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Potential conflict of interest: Nothing to report.
Entecavir (ETV) is currently recommended as a rescue therapy purely for adefovir (ADV)-resistant chronic hepatitis B virus (HBV) infections. We evaluated the efficacy of ETV in patients who were resistant to lamivudine (LAM)/ADV sequential therapy and in those resistant to LAM monotherapy. Fifty LAM/ADV-resistant and 38 LAM-resistant patients who received ETV 1 mg/day for at least 48 weeks were enrolled. Mean baseline serum HBV DNA and alanine aminotransferase (ALT) levels were significantly lower in the LAM/ADV-resistant group, compared with the LAM-resistant group (6.90 versus 7.62 log10 copies/mL and 102.6 versus 160.2 IU/L; both P < 0.05); hepatitis B e antigen (HBeAg) status and LAM-resistant mutation patterns were similar in the two groups. At week 48, mean reductions in HBV DNA and ALT levels were significantly less in the LAM/ADV-resistant group (−2.96 versus −4.86 log10 copies/mL and −68.3 versus −128.9 IU/L; both P < 0.05). Achievement of undetectable HBV DNA was also less common in the LAM/ADV-resistant group (10.0% versus 34.2%; P = 0.006), although the rates of HBeAg loss and ALT normalization did not differ between the two groups. Resistance to both LAM and ADV was an independent risk factor for failure of HBV DNA negativity at week 48 (odds ratio, 0.138; P = 0.019). In both LAM/ADV-resistant and LAM-resistant groups, primary responders (≥1 log decline in HBV DNA at week 12) achieved a significantly greater decrease in HBV DNA levels over the 48-week period, compared with primary nonresponders (−4.18 versus −0.97 and −5.37 versus −2.15 log10 copies/mL, respectively; both P < 0.05). Conclusion: The 48-week ETV treatment was less effective in LAM/ADV-resistant than in LAM-resistant patients. Continuing ETV monotherapy could be determined based on the virological response at 12 weeks in LAM/ADV-resistant patients. (HEPATOLOGY 2009.)
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Over the past decade, the development of oral nucleoside/nucleotide analogs (NAs) with favorable potencies and tolerabilities has led to substantial advances in chronic hepatitis B (CHB) therapy. The oral anti-HBV agents currently approved are lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine, clevudine (exclusively in Korea), and tenofovir.1, 2
These NAs necessitate long, and in many cases, indefinite treatment to achieve sustained viral suppression. Unfortunately, because the duration of NA treatment is prolonged, the risk of development of drug resistance increases.3 Selection for drug resistance is associated with attenuated antiviral suppression and rapid disease progression.4, 5 Previous studies in treatment-naïve patients reported a cumulative LAM resistance incidence of 70% to 80% after 4 to 5 years of therapy6, 7 and an ADV antiviral resistance rate of 29% after 5 years.8 Sequential resistance to LAM and later ADV has been reported in patients who were switched to ADV monotherapy for LAM-resistant HBV.9, 10 Furthermore, ADV mutations emerged more commonly in LAM-resistant patients than in treatment-naïve patients.9, 10 It was suggested that sequential antiviral therapy leads to selection of multidrug-resistant HBV, and evolution of mutations during continued treatment may select for mutants with increased replication fitness or maximal viral resistance.11
In terms of salvage therapy for LAM-resistant or ADV-resistant CHB, the American Association for the Study of Liver Diseases practice guideline recommended switching to ETV as an optimal strategy,12 and this protocol has been widely applied in clinical practice. Recent reports have shown that switching to ETV therapy in LAM-refractory patients with CHB resulted in superior viral suppression compared with continued LAM therapy, with a comparable safety profile.13–15 However, the cumulative probability of genotypic ETV resistance development over 4 years was 43% in LAM-refractory patients, which is considerably greater than the 1.2% probability seen in NA-naïve patients.16 In fact, a new treatment algorithm does not recommend ETV monotherapy as a rescue therapy for patients with CHB who had LAM resistance.1
There have been few reports on the effect of ETV switching in patients with multidrug resistance that developed after switching to ADV monotherapy for LAM-resistant HBV. We evaluated the virological, serological, and biochemical outcomes of ETV monotherapy over 48 weeks in patients with compensated CHB who developed resistance to both LAM and ADV after sequential therapy, compared with patients showing resistance to LAM only.
We enrolled 50 patients with CHB with or without liver cirrhosis who developed antiviral drug resistance to LAM-ADV sequential therapy (LAMr/ADVr group), and 38 individuals displaying resistance to LAM monotherapy (LAMr group). These patients received 1 mg/day ETV for at least 48 weeks at our institution. Antiviral resistance was defined as follows: persistently detectable HBV DNA after at least 36 weeks after initiation of LAM or ADV therapy14; virological breakthrough (>1 log10 increase in serum HBV DNA level from the nadir in a patient who had an initial virological response12) in patients with LAM or ADV therapy; or documented LAM-resistance or ADV-resistance mutations accompanied by HBV viremia. All patients were characterized by the presence of persistent hepatitis B surface antigen for more than 6 months and serum HBV DNA levels of greater than 4 log10 copies/mL at baseline, irrespective of hepatitis B e antigen (HBeAg) status. Patients with antibodies against hepatitis C virus or human immunodeficiency virus, or with hepatic decompensation generally associated with jaundice, ascites, encephalopathy, or gastrointestinal bleeding, were excluded. Additional criteria for exclusion were liver transplantation and past or current hepatocellular carcinomas. All patients were followed every 2 to 3 months by clinical examination and biochemical and virological assessments. Adherence to treatment was assessed during each visit to the clinic. This study was approved by the institutional review board of our hospital.
Serum Assay Methodology.
Serum HBV DNA levels from all patients were quantified using a commercially available real-time polymerase chain reaction (PCR) assay (Abbott Laboratories, Chicago, IL), according to the manufacturer's instructions, with a linear dynamic detection range of 5.1 × 101 to 3.4 × 109 copies/mL. Genotypic resistances to LAM and ADV were proven by restriction fragment mass polymorphism analysis on HBV from all individuals at baseline. Genotypical analysis for ETV resistance was performed on a subset of patients showing virological breakthrough during follow-up using restriction fragment mass polymorphism analysis as previously described.9 For restriction fragment mass polymorphism genotyping, viral DNA was amplified using PCR. Table 1 shows the sequences of forward and reverse primers used for PCR. A sequence with small letters (ggatg) is a restriction site in an amplicon, recognized by FokI and BtscI, and marking the rtL180 and rtM204 substitutions characteristic of LAM resistance. FokI recognized the rtI169, rtT184, rtS202, and rtM250 substitutions of ETV resistance. To detect ADV resistance substitutions at rtA181 and rtN236, a sequence shown in small letters is embedded in the forward primer to insert an MmeI site (tccaac) in the amplicon, and sequences with underlined letters inserted a HaeIII site (GGCC) or an SspI site (AATATT). In general, viral markers, including hepatitis B surface antigen, HBeAg, and anti-HBe, were determined using commercially available enzyme immunoassays (Abbott Laboratories, Chicago, IL). Aspartate aminotransferase, alanine aminotransferase (ALT), albumin, and total bilirubin in serum and prothrombin time were measured using standard laboratory procedures.
Table 1. Primers Used for Amplification in RFMP Assays of Mutations Imparting Resistance to Each of the Three Nucleos(t)ide Analogs
RFMP, restriction fragment mass polymorphism.
Serum HBV DNA levels were expressed on a logarithmic scale. Between-group comparisons were performed using Student t test or the Mann-Whitney U test for continuous variables, and the chi-squared test or Fisher's exact test for categorized variables, as appropriate. Covariates with P < 0.27 from univariate analysis were then included in multivariate analysis using a logistic regression model. A probability value of P < 0.05 was considered statistically significant.
The baseline characteristics of the two study groups are presented in Table 2. There were no statistically significant differences in the mean age or sex composition between the two groups. At least 70% of subjects were positive for HBeAg, and liver cirrhosis was diagnosed by clinical, radiological, or histological assessments in more than 30% of patients. Mean serum HBV DNA level was significantly lower in the LAMr/ADVr group compared with the LAMr group (6.90 versus 7.62 log10 copies/mL; P = 0.029). Mean serum aspartate aminotransferase and ALT levels were also significantly lower in the LAMr/ADVr group (75.6 versus 106.5 and 102.6 versus 160.2 IU/L; P = 0.014 and P = 0.009, respectively). Basal serum total bilirubin and albumin levels and prothrombin time did not significantly differ between the two groups. The prevalence of LAMr mutations, M204V/I substitutions with or without L180M, were similar between the LAMr/ADVr and LAMr groups (82.0% and 86.8%, respectively). In the LAMr/ADVr group, genotypical resistance to ADV was documented in a total of 27 patients (54.0%); 15 (30.0%) had the A181V/T substitution alone, four (8.0%) had the N236T substitution alone, and eight (16.0%) had both mutations.
Table 2. Baseline Patient Characteristics
LAMr/ADVr group (N = 50)
LAMr group (N = 38)
ADV, adefovir; ALT, alanine aminotransferase; ETV, entecavir; HBeAg, hepatitis B e antigen; INR, international normalized ratio; LAM, lamivudine; NA, not applicable; SD, standard deviation.
Sex (No. [%])
47.0 ± 8.3
44.7 ± 9.4
Cirrhosis present (No. [%])
Serum HBV DNA level (log10 copies/mL)
Mean ± SD
6.90 ± 1.44
7.62 ± 1.57
HBeAg-positive (No. [%])
Serum AST level (IU/L)
Mean ± SD
75.6 ± 80.4
106.5 ± 101.7
Serum ALT level (IU/L)
Mean ± SD
102.6 ± 115.7
160.2 ± 172.2
Serum total bilirubin level (mg/dL)
Mean ± SD
1.3 ± 1.1
1.3 ± 0.5
Serum albumin level (g/dL)
Mean ± SD
4.0 ± 0.4
Prothrombin time (INR)
Mean ± SD
1.06 ± 0.10
LAM-resistance mutational pattern (No. [%])
M204V + L180M
M204I + L180M
M204V/I + L180M
ADV-resistance mutational pattern (No. [%])
A181V/T + N236T
Mean changes in serum HBV DNA levels at each timepoint are shown in Fig. 1 and Table 3. The mean reduction in serum HBV DNA levels at week 24 was significantly less in the LAMr/ADVr group than in the LAMr group (−2.41 versus −3.73 log10 copies/mL; P = 0.015). At week 48, the mean decline in serum HBV DNA levels was significantly lower in the LAMr/ADVr group than in the LAMr group (−2.96 versus −4.86 log10 copies/mL; P = 0.002).
Table 3. Virological and Biochemical Responses During ETV Rescue Therapy
LAMr/ADVr Group (N = 50)
LAMr Group (N = 38)
ALT, alanine aminotransferase; ETV, entecavir; SD, standard deviation.
Defined as a decrease in HBV DNA of ≥1 log10 copies/mL from baseline at week 12.
At week 48, serum HBV DNA was undetectable by real-time PCR (<51 copies/mL) in a significantly smaller percentage of the LAMr/ADVr group compared with the LAMr group (10.0% versus 34.2%; P = 0.006; Table 3).
Serum ALT levels decreased progressively during 48 weeks of treatment in both patient groups, as shown in Table 3. Throughout the treatment period, the mean decline in serum ALT levels was significantly less in the LAMr/ADVr group compared with the LAMr group (−66.2 versus −125.9 IU/L at week 24; P = 0.005, and −68.3 versus −128.9 IU/L at week 48; P = 0.012, respectively). However, the proportions of patients showing normalized serum ALT levels at weeks 24 and 48 did not differ significantly between the two groups.
HBeAg status during treatment is summarized in Table 4. Among patients who were HBeAg-positive at baseline, 31.4% (11/35) in the LAMr/ADVr group and 30.3% (9/30) in the LAMr group were HBeAg-negative at week 48. However, this difference was not statistically significant. The rates of HBeAg seroconversion at week 48 were also similar between the two groups (14.3% versus 6.7%; P = 0.442).
Table 4. Serological Responses During ETV Rescue Therapy
All patients who tested positive for HBeAg at baseline in the LAMr/ADVr group.
All patients who tested positive for HBeAg at baseline in the LAMr group.
HBeAg seroconversion (No. [%])
HBeAg loss (No. [%])
Baseline Predictive Factors for Antiviral Efficacy at 48 Weeks.
After multivariate analysis with adjustment for baseline variables (sex; age; HBeAg status; serum HBV DNA, aspartate aminotransferase, ALT, total bilirubin and albumin levels; prothrombin time; and drug resistance profiles) for all 88 patients in both groups, resistance to both LMV and ADV was the only independent covariate to be inversely associated with serum HBV DNA negativity at week 48 (adjusted odds ratio, 0.138; 95% confidence interval, 0.027-0.722; P = 0.019; Table 5).
Table 5. Multivariate Analysis of Baseline Factors Predictive of Serum HBV DNA-Negativity After 48 Weeks of ETV Rescue Therapy
Generated by multivariate logistic regression analysis.
Resistance to LMV alone
Resistance to LMV and ADV
Age (per year)
HBV DNA level (per 1 log10 copies/mL)
AST level (per 1 IU/L)
ALT level (per 1 IU/L)
Impact of HBV DNA Reduction at 12 Weeks on Antiviral Efficacy at 48 Weeks.
Primary response, defined as a 1-log10 copies/mL or more decrease in serum HBV DNA level within 12 weeks of the commencement of antiviral therapy,1 was achieved in significantly fewer LAMr/ADVr patients (70.7%) than LAMr patients (81.6%) (P = 0.031; Table 3). In both groups, primary responders at week 12 showed a more significant decrease in serum HBV DNA levels from baseline at 48 weeks of treatment compared with individuals who had no primary response (−4.18 versus −0.97 in the LAMr/ADVr group; P < 0.001, and −5.37 versus −2.15 log10 copies/mL in the LAMr group; P = 0.009, respectively; Fig. 2A and 2C). The changes in serum ALT levels were not different between primary responders and nonresponders (−93.3 versus −27.5 in the LAMr/ADVr group; P = 0.180, and −137.4 versus −83.3 IU/L in the LAMr group; P = 0.445, respectively; Fig. 2B and 2D).
In total, two patients experienced virological breakthrough during 48 weeks of treatment, including 1 of 50 patients (2.0%) in the LAMr/ADVr group and 1 of 38 patients (2.6%) in the LAMr group; breakthrough was seen at 36 weeks of ETV therapy in both cases. However, biochemical breakthrough was noted in the patient in the LAMr group only. Both patients who experienced virological breakthrough developed the S202G ETV-resistance substitutions, accompanied by emerging LAM-resistance substitutions (M204V or L180M). The patient in the LAMr/ADVr group also had ADV resistance substitutions (A181V/T + N236T).
We found that ETV was less effective, as measured by virological and biochemical outcomes, in patients with LAM-ADV dual resistance compared with patients with LAM monoresistance. Previous in vitro studies have shown that ADV-resistant HBV strains were susceptible to ETV.17–19 Although increasing evidence supports the therapeutic efficacy of ETV in LAMr patients,13–15 limited clinical data are available on ADV-resistant patients showing prior LAM resistance.20, 21 A small cohort study showed that switching to ETV plus ADV was highly efficient in 13 CHB patients who failed sequential or combination therapy with LAM and ADV, with a 76.9% (10/13) HBV DNA clearance rate during a median 10 months of treatment (range, 4–16 months).22
ETV was reported to be effective irrespective of baseline HBV DNA levels in treatment-naïve patients.23 Studies with LAM or ADV suggested that the presence of high pretreatment HBV DNA levels was a risk factor for increased antiviral drug resistance and attenuation of virological response in treatment-naïve or LAM-resistant patients.24, 25 In the current study, pretreatment serum HBV DNA levels were lower in LAMr/ADVr patients. Given that pretreatment low viral load is predictive of HBe seroconversion,26 it was interesting to note that the rate of HBeAg loss (including seroconversion) after ETV monotherapy was no higher in LAMr/ADVr patients (with lower pretreatment viral loads) than in LAMr patients. Our study indicates that LAM-ADV dual resistance was the only covariate that could predict an adverse effect on viral clearance, independent of pretreatment HBV DNA and ALT levels (Table 5).
A possible explanation for the virological and biochemical differences between LAMr/ADVr and LAMr patients may involve unrecognized overlapping cross-resistance patterns between ETV and ADV. The antiviral sensitivity of ADV-resistant HBV containing the A181V/T mutation to ETV has not yet been determined, although amino acid substitutions that are not related to ADV resistance generally predispose to high-level ETV resistance.27, 28 This assumption is consistent with recent in vitro data showing that the A181V/T mutant has relatively reduced susceptibility to ETV compared with the N236T mutant.29 In a phenotypical analysis of both LAM-resistant and ADV-resistant HBV mutants, the L180M+M204V and L180M+M204V+N236T mutants showed no difference in susceptibility to ETV in vitro, although both displayed a 6.25-fold resistance to ETV compared with wild-type HBV.17 In the current study, the A181V/T substitution was identified in 46% of patients with documented genotypical resistance to ADV, whereas the N236T substitution was in 24% (Table 2). Preexisting YMDD (Tyr-Met-Asp-Asp) mutation also may affect clinical outcomes, because ETV shows reduced efficacy against M204V/I mutant strains27; however, the two populations in our study did not differ significantly.
Patients with ADV monoresistance may show relatively better responses to ETV treatment, unlike ADV-resistant patients with prior LAM resistance, because ETV-resistant strains are typically encountered in individuals with one or more preexisting LAMr mutations, such as L180M and M204V.27, 28 Barthe and Arab30 reported that 14 of 22 patients with CHB (63.6%) with suboptimal responses to ADV, including a single patient (4.5%) with a YMDD mutation who had prior exposure to LAM, became HBV DNA undetectable (<70 copies/mL) after 24 weeks of ETV treatment.
ETV strongly suppressed viral replication in LAM-monoresistant patients, with 34.2% achieving undetectable HBV DNA (<51 copies/mL) at 48 weeks, which was at least equivalent to the results of two recent studies (19% [<300 copies/mL] and 33% [<400 copies/mL]).14, 15 The ALT normalization rate (73.7%) at 48 weeks in our analysis was also similar to these studies (61% and 78%).14, 15 Previous studies with LAM and telbivudine demonstrated a positive correlation between early profound HBV suppression and long-term clinical outcome.25, 31, 32 Our study also showed that virological responses to ETV at week 12 of treatment determined the degree of HBV DNA reduction over 48 weeks, regardless of previous antiviral drug resistance.
Although patients with LAM-ADV dual resistance showed a lesser antiviral response to ETV than LAM-monoresistant patients in our study, virological breakthrough did not emerge more commonly. However, 2.6% (1/38) of LAM-monoresistant patients experienced virological breakthrough during 48 weeks of ongoing ETV treatment, which is slightly higher than the 1.4% (2/141) of a previous report.14 Because genotypical analyses for ETV resistance were performed only in selected patients who showed virological breakthrough during the study period, the exact time and the rate of mutation emergence were not investigated in all patients. Because most antiviral-resistant strains have inferior replication capacity initially, several months or even years may be required before a progressive increase in serum HBV DNA is noted,33 and thus lengthy follow-up is required.
Our observations indicate the need for further studies on ETV treatment of LAMr/ADVr and LAMr HBV patients, including a larger cohort size and involving thorough analysis of genotypic resistance to ETV by DNA sequencing. Currently, emergence of multidrug resistance is an increasing challenge in antiviral therapy, particularly in patients who were switched to ADV monotherapy for LAM-resistant HBV. Multidrug resistance to antiviral agents was closely associated with a reduction in susceptibility to subsequent antiviral treatments, because mutations conferring resistance to multiple antiviral agents co-locate on the viral genome.11 To prevent multidrug resistance development, the new generation NA, which have higher genetic barriers to resistance, should be used as first-line therapy, and the addition of a potent second drug, sharing no NA cross-resistance, should be preferred in patients with antiviral-resistant CHB.
In conclusion, our comparative analysis demonstrates that, in ADV-refractory HBV patients with prior LAM resistance, ETV rescue therapy over 48 weeks is less efficacious than in LAM-refractory HBV patients, as shown by virological and biochemical responses. A good antiviral response at 12 weeks after the switch to ETV monotherapy is a predictor of favorable 48-week antiviral outcomes. For patients with both LAM and ADV resistance, ETV monotherapy could be continued only in cases with the initial optimal responses at 12 weeks.