Global Hypomethylation in Cell-free DNA Enables Non-invasive Colorectal Cancer Screening: Results from a Retrospective Validation Study

Background Global DNA hypomethylation is a fundamental epigenetic hallmark of colorectal cancer, yet it remains difficult to measure in circulating cell-free DNA using existing technologies that focus on a limited number of genomic loci. As a result, global hypomethylation has not been effectively translated into non-invasive diagnostics. We evaluated a label-free electrical impedance assay that measures cell-free DNA aggregation, a biophysical property linked to genome-wide methylation state, to determine whether global hypomethylation can be detected and clinically leveraged for colorectal cancer screening and monitoring. Results Plasma samples from 46 treatment-naïve colorectal cancer patients and 33 healthy individuals were analyzed. The assay distinguished cancer from healthy samples with 95.65% sensitivity and 93.94% specificity across all disease stages, including early-stage cancers. Longitudinal analysis of six patients showed concordance between assay output and clinical course, including response to treatment and disease recurrence. Transmission electron microscopy confirmed methylation-dependent differences in cell-free DNA aggregation underlying the electrical signal. To contextualize these findings biologically, we analyzed 11 public DNA methylation datasets. Conventional whole-array analyses underestimated global hypomethylation due to overrepresentation of CpG island probes. Restricting analysis to OpenSea regions, which better represent genome-wide methylation and are enriched in cell-free DNA, revealed a significant 5.8% reduction in global methylation in colorectal cancer tissue compared to adjacent normal tissue, consistent across molecular subtypes. In contrast, blood-derived immune cell genomic DNA showed no comparable hypomethylation, supporting a predominantly tumor-derived contribution to the observed circulating signal. Conclusions These results demonstrate that global DNA hypomethylation is detectable in circulating cell-free DNA and can be captured using a rapid, amplification-free electrical assay. By measuring structural consequences of pan-genomic epigenetic alterations rather than locus-specific changes, this approach enables accurate, non-invasive detection and monitoring of colorectal cancer. The findings support global hypomethylation as a clinically actionable epigenetic biomarker and establish a new framework for cell-free DNA–based cancer diagnostics.