Systematic discovery of novel methylation biomarkers in colon cancer.

B22 The aberrant methylation of CpG islands on promoter region of genes is common epigenetic phenomenon found early in cancers. This alteration contributes to cancer formation through the gene silencing of associated tumor suppressor genes. The aim of this study were to develop a systematic and stepwise genome-wide screening system for discovery of a new panel of genes enables to represent the high frequency of promoter hypermethylation in the development of colon cancer. We firstly determined a subset of genes relatively decreased in gene expression in colon cancers compared with their normal appearing-adjacent parts from 5 patients using human 17K cDNA microarray. We secondly analyzed expression profiles to identify genes epigenetically reactivated in two colon cancer cell lines after treatment with methylating inhibiting agent, 5 aza-29-deoxycytidine. We obtained 45 genes by cross-comparison of two gene lists and eliminated 18 genes not having CpG islands. We then carried out examination of methylation status of each gene (by PCR after digestion with restriction enzymes, Hpa II and Msp I) and selected 11 genes showing positive for methylaion in at least one cell line. To increase specificity of the screen for identification of methylation-associated genes inactivated in colon cancer we validated methylation status of these genes in tumor tissues used for microarray analysis and 5 normal tissues from non-patients. We found that 6 genes showed promoter methylation frequently in tumor tissues but were completely unmethylated in normal tissues. In further clinical study, the methylation status of panel of 6 genes was determined in primary tumors and normal appearing-adjacent parts from 59 colon cancer patients with various clinico-pathological features. Each gene (GABRA1, LAMA2, MGLL, CPM, WISP2, F2RL1) promoter regions were found to be frequently methylated in primary colon tumors (73%, 61%, 57%, 44%, 42%, 27% respectively), but less in adjacent parts. Aberrant promoter hypermethylation was detected in any of these genes in 100% (59 of 59), 95% (56 of 59) and 0% (0 of 5) in tumors, normal appearing-adjacent parts, and normal tissues from non-patients, respectively. We also confirmed that gene expression of these genes was significantly repressed in 59 tumors compared with normal appearing-adjacent parts. The results provide that a panel of six genes discovered through genome-scale systematic identification and stepwise filtering approach in this study could be a potential diagnostic biomarker for early detection of colon cancer.