Development and validation of a digital PCR assay targeting plasma methylation for early detection of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, yet current surveillance with alpha-fetoprotein (AFP) and ultrasound (US) lacks sufficient accuracy, particularly for early-stage disease. This study aimed to develop and clinically validate a blood-based digital PCR (dPCR) assay targeting novel methylation biomarkers for HCC detection. Genome-wide methylation profiles from HCC, normal, and other cancer types were analysed to identify candidates, which were then screened and verified in cancer cell lines, primary tumour tissues, and plasma specimens. A total of 186 plasma samples were evaluated, including 66 healthy controls, 60 patients with chronic liver disease (CLD) without HCC, and 60 patients with HCC. methylation emerged as an HCC-specific biomarker, showing significantly higher levels in liver cancer cell lines and HCC tumour tissues compared with controls. A dPCR assay targeting achieved 70.0% sensitivity (42/60; 95% CI, 56.8%-81.2%) and 96.8% specificity (122/126; 95% CI, 92.1-99.1%), with an area under the curve (AUC) of 0.90 (95% CI, 0.85-0.96). Combining with AFP improved sensitivity to 88.3% (53/60; 95% CI, 77.4%-95.2%) while maintaining 96.8% specificity. Notably, early-stage HCC (Barcelona Clinic Liver Cancer [BCLC] stage 0-A) was detected with 85.4% sensitivity (35/41; 95% CI, 77.4%-94.4%). This blood-based dPCR assay demonstrated high diagnostic performance, underscoring its potential as a noninvasive tool to enhance early detection and clinical management of HCC in high-risk populations.