Franklin WA, LaRosa F, Folkvord J, et al: Widely dispersed p53 mutation in bronchial epithelium as a mechanism for field carcinogenesis. Submitted 26. Franklin WA, Todd S, Gemmill RM, et al: Correlative assessment of morphologic, immunophenotypic, and genetic changes in bronchial epithelium of tobacco smokers. Chest 109(3 suppl):26S, 1996 27. Frost JK, Ball WC Jr, Levin ML, et ah Early lung cancer detection: Results of the initial (prevalence) radiologic and cytologic screening in the Johns Hopkins study. Am Rev Respir Dis 130:549, 1984 28. Ganju RK, Sunday M, Tsarwhas DG, et ah CD10/NEP in non-small cell lung carcino- mas. Relationship to cellular proliferation. J Clin Invest 94:1784, 1994 29. Giaccone G, Battey J, Gazdar AF, et al: Neuromedin B is present in lung cancer cell lines. Cancer Res 52(9 suppl):2732s, 1992 30. Gray DA, lnazawa j', Gupta K, et al: Elevated expression of Unph, a proto-oncogene at 3p21.3, in human lung tumors. Oncogene 10:2179, 1995 31. Harbour JW, Lai SL, Whang-Peng J, et al: Abnormalities in structure and expression of the human retinoblastoma gene in SCLC. Science 241:353, 1988 32. Hensel CH, Hsieh CL, Gazdar AF, et al: Altered structure and expression of the human retinoblastoma susceptibili ,ty gene in small cell lung cancer. Cancer Res 50:3067, 1990 33. Hosoe S, Shigedo Y, Ueno K, et al: Detailed deletion mapping of the short arm of chromosome 3 in small cell and non-small cell carcinoma of the lung. Lung Cancer 10:297, 1994 34. Hung J, Kishimoto Y, Sugio K, et al: Allele-specific chromosome 3p deletions occur at an early stage in the pathogenesis of lung carcinoma. JAMA 273:1908, 1995 35. Islam SS, Schottenfeld D: Declining FEV1 and chronic productive cough in cigarette smokers: A 25-year prospective study of lung cancer incidence in Tecumseh, Michigan. Cancer Epidemiol Biomarkers Prev 3:289, 1994 36. Johnson BE, Sakaguchi AY, Gazdar AF, et al: Restriction fragment length polymor- phism studies show consistent loss of chromosome 3p alleles in small cell lung cancer patients' tumors. J Clin Invest 82:502, 1988

232 MILLER & FRANKLIN 37. Kawajiri K, Nakachi K, Imai K, et al: Germ line polymorphisms of p53 and CYP1A1 genes involved in human lung cancer. Carcinogenesis 14:1085, 1993 38. Kennedy T, Proudfoot S, Franklin WA, et al: Airflow obstructed patients with signifi- cant smoking histories have a high incidence of sputum cytology dysplasia. Cancer Res 56:4673, 1996 39. Kerr KM, Carey FA, King G, Lamb D: Atypical alveolar hyperplasia: Relationship with pulmonary adenocarcinoma, p53, and c-erbB-2 expression. J Pathol 174:249, 1994 40. Kishimoto Y, Sugio K, Hung JY, et al: Allele-specific loss in chromosome 9p loci in preneoplastic lesions accompanying non-small-cell lung cancers. J Natl Cancer Inst 87:1224, 1995 41. Kishimoto Y, Sugio K, Mitsudomi T, et al: Frequent loss of the short arm of chromo- some 9 in resected non-small-cell lung cancers from Japanese patients and its associa- tion with squamous cell carcinoma. J Cancer Res Clin Oncol 121:291, 1995 42. Kitamura H, Kameda Y, Nakamura N, et al: Proliferative potential and p53 overexpres- sion in precursor and early stage lesions of bronchioloalveolar lung carcinoma. Am J Pathol 146:876, 1995 43. Kok K, Hofstra R, Pilz A, et al: A gene in the chromosomal region 3p21 with greatly reduced expression in lung cancer is similar to the gene for ubiquitin-activating enzyme. Proc Natl Acad S.ci USA 90:6071, 1993 44. Kok K, Osinga J, Carritt B, et al: Deletion of a DNA sequence at the chromosomal region 3p21 in all major types of lung cancer. Nature 330:578, 1987 45. Kok K, van den Berg A, Veldhuis PM, et al: A homozygous deletion in a small cell lung cancer cell line involving a 3p21 region with a marked instability in yeast artificial chromosomes. Cancer Res 54:4183, 1994 46. Kubik A, Parkin DM, Khlat M, et al: Lack of benefit from semi-annual screening for cancer of the lung: Follow-up report of a randomized controlled trial on a population of high-risk males in Czechoslovakia. Int J Cancer 45:26, 1990 47. Lam S, MacAulay C, Palcic B: Detection and localization of early lung cancer by imaging techniques. Chest 103:12S, 1993 48. Landi MT, Caporaso NE: Heterogeneity in the associations of different histologies of lung cancer with genetic susceptibility factors [abstract]. Proc Annu Meet Am Assoc Cancer Res 35:A1737, 1994 49. Mabry M, Nelkin BD, Falco JP, et al: Transitions between lung cancer phenotypes---implications for tumor progression [review]. Cancer Cells 3:53, 1991 50. Mao L, Hruban RH, Boyle JO, et al: Detection of oncogene mutations in sputum precedes diagnosis of lung cancer. Cancer Res 54:1634, 1994 51. Melamed MR, Ftehinger BJ, Zaman MB, et al: Screening for early lung cancer. Results of the Memorial Sloan-Kettering study in New York. Chest 86:44, 1984 52. Miller YE, Mirma JD, Gazdar AF: Lack of expression of aminoacylase-1 in small cell lung cancer. Evidence for inactivation of genes encoded by chromosome 3p. J Clin Invest 83:2120, 1989 53. Miller YE, Welsh CH, Prochazka A, Aguayo SM: Bombesin-tike peptides in pulmonary diseases and lung cancer. Lung Cancer 11:211, 1994 54. Miozzo M, Sozzi G, Musso K, et al: Microsatellite alterations in bronchial and sputum specimens of lung cancer patients. Cancer Res 56:2285, 1996 55. Murata M, Tagawa M, Kimura M, et al: Analysis of a germ line polymorphism of the p53 gene in lung cancer patients; discrete results with smoking history. Carcinogenesis 17:261, 1996 56. Murata Y, Tamari M, Takahashi T, et al: Characterization of an 800 kb region at 3p22- p21.3 that was homozygously deleted in a lung cancer cell line. Human Molecular Genetics 3:1341, 1994 57. Nakachi K, Imai K, Hayashi S, Kawajiri K: Polymorphisms of the CYP1A1 and glutathione S-transferase genes associated with susceptibility to lung cancer in relation to cigarette dose in a Japanese population. Cancer Res 53:2994, 1993 58. Naylor SL, Johnson BE, Mirma JD, Sakaguchi AY: Loss of heterozygosity of chromo- some 3p markers in small-cell lung cancer. Nature 329:451, 1987 59. Olopade OI, Buchhagen DL, Malik K, et al: Homozygous loss of the interferon genes ! def'mes the critical region or suppl):2410, 1993 60. Pagano M, Tam SW, Theodo~ in regulating abundance o 269:682, 1995 61. Polak JM, Becker KL, Cutz E, Anat Rec 236:169, 1993 62. Rabbitts PH: Genetic changes 50:688, 1994 63. Rabbitts P, Bergh J, Dougia-~ D3S3 locus in a cell line isol~ Cancer 2:231, 1990 64. Rasio D, Negrini M, Manen lung adenocarcinoma: Idel 55:3988, 1995 65. Roche J, Boldog F, Robinso identification of a new hum, 66. Saccomanno G, Archer VE, as reflected in exfoliated cel: 67. Scaloni A, Jones W, Pospisc cell lung carcinoma cell line 68. Schauer I, Siriwardana S, L inactivation: Implications fe lung cancer. Proc 69. Sekido Y, Bader S, l~t;, e small cell lung cance~[~dele Proc Natl Acad Sci USA 93 70. Sekido Y, Bader S, Latif F, tumor suppressor gene in 71. Sellers TA, Bailey-Wilson J the pathogenesis of lung ca 72. Shipp MA, Tart GE, Chen bombesin-like peptides ant Proc Natl Acad Sci USA 8g 73. Sidransky D, Frost P, Von Med 326:737, 1992 74. Siegfried JM, Han YH, De by a non-small cell lung cg free conditions. J Biol Che~ 75. Sithanandam G, Latif F, D activated protein kinase k region. Mol Cell Biol 76. Skillrud DM, Offord KP, pulmonary disease. A p 105:503, 1986 77. Slaughter DP, Southwick mous epithelium. Clinical 78. Smith AL, Hung J, Walks respiratory epithelium of 79. Sozzi G, Miozzo M, Dong tic lesions of the lung. Ca 80. Sozzi G, Miozzo M, Past( multiple preneoplastic an 8l. Sozzi G, Veronese NIL, cancer. Cell 85:17, 1996 82. Sugio K, Kishimoto Y, V in the pathogenesi_~of lul 83. Sundaresan V, ~ P.

,hisms of p53 and CYP1A1 ~85, 1993 'ucted patients with signifi- cytology dysplasia. Cancer ,erplasia: Relationship with [ Pathol 174:249, 1994 in chromosome 9p loci in .racers. j" Natl Cancer inst the short arm of chromo- se patients and its associa- I 121:291, 1995 enfial and p53 overexpres- ~lar lung carcinoma. Am J . region 3p21 with greatly e for ubiquitin-activating ence at the chromosomal 3, 1987 ~s deletion in a small cell ~stabflity in yeast artificial screening for on a population ) of early lung cancer by ~f different histologies of c Annu Meet Am Assoc between lung cancer ncer Cells 3:53, 1991 ~e mutations in sputum .~rly lung cancer. Results ,:44, 1984 ~oacylase-1 in small cell chromosome 3p. J Clin peptides in pulmonary bronchial and sputum ~e polymorphism of the history. Carcinogenesis 800 kb region at 3p22- ine. Human Molecular ~ of the CYP1AI a~ld • lung cancer in relation 1993 .rozygosity of chromo- 7 of the interferon genes ! ! i i MOLECULAR EVENTS IN LUNG CARCINOGENESIS 233 defines the critical region on 9p that is deleted in lung cancers. Cancer Res 53(10 suppl):2410, 1993 60. Pagano M, Tam SW, Theodoras AM, et ah Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269:682, 1995 61. Polak JM, Becker KL, Cutz E, et al: Lung endocrine cell markers, peptides, and amines. Anat Rec 236:169, 1993 62. Rabbitts PH: Genetic changes in the development of lung cancer [review]. Br Med Bull 50:688, 1994 63. Rabbitts P, Bergh J, Douglas J, et al: A submicroscopic homozygous deletion at the D3S3 locus in a cell line isolated from a small cell lung carcinoma. Genes Chromosom Cancer 2:231, 1990 64. Rasio D, Negrini M, Manenti G, et ah Loss of heterozygosity at chromosome 11q in lung adenocarcinoma: Identification of three independent regions. Cancer Res 55:3988, 1995 65. Roche J, Boldog F, Robinson M, et ah Distinct 3p21.3 deletions in lung cancer and identification of a new human semaphorin. Oncogene 12:1289, 1996 66. Saccomarmo G, Archer VE, Auerbach O, et al: Development of carcinoma of the lung as reflected in exfoliated cells. Cancer 33:256, 1974 67. Scaloni A, Jones W, Pospischil M, et ah Deficiency of acylpeptide hydrolase in small- cell lung carcinoma cell lines [see comments]. J Lab Clin Med 120:546; 1992 68. Schauer I, Siriwardana S, Langan TA, Sclafani RA: Cyclin D1 overexpression vs. Rb inactivation: Implications for growth control evasion in non-small cell and small cell lung cancer. Proc Natl Acad Sci USA 91:7827, 1994 69. Sekido Y, Bader S, Latif F, et al: Human semaphorins A(V) and IV reside in the 3p21.3 small cell lung cancer deletion region and demonstrate distinct expression patterns. Proc Natl Acad Sci USA 93:4120, 1996 70. Sekido Y, Bader S, Latif F, et ah Molecular analysis of the von Hippel-Lindau disease tumor suppressor gent in human lung cancer cell lines. Oncogene 9:1599, 1994 71. Sellers TA, Bailey-Wilson JE, Elston RC, et al: Evidence for mendelian inheritance in the pathogenesis of lung cancer [see comments]. J Natl Cancer Inst 82:1272, 1990 72. Shipp MA, Tarr GE, Chen CY, et al: CD10/neutral endopeptidase 24.11 hydrolyzes bombesin-like peptides and regulates the growth of small cell carcinomas of the lung. Proc Natl Acad Sci USA 88:10662, 1991 73. Sidransky D, Frost P, Von Eschenbach A, et al: Clonal origin bladder cancer. N Engl J Med 326:737, 1992 74. Siegfried JM, Han YH, DeMichele MA, et ah Production of gastrin-reteasing peptide by a non-small cell lung carcinoma cell line adapted to serum-free and growth factor- free conditions. J Biol Chem 269:8596, 1994 75. Sithanandam G, Latif F, Duh FM, et al: 3pK, a new mitogen-activated protein kinase-- activated protein kinase located in the small cell lung cancer tumor suppressor gene region. Mol Cell Biol 16:868, 1996 76. Skillrud DM, Offord KP, Miller RD: Higher risk of lung cancer in chronic obstructive pulmonary disease. A prospective, matched, controlled study. Ann Intern Med 105:503, 1986 77. Slaughter DP, Southwick HW, Smejkal W: Field cancerization in oral stratified squa- mous epithelium. Clinical implications of multicentric origin. Cancer 6:963, 1953 78. Smith AL, Hung J, Walker L, et ah Extensive areas of aneuploidy are present in the respiratory epithelium of lung cancer patients. Br J Cancer 73:203, 1996 79. Sozzi G, Miozzo M, Donghi R, et ah Deletions of 17p and p53 mutations in preneoplas- tic lesions of the lung. Cancer Res 52:6079, 1992 80. Sozzi G, Miozzo M, Pastorino U, et al: Genetic evidence for an independent origin of multiple preneoplastic and neoplastic lung lesions. Cancer Res 55:135, 1995 81. Sozzi G, .Veronese ML, Negrini M, et al: The FHIT gent 3p14.2 is abnormal in lung cancer. Cell 85:17, 1996 82. Sugio K, Kishimoto Y, Virmani AK, et al: K-ras mutations are a relatively late event in the pathogenesis of lung carcinomas. Cancer Res 54:5811, 1994 83. Sundaresan V, Ganly P, Hasleton P, et al: p53 and chromosome 3 abnormalities,

234 MILLER & FRANKLIN characteristic of malignant lung tumours, are detectable in preinvasiv.e lesions of the bronchus. Oncogene 7:1989, 1992 84. Tockman MS, Anthonisen NR, Wright EC, Donithan MG: Airways obstruction and the risk for lung cancer. Ann Intern Med 106:512, 1987 85. Todd S, Roche J, Hahner L, et al: YAC contigs covering an 8-megabase region of 3p deleted in the small-cell lung cancer cell line U2020. Genomics 25:19, 1995 86. Traver RD, Siegel D, Beall HD, et al: Characterization of polymorphism of NAD(P)H: Quinone oxidoreductase (DT-diqphorase). Br J Cancer 75:69, 1997 87. Vogelstein B, Kinzler KW: The multistep nature of cancer. Trends Genet 9:138, 1993 88. Whang-Peng J, Kao-Shan CS, Lee EC, et al: Specific chromosome defect associated with human small-cell lung cancer; deletion 3p(14-23). Science 215:181, 1982 89. Wiest JS, Franklin WA, Otstot JT, et al: Identification of a novel region of homozygous deletion on chromosome 9p in squamous cell carcinoma of the lung: the location of a putative tumor suppressor gene. Cancer Res 57:1, 1997 90. Wingo PA, Tong T, Bolden S: Cancer statistics, 1995 [published erratum appears in CA Cancer J Clin 45:127-128, 1995]. CA Cancer J Clin 45:8, 1995 91. Xiao S, Li D, Corson JM, et al: Codeletion of p15 and pl6 genes in primary non-small cell lung carcinoma. Cancer Res 55:2968, 1995 92. Yamakawa K, Takahashi T, Horio Y, et al: Frequent homozygous deletions in lung cancer cell lines detected by a DNA marker located at 3p21.3-p22. Oncogene 8:327, 1993 Address reprint requests to York E. Miller, MD Respiratory 111A Denver Veterans Affairs Medical Center 1055 Clermont St. Denver, CO 80220 MULTIDISCIPLINARY CARE LUNG CANCER PATIENTS INTE< O James L. Mul PhD, In 1996, a prolectt United States resultin, most other cancers h: has nullified any impt mortality benefit frorc lights the need for a While new understan~ lated into advances in directed at empoweri: so they can reduce United States are alre, "managed lifestyle." burden of chronic di,, emphasizing such prc This article consi, nal prevention-orientt tion of new molecul costs to our health c, ': .... Strategic applica! future health care vi [:tom ti~e Biomarkers an National Cancer Inst Department of Envir( and Public Health, Bc I tt:M..VFOLOGY \ ~+ ~I U +",.1I+~ l I • NUMBER 2 • L