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[Cancer Research 65, 1664-1669, March 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

Loss of Heterozygosity Patterns Provide Fingerprints for Genetic Heterogeneity in Multistep Cancer Progression of Tobacco Smoke–Induced Non–Small Cell Lung Cancer

Hongjie Pan1,2, Joseph Califano4, Jose F. Ponte1,2, Andrea L. Russo1, Kuang-hung Cheng1,2,3, Arunthathi Thiagalingam1, Pratima Nemani1, David Sidransky4 and Sam Thiagalingam1,2,3

Departments of 1 Medicine (Genetics Program and Cancer Research Center), 2 Genetics & Genomics, and 3 Pathology & Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts; and 4 Head and Neck Cancer Research Division, Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, Maryland

Requests for reprints: Sam Thiagalingam, Department of Medicine (Genetics Program and Cancer Research Center), Boston University School of Medicine, 715 Albany Street, L320, Boston, MA 02118. Phone: 617-638-6013; Fax: 617-638-4275; E-mail: samthia{at}bu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dilution end point loss of heterozygosity (LOH) analysis, a novel approach for the analysis of LOH, was used to evaluate allelic losses with the use of 21 highly polymorphic microsatellite markers at nine chromosomal sites most frequently affected in smoking-related non–small cell lung cancers. Allelotyping was done for bronchial epithelial cells and matching blood samples from 23 former and current smokers and six nonsmokers as well as in 33 adenocarcinomas and 25 squamous cell carcinomas (SCC) and corresponding matching blood from smokers. Major conclusions from these studies are as follows: (a) LOH at chromosomal sites 8p, 9p, 11q, and 13q (P > 0.05, Fisher's exact test) are targeted at the early stages, whereas LOH at 1p, 5q, 17p, and 18q (P < 0.05, Fisher's exact test) occur at the later stages of non–small cell lung cancer progression; (b) LOH at 1p, 3p, 5q, 8p, 9p, 11q, 13q, 17p, and 18q occurs in over 45% of the tobacco smokers with SCC and adenocarcinoma; (c) compared with bronchial epithelial cells from smokers, there is a significantly higher degree of LOH at 1p, 5q, and 18q in adenocarcinoma and at 1p, 3p, and 17p in SCC (P < 0.05, Fisher's exact test). We propose that lung cancer progression induced by tobacco smoke occurs in a series of target gene inactivations/activations in defined modules of a global network. The gatekeeper module consists of multiple alternate target genes, which is inclusive of but not limited to genes localized to chromosomal loci 8p, 9p, 11q, and 13q.

Key Words: LOH • BEC • SCC • ADC • NSCLC • tobacco smoke • gatekeeper • network • allelic loss • tumor progression • lung cancer


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lung cancer is the leading cause of cancer-related death for which the major etiologic factor is tobacco smoke (1, 2). There are two major treatment groups, non–small cell lung cancer (NSCLC), which constitutes 75% of the cases, and small cell lung cancer (SCLC), which accounts for 25% of affected individuals (3–5). The overall 5-year survival of the disease is only 14%, which is mostly due to the low rate of detection before distant metastasis (1, 2). The major problem associated with the treatment of lung cancer is recurrence and relapse resulting from micrometastases that have already occurred due to late diagnosis.

Human cancers are now widely accepted as genetic diseases developing through the accumulation of genetic alterations in critical genes. It is currently known that genetic changes in tumor suppressor genes, such as p53, RB, FHIT, p16, RASSF1A, APC, and PTEN and deregulation or activating mutations of oncogenes, such as EGFR, MYC, K-ras, Her-2/neu, Cyclin D1, and BCL-2 are some of the prominent alterations that contribute to the genesis of lung cancer (6–8). One of the major challenges in cancer diagnosis is the use of cancer-specific markers for the early detection of sporadic cancer. Although the analysis of genetic changes that target specific gene alterations provide an accurate molecular basis for assessment of the cancer stage, most of these alterations could only be detected at an advanced stage and could be a laborious and expensive undertaking with a low return in treating the disease.

On the contrary, it is clear from various studies that a consistent loss of heterozygosity (LOH) could be used as an indicator for the targeted deletion of tumor suppressor genes (9). Thus, LOH of critical chromosomal regions could manifest susceptibility to or the presence of cancer and play an etiologic role in its initiation and progression to carcinoma. Furthermore, LOH could also be observed in neoplastic and apparent phenotypically normal preneoplastic cells that may eventually progress to become cancer.

Allelic loss and cytogenetic studies have pointed to several candidate regions in different chromosomes of the genome, such as 1p, 3p, 5q, 8p, 9p, 11q, 13q, 17p, 18q, and X, for the involvement of tumor suppressor genes in lung cancer susceptibility (6, 10, 11) . A high LOH score based on detectability of LOH at multiple sites in a patient sample has been shown to correlate with positive diagnosis of lung and colon cancer due to their localization at loci that are known to or predicted to harbor tumor suppressor genes (12, 13). Recent studies suggest that fractional allelic loss occurs at higher levels in lung cancers of smokers than in nonsmokers (14–16). Similarly, activation of point mutations in codon 12 of the K-ras gene has been described at a higher frequency (30%) in smokers than in nonsmokers (7%; ref. 17). These studies suggest that tobacco smoke may have a direct role in enhancing both tumor suppressor gene inactivation and oncogene activation in lung cancer.

In this study, we describe the application of a novel LOH analysis method known as dilution end point LOH analysis (DELA) to detect LOH at multiple sites with increased accuracy to analyze microsatellite or other markers localized to chromosomal loci that are frequently affected by smoking-induced lung cancer. Our goal was to find early genetic lesions induced by tobacco smoke exposure and to establish its relationship to subsequent changes in determining the progression of lung cancer. We show that LOH patterns could be used to not only detect genetic alterations that occur in smokers before neoplastic disease but also could be used to differentiate between adenocarcinoma and squamous cell carcinoma (SCC), the two major histologic types of NSCLC occurring at ~30% each of all lung cancer cases, respectively (4). These studies also enabled us to predict a novel mutimodular, multistep cancer progression model reflecting the genesis of lung cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subject Enrollment and Tissue Collection. Volunteers who were nonsmokers, former smokers, and current smokers, including those suspected of having lung cancer, were recruited for this study with the use of a protocol approved by the Boston University Medical Center Institutional Review Board (Table 1). Subjects (n = 29) provided information on smoking history and family cancer incidence. The individuals who consented were asked to undergo bronchoscopy to collect bronchial brushings in addition to providing 15 mL blood.


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  		Table 1. Demographic data regarding the lung cancer and epithelial samples

 
Bronchoscopy was done according to the standard clinical procedure. Briefly, a sterile i.v. catheter was placed to give the patient fluids and medication, such as Midazolam (Versed), and in some cases a narcotic, such as Meperidine (Demerol). The patient's nose, mouth, throat, and vocal cords were numbed by inhaling a mist of lidocaine from a nebulizer followed by a few sprays to the back of the throat and nose. Then, a bronchoscope with an attached brush was passed through the nose or mouth into the lung. The pulmonary physician collected brushings from the lungs.

Tumor Specimens. Primary lung carcinoma tissues and pretreatment blood were collected from participants who consented and enrolled with institutional review board approval from the Johns Hopkins Hospital (Table 1; ref. 15). Primary tumors were snap frozen immediately after resection until further analysis. Microdissection was done on a cryostat with the use of fresh frozen tissues. Initially, 7 µm H&E sections were evaluated by a pathologist and extra nonneoplastic tissue was removed from the specimen on the cryostat block. Greater than 75% purity was confirmed in an additional 20 sections of 20 µm thickness that were harvested for isolation of genomic DNA. Maintenance of purity of neoplastic tissue was again confirmed by a subsequent H&E staining of these sections after removal of the neoplastic tissue.

Genomic DNA Isolation. Genomic DNA was isolated from blood by first combining upper layer serum with 1 mL PBS in a 15 mL tube and by centrifugation at 1,200 rpm for 25 minutes. The pellet was resuspended in genomic DNA sample buffer [100 mmol/L NaCl, 25 mmol/L EDTA (pH 8), 10 mmol/L Tris-Cl (pH 8), and 0.5% SDS] and digested with Proteinase K in 0.1% SDS. Overnight incubation at 58°C was followed by standard phenol-chloroform extraction and sodium acetate and ethanol precipitation (18).

Bronchial epithelial cell (BEC) DNA was isolated from the DNA fraction of the Trizol RNA preparation procedure for the BEC RNA isolation. The DNA fraction was processed by three sodium citrate washes in 10% ethanol. After the final wash, DNA was suspended in 75% ethanol, centrifuged at 2,000 x g for 5 minutes, air dried, and dissolved in 8 mmol/L NaOH.

Tumor cells were microdissected and DNA extraction was done with the use of standard phenol-chloroform extraction and ethanol precipitation. Corresponding normal DNA for these specimens were derived from peripheral WBC nuclei according to previously published procedures (15).

Allelic Loss Analysis By Using DELA with Radiolabeled Markers. We found that the sensitivity for accurate determination of LOH could be highly enhanced by the use of the template genomic DNA that is progressively diluted to the end point of signal loss (Fig. 1A). Therefore, we decided to perform LOH analysis with the use of DELA.



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  		Figure 1. DELA analysis. A, a representative example of DELA analysis of normal (N1 and N2) and tumor (T1, T2, T3, and T4) samples for D5S816. 0 (1), 1/10 (2), 1/100 (3), and 1/1,000 (4) dilutions of the template from the test sample (10 ng/µL) were used. Arrows, allele showing loss in the test sample. B, representative examples of the LOH analysis using the microsatellite markers D1S226, D3S1300, D5S816, and D18S851 for the indicated samples. Progressive dilutions: 1, normal control (undiluted); 2, tumor/BEC (undiluted); and 3, tumor/BEC (dilution, 1/100). Numbers above the dilutions, codes for the BEC/matching blood (markers D3S1300 and D18S851A) or tumor/matching blood (markers D1S226, D5S816, and D18S851B) samples. Arrows, LOH.

 
The microsatellite markers used for LOH analysis in our test samples are as follows: D1S226 (1p22), D1S255 (1p34), D1S2672 (1p36), D1S199 (1p36), D1S1597 (1p36), D3S1300 (3p14), D3S1578 (3p14-21), D5S816 (5q31), LNCA (5q31), D8S255 (8p11), D8S505 (8p12), IFNA (9p21-22), D9S974 (9p21-22), D11S1987 (11q23), D11S934 (11q24), RB1 (13q14), D13S135 (13q14), D17S974 (17p13), p53 (17p13), D18S535 (18q12), and D18S851 (18q21). The primers for each of the markers analyzed were synthesized based on the sequence information available from Cooperative Human Linkage Center or obtained from Invitrogen (Carlsbad, CA). The PCR reactions for microsatellite marker analysis were set up as follows (18). One primer of each pair was end-labeled with 32P-{gamma}-ATP and T4 polynucleotide kinase and PCR amplifications were carried out in 96-well plates in a 10 µL reaction. The reaction mixture contained 67 mmol/L Tris-HCl (pH 8.8); 16.6 mmol/L ammonium sulfate; 6.7 mmol/L magnesium chloride; 10 mmol/L ß-mercaptoethanol; 6% dimethyl sulfoxide; 100 µmol/L each of dATP, dGTP, dCTP, and dTTP; 0.02 mmol/L each of the primers; 10 ng DNA template and appropriate progressive dilutions as indicated; and 0.1 units of Platinum Taq (Invitrogen). An initial denaturation at 95°C for 2 minutes was followed by 30 cycles, each carried out at 95°C for 30 seconds, 55°C to 60°C for 1 minute, and 72°C for 1 minute.

Statistical Analysis. LOH at all microsatellite markers for the various samples were recorded in a spreadsheet format. The statistical differences were compared with the use of Fisher's exact test (2 x 2 tables). No adjustments were made for multiple comparisons. The percentages of LOH were compared by using the t test. All tests were two-sided. P ≤ 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Samples and Loss of Heterozygosity Analyses. We analyzed bronchial brushings and matching blood samples from 23 former/current smokers and six nonsmokers and tumor and corresponding pretreatment blood samples from 58 smokers for allelic loss at defined loci. Eight chromosomal loci (1p, 3p, 5q, 8p, 9p, 11q, 13q, and 17p) that exhibit increased frequency of allelic loss and one (18q) that exhibited similar levels of allelic loss in tobacco smoke–induced lung cancer when compared with non–tobacco smoke–related lung cancer were chosen in this study (15). We wished to establish a relationship between the early allelic losses that are observed in morphologically normal BECs and NSCLC from the smokers to determine the progression in genetic alterations induced by tobacco smoke exposure during the genesis of lung cancer.

We compared the frequency of LOH using 21 highly polymorphic markers with a minimum of two markers corresponding to each locus. We used a novel approach, DELA, which enables accurate determination of allelic loss by visual inspection/densitometry. When we did progressive dilution of the template DNA before LOH analysis, we were able to accurately determine the presence of cells with LOH in a sample that consist predominantly of normal cells (Fig. 1A). Representative examples of DELA for various loci using both nonneoplastic as well as neoplastic test samples along with their corresponding matching normal samples are shown in Fig. 1B.

Despite the small sample size (n = 6) of BECs from nonsmokers compared with current or former smokers (n = 23), our study showed increased frequency of allelic loss in smokers compared with nonsmokers, which is consistent with the previous results reported in the literature (15, 16). Overall, we detected a significantly higher frequency of LOH in smokers (27%; 89 of 324) than in the nonsmokers (13%; 11 of 84) in all the informative determinations (P = 0.007, Fisher's exact test).

Tobacco Smoke–Exposed Bronchial Epithelial Cells and NSCLC Share Allelic Loss from Some Common Loci. Chromosomal sites 1p, 5q, 17p, and 18q exhibited a statistically higher frequency of LOH (P < 0.05, Fisher's exact test), whereas 3p, 8p, 9p 11q, and 13q exhibited statistically insignificant level of differences in the frequency of LOH (P > 0.05, Fisher's exact test; Table 2). Although a comparable level of allelic loss at 3p was observed in both BECs and adenocarcinoma, a statistically significant increase in LOH at 3p was found in SCC (P < 0.05, Fisher's exact test; Table 3). Thus, in summary, these results suggest that LOH at chromosomal sites 8p, 9p, 11q, and 13q could be early events of genetic alterations in tobacco smoke–induced lung cancer. On the other hand, LOH at 1p, 3p, 5q, 17p, and 18q could occur at the later stages of NSCLC progression (Tables 2 and 3).


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  		Table 2. Comparison of the incidences of LOH between tobacco smoke–exposed BECs and neoplastic samples from smokers

 

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  		Table 3. Tobacco smoke–exposed BECs, adenocarcinoma, and SCC exhibit common and distinct allelic loss patterns

 
Similar and Distinct Allelic Loss Patterns in Adenocarcinoma and Squamous Cell Carcinoma in Smokers Compared with Bronchial Epithelial Cells of Smokers Suggest Progression to Specific Histologic Types Could Occur as Late Events. LOH at the chromosomal loci 8p, 9p, 11q, and 13q occurs at comparable levels in preneoplastic BECs as well as in SCC and adenocarcinoma (Table 3). When we compared the frequency of LOH at all nine sites tested in this study between either BECs and adenocarcinoma or between BECs and SCC, we noticed that although both histologic subtypes exhibit increased frequency of LOH (>45%), there may be subtle differences in the rates of loss for specific loci (Table 3). There was a significantly higher frequency of loss at 1p, 5q, and 18q in adenocarcinomas and at 1p, 3p, and 17p in SCC (P < 0.05, Fisher's exact test; Table 3).

Tobacco Smoke–Induced Loss of Heterozygosity Patterns in NSCLC Is Diagnostic of the Degree of Cancer Progression. In summary, the overall frequency of LOH determined from 21 hotspot markers in various chromosomes showed that 55% (32 of 58) of cancer patients had >50% loss of informative markers compared with 17% (4 of 23) in BECs from likely preneoplastic patients. Here, the percentage of LOH is specified as the number of LOH divided by the total number of LOH and retention observed for informative markers in individual patients. The t test showed that the percentage of LOH in the tumor group is significantly higher than that in the nontumor group (P < 0.01; Fig. 2).



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  		Figure 2. Comparison of the frequency of LOH in the tumor group versus nontumor group. Tumor samples from cancer patients (n = 58) exhibited significantly higher frequency of multiple LOH than that in the nontumor epithelial group (n = 23; P < 0.01). The percentage of LOH is specified as the number of LOH divided by the total number of informative samples that included samples that exhibited LOH or retention for the markers analyzed for individual patients. The percentages of LOH between two groups were compared by using the t test.

 
Furthermore, our results are also consistent with the hypothesis that there are three types of genetic lesions that define tobacco smoke–induced progression to lung cancer: (a) precursor lesions, (b) common lesions in all lung cancers, and (c) unique lesions that determine the development of each histologic type of lung cancer.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The lack of histologically well defined early neoplasms for lung cancer has frustrated effective prevention, diagnosis, and therapy of lung cancer. Recent studies suggest that cigarette smoking is the predominant etiologic risk factor for lung cancer, which could be directly correlated to increased levels of chromosomal alterations (14–16, 19). In this study, we compared allelic loss induced by tobacco smoke in histologically normal BECs to NSCLC to determine early and late genetic alterations that are responsible for lung cancer to shed light on the molecular changes that may help to define the stages and histologic subtypes of NSCLC. The novel DELA method enabled accurate determination of allelic loss even in few cells by visual inspection/densitometry in samples that consisted predominantly of normal cells. The DELA method enables unambiguous detection of the presence of the retained allele and the absence of the lost allele in the affected cells at the dilution end point.

We found that LOH at chromosomal sites 8p, 9p, 11q, and 13q (P > 0.05, Fisher's exact test) are targeted at early stages of lung cancer progression, suggesting that these loci may harbor gatekeeper tumor suppressor genes for lung cancer (Table 2; Fig. 3; ref. 20). Two of the prominent tumor suppressor genes known to map to these sites and associated with lung cancer are p16 and RB, which localize to 9p and 13q, respectively (21, 22) . The p16 gene, localized to 9p21, encodes a negative regulator of the G1-S transition of the cell cycle by inhibiting the activity of cyclin-dependent kinase 4 or cyclin-dependent kinase 6 (23). The loss of p16 was observed in 50% of advanced NSCLC and the predominant mechanism for its inactivation has been shown to result from gene silencing via promoter methylation (24). Additionally, abnormalities in the RB gene product, a key cell cycle regulator that senses and integrates proliferation and antiproliferation signals allowing the commitment to enter the DNA synthetic phase (S), have been reported in 15% to 30% of NSCLC (22, 25–27).



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  		Figure 3. Progressive genetic alterations in modules of subnetworks in a global network define multistage tobacco smoke–induced lung cancer. Lung cancer could be induced by tobacco smoke by mediating genetic alterations at multiple steps. The early genetic alterations at chromosomal sites 8p, 9p, 11q, and 13q could mediate inactivation of targeted gatekeeper tumor suppressor genes that act alone or in combination in a specific gatekeeper module. The cancer precursor cells harboring the initial critical alterations are receptive to additional genetic alterations induced by continued tobacco smoke exposure leading to the genesis of lung cancer. The unique sets of genetic alterations that occur depending on the differences in the constituent carcinogens and genetic differences in the patients determine the subsequent progression of lung cancer into specific histologic subtypes. Similar frequencies of LOH are observed at some chromosomal loci (e.g., 1p); increased frequencies of LOH at other sites [e.g., 3p and 17p (SCC) and 5q and 18q (adenocarcinoma)] could be responsible for the common alterations and changes leading to the development of specific histologic subtypes of lung cancer. Although the genetic alterations described here could be involved in lung cancer as suggested from our study, it should be noted that other genetic and epigenetic alterations that we did not analyze in this study could also have a role in these processes. In summary, the development of lung cancer from BECs occurs in a multistep process involving early gatekeeper pathway alterations followed by common and unique lesions that define the genesis of specific subtypes of lung cancer.

 
Interestingly, our studies further show that LOH at 9p and 13q occurs frequently in nonoverlapping samples, suggesting the possibility that the initial alterations may be directed to multiple alternate targets (data not shown). These results prompted us to hypothesize that the inactivation of gatekeeper function could be targeted to a module that consists of multiple interconnected pathways in a network rather than individual targets of tumor suppressor genes for lung and other cancers. The existence of a p16INK4-CyclinD1-CDK4-RB axis in cell cycle regulation provides legitimacy to this notion (28). However, the role(s) of one or more of these gene(s) from affected chromosomal loci and their involvement in a gatekeeper function for lung cancer requires further study.

Our data also revealed that LOH at 1p, 5q, 17p, and 18q (P < 0.05, Fisher's exact test) occurs at the later stages of NSCLC (Table 3). The exception was 3p allelic loss, which seemed to occur at statistically similar levels in both BECs from smokers and adenocarcinoma, whereas it occurred at a significantly higher frequency in SCC when compared with BECs (P < 0.05, Fisher's exact test; Table 3). The prominent putative tumor suppressor gene that maps to chromosome 3p14.2 spanning the FRA3B common fragile site and associated with lung cancer is FHIT (29). The fragile histidine triad (FHIT) gene product is involved in mediating the Mg2+-dependent hydrolysis of P1-5'-O-adenosine-P3-5'-O-ATP, Ap(3)A, to AMP and ADP (30, 31). Frequent allelic losses, homozygous deletions, and aberrant transcripts of the FHIT gene have been described in a high percentage of lung tumors (30). There is precedence from the studies of colon cancer that allelic loss at 17p and 18q are associated with later stages of cancer and harbor p53 and SMAD4, respectively, as the major target genes (32, 33). Although p53 is a well-established target gene for mutational inactivation in >50% of lung cancers, SMAD4 is rarely targeted for mutational inactivation in lung cancer (34–36). Furthermore, p73 and APC are prominent tumor suppressor genes localized to 1p and 5q, respectively, and associated with lung cancer; however, the roles of these target genes or others in advanced lung cancer require systematic studies (37, 38).

Previous studies have attempted to compare patterns of allelic losses between SCC and adenocarcinoma, the two major histologic types of NSCLC (4, 39, 40). These studies reported that there is a relatively higher frequency of allelic losses in SCC than in adenocarcinoma (39–42). Our analysis of tobacco smoke–induced genetic alterations in SCC and adenocarcinoma suggested that although the overall LOH frequencies for SCC are higher than in adenocarcinoma, specific loci exhibit unique and differential LOH patterns (Table 3; Fig. 3). Overall, although LOH at 1p, 3p, 5q, 8p, 9p, 11q, 13q, 17p, and 18q occurs in ≥45% of the tobacco smokers with SCC or adenocarcinoma, there seems to be a significantly higher degree of preferential LOH at 1p, 5q, and 18q in adenocarcinoma and at 1p, 3p, and 17p in SCC (P < 0.5; Table 3; Fig. 3).

In summary, our data suggest that tobacco smoke induces allelic losses at a specific set of chromosomal loci including 8p, 9p, 11q, and 13q, possibly targeting individual or multiple gatekeeper tumor suppressor gene(s) that act in one or more interconnected axes of events in a defined module of a network to generate the neoplastic precursor cells that are receptive to additional genetic alterations leading to the genesis of lung cancer (Fig. 3). We propose that multistep cancer progression in lung and other cancers is mediated by dysregulation/inactivation of a series of functional subnetwork modules. The first module in the series of a global network represents the gatekeeper module and the suggestion for the existence of multiple targets in each module explains genetic and epigenetic heterogeneity observed during progression to similar pathologic subtypes of cancer. We propose that the gatekeeper modular alterations during NSCLC progression is succeeded by modules representing intermediary stages (represented by LOH at 1p) and the terminal histologically differentiated stages characteristic of each subtype of NSCLC (represented by LOH at 5q and 18q in adenocarcinoma and at 3p and 17p in SCC). The extent of tobacco smoke exposure and the ratios of active carcinogenic ingredients present in tobacco smoke as well as the genetic differences of the subjects may define the common and unique changes that determine the development of the specific histologic type of lung cancer. Our observations provide a road map for further research to develop routine early diagnostic and prognostic tests as well as a blueprint for the discovery of critical points for therapeutic intervention in lung cancer.


   Acknowledgments
 
Grant support: National Institute of Environmental Health Sciences grant ES10377 (S. Thiagalingam), NIH training grant T32HL07035 (J.F. Ponte), and The Medical Foundation (S. Thiagalingam).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Avrum Spira, Gang Liu, and Jerome Brody (Pulmonary Center, Boston University School of Medicine, Boston, MA) for generously helping to collect clinical samples and Jerome Brody for critical reading of the manuscript.


   Footnotes
 
Note: A. Thiagalingam is currently in Bayer Corporation, East Walpole, MA 02032. S. Thiagalingam is a Dolphin Trust investigator.

Received 9/13/04. Revised 12/ 1/04. Accepted 12/15/04.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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