HBV Treatment – Oct. 30

Antiviral Treatments for Hepatitis B

#966.  Mutations at HBV-Polymerase Gene Associated with Entecavir Drug Resistance in Patients Not Undergoing Entecavir Therapy

R. Jardi; F. Rodriguez-Frias1; M. Buti; M. Schaper; R. Esteban; J. Guardia



The incidence and risk factors for the development of entecavir (ETV)-resistant hepatitis B virus (HBV) have not been clearly defined. Resistance requires substitutions at residues I169T, T184G, S202I and/or M250V. To develop ETV-substitutions, prior 3TC (lamivudine)-resistant L180M and/or M204VI changes are required and it has been suggested that 3TC alone can pre-select for ETV-changes.



To determine the incidence of ETV-resistant variants in patients with 3TC drug-resistance, investigate factors associated with ETV-resistance and determine whether drug-resistant substitutions occur in nucleose analog-naïve patients.


Patients and Methods:

This retrospective study includes 111 untreated patients under 3TC- therapy for at least 24 months (18-60 months). Serum samples were obtained at pre-treatment and during follow-up. Resistance-associated mutations to ETV ant 3TC were analyzed by amplification of a fragment of 747 bp. of polymerase gene. To confirm the detection of ETV-resistance-associated mutations both strands of the nested-amplification product (amino-ac58-257) were sequenced. The same sequence was used to determine HBV-genotypes. Genotypic analysis was performed comparing DNA sequences from patients at study entry with sequences obtained during 3-TC-therapy and with 185 complete HBV sequences from the GenBank.



At baseline, 5(4.5%) of 111 patients presented substitutions in the HBV polymerase-gene associated with nucleose-analogue resistance, two with 3TC- resistance (L180LM, and L180M plus M204MI) and 3 with the ETV-resistance, mutation S202SI. None of the patients with ETV-substitutions had 3TC-resistant variants. During 3TC-treatment, emergence of ETV-resistant mutations was observed in 11(10%) additional patients, 10 of whom were non-responders: the S202SI variant was observed in 6 cases (3 cases was found in conjunction with I169IT), 2 cases had T184TA, 1 M250MV and the remaining case was constituted by a pattern of S202I and T184A variants. All of them presented ETV and 3TC resistances at the same time. The responder patient showed the ETV variant S202SI in absence of 3TC variants.



The mixture of wild-type and mutated sequences for ETV-resistance observed prior to therapy in patients who had never received nucleose-analogues suggests that ETV-variants are present in a significant proportion of the complex HBV genome quasispecies. ETV-resistant mutations can also emerge during 3TC therapy in the absence of ETV treatment.


#967. Impact of Nucleoside Treatment on Antiviral T-Cell Reactivity in Chronic Hepatitis B: Major Differences Depending on Early Viral Suppression, HBeAg Status and HBV Genotype

N. V. Naoumov; H. Cooksley; J. Hou; L. Vitek;. Urbanek; W. Abbott; E. Gane; P. Hofmann; S. Zeuzem; H. Wedemeyer; M. Buti; D. Standring; L. van Doorn; G. C. Chao; N. A. Brown



Weak and functionally impaired antiviral T-cell responses are a dominant cause for HBV persistence in chronic hepatitis B (CHB). Antiviral therapy enhances T-cell reactivity in some patients but underlying mechanisms are unknown. Telbivudine (LdT) showed more rapid and significantly greater anti-HBV activity compared with lamivudine in a phase III randomized trial in HBeAg positive (HBe+) and HBeAg negative (HBe-ve) CHB (GLOBE trial). In a GLOBE substudy we investigated the impact of viremia reduction and HBV genotype on antiviral T-cell reactivity.



Peripheral blood mononuclear cells (PBMC) were collected prospectively at protocol time points from 63 patients (39 HBe+/24 HBe-ve) at 7 centres. PBMC were analyzed with Elispot assays and flow cytometry to characterize CD4+/CD8+ T-cells specific for HBV core and surface antigens. HBV DNA sequencing identified 5 HBV genotypes in patients studied (A, n=13; B, 13; C, 18, D, 18, E, 1). T-cell reactivity according to HBV genotype was assessed with genotype-specific peptides (15-mer overlapping by 10aa) spanning the entire core and surface proteins. Serum HBV DNA was quantified by COBAS PCR assay.



In HBe+ patients, rapid and profound viral suppression [baseline to week 8 (W8) >4.0 log10 copies/ml] led to significant increase in core-specific T-cells at W8 (p=0.017). This increase was not seen with viral load decline <3.9 log10 copies/ml or in surface-specific T-cells. Linear regression analysis revealed significant correlation between early HBV DNA decline and core-specific T-cell frequency at W4 (p=0.024), independent of age, gender or treatment type. In HBe-ve CHB there were no significant relations between viral suppression and T-cell frequencies.


T-cell epitope mapping with genotype-specific peptides showed no differences at baseline comparing HBV genotypes. However, treatment-induced viral suppression increased the breadth of T-cell reactivity in patients with HBV-A and D genotypes, particularly at W8 and W24, recognizing significantly more HBV core peptides, compared with HBV-B (p<0.005) or C (p<0.02). HBV genotype was a significant variable for HBV core-specific T-cell reactivity both in HBe+ patients at W24 (p=0.026) and W48 (p=0.01), and in HBe-ve patients at W48 (p=0.01).



This largest investigation of HBV-specific T-cell reactivity during antiviral therapy reveals the major impact of viremia suppression for recovery of antiviral T-cell reactivity. The impact of HBV genotypes most likely reflects differences in route of transmission and/or duration of HBV infection. These results prove a basis for developing individualized combination therapies.


#968. Emergence of Predominant Adefovir Resistant Mutation during Additive Lamivudine Therapy for Adefovir Resistance in Chronic Hepatitis B Patients with Prior Resistance to Lamivudine

S. Kwon; W. Choe; J. Yeon; K. Byun; Y. Kim; Y. Seo; S. Hong; S. Kim; W. Yoo; C. Lee


Background and Aim:

Lamivudine (LAM) is commonly used for the treatment of chronic hepatitis B(CHB) as a first-line antiviral agent. Adefovir-dipivoxil (ADV) therapy is effective, but viral resistance is common in LAM resistant patients. It has been reported that sequential treatment of antiviral agents may increase the risk of viral resistance to multiple agents. Antiviral efficacy of additive LAM treatment for ADV resistance in patients with prior LAM resistance is uncertain. We investigated virologic response and emerging pattern of resistant mutations during administration of additive LAM therapy for ADV resistance in chronic hepatitis B patients with prior resistance to LAM.


Patients and Methods:

Ten patients of CHB with an additive LAM therapy for at least 6 months in addition to ongoing ADV treatment were included (M/F:9/1, mean age 47.6 yrs, HBeAg+ 7cases , HBeAg- 3cases). All of them have received LMV and ADV monotherapy sequentially. Laboratory tests including HBV-DNA level (real-time PCR) and ADV-resistant mutation (RFMP) were performed in stored sera at the start of ADV and every 3 months thereafter. Virologic response was defined as HBV-DNA <5log10 copies/ml or decrease in HBV-DNA by >2log10 copies/ml, and viral breakthrough as an increase in HBV-DNA level>1 log10 from the nadir in virologic responders.



All the patients except one had liver cirrhosis, and two of them had hepatocellular carcinoma. All of them revealed YMDD mutation(5 YD cases, 3 YVDD cases, and 1 mixed case) during prior LAM therapy. After commencement of ADV monotherapy for the LAM resistance, YMDD mutations were not detected by RFMP. The patients showed viral resistance to ADV with emergence of ADV resistant mutations (rtA181V/T in 7 cases, rtN236T in 1 case) as a mixed pattern of mutant and wild type. LAM was started in addition to ongoing ADV therapy. Mean follow up period of the additive LAM therapy was 9.6 months. Two HBeAg-negative patients showed viral response with ALT normalization. However, 8 patients did not achieve viral response at all. ADV resistant mutations persisted as a predominant pattern in 6 patients among the 8 non-responders. ALT level was increasing during the additive LAM treatment in non-responders, 2 of them showed ALT flare (> 5 times UNL) with clinical deterioration. Three patients were switched to tenofovir/LAM for salvage therapy.



Additive LAM therapy for ADV resistance may potentiate pre-existing ADV resistant mutation which developed during the sequential treatment of ADV monotherapy for LMV resistance. ADV resistant mutation might be selected as a predominant pattern during the sequential antiviral treatment.


#969. Long-term Efficacy and Safety of Adefovir Dipivoxil for the Treatment of HBeAg-Positive Chronic Hepatitis B (CHB) Patients

P. Marcellin1; T. Chang; S. Lim; W. Sievert; M. Tong; S. Arterburn; K. Borroto-Esoda; S. Chuck



Treatment of 171 patients with HBeAg(+) CHB with ADV 10 mg over 48 weeks resulted in significant histological, virological, serological, and biochemical improvement compared to placebo in the first year of this study (NEJM 348:808-816, 2003). The long-term efficacy and safety of ADV in HBeAg(+) CHB patients was investigated for up to five years.



Entry criteria were HBsAg(+) ≥6 months, HBeAg(+), serum HBV DNA ≥6 log10 copies/mL (Roche Amplicor Monitor PCR, LLQ 1000 copies/mL), and ALT 1.2-10X ULN. Patients were randomized to receive ADV 10 mg, ADV 30 mg, or placebo in year 1. Most patients had multiple doses of placebo in year 2 due to an error in drug allocation. Patients with confirmed HBeAg loss or seroconversion were followed off treatment. Patients given ADV 10 mg in year 1 who dnot seroconvert in years 1 and 2 could continue in a long-term, safety and efficacy study (LTSES) with assessments every 3 months for 3 years. A two-state Markov model was used to estimate the percentage of patients with HBeAg loss and seroconversion over time and to account for seroconversion.



Baseline characteristics of the 65 LTSES patients were: median age 34 years, 83% male, 74% Asian, median serum HBV DNA 8.45 log10 copies/mL, median ALT 2.0 x ULN. At 5 years, the median change from baseline in serum HBV DNA and ALT for the 41 patients still on ADV was -4.05 log10 copies/mL and -50 U/L. Markov two-state probabilities of HBeAg loss and seroconversion at 240 weeks were 60% and 48%, respectively. Four patients (2%) lost HBsAg and seroconverted to anti-HBs. Fifteen patients had baseline and LTSES liver biopsies. Ranked assessments of the baseline and LTSES liver biopsies showed improvements in necroinflammation and fibrosis in 67% and 60%, respectively. Median changes in Knodell necroinflammation and Ishak fibrosis scores were -4 (p=0.014) and -1 (p=0.022), respectively. Adefovir-resistance mutations A181V and/or N236T were not detected in year 1 (0/171) but developed in 13 LTSES patients (20%) beginning at study week 195; 12 patients (12/65; 18%) also had virologic resistance (VR= mutation + confirmed rebound ≥ 1 log10 copies/mL from nadir or never < 3 log10 copies/mL) and 5 patients (5/65; 8%) had clinical resistance (VR and ALT>ULN after normalizing ALT). There were no serious adverse events related to ADV. Six patients (9%) had a confirmed increase in serum creatinine of ≥0.5 mg/dL beginning after 3.5 years; 2 (3%) discontinued ADV.



Treatment with ADV beyond 48 weeks produced long-term virologic, biochemical, serologic and histologic improvements and was safe and well-tolerated in patients with HBeAg(+) CHB.


# 971. No Evidence for Tenofovir Resistance in Patients with Lamivudine-Resistant HBV Infection During Long-Term Treatment for Up to 5 Years

F. van Bömmel; S. Mauss; D. Schürmann; D. Hüppe; R. Neuhaus; P. Reinke; S. Christoph; U. Spengler; B. Möller; B. Zöllner; B. Wiedenmann; T. Berg



Tenofovir disoproxil fumarate (TDF), an acyclic nucleote reverse transcriptase inhibitor is used in the treatment of HIV infections and has also potent activity against hepatitis B virus (HBV). In this retrospective study we analyzed the long-term effectiveness of tenofovir with respect to virologic parameters and resistance development.



Sixty-nine patients with chronic lamivudine-resistant HBV infection (m/f 59/10, mean age 48±12 years [18-76], 61 HBeAg positive) and different co-morbities were treated with TDF 300 mg daily (except one patient who received 300 mg TDF every second day due to renal insufficiency) and followed for at least 6 and up to 59 months (mean 33±16 months; 6, 18, 9, 26 and 10 patients for up to 1, 2, 3, 4, and 5 years). 24 out of the 69 patients were HIV/HBV co-infected and received TDF as part of their anti-retroviral therapy; 5 and 7 patients were treated in the course of kidney and liver transplantation. The remaining 33 patients had no co-morbidities. At baseline the HBV polymerase gene was screened for mutations by direct sequencing from codon rt88 to rt282 in all patients. The mean HBV DNA level at baseline was 7.5±1.3 log10 copies/mL (range, 4.0-10.4), and 52 patients had elevated ALT levels. ALT, creatinine, and HBV DNA levels were measured on a three-monthly basis (HBV Monitor, Roche Diagnostics, detection limit 400 copies/mL).



At the end of the observation period, a reduction of HBV DNA levels below the limit of detection was found in 68 out of the 69 patients. The one patient with detectable viremia received a reduced TDF dose due to renal insufficiency. The mean HBV DNA decline in this patient was -3.17 log10 copies/mL at month 18. There was no evidence of TDF resistance development as a re-increase of HBV DNA from undetectable levels was not observed in any of the patients studied. HBV polymerase sequencing during treatment could not be performed because HBV DNA could not be amplified by sensitive PCR assays. HBeAg seroconversion was documented in 36% of the patients after a mean duration of 14±9 (range, 3-36) of TDF treatment, and HBsAg loss in 8% after 16±6 (range, 9-25) months. The presence of co-morbidities or HIV co-infection as well as HBV polymerase gene mutations at baseline did not influence the response to TDF. No significant side effects associated with TDF were noted.



In this long-term observation no evidence for HBV resistance against tenofovir could be found. The results demonstrate the high long-term efficacy and the safety profile of tenofovir.


#973. The Effect of Lamivudine on Preventing Hepatocellular Carcinoma in Chronic Hepatitis B: A Retrospective Study of 2,518 Patients

J. Eun; B. Jang; T. Kim; H. Lee; K. Lee



This retrospective, case-controlled study was conducted to evaluate the effect of lamivudine on preventing hepatocellular carcinoma(HCC) in chronic hepatitis B.


Patients and Methods:

Of the 2518 patients who satisfied our inclusion criteria for analysis, 866 had lamivudine therapy and 1632 patients had not. In our results using Cox regression model, 4 factors were related with hepatocellular carcinoma; sex(female, Odds ratio(OR)=0.55, p=.009), age(≥40 years, OR=4.44, p<.001), platelet count(≥100×103/mm3, OR=0.42, p<.001), alcohol(≥80 g/day, OR=1.7, p=.009). Enrolled patients were dived into 144 categories for case-controlled study by 6 factors; sex (male, female), age(<39, 40-49, ≥50 years), platelet count(<99, 100-149, ≥150×103/mm3), serum albumin(<3.5, ≥3.5 g/dL), alcohol consumption(<20, ≥20 g/day), ascites (yes or no). When the cutoff was defined at 7 years of follow-up, 561 patients in the lamivudine-treated group and 385 patients in the lamivudine-untreated group were selected for matched case-controlled study. The mean follow-up duration was 2.9 years in the lamivudine-treated group and 3.3 years in the control group. The age, sex, platelet count, serum albumin, alcohol history, ascites were not different between two groups.



In the lamivudine group (n=561), HCC occurred in 13 patients(2.3%) with an annul incidence rate of 0.8% (patient/year), whereas in the control group(n=385) HCC occurred in 43 patients(11.2%) with an annual incidence rate of 3.4%(patient/ year). The cumulative incidence of HCC in the lamivudine group was lower than in the control group (p<.001, log-rank test).



Lamivudine may reduce the increase of hepatocellular carcinoma in patients with chronic hepatitis B.



Comparison of the cumulative incidence of HCC between the lamivudine group and the control group by the Kaplan-Meier method.


#977. HBV cccDNA, Pregenomic RNA and Total HBV DNA Levels in the Liver of Chronic Hepatitis B Patients 4-5 Years from Start of Effective Long-Term Adefovir Dipivoxil Treatment

S. J. Hadziyannis; A. Laras; A. Costamena



Long term adefovir dipivoxil (ADV) therapy of HBeAg-negative chronic hepatitis B (CHB) results in effective suppression of hepatitis B virus (HBV) replication with PCR-undetectable serum HBV DNA levels in approximately 2/3 of treated patients. Relapse after stop of treatment is a major concern linked with residual HBV replication and persistence of its covalently closed circular DNA (cccDNA).



To evaluate HBV cccDNA, pregenomic (pg) RNA and virion DNA levels in the liver of HBeAg-negative patients with CHB, treated effectively with ADV for 4-5 years.



Twenty-four patients submitted to long-term treatment with ADV (10mg), were subjected to liver biopsy at 4-5 years after initiation of therapy. All patients harbored genotype D G1896A mutant HBV, had serum HBV DNA levels at start of therapy >106 cp/ml and achieved sustained virological response with HBV DNA nondetectable by Cobas TaqMan real-time PCR. The results were compared to those in a similar number of HBeAg-negative CHB patients prior to start of antiviral treatment. Total DNA and RNA were extracted from liver biopsy samples of the 24 ADV treated and controls and intrahepatic HBV cccDNA, pgRNA and total HBV DNA levels were quantified by real-time PCR.



After 4-5 years of ADV treatment, HBV cccDNA levels were found to be low in all patients, with median value 0.045 cp/cell (0.03-0.11), 38-fold lower compared to the median cccDNA levels 1.69 cp/cell (0.13-6.80) in the control group. Total HBV DNA levels in the liver were 110-fold lower in the treated patients compared to the controls, [median 0.085 cp/cell (0.02-0.36) vs 9.4 cp/cell (1.40-113)]. The production of intrahepatic genomic viral DNA in the ADV treated patients was found to be dramatically lower (530-fold) compared to that in the untreated group [0.016 cp/cell (0-0.28), vs, 8.21 cp/cell, (1.29-109)]. Median pregenomic RNA levels in the ADV treated patients were 56-fold lower compared to the untreated group, [0.102 cp/cell (0.03-1.06) vs 5.73 cp/cell (1.40-44.9).



1) Long term suppression of HBV replication with ADV in HBeAg(-) CHB patients leads to a significant reduction-but not elimination- of intrahepatic HBV cccDNA and to a parallel decrease of pgRNA. 2) Production of genomic HBV DNA in the liver is markedly impaired in such patients. 3) Evaluation of these variables as potential predictors/determinants of sustained response/relapse after stop of therapy is under way.


#982. Entecavir Results in Higher HBV DNA Reduction vs Adefovir in Chronically-Infected HBeAg(+) Antiviral-Naive Adults: 24 WK Results (E.A.R.L.Y. Study)

N. Leung; C. Peng; J. Sollano; L. Lesmana; M. Yuen; L. Jeffers; H. Han; M. Sherman; J. Zhu; K. Mencarini; R. J. Colonno; A. Cross; R. Wilber; J. Lopez-Talavera



Elevated HBV DNA is associated with increased risk of disease progression and complications in patients with chronic hepatitis B (CHB). The aim of antiviral therapy is to rapidly and extensively reduce viral load (VL). We present the Week 24 results of a randomized, open-label, comparative study comparing entecavir (ETV) to adefovir (ADV) in CHB patients, and the evaluation of early viral suppression as a predictor of Week (Wk) 24 results.



Sixty-nine HBeAg(+), antiviral-naïve, CHB patients were randomized 1:1 to receive either ETV (0.5 mg) or ADV (10 mg) QD for a minimum of 52 weeks. Measurements of serum HBV DNA by PCR assay were obtained through Wk 12 for assessment of VL reduction and viral kinetics. Additional serum samples were obtained at Wk 24 for determination of HBV DNA by PCR. Evaluable patients were those who had baseline, Wk 12 and Wk 24 HBV DNA by PCR and received their assigned treatment. The difference in reduction of VL between treatments was based on a linear regression model adjusted for baseline. Prediction of Wk 24 VL was based on the Cochran-Armitage trend test.



Mean baseline HBV DNA was 10.26 (ETV) and 9.88 (ADV) log10 copies/mL. ETV demonstrated superior early antiviral activity and viral kinetic profiles compared to ADV as early as Day 10, with superior reduction in HBV DNA at Wk 12 (-6.23 vs. -4.42 log10 copies/mL, p<0.0001) Mean HBV DNA change from baseline at Wk 24 was -6.97 (ETV) vs. -4.84 log10 copies/mL (ADV) [Difference (95% CI): -1.86 (-2.51, -1.20)]. Undetectable HBV DNA (<300 copies/mL) by PCR was achieved by Wk 24 in 45% of ETV- vs.13 % of ADV-treated patients.


The trend test showed that at day 10 VL was predictive of Wk 24 PCR results: among those patients achieving HBV DNA <106 copies/mL by Day 10, 91% of ETV vs 50% of ADV patients subsequently achieved undetectable VL at Wk 24. Both antivirals had comparable safety profiles throughout the study.



At Week 24, ETV-treated patients achieved a mean 1.86 log10 (72-fold) greater reduction in HBV DNA, with a higher proportion of patients achieving undetectable VL compared to ADV. The extent of HBV DNA reduction at Day 10 was predictive of achieving undetectable HBV DNA at Wk 24. Both antivirals were generally well tolerated.

#985. Initial Viral Response Is the Most Powerful Predictor of the Emergence of YMDD Mutant Virus in Chronic Hepatitis B Patients Treated with Lamivudine

N. Kurashige; N. Hiramatsu; K. Ohkawa; T. Oze; T. Yakushijin; H. Uyama; T. Igura; S. Kiso; T. Kanto; T. Takehara; S. Tamura; M. Oshita; T. Hijioka; K. Katayama; I. Yabuuchi; H. Yoshihara; E. Hayashi; Y. Imai; M. Kato; N. Hayashi


Background and Aim:

Lamivudine (LAM) is widely used for the treatment of chronic hepatitis B (CHB) patients. However, the emergence of the YMDD mutant virus greatly limits long-term therapeutic efficacy. In this study, we investigated factors affecting the emergence of this mutant virus in CHB patients treated with LAM.



The subjects were 190 CHB patients (139 males and 51 females, median age 52 years) who underwent continuous LAM therapy. Of them, 87 (46%) were HBeAg-positive and 77 (41%) had cirrhosis. With 33 (18%) patients, combination therapy with interferon was carried out for the initial 6 months. The median duration of follow-up was 38 (range 12-104) months. Serum HBV DNA was detected by PCR or transcription mediated amplification method. The YMDD mutant virus was examined using the PCR-enzyme-linked minisequence assay. The initial viral response (IVR) was defined as HBV DNA < 104 copies/ml at 6 months. Twenty-seven (14%) patients were judged as IVR-positive. Statistical analyses were performed using the log-rank test and the Cox proportional hazards regression.



The cumulative emergence rates of the YMDD mutant virus were 10% at 1 year, 30% at 2 years and 46% at 3 years. In the univariate analysis, there were two pretreatment factors contributing to the emergence of the mutant virus: pretreatment HBV DNA > 106.5 copies/ml (p = 0.0044) and being HBeAg-positive (p = 0.0062). In addition, the IVR was found to be closely correlated to the emergence of the mutant virus (p < 0.0001); the cumulative emergence rates of the mutant virus in IVR-positive and -negative patients were 4% and 41% at 1 year, 25% and 69% at 2 years, and 41% and 79% at 3 years. This contribution of IVR to the emergence of the mutant virus was event in subgroups of patients having pretreatment HBV DNA > 106.5 copies/ml (p < 0.0001), or HBeAg-positive patients (p < 0.0001). In multivariate analysis including nine factors (age, gender, liver disease, pretreatment ALT level, pretreatment HBV DNA, HBeAg positivity, combination therapy with IFN, the presence of acute exacerbation, and IVR) as variables, only IVR was an independent factor affecting the emergence of the YMDD mutant virus (p < 0.0001).



IVR is a useful factor for predicting the emergence of the YMDD mutant virus in CHB patients treated with LAM. For IVR-negative patients, therapeutic options other than LAM monotherapy should be carried out because of high incidence of the emergence of the YMDD mutant virus.


#986. Selection of the A181T/V Substitution in HBV Chronically-Infected Patients Who Developed a Resistance to Lamivudine and/or Adefovir

F. Zoulim; S. Villet; C. Pichoud; C. Trepo



Adefovir (ADV) therapy in HBV chronically infected patients may lead to the selection of a rtN236T or rtA181V mutation in the polymerase gene. Although the rtN236T mutation has been well phenotypically characterized, only a few data are available for the rtA181V mutation. In this study, based on 15 patients who developed a resistance to lamivudine (LAM) or ADV, we analyzed the emergence of the rtA181T/V substitution and characterized the main variants harbouring this mutation in cell culture for their resistance to nucleose analogues.



Genotypic analysis, performed on HBV polymerase isolated from patients’ serum samples, revealed that approximatively half of the patients included in this study developed a resistance with a rtA181T/V substitution when they were treated by a LAM+ADV bitherapy. This rtA181T/V substitution also emerged in 25% and 30% of patients treated by LAM and ADV monotherapies respectively. Moreover, this analysis showed that the rtA181V, like the rtA181T, substitution can emerge with the same ratio under LAM or ADV therapy.


Clonal analysis showed that the rtA181T/V mutation can be associated with other mutations on the same genome to confer a resistance to LAM or ADV. Resistance to ADV was associated in 50% of the patients with the emergence of a rtA181T/V+N236T variant, although this variant was not observed under LAM+ADV bitherapy. Moreover, the rtA181V, in contrast to the rtA181T substitution, was frequently associated with rtL180M and/or rtM204V/I LAM-resistance mutations, when LAM was administrated in mono- or bi-therapy.


Phenotypic analysis of these variants in Huh7 cell line showed that the rtA181T/V substitution induced a slight decrease of susceptibility to LAM (<10 fold) as compared to rtM204V/I, a slight decrease of susceptibility to tenofovir (~2 fold), a 3 to 6 fold resistance to ADV, but remains sensitive to entecavir. The association of the rtA181T/V+N236T mutations increased the level of resistance to all the tested drugs as compared to the rtA181T/V mutation alone, except for entecavir.



The rtA181T/V substitution is associated to a decreased susceptibility to both LAM and ADV. These data emphasize the clinical relevance of genotypic and phenotypic analysis in the management of antiviral drug resistance to tailor antiviral therapy to the virologic situation. This knowledge may help to design new treatment algorithm depending on the profile of mutations in patients failing antiviral therapy.


#992. Hepatitis B Surface Antigen Loss in Antiviral-Treated Patients with HBeAg(+) Chronic Hepatits B (CHB) Infection: Observations from Antiviral-Naïve Patients Treated with Entecavir (ETV) or Lamivudine (LVD)

R. Gish; T. Chang; C. Lai; R. de Man; A. Gadano; F. Poordad; J. Zhu; J. Yang; H. Brett-Smith



Hepatitis B surface antigen (HBsAg) loss and HBsAg seroconversion are considered an important goal of HBV therapy but are seldom achieved with current treatments. We describe baseline characteristics and Week 24 treatment responses of patients who demonstrated confirmed HBsAg loss by Week 120 (on-treatment or during the 24-week follow-up period).



A total of 709 nucleose-naïve, HBeAg(+) patients received ETV 0.5 mg (n=354) or LVD 100 mg (n=355) once daily for a minimum of 52 Wks and maximum of 96 Wks in the randomized, double-blind ETV-022 trial. Entry criteria included: biopsy-confirmed liver disease, serum HBV DNA by bDNA levels 3 MEq/mL, ALT levels 1.3-10 x ULN and no prior nucleose therapy >12 Wks. Serum HBV DNA, HBV serology (HBeAg/anti-HBe) and ALT were measured on treatment and through 24 weeks off-treatment.



HBsAg loss was confirmed in 28/709 (4%) patients (18 for ETV and 10 for LVD) by Wk 120. Baseline characteristics and Week (Wk) 24 treatment responses for patients with confirmed HBsAg loss by Wk 120 are presented below.



Patients with confirmed HBsAg loss by Wk 120 were characterized by genotype A or D, male sex and Caucasian race at baseline and by HBeAg loss and HBeAg seroconversion at Wk 24.


Baseline characteristics

Patients with confirmed HBsAg loss by Wk 120


Overall (N=28)









Mean viral load (log10 copies/ml)


Genotype A


Genotype B


Genotype C


Genotype D


Knodell necroinflammatory score (mean)


Mean and Median ALT (U/L)

226, 163

Week 24 Responses


HBeAg loss


HBeAg seroconversion


ALT normalization (≤1 x ULN)


HBV DNA <300 copies/mL



#994. Combination of Adefovir Dipivoxil with Lamivudine vs. Adefovir Alone in Lamivudine-Resistant HBeAg-Negative Chronic Hepatitis B Patients

S. J. Hadziyannis; I. N. Rapti; E. Dimou; P. Mitsoula



Adefovir dipivoxil (ADV) is effective in most lamivudine resistant (LAM-R) chronic hepatitis B (CHB) patients but ADV resistance develops particularly in HBeAg (+) pts. There are no prospective data on the long-term comparisons of adefovir alone vs. its combination with LAM in HBeAg(-)CHB.



To evaluate prospectively the long-term efficacy of the combination treatment with ADV and LAM vs. LAM alone in LAM-R HBeAg (-) CHB patients.


Patients and Methods:

Total of 46 HBeAg (-) patients, from a single centre, with histologically confirmed CHB resistant to LAM, were randomly allocated in a two arm study of the efficacy and possible development of ADV resistance of ADV+LAM combination therapy vs. ADV monotherapy. At baseline and during treatment HBV-DNA was tested by a R-T PCR assay (sensitivity level<1000 copies/ml), genotyped and sequenced for LAM and ADV resistant mutations.



All patients were Caucasian [median age 55.5 years (range 39-76); M/F: 41/5]. Median duration of LAM before development of resistance was 31 months (range 12-84). The majority (26/46) of the pts had either M204I or M204I/L180M mutation, the rest had M204V/L180M. 18 pts had cirrhosis on liver histology. Median HBV-DNA levels before start of ADV treatment were 1.1E+07 cps/ml (range 15500-6.4E+08). Thirty-two pts were treated with combination of ADV and LAM, and 14 with ADV alone. Median hitherto duration of treatment is 34 months (range 5-53). Virologic response was favorable since median HBV-DNA levels at baseline, 6months, 12months and 24months were: 1.1E+0.7 cps/ml (range: 15500-6.4E+0.8), 1000 cps/ml (1000-13000000), 1000 cps/ml (range: 1000-12000000), 1000 cps/ml (range: 1000-4957000). The percentage of patients with undetectable HBV-DNA ( 1000 cps/ml) at 6, 12 and 24 months of treatment were 55%, 71% and 80% respectively. HBV DNA became undetectable at month 12 and ALT normalized in 80% of pts in both groups and in 100% of those with HBV DNA levels <107 cps/ml at baseline. During year two of the study, 3/14 monotherapy pts (22%) developed resistance to ADV and were successfully switched to the combination scheme. On the contrary, none of the pts under combination treatment of up to 4 years developed ADV resistance.



1) Combination treatment with ADV and LAM seems to be the treatment of choice in HBeAg-negative CHB patients with resistance to lamivudine. 2) More than 80% of patients achieve PCR non-detectable serum HBV DNA levels maintained for >3.5 years 3) Switching from LAM to ADV is associated with development of ADV resistance in the second year in approximately 20% of them.


#997. Entecavir Achieves Superior Virologic Response Compared to Lamivudine for the Treatment of Chronic Hepatitis B: 2-year Results from a Phase 3 Study in Nucleose-naïve Patients in China (ETV-023)

G. Yao; C. Chen; W. Lu; H. Ren; D. Tan; Y. Wang; D. Xu; J. Liu; D. Xu; L. MacDonald



Entecavir (ETV) demonstrated superior virologic and biochemical benefit compared to lamivudine (LVD) after 48 weeks of therapy in nucleose-naïve chronic hepatitis B (CHB) patients in China.



Patients with compensated CHB who were either HBeAg+ or HBeAg-/HBeAb+ were randomized to receive ETV 0.5 mg (n=258) or LVD 100 mg (n=261) QD for at least 52 weeks. At Wk 52, patients who at Wk 48 had (a) a consolated response (HBV DNA <0.7 MEq/mL by bDNA assay and HBeAg-negative both for at least six months and ALT <1.25 x ULN) stopped therapy, (b) HBV DNA <0.7 MEq/mL but had not yet achieved a consolated response (CR) continued blinded therapy, (c) HBV DNA 0.7 MEq/mL stopped therapy.



Cumulative confirmed responses through 2 years for all treated patients are shown in the table. At the end of dosing (EOD) through Year 2, the mean change in HBV DNA from baseline was significantly greater for ETV than LVD (–5.89 log10 copies/mL for ETV and –3.55 log10 copies/mL for LVD; p<0.0001). The total proportions of ETV- and LVD-treated patients achieving a CR at Wk 48 or EOD in Year 2 was 30% and 24%, respectively. The response was sustained at 24 weeks of off-treatment follow-up for 70% of ETV patients and 48% of LVD patients. Among patients who continued to the second year of therapy (ETV n=193, LVD n=145), 74% of ETV- and 41% LVD-treated patients had HBV DNA levels <300 copies/mL at the EOD; and 96% of ETV- and 82% LVD-treated patients had ALT levels 1 x ULN at the EOD. The proportion of patients with any adverse event on-treatment was 64% for ETV and 60% for LVD. ALT flares were observed in low proportions of patients in both treatment groups on-treatment (ETV 4%, LVD 7%).



This study supports the use of ETV 0.5 mg once daily as first line therapy for the treatment of CHB in nucleose-naïve patients. Treatment with ETV for up to 2 years proved significant clinical benefit compared to LVD. ETV was well-tolerated with a safety profile comparable to that of LVD. Cumulative confirmed end points through Year 2:




Difference estimate between treatments

(95% CI)


ETV 0.5 mg

LVD 100 mg

HBV DNA <300 copies/mL by PCR assay



33.5(25.9, 41.1)


ALT ≤1.0 x ULN



3.9(–0.1, 7.9)


Loss of HBeAg



0.4(–7.8, 8.6)



#998. Viral Breakthrough after Emergence of YMDD Mutant Hepatitis B Virus Always Goes to Clinical Breakthrough: Is It Correct?

D. Kim, 3; S. Ahn; H. Lee; J. Kim; C. Lee; Y. Paik; K. Lee; K. Han; C. Chon; Y. Moon



A proportion of chronic hepatitis B (CHB) patients treated with lamivudine maintain relatively low levels of serum alanine aminotransferase (ALT) in spite of the emergence of YMDD mutation and re-appearance of serum HBV DNA. Still is in question whether additional antiviral agent against YMDD mutants is mandatory in these patients. The natural history and clinical characteristics from viral breakthrough (VB) to clinical breakthrough (CB) in CHB patients with YMDD mutants were sequentially investigated.



A total of 93 HBeAg-positive CHB patients treated with lamivudine (100 mg/day) have developed YMDD mutants. Measurement of quantitative HBV DNA, HBeAg, anti-HBe, and biochemistry were performed every 3 months with maintenance of lamivudine therapy. CB was defined by elevation of ALT ( 2 times upper limit of normal, ULN) plus VB, that is, the re-appearance of HBV DNA in serum ( 105 copies/mL). Enrolled patients were dived into two groups according to the time sequence of two breakthroughs; group I (simultaneous VB and CB, n=25), group II (VB followed by CB, n=68)



The median age of the patients was 47 years with 76 males and 17 females. The median duration of lamivudine therapy was 39 months (12-60), and the median value of pre-lamivudine ALT and HBV DNA was 165 IU/L and 1.2 X 108 copies/mL, respectively. The rate of HBV DNA loss and ALT normalization was 72.4%, 95.2% and 48.9%, 75.0% at 3, 6 months of lamivudine therapy, respectively. Interestingly, CB occurred at the same time or with some time interval in all the 93 patients with VB. There was no difference in age, gender, ALT and HBV DNA level at the time of baseline and CB between two groups. The median time of HBV DNA loss and VB in group I was 3 and 18 months, respectively. Similar results were obtained in group II. The median time from VB to CB was 8 months (3-36) in group II. Early occurrence of CB (less than 3 months after VB) in group II was strongly associated with acute flare-up of ALT level ( 10 times ULN).



Our data suggest that viral breakthrough with YMDD mutant HBV subsequently goes to clinical breakthrough, sometimes flare-up, with time interval. Early rescue therapy against YMDD mutants should be considered at the time of viral breakthrough with or without elevation of ALT level.


#999. Pre-existing or RapEmergence of A181T Polymerase Mutation in Lamivudine-Resistant Chronic Hepatitis B Patients Treated with Adefovir

Y. Kim; J. Yeon; W. Han; S. Oh; Y. Seo; H. Yim; W. Choe; S. Kwon; K. Byun; C. Lee



The known HBV polymerase mutations that confer to resistance to adefovir (ADV) were rtN236T and rtA181V. The rtA181T mutation, which was detected during lamivudine (LMV) treatment, is also reported to increase resistance to ADV by 4 fold in vitro. The aim of our study was to define the clinical significance of rtA181T mutation in ADV treated LMV-resistant chronic hepatitis B patients (LMV-R CHB).



Two hundred and four LMV-R CHB treated with ADV were included; the mean treatment duration was 18 months (range 6 months-39 months). All patients enrolled in our study were treated with ADV monotherapy with (n=83) or without (n=121) an initial short course of LMV combination. Serum HBV DNA was quantified by real time PCR. Biochemical and virologic parameters were measured every 2~3 months. Genotypic mutation to ADV was detected using mass spectrometry-based genotyping assay (RFMP). Antiviral efficacy to ADV, virologic breakthrough (VBT) and ALT flare were compared between patients carrying A181T and N236T/A181V.



During the course of ADV therapy, rtA181T, rtA181V, and rtN236T mutations were detected in 40 (20%), 24 (12%), and 20 (10%) patients, respectively. At 12 months of treatment, rtA181V was detected in 4 of 24 patients (17%), and rtN236T was detected in 3 of 20 patients (15%). On the contrary, 31 of 40 patients (77%) with rtA181T mutation were detected within 12 months of treatment. At the initiation of ADV treatment, 6 patients had A181T mutation. There were no significant differences in the detection rates of genotypic mutation in patients treated with or without an initial short course of LMV treatment. No significant differences were found in the rate of VBT (14% vs 15%) and ALT flare (0% vs 9%) in patients with A181T and with N236T/A181V.



A high prevalence of rtA181T mutation was detected in LMV-R CHB treated with ADV. The rtA181T mutation was detected at the initiation of ADV treatment or during the early course of ADV treatment. The clinical consequences of patients with rtA181T mutation were not significantly different from those with rtN236T mutation or rtA181V mutation. Further studies are needed to define the role of rtA181T mutation in prolonged ADV therapy.


#1002. Virologic Response Can Be Durable in HBeAg Positive Patients with Chronic Hepatitis B after Lamivudine Monotherapy during Long-term Follow-up?

H. Lee; K. Han; S. Myoung; Y. Chung; J. Park; J. Lee; J. Kim; S. Ahn; Y. Paik; K. Lee; C. Chon; Y. Moon



Many short-term observations showed that lamivudine induced HBeAg responses are durable for months after successful therapy. However, the question remains whether such HBeAg responses can be maintained over extended follow-up period. The aim of our study was firstly to evaluate posttreatment durability for up to 5 years of virologic responses to lamivudine monotherapy in patients who experienced HBeAg loss. Secondly, it was to investigate the long-term clinical outcome of lamivudine monotherapy in 297 Korean patients with HBeAg positive chronic hepatitis B.



A total of 297 patients (234 men and 63 women) with HBeAg positive chronic hepatitis B were treated with lamivudine monotherapy and followed up for more than 24 months (range, 24-100) between January 1998 and December 2003. Mean age was 43 years (range, 21-65). The mean of baseline serum HBV-DNA level was 3.8 ± 7.5 x 100,000,000 copies/ml. The mean of ALT level was 236.8 IU/L. The mean duration of lamivudine monotherapy was 36 months (range, 12-96). The definition of complete response (CR) was serum HBeAg loss, undetectable HBV-DNA, and normal ALT. Additional lamivudine treatment was continued at least more than 6 month after CR.



The cumulative HBeAg loss rates at 1, 2, 3, 4 and 5 years were 37.7%, 50.6%, 62.9%, 68.2% and 74.1%, respectively. The mean time to HBeAg loss after lamivudine therapy was 15 months (range, 3-48). The cumulative virological breakthrough rates at 1, 2, 3, 4 and 5 years were 15.2%, 31.8%, 43.5%, 47% and 49.6%, respectively. The mean time to breakthrough after lamivudine therapy was 19 months (range, 4-54). In multivariate analysis, age was the only independent predictive factor for CR (p = 0.009). The rate of CR was significantly higher in younger patients under 40 years old (60.0% vs 40.6%, p = 0.001). Among 110 patients, who discontinued lamivudine monotherapy after CR, 70 patients (63.6%) maintained sustained virological response. The cumulative durability of CR at 1, 2, 3, 4, and 5 years were 77.8%, 71.8%, 69.8%, 66.6 %, and 63.6%, respectively. The mean time to relapse after cessation of lamivudine was 9 months (range, 2-41). Most relapses were occurred within 2 years after discontinuation of lamivudine (31/40, 77.5%). No benefit according to duration of additional lamivudine treatment after HBeAg loss was observed in our study (p = 0.082).



Although one-third of complete responders relapsed within two years, two thirds of patients maintained sustained virological response during long-term follow-up (>24 months). Also, our results showed significantly higher CR rates in younger patients under 40 years old.


#1004. Hepatitis B Virus Core-related Antigen as an Indicator of Safe Discontinuation of Lamivudine Therapy

M. Fukuda; H. Yatsuhashi; R. Hamada; R. Nakao; N. Hai; M. Miyazato; E. Ozawa; T. Kamihira; S. Nagaoka; N. Taura; K. Ohata; S. Abiru; K. Yano; A. Komori; M. Daikoku; M. Nakamura; K. Migita; H. Fujioka; H. Ishibashi



The short term lamivudine (LMV) application leads to post treatment flares (PTFs) while the long term application of the agent results in a high rate emergence of resistant strains (YMDD mutant). Hepatitis B virus core-related antigen (HBVcrAg) is a new marker of hepatitis B virus (HBV) infection and consists of HBV core and e-antigens (T. Kimura, et al., J Clin Microbiol, 2002).



The aims of this study were to examine clinical usefulness of the HBVcrAg assay during lamivudine (LMV) therapy and to clarify whether it can be a predicator of PTFs after stopping LMV therapy.


Methods and Patients:

HBVcrAg and HBV-DNA levels were measured in untreated 101 patients. Subsequently, 25 patients (19 HBeAg-positive and 6 HBeAg-negative) who initiated LMV therapy in our hospital during 2000 and 2004 were enrolled to this study. The mean observation period was 4.4 years (3.0-5.5 years). Eight patients got YMDD mutation (=group A) while 17 patients dnot during these 4 years (=group B). Samples at pretreatment, 6 months, 1 year, 2 years, 3 years, and 4 years under LMV therapy were collected for assay.



Serum HBVcrAg concentrations were well correlated with HBV-DNA levels in our untreated 101 patients infected with HBV (r=0.8855, P<0.001). The median of HBV-DNA levels were 7.0, 2.9, 3.1, 6.5, 7.6, and 7.6 log copies/ml and 7.2, 2.6, 2.6, 2.6, 2.6, and 2.6 log copies/ml in group A and B, respectively (P<0.001). The median of the HBVcrAg levels is 6.7, 5.6, 5.5, 6.6, 7.1, and 7.3 log U/ml and 7.3, 5.1, 4.6, 4.1, 4.0, and 3.6 log U/ml in group A and B, respectively (P<0.001). In the patients without LMV resistance during observations, levels of both HBVcrAg and HBV-DNA reduced constantly after the initiation of LMV. The reduction rate of HBVcrAg levels, however, was significantly lower than that of HBV-DNA levels. (-3 log / 4 years vs -4 log / 6 months). All patients who showed low levels of HBVcrAg (less than 3.0 log U/ml) at the cessation of LMV dnot experience PTFs. In contrast, patients with LMV resistance or PTFs remained high level of HBVcrAg (no less than 4.7 logU/ml) during observations.



This study indicates that a sufficiently long term LMV administration (may be more than 4-5 years) is needed to establish the HBVcrAg level of less than 3.0 log U/ml, which may be the critical level to avoPTFs. Several Japanese studies indicated that lower serum HBVcrAg levels may reflect lower levels of cccDNA in hepatocytes, because the mRNAs of HBVcrAg are transcribed from the cccDNA. In patients who show declined levels of HBVcrAg, cessation of LMV treatment can be considered in view of low risk of PTFs.


#1005. A Randomized Trial of Telbivudine (LdT) vs. Adefovir for HBeAg-Positive Chronic Hepatitis B: Final Week 52 Results

N. Bzowe; H. Chan; C. Lai; M. Cho; Y. Moon; Y. Chao; E. J. Heathcote; R. Myers; G. Minuk; P. Marcellin; L. Jeffers;  Sievert; R. Kaiser; G. Harb; G. C. Chao; N. A. Brown; S. The 018



Telbivudine (LdT) demonstrated greater antiviral efficacy vs lamivudine in Phase II and III trials. Adefovir dipivoxil (ADV) was superior to placebo in Phase III but has not been evaluated in active-comparator trials. Here we report final results at week 52 (W52) of a randomized trial of LdT vs ADV in adults with HBeAg-positive chronic hepatitis B (CHB), including the primary antiviral efficacy analysis at week 24 and the results of a randomized switch from ADV to LdT.



The study enrolled 135 adults with HBeAg-positive compensated CHB with HBV DNA >6 log10 copies/mL by COBAS Amplicor PCR assay (LLOQ = 300 copies/mL), serum ALT 1.3-10 x ULN. Patients were initially randomized (2:1) to ADV 10 mg/d or LdT 600 mg/d for 24 weeks, with a secondary randomization (1:1) of ADV recipients to either continue ADV or switch to LdT after Week 24. The primary endpoint is HBV DNA reduction at Week 24, with secondary efficacy and safety endpoints assessed at Week 24 and Week 52.



Treatment groups were matched at baseline. At Week 24 mean HBV DNA reduction from baseline was significantly greater with LdT vs ADV (-6.30 vs. -4.97 log10 copies/mL; p<0.01). Compared with 52 wks of ADV treatment, patients treated with or switched to LdT (1) achieved greater HBV DNA reductions; (2) were significantly more likely to achieve HBV <5log10 (91% and 87% vs 66%, p<0.05); and (3) achieved proportionally greater PCR-negativity (58% and 54%, vs 39%) and HBeAg loss (31% and 26%, vs 21%). In the ADV/LdT switch group, mean viral load decreased rapidly following Week 24; by Week 40 the mean viral load was similar to the group receiving LdT from baseline (Figure). All regimens were well-tolerated. Resistance is being analyzed and will be presented.



At 24 wks, LdT produced significantly greater and more consistent antiviral efficacy than ADV. At one year, patients treated continuously with LdT, or switched from ADV to LdT, showed proportionally better results on all measures of antiviral efficacy compared to continuous ADV.



#1007. A Randomized Trial of Telbivudine (LdT) versus Lamivudine in Lamivudine Experienced Patients – Week 24 Primary Analysis

E. Gane; R. Safadi; Q. Xie; Y. Chen; Y. Yin; L. Wei; S. Hwang; E. Zuckerman; J. Jia; M. Temam; X. Qiao; G. Harb; N. A. Brown



There is growing recognition that maximizing viral suppression is an important therapeutic objective for treatment of chronic hepatitis B. Telbivudine (LdT) achieved significantly greater viral suppression vs. lamivudine in phase II and III trials. This current study investigates whether patients (pts) receiving lamivudine therapy would benefit from conversion to telbivudine.



Eligible patients previously received lamivudine for 3-12 mos at the time of screening, and were HBsAg-positive, HBeAg-positive or -negative, with HBV DNA >3 log10 copies/mL and compensated liver disease. Pts were randomized (1:1) to either continue lamivudine (100mg/d) or switch to telbivudine (600mg/d) for 1 yr. Patients were stratified according to HBeAg status and duration of prior lamivudine treatment (12-24 weeks or 25-52 weeks). The primary efficacy endpoint is HBV DNA reduction at Week 24.



Median HBV DNA at Baseline was 5.04 and 5.27 log10 copies/mL for the telbivudine (n=121) and lamivudine (n=124) arms respectively. At Week 24, patients switched to telbivudine showed significantly greater reduction of serum HBV DNA compared to patients who continued lamivudine (median -1.66 log10 copies/mL vs. -0.95 log10 copies/mL respectively, p<0.01). HBV DNA was suppressed to below 5 log10 copies/mL in 80% of patients switched to telbivudine vs. 56% in the lamivudine group (p<0.001), and became non-detectable by PCR in 41% vs 31% respectively (p=0.14). Observations were similar in all four randomization strata. In patients who were HBeAg positive at study entry, 9% and 7% lost HBeAg by Week 24 in the telbivudine and lamivudine groups respectively (p=0.77). Both treatments were well tolerated.



In patients with persistent viraemia during lamivudine therapy, switching to telbivudine is associated with significantly improved HBV suppression. Longer follow-up will determine whether this results in continued clinical benefits.



#1008. Virologic Response at Treatment Months 12 May Predict Sustained Antiviral Efficacy in Adefovir-Treated Lamivudine-Resistant Chronic Hepatitis B

W. Han; J. Yeon; Y. Kim; S. Oh; Y. Seo; H. Yim; W. Choe; S. Kwon; K. Byun; C. Lee



In lamivudine-treated chronic hepatitis B patients (CHB), maximal HBV DNA reduction early in treatment has been associated with better clinical outcome and lower resistance rate. Aim of our study is to define the potential role of virologic response at treatment 12 month in predicting subsequent virologic and clinical outcome in adefovir (ADV) treated LMV-resistant CHB (LAM-R).



A total of 204 LMV-R CHB treated with ADV were included; mean treatment duration was 18 months (range 6-39). Baseline HBV DNA represented as log10 copies/ml was 7.4. Serum HBV DNA was quantified by real time PCR. Biochemical and virologic parameters were measured every 2~3 months. Virologic response at 12 month (VR12) was defined as HBV DNA less than 4 log after 12 months. Virologic breakthrough (VBT) was defined as HBV DNA elevation more than 1 log10 from nadir. Genotypic mutation to ADV was detected using mass spectrometry-based genotyping assay (RFMP).



VR12 occurred in 110 of 204 patients (54%). In comparison with patients between presence and absence of VR12, mean HBV DNA reduction from baseline at treatment month 12 (Tx.12) and 24 (Tx.24) were 3.8 vs 1.9, and 3.5 vs 2.4 respectively (p=0.001). HBeAg seroconversion rate at Tx.12 and Tx.24 was 32 vs 14% and 40 vs 27% in each group respectively (p<0.05). The genotypic mutation to ADV at Tx.12 and Tx.24 was 0 vs 6.0% and 21 vs 42% respectively (p=0.014). VBT at Tx.12, Tx.24 was 0 vs 7.0% and 9.0 vs 25% (p=0.002). Factors associated with VR12 were baseline HBV DNA level, pretreatment ALT, presence or absence of HBeAg.



In treating LMV-R CHB with ADV, 50% of patients were failed to achieve VR12. Absence of VR12 is likely to have less potent viral suppression, lower HBeAg seroconversion and frequent genotypic mutation. Add on or switch to other potent antivirals may need in patients failed to have VR12.


#1011. Dynamics of Hepatitis B Virus Resistance to Adefovir Dipivoxil Unraveled by a Thorough Quasispecies Analysis

J. Pawlotsky; C. Pallier; C. Rodriguez; R. Brillet


Adefovir dipivoxil administration significantly reduces HBV replication and improves liver disease in patients with chronic hepatitis B. However, adefovir selects for resistant HBV variants in up to 29% of cases after 5 years, and this is associated with virological and biochemical rebound in 20% and 11% of cases, respectively.



In order to understand the molecular mechanisms underlying adefovir resistance, we extensively characterized the dynamics of HBV quasispecies variant populations in HBV-infected patients who developed such resistance. METHODS: 26 to 39 clones per time point were generated for each patient (patient A: 405 clones at 14 time points; patient B: 338 clones at 12 time points; patient C: 422 clones at 14 time points). For each clone, the full-length HBV rt sequence was determined. Amino acid sequences were aligned and the dynamics of quasi-species variants were analyzed.



(i)                Patient A: The virological breakthrough occurred at month 26 and the A181V mutation was detected simultaneously by direct sequence analysis. Quasispecies analysis showed the emergence of A181V variants as minor variants as early as 6 months before the virological breakthrough and their progressive growth, together with the emergence of minor variants bearing the N236T substitution at month 23 that subsequently disappeared. No additional position showed selection of a specific amino ac.

(ii)              Patient B: The virological breakthrough occurred at month 34 and N236N/T mixtures were detected simultaneously by direct sequence analysis. Quasispecies analysis showed the simultaneous emergence of variants bearing A181V and N236T substitutions as minor viral populations. They progressively grew to become predominant at month 37 and thereafter. (iii)    Patient C: This patient received adefovir until month 10, stopped therapy for 3 months and restarted it until month 41. The virological breakthrough occurred at month 27. A mixture of A181V and N236N/T was detected 5 months later only by direct sequence analysis. Quasispecies analysis showed no change during the first course of therapy. N236T variants emerged as minor populations at month 21 and A181V variants at months 27. Both populations progressively grew but the A181V population took over after month 32.



Adefovir dipivoxil selects variants bearing mutations at both positions 181 and 236 of the HBV rt. Their emergence precedes the virological breakthrough but their growth is very low, suggesting poor in vivo fitness. Subsequent changes may appear, driven by replicative advantages conferred by additional mutations and the changing environment in which these variants replicate.


# 1012. Sequential Lamivudine and Adefovir Therapy Maintains Seroconversion Rates In Patients With HBeAg+ Chronic Hepatitis B

S. Lim; M. Aung; B. Mak; D. Sutedja; Y. Lee; Y. Dan; D. Wai; E. Koay


Even with the advent of more nucleose analogues, the optimal strategy for treatment of chronic hepatitis B is still being explored. Studies have focused on the efficacy of indivual nucleoses or combinations, but little data is available on the strategy of sequential therapy. We examined the seroconversion rate, viral breakthrough and renal impairment in all patients with HBeAg+ chronic hepatitis B treated at a single centre.



Data was collected prospectively on all chronic hepatitis B patients prescribed nucleose analogues lamivudine (LAM) or LAM followed by adefovir (ADV) rescue in this cohort study. Only patients who were HBeAg+ were selected for this analysis. Patients receiving nucleoses had Hepatitis serology, LFTS, and HBV DNA and serum creatinine (only ADV) performed at baseline and 3 monthly. Nucleoses were discontinued 6 months after seroconversion in all patients. Viral breakthrough occurred was defined as >1log rise in HBV DNA. HBV DNA was tested using RT-PCR. Kaplan Meier analysis was performed and differences determined by log rank test. Patients with sero-reversion of HBeAg were excluded from the seroconversion analysis.



A total of 166 patients were followed for a mean of 28.15 months. 42 patients seroconverted during this time but 19% of patients sero-reverted leaving the durability of seroconversion as 81%. The overall seroconversion rate was 31.48% based on KM analysis in the entire cohort based on first line therapy with LAM and ADV rescue upon development of breakthrough. When seroconversion KM curves of LAM and ADV were analysed, no difference was found (p=0.299), indicating that seroconversion rates were similar. LAM breakthrough occurred in 80% of patients over 42 months. ADV breakthough occurred in 29.4% of pts over 21 months. An increased creatinine above normal was seen in 16.9% of patients, which was similar in patients with and without cirrhosis.



Sequential therapy for HBeAg+ CHB appears to be a suitable option as seroconversion rates are maintained. However with a switch over to ADV, 16.9 % of patients developed renal impairment and 29.4% developed viral breakthrough.

#1013. Selection of Multidrug-Resistant HBV: Clonal Analysis To Investigate the Complex Evolution of Multiple HBV Mutations

A. Bartholomeusz; A. Ayres; M. Littlejohn; D. Colledge; L. Yuen; P. Angus; S. Locarnini



Sequential monotherapy, involving both rescue/ add-on combination therapies and/or switching approaches has resulted in the selection of multrug resistant HBV. The aim of this study was to perform clonal analysis on HBV genomes from patients failing combination therapy in order to study the pattern of emergence of HBV mutations in the polymerase gene, define the complexity of the HBV quasispecies and to gain further insights into the potential co-selection bias of HBV mutations detected during breakthrough on combination treatment.



The HBV polymerase gene was amplified by PCR and sequenced from three patients who failed the combination therapy of adefovir (ADV) and lamivudine (LMV). HBV DNA was isolated from the patients’ samples taken at multiple time points, cloned, and up to 30 individual clones sequenced. A software program that detects specific HBV mutations (SeqHepB) linked to a relational database was used to analyze therapy-associated mutations.


Results and Discussion:

Direct sequencing and clonal analysis of HBV genomes isolated from the serum samples of the three patients revealed complex mutation profiles. During virological breakthrough on combination therapy, Patient A selected HBV encoding new antiviral resistance mutations rtM250L +rtI233V in the context of pre-existing ADV resistance (rN236T +rtA181T). In response to combination treatment, Patients B and C selected HBV encoding both primary LMV and ADV resistance mutations. Clonal analysis of HBV from these two patients revealed that two different populations were present and the primary resistance mutations were detected on separate genomes. One population consisted of HBV encoding the LMV resistant mutations at rtL180M+ rtM204V/I and the second population consisted of HBV encoding rN236T +/- rtA181V. No clones encoding both the rtN236T and rtM204I/V were detected on the same genome. In vitro analysis of the replication yield phenotype for HBV encoding dual ADV and LMV resistance revealed low replication capacity.



The complex HBV mutation profile detected in patients on combination therapy demonstrates the need for careful monitoring of patients. Clonal analysis proves an adjunct to in vitro phenotypic studies in order to understand complex mutation profiles. Clonal analysis during different treatment regimens may aid in our understanding of the evolution of different mutation profiles, and prove an insight into assessing therapeutic options for patients failing combination therapies.


#1016. Is Tenofovir Effective in Treatment of Adefovir-Resistant Hepatitis B Virus (HBV) Infections?

F. van Bömmel; B. Zöllner; B. Möller; D. Hüppe; H. Feucht; B. Wiedenmann; T. Berg


Development of HBV resistance against adefovir (ADV) is rare during the first two years of treatment. With increasing treatment duration ADV-resistant HBV mutants are selected (rtN236T; 28% after 5 years) and associated with the risk of a viral breakthrough. In vitro, these mutations cause a 3-14-fold decrease of ADV efficacy. Tenofovir (TDF), a substance closely related to ADV possesses equipotent antiviral activity against HBV on molar basis and is cross resistant to ADV in vitro. However, due to the 25-fold higher dosage of TDF as compared to ADV, TDF might still be effective in ADV resistance.



The efficacy of TDF was evaluated in 3 HBeAg-positive patients (no HIV coinfection) with initial lamivudine resistance and consecutive development of ADV resistance after 22-24 months of ADV monotherapy. When ADV (10 mg/d) was switched to TDF (300 mg/d), the levels of HBV DNA were 7.9, 9.4 and 8 log10 copies/mL (HBV Monitor, Roche) and all patients had elevated ALT levels. After start of TDF treatment, HBV DNA levels were measured 2-monthly. Additionally, during the course of treatment the HBV polymerase gene was sequenced from codon rt88 to rt282 after PCR products were cloned in E. coli (TOPO-TA cloning system, Invitrogen; 12 to 20 clones were sequenced each).



Within the observation period of 12 months, in all 3 patients (HBV genotype A, D und D) a decrease of HBV DNA of -3.8, -5.4, -3.5 log10 copies/mL was demonstrated, but none of the three patients became HBV DNA negative. HBV DNA levels at month 12 of TDF treatment were 4.2, 4.1 and 4.4 log10 copies/mL. The percentage of clones bearing the rtN236T variants at baseline was 50% in patient 1, 30% in patient 2, and 100% in patient 3. However, at the end of observation, the percentage of the rtN236T variants reached 100% in all three patients. The mutation rtA181T was present in 2 patients from the beginning and was also selected by month 7 in the remaining patient. ALT levels normalized only in one patient. Combination therapy with TDF and lamivudine was started in one patient at month 12 (HBV DNA 4.5 log10 copies/mL, ALT 47 IU/mL), which after 2 months lead to undetectable HBV DNA levels and normal ALT values.



Although TDF has significant antiviral efficacy in patients with ADV resistance, it neither could induce complete suppression of HBV DNA in any of the three patients studied nor prevent the further selection of ADV associated resistance mutations. Therefore, a combination of TDF plus a nucleose analogue (i.e. lamivudine) seems to be a more promising therapeutic approach to address this clinical problem.


#1017. Amino AcVariability of the Overlapping Reverse Transcriptase Region of Hepatitis B Surface Antigen in Chronic HBV Carriers with Concomitant Anti-HBs Antibodies in Serum

P. Colson; P. Borentain; A. Motte; V. Moal; M. Henry; D. Botta; C. Tamalet; R. Gérolami


Background and Aims:

Mutations in the HBV reverse transcriptase (rt) have been described to occur as a consequence of vaccine or HBIg induced amino ac(aa) changes in the overlapping HBsAg. Some have been shown to modulate HBV replication. We previously described increased variability and particular mutations patterns in the S protein of HBV from chronic HBsAg carriers with concomitant anti-HBs antibodies in serum. In the present study, we aimed to analyse the aa variability within the HBV rt of these HBV strains.



Among 459 HBsAg chronic carriers followed-up in Marseilles public hospitals from 2003/11 to 2005/04, 14 (3.1%) concomitantly harbored anti-HBs. HBV rt aa sequences from these 14 pts were compared with those from 51 newly diagnosed HBV chronically-infected patients (pts) without anti-HBs (control group). HBV rt gene was amplified/directly sequenced with in-house protocols (ABI 3100). Analysis of variability within rt areas overlapping the different domains of HBsAg was performed.



HBV rt sequences were obtained from 12/14 (86%) HBsAg+/HBsAb+ pts (vs 41% in controls; p=0.004). Non-A genotypes were significantly more frequent vs controls (91% vs 51%, p=0.043). Rt regions overlapping the C terminal region of HBsAg and domain 3 of its major hydrophilic region (MHR) were the most variable. The mean proportion of mutated sequences per aa position and the number of mutations for rt regions overlapping the C terminal domain of HBsAg and its “a” determinant were 5.6 and 1.9-times higher than for the control group, respectively (3.9% vs 0.7%; 6.6% vs 3.4%). Aa rt16I/T, rt134D/E, rt135T/Y/N, and rt138N/R/K were significantly more frequent in HBV from pts with concomitant HBsAg and anti-HBs antibodies than from controls (p<0.05); 3 of them overlap the “a” determinant. Two HBsAg+/HBsAb+ pts were receiving LMV, 1 LMV+ADF, and 1 TDF, but only 1 pt on LMV harbored drug-resistant HBV (180M/204V). These latter strains were also the only one without mutation within the “a” determinant. Mutations rtT128N and rtW153Q (corresponding to sP120T and sG145R) previously found to partially restore the in vitro replication of LMV-resistant HBV were found in HBV from one and three HBsAg+/HBsAb+ pts (including one on LMV), respectively; rtW153K was found in another pt.



HBsAg variability observed in chronic HBV carriers with concomitant anti-HBs was found to have significant consequences on the aa composition of the HBV polymerase. Whether or not these HBV rt mutations might alter its structure and functional activity either in the absence or in the presence of drug-resistance mutations warrant further studies.

#lB5. Adefovir and Lamivudine Combination Therapy Is Superior to Adefovir Monotherapy for Lamivudine-resistant Patients with HBeAg-Negative Chronic Hepatitis B

P. Lampertico; A. Marzano; M. Levrero; T. Santantonio; V. Di Marco; M. Brunetto; P. Andreone; E. Sagnelli; S. Fagiuoli; G. Mazzella; G. Raimondo; G. Gaeta; A. Ascione


To assess whether adefovir dipivoxil (ADV) should be “switched” or “added” to lamivudine in lamivudine resistant (LAM-R) patients, we compared the long-term virological response and adefovir resistance (ADV-R) rates of LAM-R, HBeAg-negative patients under ADV+LAM combination or ADV monotherapy.


Material and Methods:

A total of 588 LAM-R patients with HBeAg-negative chronic hepatitis B who started ADV treatment between 2002 and 2004 in 31 Italian centers were enrolled in a prospective cohort study and followed for 24 months, on average. Mean age was 54 years, 85% were men, 49% cirrhotics; 303 (52%) patients switched from LAM to ADV (ADV mono group) while 285 (48%) added ADV to LAM (combo group). HBV-DNA was quantified by sensitive assays (LLQ: 2 o 3 log copies/mL); a virological response was an undetectable HBV-DNA, a virological rebound was a confirmed > 1 log increase of HBV-DNA; ADV-R was confirmed by molecular analysis.



Baseline demographic, clinical and virological characteristics as well as duration of follow-up were similar between treatment groups. The 6 and 12-month rates of HBV DNA undetectability were 47% and 66% in the ADV mono group and 49% and 64% in the combo group, respectively (p=ns). After a median follow-up of 24 months, 67% of the patients in the former group cleared HBV DNA compared to 69% of those in the latter one (p=ns). Residual viremia was similar in incomplete responders from both groups (4.8 vs 4.9 log copies/ml of HBV DNA). By converse, the rates of virological breakthrough were significantly higher in the ADV mono than in the combo group (9% vs 2%, p<0.001) and ADV-related signature mutations were identified more frequently in the former than in the latter group (5% vs 0.8%, p<0.01). At multivariate analysis, patients treated with ADV monotherapy had higher chances of experiencing a virological rebound (p<0.001), indicating that although ADV-LAM combination therapy does not suppress HBV replication more rapidly than ADV monotherapy, it significantly reduces the risk of virological breakthrough and genotypic resistance to ADV.



In HBeAg-negative LAM-R patients, ADV should be added to LAM and both drugs should be continued to reduce the risk of ADV-related secondary treatment failure.


#D-91  Two-Year Results from the GLOBE Trial in Patients with Hepatitis B: Greater Clinical and Antiviral Efficacy for Telbivudine (LdT) vs. Lamivudine

C. Lai; E. Gane; C. Hsu; S. Thongsawat; Y. Wang; Y. Chen; E. J. Heathcote; Rasenack; N. Bzowej; N. Naoumov; S. Zeuzem; A. Di Bisceglie; G. C. Chao; B. A. Fielman Constance; N. A. Brown; S. Globe



First-year results from the GLOBE trial, reported in 2005, indicated superior efficacy for telbivudine vs. lamivudine on all measures of direct antiviral efficacy and on several key clinical efficacy measures. Here we report the 2-year results from this large trial.



The GLOBE study is a randomized, blinded Phase III trial comparing telbivudine (600 mg/d PO) vs standard lamivudine (Lam) treatment (100 mg/d PO) for 2 years, in an intent-to-treat population of 1,367 patients (pts) with chronic hepatitis B recruited from 20 countries. Key entry criteria were HBsAg+, HBV DNA >6 log10 copies/mL by COBAS Amplicor PCR assay, ALT 1.3-10 xULN, and compensated liver disease. Patients were pre-stratified for HBeAg status (+/-) and ALT < or > 2.5 xULN. Follow-up liver biopsies were performed at 1 year but were not repeated at 2 years.



Treatment groups were well-matched at Baseline. Efficacy data (ITT) at Week 104 are shown below. At 2 years, LdT was superior to Lam for the primary efficacy measure (Therapeutic Response; HBV DNA <5 log10 with HBeAg loss or ALT normalization) and for all direct measures of antiviral efficacy in both HBeAg+ and HBeAg- pts. HBeAg loss was significantly better for LdT for the subgroup of pts recommended for treatment by AASLD and Asia-Pacific guidelines, i.e. baseline ALT ≥ 2xULN. ALT normalization was proportionally greater for LdT in both HBeAg+ and HBeAg- pts (p=0.08). Treatment failure was significantly more common with Lam in both groups. Ongoing analyses of genotypic resistance will be available at the meeting. Both study drugs were generally well-tolerated, with similar patterns of clinical adverse events.



During 2 years of treatment, telbivudine produced significantly greater antiviral efficacy than lamivudine and was associated with greater and better-maintained clinical efficacy, in HBeAg+ and HBeAg- patients with chronic hepatitis B.



HBeAg Positive

HBeAg Negative






n †





Mean log10 HBV DNA ↓





% PCR negative





% ALT normalization





% Therapeutic Response





% HBeAg loss





% HBeAg loss (baseline ALT ≥2 xULN)





% HBeAg seroconversion





% Primary Treatment Failure‡





*p ≤ 0.05, LdT vs. Lam Analysis includes entire ITT population that initiated trial HBV DNA never < 5 log10 copies/mL (AASLD guidelines)

#94. Inhibitory Activity of the 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]-pyrimidine (PMEO) Against Wild Type and Drug-Resistant Mutants of HBV

F. Zoulim; M. Brunelle; J. Lucifora; S. Villet; J. Neyts; C. Trepo



Successive anti-human hepatitis B virus (HBV) therapy with nucleoside analogs leads to the emergence of resistant HBV strains harboring complex pattern of mutations within HBV polymerase gene. The development of new HBV inhibitors is thus needed. The 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]-pyrimidine (PMEO), a novel acyclic pyrimidine nucleoside phosphonate analog, was shown to have an anti-HBV effect comparable to adefovir (ADV) in hepatoma cells expressing permanently wild-type (wt) or lamivudine-resistant (LAM-R) HBV. In this study, we investigated the cross-resistance profile of multiresistant HBV strains against PMEO in comparison to LAM, ADV, entecavir (ETV) and tenofovir (TDF).



The anti-HBV activity of the drugs was evaluated after transfection of Huh7 cells with HBV genomes cloned in a pTriex-HBV vector. These genomes are from a) laboratory HBV strains [wt, LAM-R (L180M/M204V), ADV-R (N236T) or LAM+ADV-R (L180M/M204V/N236T)], b) the viral quasispecies of a chronically infected patient failing successive therapy with LAM and LAM+ADV (patient 1) [wt, LAM-R (L180M/M204V; L180M/A181V) or LAM+ADV-R (V173L/L180M/A181V; V173L/L180M/A181V/M204V; V173L/L180M/A181V/N236T; V173L/L180M/A181V/M204V/N236T)], c) a chronically infected patient failing successive therapy with LAM and ETV (patient 2) [wt, LAM-R (V173L/L180M/M204V) or ETV-R (L180M/M204V/S202G)]. Drugs were administrated daily from day 4 to day 9 post transfection. Cells were lysed at day 9 for analysis of intracellular viral DNA by Southern Blot hybridization.



The in vitro drug potency to inhibit wt HBV replication, whatever the strain origin, was ranked in the following order: ETV>LAM>PMEO>ADV≈TDF. Regarding laboratory strains and from patient 2, PMEO inhibits similarly the replication of wt and mutant HBV strains whereas the activity of ADV/TDF is decreased against ADV-R and LAM+ADV-R mutants. The mutation M204V leads to a loss of susceptibility to ETV and a resistance to LAM that inhibit efficiently only the ADV-R mutant. For patient 1, all mutant strains are resistant to LAM, sensitive to TDF and retain susceptibility to PMEO, the V173L/L180M/A181V/N236T mutant displaying the highest fold resistance to PMEO (FR=5.1, IC50= 28.6±5.34 µM).



The antiviral potency and the cross-resistance profile of PMEO suggest that it represents a new candidate for the treatment of chronic HBV carriers who have developed resistance to currently approved drug regimen, and for the design of combination therapy to delay the emergence of resistance. PMEO warrants further evaluation in animal models and clinical trials.