Fragmentation Analysis of the MYC Enhancer in Cell-Free DNA by qPCR for Early Detection of Hepatocellular Carcinoma

The early detection of hepatocellular carcinoma (HCC) remains a significant challenge. This study aimed to develop a quantitative PCR (qPCR) assay to analyze the fragment size distribution of cfDNA at the enhancer region of the oncogene MYC, evaluate its potential ability as a diagnostic biomarker for HCC.

In this retrospective case-control study, we enrolled 40 patients with newly diagnosed HCC, 45 patients with cirrhosis, and 130 healthy volunteers. We designed two overlapping primer pairs to amplify a long (432 bp) and a short (65 bp) fragment via qPCR targeting MYC enhancer region. The Fragment Index (FI-myc), defined as the ratio of short to long fragment, was calculated to infer regional chromatin accessibility, and the diagnostic performance of FI-myc was evaluated using receiver operating characteristic (ROC) curve and logistic regression analyses.

FI-myc levels were significantly elevated in HCC patients compared to those with cirrhosis and healthy controls (p < 0.01). Using the cutoff of 1.87, FI-myc demonstrated a sensitivity of 85.02% for discriminating HCC from cirrhosis and healthy controls. The area under the ROC curve (AUC) for FI-myc was 0.81, which was significantly superior to that of alpha-fetoprotein (AFP) (AUC = 0.49, p < 0.001). FI-myc maintained high sensitivity of 80.09% in early-stage HCC, and could detect 81.49% of HCC patients with negative AFP. Multivariate analysis demonstrated FI-myc as an independent indicator for HCC (OR = 2.82, p < 0.05).

We developed a qPCR-based assay to infer MYC enhancer accessibility. The FI-myc index represents a promising liquid biopsy biomarker for early HCC detection, particularly as a complementary tool in AFP-negative cases, showing potential for clinical translation.