Clonal proliferation of hepatocytes during chronic hepatitis B virus infection.

Abstract

Chronic hepatitis B virus (HBV) infection causes liver disease that can progress to cirrhosis and hepatocellular carcinoma (HCC). Changes in the hepatocyte population that occur from the early immune-tolerant stage of infection to late-stage disease outcomes remain unclear. We hypothesised that some hepatocytes lose HBV antigen expression and escape the HBVspecific immune response, allowing them to undergo clonal proliferation. Clonal proliferation of altered hepatocytes may be a marker of disease progression and may have a direct role in the development of HCC. Liver tissues from 30 patients were analysed, including patients with early-stage HBV infection, late-stage infection with cirrhosis, or with HCC. Unique virus-cell DNA junctions formed by the integration of HBV DNA into the host cell genome were detected using inverse nested PCR (invPCR). The copy number of unique virus-cell DNA junctions was used as a measure of clonal proliferation of hepatocytes. A computer simulation of a liver undergoing stochastic liver turnover was used to determine if the hepatocyte clones observed by invPCR could have been formed by random chance. Immunohistochemistry for HBV surface antigen (HBsAg) expression and Imaging Mass Spectrometry (IMS) for cellular protein expression were carried out to detect cellular changes that may be associated with clonal proliferation. Significantly (p<0.01) larger clones were observed by invPCR in liver DNA extracts of patients with late-stage HBV-associated disease (≤280000 hepatocytes) compared to patients in early-stage HBV infection (8-1124 hepatocytes). Computer simulations indicated that stochastic turnover could not produce clones of >10000 hepatocytes, suggesting that the hepatocytes that had formed large clones had a survival advantage. No significant difference in the extent of clonal proliferation was observed in foci of HBsAg-positive and –negative hepatocytes isolated by laser-microdissection. Heterogeneous expression of cellular proteins was detected using IMS in hepatocytes with apparently normal histology. These results indicate that clonal proliferation of hepatocytes with survival advantage does occur in the hepatocyte population during chronic HBV infection and can be detected before histological changes are evident in the hepatocytes of patients with both early- and late-stage disease. Consistent with our hypothesis, larger hepatocyte clones were associated with disease progression. The cause of the clonal proliferation remains unknown. Contrary to our hypothesis, loss of HBsAg expression was not associated with increased clonal proliferation, suggesting that escape from HBsAg-specific immune attack is not a survival advantage. While not investigated in this thesis, the loss of expression of other HBV antigens may provide a survival advantage. Heterogeneous cellular protein expression suggests that hepatocyte phenotype has been altered in some hepatocytes. However, we could not show using invPCR approaches that the foci of hepatocytes with altered cellular protein expression were clonal. In conclusion, this research has provided groundwork in determining the relationship between the clonal proliferation of hepatocytes, altered hepatocyte phenotype and HBV-associated disease progression. Further studies into the molecular causes of clonal proliferation of hepatocytes with survival advantages could elucidate pathways of HBV-associated disease progression and novel ways to curb the evolution of the hepatocyte population to a less pathogenic state.