Identification of a G-quadruplex-forming cell-free DNA fragment as a biomarker for the precise diagnosis of hepatocellular carcinoma [cfDNA]
Early detection of hepatocellular carcinoma (HCC) remains challenging, as the currently recommended surveillance strategy based on ultrasound combined with alpha-fetoprotein (AFP) is limited by suboptimal sensitivity and accessibility. Cell-free DNA (cfDNA) provides a minimally invasive avenue for cancer detection. However, most existing cfDNA-based approaches either perform unreliably in low-input samples or require analytically complex workflows. Here, we systematically profiled serum cfDNA from patients with HCC and healthy controls and identified a high-abundance tumor-associated single cfDNA fragment at the FAM230F genomic region. Integrative analysis of liver ATAC-seq datasets revealed consistent tumor-specific chromatin accessibility at this locus, suggesting a tumor-derived origin. Structural characterization further demonstrated enrichment of G-quadruplex (G4) features within the target sequence, which may increase resistance to serum nuclease degradation and promotes its preferential retention in circulation. Based on these properties, we established a standardized qPCR-based detection workflow with clinical accessibility. In an independent validation cohort, this assay achieved a sensitivity of 94.5% and a specificity of 91.5% for discriminating HCC from healthy controls. Collectively, our study identifies FAM230F as a structurally stable tumor-associated cfDNA fragment and establishes a simple and scalable qPCR-based assay for HCC detection, providing a practical framework for translating cfDNA fragment analysis into clinically actionable biomarkers.