Early Diagnosis of Colorectal Cancer Based on Bisulfite-free Site-specific Methylation Identification PCR Strategy: High-Sensitivity, Accuracy, and Primary Medical Accessibility.

ABSTRACT

Due to its decade-long progression, colorectal cancer (CRC) is most suitable for population screening to achieve a significant reduction in its incidence and mortality. DNA methylation has emerged as a potential marker for the early detection of CRC. However, the current mainstream methylation detection method represented by bisulfite conversion has issues such as tedious operation, DNA damage, and unsatisfactory sensitivity. Herein, a new high-performance CRC screening tool based on the promising specific terminal-mediated polymerase chain reaction (STEM-PCR) strategy is developed. CRC-related methylation-specific candidate CpG sites are first prescreened through The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases using self-developed bioinformatics. Next, 9 homebrew colorectal cancer DNA methylated STEM‒PCR assays (ColoC-mSTEM) with high sensitivity (0.1%) and high specificity are established to identify candidate sites. The clinical diagnostic performance of these selected methylation sites is confirmed and validated by a case-control study. The optimized diagnostic model has an overall sensitivity of 94.8% and a specificity of 95.0% for detecting early-stage CRC. Taken together, ColoC-mSTEM, based on a single methylation-specific site, is a promising diagnostic approach for the early detection of CRC which is perfectly suitable for the screening needs of CRC in primary healthcare institutions.