Hypermethylation of cytosines in CpG-rich islands of the promoter regions of regulatory genes has been discovered as a common mechanism of gene silencing during carcinogenesis. We analysed 64 primary lung carcinomas for promoter methylation of the tumour suppressor genes (TSGs) p16 (p16 INK4a /CDKN2A) and p14 (p14 ARF ) by methylation-specific PCR, in order to evaluate aberrant methylation as a potential biomarker for epigenetic alterations in tobacco-related lung cancer. Methylation of p16 was observed in 34% (22/64) of the lung tumours examined. In particular, p16 methylation occurred in nonsmall cell lung cancer (NSCLC) only, with 41 % (22/54) of the tumours being positive. The highest frequency was found in large cell carcinoma (5/7, 71%), followed by adenocarcinoma (9/25, 36%) and squamous cell carcinoma (7/21, 33%). Methylation of the p14 gene was less frequent in lung cancer (4/52, 8%). When association with tobacco smoking was analysed, 42% (21/50) of NSCLC from ever smokers exhibited p16 methylation. Interestingly, the analysis revealed a significantly higher risk of p16 methylation in former smokers as compared to current smokers [odds ratio (OR) 5.1; 95% confidence interval (CI) 1.3-22]. The difference was retained after adjustment for age (OR 3.7; 95% CI 0.9 -17). The promoter methylation results were then combined with data on genetic alterations determined previously in the same set of tumours. This data similarly showed that p16 methylation in parallel with p53 gene mutation or p14 methylation occurred more frequently in former smokers than in current smokers (44% vs. 14%; P ‫؍‬ 0.035). Taken together, our data suggest that analysis of promoter methylation in TSGs may provide a valuable biomarker for identification of groups with an elevated risk of cancer, such as smokers and ex-smokers.

Studies on somatic mutations in lung cancers associated with cigarette smoking and asbestos exposure are few. We investigated prevalence of mutations in the p53 and K-ras genes in lung tumors from smokers with and without asbestos exposure at work. For K-ras mutations, the study was an extension of an earlier analysis. Nearly all of the 105 consecutive patients examined were smokers and had non-small-cell carcinoma of the lung with squamous-cell carcinoma or adenocarcinoma histology. Exposure to asbestos was estimated by pulmonary fiber counts and occupational histories. A pulmonary burden of >/= 1 x 10(6) asbestos fibers per gram of lung tissue, indicating work-related exposure, was found in 32% of the patients for whom fiber-analysis data were available (33 of 102 patients, all men). The statistical analysis showed pulmonary fiber count as the only significant predictor of adenocarcinoma histology, in contrast to squamous-cell carcinoma (smoking-adjusted odds ratio [OR] 3.0, 95% confidence interval [CI] 1.1 to 8.5). The frequency of p53 mutations was 39% (13 of 33) among the asbestos-exposed cases, as compared with 54% (29 of 54) among the nonexposed cases; the difference was not significant, however. In male ever-smokers, a long duration of smoking was associated with p53 mutation (OR 3.2, 95% CI 1.2 to 8.8). In adenocarcinoma, p53 mutations were less prevalent (10 of 30, 33%) as compared with squamous-cell carcinoma (28 of 46, 61%; P = 0.02), whereas a strong and significant association was found between adenocarcinoma and K-ras mutation (OR 37, 95% CI 5.8 to 232, adjusted for smoking and asbestos exposure). Asbestos exposure alone was not significantly associated with increased occurrence of K-ras mutations. In conclusion, the results may primarily reflect the observed excess of adenocarcinoma in the asbestos- exposed patients, and hence the decrease in p53 mutations and increase in K-ras mutations.

We report here a compilation of p53 mutation results from two smoking-related cancers, lung cancer and bladder cancer. The overall mutation frequencies reported for these two types of cancer were relatively similar-50% in lung cancer and 40% in bladder cancer. The compiled data from lung cancer and bladder cancer suggest an increasing proportion of patients with p53 mutations in nonsmokers, former smokers, and current smokers, in that order, in both cancer groups. Taken together, more than half (55% and 56% for lung cancer and bladder cancer, respectively) of the patients who continued smoking (CS), less than 40% (38% and 38%) of those who had stopped smoking before (.1 or .5 years) clinical diagnosis (ES), and less than 30% (25% and 29%) of those who were nonsmokers (NS) had a p53 mutation. The differences seen in the mutation frequencies between the three smoking groups did not, however, reach statistical significance (lung cancer-CS vs ES: odds ratio [OR] = 2.0, 95% Cl 0.7-5.4; CS vs NS: OR=3.7, 95% Cl 0.4-37; bladder cancer-CS vs ES: OR=2.1, 95% Cl 0.6-7.9; CS vs NS: OR=3.1, 95% Cl 0.7-13). Guanine to thymine transversions were the most common type in lung cancer followed by guanine to adenine transitions. In bladder cancer, on the contrary, G:C to A:T transitions at cytosine-guanine dinucleotide sites were the most frequently detected base substitutions. Analysis of the compiled p53 mutation data suggests that, in addition to lifetime cumulative exposure to cigarette smoke, also stopping smoking for years prior to clinical manifestation of the disease may affect the incidence of p53 mutations. The differences in the mutation profiles appear to support the view that the main genotoxic agents from cigarette smoke exposure may be different in bladder cancer as compared to lung cancer, as suggested previously by DNA adduct studies. Environ Health Perspect 104(Suppl 3): 553-556 (1996)

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