Sensitive detection of circulating methylated SEPT9 in hepatocellular carcinoma patients using a novel quantitative PCR assay†

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. Early detection is crucial, yet reliable biomarkers are limited. Methylated SEPT9 (mSEPT9) has emerged as a promising biomarker for HCC. Building upon previous ExBP technology, we enhanced the semi-nested realtime PCR assay by integrating TaqMan probes, enabling quantitative detection of mSEPT9 in plasma samples of HCC patients. The assay was validated using synthetic DNA standards and plasma samples from 49 HCC patients, 20 chronic liver disease (CLD) patients, and 32 healthy donors (HDs). Our assay demonstrated sensitivity in detecting methylation ratios as low as 1 : 100 000. The assay showed a strong linear correlation between Ct values and methylation levels over four orders of magnitude (R² = 0.96178), indicating robust quantification. Clinically, the assay revealed significant differences in ΔCt values between HCC patients (median ΔCt = 19.55) and controls (CLD: 29.32 and HDs: 26.19, p < 0.005). ROC analysis for HCC vs. controls yielded an AUC of 0.729, with 77.55% sensitivity and 59.62% specificity at the optimal cutoff (≤25.98). Notably, the assay identified 72.73% of HCC cases with AFP levels below 20 ng mL⁻¹, underscoring its potential in detecting AFP-negative cases. These findings suggest that the novel mSEPT9 assay is a sensitive and specific tool for early HCC detection, offering prognostic value for clinical monitoring.