Two-year assessment of entecavir resistance in Lamivudine-refractory hepatitis B virus patients reveals different clinical outcomes depending on the resistance substitutions present

Abstract

Entecavir (ETV) is a deoxyguanosine analog approved for use for the treatment of chronic infection with wild-type and lamivudine-resistant (LVDr) hepatitis B virus (HBV). In LVD-refractory patients, 1.0 mg ETV suppressed HBV DNA levels to below the level of detection by PCR (<300 copies/ml) in 21% and 34% of patients by Weeks 48 and 96, respectively. Prior studies showed that virologic rebound due to ETV resistance (ETVr) required preexisting LVDr HBV reverse transcriptase substitutions M204V and L180M plus additional changes at T184, S202, or M250. To monitor for resistance, available isolates from 192 ETV-treated patients were sequenced, with phenotyping performed for all isolates with all emerging substitutions, in addition to isolates from all patients experiencing virologic rebounds. The T184, S202, or M250 substitution was found in LVDr HBV at baseline in 6% of patients and emerged in isolates from another 11/187 (6%) and 12/151 (8%) ETV-treated patients by Weeks 48 and 96, respectively. However, use of a more sensitive PCR assay detected many of the emerging changes at baseline, suggesting that they originated during LVD therapy. Only a subset of the changes in ETVr isolates altered their susceptibilities, and virtually all isolates were significantly replication impaired in vitro. Consequently, only 2/187 (1%) patients experienced ETVr rebounds in year 1, with an additional 14/151 (9%) patients experiencing ETVr rebounds in year 2. Isolates from all 16 patients with rebounds were LVDr and harbored the T184 and/or S202 change. Seventeen other novel substitutions emerged during ETV therapy, but none reduced the susceptibility to ETV or resulted in a rebound. In summary, ETV was effective in LVD-refractory patients, with resistant sequences arising from a subset of patients harboring preexisting LVDr/ETVr variants and with approximately half of the patients experiencing a virologic rebound.

Figures

FIG. 1.

Relative potencies of anti-HBV agents against WT and LVDr HBV. The relative potency was expressed as a function of the average in vitro HBV RT IC50 and intracellular cell culture levels of triphosphates (diphosphate for ADV) at clinical exposure levels. IC50s (n ≥ 3) and intracellular triphosphate levels at clinical exposures (n = 2) were determined as described in Materials and Methods. The higher the number is, the greater the level of potency was. WT, wild-type HBV polymerase; LVDr, lamivudine-resistant HBV polymerase with M204V and L180M substitutions.

FIG. 2.

HBV DNA levels in LVD-refractory patients treated with ETV or LVD. (A) Time point analysis. Gray and white circles, all 1.0-mg ETV- and 100 mg LVD-treated LVD-refractory patients, respectively. The size of the circles at each log10 interval is reflective of the percentage of patients (each column adds up to 100%) with the indicated HBV DNA levels at that treatment time point (week 0, baseline; Week 24 treatment period, weeks 12 to 30; Week 48 treatment period, weeks 42 to 72; Week 96 treatment period, weeks 90 to 102). The lowest circle represents patients with HBV DNA levels below the level of detection (300 copies/ml). N, number of patients included in the analysis at each time interval. (B) Cumulative percentage of ETV-treated (diamonds) and LVD-treated (triangles) patients who experienced HBV DNA reductions to undetectable levels (<300 copies/ml) by the week indicated. N, number of patients included in each data set.

FIG. 3.

Phenotypic susceptibilities of population isolates from patients with virologic rebounds. Population phenotypes of baseline and rebound isolates were determined as described in Materials and Methods. (A) ETV EC50s for isolates from patients at baseline and the time of rebound (Rb); (B) fold change in ADV susceptibilities of rebound isolates. Open circles, patient isolates with WT or LVDr-encoding sequences at the time of rebound; filled circles, patient isolates with ETVr-encoding substitutions at the time of rebound. The median ADV susceptibilities (EC50s) at the baseline and the time of rebound were both 2.7 μM. Isolates from patient 158 were not tested for their susceptibilities to ADV.

FIG. 4.

HBV DNA profiles for patients with virologic rebounds while receiving ETV. Patient HBV DNA levels over time are shown for patients exhibiting virologic rebounds while receiving ETV. Patient isolates had sequence evidence of ETVr-encoding substitutions at baseline (A), substitutions that emerged by Week 48 (B), substitutions that emerged by Week 96 (C), or no ETVr-encoding substitutions or reduced ETV susceptibility (D). Time points marked with a boxed X in panel D represent off-treatment visits.