© 1999 by Oxford University Press
Journal of the National Cancer Institute, Vol. 91, No. 21, 1863-1868,
November 3, 1999
© 1999 Oxford University Press
REPORTS |
Multiple Clonal Abnormalities in the Bronchial Epithelium of Patients With Lung Cancer
Affiliations of authors: I.-W. Park, Hamon Center for Therapeutic Oncology and Research, University of Texas Southwestern Medical Center, Dallas, and Department of Internal Medicine, Chung-Ang University Medical School, Seoul, Korea; I. I. Wistuba, A. K. Virmani, Hamon Center for Therapeutic Oncology and Research, University of Texas Southwestern Medical Center; A. Maitra, A. F. Gazdar, Hamon Center for Therapeutic Oncology and Research and Department of Pathology, University of Texas Southwestern Medical Center; S. Milchgrub, Department of Pathology, University of Texas Southwestern Medical Center; J. D. Minna, Hamon Center for Therapeutic Oncology and Research and Departments of Medicine and Pharmacology, University of Texas Southwestern Medical Center.
Correspondence to: Adi F. Gazdar, M.D., Hamon Center for Therapeutic Oncology and Research, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX (e-mail: gazdar{at}simmons.swmed.edu).
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionNotesReferences |
|---|
BACKGROUND: Several molecular changes, including loss of heterozygosity (i.e., deletion of one copy of allelic DNA sequences) and alterations in microsatellite DNA, have been detected early in the pathogenesis of lung cancer, even in histologically normal epithelium. In the bronchial epithelium of patients with lung cancer, we have determined the frequency, size, and patterns of molecularly abnormal clonal patches. METHODS: We studied formalin-fixed, paraffin-embedded samples from 16 surgically resected lung carcinomas (five squamous cell carcinomas, four small-cell carcinomas, six adenocarcinomas, and one large-cell carcinoma). From each carcinoma, we microdissected foci (each containing about 200 cells) of tumor tissue and equivalent samples of histologically normal and abnormal epithelium. Furthermore, multiple discontinuous foci of bronchial epithelium were analyzed from methanol-fixed samples from three additional patients with lung cancer (two with squamous cell carcinoma and one with adenocarcinoma). We used two-step polymerase chain reaction-based assays involving 12 microsatellite markers at seven chromosomal regions frequently deleted in lung cancer. RESULTS: Two hundred eighteen foci of nonmalignant bronchial epithelium (195 of histologically normal or slightly abnormal epithelium and 23 of dysplastic epithelium) were studied from the 19 surgically resected lobectomy specimens. Thirteen (68%) of the 19 specimens had at least one focus of bronchial epithelium with molecular changes. At least one molecular abnormality was detected in 32% of the 195 histologically normal or slightly abnormal foci and in 52% of the 23 dysplastic foci. Extrapolating from our two-dimensional analyses, we estimate that most clonal patches contain approximately 90 000 cells. Although, in a given individual, tumors appeared homogeneous with respect to molecular changes, the clonally altered patches of mildly abnormal epithelium were heterogeneous. CONCLUSIONS: Our findings indicate that multiple small clonal or subclonal patches containing molecular abnormalities are present in normal or slightly abnormal bronchial epithelium of patients with lung cancer.
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNotesReferences |
|---|
As with other epithelial malignancies, lung cancers are believed to arise after a series of progressive histopathologic changes (preneoplastic lesions) in the bronchial epithelium. In centrally arising squamous cell carcinomas, the changes include (from the earliest to the most advanced) hyperplasia, metaplasia, dysplasia of increasing severity, carcinoma in situ, invasive cancer, and metastatic cancer (1-3). However, the sequence of events preceding small-cell lung carcinomas and peripherally arising adenocarcinomas are not fully understood (4,5). Preneoplastic changes frequently are extensive and multifocal, occurring throughout the respiratory tree, a phenomenon referred to as field cancerization (6). Tests for loss of heterozygosity (LOH) by use of polymorphic microsatellite DNA markers are frequently used to identify allelic losses at specific chromosomal loci. Allelic losses at the short arms of chromosomes 3, 9, and 17 (3p, 9p, and 17p, respectively) occur relatively early in the multistage development of invasive lung cancer (7-11). Microsatellite alterations (representing changes in the size of one or both alleles) are found in many human cancers, including lung cancer, and may serve as clonal markers for early cancer detection (12,13). Very high incidences of LOH and microsatellite alterations have been detected in the histologically normal and mildly abnormal bronchial epithelia (hyperplasia or squamous metaplasia) of most subjects with lung cancer and in current and former smokers (8,14-16).
The purpose of this study was to determine the frequency, size, and patterns of molecularly abnormal clonal patches in the bronchial epithelium of patients with lung cancer. A determination of the size of the clonal patches with such changes may be important for understanding tumorigenesis and for the evaluation of chemoprevention studies.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNotesReferences |
|---|
Tumor Specimens
Specimens from 19 surgically resected small-cell lung carcinomas and non-small-cell lung carcinomas were studied. These specimens were from 13 male patients and six female patients who ranged from 37 to 77 years of age (mean, 58 years). All specimens were from curative intent lung cancer resections (lobectomies). Patients were staged by use of the International System for Staging Lung Cancer, and all had stage I and II disease (17). Specimens were obtained as part of an Institutional Review Board-approved research study. The 15 patients for whom data were available were current or past heavy smokers (>20 pack-years of smoking exposure [1 pack-year = one pack smoked per day for 1 year]). Archival microslides were obtained from 16 carcinomas (five squamous cell carcinomas, four small-cell carcinomas, six adenocarcinomas, and one large-cell carcinoma). From each carcinoma, a single cross-section of one lobar, segmental, or subsegmental bronchus with partially or completely intact histologically normal or abnormal bronchial epithelium was selected. Whenever possible, the cross-section was selected as close to the proximal resection margin, provided that the tumor was not identified in the same microsection. Serial 5-µm sections were cut from formalin-fixed, paraffin-embedded tissues. All slides were stained with hematoxylin-eosin, and one of the slides was coverslipped. The coverslipped slide was used as a guide to localize regions of interest for microdissection of the other slides. Contiguous foci of approximately 200 cells along the entire epithelial surface were carefully counted and microdissected. We selected this cell number because it was the minimum number of cells required for multiplex analysis of the multiple markers studied and because it was the minimum number of epithelial cells present in bronchial biopsy specimens with an intact layer of histologically normal cells (Gazdar AF: unpublished observations).
In addition, methanol-fixed samples prepared by the epithelial aggregate separation and isolation method (EASI preparations) (19) were available from three more patients (two with squamous cell carcinomas and one with adenocarcinoma). For EASI preparations, the surfaces of large bronchi near the proximal resection margin were gently scraped with the edge of a plain uncharged microscope slide. The cellular materials so obtained were evenly spread onto the surface of one or more uncharged slides. The slides were immediately fixed in 95% methanol, stained with hematoxylin-eosin, and not covered with a coverslip. Tumors and preneoplastic lesions were identified by three pathologists (S. Milchgrub, A. Maitra, and A. F. Gazdar) who used standard published criteria (18).
We used laser-captured microdissection (20) or a manual
micromanipulator, as previously described (7), for the serial
microdissection of foci, each containing approximately 200 cells (Figs. 1
and 2). Stromal cells or lymphocytes from the same slides were used as a
source of constitutional (normal) DNA from each specimen. Multiple foci of a similar size were
also microdissected from the EASI preparations. To investigate the heterogeneity of tumor tissue,
we microdissected several areas of 200-1000 cells. After DNA extraction, 5 µL of the
proteinase K-digested samples was used for each multiplex polymerase chain reaction (PCR).
|
|
Polymorphic DNA Markers and PCR-Based LOH Analysis
To evaluate LOH and microsatellite alterations, we used primers flanking 12 dinucleotide and multinucleotide microsatellite repeat polymorphisms located at the following seven gene or chromosomal locations: 3p12 (D3S1274), 3p14-21 (D3S1766), 3p21 (D3S1029), 3p22-24.2 (D3S2432, D3S1351, and D3S1537), 3p24.3 (D3S1244 and D3S1293), 9p21 (IFNA and D9S1748), and 17q13.1 (the dinucleotide and the pentanucleotide repeats of the TP53 [p53] gene). These genes or locations were selected because they are sites of frequent allelic losses in lung cancers (21). Primer sequences can be obtained from the Genome Database, with the exceptions of two pentanucleotide and dinucleotide repeats in the p53 gene (22). Nested PCR (8) or two-round PCR (15) methods were used. Multiplex PCR was performed during the first amplification, followed by uniplex PCR for individual markers. LOH was scored visually as more than a 50% reduction of the autoradiographic signal corresponding to one of the two alleles in informative specimens (in most cases, loss was complete). Microsatellite alterations were detected by a shift in the mobility of one or both alleles with respect to the parental alleles.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNotesReferences |
|---|
Microdissection of Foci of Bronchial Epithelium
From the 19 surgical resection specimens, a total of 218 foci of
bronchial epithelium (each containing approximately 200 cells) were
dissected (175 from archival paraffin-embedded slides and 43 from
methanol-fixed EASI preparations). The molecular findings are
summarized in Table 1. For the archival materials,
the foci were dissected from the entire bronchial circumference
selected for study and were consecutively numbered from a preselected
starting point (Fig. 2
), although, in some cases, the mucosa was not
intact throughout the cross-section. The number of foci per specimen
varied from five to 25 (mean = 11.4 foci per specimen). The foci from
EASI preparations represented isolated epithelial cell clumps, and
their spatial relationship to each other could not be determined. Foci
of squamous metaplasia were present in five of the 19 specimens, and
dysplasia (mild or moderate) was present in four. Of the 218 individual
foci microdissected from both types of preparation, 23 were dysplastic
and the remainder were histologically normal or had mild changes (i.e.,
metaplasia or hyperplasia). At least one focus was dissected from all of the
corresponding tumors, and multiple foci were dissected from four tumors.
|
Identification of Clonal (or Subclonal) Patches of Bronchial Epithelium
Molecular changes were detected in at least one focus of
nonmalignant epithelium from 13 (68%) of the 19 specimens studied. Of
the 16 paraffin-embedded preparations, 60 (34%) foci from 10 specimens
had at least one molecular change (either LOH or microsatellite
alteration; Fig. 2). When adjacent foci contained identical changes,
the contiguous foci were regarded as a single patch of altered cells
(Fig. 2). A total of 54 patches were identified that consisted of 49
single foci, and five larger patches were identified that each had two
or three foci. Thus, by two-dimensional analysis, the vast majority
(91%) of the patches contained a single focus of about 200 cells, and
the remainder contained 400-600 cells. Of three EASI preparations, 15
(35%) of 43 foci from all preparations had one or more molecular
changes. Because the spatial relationship between the foci could not be
determined, each altered focus was regarded as a separate clonal patch.
Molecular Changes in Clonal Patches
At least one molecular change was identified in 12 (52%) of the 23 total dysplastic foci and in 63 (32%) of the remaining 195 foci with normal or mildly abnormal histology. As discussed previously, these 75 foci with molecular changes were believed to represent 69 clonal or subclonal patches. The most frequently detected abnormality was LOH at one or more regions of 3p (49 [71%] of the molecularly altered patches). Microsatellite alterations were detected in 22 patches (32%). LOH at p53 gene was detected in seven patches (10%). Although we failed to detect LOH of 9p in any patch, only five of the 16 samples were informative for either of the two 9p markers used.
Relationship Between Changes in Clonal Patches and in Corresponding Tumors
Molecular changes were present in all 13 tumor specimens, with one
or more altered clones present in the accompanying nonmalignant
epithelium (Figs. 2 and 3). We compared the
pattern
of changes in the clonal patches with those present in their
corresponding tumors. If the changes in the clonal patches were
identical with those present in the corresponding tumor (even if the
tumor had more extensive changes), we regarded the clonal patches as
possibly representing direct precursor lesions. We regarded the changes
in the clonal patches as different from the corresponding tumor if 1)
both lesions had LOH with a particular marker but the losses involved
different parental alleles or if 2) LOHs or microsatellite alterations
present in the patches were not present in the corresponding tumor. If
we apply these criteria to the 69 clonal patches, nine (13%) would
represent possible precursor lesions and 60 (87%) would represent
clones independent of the corresponding tumor.
|
Clonal Changes in Tumors
To determine whether heterogeneity was present in tumor tissue, we compared the pattern of molecular changes present in multiple areas. From four tumors (one squamous cell, one small-cell lung carcinoma, and two adenocarcinomas), we microdissected six to 11 foci per tumor, each containing 200-1000 cells. Foci were selected from several individual tumor blocks and as far away from each other as possible on individual microslides. In all cases, all foci from individual tumors had identical patterns of molecular changes (data not shown).
Validation of Results of Molecular Analyses
PCR-based assays by use of small quantities of DNA from formalin-fixed, paraffin-embedded materials may result in preferential amplification of the smaller of a pair of alleles and spurious band shifts, resulting in the artifactual appearance of apparent allelic losses and microsatellite alterations. For these reasons, we did further analyses to confirm the validity of our results. As mentioned above, molecular analyses of 32 independently dissected areas of four tumors were 100% reproducible. Allelic losses or alterations in foci of bronchial epithelium were confirmed by redissection and reanalysis of the same foci from replicate slides (24 of 24 foci). Electronic capture of images of the bronchial cross-sections analyzed aided precise redissection of the foci. Of all specimens with allelic losses, the larger allele was lost in 57% and the lower allele was lost in 43%.
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNotesReferences |
|---|
We determined the size of clonal patches of bronchial epithelium (both normal and abnormal) by studying molecular changes (allelic losses and microsatellite alterations) at several locations that are frequently involved in lung cancer pathogenesis. We used a two-dimensional model and carefully microdissected foci of epithelium from the circumference of cross-sections of large bronchi, usually near the proximal surgical resection margin.
We dissected epithelial foci, each containing approximately 200 cells, because, in our experience, this is the minimum number of epithelial cells present in a single 5-µm section of a histologically normal biopsy specimen obtained by fluorescence bronchoscopy. The major finding of our study is that multiple small clonal patches of molecular abnormalities are present in the histologically normal, as well as abnormal, bronchial epithelium of patients with lung cancer. At least one molecular abnormality was detected in 32% of histologically normal or slightly abnormal foci and in 52% of dysplastic foci. Thus, about one third of the epithelial surface of large bronchi of patients with lung cancer may have molecular damage. We had previously demonstrated at least one molecular abnormality in 48% of histologically normal bronchial biopsy specimens from smokers without cancer (16). However, areas of abnormal fluorescence were preferentially selected. In this study, the somewhat lower percentage (32%) of changes in histologically normal or slightly abnormal epithelium from lung cancer patients may reflect the nonselected circumferential analysis of a single bronchus. As described in the "Results" section, our data were confirmed and validated by redissection and reanalysis of epithelial foci. This study and previous studies (14,16) support the concept that numerous small clonally altered foci are present in the bronchial epithelium of the majority of smokers and patients with lung cancer.
Previous studies (14-16) have demonstrated multiple molecular abnormalities, especially allelic losses at 3p and 9p, in bronchial biopsy specimens from smokers and patients with squamous cell lung cancer. Losses at chromosome 3 were the most common molecular change observed in this study. Because relatively few of our specimens were informative for the two 9p loci studied, we could not evaluate the role of 9p losses fully. The clonal patches, as analyzed in our two-dimensional examination, indicated that the patches were relatively small, usually between 200 and 400 cells. The largest clonal patch identified was 600 cells. If we assume that the surface dimensions of the patches are approximately equal in size and accept 300 cells as the average number of cells in a patch as measured in two dimensions, then the approximate number of cells per clonal patch would be 90 000. This number is less than the average number of epithelial cells present in hyperplastic or dysplastic biopsy specimens (Gazdar AF, MacAulay C, Smith A, Lam S: unpublished results).
Our previous studies (15,16,23) have indicated a possible clonal relationship between the molecularly altered foci of bronchial epithelium present in smokers and patients with lung cancer. For allelic losses, the same parental allele was lost in biopsy specimens obtained from widely dispersed parts of the bronchial epithelium, a phenomenon that we have referred to as allele-specific loss (7). Allele-specific loss may indicate the widely dispersed presence of individual molecularly altered clones of cells (24). A recent report (23) described a single identical point mutation in multiple areas of histologically abnormal bronchial epithelium in both lungs of a smoker without lung cancer and provided further evidence that individual clones may disperse progeny cells throughout the bronchial epithelium. In this study, we failed to find a clonal relationship (or allele-specific losses) between the molecularly altered patches or between the patches and the corresponding tumors. Thus, our current findings appear to contradict our previous studies. However, in this study, we blindly selected small contiguous patches of epithelium, whereas, in the previous studies, we selected, in general, patches that had abnormal histology or fluorescence patterns. This study is consistent with the findings of Hittelman (25,26) who found evidence for the presence of numerous small monosomic or trisomic clonal and subclonal patches in smoking-damaged upper aerodigestive tract epithelium, as determined by fluorescense in situ hybridization analyses. One possible explanation for these apparently contradictory findings is that numerous small individual clonal patches of histologically or molecularly altered epithelium are present in the lungs of smokers. Subsequently, a small number of more advanced clones acquire the ability to become widely dispersed, and these clones are the direct precursors of invasive tumors. A possible problem with interpretation of the studies cited above is that evidence directly linking the changes to smoking exposure is limited. Equivalent specimens from aged-matched nonsmoking controls are exceedingly difficult to obtain. Examination of a modest number of bronchial biopsy specimens from lifetime nonsmokers identified few (14) or no (16) molecular changes. However, further studies on specimens from nonsmokers are required for confirmation.
It is of interest to compare our findings with observations from other cancers arising in widely damaged epithelia that show a field effect. In Barrett's disease, a precursor lesion of esophageal adenocarcinoma, large patches of epithelium have metaplastic and dysplastic changes. Specimens of Barrett's epithelium from separate sites may have identical p53 mutations, suggesting a clonal origin (27). Thus, the clonally altered patches with molecular abnormalities are relatively large. These findings are in contrast to observations from sun-damaged skin, which predisposes to nonmelanotic skin cancer. Sun-exposed human skin contains clonal patches of p53-mutated keratinocytes, arising from the dermal-epidermal junction and from hair follicles (28). These clones of 60-3000 cells were present at frequencies exceeding 40 clones/cm2 and together may involve as much as 4% of the epidermis. Clones in sun-exposed skin are both more frequent and larger than clones in sun-shielded skin. The authors concluded that, in addition to being a tumorigenic mutagen, sunlight acts as a tumor promoter by favoring the clonal expansion of p53-mutated cells (28). These combined actions of sunlight result in normal individuals carrying a substantial burden of keratinocytes predisposed to cancer. The findings in smoke-damaged epithelium may be more comparable to those present in sun-damaged skin than to Barrett's esophagus and suggest that exposure to carcinogens present in tobacco smoke results in multiple molecularly altered cells that have a growth advantage for clonal expansion. Larger patches of molecularly altered epithelium, as identified by fluorescence bronchoscopy, may be more likely to be clonally related to each other and to any subsequently arising invasive tumor. As expected, all foci microdissected from tumor cells appeared to be clonally related, consistent with the theory that most if not all tumors are clonally derived.
In conclusion, our findings indicate that multiple small clonal or subclonal patches of molecular abnormalities (usually smaller in size than the average bronchial biopsy specimen obtained by fluorescence bronchoscopy) can be detected in normal or slightly abnormal bronchial epithelium of patients with lung cancer. The clonal patches of bronchial epithelium with molecular changes were usually small. Although the tumors were homogeneous for molecular changes, the clonally altered patches of mildly abnormal bronchial epithelium were usually heterogeneous. The presence of numerous small clonal patches of molecularly altered cells indicates that either arbitrarily selected (14) or fluorescence-guided (16) biopsy specimens of smoking-damaged bronchial epithelium will frequently contain the presence of such clones. Our findings may help in the design of chemoprevention studies by use of sequential bronchial biopsy specimens for the monitoring of intermediate biomarkers as end points.
|
NOTES |
|---|
Supported by a Public Health Service grant P50CA70907 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNotesReferences |
|---|
1 Saccomanno G, Archer VE, Auerbach O, Saunders RP, Brennan LM. Development of carcinoma of the lung as reflected in exfoliated cells. Cancer 1974;33:256-70.[CrossRef][Web of Science][Medline]
2 Auerbach O, Hammond EC, Garfinkel L. Changes in bronchial epithelium in relation to cigarette smoking, 1955-1960 vs. 1970-1977. N Engl J Med 1979;300:381-5.[Abstract]
3 Saccomanno G, Archer VE, Saunders RP, Auerbach O, Klein MG. Early indices of cancer risk among uranium miners with reference to modifying factors. Ann N Y Acad Sci 1976;271:377-83.[CrossRef][Web of Science][Medline]
4 Weng SY, Tsuchiya E, Kasuga T, Sugano H. Incidence of atypical bronchioloalveolar cell hyperplasia of the lung: relation to histological subtypes of lung cancer. Virchows Arch A Pathol Anat Histopathol 1992;420:463-71.[CrossRef][Web of Science][Medline]cancerlit;92303122
5 Nakanishi K. Alveolar epithelial hyperplasia and adenocarcinoma of the lung. Arch Pathol Lab Med 1990;114:363-8.[Web of Science][Medline]cancerlit;90210848
6 Strong MS, Incze J, Vaughan CW. Field cancerization in the aerodigestive tract—its etiology, manifestation, and significance. J Otolaryngol 1984;13:1-6.[Web of Science][Medline]cancerlit;84188620
7
Hung J, Kishimoto Y, Sugio K, Virmani A, McIntire DD, Minna
JD, et al. Allele-specific chromosome 3p deletions occur at an early stage in the pathogenesis of
lung carcinoma [published erratum appears in JAMA 1995;273:1908]. JAMA 1995;273:558-63.
8
Kishimoto Y, Sugio K, Hung JY, Virmani AK, McIntire DD,
Minna JD, et al. Allele-specific loss of chromosome 9p loci in preneoplastic lesions accompanying
non-small-cell lung cancers. J Natl Cancer Inst 1995;87:1224-9.
9 Sundaresan V, Ganly P, Hasleton P, Rudd R, Sinha G, Bleehen NM, et al. p53 and chromosome 3 abnormalities, characteristic of malignant lung tumours, are detectable in preinvasive lesions of the bronchus. Oncogene 1992;7:1989-97.[Web of Science][Medline]cancerlit;93026374
10
Thiberville L, Payne P, Vielkinds J, LeRiche J, Horsman D,
Nouvet G, et al. Evidence of cumulative gene losses with progression of premalignant epithelial
lesions to carcinoma of the bronchus. Cancer Res 1995;55:5133-9.
11
Chung GT, Sundaresan V, Hasleton P, Rudd R, Taylor R,
Rabbitts PH. Clonal evolution of lung tumors. Cancer Res 1996;56:1609-14.
12
Mao L, Lee DJ, Tockman MS, Erozan YS, Askin F, Sidransky
D. Microsatellite alterations as clonal markers for the detection of human cancer. Proc Natl
Acad Sci U S A 1994;91:9871-5.
13
Miozzo M, Sozzi G, Musso K, Pilotti S, Incarbone M, Pastorino
U, et al. Microsatellite alterations in bronchial and sputum specimens of lung cancer patients. Cancer Res 1996;56:2285-8.
14
Mao L, Lee JS, Kurie JM, Fan YH, Lippman SM, Lee JJ, et al.
Clonal genetic alterations in the lungs of current and former smokers. J Natl Cancer Inst 1997;89:857-62.
15 Wistuba II, Behrens C, Milchgrub S, Bryant D, Hung J, Minna JD, et al. Sequential molecular abnormalities are involved in the multistage development of squamous cell lung carcinoma. Oncogene 1999;18:643-50.[CrossRef][Web of Science][Medline]cancerlit;99142920
16
Wistuba II, Lam S, Behrens C, Virmani AK, Fong KM, LeRiche
J, et al. Molecular damage in the bronchial epithelium of current and former smokers. J
Natl Cancer Inst 1997;89:1366-73.
17
Mountain CF. Revisions in the International System for Staging
Lung Cancer. Chest 1997;111:1710-7.
18 Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E, and collaborators from 14 countries. World Health Organization Classification of Lung and Pleural Tumors. 3rd ed. Berlin (Germany): Springer-Verlag; 1999.
19 Maitra A, Wistuba II, Virmani AK, Sakaguchi M, Park I, Stucky A, et al. Enrichment of epithelial cells for molecular studies. Nat Med 1999;5:459-63.[CrossRef][Web of Science][Medline]cancerlit;99217590
20
Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang
Z, Goldstein SR, et al. Laser capture microdissection. Science 1996;274:998-1001.
21 Virmani AK, Fong KM, Kodagoda D, McIntire D, Hung J, Tonk V, et al. Allelotyping demonstrates common and distinct patterns of chromosomal loss in human lung cancer types. Genes Chromosomes Cancer 1998;21:308-19.[CrossRef][Web of Science][Medline]cancerlit;98220096
22 Cawkwell L, Lewis FA, Quirke P. Frequency of allele loss of DCC, p53, RBI, WT1, NF1, NM23 and APC/MCC in colorectal cancer assayed by fluorescent multiplex polymerase chain reaction. Br J Cancer 1994;70:813-8.[Web of Science][Medline]cancerlit;95034041
23 Franklin WA, Gazdar AF, Haney J, Wistuba II, La Rosa FG, Kennedy T, et al. Widely dispersed p53 mutation in respiratory epithelium. A novel mechanism for field carcinogenesis [published erratum appears in J Clin Invest 1997;100:2639]. J Clin Invest 1997;100:2133-7.[Web of Science][Medline]cancerlit;97474785
24
Sidransky D. Importance of chromosome 9p loss in human lung
cancer [editorial]. J Natl Cancer Inst 1995;87:1201-2.
25 Hittelman WN. Genetic instability assessments in the lung cancerization field. In: Brambilla C, Brambilla E, editors. Lung tumors: fundamental biology and clinical management. New York (NY): Marcel Dekker, Inc.; 1998. p. 255-67.
26 Hittelman WN. Molecular cytogenetic evidence for multistep tumorigenesis: implications for risk assessments and early detection. In: Srivastava S, Hensen DE, Gazdar A, editors. Molecular pathology of cancer. Amsterdam (The Netherlands): IOS Press; 1999. p. 385-404.
27
Casson AG, Mukhopadhyay T, Cleary KR, Ro JY, Levin B,
Roth JA. p53 gene mutations in Barrett's epithelium and esophageal cancer. Cancer
Res 1991;51:4495-9.
28
Jonason AS, Kunala S, Price GJ, Restifo RJ, Spinelli HM,
Persing JA, et al. Frequent clones of p53-mutated keratinocytes in normal human skin. Proc Natl Acad Sci U S A 1996;93:14025-9.
Manuscript received June 16, 1999; revised September 2, 1999; accepted September 8, 1999.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Rhiner and E. Moreno Super competition as a possible mechanism to pioneer precancerous fields Carcinogenesis, May 1, 2009; 30(5): 723 - 728. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Wang, M. Wang, G. T. MacLennan, F. W. Abdul-Karim, J. N. Eble, T. D. Jones, F. Olobatuyi, R. Eisenberg, O. W. Cummings, S. Zhang, et al. Evidence for Common Clonal Origin of Multifocal Lung Cancers J Natl Cancer Inst, April 15, 2009; 101(8): 560 - 570. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Walser, X. Cui, J. Yanagawa, J. M. Lee, E. Heinrich, G. Lee, S. Sharma, and S. M. Dubinett Smoking and Lung Cancer: The Role of Inflammation Proceedings of the ATS, December 1, 2008; 5(8): 811 - 815. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Herbst, J. V. Heymach, and S. M. Lippman Lung Cancer N. Engl. J. Med., September 25, 2008; 359(13): 1367 - 1380. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Lam, B. Standish, C. Baldwin, A. McWilliams, J. leRiche, A. Gazdar, A. I. Vitkin, V. Yang, N. Ikeda, and C. MacAulay In vivo Optical Coherence Tomography Imaging of Preinvasive Bronchial Lesions Clin. Cancer Res., April 1, 2008; 14(7): 2006 - 2011. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Gray, J. T. Mao, E. Szabo, M. Kelley, J. Kurie, and G. Bepler Lung Cancer Chemoprevention: ACCP Evidence-Based Clinical Practice Guidelines (2nd Edition) Chest, September 1, 2007; 132(3_suppl): 56S - 68S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Lippman and J. V. Heymach The Convergent Development of Molecular-Targeted Drugs for Cancer Treatment and Prevention Clin. Cancer Res., July 15, 2007; 13(14): 4035 - 4041. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Angeloni Molecular analysis of deletions in human chromosome 3p21 and the role of resident cancer genes in disease Brief Funct Genomic Proteomic, May 24, 2007; (2007) elm007v1. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Li, N. W. Todd, Q. Qiu, T. Fan, R. Y. Zhao, W. H. Rodgers, H.-B. Fang, R. L. Katz, S. A. Stass, and F. Jiang Genetic Deletions in Sputum as Diagnostic Markers for Early Detection of Stage I Non-Small Cell Lung Cancer Clin. Cancer Res., January 15, 2007; 13(2): 482 - 487. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Vineis and K. Husgafvel-Pursiainen Air pollution and cancer: biomarker studies in human populations Carcinogenesis, November 1, 2005; 26(11): 1846 - 1855. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. E. Miller Pathogenesis of Lung Cancer: 100 Year Report Am. J. Respir. Cell Mol. Biol., September 1, 2005; 33(3): 216 - 223. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Safar, H. Spencer III, X. Su, M. Coffey, C. A. Cooney, L. D. Ratnasinghe, L. F. Hutchins, and C.-Y. Fan Methylation Profiling of Archived Non-Small Cell Lung Cancer: A Promising Prognostic System Clin. Cancer Res., June 15, 2005; 11(12): 4400 - 4405. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Meuwissen and A. Berns Mouse models for human lung cancer Genes & Dev., March 15, 2005; 19(6): 643 - 664. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. I. Wistuba Histologic Evaluation of Bronchial Squamous Lesions: Any Role in Lung Cancer Risk Assessment? Clin. Cancer Res., February 15, 2005; 11(4): 1358 - 1360. [Full Text] [PDF]
|
|
|
|
 |
|
 D. Moro-Sibilot, F. Fievet, M. Jeanmart, S. Lantuejoul, F. Arbib, M.H. Laverriere, E. Brambilla, and C. Brambilla Clinical prognostic indicators of high-grade pre-invasive bronchial lesions Eur. Respir. J., July 1, 2004; 24(1): 24 - 29. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Kaminski and M. Krupsky Gene Expression Patterns, Prognostic and Diagnostic Markers, and Lung Cancer Biology Chest, May 1, 2004; 125(5_suppl): 111S - 115S. [Full Text] [PDF]
|
|
|
|
|
|
H. Kim, G.-L. Xu, A. C. Borczuk, S. Busch, J. Filmus, M. Capurro, J. S. Brody, J. Lange, J. M. D'Armiento, P. B. Rothman, et al. The Heparan Sulfate Proteoglycan GPC3 Is a Potential Lung Tumor Suppressor Am. J. Respir. Cell Mol. Biol., December 1, 2003; 29(6): 694 - 701. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K M Fong, Y Sekido, A F Gazdar, and J D Minna Lung cancer * 9: Molecular biology of lung cancer: clinical implications Thorax, October 1, 2003; 58(10): 892 - 900. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. M. Braakhuis, M. P. Tabor, J. A. Kummer, C. R. Leemans, and R. H. Brakenhoff A Genetic Explanation of Slaughter's Concept of Field Cancerization: Evidence and Clinical Implications Cancer Res., April 15, 2003; 63(8): 1727 - 1730. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Sutedja New techniques for early detection of lung cancer Eur. Respir. J., January 1, 2003; 21(39_suppl): 57S - 66s. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. H. Dragnev, D. Stover, and E. Dmitrovsky Lung Cancer Prevention: The Guidelines Chest, January 1, 2003; 123 (2009): 60S - 71S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. C. Slebos, D. S. Oh, D. M. Umbach, and J. A. Taylor Mutations in Tetranucleotide Repeats following DNA Damage Depend on Repeat Sequence and Carcinogenic Agent Cancer Res., November 1, 2002; 62(21): 6052 - 6060. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Cave-Riant, B. Cuillerier, M. Beau-Faller, N. Martinet, F. Alla, C. Bronner, A. Schneider, P. Oudet, and M. P. Gaub Association of Genetic Defects in Primary Resected Lung Adenocarcinoma Revealed by Targeted Allelic Imbalance Analysis Am. J. Respir. Cell Mol. Biol., October 1, 2002; 27(4): 495 - 502. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Ji, M. Nishizaki, B. Gao, D. Burbee, M. Kondo, C. Kamibayashi, K. Xu, N. Yen, E. N. Atkinson, B. Fang, et al. Expression of Several Genes in the Human Chromosome 3p21.3 Homozygous Deletion Region by an Adenovirus Vector Results in Tumor Suppressor Activities in Vitro and in Vivo Cancer Res., May 1, 2002; 62(9): 2715 - 2720. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Belinsky, W. A. Palmisano, F. D. Gilliland, L. A. Crooks, K. K. Divine, S. A. Winters, M. J. Grimes, H. J. Harms, C. S. Tellez, T. M. Smith, et al. Aberrant Promoter Methylation in Bronchial Epithelium and Sputum from Current and Former Smokers Cancer Res., April 1, 2002; 62(8): 2370 - 2377. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. O'Shaughnessy, G. J. Kelloff, G. B. Gordon, A. J. Dannenberg, W. K. Hong, C. J. Fabian, C. C. Sigman, M. M. Bertagnolli, S. P. Stratton, S. Lam, et al. Treatment and Prevention of Intraepithelial Neoplasia: An Important Target for Accelerated New Agent Development : Recommendations of the American Association for Cancer Research Task Force on the Treatment and Prevention of Intraepithelial Neoplasia Clin. Cancer Res., February 1, 2002; 8(2): 314 - 346. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Uematsu, A. Yoshimura, A. Gemma, H. Mochimaru, Y. Hosoya, S. Kunugi, K. Matsuda, M. Seike, F. Kurimoto, K. Takenaka, et al. Aberrations in the Fragile Histidine Triad (FHIT) Gene in Idiopathic Pulmonary Fibrosis Cancer Res., December 1, 2001; 61(23): 8527 - 8533. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Aoyagi, T. Yokose, Y. Minami, A. Ochiai, T. Iijima, Y. Morishita, T. Oda, K. Fukao, and M. Noguchi Accumulation of Losses of Heterozygosity and Multistep Carcinogenesis in Pulmonary Adenocarcinoma Cancer Res., November 1, 2001; 61(21): 7950 - 7954. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Vilkki, J.-L. Tsao, A. Loukola, M. Poyhonen, O. Vierimaa, R. Herva, L. A. Aaltonen, and D. Shibata Extensive Somatic Microsatellite Mutations in Normal Human Tissue Cancer Res., June 1, 2001; 61(11): 4541 - 4544. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. G. Burbee, E. Forgacs, S. Zochbauer-Muller, L. Shivakumar, K. Fong, B. Gao, D. Randle, M. Kondo, A. Virmani, S. Bader, et al. Epigenetic Inactivation of RASSF1A in Lung and Breast Cancers and Malignant Phenotype Suppression J Natl Cancer Inst, May 2, 2001; 93(9): 691 - 699. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Roth, N. Hu, M. R. Emmert-Buck, Q.-H. Wang, S. M. Dawsey, G. Li, W.-J. Guo, Y.-Z. Zhang, and P. R. Taylor Genetic Progression and Heterogeneity Associated with the Development of Esophageal Squamous Cell Carcinoma Cancer Res., May 1, 2001; 61(10): 4098 - 4104. [Abstract] [Full Text]
|
|
|
|
|
|
J. O. Boyle, F. Lonardo, J. H. Chang, D. Klimstra, V. Rusch, and E. Dmitrovsky Multiple High-Grade Bronchial Dysplasia and Squamous Cell Carcinoma: Concordant and Discordant Mutations Clin. Cancer Res., February 1, 2001; 7(2): 259 - 266. [Abstract] [Full Text]
|
|
|
|
|
|
M. I. Lerman and J. D. Minna The 630-kb Lung Cancer Homozygous Deletion Region on Human Chromosome 3p21.3: Identification and Evaluation of the Resident Candidate Tumor Suppressor Genes Cancer Res., November 1, 2000; 60(21): 6116 - 6133. [Abstract] [Full Text]
|
|
|
|
 |
|
 K. H. Dragnev, J. R. Rigas, and E. Dmitrovsky The Retinoids and Cancer Prevention Mechanisms Oncologist, October 1, 2000; 5(5): 361 - 368. [Abstract] [Full Text]
|
|
|
|
 |
|
 Y. Yatabe, H. Konishi, T. Mitsudomi, S. Nakamura, and T. Takahashi Topographical Distributions of Allelic Loss in Individual Non-Small-Cell Lung Cancers Am. J. Pathol., September 1, 2000; 157(3): 985 - 993. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. I. Wistuba, J. Berry, C. Behrens, A. Maitra, N. Shivapurkar, S. Milchgrub, B. Mackay, J. D. Minna, and A. F. Gazdar Molecular Changes in the Bronchial Epithelium of Patients with Small Cell Lung Cancer Clin. Cancer Res., July 1, 2000; 6(7): 2604 - 2610. [Abstract] [Full Text]
|
|
|
|
|
|
X. Matias-Guiu, E. Bussaglia, L. Catasus, H. Lagarda, E. Gras, P. Machin, J. Prat, W. M. Lin, and C. Y. Muller Correspondence re: W. M. Lin et al., Loss of Heterozygosity and Mutational Analysis of the PTEN/MMAC1 Gene in Synchronous Endometrial and Ovarian Carcinomas. Clin. Cancer Res., 4: 2577-2583, 1998. Clin. Cancer Res., April 1, 2000; 6(4): 1598 - 1600. [Full Text]
|
|
|
|
|
|
W. N. Hittelman Clones and Subclones in the Lung Cancer Field J Natl Cancer Inst, November 3, 1999; 91(21): 1796 - 1799. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||