The efficacy of tenofovir-based therapy in patients showing suboptimal response to entecavir-adefovir combination therapy

Patients

We retrospectively reviewed the medical records of CHB patients treated with a combination therapy with ETV 1 mg plus ADV 10 mg as rescue therapy for drug resistance more than 6 months at Konkuk University Hospital in Seoul, South Korea. The ETV-ADV combination therapy was started between July 2008 and October 2011, and final follow-ups were conducted until December 2014. Only patients who did not respond to more than 2 years of prior rescue therapy other than the ETV-ADV combination therapy were enrolled. In case of hepatitis C or human immunodeficiency virus co-infection, patients who were receiving immunosuppressant therapy such as chemotherapy were excluded.

Serial serum samples were collected from each patient at the time of initiation of the ETV-ADV combination treatment and every 6 months during treatment and stored frozen at -80℃. Written informed consent for the collection of serum samples was obtained from all of the patients. The study protocol was approved by the Institutional Review Board of Konkuk university hospital (KUH1010496) and was conducted in accordance with the ethical guidelines of the 1975 Declaration of Helsinki.

Clinical and laboratory assessments

Laboratory data including serum alanine amino transferase (ALT), albumin, bilirubin, creatinine, HBV DNA, hepatitis B envelope antigen (HBeAg), and anti-HBe were measured at baseline and every 3 months during treatment. Serum HBV DNA levels were assessed by using the COBAS Amplicor polymerase chain reaction (PCR) assay (lower detection limit, 20 IU/mL; Roche Molecular Systems, Branchburg, NJ, USA). Before initiation of the ETV-ADV combination therapy, antiviral-resistant mutations were tested by using a restriction fragment mass polymorphism (RFMP; Genematrix, Youngjin, South Korea) [18].

VR was defined as an undetectable HBV DNA level (< 20 IU/mL), and biochemical response (BR) was defined as a normalized ALT level. Nonresponse was defined as <2 log₁₀IU/mL decline in the HBV DNA level from baseline after 6 months of therapy.

Production of the HBV replicons

We selected four patients with nonresponse and with HBV DNA > 4 log₁₀IU/mL at 1 year. Sera collected from the patients at 1 year of treatment were studied.

The gene encoding the HBV polymerase reverse transcriptase (RT) domain was analyzed in these patients. The HBV DNA was extracted from sera samples obtained from the four patients by using the QIAamp MinElute virus spin kit (Qiagen) according to the manufacturer’s protocol. The RT domains of wild-type (WT) HBV and HBV mutants isolated from sera were amplified and converted into the replication-competent HBV 1.2 mer constructs. Eight to 10 clones were obtained from each case, and sequencing of the RT domain was performed. In these clones, we generated several patient-derived HBV 1.2 mer replicons by switching the RT gene from WT HBV 1.2 mer replicon as previously described [19,20].

Cell culture and transfection

Huh7 human hepatoma cancer cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, GrandIsland, NY, USA), with additional condition of 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco), and incubated at 37°C in a 5% CO₂ environment. The cells were seeded 3×10⁵ in a six-well plate and transiently transfected with 2 µg of the HBV 1.2mer replicons by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Antiviral drugs and treatment condition

ETV was purchased from Moravek (Brea, CA, USA), and ADV and TDF were obtained from Gilead Science (Foster City, CA, USA). After 4-6 h of post-transfection, the media were changed to fresh DMEM mixed with appropriate concentration of antiviral drugs. Each drug was treated every 4 days with daily change with fresh DMEM. The concentrations of the drugs were 20 µM for TDF and 1 µM for ETV.

In vitro susceptibility and replication assay

To analyze the HBV DNA replication capacity, Southern blot analysis was performed as described previously [19,20]. We concisely summarize the procedure as follows: The transfected Huh7 cells were harvested at 4 days post-transfection for detection of HBV DNA intermediates. The cells were lysed with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (10 mM HEPES at pH 7.5, 100 mMNaCl, 1 mM EDTA, and 1% NP-40). For elimination of transfected plasmid or cellular chromosomal DNA, the lysates were treated with DNaseI (Clontech/Takara Bio, Mountain View, CA, USA) and mung bean nuclease (Clontech/Takara Bio) at 37°C for 15 minutes. Cytoplasmic core particles were precipitated with polyethylene glycol (PEG) solution (1.2 M NaCl, 60 mM EDTA, 30% sucrose, 26% PEG 8000), and the capsid protein was digested with proteinase K (20 mg/mL; Roche Applied Science, Indianapolis, IN, USA) in the presence of sodium dodecyl sulfate (SDS) at 37°C for 2 hours. To obtain purified HBV DNA, phenol extraction and ethanol precipitation was performed. The HBV DNA was separated in 0.8% agarose gel. After transfer onto a Hybond-N+ membrane (GE Healthcare, Buckinghamshire, UK), HBV DNA was detected with a highly pure randomized HBV probe with labeled isotope.

Statistical analyses

Statistical testing was performed by using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Data were expressed as median (range) and number (percentile). Continuous variables were compared by using the Mann-Whitney U-test. Categorical data were compared by using the chi-squared test or Fishcer exact test. A p < 0.05 was considered statistically significant.

Baseline Characteristics

For the analysis, 48 patients were enrolled (Fig. 1). Of the patients, 41 (85.4%) were male, and the median age was 48 years. Cirrhosis and HCC were detected in 12 (25.0%) and 3 patients (6.3%) respectively. All of the HCC patients were treated with radiofrequency ablation before ETV-ADV combination treatment. Most patients were HBeAg positive (47, 97.9%), and the median HBV DNA level was 5.8 log₁₀IU/mL. The initial treatment agents were LMV in 41 patients (85.4%), CLV in 6 patients (12.5%), and ETV in 1 patient (2.1%); (Table 1).

Figure 1.

Flow chart of the enrolled patients.

Table 1.

Baseline characteristics

Treatment response to ETV-ADV combination therapy within 3 years

Twelve patients achieved VR, and no difference in baseline characteristics were observed between the groups with or without VR (Table 1).

The VR rates were 6.5% (3/46) at month 6, 18.6% (8/43) at month 12, 26.7% (8/30) at month 24 and 52.9% (9/17) at month 36. The BR rates were 60.0% (27/45) at month 6, 65.9% (29/44) at month 12, 80.0% (24/30) at month 24, and 100% (17/17) at month 36. Nonresponse was observed in 19 patients, 39.6% of all the patients and 52.8% of those without VR.

Clinical courses of the patients without VR within 3 years

Fifteen patients were lost to follow-up. Two patients continued the ETV-ADV combination regimen, and one patient achieved VR at month 42. Ten patients switched to TDF monotherapy (300 mg/day), mutation test before switch showed no mutation in 4, L180M+M204I+M204V in one, L180M+M204I+M204V+M250L in one, L180M+M204V+M250V in one, L180M+M204V+T184L in two, M204I+A181S+T184I in one, and 9 of whom achieved VR within 1 year except the patients with L180M+M204V+M250V mutation. Seven patients switched to LMV (100 mg/day) plus TDF combination therapy, mutation test before switch showed no mutation in two, L180M+M204I in two, L180M+M204V in one, M204I in two, and 6 of whom achieved VR within 1 year except the patients with M204I. Two patients switched to ETV (1 mg/day) plus TDF combination therapy, mutations detected before switch were L180M+M204V+I169T+T184L, L180M+M204V+T184L+S202G, and all of whom achieved VR within 1 year (Fig. 1).

Sequence evolution of HBV polymerase RT

Four patients were selected for clonal evaluation (Table 2). Six clones were selected from the four patients and generated to 1.2 mer replicon by swapping the RT region of the WT 1.2 mer clone. The sequences were compared against the sequence from genotype C HBV (NCBI GenBank accession no. GQ872210). The sequence showed rtM204I/V with or without rtL180M mutation, and no ADV-, TDF-, nor ETV-resistant mutation (Fig. 2A).

Table 2.

Demographic data of the selected patients

Figure 2.

The in vitro sequence and drug susceptibility assay of the RT mutants isolated at 1-year treatment from the serum of CHB patients refractory to the ETV-ADV combination therapy. (A) The HBV RT genes from each serum sample were converted into HBV 1.2mer replicons, and the substitutions were compared with the wild type (NCBI GQ872210). (B-D) Cloned HBV DNAs were transfected into Huh7 cells, and the cells were treated for 4 days with 20 µM TDF or 20 µM TDF and 1 µM ETV. The intracellular HBV DNA was analyzed by Southern blotting. The YI/VDD mutation in HBV RT region had a replication-defect.

In vitro drug susceptibility test

Next, we checked the replication capacity of these clones by Southern blotting. The YI/VDD mutation in the HBV RT region is well known to have a replication defect, and our cases also had replication-defective clones (Fig. 2B) [21]. To investigate the susceptibility to antiviral agents, TDF monotherapy and ETV-TDF combination treatment were initiated. Replication decreased after both treatments. However, replication was still detected after the long exposure in the case of TDF monotherapy despite the weak replication (Fig. 2C, D).