Aberrant methylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers. There are major smoke exposure, histology, geography and gender-related changes in non-small cell lung cancer (NSCLC). We investigated smoking-related, histologic, geographic and gender differences in the methylation profiles of resected NSCLCs. We examined 514 cases of NSCLC and 84 corresponding nonmalignant lung tissues from 4 countries (USA, Australia, Japan and Taiwan) for the methylation status of 7 genes known to be frequently methylated in lung cancers [p16, RASSF1A (RAS association domain family 1), APC, RAR␤, CDH13, MGMT and GSTP1]. Multivariate analyses were used for data analysis. Adenocarcinoma was the major histologic type in women and never smokers; analyses that involved smoke exposure and gender were limited to this histology. Our major findings are a) methylation status of any single gene was largely independent of methylation status of other genes; b) the rates of methylation of p16 and APC and the mean Methylation Index (MI), a reflection of the overall methylation status, were significantly higher in ever smokers than in never smokers; c) the mean MI of tumors arising in former smokers was significantly lower than the mean of current smokers; d) the methylation rates of APC, CDH13 and RAR␤ were significantly higher in adenocarcinomas than in squamous cell carcinomas; e) methylation rates of MGMT and GSTP1 were significantly higher in the USA and Australian cases than in those from Japan and Taiwan; and (f) no significant gender-related differences in methylation patterns were noted. Our findings demonstrate important smoke exposure, histologic type and geography-related differences in the methylation profiles of NSCLC tumors.

No abstract

Summary We examined 159 consecutive cases of non-small-cell lung cancer (NSCLC) for a mutation at codon 12 of the K-ras gene and for a mutation of the p53 gene occurring in exons 5-8. Eleven (6.9%) had mutations of the K-ras (ras+) and 57 (35.8%) had mutations of the p53 (p53+). There were 95 cases (59.7%) with ras -p53 -, seven cases (4.4%) with ras+/p53 -, 53 cases (33.3%) with ras -/p53+ and four cases (2.5%) with ras+/p53+. The ras+ group had a worse prognosis than the ras -group in all cases and in 107 early-stage cases (stage l-ll, P<0.05). The p53+ group had a worse prognosis in 107 early-stage cases (P<0.01), but there was no statistically significant difference when 52 advanced-stage cases (stage III-IV) or all patients were considered. Both ras and p53 mutations were unfavourable prognostic factors in 94 cases with adenocarcinoma, but there was no statistical significance in 57 cases with squamous cell carcinoma. According to Cox's model, the pathological stage, ras mutation and p53 mutation were found to be independent prognostic factors. Our results suggest that ras and p53 mutations were independent unfavourable prognostic markers especially in the early stage of NSCLC or in adenocarcinoma.

Dear Sir,Tobacco smoking is the most important risk factor for lung cancer worldwide. Cancers arise following an accumulation of genetic and epigenetic alterations, including point mutations of genes such as p53 1,2 and Ras, chromosomal deletions 3 and methylation of multiple genes; 4,5 and many of these changes occur in tobacco-related lung cancers. In addition, tobacco smoking is associated with a dose-response increase in p53 mutations. 3 We previously demonstrated that the aberrant methylation status of p16, RASSF1A, APC, RAR␤ and CDH13 genes in lung cancer is related to smoke exposure and histologic type but not to gender. 6 Here, we report the results of further examination for methylation status in 383 Japanese non-small cell lung cancer (NSCLC) cases. The characteristics of our cases are shown in Table I. These samples have extensive data for dosage of tobacco exposure, so we examined the relationship between smoking dosage and methylation status of the 5 genes related to tobacco exposure. Methylation-specific PCR was used for determining the methylation status of samples. 7 Methylation-specific PCR, with sensitivity determined as previously described, could detect one methylated allele in the presence of 10 3 -10 4 unmethylated alleles. 6 DNA from lymphocytes of a healthy volunteer artificially methylated by treatment with Sss1 (New England BioLabs, Beverly, MA) was used as a positive control for methylated alleles. We also used cell lines as a positive control, which we reported previously. 5 PCR products were visualized on 2% agarose gels stained with ethidium bromide.Based on smoking status, we divided the cases into 3 groups: never-smokers (Ͻ100 cigarettes/lifetime), smokers with exposure of Ͻ30 pack-years and smokers with exposure Ն30 packyears. The smoking effects on methylation were examined by the Cochran-Armitage trend test, and the results are shown in Figure 1. In all cases, methylation of p16 and RASSF1A and the methylation index (MI), which was defined as a fraction representing the number of genes methylated/the number of genes tested, 4 showed a dose-related response. Because we previously noted that histologic differences affect the profile of aberrant methylation, 6 we also examined the effects of the degree of smoke exposure on both adenocarcinoma and squamous cell carcinoma, the major subtypes of NSCLC. As most lung cancers in never-smokers are adenocarcinomas, our study was limited by the relatively small number of squamous cell carcinomas (n ϭ 8) in never-smokers. However, for both histologic types, methylation of p16 and MI showed a doserelated response. For adenocarcinoma, RASSF1A and APC methylation also demonstrated a dose-dependent response. While a similar tendency for squamous cell carcinomas was noted, the effect was not statistically significant. For CDH13 and RAR␤, there was no relationship to extent of smoke exposure.Our present findings confirm and extend our previous findings that p16 and APC methylation are more frequent in eversmokers than in never-smokers. 6 While me...

No abstract