Introduction: Splicing of the HBV pregenome, resulting in the generation of spliced HBV transcripts (spDNA), may facilitate replication of wild type (wt) HBV, promoting development of hepatocellular carcinoma (HCC). In this study we have analysed changes in HBV splicing over time; providing the first longitudinal analysis of HBV spDNA in relation to the development of HCC. Our in vivo studies are supplemented by in vitro examination of HBV splicing and replication.
Methods: Serial serum samples were collected from 165 patients with chronic HBV monoinfection, including 58 patients who later developed HCC. Real time PCR was used to amplify and quantify wt and sp DNA loads. Huh‑7 hepatoma cells were transiently transfected with plasmids containing wtHBV (1.3WT) and spHBV (pCI2.2). Cell lysates and supernatents were harvested and purified and Southern Blot and PCR analysis were used to compare production of DL DNA as well as wt and spDNA loads.
Results: Univariate analysis revealed HBV viral load to be highest in HCC patients prior to diagnosis of HCC ((τ= -0.306; p< 0.001) and strong correlations between time and HBV spDNA levels (τ= 0.203; p= 0.016). Asian HBV genotype (p= 0.025) and increased viral load (p< 0.001) were also significantly associated with increased HBV spDNA. Multiple regression analysis revealed time to diagnosis of HCC, Asian HBV genotypes and viral load to be associated with increased HBV spDNA (model p< 0.001; R2= 0.189). Preliminary in vitro results suggest that co-transfection of Huh-7 cells with 1.3WT and pCI2.2 significantly increases production of HBV wtDNA.
Conclusions: HBV spDNA is abundant in the setting of HCC and increases prior to diagnosis of clinical disease. Taken together these findings suggest that HBV spDNA may be contributing to HCC through increased HBV replication.