Integrated DNA estimation in tissue biopsy and detection in liquid biopsy by HBV-targeted NGS assay.

Background & aims: Integrated HBV DNA (iDNA) plays a critical role in HBV pathogenesis, particularly in predicting treatment response and HCC. This study aimed to use an HBV hybridization-capture next-generation sequencing (HBV-NGS) assay to detect HBV-host junction sequences (HBV-JS) in a sensitive nonbiased manner to detect and estimate the iDNA fraction in tissue biopsies and HBV genetics by liquid biopsy.

Methods: HBV DNA from plasmid monomers, HBV-HCC cell line (SNU398, Hep3B, and PLC/PRF/5), tissue biopsies of patients with serum HBV DNA <4 log IU/ml, and matched urine and plasma of HBV patients were assessed by HBV-NGS. Junction-specific qPCR (JS-qPCR) assays were developed to quantify abundant HBV-JS.

Results: We demonstrated high coverage uniformity, reproducibility across all HBV genotypes A-D, and 0.1% sensitivity for detecting iDNA by the HBV-NGS assay. The sequence and structures of iDNA molecules from SNU398 and Hep3B are reported. An iDNA estimation model was developed using six abundant HBV-JS sequences identified from tissue biopsies by HBV-NGS assay and validated using total DNA of SNU398 and Hep3B cells. Furthermore, the utility of the HBV-NGS assay for HBV genetic analysis in liquid biopsies was explored using matched plasma-urine samples from three patients with serum HBV DNA levels ranging from high to undetectable. HBV-JS was detected in all body fluids tested, regardless of viral load.

Conclusion: These findings suggest that the iDNA fraction in tissue biopsies from patients with limited or undetectable serum HBV DNA can be estimated using a robust HBV-NGS assay, and a sensitive HBV genetics liquid biopsy can be obtained. This study highlights the potential of NGS-based methods to advance HBV management.