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). We analyzed cfDNA from 37 treatment-naive CRC patients and 32 healthy controls using duet evoC 6-base whole genome sequencing (A, C, T, G, 5mC, 5hmC). We then built CRC classifiers using differentially methylated regions identified in stage IV CRC tissue samples. We compared the performance of models using 5mC-only, 5hmC-only, modified cytosine, or 5mC and 5hmC features. Here we show that models combining measurements of 5mC and 5hmC significantly enhance diagnostic accuracy (AUC = 0.95) compared to traditional approaches that conflate these markers (modified C, AUC = 0.66). Notably, of all the DMRs considered, almost half show an increase in 5hmC at stage I and a corresponding decrease in 5mC at 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 improves the sensitivity of liquid biopsy tests for early-stage cancer detection. Colorectal cancer is one of the most common cancers and detecting it early can greatly improve treatment success. Liquid biopsy tests use blood samples to detect tumors by identifying DNA from tumor cells amongst a large background of DNA from healthy cells. One promising approach is to examine DNA methylation marks, chemical tags that control whether genes are turned on or off. Two important marks are 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Current tests often combine these into a single measure, which can miss key differences. In this study, we found that measuring 5mC and 5hmC separately made detection much more accurate for early-stage cancer. This could lead to better blood tests, helping more people receive timely treatment. Puddu et al. investigate the role of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) as biomarkers for early-stage colorectal cancer (CRC) detection in cell-free DNA (cfDNA). 6-base genome sequencing, using both 5mC and 5hmC biomarkers, increases the detection of stage I CRC.