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Purpose: Metastasis is the leading cause of death for gastric carcinoma. An epigenetic biomarker panel for predicting gastric carcinoma metastasis could have significant clinical impact on the care of patients with gastric carcinoma. The main purpose of this study is to characterize the methylation differences between gastric carcinomas with and without metastasis.Experimental Design: Genome-wide DNA methylation profiles between 4 metastatic and 4 nonmetastatic gastric carcinomas and their surgical margins (SM) were analyzed using methylated-CpG island amplification with microarray. The methylation states of 73 candidate genes were further analyzed in patients with gastric carcinoma in a discovery cohort (n = 108) using denatured high performance liquid chromatography, bisulfite-sequencing, and MethyLight. The predictive values of potential metastasis-methylation biomarkers were validated in cohorts of patients with gastric carcinoma in China (n = 330), Japan (n = 129), and Korea (n = 153).Results: The gastric carcinoma genome showed significantly higher proportions of hypomethylation in the promoter and exon-1 regions, as well as increased hypermethylation of intragenic fragments when compared with SMs. Significant differential methylation was validated in the CpG islands of 15 genes (P < 0.05) and confirmed using bisulfite sequencing. These genes included BMP3, BNIP3, CDKN2A, ECEL1, ELK1, GFRA1, HOXD10, KCNH1, PSMD10, PTPRT, SIGIRR, SRF, TBX5, TFPI2, and ZNF382. Methylation changes of GFRA1, SRF, and ZNF382 resulted in up- or downregulation of their transcription. Most importantly, the prevalence of GFRA1, SRF, and ZNF382 methylation alterations was consistently and coordinately associated with gastric carcinoma metastasis and the patients' overall survival throughout discovery and validation cohorts in China, Japan, and Korea.Conclusion: Methylation changes of GFRA1, SRF, and ZNF382 may be a potential biomarker set for prediction of gastric carcinoma metastasis. Clin Cancer Res; 20(17); 4598–612. ©2014 AACR.

Supplementary Figure S4. Methylation levels of GFRA1, SRF, and ZNF382 by DHPLC are confirmed by MethyLight and inversely correlated with their transcription levels. (A) Correlation between DNA methylation levels measured by DHPLC and MethyLight. The methylation levels of GFRA1, SRF, and ZNF382 measured by DHPLC positively correlated with the results obtained using MethyLight assay. RCN, relative copy number relative to the reference gene COL2A1 for bisulfite-modified DNA; (B) The methylation levels of these genes were inversely correlated with their mRNA levels. The mRNA levels were determined by quantitative RT-PCR assays in matched GC tissues. Statistically significant correlations were obtained using Spearman test. RCN, relative copy number relative to the reference gene GAPDH.