5-methylcytosine and 5-hydroxymethylcytosine are synergistic biomarkers for early detection of colorectal cancer

Early cancer detection has the potential to significantly improve treatment outcomes and survival rates. This study investigates the roles of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) as biomarkers for early-stage colorectal cancer (CRC) detection in cell-free DNA (cfDNA). Using whole genome sequencing, we analyzed cfDNA from 37 treatment-naive CRC patients and 32 healthy controls. Our findings indicate that combining measurements of 5mC and 5hmC significantly enhances diagnostic accuracy (AUC = 0.95) compared to traditional approaches that conflate these markers (modified C, AUC = 0.66). Notably, 71.7% of differentially methylated regions (DMRs) exhibiting an increase in 5hmC in stage I cfDNA also showed a corresponding decrease in 5mC in stage IV, suggesting that 5hmC can effectively track regions undergoing demethylation during tumor development. These results support the hypothesis that distinguishing between 5mC and 5hmC can improve the sensitivity of liquid biopsy tests for early cancer detection.