Exploratory analysis of transcriptionally active HBV-host chimeric transcript burden in patients with increasing HBsAg during long-term antiviral therapy

Abstract Background and aims The longitudinal relationship between transcriptionally active hepatitis B virus (HBV) integration dynamics and serum hepatitis B surface antigen (HBsAg) during long-term antiviral therapy remains unclear. Methods Patients with HBV-related fibrosis/cirrhosis who underwent at least one liver biopsy at baseline, week 78, or week 260 were enrolled. HBsAg increase was defined as either a ≥5% increase from baseline to week 208 or at least two ≥5% increases between consecutive available visits. A subset of 10 patients with serial liver RNA sequencing underwent integrated clinical, transcriptomic, and histopathological analyses. HBV-host chimeric transcripts were identified using ChimericSeq and verified by BLAST, and intrahepatic HBsAg was assessed by immunohistochemistry. Results Overall, 31.8% (99/311) of patients met the criteria for HBsAg increase, while others were classified into the stable/decrease group. Lower baseline HBsAg and higher histologic activity index (HAI) were independently associated with HBsAg increase (both p < 0.05). In the serial-biopsy subset ( n = 10), patients in the HBsAg increase group (4/4) showed increased HBV-host chimeric transcript burden at week 260 versus week 78, whereas the six patients in the stable/decrease group showed stable or reduced burden. Intrahepatic HBsAg immunohistochemistry showed persistent clustered cytoplasmic positivity, with transcriptomic analysis indicating downregulation of multiple immune-related pathways in the HBsAg increase group. Conclusion About 31.8% of patients had HBsAg increase during antiviral therapy. Baseline HBsAg and HAI were associated with HBsAg increase during antiviral therapy. In the exploratory serial-biopsy subset, patients with HBsAg increase tended to exhibit an increase in transcriptionally active HBV-host chimeric transcript burden and persistent clustered cytoplasmic HBsAg positivity.