Profound reduction of HBV covalently closed circular DNA with long-term nucleoside/tide analogue therapy

Abstract

BACKGROUND: Long-term nucleoside/tide analogue (NA) treatment suppresses serum HBV DNA to undetectable levels in a majority of patients. We aimed to investigate the effect of longterm NA on the suppression of covalently closed circular DNA (cccDNA) and intrahepatic HBV DNA (ihHBV-DNA). METHODS: We recruited 40 patients (median age 44.2 years, range 24.3-63.2) who had been on continuous long-term (5 – 10 years) NA. All patients had 3 liver biopsies: at baseline, after 1 year of treatment and at the last follow-up. Serum HBV DNA and HBsAg were measured by the COBAS TaqMan HBV Test and the Elecsys HBsAg II Assay, respectively (both Roche Diagnostics). ihHBV-DNA and cccDNA were assayed by real-time PCR, with lower limits of detection of 0.001 and 0.005 copies/cell, respectively. RESULTS: The median duration of treatment was 10.5 years (range: 6.0 – 11.9 years). At baseline, 13 patients had 100mg lamivudine, 11 had 600mg telbivudine, 9 had 0.5mg entecavir, 4 had 30mg clevudine, and 3 had 10mg adefovir. At the last follow up, these patients were on 0.5-1.0mg entecavir (n=23), 600mg telbivudine (n=9), 10mg adefovir (n=4), 300mg tenofovir (n=2), or combination therapy of lamivudine plus adefovir/tenofovir (n=2). Histology of the third biopsy showed complete resolution of interface hepatitis in 60% of patients with the remainder showing mild-to-moderate activity. Persistent immunoreactivity for HBsAg was found in 80%, the mean number of hepatocytes positive for HBsAg being 10.4% (range 1-80%). All but 1 (2.5%) was immunoreactive for HBcAg. At baseline, the median serum HBV DNA, HBsAg, ihHBV-DNA and cccDNA levels were 6.84 logIU/mL, 3.38 logIU/mL, 286 copies/cell, and 7.3 copies/cell, respectively. At the time of the last biopsies, 36 (90%) patients had undetectable serum HBV DNA (<20 IU/mL), all but one patient still had detectable HBsAg (median: 2.74 logIU/mL), all had detectable ihHBV-DNA (median: 0.4 copies/cell), but 18 (45%) patients had undetectable cccDNA. There was a trend of reduction of HBsAg, ihHBV-DNA and cccDNA levels from baseline to 1 year to last follow-up (all p<0.0001). The median log drop of HBsAg at last biopsy was 0.55 logIU/mL. The median percentage reductions of HBsAg, ihHBV-DNA and cccDNA at last biopsies were 71.46%, 99.85% and 99.89%, respectively. CONCLUSIONS: Long-term NA treatment significantly reduced cccDNA and ihDNA. 45% of patients had undetectable cccDNA, although small amount of ihHBV-DNA were still detectable in all patients. Integrated HBV DNA may be a possible source of detectable ihHBV-DNA and HBsAg. Continuous long-term NA therapy can reduce cccDNA to undetectable levels, suggesting a possible end-point of treatment.