Carcinogenesis

Carcinogenesis Advance Access originally published online on May 4, 2006
Carcinogenesis 2006 27(10):2028-2033; doi:10.1093/carcin/bgl047

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MDM2 gene promoter polymorphisms and risk of lung cancer: a case–control analysis

Guojun Li, Xiaodong Zhai, Zhengdong Zhang, Robert M. Chamberlain, Margaret R. Spitz and Qingyi Wei^*

Department of Epidemiology, The University of Texas M.D. Anderson Cancer Center Houston, TX 77030, USA

^*To whom correspondence should be addressed at: Department of Epidemiology, Unit 1365, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. Tel: +1 713 792 3020; Fax: +1 713 792 0807; Email: qwei{at}mdanderson.org

	Abstract

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The MDM2 protein negatively regulates p53 expression level in modulating DNA repair, cell-cycle control, cell growth and apoptosis. Polymorphisms in the promoter region of the MDM2 gene have been shown to alter protein expression and may, thus, play a role in carcinogenesis. To test our hypothesis that the MDM2 promoter polymorphisms are associated with risk of lung cancer, we conducted a hospital-based, case–control study of 1026 non-Hispanic white patients newly diagnosed with lung cancer and 1145 cancer-free controls who were frequency-matched by age (±5 years), sex, ethnicity and smoking status. We genotyped for the MDM2 promoter G2580T (also called SNP309) and G2164A polymorphisms that have a minor allele frequency >0.05. The distributions of the MDM2-2580G variant allele and genotypes were significantly less common among the cases than among the controls (P = 0.038 and 0.045, respectively), but this was not evident for MDM2-2164G (P = 0.865 and 0.614, respectively). Compared with the MDM2-2580TT genotype, the MDM2-2580G variant genotypes were associated with a decreased risk of lung cancer [odds ratio = 0.81 and 95% confidence interval = 0.67–0.98 for GT, 0.83 (0.63-1.08) for GG, and 0.81 (0.68-0.97) for the combined GT/GG genotype]. However, no significant association was observed between the MDM2-2164G variant genotypes and lung cancer risk. Our results suggest that the MDM2-2580G allele may be a marker of reduced genetic susceptibility to lung cancer in the non-Hispanic white population, a finding that seems to contradict previous reports.

Abbreviations: bp, base pair; CI, confidence interval; MAF, minor allele frequency; PCR, polymerase chain reaction; OR, odds ratio; SNP, single nucleotide polymorphism

	Introduction

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Cigarette smoking constitutes 80% of the attributable risk to lung cancer, but only a small proportion of smokers will develop lung cancer, suggesting that there is an interindividual variation in genetic susceptibility to lung cancer in the general population (1). Tobacco smoke contains numerous carcinogens that can cause various kinds of DNA damage after bioactivation by the host's metabolic systems. The damage to DNA initiates a series of signals that call upon an arrest of cell cycle for the completion of repair activities or activate the apoptotic pathway if the damage to DNA is unrepairable. It is conceivable that genetic variation in critical pathways of DNA repair (1,2), cell-cycle control (3) or apoptosis (4) may contribute to interindividual variation in susceptibility to lung cancer among the smokers. Delineating genetic variations related to these pathways is crucial for identifying at-risk populations and for the early detection and prevention of lung cancer.

The tumor suppressor gene p53 plays a central role in response to DNA damage, maintaining host-cell genomic stability after carcinogenic exposure. The p53 protein can be activated by or interact with many other proteins in the signaling pathway network. For example, a functional p53 protein protects cells from malignant transformation by activating downstream genes in cell-cycle control and DNA repair (5,6), and inhibits cell growth by inducing apoptosis or G₁ cell-cycle arrest (7). Therefore, p53 may modulate cellular response to carcinogens (8).

The human MDM2 gene located on chromosome 12q13-14 with genomic size of 34 kb is a negative regulator of p53 (9). MDM2 promotes rapid degradation of p53, inhibits growth arrest or p53-mediated apoptosis and masks the transactivation domain of p53 that interacts with the transcriptional machinery, resulting in the inactivation of p53 (10). Furthermore, as an oncogene, MDM2 plays a regulatory role in its interaction with other tumor-related genes that are important for cell-cycle control. It also contributes to carcinogenesis independently of p53 through interaction with transcriptional factors of the E2F family (11), inhibition of the Rb growth regulatory function (12) and inhibition of G₀/G₁-S-phase transition in normal cells (13).

In humans, the expression levels of MDM2 seem to be critical for regulating p53 in response to DNA damage, and p53 inactivation is often caused by point mutations within its coding regions. However, p53 inactivation can also occur through amplification of MDM2 (14) in many sporadic tumors, including lung cancer (15,16). One study reported that the accumulation of MDM2 in preneoplastic lung lesions was an early event of lung carcinogenesis (17). In tumors, the overexpression of MDM2 mRNA and proteins can substitute for p53 inactivation in the absence of p53 mutations (14) and is, thus, often associated with the clinical behavior of the tumors, such as cancer progression and treatment response (18).

Although MDM2 is rarely mutated, it is highly polymorphic. For example, there are at least 152 single-nucleotide polymorphisms (SNPs) and 18 insertion polymorphisms in the MDM2 gene, of which 68 have a minor allele frequency (MAF) > 5%, but none of these cause amino acid changes (http://egp.gs.washington.edu/data/mdm2/mdm2x.csnps.txt) (19). Therefore, MDM2 polymorphisms in a regulatory region, such as the promoter, may alter its transcriptional activities, thereby affecting p53 tumor suppression and carcinogenesis in humans. Therefore, MDM2 functional promoter polymorphisms probably contribute to interindividual variation in susceptibility to lung cancers as evidenced by an early age of onset (20).

To investigate the effect of the MDM2 promoter polymorphisms on the risk of lung cancer, we first chose the recently described functional SNP, also named SNP309 (T to G change at nucleotide 309) (20), designated G2580T in a published SNP database (see http://egp.gs.washington.edu/data/mdm2/mdm2x.csnps.txt) (19). From the same database, we also identified another three SNPs (C1797G, G2164A, and T2326C) that have an MAF > 10%. In a pilot study of 400 cases and 400 controls, however, we found that SNPC1797G and SNPT2326C were rare (MAF < 5%). Therefore, we only used the common (i.e. MAF > 5%) SNPG2580T (i.e. SNP309) and SNPG2164A to test our hypothesis that they are associated with risk of lung cancer by conducting a hospital-based case-control study of 1026 patients with lung cancer and 1145 cancer-free controls.

	Materials and methods

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Study subjects
The detailed methods of recruiting participants for this case–control study have been described elsewhere (2,21). Briefly, the participants were recruited consecutively from an ongoing molecular epidemiological study of lung cancer conducted by the Department of Epidemiology at The University of Texas M.D. Anderson Cancer Center. The 1026 case patients in this study had newly diagnosed, histopathologically confirmed, previously untreated (i.e. by radiotherapy or chemotherapy) lung cancer without restrictions on age, sex, stage or histology, and all were non-Hispanic whites. All patients were Texas residents recruited between 12 July 1996 and 24 September 2003.

The 1145 control participants were recruited from a pool of cancer-free volunteers from multiple metropolitan-area clinics of Houston's largest multispecialty physician practice, the Kelsey Seybold Foundation, during the period from 26 February 1996 through 1 October 2003. All controls, like the cases, were the residents in Texas who were enrolled as participants of the Kelsey Seybold Clinics. The controls were frequency matched to the cases on age (±5 years), sex, ethnicity and smoking status (i.e. ever and never). The response rate was 77.4% for the cases and 73.3% for the controls. The exclusion criteria included having had previous radiotherapy or chemotherapy, previous cancer or recent (in last 6 months) blood transfusions. After informed consent was obtained, each participant was interviewed, and a structured questionnaire on demographic data and selected risk factors was given and facilitated by interviewers. The study was approved by the institutional review boards of the M.D. Anderson Cancer Center and the Kelsey Seybold Foundation.

MDM2 polymorphism genotyping
We extracted genomic DNA from the buffy-coat fraction of the blood samples using a DNA blood mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. We performed genotyping for the MDM2G2580T genotypes using a primer-introduced restriction analysis polymerase chain reaction (PCR) method to create a Taq I restriction site. We used primers 5'-gttttgttggactggggcta-3' (sense) and 5'-tgcgatcatccggacctcccgcgtc-3'(antisense), for which a mismatched A was introduced to replace G 2 base pairs (bp) downstream from the polymorphism site so as to create a Taq I restriction site. We performed the PCR analysis with a PTC-200 DNA engine Peltier thermal cycler (MJ Research, Waltham, MA) in 10 µl of PCR mixture containing 20 ng of genomic DNA, 0.1 mM deoxyribonucleoside triphosphates (dNTPs), 10x PCR buffer (50 mM KCl, 10 mM Tris–HCl and 0.1% Triton X-100), 1.5 mM MgCl₂, 0.5 units of Taq polymerase (Sigma-Aldrich, St. Louis, MO) and 2 pmol of each primer. The amplification conditions were 5 min of initial denaturation at 95°C; 40 cycles of 30 s at 95°C, 45 s at 67°C and 60 s at 72°C; and a final 10 min extension step at 72°C. The PCR product (175 bp) was digested by Taq I and resolved on 3% metaphor gel containing ethidium bromide. The MDM2G2580T genotype was indicated by the presence of a T allele with fragment lengths of 26 and 149 bp and a G allele with a fragment length of 175 bp.

For the MDM2G2164A polymorphism, we designed a nested PCR with two pairs of primers and used the first PCR products as a template for the second PCR reaction instead of directly using the genomic DNA as PCR templates. Because the GC-rich sequence near the polymorphism site forms highly complex structures, the direct use of genomic DNA as PCR templates makes the PCR reaction more difficult and less efficient. We first performed the PCR with 5'-tgaccgagatcctgctgctttc-3' (sense) and 5'-tgagtcaacctgcccactgaac-3'(antisense) primers in a reaction mixture containing 20 ng of genomic DNA, 0.1 mM dNTPs, 10x PCR buffer (50 mM KCl, 10 mM Tris–HCl and 0.1 % Triton X-100), 1.5 mM MgCl2, 0.5 units of Taq polymerase (Denville Scientific, Inc, Metuchen, NJ) and 2 pmol of each primer. The amplification conditions were 5 min of initial denaturation at 95°C; 30 cycles of 30 s at 95°C, 30 s at 65°C and 45 s at 72°C; and a final 10 min extension step at 72°C. We then performed the second PCR with 5'-tgaccgagatcctgctgctttc-3' (sense) and 5'-tgagtcaacctgcccactgaac (antisense) primers in a mixture containing 2 ng of first PCR product DNA (640 bp), 0.1 mM dNTPs, 10x PCR buffer (50 mM KCl, 10 mM Tris–HCl and 0.1 % Triton X-100), 1.5 mM MgCl2, 0.5 units of Taq polymerase (Denville Scientific, Inc, Metuchen, NJ) and 2 pmol of each primer. The amplification conditions were 5 min of initial denaturation at 95°C; 16 cycles of 30 s at 95°C, 30 s at 65°C and 30 s at 72°C; and a final 10 min extension step at 72°C. The 115 bp PCR product was digested by Bcl I and separated on 3% metaphor gel containing ethidium bromide. The MDM2G2164A genotype was indicated by the presence of A allele with fragment lengths of 17 and 98 bp and a G allele with a fragment length of 115 bp. All the primer designs were based on the genomic DNA sequences (GenBank accession no. AF527840 [GenBank] ). We performed the PCRs and evaluated the results without knowing the samples' case or control status. At least 10% of the random samples were retested, and the results of retesting were 100% concordant.

Statistical analysis
Differences in select demographic variables, smoking status, pack-years smoked and MDM2 genotype frequencies between the cases and controls were evaluated by using the ² test. The associations between MDM2 variant genotypes and risk of lung cancer were estimated by computing the odds ratios (ORs) and their 95% confidence intervals (CIs) from both univariate and multivariate logistic regression analyses. We used stratification analysis to estimate risk for subgroups by age, sex, smoking status, pack-years smoked and lung cancer histology. Those who had smoked <100 cigarettes in their lifetime were defined as never smokers and others as ever smokers. We also evaluated the association between lung cancer risk and the combined genotypes of these two polymorphisms. Logistic regression was used to assess the potential interaction effects by evaluating departures from the models of additive and multiplicative interactions between the two polymorphisms and age, sex and smoking variables. A more-than-additive interaction was suggested when OR₁₁ > OR₁₀ + OR₀₁ –1, for which OR₁₁ = OR when both factors were present, OR₁₀ = OR when only factor 1 was present and OR₀₁ = OR when only factor 2 was present. A more-than-multiplicative interaction was suggested when OR₁₁ > OR₁₀ x OR₀₁. Statistical difference was considered significant for P values <0.05. All tests were two-sided, and all statistical analyses were performed with statistical analysis system software (Version 8e, SAS Institute, Cary, NC).

	Results

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The demographic variables, smoking status and pack-years smoked for the cases and controls are summarized in Table I. There were five mixed tumors that did not fit in any of the categories, and we excluded these five patients from the analysis. Therefore, in our final analysis we had 1026 case patients and 1145 cancer-free controls, all of whom were non-Hispanic white. Because we used frequency matching, there were no significant differences in the distributions of age, sex and smoking status between the cases and controls (P = 0.983 for age, P = 0.100 for sex and P = 0.703 for smoking status). The median age for both the cases (mean = 61.2 years, range = 33–87 years) and the controls (mean = 61 years, range = 32–91 years) was 62 years. There were more heavy smokers (pack-years 38) and fewer light smokers (pack-years <38) among the cases than among the controls, and the difference was statistically significant (P < 0.001). Because the matching on smoking status for this analysis was relaxed to ever versus never (2), it appeared that this analysis included more current and heavy but fewer former smokers among the cases than among controls, not achieving adequate frequency matching on smoking status (current, former, and never smokers) (P = 0.024). To control for any residual effect of imperfect frequency matching, these variables were adjusted for in later multivariate logistic regression analyses.

View this table: [in this window] [in a new window]   Table I Frequency distributions of selected variables in lung cancer cases and control subjects

 
The genotype and allele frequencies of the MDM2G2580T and MDM2G2164A polymorphisms and their association with lung cancer risk are shown in Table II. The genotype distributions among the controls for both MDM2G2580T and MDM2G2164A polymorphisms were in Hardy–Weinberg equilibrium (P = 0.101 and 0.227, respectively). The MDM2-2580G variant allele frequency was statistically significantly less common among the cases (0.362) than among the controls (0.393) (P = 0.038), suggesting that the MDM2-2580G allele had a protective effect on risk for lung cancer or, alternatively, the 2580T allele may be a risk allele. However, the frequency of the MDM2-2164G variant allele was not significantly different between the cases (0.411) and the controls (0.408) (P = 0.865). For subjects with the MDM2G2580T polymorphism, the distribution of the TT, GT and GG genotypes between the cases and controls was statistically significantly different (P = 0.045), but the distribution of the MDM2-2164 AA, GA and GG genotypes was not (P = 0.614).

View this table: [in this window] [in a new window]   Table II Risk of lung cancer associated with the MDM2 genotypes and allele frequencies

 
Compared with the MDM2-2580TT genotype, there was a significantly (19%) reduced lung cancer risk associated with the MDM2-2580GT (adjusted OR = 0.81, 95% CI = 0.67–0.98) and the combined MDM2-2580GT/GG (OR = 0.81, 95% CI = 0.68–0.97) genotypes (Table II). However, compared with the MDM2-2164AA genotype, there was no significant association between lung cancer risk and the MDM2-2164GA (OR = 0.92, 95% CI = 0.76–1.11), MDM2-2164GG (OR = 1.06, 95% CI = 0.82–1.36) and combined MDM2-2164GA/GG (OR = 0.95, 95% CI = 0.79–1.14) genotypes.

We further stratified the data by subgroups of age, sex, smoking status, pack-years smoked and cancer histology (Table III). When we used the combined variant MDM2-2580GT/GG genotype as the reference, the risk associated with the TT genotype was more evident for younger (60 years) individuals (OR = 1.28, 95% CI = 0.99–1.67), men (OR = 1.32, 95% CI = 1.03–1.69), ever smokers (OR = 1.34, 95% CI = 1.10–1.63), light smokers (1–37 pack-years) (OR = 1.58, 95% CI = 1.17–2.14) and subjects with squamous cell lung cancer (OR = 1.37, 95% CI = 1.01–1.85) or non-small cell lung cancer not otherwise classified. (OR = 1.38, 95% CI = 1.01–1.88). When we used the combined variant MDM2-2164GA/GG genotype as the reference, we did not find any significant association between the AA genotype and lung cancer risk in any subgroup. Furthermore, there was no evidence for an interaction between these MDM2 variant genotypes and age, sex, smoking status and pack-years smoked on the risk of lung cancer (data not shown).

View this table: [in this window] [in a new window]   Table III Associations and stratification analysis of MDM2 polymorphisms and lung cancer risk

 
Because both the MDM2-2580T and MDM2-2164A alleles appeared to be associated with an increased risk of lung cancer, we evaluated their combined effect by grouping the participants into four categories based on the number of risk alleles each possessed. We performed a linkage disequilibrium test between these two SNPs (D' = 0.777, R² = 0.255 and P < 0.01) before we combined them for the analysis. Group 1 had zero or one risk allele of either polymorphism (i.e. genotypes MDM 2-2580GT/2164GG, MDM2-2580GG/2164GA or MDM2-2580GG/2164GG); group 2 had two risk alleles of either polymorphism (i.e. genotypes MDM2-2580TT/2164GG, MDM2-2580GT/2164GA or MDM2-2580GG/2164AA); group 3 had three risk alleles of either polymorphism (i.e. genotypes MDM2-2580TT/2164GA or MDM2-2580GT/2164AA) and group 4 had four risk alleles of either polymorphism (i.e. genotypes MDM2-2580TT/2164AA). As shown in Table IV, the difference in the distribution of the combined genotypes between the cases and controls was statistically significant (P = 0.023). Compared with group 1, groups 2, 3 and 4 had a significantly increased risk of lung cancer (adjusted OR = 1.78, 95% CI = 1.16–2.72 for group 2, 1.71, 1.11–2.64 for group 3 and 2.26, 1.33–3.85 for group 4), but the trend was only borderline statistically significant (P_trend = 0.070).

View this table: [in this window] [in a new window]   Table IV ORs and 95% CIs for the combined MDM2G2580T and MDM2G2164A polymorphism genotypes associated with lung cancer risk

 

	Discussion

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In the present study, we examined the association of the MDM2G2580T and MDM2G2164A polymorphisms with the risk of lung cancer. We observed a nearly 20% reduced lung cancer risk associated with the MDM2-2580GT and combined MDM2-2580GT/GG variant genotypes compared with the MDM2-2580TT genotype. Conversely, the MDM2-2580TT genotype was associated with a 25% increased risk for lung cancer, compared with the other genotypes, and this risk was more evident for younger (60 years) individuals, men, ever smokers, light smokers and patients with squamous cell or non-small cell lung cancer not otherwise classified. We observed no association between the MDM2G2164A polymorphism and the risk of lung cancer, but the polymorphism did appear to interact with the MDM2G2580T polymorphism. Indeed, we found that participants who were homozygous for both risk alleles had a nearly 2.3-fold increased risk, although such individuals were relatively uncommon.

It is plausible that MDM2 polymorphisms might affect cancer risk, as MDM2 has been shown to interact with several key tumor suppressors, including Rb (12), p53-family protein, p73 (22), the growth suppressor p14/p19 (23) and p53 (9). These interactions contribute to the role of MDM2 in inactivating tumor suppressor genes that lead to negatively regulated cell proliferation, which is a critical factor in human tumorigenesis. Overexpression of MDM2 and the subsequent expression of p21 are induced by p53 in response to DNA damage caused by exposure to environmental carcinogens, leading to an arrest of cell cycle, which in turn allows sufficient time for DNA repair (24). The MDM2G2580T polymorphism is near the element believed to direct the intronic p53-response promoter activity of the gene, resulting in increased levels of MDM2 RNA and protein in an in vitro functional assay (20). Therefore, the variant G allele is correlated with a higher promoter activity and, thus, subsequently a lower level of p53 compared with the T allele, because the variant G allele increases the affinity of the transcriptional activator Sp1 and, subsequently, attenuates the p53 pathway (20). However, in the current study, we observed that the variant G allele was associated with a significantly reduced risk of lung cancer, which seems to contradict the findings of the in vitro functional assay (20).

Although experimental approaches can provide biological evidence of the functional role of the SNP309G allele, which predicts a higher risk of cancer compared with the SNP309T allele (20), epidemiological study of complex human traits may unravel more complicated interactions among all variants in the MDM2 gene or variants in other candidate genes that may have an effect on carcinogenic outcomes. For example, in studies of CYP3A4 on metabolism of drugs, experimental evidence of genetic variant functions was also not consistent with results of epidemiological studies (25). Thus, it might not be possible to make a clear inference in humans from the in vivo allele function based on experimental data (25). Even in humans, the same gene might play different roles in different carcinomas. For example, overexpression of MDM2 was correlated with a decreased level of p21 expression in epithelial breast cancer but with an increased level of p21 in oral squamous cell carcinoma (26).

Furthermore, most of the published studies on the association between the MDM2G2580T polymorphism and risk of cancers did not show that the G allele was associated with a younger age of onset or increased risk of cancer (27–30), although only one Chinese lung cancer study did find a positive association (31). Specifically, one lung cancer study found no association between the MDM2G2580T polymorphism and risk of lung cancer in a Chinese population (27), and another found no significant association between this polymorphism and risk of breast or ovarian cancer among women in the UK (28). In a Finnish population, no significant positive association was found between the MDM2G2580T polymorphism and risk of uterine leiomyosarcoma, colorectal cancer or squamous cell carcinoma of the head and neck, and nor was such an association found with colorectal cancer in an American study (30).

The conflicting results lead us to think that although the MDM2-2580G variant alleles may be functionally relevant, epidemiological findings may also imply their linkage disequilibrium with other unidentified functional variants of MDM2 or with alleles at other nearby loci of other candidate genes. However, this hypothesis remains to be tested. Furthermore, the effect of a low-penetrance susceptibility gene on disease risk might be influenced by other modifying genes and environmental factors through gene–gene or gene–environment interactions. Therefore, different genetic backgrounds and different risk factors might explain, to some extent, the somewhat conflicting results in risk estimates associated with this MDM2 polymorphism in different cancers and different study populations. For example, in a previous study, we found that an increased risk of lung cancer was significantly associated with the variant alleles of the p73G4C14-to-A4T14 polymorphism in an American, non-Hispanic population, whereas the same variant allele was found to be protective against lung cancer in a Chinese population (32). Other factors in the studies such as small sample size, inclusion of a single polymorphism or different ethnic groups in a single study, or inadequate adjustment for confounding factors could also cause the inconsistent results.

We found that the greater risk associated with the risk allele genotype MDM2-2580TT compared with genotype MDM2GT/GG was in younger (60 years) participants and light smokers, suggesting an early age of onset and genetic susceptibility to lung cancer in this study population. On the other hand, it is possible that high-level exposure to cigarette smoke (i.e. heavy smoking) may cause severe DNA damage and, subsequently, lead to more cell death through the apoptotic pathway, which if true, would reduce the probability of an oncogenic process. We also observed a significantly higher risk in men than women, particularly among smokers; however, the interaction between sex and the MDM2 polymorphism was not statistically significant (data not shown), which warrants further investigation with larger sample sizes. In addition, we found that the risk was higher for squamous cell lung cancer or non-small cell lung cancer not otherwise classified that was probably induced by tobacco smoke (33), which is consistent with the greater risk in ever smokers than in never smokers we observed in the present study. However, the significance of this finding may be limited because of the small number of patients with squamous cell or non-small cell lung cancer not otherwise classified included in this study. Future studies with larger sample sizes of these two subtypes of lung cancer are needed to confirm our finding.

In conclusion, we found that the MDM2-2580G (or SNP309) variant genotypes were associated with a reduced risk for lung cancer, a finding that seems to contradict an earlier functional study of this variant (20). Because this was a hospital-based, case–control study and the controls were not selected from the same population from which the cases arose, we could not rule out possible selection bias. However, by matching on age, sex, ethnicity and smoking status, we hope to have minimized potential confounding factors. To confirm the role of these MDM2 polymorphisms in cancer risk, the findings need to be verified in large population-based prospective studies for more rigorous analyses of subgroups and gene–environment and gene–gene interactions.

	Acknowledgments

 
We thank Margaret Lung and Peggy Schuber for assistance in recruiting the subjects; Li-E Wang, Zhaozheng Guo, Yawei Qiao, Jianzhong He and Kejin Xu for laboratory assistance; and Betty J.Larson and Joanne M.Sider for manuscript preparation and Chris J.Yeager for scientific editing. This study was supported in part by National Institutes of Health grants ES 11740 (to Q.W.), CA 57730 (to R.M.C.), CA55769 (to M.R.S.), Cancer Center Support Grant CA 16672, ES 11074 (to M.D. Anderson) and a Flight Attendants Medical Research Institute Professorship (to M.R.S.).

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Received December 9, 2005; revised March 2, 2006; accepted March 31, 2006.

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