A targeted DNA methylation method for detecting gastrointestinal cancer in circulating cell-free DNA

Colorectal cancer (CRC) and gastric cancer (GC) are leading causes of cancer-related mortality. However, cost-effective methods for simultaneous detection of CRC and GC in circulating cell-free DNA (cfDNA) remain insufficiently explored. To address this, we developed targeted methylated CpG tandem amplification and sequencing (tMCTA-seq), a PCR-based method utilizing a set of locus-specific primers with a universal CGCGCGG primer, and targeted a panel of 110 loci. The method demonstrated high technical sensitivity below one haploid genome equivalent. Using a repeated nested cross-validation framework, the ensemble model, applied to 448 plasma samples (170 CRC, 101 GC, and 177 control participants), achieved areas under the curve (AUCs) of 0.928 (88.2% sensitivity and 90.7% specificity) for CRC and 0.926 (86.7% sensitivity and 94.4% specificity) for GC on the test set. Furthermore, tMCTA-seq differentiated between CRC and GC (AUC = 0.819). Thus, tMCTA-seq is a cost-effective, methylation-based approach for simultaneous detection of and differentiation between two major gastrointestinal cancers in blood.