Methylation plus alpha-fetoprotein blood test for early detection of HCC in at-risk populations.

Background Semi-annual abdominal ultrasound plus alpha-fetoprotein (AFP) is recommended for HCC surveillance but has limited performance, particularly in patients with metabolic dysfunction-associated steatotic liver disease. We aimed to validate a blood-based test combining cell-free DNA methylation profiling with AFP to improve HCC detection in at-risk individuals. Methods Between September 1, 2023, and April 30, 2025, 135 patients with HCC and 167 at risk for HCC were prospectively recruited at 2o medical centers; archived samples (46 HCC and 32 at-risk) were also included. A cell-free DNA methylation assay was performed to generate methylation scores, and AFP testing was conducted as part of standard of care. A pretrained model integrated methylation scores and AFP levels for HCC detection. Results The combination of methylation and AFP tests achieved an AUC of 0.94 (95% CI, 0.92-0.96) for detecting HCC in at-risk individuals, significantly outperforming both methylation alone (AUC: 0.92, 95% CI, 0.89-0.95; p=0.001) and AFP alone (AUC: 0.83, 95% CI, 0.79-0.87; p<0.0001). In patients with early-stage HCC (within Milan Criteria), the combined test achieved an AUC of 0.90 (95% CI, 0.86-0.94), outperforming methylation alone (AUC: 0.88, 95% CI, 0.83-0.92; p=0.002) and AFP alone (AUC: 0.81, 95% CI, 0.76-0.86; p<0.0001). At 92.0% specificity, sensitivity for early-stage HCC was 73% (95% CI, 64%-80%) with the combined test, compared with 62% (95% CI, 52%-71%; p=0.025) for methylation alone and 52% (95% CI, 43%-62%; p<0.0001) for AFP alone. Sensitivity improvements were consistent across major demographic and etiologic subgroups. Conclusions The combination of cell-free DNA methylation with AFP shows promising performance for early-stage HCC detection in this case-control validation study and warrants further validation.