Fragmentation Analysis of the MYC Enhancer in Cell‐Free DNA by qPCR for Early Detection of Hepatocellular Carcinoma
Yan Jiang, Xuchu Wang, Yibei Dai, Pan Yu, Yue Cheng, Depeng Wang, Tao ChenABSTRACT
Background
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.
Methods
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.
Results
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).
Conclusion
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.