Discovering methylation markers and development of a sense-antisense and dual-MGB probe PCR assay in plasma for colorectal cancer early detection

Screening for colorectal cancer (CRC) using plasma methylation is challenging due to the low abundance of cell-free DNA (cfDNA). Therefore, the development of signal amplification assays based on appropriate markers is essential to increase sensitivity. In this study, we employed an epigenome-wide approach for de novo identification differentially methylated CpGs (DMCs) common to CRC and adenoma using 17 public datasets. A sense-antisense and dual MGB probe (SADMP) assay was then developed. Subsequently, the biomarkers were validated in 712 plasma samples based on SADMP. A total of 2237 DMCs showed overlap between CRC and adenoma. Of these, 75 were hypomethylated in 30 other non-CRC cancers. Following LASSO regression, WBC validation and primer/probe design evaluation, NTMT1 and MAP3K14-AS1 were identified as the most informative candidate biomarkers. At preset template concentrations, the SADMP assay for NTMT1 or MAP3K14-AS1 could reduce the cycle threshold by 1. The NTMT1 and MAP3K14-AS1 dual-target SADMP assay demonstrated a sensitivity of 84.8% for CRC (stage I: 75.0%), a sensitivity of 32.0% for advanced adenomas (AA), and a specificity of 91.5% in controls. The dual-target assay showed high performance for CRC early detection in plasma, suggesting that it may serve as a promising noninvasive tool for CRC screening.