Apoptosis and Hepatitis B: The Role of Apoptosis in Patients Who Are HBeAg Negative

Through a retrospective chart review, patients with HBsAg who are HBeAg negative, have normal ALT, and HBV DNA >10,000 copies/mL and who have or who are about to undergo liver biopsy will be identified. These individuals will be patients at the Toronto Western Hospital Liver Clinics. Inclusion criteria:(1) HBsAg positive.(2) Stable HBeAg status for at least one year prior to biopsy.(3) Normal ALT levels (defined as <1.5 x the upper limit of normal) for at least 90 days prior to biopsy.(4) HBV DNA ≥10,000 copies/mL by PCR measured within 90 days of liver biopsy.Exclusion criteria:(1) Coinfection with viral hepatitis A, C or D.(2) Coinfection with HIV.(3) Presence of Hepatoma.(4) Known presence of other malignancy.(5) Previous antiviral treatment.The diagnosis of hepatitis B will be based on standard serological assay results and HBV DNA detected with polymerase chain reaction using the Cobas Amplicor HBV Monitor Test (Roche Diagnostics inc.). This testing is part of the usual blood work regularly performed on these patients.We intend to stain the liver biopsies for quantitative assessment of apoptotic activity using three staining techniques:(1) TUNEL.(2) Immunohistochemistry for activated caspase-3, caspase-9 and cytochrome-C release.(3) Western blotting for caspase 3,6,7,8 and 9. Liver biopsies will only be performed if clinically indicated independent of this study protocol.At the time of liver biopsy, liver tissue was/will be buffered in formalin and embedded in paraffin. Tissue will also be frozen at minus 80 degrees for Western blotting. Two age-matched control groups of patients will also be randomly selected and compared to the study population. These control liver biopsies will be stained for apoptotic activity and for disease activity as well. These control groups will include patients who are:(1) HBeAg negative, unstable (elevated) ALT.(2) Healthy controls (living donor biopsies). All sections will be assessed for apoptotic activity using the appropriate technique that is specific for TUNEL, immunohistochemistry and Western blotting respectively. Liver histology will also be reviewed for grade and stage by an experienced hepatopathologist at the UHN. Necroinflammatory activity will be assessed using Ishak's hepatitis activity index and the Laennec grading system (a minor modification of the METAVIR system) will be used for assessment of hepatic fibrosis. Liver cell apoptosis assessed by the various techniques will be reported for the study group and the control groups as means +/- SEM. To compare the means between groups, ANOVA or Student's t test will be performed. Pearson's correlation coefficient will be used to measure the degree of association between apoptosis and histopathological activity on liver biopsy. Only the principal investigator will have access to personal and demographic information relating to the individuals included. Those performing pathological review and statistical analysis will be blinded to patient information.