Early detection of gastric cancer via multiplex blood assay targeting CfDNA methylation signatures.

BACKGROUND: High heterogeneity of gastric cancer (GC) drives cellular diversity. This complicates liquid biopsies, as detecting tumor markers in bodily fluids may not fully represent the tumor due to uneven marker distribution, compromising test accuracy and sensitivity. METHODS: Integrative analysis of 43 paired tissues, TCGA, and GEO databases identified 24 key GC-specific DMPs. Combined analysis with cfDNA bisulfite sequencing (79 normal controls and 70 GC) revealed four GC-specific DMPs. We developed GC-mqMSP using cfDNA from 70 normal controls, 106 GC, 41 lung cancer, and 23 colorectal cancer patients, was further validated in an independent cohort (45 GC and 13 normal controls). dCas9-Tet1-CD targeted demethylation assessed functional roles in GC development. RESULT: Based on four GC-specific DMPs common to both tissue and cfDNA, we developed an efficient and convenient GC-mqMSP assay for detection. This assay demonstrated the ability to distinguish early-stage GC patients from normal controls and specifically differentiated GC patients from those with other tumors, achieving an AUCs (0.932 and 0.927), sensitivity (90.54% and 84.38%), and specificity (83.67% and 90.48%) in the training and validation sets. We selected ZNF154 for further study for the high coefficient weights of its two DMPs (Cg05661282 and Cg03234186). DNMT1/UHRF1 complex bind to promoting their hypermethylation and suppressing ZNF154 expression. Hypermethylation of Cg05661282 and Cg03234186 facilitated GC cell proliferation, migration, and tumor formation both in vitro and in vivo. Targeted demethylation at these DMPs reversed tumorigenesis and progression. CONCLUSION: A cfDNA methylation-based integrated score was constructed in this study to predict GC in patients clinically.