Clonal expansion of hepatocytes with a selective advantage occurs during all stages of chronic hepatitis B virus infection

Abstract

Hepatocyte clone size was measured in liver samples of 21 patients in various stages of chronic hepatitis B virus (HBV) infection and from 21 to 76 years of age. Hepatocyte clones containing unique virus-cell DNA junctions formed by the integration of HBV DNA were detected using inverse nested PCR. The maximum hepatocyte clone size tended to increase with age, although there was considerable patient-to-patient variation in each age group. There was an upward trend in maximum clone size with increasing fibrosis, inflammatory activity and with seroconversion from HBV e-antigen (HBeAg)-positive to HBeAg-negative, but these differences did not reach statistical significance. Maximum hepatocyte clone size did not differ between patients with and without a coexisting hepatocellular carcinoma. Thus, large hepatocyte clones containing integrated HBV DNA were detected during all stages of chronic HBV infection. Using laser microdissection, no significant difference in clone size was observed between foci of HBV surface antigen (HBsAg)-positive and HBsAg-negative hepatocytes, suggesting that expression of HBsAg is not a significant factor in clonal expansion. Laser microdissection also revealed that hepatocytes with normal-appearing histology make up a major fraction of the cells undergoing clonal expansion. Thus, preneoplasia does not appear to be a factor in the clonal expansion detected in our assays. Computer simulations suggest that the large hepatocyte clones are not produced by random hepatocyte turnover but have an as-yet-unknown selective advantage that drives increased clonal expansion in the HBV-infected liver.

Synthetic RNAi triggers and their use in chronic hepatitis B therapies with curative intent

“…Treatment with IFN results in a somewhat greater frequency of HBsAg seroclearance, with HBsAg loss reported in 23% of HBeAg(+) patients over an eight-year period (van Zonneveld et al, 2004). The decline in on-therapy HBsAg levels is more rapid and pronounced in patients treated with IFN as compared to NUCs, and a more rapid decline in HBsAg levels in patients undergoing IFN therapy is associated with a greater likelihood of HBsAg seroclearance (Moucari et al, 2009;Reijnders et al, 2011;Sonneveld et al, 2013).…”

“…In addition, certain HCC cell lines grown in culture that contain integrated HBV DNA can support the production of HBsAg-containing hepatitis D virus (HDV) virions, suggesting that these cells can produce at least low levels of functional HBsAg (Freitas et al, 2014). HBV integrants have also been detected in normal-appearing hepatocytes in both chimpanzees and humans with CHB (Mason et al, 2009(Mason et al, , 2010Tu et al, 2015). Regardless of their origin, transcripts containing HBV sequences could still be targeted using RNAi-based approaches.…”

HBV DNA Integration: Molecular Mechanisms and Clinical Implications

“…HBV DNA integration has been generally described in both HCC and cirrhotic patients with long-term CHB [42,46,60,61,62,63,64,65,66,67,68,69], but recently also prior to histologically observable liver damage in CHB patients [7,70]. Even earlier integration of HBV DNA has been reported in congenitally-infected children (as young as 5 months) with severe liver disease [71,72] and in patients with acute HBV infection [73].…”

In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

“…Our study also has its limitations. Although integration is not strictly required for the HBV life cycle and was not detectable by Southern blot analysis in CHB patients without cirrhosis or hepatocellular carcinoma (39), hepatocyte clones harboring an integrated HBV sequence do exist (40), and our cccDNA-specific probe set was unable to differentiate these events from episomal DNA. Second, since our probe sets were designed for the genome sequence of subtype C HBV, the sensitivity of our assay was influenced by sequence complementarity between viral and probe sequences.…”