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[Cancer Research 64, 8702-8707, December 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Loss of Heterozygosity of Chromosome 3p21 Is Associated with Mutant TP53 and Better Patient Survival in Non–Small-Cell Lung Cancer

Carmen J. Marsit1, Masayuki Hasegawa4, Tomoko Hirao5, Duk-Hwan Kim6, Kenneth Aldape7, Philip W. Hinds3, John K. Wiencke8, Heather H. Nelson2 and Karl T. Kelsey1,2

1 Department of Genetics and Complex Diseases, and 2 Department of Environmental Health, Harvard School of Public Health, and 3 Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, Massachusetts; 4 Department of Surgery, Chiba Social Insurance Hospital, Chiba, Japan; 5 Department of Surgery, Nara University, Kashihara, Japan; 6 Center for Genomics Research, Samsung Biomedical Research Institute, Sungkyunkwan University, Seoul, Republic of Korea; 7 Department of Pathology, University of Texas M. D. Anderson Cancer Center, Houston, Texas; and 8 Laboratory for Molecular Epidemiology, University of California-San Francisco, San Francisco, California


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Allelic loss of chromosome region 3p21.3 occurs early and frequently in non–small-cell lung cancer, and numerous tumor suppressor genes at this locus may be targets of inactivation. Using an incident case series study of non–small-cell lung cancer, we sought to determine the prevalence of loss of heterozygosity (LOH) in the 3p21.3 region and to examine the associations between this alteration and patient outcome, exposure to tobacco smoke, occupational asbestos exposure, and additional molecular alterations in these tumors. We examined LOH at 7 microsatellite markers in the chromosome 3p21.3 region, and LOH was present in at least one of the loci examined in 60% (156 of 258) of the tumors, with the prevalence of LOH at individual loci ranging from 15 to 56%. Occupational asbestos exposure and TP53 mutation were significantly associated with more extensive 3p21 LOH. In squamous cell carcinomas, measures of cumulative smoking dose were significantly lower in patients with LOH at 3p21, particularly in TP53 mutant tumors. Examining patient outcome, we found that in squamous cell carcinomas, having any LOH in this region was associated with a better overall survival (log-rank test, P < 0.04). Together, these results indicate that allelic loss at 3p21 can affect patient outcome, and that this loss may initially be related to carcinogen exposure, but that extension of this loss is related to TP53 mutation status and occupational asbestos exposure.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lung cancer is among the most common neoplastic diseases in the United States and leads in the number of cancer-related deaths of all tumor types (1) . Specific genetic alterations found in lung cancer are thought to occur in premalignant clones long before the appearance of disease, and these changes may persist for many years after smoking cessation (2) . Some of the earliest and most often observed of these alterations are allelic losses at the chromosome 3p21.3 region, where loss can be detected in nonmalignant cells of smokers (3 , 4) . Detailed analysis of this region has identified two minimal deletion regions, which are thought to harbor one or more tumor suppressor genes targeted for alteration in lung cancer and other epithelial malignancies (5, 6, 7) . Functional inactivation of these genes, through allelic loss or promoter hypermethylation, may be critical for lung tumorigenesis (8, 9, 10, 11, 12, 13, 14, 15, 16, 17) .

Mutations in the tumor suppressor TP53 also occur frequently in lung cancer development (18 , 19) , and alterations in TP53 are thought to lead to genomic instability in TP53-altered cells (20, 21, 22, 23) . Zienolddiny et al. (24) showed an association between TP53 mutation and loss of heterozygosity (LOH) at 3p21, whereas Geradts et al. (25) found no association when looking at TP53 alteration with only one microsatellite marker in 3p21. These conflicting results indicate a need for more thorough examination of allelic loss at 3p21 to more completely understand the relationship of allele loss in this region and TP53 mutation.

It is clear that alterations of 3p21.3 occur early in lung tumorigenesis, and putative tumor suppressors in this region have been identified; yet, the relationship between their loss and exposure to tobacco smoke and other lung carcinogens, such as asbestos, has been poorly explored. We wanted to more closely examine these alterations to determine the predictive role of tobacco or occupational asbestos exposure, as well as additional alterations affecting cell phenotype, such as TP53 mutation. In addition, we sought to identify any clinical or predictive value these alterations may have on patient outcome using a case series study of primary, surgically resected non–small-cell lung cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Population and Tumor Specimens.
Eligible cases consisted of all of the newly diagnosed patients with resectable lung cancer who received treatment at the Massachusetts General Hospital Thoracic Surgery Service from November 1992 through December 1996 (26) . All of the patients involved provided written informed consent in accordance with the appropriate institutional review boards. Patients with recurrent disease or nonoperable tumors were excluded. A random subset of 260 was analyzed for somatic loss at 3p21. For these, tumor-derived DNA was obtained from archived pathology specimens as described previously (27) , and matched constitutive DNA was derived from circulating blood lymphocytes with the QIAmp DNA Blood Mini Kit (Qiagen, Valencia, CA). Demographic and epidemiologic data, including all of the data on tobacco use and occupational asbestos exposure, were gathered by interviewer review of a self-administered questionnaire completed by patients and reviewed by a single reviewer during the hospitalization for thoracic surgery.

LOH Analysis.
To evaluate LOH spanning 3p21, the microsatellites D3S1029, D3S3582, D3S3667, D3S3640, D3S1568, and D3S3026 were amplified with fluorescently labeled forward primers (Invitrogen, Carlsbad, CA). LOH analysis for D3S1478 has been previously described on these cases (28) . Each PCR mixture contained 10 ng of template DNA, 12.5 pmol/L of each primer, 0.2 mmol/L deoxynucleoside triphosphates, AmpliTaq Gold reaction buffer, and 1.25 units of AmpliTaq Gold (Applied Biosystems, Carlsbad, CA) in a final volume of 25 µL. PCR amplification was done with a hot start of 95°C for 5 minutes followed by 40 cycles of amplification of 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute. A final extension at 72°C was done for 5 minutes.

Amplified PCR products were separated on a 4% denaturing polyacrylamide gel for the ABI PRISM 377 (Applied Biosystems) with a labeled marker (TAMRA 350) as an internal size standard. The data were then analyzed with the Genescan 2.1 software (Applied Biosystems). LOH was determined for those loci that were heterozygous in blood samples. The allelic ratio was derived as the ratio of the peak heights in the blood-derived samples divided by the peak heights of the alleles from the tumor. Ratio values of <0.5 or >1.5 were taken to be indicative of LOH.

TP53 Analysis.
TP53 mutations were detected by PCR-single-strand conformational polymorphism (SSCP) of exons 5 to 10 in these tumors, as described previously by Nelson, et. al (27) with previously reported primer sequences (29) .

TP53 Immunohistochemistry.
Immunostaining was done as described previously (30) . Briefly, 5-µm sections were mounted on positively charged slides, deparaffinized, and rehydrated in PBS. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in PBS containing 0.05% Tween 20 for 20 minutes. Sections were then washed in PBS and blocked for 20 minutes in the appropriate serum from the same species as the secondary antibody diluted to 10% in PBS. Microwave antigen retrieval was done by placing the slides in 50 mmol/L citrate buffer (pH 6.0) and microwaving for 12 minutes at full power and 10 minutes at 20% power, followed by cooling for 15 minutes and two to three 5-minute washes in PBS. Primary antibody in PBS with 10% serum were applied to the sections in a humid chamber overnight at 4°C. After washing two to three times in PBS, secondary antibody was applied with the Dako Envision kit (Carpinteria, CA) according to the manufacturer’s instructions. Detection of bound secondary antibody was done with diaminobenzadine for 5 minutes. Sections were then counterstained with light hematoxylin and coverslipped. The primary antibody used for TP53 staining was anti-TP53 (DO-7, Oncogene Research, San Diego, CA; 1:150 dilution). Scoring for p53 was based on the percentage positive nuclei (0 = <1% positivity, 1 = between 1 to 5%, 2 = 5 to 30%, and 3 = >30%).

Statistical Analysis.
Statistical analyses of 3p21 LOH at individual markers, patient demographics, exposure information, and tumor traits were done with SAS software (SAS Institute, Cary, North Carolina). Pack-years were calculated by multiplying the self-reported number of years smoked by the number of packs of cigarettes smoked per day.

To examine the extent of 3p21 loss in the samples, a fractional allelic loss score was also calculated, as previously described by Wistuba et al. (31) . The 3p21 fractional allelic loss was determined with the following formula: total number of 3p21 markers with allelic loss per sample/total number of informative 3p21 markers per sample.

Thus, individuals with a score of zero had no LOH at any informative loci, whereas those with the maximum score of one had LOH at all of the informative loci. Those individuals with no informative loci were restricted from analysis. To preserve power, this value was categorized into three groups, those with a score of 0, those >0 but ≤ the median score of 0.2, and those >0.2. To examine any LOH at 3p21, the score was dichotomized with groups of scoring 0, indicative of no LOH at any informative loci, or >0, indicating LOH at one or more informative loci.

The {chi}2 or Fisher’s exact tests were used to examine categorical data, whereas nonparametric tests (Wilcoxon ranks sums) were used to examine continuous data. To examine the relationship of multiple covariates to extensive 3p21 LOH (fractional allelic loss score > the median of 0.2 compared with ≤0.2), unconditional logistic regression was used to adjust for the simultaneous effect of multiple variables on the prevalence of extensive 3p21 LOH. For survival analysis, Kaplan-Meier survival curves were constructed for groups based on univariate predictors, and differences between the groups were tested by the log-rank test. To examine the simultaneous effects of several variables on patient outcome, the Cox proportional hazards model was used, and all of the variables examined were consistent with the assumptions of Cox proportional modeling. All tests of significance were two-sided and considered significant at a P < 0.05 level.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LOH at 3p21.
Heterozygosity exceeded 50% at each of the seven microsatellite markers, and LOH occurred with prevalences ranging from 15 to >50% at each of the sites (Table 1)Citation . Overall, 60% (156 of 258) of tumors examined and informative at one or more loci showed LOH for at least one marker. Fractional allelic loss scores ranged from 0 to 1 with a median score of 0.2.


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  		Table 1 Frequency of LOH at microsatellite markers on 3p21

 
Alterations of 3p21 with Demographic and Clinical Characteristics.
Allelic loss at any site individually or at one or more sites in 3p21 showed no statistically significant association with gender or age of patients (Table 2)Citation ; however, the prevalence of 3p21 loss differed significantly between the two major histologic subtypes of non–small-cell lung cancer, squamous cell cancer and adenocarcinoma. LOH at any site in 3p21 occurred in 59 of 81 (73%) squamous cell cancer tumors compared with 83 of 145 adenocarcinoma (57%; P ≤ 0.02 for squamous cell cancer versus adenocarcinoma).


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  		Table 2 Correlations of any loss at 3p21 with case characteristics and tumor molecular alterations

 
There was no significant association between LOH at 3p21 with disease pathological stage, yet 52 of 107 (49%) tumors classified as poorly differentiated or completely undifferentiated compared with 41 of 128 (32%) tumors classified as moderately to completely differentiated had a higher than median 3p21 fractional allelic loss (P ≤ 0.01 for {chi}2 test, two-sided), suggesting that tumors with the greater extent of LOH are less differentiated.

LOH at 3p21 and RASSF1A Methylation.
We have previously studied hypermethylation of the RASSF1A gene in these tumors, and as this gene is located at 3p21.3, we sought to determine whether this alteration was associated with allelic loss in this region.9 When analysis was restricted to squamous cell cancer, any LOH at 3p21 was significantly associated with hypermethylation of the RASSF1A promoter (Table 2Citation ; P = 0.04).

LOH at 3p21, TP53 Mutation, and Occupational Asbestos Exposure.
In the total case series, 3p21 LOH was associated with TP53 status. LOH at any site in 3p21 occurred in 50 of 66 (76%) tumors with mutant TP53 compared with 98 of 177 (55%) tumors with wild-type TP53 (P ≤ 0.003; Table 2Citation ). A similar trend, although not statistically significant, was seen in the squamous cell cancer subgroup, whereas the association between 3p21 LOH and TP53 mutation was significant in the adenocarcinoma subgroup (P ≤ 0.01). No significant association was found between aberrant staining of TP53 by immunohistochemistry and 3p21 LOH (Table 2)Citation . A significant association between any LOH at 3p21 and occupational asbestos exposure was also observed (P ≤ 0.05; Table 2Citation ).

To investigate how the extent of LOH was related to TP53 mutation as well as carcinogen exposures, the 3p21 fractional allelic loss score was calculated, and the tumors were dichotomized as those with no or less extensive LOH (fractional allelic loss score 0 to 0.2) compared with those with extensive LOH (fractional allelic loss score >0.2). Those tumors with the 3p21 fractional allelic loss score higher than the median score had more than twice the prevalence of TP53 mutation than those with no LOH or those with 3p21 fractional allelic loss below the median (P ≤ 0.001 for {chi}2 test, 2-sided; Table 3Citation ). A similar association was observed between tumors with a > median fractional allelic loss score and occupational asbestos exposure, with the prevalence of extensive 3p21 LOH being twice as high in the asbestos-exposed individuals compared with unexposed cases (P ≤ 0.02; Table 3Citation ). In these patients, there was no statistically significant association observed between TP53 mutation status and occupational asbestos exposure (data not shown).


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  		Table 3 Extensive 3p21 LOH is associated with TP53 mutation and occupational asbestos exposure

 
Because TP53 mutation status and occupational asbestos exposure were both associated with extensive 3p21 LOH in univariate analysis, we used a multivariate logistic regression model to clarify the associations controlling for additional variables in the model. Histology and gender were included in the model as potential confounders. Age and pack-years were initially included in the model, but they were removed as neither was statistically significant, nor did their inclusion have an effect on the coefficients of the other variables in the model. Using this model, TP53 mutation was significantly associated with extensive 3p21 LOH, having an odds ratio of 3.36 (95% confidence interval 1.72 to 6.55; Table 4Citation ), whereas occupational asbestos exposure was not significantly associated with 3p21 fractional allelic loss score >0.2.


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  		Table 4 Multivariate models of association between extensive 3p21 LOH, TP53 mutation status, and occupational asbestos exposure * (n = 214) {dagger}

 
Alterations of 3p21 and Smoking Measures.
Neither LOH at one or more sites nor LOH at any individual site at 3p21 was associated with use of tobacco defined as never compared with ever smoking (Table 2)Citation . In examining metrics of smoking, patients with LOH at 3p21 had a higher median number of cigarettes smoked per day than did those with retention of this region, although this difference was not statistically significant. However, in squamous cell cancer where loss was more prevalent, patients with tumors that retained 3p21 smoked a median of 40 cigarettes per day, whereas those with loss at one or more sites of 3p21 smoked a median of 30 cigarettes per day (P ≤ 0.04, Wilcoxon rank-sum test; Table 5Citation ). In these squamous cell cancers, this association was statistically significant in TP53 WT tumors, where patients with retained 3p21 smoked a median of 40 cigarettes per day, compared with 25 cigarettes per day in patients with tumors having any loss of 3p21 (P ≤ 0.005, Wilcoxon rank-sum test). The difference was smaller (and not statistically significant) in TP53 mutant squamous cell cancer tumors (Table 5)Citation .


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  		Table 5 In SCC, cigarette dose is inversely related to LOH at 3p21 only in TP53 WT tumors

 
Alterations at 3p21 and Patient Outcome.
LOH at one or more sites in 3p21 was significantly associated with a better patient outcome in squamous cell cancer (P ≤ 0.04, log-rank test; Fig. 1Citation ). In the squamous cell cancer subgroup, to control for additional variables known to predict patient outcome, we did multivariate analysis with the Cox proportional hazards model including, as covariates, age, gender, and stage (I or II versus III or IV). This modeling will allow for a measure of the association between multiple variables and patient outcome and is presented as a hazard ratio, which is interpreted as the relative risk of death at a given time for a patient with the alteration compared with one without controlling for all of the other variables in the model. Controlling for these covariates, LOH at one or more sites in 3p21 was a strong, significant predictor of more favorable patient outcome, with a hazard ratio of 0.53 (95% confidence interval = 0.30 to 0.94; Table 6Citation ). When the model was stratified by stage (I or II versus III or IV), the protective effect was stronger in higher stage disease (hazard ratio = 0.4) compared with lower stage disease (hazard ratio = 0.7), although the point estimates were unstable as a result of small sample sizes in these strata.



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  		Fig. 1. Kaplan-Meier survival probability curves in squamous cell carcinomas of the lung by 3p21 LOH or 3p21 retention (n = 158). Survival time was defined as the time from surgery until the death of the patient, known recurrence, or the last time the patient was known to be alive. We used the log-rank method to test differences between the groups, and a significant difference is observed (P ≤ 0.04). Tick marks represent censored values.

 

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  		Table 6 Cox proportional hazards models for multivariate analysis of patient outcome in SCC *

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We characterized the epidemiology of 3p21 allele loss in non–small-cell lung cancer. Loss at one or more of the seven informative loci occurred in ~60% of tumors, with loss at individual loci occurring in between 15 to >50% of informative samples. These results are consistent with previous reports of LOH at this region in non–small-cell lung cancer (3 , 4 , 7) .

As methylation may be a second "hit" for inactivation of a tumor suppressor, we expected that there should be some association between LOH and methylation, indicating that these are the two modes of inactivation of this tumor suppressor. Consistent with a previous report, there was an association in squamous cell cancer between any LOH at 3p21 and RASSF1A methylation, with almost all of the methylation positive tumors showing LOH at 3p21 (11) . However, this association was not as apparent at loci surrounding the RASSF1A gene (D3S1568 and D3S3667), indicating that the loss may not be targeting RASSF1A specifically, and that this association may indicate a relationship between methylation of RASSF1A and loss of another tumor suppressor gene in this region.

Patterns describing 3p21 LOH were strongly influenced by histology, with squamous cell cancer having more frequent LOH compared with adenocarcinoma, consistent with previous reports (24 , 32) . This histologic influence may also be related to the limited association between LOH and methylation. Such results may indicate that the cells from which these tumors arise may have differential susceptibilities to either methylation or LOH or have cell-type specific phenotypes, which select for one type of alteration over the other. It should be noted that both LOH and methylation can be found in either squamous cell cancer or adenocarcinoma, and so the cell-specific factor is not exclusive. Similar differences in somatic alteration by histology have been observed in KRAS mutations, which occur almost exclusively in adenocarcinomas (26 , 33) .

One factor that appears to be related to 3p21 LOH is mutation of the TP53 tumor suppressor. Previous studies examining the association between TP53 mutation and 3p21 LOH differ in their results dependent on the method used to evaluate TP53 inactivation. Geradts et al. (25) used TP53 immunohistochemistry to evaluate aberrant TP53 expression and found no association between aberrant TP53 staining and 3p21 LOH. On the other hand, Zienolddiny et al. (24) used PCR-SSCP to examine TP53 mutation and did find a significant association between TP53 mutation and 3p21 LOH. Our data are consistent with both of these studies, as TP53 mutant tumors, as determined by PCR-SSCP, were significantly more likely to have LOH at one or more loci in 3p21 than were TP53 wild-type tumors. On the other hand, if TP53 aberrant staining by immunohistochemistry is used, no significant association was observed. Our group and others have reported discordance between TP53 mutation and immunohistochemistry-positivity (34, 35, 36, 37, 38) in various tumor types. Although aberrant nuclear staining of TP53 can indicate that the protein is mutant, functionally inactivated TP53 occurring as a result of alterations in the pathway that normally leads to its regulation and activation can also be the cause of the abnormal staining. Some possibilities along these lines include loss of TP53 transcriptional activity, thereby loss of MDM2 activation and appropriate ubiquitin-dependent degradation of TP53 (38) , phosphorylation and stabilization of TP53 at its NH2-terminal transactivation domain (39) , phosphorylation and inactivation of MDM2 by ATM (40) , or sequestration of MDM2 by ARF (41) . If the overexpression of TP53 is because of posttranslational modifications of TP53 or MDM2, or the activity of ARF, this may indicate that DNA damage response pathways, such as those initiated by ATM, may still be responsive to DNA damage, and, thus, these cells are less likely to exhibit LOH at 3p21, although the TP53 response is abnormal. Mutations of the TP53 gene may indicate a predisposition to damage events due with to the complete inactivation of the TP53 pathway, or to some inherent susceptibility to DNA damage, and thus the association with 3p21 LOH.

We also observed that the extent of LOH (as measured by fractional allelic loss score) in these tumors is greater in TP53 mutant tumors, as well as in tumors from patients having occupational asbestos exposure. Altered TP53 function is known to lead to genomic instability and increased recombination, thereby predisposing cells to events leading to allelic loss (20, 21, 22, 23) . Asbestos exposure, in in vitro studies has been shown to be clastogenic, cytotoxic, and able to induce chromosomal aberrations and large chromosomal deletions (42, 43, 44) . In univariate analysis, both TP53 mutation and occupational asbestos exposure are significantly related to extensive 3p21 LOH, consistent with the hypothesis that coding change mutations of TP53 through enhanced genomic instability and chromosomal aberrations caused by asbestos fibers may lead to extensive loss at 3p21. When this extensive LOH is modeled with both TP53 mutation and occupational asbestos exposure, only TP53 mutation remains significantly associated with higher than median fractional allelic loss score, although asbestos is a clear confounder in this model as removing the term greatly alters the coefficient of the TP53 mutation term. Larger studies including asbestos-exposed individuals will be needed to clarify the complex relationship between asbestos, TP53, and 3p21 LOH.

A greater extent of LOH at 3p21, as denoted by a 3p21 fractional allelic loss score, was also associated with more poorly differentiated tumors. This is consistent with results of previous studies, which observed an increased extent of 3p21 LOH with histologic severity and may indicate that inactivation of several tumor suppressor genes in the 3p21 region is necessary for tumor progression (3 , 4) .

We have previously shown that a younger age at smoking initiation was associated with LOH at one of the microsatellites (D3S1478) in squamous cell cancer. We did not see similar associations with the other microsatellites examined in this study nor with overall LOH or extent of LOH. However, in these squamous cell cancers, we found an inverse dose response of LOH at 3p21 with increasing number of cigarettes smoked per day, which is apparent only in TP53 wild-type tumors. This finding together with our data on LOH extent, TP53 mutation, and tumor differentiation indicate that exposure to tobacco carcinogens while the lung is still developing may cause discreet deletion events harbored in premalignant clones. Additional exposures in a TP53 wild-type background may have little additional effect on LOH, but once TP53 is inactivated, the extent of LOH is increased. Our data indicating that LOH extent is greater in poorly differentiated tumors supports this hypothesis.

Interestingly, in squamous cell cancer, LOH at one or more site at 3p21 was a marker of better prognosis, and when controlled for age, gender, and stage, providing a 50% reduced risk for adverse outcome compared with those with retention of 3p21. Although the statistical power in the present study is low because of small numbers of samples in the strata, the hazard ratio is lower in tumors of high stage compared with those of low stage, indicating that the protective effect of LOH is stronger in higher stage disease. Because of the loss of specific genes in this region, or possibly because of increased overall genetic instability, tumors with LOH at 3p21 may be more likely to respond to the surgical treatment that they received, leading to the overall increased survival of these patients. If the LOH at 3p21 is any marker of genomic instability, these results may also suggest that these more unstable tumors are less likely to metastasize, or do so more slowly, thus the increased survival. Patient survival, however, can be affected by a number of clinical, biological, and lifestyle factors, many of which are not measured or controlled for in this analysis; thus, the hazard ratio obtained in these analyses may be influenced by uncontrolled confounding that can be having a great impact on the direction and magnitude of the result. Looking at additional areas of allelic loss outside of 3p21 could help to clarify if there is something specific about loss in this region, or if it is a susceptibility to allelic loss in general that provides some survival advantage to these patients.

Alterations at chromosome 3p21.3 occur frequently in non–small-cell lung cancer. Although the initiation of these events may occur with exposure to tobacco smoke during adolescence, and these early tobacco exposures may lead to the creation of altered cellular fields in which clones containing 3p21 LOH are not present and are more susceptible to additional carcinogen damage (28 , 45 , 46) . Extensions of loss in this region may require additional alterations to other tumor suppressor pathways as additional exposures continue to alter these cellular fields and select the malignant clones. These results suggest that the timing of silencing of specific tumor suppressor genes in this region may be important to lung carcinogenesis, and that loss of this region may serve as a marker of response to treatment, as well as a marker of early carcinogenesis, and may thus be useful in identifying patients at enhanced risk for tumor development. Additional studies are needed to clarify when in the process these events occur, and if examining LOH at 3p21 is specific enough to be used as a marker of early and more treatable lung cancer.


   FOOTNOTES
 
Grant support: NIH Grants P42ES05947, R01CA100679, ES000002, and T32CA09078 (C. Marsit).

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.

Requests for reprints: Karl T. Kelsey, Harvard School of Public Health, Building I, Room 607, 665 Huntington Avenue, Boston, MA 02115. Phone: (617) 432-3313; Fax: (617) 432-0107; E-mail: kelsey{at}hsph.harvard.edu

9 C. J. Marsit, D. Kim, M. Liu, P. W. Hinds, J. K. Wiencke, H. H. Nelson, and K. T. Kelsey. Hypermethylation of RASSF1A and BLU tumor suppressor genes in non-small cell lung cancer: implications for tobacco smoking during adolescence, submitted for publication. Back

Received 7/16/04. Revised 8/19/04. Accepted 9/23/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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