The genomic and epigenomic abnormalities of plasma cfDNA as liquid biopsy biomarkers to detect hepatocellular carcinoma: a multicenter cohort study.

Circulating tumor DNA (ctDNA) is a promising biomarker for early cancer detection; however, the optimal biomarker approach for early detection of hepatocellular carcinoma (HCC) remains unclear. Furthermore, current next-generation sequencing-based detection methods remain costly and complex, limiting their clinical utility for high-risk population screening. Hence, this study compared ctDNA mutation and methylation for HCC detection and aimed to develop a clinically accessible assay. A total of 1965 participants, including 629 HCC and 1336 control participants, were enrolled in five centers. Parallel ultra-deep targeted sequencing and targeted bisulfite sequencing revealed that a methylation-based ctDNA model significantly outperformed the mutation model in detecting HCC (sensitivity, 92.1% vs. 63.7%; P < 0.001), with no added benefit from combining both markers. We subsequently developed a multiplex PCR-based bisulfite amplicon sequencing assay (MBA-seq) using 25 selected methylation markers, achieving high diagnostic accuracy (AUC = 0.958, sensitivity = 86.7%, specificity = 90.1%) in the testing set. Further refinement through AUC-based selection yielded a two-marker panel (OTX1 and HIST1H3G) adapted into a quantitative methylation-specific PCR assay, designated HCCtect. This optimized model showed robust performance (AUC = 0.925, sensitivity = 78.4%, specificity = 93.0%), significantly surpassing alpha-fetoprotein (P < 0.001) while comparable with MBA-seq. HCCtect also demonstrated efficacy in early-stage HCC detection (sensitivity = 69.5%) and discrimination from chronic hepatitis B or liver cirrhosis (specificity = 93.0%). These findings establish ctDNA methylation as a superior approach over mutation analysis and highlight HCCtect as a promising non-invasive tool for HCC detection and high-risk population monitoring.