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.

The mechanisms underlying cellular drug resistance have been extensively studied, but little is known about its regulation. We have previously reported that activating transcription factor 4 (ATF4) is upregulated in cisplatinresistant cells and plays a role in cisplatin resistance. Here, we find out a novel relationship between the circadian transcription factor Clock and drug resistance. Clock drives the periodical expression of many genes that regulate hormone release, cell division, sleep-awake cycle and tumor growth. We demonstrate that ATF4 is a direct target of Clock, and that Clock is overexpressed in cisplatin-resistant cells. Furthermore, Clock expression significantly correlates with cisplatin sensitivity, and that the downregulation of either Clock or ATF4 confers sensitivity of A549 cells to cisplatin and etoposide. Notably, ATF4-overexpressing cells show multidrug resistance and marked elevation of intracellular glutathione. The microarray study reveals that genes for glutathione metabolism are generally downregulated by the knockdown of ATF4 expression. These results suggest that the Clock and ATF4 transcription system might play an important role in multidrug resistance through glutathione-dependent redox system, and also indicate that physiological potentials of Clock-controlled redox system might be important to better understand the oxidative stress-associated disorders including cancer and systemic chronotherapy.

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A strong nuclear expression of ERbeta correlates with EGFR mutations, and its favorable prognostic significance was influenced by the EGFR mutations in adenocarcinoma of the lung.

Purpose: Somatic mutations of the epidermal growth factor receptor (EGFR) gene are associated with an increased response to gefitinib in patients with non^small cell lung cancer. We have examined the impact of gefitinib on progression-free survival and overall survival in patients with EGFR mutation^positive non^small cell lung cancer. Experimental Design: We searched for all clinical trials that prospectively evaluated the efficacy of gefitinib for advanced non^small cell lung cancer with EGFR mutations in Japan. We did a combined analysis based on individual patient data from the identified trials. Results: Seven eligible trials were identified for a total of 148 non^small cell lung cancer patients with EGFR mutations. The overall response rate to gefitinib was 76.4% [95% confidence interval (95% CI), 69.5-83.2]. The median progression-free survival and overall survival were 9.7 months (95% CI, 8.2-11.1) and 24.3 months (95% CI, 19.8-28.2), respectively. Good performance status and chemotherapy-naI« ve status were significantly associated with a longer progression-free survival or overall survival. Of the 148 patients, 87 received gefitinib as a first-line therapy, whereas 61received systemic chemotherapy before gefitinib treatment. The median progression-free survival after the start of first-line therapy was significantly longer in the gefitinib-first group than in the chemotherapy-first group (10.7 versus 6.0 months; P < 0.001), whereas no significant difference in median overall survival was apparent between the two groups (27.7 versus 25.7 months; P = 0.782). Conclusions: Gefitinib monotherapy confers substantial clinical benefit in terms of progressionfree survival and overall survival in non^small cell lung cancer patients with EGFR mutations. Randomized trials comparing chemotherapy with gefitinib as a first-line treatment are warranted in such patients.Non-small cell lung cancer is the leading cause of death related to cancer worldwide (1). Cytotoxic chemotherapy remains the mainstay of treatment for patients with metastatic non -small cell lung cancer on the basis of the associated moderate improvement in survival and quality of life (2 -4). The poor outlook even for patients with advanced non -small cell lung cancer who receive such chemotherapy has prompted a search for new therapeutic approaches.The epidermal growth factor receptor (EGFR) is frequently overexpressed in non -small cell lung cancer and has been implicated in the pathogenesis of this disease (5, 6). Given the biological importance of EGFR signaling in non -small cell lung cancer, EGFR-specific tyrosine kinase inhibitors, including gefitinib and erlotinib, have been extensively studied in patients with this condition (7 -10). We and others have shown that a clinical response to these agents is more common in women than in men, in Japanese than in individuals from Europe or the United States, in patients with adenocarcinoma than in those with other histologic subtypes of cancer, and in individuals who have never

LOH at 9p loci occurs at the earliest stage in the pathogenesis of lung cancer and involves all regions of the respiratory tract. LOH in NSCLC is not random but targets a specific allele in individuals. Studying preneoplastic lesions may help identify intermediate markers for risk assessment and chemoprevention.

Somatically acquired mutations in the epidermal growth factor receptor (EGFR) gene in lung cancer are associated with significant clinical responses to gefitinib, a tyrosine kinase inhibitor that targets EGFR. We screened the EGFR in 469 resected tumours of patients with lung cancer, which included 322 adenocarcinomas, 102 squamous cell carcinomas, 27 large cell carcinomas, 13 small cell carcinomas, and five other cell types. PCR with a specific condition was performed to identify any deletion in exon 19, while mutantallele-specific amplification was performed to identify a mutation in codon 858 of exon 21. EGFR mutations were found in 136 cases (42.2%) with adenocarcinoma, in one case with large cell carcinoma, and in one case with pleomorphic carcinoma. An in-frame deletion in exon 19 was found in 62 cases while an L858R mutation was found in 77 cases. In the 322 cases with adenocarcinoma, these mutations were more frequently found in women than in men (P ¼ 0.0004), in well differentiated tumours than in poorly differentiated tumours (P ¼ 0.0014), and in patients who were never smokers than in patients who were current/former smokers (Po0.0001). The mutation was more frequently observed in patients who smoked p20 pack-year, and in patients who quit at least 20 years before the date of diagnosis for lung cancer. The K-ras mutations were more frequently found in smokers than in never smokers, and in high-dose smokers than in low-dose smokers. In conclusion, the mutations within the tyrosine kinase domain of EGFR were found to specifically occur in lung adenocarcinoma patients with a low exposure of tobacco smoking.

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