Plasma-based digital PCR assay for early detection of gastric cancer using multiple methylation biomarkers

Endoscopy is the gold standard for diagnosing gastric cancer (GC), but its invasiveness limits widespread participation and has not substantially reduced GC-related mortality. This study developed and validated a blood-based digital PCR assay for early GC detection using DNA methylation biomarkers. Genome-wide methylation profiles from over 10,000 samples were screened, and two candidates were validated in GC cell lines, tumors, matched non-cancerous tissues, and plasma. Plasma from 60 GC patients, including 38 with stage I disease, and 40 healthy controls was analyzed with a digital PCR assay targeting the selected biomarkers, using ACTB as a reference. GHR and GLRB methylation were identified as novel GC biomarkers, showing consistent hypermethylation in GC cell lines and tumor tissues. In plasma, the two-marker assay achieved 83.3% (95% CI, 71.5%-91.7%) sensitivity and 90% (95% CI, 76.3%-97.2%) specificity, clearly outperforming carcinoembryonic antigen (CEA) testing (10.0%; 95% CI, 3.8%-20.5%). Incorporation of GATM methylation as a third marker increased sensitivity to 86.7% (95% CI, 75.4%-94.1%) overall and 81.6% (95% CI, 65.7%-92.3%) for stage I disease, while maintaining 90.0% specificity. This methylation-based digital PCR assay enabled accurate, non-invasive detection of GC, particularly at early stages, and may facilitate timely diagnosis and curative treatment.