Highly specific multiplex DNA methylation detection for liquid biopsy of colorectal cancer

Background: Circulating tumor DNA (ctDNA) has emerged as a useful biomarker for cancer detection and prognosis. In this study, we developed a strategy for developing a highly specific multiplex qPCR assay to detect methylated ctDNA in the blood of colorectal cancer (CRC) patients and investigated the potential use for the detection and prognosis of CRC. Methods: Bisulfite conversion and amplicon sequencing were used to confirm potential CRC-specific DNA methylation markers. The selected DNA methylation candidates were validated by qMSP. The six best- performing markers were used to develop a new single-tube multiplex quantitative methylation-specific PCR assay (mqMSP). The mqMSP assay was applied to analyze plasma samples from 114 CRC patients, 47 patients with advanced adenoma, 45 patients with benign polyps, and 57 healthy controls. The clinical performance of the assay and associations with clinical outcomes were assessed. Results: Six DNA methylation biomarkers were confirmed to be specifically hypermethylated in CRC tumor tissues. The newly developed mqMSP assay detected CRC with extremely high specificity (specificity of 98.2 %, with sensitivity of 67.5 %). The detection rate of ctDNA was significantly correlated with tumor size and clinical stage, with ctDNA methylation levels in the blood markedly increased with larger tumor size, poor differentiation, and advanced stage. Moreover, high preoperative methylated ctDNA level was associated with worse recurrence-free survival and overall survival. Conclusion: We provided a strategy for identification of multiple highly-specific DNA methylation markers for designing multiplex DNA methylation assays for liquid biopsies of CRC. The newly developed assay has potential for CRC early detection, and prognosis.