Abstract 2290: DNA methylation markers for sensitive detection of circulating tumor DNA in plasma from patients with gastroesophageal adenocarcinomas

Abstract

Abstract Background: We have previously shown that detection of methylated ctDNA in the three genes c9orf50, KCNQ5, and CLIP4 is a highly sensitive method (named the TriMeth assay) to discriminate patients with colorectal cancer from healthy individuals (1). The publicly available Infinium HumanMethylation450K BreadChip DNA methylation array indicates that these three markers are also highly methylated in gastric and esophageal cancer. In this study, we investigate the ability of the TriMeth assay to detect ctDNA in patients with gastric and gastroesophageal junction (GEJ) adenocarcinomas. Methods: Purified DNA was extracted from tumor tissue and plasma from 63 patients with gastric/GEJ adenocarcinomas. Sodium bisulfite conversion was performed to distinguish methylated from unmethylated cytosines. Droplet digital PCR (ddPCR) with methylation-specific primers and probes was performed to detect hypermethylated sites in the three genes c9orf50, KCNQ5, and CLIP4. The TriMeth assay was considered positive if at least two out of three markers were methylated according to a predefined cut-off. Eleven plasma samples from healthy individuals were used as negative controls.Results: In tumor tissue, methylated copies were detected in all 29 surgical samples (100% sensitivity), with varying allele frequencies. In plasma, methylated copies were detected in 14 out of 18 samples (78% sensitivity) from patients with advanced disease, with varying sensitivity amongst the three markers (AUC for the three markers CLIP4, C9orf50, and KCNQ5 was 0.61, 0.86, and 0.94, respectively). In plasma from patients with resectable disease, methylated copies were detected in 10 of 17 samples (59% sensitivity), although only 7/17 (41% sensitivity) were positive according to the predefined cut-off. All 11 controls were negative for methylation signals (100% specificity). Conclusion and Further Perspectives: Using a pre-defined cut-off value, the TriMeth assay detected methylated DNA in all tumor samples. The sensitivity of the TriMeth assay in plasma was 78% in advanced disease and 41% in resectable disease. Further investigations of the TriMeth assay in patients with resectable gastric and GEJ adenocarcinomas are needed. We plan to validate the sensitivity of the TriMeth assay for treatment monitoring and detection of residual disease.