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

Background: Screening for colorectal cancer (CRC) using plasma cell-free DNA (cfDNA) methylation is more challenging than stool testing due to the low abundance of cfDNA. Therefore, the development of signal amplification assays based on appropriate markers is essential to increase sensitivity. Methods: A total of 17 existing 450K microarray datasets including tissue, healthy white blood cell (WBC) and plasma cfDNA data from public databases were used to identify differentially methylated CpGs (DMCs) common to CRC and adenoma. The methylation status of candidate DMCs was confirmed by Sanger sequencing with CRC and normal tissues. A sense-antisense and dual MGB probe (SADMP) assay was then developed. Subsequently, the biomarkers were validated in 712 plasma samples using the SADMP method. Results: A total of 2237 DMCs showed overlap between the cancer vs. normal and adenoma vs. normal groups. Of these, 75 were hypomethylated in 30 other non-CRC cancers. After LASSO regression, this number was reduced to eight. Two of these, NTMT1 and MAP3K14-AS1, were identified as promising candidate markers following WBC validation and primer/probe design evaluation. The SADMP technology demonstrated the ability to amplify the detection signal to approximately twice the original level. Overall, the 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. Conclusions: The dual-target assay demonstrated high performance for CRC and AA detection in plasma-based tests, suggesting that it may serve as a promising noninvasive tool for CRC detection.