Abstract LB-176: Identification of DNA methylation markers to predict esophageal cancer risk for people with carcinogen exposure
Authors: Kishino Takayoshi , Takahashi Takamasa , Lee Yi-Chia , Yamashita Satoshi , Ushijima Toshikazu 
Published: May 2015

Abstract
Aberrant DNA methylation of promoter CpG islands (CGIs) is induced in normal cells by aging, chronic inflammation, and exposure to specific carcinogenic factors, and the accumulation level of aberrant DNA methylation can be correlated with risk of cancer development. Also for esophageal squamous cell carcinoma (ESCC), the predominant histologic type in Asian countries, aberrant methylation is present in non-cancerous esophageal mucosae of ESCC patients, and methylation of specific genes is associated with smoking history [Oka et al. Cancer. 2009] and cancer risk [Lee et al. Cancer Prev Res. 2011]. However, for clinical application, markers informative even among drinkers, users of betel quid, and smokers, are required. Here, we aimed to isolate such markers by genome-wide methylation analysis. A total of 311 specimens [51 normal mucosae from healthy subjects (Group 1), 88 normal mucosae of healthy subjects exposed to carcinogen (alcohol, betel quid and tobacco) (Group 2), 94 non-cancerous background mucosae from ESCC patients (Group 3), and 78 cancer tissues from ESCC patients (Group 4)] were collected by endoscopic biopsy. A genome-wide methylation analysis was performed using an Infinium HumanMethylation450 BeadChip array that covered 485,577 CpG sites. Four pooled samples of Group 2 and four pooled samples of Group 3, each pool consisting of four specimens, was prepared. A methylation level of a CpG site for a group of samples was defined as the median of the beta-values of the samples, and that of a locus was defined as a mean of the methylation levels of the probes within the locus. First, we searched for CGI loci differentially methylated between the four pooled samples from Group 2 and those from Group 3 (difference in beta-value &gt; 0.1), and 37 loci were isolated. We were able to design primers for real-time MSP for 12 regions, and measured their methylation levels in the screening set (Group 2, n=38; Group 3, n=40). Seven regions demonstrated higher methylation levels in Group 3 than in Group 2 (P&lt;0.05), and cut-off methylation levels for these regions were determined based upon their ROC. The validation set (Group 2, n=34; Group 3, n=38) was analyzed with the prefixed cut-offs, and the methylation levels of three CGI loci predicted esophageal cancer risk at odds ratios of 5.3 to 6.2 (P&lt;0.05). The results of the present study indicated that the methylation status of the three loci is a candidate marker to predict the risk of ESCC in healthy subjects exposed to carcinogen.
Citation Format: Takayoshi Kishino, Takamasa Takahashi, Yi-Chia Lee, Satoshi Yamashita, Toshikazu Ushijima. Identification of DNA methylation markers to predict esophageal cancer risk for people with carcinogen exposure. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-176. doi:10.1158/1538-7445.AM2014-LB-176