Global Hypomethylation in Cell-Free DNA Enables Noninvasive Colorectal Cancer Screening: Results from a Retrospective...

Global DNA hypomethylation is a defining hallmark of colorectal cancer (CRC) but is poorly captured by existing cell-free DNA (cfDNA) technologies, which typically interrogate only a fraction of CpG sites and are biased toward CpG islands. Asima Rev is a rapid, label-free, whole-genome electrical impedance cfDNA assay previously characterized across 216 clinical samples spanning 15 cancer types, from which a diagnostic threshold was established. The assay differentiates healthy individuals from those with cancer by measuring cfDNA aggregation patterns associated with methylation state, enabling functional detection of genome-wide hypomethylation. In this study, this prespecified threshold (adjusted to the present electrode geometry via cell constant normalization) is applied without modification to an independently collected cohort of 46 treatment-naïve patients with CRC and 33 controls, constituting a disease-specific retrospective validation of the assay. Only 4 samples overlapped between the 2 cohorts; all remaining samples were independently collected, processed, and analyzed. Asima Rev achieved 95.65% sensitivity (95% confidence interval, 85.47% to 99.23%) and 93.94% specificity (95% confidence interval, 80.39% to 98.92%), with longitudinal monitoring in 6 patients fully concordant with clinical outcomes. Interrogation of 11 public methylation array datasets showed that whole-array analyses underestimate global changes. Restricting analyses to OpenSea regions, where cfDNA is enriched, however, revealed patterns consistent with Asima Rev and a significant 5.8% global hypomethylation in CRC tissue compared to adjacent normal tissue. Hypomethylation was not observed in immune cell genomic DNA, a major contributor to cfDNA, supporting a predominantly tumor-derived contribution to the observed cfDNA signal. Together, these results demonstrate that Asima Rev captures a cfDNA signal consistent with true global methylation loss and outperforms locus-specific assays by measuring structural consequences of pan-genomic epigenetic alterations.