Application of a modified MSRE-qPCR method for detecting circulating cell-free DNA methylation in cervical cancer

BackgroundThe methylation status of cell-free DNA (cfDNA) shows promise for the clinical detection of cervical cancer and its precursors; however, its measurement in liquid biopsies is hindered by low cfDNA yields and technically demanding protocols.MethodsWe developed an enhanced Methylation-sensitive restriction enzyme-quantitative polymerase chain reaction (MSRE-qPCR) method to create a combinatorial methylation assay with high sensitivity and specificity. Incorporating T4 DNA ligase following MSRE digestion improves the detection of short cfDNA fragments. This T4-assisted system enables methylation analysis of RXFP3-L1, RXFP3-L2, ZNF671, PAX1, and SOX1 for cervical cancer (CESC) evaluation with only 1.5 ng of DNA input; furthermore, lower input is sufficient for assessing the hypermethylated PAX1 gene.ResultsWhen applied to 24 blood samples from CESC patients and 21 matched tissues, the modified MSRE-qPCR method achieved 87.5% sensitivity and 90.3% specificity using 1.5ng of DNA, outperforming MethyLight without requiring more sensitive quantitative techniques. A methylation panel comprising RXFP3-L1, RXFP3-L2, ZNF671, and SOX1 yielded an AUC of 0.917 (95% CI: 0.830–1.000) in the development set. We constructed a logistic regression model using this gene panel to predict CESC status.ConclusionThis methodological improvement overcomes the limitation of low cfDNA abundance and provides a practical methylation-based tool for CESC detection.Clinical SignificanceThis non-invasive cfDNA methylation assay shows promise as an adjunct for the early detection of cervical cancer. It could also enhance screening adherence relative to conventional cytology, especially in resource-constrained environments.