GLUTATHIONE-S-TRANSFERASE POLYMORPHISMS AND RISK OF SQUAMOUS-CELL CARCINOMA OF THE HEAD AND NECK

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1 Int. J. Cancer (Pred. Oncol.): 84, (999) 999 Wiley-Liss, Inc. GLUTATHIONE-S-TRANSFERASE POLYMORPHISMS AND RISK OF SQUAMOUS-CELL CARCINOMA OF THE HEAD AND NECK Publication of the International Union Against Cancer Publication de l Union Internationale Contre le Cancer Lie CHENG, Erich M. STURGIS,2, Susan A. EICHER 2, David CHAR, Margaret R. SPITZ and Qingyi WEI * Department of Epidemiology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA 2 Department of Head and Neck Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Differences in genetic susceptibility to tobacco-induced carcinogenesis appear to modulate an individual s risk of squamous-cell carcinoma of the head and neck (SCCHN). Risk for SCCHN may be associated with the null alleles of the carcinogen-metabolizing genes glutathione-s-transferase (GST) T and GSTM. In this study, we evaluated the association between GSTM and GSTT null genotypes and risk of SCCHN in a matched case-control study of 62 patients with SCCHN and 35 healthy controls. Our results showed that 53.% of cases and 42.9% of controls were null for GSTM, whereas 32.7% of cases and 7.5% of controls were null for GSTT (p F 0.05 and p F 0.00, respectively). Furthermore, 9.8% of cases but only 7.9% of controls were null for both genes (p F 0.00). Multivariate analysis using logistic regression models, including age, sex, ethnicity, smoking status, alcohol status and GST genotypes, showed that both of these genotypes remained independent risk factors for disease [adjusted odds ratios (ORs).50 and 2.27, respectively; 95% confidence intervals (CIs) and , respectively). When the genotypes were divided into neither null, either null or both null, there was a dose-response relationship (adjusted OR.50, 95% CI ) for the either-null group and (adjusted OR 3.64, 95% CI ) for the both-null group (p F 0.00, trend test). Our findings suggest that the GSTM and GSTT null genotypes are independent risk factors for SCCHN and markers for genetic susceptibility to tobacco-induced carcinogenesis. Int. J. Cancer (Pred. Oncol.) 84: , Wiley-Liss, Inc. Squamous-cell carcinoma of the head and neck (SCCHN), including the oral cavity, pharynx and larynx, is the 5th most common non-cutaneous malignancy worldwide and 2nd only to stomach cancer in the developing world (Parkin et al., 993). The vast majority of SCCHNs are induced by tobacco and alcohol use, but while approximately 46 million Americans smoke (Office of Smoking and Health, CDC, 994), only 40,000 to 50,000 SCCHNs are diagnosed annually in the United States (Landis et al., 998). This disparity suggests that there is a spectrum of genetic susceptibility to upper aerodigestive tract carcinogenesis. Further support for susceptibility to this disease is evidenced by the increased risk of SCCHN in first-degree relatives of SCCHN patients (Foulkes et al., 996), by patients who develop SCCHN at unusually young ages (Schantz et al., 988) and by SCCHN patients who had no exposure to known carcinogens (Koch and McQuone et al., 997). Differences in carcinogen metabolism and DNA repair due to genetic variations (polymorphisms) have been suggested to underlie this variance in susceptibility. Phenotypic assays, such as the mutagen-sensitivity assay (Spitz et al., 989; Wang et al., 998) and the host cell re-activation assay (Cheng et al., 998), and measurement of DNA repair gene transcript levels (Wei et al., 998) have been shown to be useful for assessing risk for SCCHN. These assays suggest that differences in DNA repair ability may partially underlie differences in risk; however, the results of such phenotypic assays vary widely for unknown reasons. Genotypic studies of DNA repair genes have not been practical because little is known about genetic variation in these genes. In fact, only recently have polymorphisms in several DNA repair genes in the normal population been reported (Broughton et al., 996; Shen et al., 998). However, polymorphisms in carcinogen-metabolizing genes have been well documented in several molecular epidemiology studies (Rebbeck, 997; Puga et al., 997). Tobacco smoke comprises at least 50 known carcinogens, the most important being the polycyclic aromatic hydrocarbons, aromatic amines and nitroso compounds (Ryberg et al., 997). Typically, these compounds are oxidized by the phase I enzymes of the cytochrome P-450 system to yield reactive epoxide intermediates such as benzo[a]pyrene diol epoxide (BPDE) (Gelboin, 980; Cosman et al., 992; Ryberg et al., 997). BPDE has toxic electrophilic and free-radical properties and can modify DNA by covalent binding and oxidation. BPDE is detoxified by various phase II enzymes, including the glutathione-s-transferases (GSTs) (Puga et al., 997; Ryberg et al., 997). The GSTs catalyze glutathione conjugation of carcinogenic electrophilic epoxide intermediates to protect the cell against DNA damage and adduct formation. The GSTs comprise 4 different gene classes (,µ, and ), and each class includes several genes. For instance, the GST µ family (GSTM) includes GSTM, GSTM2, GSTM3, GSTM4 and GSTM5. Overlap in substrate specificity and variation in tissue expression of these genes results in a complex phenotypic picture. However, genotypic differences have been studied on a number of GSTs, particularly, GSTM and GST (GSTT) (Rebbeck, 997; Puga et al., 997; Ryberg et al., 997). Each of these 2 isoenzymes has a common null allele without enzyme activity. Approximately 50% and 5% of Caucasians in the United States are homozygous for the GSTM and GSTT null genotypes, respectively, but considerable ethnic differences exist in the allele frequencies (Rebbeck, 997). Several studies have evaluated the risk of SCCHN in individuals with the GSTM null genotype (Katoh, 994; Trizna et al., 995; Jahnke et al., 996; Deakin et al., 996; Coutelle et al., 997; Jourenkova et al., 998; Ophuis et al., 998) or phenotype (Lafuente et al., 993) and the GSTT null genotype (Katoh, 994; Trizna et al., 995; Jahnke et al., 996; Deakin et al., 996; Jourenkova et al., 998; Ophuis et al., 998). Most of these case-control studies have found that those with either null genotype have slightly increased risk of SCCHN, but the combined effect of the 2 genotypes on risk was examined by only one group (Jourenkova et al., 998). Therefore, we performed a case-control study of SCCHN to explore the contribution of GSTM and GSTT null genotypes to the risk of SCCHN and to test the hypothesis that lack of such tobacco carcinogen detoxifying enzymes contributes to host genetic susceptibility. Furthermore, we explored the combined effect of the genes by controlling for smoking and alcohol use and matching for age, sex and ethnicity. MATERIAL AND METHODS Study subjects Cases were recruited from outpatients who registered in the Department of Head and Neck Surgery at The University of Texas Grant sponsor: National Institutes of Health; Grant numbers: CA70334; CA75840; CA55769; CA *Correspondence to: Department of Epidemiology 89, The University of Texas M.D. Anderson Cancer Center, 55 Holcombe Blvd., Houston, TX 77030, USA. Fax: QWEI@NOTES.MDACC.TMC.EDU Received 7 August 998; Revised 6 December 998

2 GSTM, GSTT AND HEAD-AND-NECK CANCER 22 M.D. Anderson Cancer Center between May 995 and April 998. Blood samples were obtained before treatment started. Medical records were reviewed to acquire the following information on each patient: final histo-pathologic diagnosis, T stage, N stage, overall stage, primary tumor site and number of primary tumors. Patients with primary tumors of the nasopharynx, with primary tumors outside the upper aerodigestive tract or with histopathologic diagnoses other than SCC were excluded. Cancer-free controls were obtained from 2 selected control populations: outpatients in clinics of a large local health maintenance organization (Hudmon et al., 997) and spouses of outpatients at M.D. Anderson Cancer Center. After informed consent was obtained, each participant completed a questionnaire that elicited information on age, sex, ethnicity and tobacco and alcohol use. Efforts were made to match controls to cases on age ( 5), sex, ethnicity and tobacco use. In addition, each subject donated 0 ml of blood in heparinized tubes for genotyping assays. The research protocol was approved by the M.D. Anderson Institutional Review Board. Genotypying of GSTM and GSTT The leukocyte cell pellet obtained from the buffy coat by centrifugation of whole-blood samples was used for DNA extraction with a DNA extraction kit (Qiagen, Santa Clarita, CA). A multiplex PCR assay was established to simultaneously genotype GSTM and GSTT plus the dihydrofolate reductase (DHFR) gene as an internal control for amplification failure due to DNA degradation. The primers used for these genes were 5 -GAA CTC CCT GAAAAG CTAAAG C-3 and 5 -GTT GGG CTC AAA TAT ACG GTG G-3 for GSTM, which generate a 480-bp fragment; 5 -TTC CTT ACT GGT CCT CAC ATC TC-3 and 5 -TCA CCG GAT CAT GGC CAG CA-3 for GSTT, which generate a 25-bp fragment; and 5 -CAT CGG CAA GAA CGG GGA CCT-3 and 5 -ACC GAA GCC TCC ACC CAG TTG-3 for DHFR, which generate a 280-bp fragment. In this PCR assay, the absence of a 480-bp band or a 25-bp band indicates the GSTM null and GSTT null genotypes, respectively. When none of these fragments was present, either the PCR was not successful or the DNA was degraded because DHFR is an essential gene and normally should be easily amplified. GSTM, GSTT and DHFR were co-amplified in a 40-µl reaction mixture containing 00 ng of genomic DNA as the template, 3.5 pmol of each GSTM primer, 2.9 pmol of each GSTT primer, 6.2 pmol of each DHFR primer, 0. mm each dntp, PCR buffer [500 mm KCl, 00 mm Tris HCl (ph 9.0), % Triton X-000 and 2.5 mm MgCl 2 ) and.5 units of Taq polymerase (Promega, Madison, WI). The PCR profile consisted of an initial melting step at 95 C for 5 min followed by 30 cycles at 95 C for 30 sec, 56 C for 45 sec and 72 C for 45 sec, with a final step at 72 C for 0 min for elongation. PCR products were separated on.2% agarose gels and photographed using Polaroid film. Statistical analysis The GST genotypes were analyzed as a dichotomized variable, with 0 being the null genotype and being the other genotype. Univariate analysis was first performed to calculate crude odds ratios (ORs) and confidence intervals (CIs) for various strata for the GSTM and GSTT genotypes. Those who had smoked less than 00 cigarettes in their lifetime were defined as never users of tobacco, those who were smokers but had quit smoking for more than year previously as former smokers and the rest as current smokers. Those who drank alcoholic beverages less than once a week for more than year were defined as never users of alcohol, those who were drinkers but had quit drinking for more than year previously as former users of alcohol and the rest as current users. Ethnicity was recorded as non-hispanic white, African-American or Mexican-American. For logistic regression analysis, GST genotype was recoded as a dummy variable (0.0 for both null, 0. for GSTM null and.0 for GSTT null). To evaluate the dose-response relationship, subjects were trichotomized into a value of 2 for both GSTM and GSTT null genotypes, for either null genotype and 0 for neither null genotype. To assess trend, these trichotomized variables were treated as a continuous variable and fit into the logistic regression model. All statistical analyses were performed with Statistical Analysis System software (version 6; SAS Institute, Cary, NC). RESULTS In our study, 77 patients and 35 controls were recruited and genotyped. Eleven cases were excluded (0 for having a final diagnosis other than SCC and for having a nasopharyngeal primary tumor). Four Asian patients were excluded because there were no Asians in the control population. Therefore, 62 patients and 35 controls met the inclusion criteria for this study. The characteristics of the subjects are given in Table I. Mean ages were 6.6 years (median 6, range 24 89) for patients and 60.4 years (median 63, range 27 84) for controls, and the groups were well matched on sex ratio. Perhaps most important for a case-control study of genotype frequencies, the groups were very well matched on ethnicity. As seen in Table I, tobacco and alcohol users were more prevalent in cases than in controls, indicating that matching on smoking status was not complete and needed further adjustment in later analysis. The percentage of cases and controls who were GSTM null was 53.% and 42.9% (p 0.034), respectively, whereas 32.7% of cases and 7.5% of controls were GSTT null (p 0.00) and 9.8% of cases and 7.9% of controls were null for both genes (p 0.00) (Table II). After stratification for the potential confounding variables mentioned in Table I, some interesting differences in genotype frequencies were noted between subgroups of patients (Table II). Among patients, those who were over the age of 65, never smokers and never drinkers demonstrated the highest frequencies of the GSTM null genotype. There was a trend of increasing GSTM null genotype frequency with decreasing use of alcohol among patients but not controls. Furthermore, among patients, those who were 45 years of age, never smokers and former drinkers had higher frequencies of the GSTT null genotype. There was also a trend of increasing GSTT null genotype frequency with decreasing age and decreasing tobacco use in patients. Similarly, young patients and those who were former drinkers also had exceptionally high frequencies of being null for both genes. The GSTM and GSTT distributions of controls for current, former and never smokers were in different directions from that for cases (Table II), though these results may not represent those in the TABLE I FREQUENCY DISTRIBUTION OF SEVERAL DEMOGRAPHIC VARIABLES AND RISK FACTORS OF SCCHN IN CASES AND CONTROLS Variable Cases Controls Number (%) Number (%) p value Age (years) Sex Male Female Ethnicity Non-Hispanic white African-American Mexican-American Smoking status Current Former Never Alcohol status Current Former Never Two-sided 2 test.

3 222 CHENG ET AL. TABLE II GST GENOTYPE FREQUENCIES OF CASES AND CONTROLS STRATIFIED BY SELECTED FACTORS GSTM null (%) GSTT null (%) Both null (%) Cases Controls p Cases Controls p Cases Controls p All subjects Age (years) Sex Male Female Ethnicity White Other Smoking status Current Former Never Alcohol status Current Former Never Two-sided 2 test. 2 Non-Hispanic whites. 3 African-Americans and Mexican-Americans. Genotype TABLE III LOGISTIC REGRESSION ANALYSIS OF GST GENOTYPES IN CASES AND CONTROLS Number (%) of Cases Controls Crude OR (95% CI) Adjusted OR (95% CI) Dichotomized GSTM 76 (46.9) 80 (57.) GSTM null 86 (53.) 35 (42.9).5 ( ).50 ( ) GSTT 09 (67.3) 260 (82.5) GSTT null 53 (32.7) 55 (7.5) 2.30 ( ) 2.27 ( ) Trichotomized Neither null 55 (34.0) 50 (47.6) Either null 75 (46.3) 40 (44.4).46 ( ).50 ( ) Both null 32 (9.7) 25 (7.9) 3.49 ( ) 3.64 ( ) Trend test p 0.00 p 0.00 Adjusted in multivariate logistic regression models including age, sex, ethnicity, smoking status, alcohol use and GST genotypes. general population due to the matching design. Compared with controls, however, cases also consistently had higher frequencies of the GSTM null, the GSTT null and the combined GSTM/GSTT null genotype for all subgroups examined except for the relatively small African-American/Mexican-American group (combined as Other in Table II). While cases consistently had a higher frequency of the null genotypes, many of the differences were not statistically significant, probably because of the small numbers of subjects in each subgroup. However, these differences justified the need for further adjustment of these potential confounding variables in the logistic regression model. After the data were dichotomized into null and other genotypes, the crude OR for the GSTM null genotype as a risk for SCCHN was.5 (95% CI ) and that for the GSTT null genotype was 2.30 (95% CI ) (Table III). When the data sets were divided into 3 groups (neither GSTM nor GSTT null, either GSTM or GSTT null and both GSTM and GSTT null), there was a dose-response relationship with risk for disease (trend test p 0.000) (Table III). Logistic regression analysis was then performed to adjust for the residual effects of the variables listed in Table I. The results of a logistic regression model, which included age, sex, ethnicity, smoking status, alcohol status and GSTM and GSTT null genotypes, are summarized in Table III. After the adjustment, both GSTM and GSTT null genotypes remained significant independent risk factors for SCCHN (ORs.50 and 2.27, respectively). The dose-response relationship between genotype frequencies was further evaluated and confirmed in a similar logistic regression model using the trichotomized variables (trend test p 0.000) (Table III). Among patients with newly diagnosed SCCHNs, there was no substantial difference in genotype frequencies related to T stage, N stage or overall stage, except for a significantly lower frequency of the GSTM null genotype in N patients (but not in N2 or N3 patients) compared with N0 patients (p 0.05) (data not shown). In addition, patients with primary tumors of the oral cavity had a significantly higher frequency of the GSTM null genotype (67.2%) than patients with laryngeal (44.9%, p 0.05, 2 test) or pharyngeal cancer (38.%, p 0.05, 2 test), whereas patients with laryngeal cancer had a higher frequency of the GSTT null genotype (38.%) than patients with pharyngeal (36.7%) or oral (3.%) cancer, though the differences were not statistically significant. One hundred thirty-seven patients had only a single malignancy, whereas 25 had multiple primary tumors. Those with multiple primary tumors had higher frequencies of the GSTM and the combined null genotypes (68.0% and 28.0%, respectively) than those with only a single primary tumor (50.4% and 9.0%, respectively), but this difference was not statistically significant (p 0.59 and 0.58, respectively, 2 test). DISCUSSION In our case-control study of 62 patients with SCCHN and 35 matched controls, we demonstrated a significantly elevated risk of disease in patients with both the GSTM and GSTT null genotypes after adjustment for age, sex, ethnicity, smoking and alcohol use in multivariate logistic regression models. The risk was especially elevated in those with both null genotypes.

4 GSTM, GSTT AND HEAD-AND-NECK CANCER 223 Support of the GSTM and GSTT null genotypes as markers of genetic susceptibility was evident upon analysis of frequency differences within control subgroups. First, there were no significant differences among controls in genotype frequencies related to smoking status or alcohol use. This is consistent with the genotypes being genetic markers that segregate with the disease and not with the risk factors for disease. Second, non-hispanic whites had higher GSTM and lower GSTT null genotype frequencies than the combined group of African-Americans and Mexican-Americans (p and 0.008, respectively, 2 test). This finding reflects the different genetic backgrounds of these groups and is consistent with a prior report (Rebbeck, 997). Finally, that older controls had lower frequencies of being GSTM, GSTT and both null may reflect some selection bias in the study population, such as a possible survivor effect. In theory, the most susceptible are those who develop SCCHN at a young age. In our study, it was the young ( 45 years old) who had the highest null genotype frequencies, and a clear trend was found with increasing GSTT null frequency with decreasing age. While we defined young patient as those 45 years of age, most clinical reviews have defined it as those 40 years of age (Schantz et al., 988). Our study included only such patients, but they did have exceptionally high frequencies of the GSTM null, GSTT null and combined null genotypes (55%, 55% and 36%, respectively). These findings are consistent with those of a prior study of GSTM phenotype in which 72% of young larynx cancer patients, but only 63% of older patients, lacked the GSTM isoenzyme (Lafuente et al., 993). We also might expect that more susceptible individuals would develop SCCHN even in the absence of carcinogen exposure. In fact, non-smoking patients had the highest frequencies of both the GSTM and GSTT null genotypes and non-drinking and former-drinker patients had higher frequencies of the GSTM, GSTT and combined null genotypes. Others have also found a similar connection between less tobacco exposure and higher GSTM genotype frequencies among SCCHN patients (Jourenkova et al., 998). While this subgroup analysis is obviously hampered by its lack of power, 3 groups of patients (young, non-smokers and non-drinkers) who we hypothesize are particularly susceptible to developing SCCHN also appear to have higher frequencies of the null genotypes. If the GST genotype is indeed a marker of genetic susceptibility, its frequency should not segregate with the stage of disease. Aside from one exception, there was no significant difference in genotype frequency among the various stage groups. One might hypothesize that a certain genotype may confer a greater susceptibility to disease at particular sites. Indeed, patients with oral-cavity primary tumors had higher rates of the GSTM null genotype, whereas patients with laryngeal primary tumors had higher frequencies of the GSTT null genotype. Similarly, Ophius et al. (998) found a higher frequency of the GSTM null genotype in those with oral-cavity and pharynx primary tumors than in those with laryngeal primary tumors. A large number of tumors may also indicate a more susceptible individual. Indeed, we found that more than two-thirds of the patients with multiple tumors were null for the GSTM gene, but the number of such patients was relatively small. Clearly, larger studies are needed to explore these differences in genotype frequency in patients with tumors at different sites and with different numbers of tumors. A plethora of studies have examined the risk for cancer (particularly lung and bladder) among individuals possessing the GSTM or GSTT null genotype (for review: Rebbeck, 997). Most previous studies of the GSTM null genotype (Katoh, 994; Trizna et al., 995; Jahnke et al., 996; Deakin et al., 996; Coutelle et al., 997; Jourenkova et al., 998; Ophuis et al., 998) or phenotype (Lafuente et al., 993) and the GSTT null genotype (Katoh, 994; Trizna et al., 995; Jahnke et al., 996; Deakin et al., 996; Jourenkova et al., 998; Ophuis et al., 998) in SCCHN patients have reported small or no effects. While 4 of these studies showed no significant difference in the frequency of the GSTM null genotype, all but one found a trend toward a higher frequency in SCCHN patients and 3 of the negative studies (Jahnke et al., 996; Deakin et al., 996; Ophuis et al., 998) made no attempt at matching or multivariate adjustment. Furthermore, when all results are combined, 59% of the 896 SCCHN patients and 52% of the,342 controls have the GSTM null genotype (Katoh, 994; Trizna et al., 995; Jahnke et al., 996; Deakin et al., 996; Coutelle et al., 997; Jourenkova et al., 998; Ophuis et al., 998). While none of the 5 studies performing GSTT genotyping demonstrated significant differences between cases and controls alone, when combined, 24% of the 744 SCCHN patients and 8% of the,46 controls have the GSTT null genotype (Trizna et al., 995; Jahnke et al., 996; Deakin et al., 996; Jourenkova et al., 998; Ophuis et al., 998). In addition, only one study calculated ORs adjusted for sex, age and multiple potential risk variables (Jourenkova et al., 998). These discrepancies in study design may have contributed to the differences between our results and the published data. In this study, we examined all 3 phenotypic markers of susceptibility (young age at onset and limited tobacco or alcohol exposure) in a group of SCCHN patients in relation to the genotypic markers (GSTM and GSTT null genotype) of susceptibility. We intend to increase our efforts to recruit more young patients, non-smokers and non-drinkers with SCCHN to further explore these interactions. In conclusion, we examined the risk of SCCHN associated with GSTM and GSTT null genotypes in a matched case-control study. There are multiple substrates in tobacco smoke that undergo metabolism by GSTM and GSTT enzymes. Lack of these enzymes involved in detoxification of activated tobacco carcinogens should theoretically predispose an individual to a tobaccoinduced cancer. Overlap in substrate specificity of these enzymes further predicts an additive effect due to loss of both GSTM and GSTT enzymes. Several lines of evidence presented herein support this biological plausibility. First are the differences between cases and controls in the GSTM, GSTT and combined null genotypes, a finding consistent across virtually all subgroups. Furthermore, the highest risk associated with combined null genotypes was consistent with susceptibility. Second, the GST genotype frequencies of the controls were not different in the smoking or alcohol subgroups, consistent with a marker of susceptibility that is independent of other risk factors. The ethnic differences in GST allele frequencies are also consistent with a marker of genetic differences. Third, some of the highest null allele frequencies were in hypothetically more susceptible groups (those 40 years of age, those with less tobacco and alcohol exposure and those with multiple tumors). Finally, the null allele frequencies appeared to be unaffected by stage of disease, once again suggesting that this is a marker of risk, not of disease. However, our results may be biased by the relatively small number of subjects in the various subgroups and, therefore, must be validated. Further studies with a larger sample (particularly of young patients, non-smoking and non-drinking patients and those with more than one primary tumor) and more complete measures of tobacco exposure are needed to clarify these complex interactions among genotype, phenotype, exposure and demographic subgroup. We plan to include such genotyping analysis of metabolic polymorphisms and DNA repair polymorphisms in future molecular epidemiologic studies. We feel these markers will complement one another and allow us to identify those individuals most susceptible to tobacco-induced carcinogenesis. ACKNOWLEDGEMENTS We thank Drs. R. Lotan and G. Clayman for critical review of the manuscript; Dr. M. Goode (Department of Scientific Publications) for editing the manuscript; Ms. Y. Guan and Ms. M. Chen for laboratory assistance; Ms. L. Young and Ms. S. Honn for assistance in recruiting study subjects; Mr. W. Gosbee, Ms. P. Pillow and Ms. C. Miller-Duphorne for data management; and Ms. J. Sider and Ms. J. Brown for assistance in preparing the manuscript.

5 224 CHENG ET AL. REFERENCES BROUGHTON, B.C., STEINGRIMSDOTTIR, H. and LEHMANN, A.R., Five polymorphisms in the coding sequence of the xeroderma pigmentosum group D gene. Mutation Res., 362, (996). CHENG, L., EICHER, S.A., GUO, Z., HONG, W.K., SPITZ, M.R. and WEI, Q., Reduced DNA repair capacity in head and neck cancer patients. Cancer Epidemiol. Biomarkers Prevent., 7, (998). COSMAN, M., DE LOS SANTOS, C., FIALA, R., HINGERTY, B.E., SIGH, S.B., IBANEZ, V., MARGULIS, L.A., LIVE, D., GEACINTOV, N.E., BROYDE, S. and PETEL, D.J., Solution conformation of the major adducts between the carcinogen ( )-anti-benzo[a]pyrene diol epoxide and DNA. Proc. nat. Acad. Sci. (Wash.), 89, (992). COUTELLE, C., WARD, P.J., FLEURY, B., QUATTROCCHI, P., CHAMBRIN, H., IRON, A., COUZIGOU, P. and CASSAIGNE, A., Laryngeal and oropharyngeal cancer, alcohol dehydrogenase 3 and glutathione S-transferase M polymorphisms. Hum. Genet., 99, (997). DEAKIN, M. and 4 OTHERS, Glutathione S-transferase GSTT genotypes and susceptibility to cancer: studies of interactions with GSTM in lung, oral, gastric and colorectal cancers. Carcinogenesis, 7, (996). FOULKES, W.D., BRUNET, J.S., SIEH, W., BLACK, M.J., SHENOUDA, G. and NAROD, S.A., Familial risks of squamous cell carcinoma of the head and neck: retrospective case-control study. Brit. Med. J., 33, (996). GELBOIN, H.V., Benzo[a]pyrene metabolism, activation, and carcinogenesis: role and regulation of mixed function oxidases and related enzymes. Physiol. Rev., 60, (980). HUDMON, K.S., HONN, S.E., JIANG, H., CHAMBERLAIN, R.M., XIANG, W., FERRY, G., GOSBEE, W. and SPITZ, M.R., Identifying and recruiting healthy control subjects from a managed care organization: a methodology for molecular epidemiologic case-control studies of cancer. Cancer Epidemiol. Biomarkers Prevent., 6, (997). JAHNKE, V., MATTHIAS, C., FRYER, A. and STRANGE, R., Glutathione S-transferase and cytochrome P-450 polymorphism as risk factors for squamous cell carcinoma of the larynx. Amer. J. Surg., 72, (996). JOURENKOVA, N., REINIKAINEN, M., BOUCHARDY, C., DAYER, P., BENHAMOU, S. and HIRVONEN, A., Larynx cancer risk in relation to glutathione S-transferase M and T genotypes and tobacco smoking. Cancer Epidemiol. Biomarkers Prevent., 7, 9 23 (998). KATOH, T., The frequency of glutathione-s-transferase M (GSTM) gene deletion in patients with lung and oral cancer. Jpn. J. Ind. Health, 36, (994). KOCH, W.M. and MCQUONE, S., Clinical and molecular aspects of squamous cell carcinoma of the head and neck in the nonsmoker and nondrinker. Curr. Opin. Oncol., 9, (997). LAFUENTE, A., PUJOL, F., CARRETERO, P., VILLA, J.P. and CUCHI, A., Human glutathione S-transferase µ (GSTµ) deficiency as a marker for the susceptibility to bladder and larynx cancer among smokers. Cancer Lett., 68, (993). LANDIS, S.H., MURRAY, T., BOLDEN, S. andwingo, P.A., Cancer statistics, 998. CA Cancer J. Clin., 48, 6 29 (998). OFFICE OF SMOKING AND HEALTH, NATIONAL CENTER FOR CHRONIC DISEASE PREVENTION AND HEALTH PROMOTION, CENTERS FOR DISEASE CONTROL AND PREVENTION, Cigarette smoking among adults United States, 993. Morb. Mortal. Wkly. Rep., 43, (994). OPHUIS, M.B.O., VAN LIESHOUT, E.M.M., ROELOFS, H.M.J., PETERS, W.H.M. and MANNI, J.J., Glutathione S-transferase M and T and cytochrome P450A polymorphisms in relation to the risk for benign and malignant head and neck lesions. Cancer, 82, (998). PARKIN, D.M., PISANI, P. and FERLAY, J., Estimate of the worldwide incidence of eighteen major cancers in 985. Int. J. Cancer, 54, (993). PUGA, A., NEBERT, D.W., MCKINNON, R.A. and MENON, A.G., Genetic polymorphisms in human drug-metabolizing enzymes: potential uses of reverse genetics to identify genes of toxicological relevance. Crit. Rev. Toxicol., 27, (997). REBBECK, T.R., Molecular epidemiology of the human glutathione S- transferase genotypes GSTM and GSTT in cancer susceptibility. Cancer Epidemiol. Biomarkers Prevent., 6, (997). RYBERG, D., SKAUG, V., HEWER, A., PHILLIPS, D.H., HARRIES, L.W., WOLF, C. R., ØGREID, D., ULVIK, A., VU, P. and HAUGEN, A., Genotypes of glutathione transferase M and P and their significance for lung DNA adduct levels and cancer risk. Carcinogenesis, 8, (997). SCHANTZ, S.P., BYERS, R.M., GOEPFERT, H., SHALLENBERGER, R.C. and BEDDINGFIELD, N., The implication of tobacco use in the young adult with head and neck cancer. Cancer, 62, (988). SHEN, M.R., JONES, I.M. and MOHRENWEISER, H., Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair genes in healthy humans. Cancer Res., 58, (998). SPITZ, M.R., FUEGER, J.J., BEDDINGFIELD, N.A., ANNEGERS, J.F., HSU, T.C., NEWELL, G.R. and SCHANTZ, S.P., Chromosome sensitivity to bleomycininduced mutagenesis, an independent risk factor for upper aerodigestive tract cancers. Cancer Res., 49, (989). TRIZNA, Z., CLAYMAN, G.L., SPITZ, M.R., BRIGGS, K.L. and GOEPFERT, H., Glutathione S-transferase genotypes as risk factors for head and neck cancer. Amer. J. Surg., 70, (995). WANG, L.E., STURGIS, E.M., EICHER, S.A., SPITZ, M.R., HONG, W.K. and WEI, Q., Mutagen sensitivity to benzo[a]pyrene diol epoxide and the risk of squamous cell carcinoma of the head and neck. Clin. Cancer Res., 4, (998). WEI, Q., EICHER, S., GUAN, Y., CHENG, L., XU, J., YOUNG, L., SAUNDERS, K.C., JIANG, H., HONG, W.K., SPITZ, M.R. and STROM, S.S., Reduced expression of hmlh and hgtbp: a risk factor for head and neck cancer. Cancer Epidemiol. Biomarkers Prevent., 7, (998).