Performance of a five-gene plasma cfDNA methylation assay for colorectal cancer detection: a single-center real-world analysis

Blood-based circulating cell-free DNA (cfDNA) methylation assays have been proposed as minimally invasive tools for colorectal cancer (CRC) detection, but their performance under routine clinical conditions may differ from model-development studies. We evaluated the real-world diagnostic utility and limitations of a five-gene plasma cfDNA methylation assay targeting SEPTIN9, BCAT1, IKZF1, BCAN and VAV3, and compared it with carcinoembryonic antigen (CEA). This single-center study comprised 412 patients from a colorectal surgery department, all of whom had plasma cfDNA methylation assessed and routine serum tumor-marker evaluations conducted either before surgery or prior to treatment. Pathology was used as the reference standard. The primary analysis compared CRC with non-CRC diagnoses following pathology-based adjudication. Diagnostic performance was assessed using sensitivity, specificity, and receiver operating characteristic area under the curve (AUC). The cohort comprised 219 CRC patients and193 non-CRC controls. For CRC detection, methylation testing achieved a sensitivity of 73.5% (95% CI, 67.7%-79.4%), specificity of 91.2% (95% CI, 87.2%-95.2%), and AUC of 0.873 (95% CI, 0.841–0.904). CEA yielded substantially lower sensitivity of 35.9% (95% CI, 29.6%-42.3%), specificity of 96.7% (95% CI, 94.1%-99.3%), and AUC of 0.730 (95% CI, 0.682–0.779). The AUC difference between methylation testing and CEA was statistically significant ( p < 0.001, DeLong test). The “either-positive” combination increased sensitivity to 79.9% (95% CI, 74.6%-85.2%) while reducing specificity to 88.6% (95% CI, 84.1%-93.1%), with an AUC of 0.843 (95% CI, 0.808–0.877). Performance of methylation testing increased with tumor burden, from 56.8% (95% CI, 42.2%-71.5%) in stage I to 100% (95% CI, 70.1%-100%) in stage IV disease, from 38.1% (95% CI, 20.8%-59.1%) in T1 to 94.1% (95% CI, 73.0%-99.0%) in T4 disease, and from 53.6% (95% CI, 35.8%-70.5%) in tumors < = 2 cm to 95.0% (95% CI, 76.4%-99.1%) in tumors > 6 cm. In this single-center, single-department retrospective cohort, the five-gene plasma cfDNA methylation assay demonstrated substantially higher sensitivity and overall better discriminatory ability than CEA for colorectal cancer detection, albeit with slightly lower specificity. As expected for an emerging blood-based biomarker, sensitivity varied across tumor subgroups—reflecting the inherent biological constraints of circulating tumor DNA shedding. These preliminary findings from a real-world surgical cohort support the potential of plasma methylation analysis as an adjunctive risk-stratification tool to complement existing modalities, while underscoring the need for large-scale, multi-center prospective studies to refine its clinical utility across diverse screening and diagnostic settings.