Discovery and validation of a novel dual-target blood test for the detection of hepatocellular carcinoma across stages from cirrhosis.

Background: Hepatocellular carcinoma (HCC) is one of the most common cancers. Early detection of HCC helps improve the patients' 5-year survival rate. Our goal was to identify superior methylation biomarkers to develop a methylation-specific quantitative PCR (MS‒qPCR) assay.

Methods: A five-phase case-control study identified HCC methylation biomarkers via capture sequencing, TCGA/RNA-seq filtering, technical (MS-qPCR/Sanger) and biological (quadruplex MS-qPCR) validation. Methylated biomarkers were selected based on differential methylation expression using a tissue discovery cohort (43 HCC, 32 normal) and validated in plasma validation cohorts (Phase 1: 53 HCC, 52 cirrhosis, 20 benign, 50 healthy; Phase 2: 67 HCC, 81 cirrhosis). Then, the final assay's HCC detection performance was compared with existing blood-based surveillance methods.

Results: Two methylated genes, OSR2 and TSPYL5, and a novel internal reference gene, SDF4, were identified and developed into an MS‒qPCR assay named Qliver. Qliver had an AUC of 0.955 (95% CI: 0.924-0.987) for distinguishing HCC patients from non-HCC patients in the Phase 1 plasma cohort, with a sensitivity of 88.68% (95% CI: 76.97%-95.73%) and a specificity of 89.34% (95% CI: 82.47%-94.20%), and 0.958 (95% CI: 0.927-0.989) for distinguishing HCC patients from cirrhosis patients in the Phase 2 plasma cohort, with a sensitivity of 88.06% (95% CI: 77.82%-94.70%) and a specificity of 92.59% (95% CI: 84.57%-97.23%). For the Phase 1 plus Plasma 2 cohort, Qliver had an AUC of at least 0.958 for detecting HCC in healthy individuals, cirrhosis patients and patients with benign liver diseases, which was superior to that of the GALAD score (AUC: 0.777 to 0.849). For BCLC stage 0 and A HCC patients, the sensitivity of Qliver ranged from 62.50% (95% CI: 24.49%-91.48%) to 72.73% (39.03%-93.98%), with a specificity of 90%. Overall, Qliver was superior to the AFP, AFP-L3, DCP and the GALAD score in terms of cirrhosis history, tumor stage, tumor size and tumor count.

Conclusions: Qliver demonstrated superior performance in detecting HCC compared with currently widely used blood biomarkers, suggesting its potential clinical benefit in HCC surveillance in high-risk populations.