Extensive methylation analysis of circulating tumor DNA in plasma of patients with gastric cancer

DNA methylation is known to be involved in tumor progression. This is the first study to perform an extensive methylation analysis of plasma circulating tumor DNA (ctDNA) using targeted bisulfite sequencing in gastric cancer (GC) patients to evaluate the usefulness of ctDNA methylation as a new biomarker. Sixteen patients who received chemotherapy for recurrent GC were included. After confirmation of the methylation status of 63 genes using the Cancer Genome Atlas (TCGA) dataset, the methylation status in paired tumor and non-tumor tissues and plasma were investigated using targeted bisulfite sequencing in these genes. Forty-four of the 63 genes were significantly hypermethylated in GC patients in the TCGA cohort. Of these 44 genes, hierarchical clustering showed that five (SPG20, FBN1, SDC2, TFPI2, SEPT9) were particularly hypermethylated in tumor compared to non-tumor tissues in our GC cohort. In plasma methylation analysis, patients with high methylation of these genes had significantly worse overall survival than those with low methylation (log-rank P = 0.009). In a patient who underwent blood sampling at multiple points, the methylation levels of these five genes varied closely with clinical tumor status. The plasma ctDNA methylation levels of these five genes could be useful as a noninvasive prognostic biomarker for GC.