946.Characterization of Hepatitis B Virus Polymerase Mutations Detected During Antiviral Treatment with Adefovir Dipivoxil (ADV)

T. Bock; C. Walker; B. Koeberlein; C. Fischer; R. Kandolf; J. Torresi

 

Background/Aim

Recent studies have shown that, in contrast to lamivudine, adefovir dipivoxil (ADV) therapy is associated with delayed and infrequent selection of drug resistant hepatitis B virus reverse transcriptase (RT) mutations. To date, apart from rtN236T and rtA181V ADV-resistant mutations, the impact of other mutations in the HBV RT region on the susceptibility to ADV is unknown. The aim of this study is to genotypically and phenotypically analyse emerging mutations in the HBV RT during ADV therapy.

 

Patients/Methods:

68 patients (44 ±14 years) with chronic HBV (CHB)-infection treated with ADV (18 ± 13 months) demonstrating persistent viraemia (5.5x10E+7 ± 1.4x10E+6 HBV IE/ml) were included in this analysis. The RT-region was amplified by PCR after extraction of viral DNA from patient sera. Mutational analysis of the RT-region was done by sequencing followed by genotyping. Selected mutations were introduced into HBV replication competent constructs using site-directed mutagenesis and analyzed for sensitivity to ADV (IC50) in HepG2 cell culture experiments.

 

Results

The ADV resistant mutations rtA181V and/or rtN236T were detected in 11 patients. Sequence exchanges at positions rtL122(F), rtN124(H), rtP130(Q), rtD131(N) in the RT-fingers domain were detected in 60% of the patients. However, these changes could be also detected in treatment naïve patients and represent polymorphisms. Additionally, the exchange rtN248H in the E-domain was frequent (26/72; 36%). Statistical analysis revealed that rtN248H positively correlated with ALT (r=0.65, p<0.0001), rtP130Q (r=0.5, p<0.0001) and rtN124H (r=0.42, p<0.0001), and negatively correlated with rtL180M (r=0.45, p=0.001). The phenotypic cell culture analysis of rtN248H showed a moderate reduction of susceptibility to ADV (IC50 <6µM ADV) compared to wild type HBV (IC50 <3µM ADV) and rtN236T (IC50 >10µM). Notably, the recently published mutation rtI233V could not be detected in any of our ADV treated patients.

 

Conclusion

Apart from the previously described rtN236T and rtA181V ADV resistant mutants, our analysis shows no evidence for further distinct ADV resistant mutants.. However, antiviral treatment of CHB patients with ADV can select further exchanges in the HBV polymerase. Exchanges in the RT-fingers domain, e.g. rtL122F, rtN124H, rtP130Q, and rtD131N, may represent polymorphisms and have little or no impact on antiviral susceptibility. Phenotypic analysis of exchanges in the RT E-domain, rtN248H, showed only a slight reduction in sensitivity to ADV. Suspected ADV resistant mutations, e.g., rtQ215S and rtI233V, were extremely rare or not detectable in our patients.

 


955. Drug Susceptibility Testing of a Novel Mutation Pattern (A181S+M204I) Conferring Resistance to Lamivudine and Adefovir Dipivoxil

A. Bozdayi; E. Karatayli; S. Karayalcin; H. Karaaslan; H. Kayhan; A. R. Turkyilmaz; F. Sahin; C. Yurdaydin

 

Background and Aim

We recently described a cross-resistance pattern to adefovir (ADV) in a patient with lamivudine (3TC) resistance (EASL 2005). We aimed to further analyse the drug susceptibility of this novel mutation pattern (A181S+M204I) against 3TC, ADV, tenofovir (PMPA), clevudine (L-FMAU) and embricitabine (FTC).

 

Methods

Successful suppression of HBV replication by sequential therapy of 9 MU/TIW interferon (IFN) and 3TC was followed by the genotypic resistance detected at the 19th month of treatment. ADV was added to 3TC therapy on the 44th month of antiviral treatment. However, neither ALT normalization nor a stable decrease in the HBV viral load was observed although ADV was used for more than 40 months. HBV pol region was amplified from 8 serum samples obtained before and after ADV treatment. Out of 8, complete genome was amplified in 3 serum samples and was cloned into a TA vector. DNA sequencing was performed for 8 PCR products of HBV pol gene and for 9-10 clones from 3 cloned constructs. Full genome of A181S+M204I variant was also cloned into an expression vector and its in vitro susceptibility to 3TC, ADV, PMPA, L-FMAU and FTC was determined in transiently transfected Huh7 cells.

 

Results

Of the 29 clones sequenced, A181S + M204I pattern was detected in 14. The novel A181S mutation in the reverse transcriptase gene was associated with W172C mutation in the overlapping surface antigen gene. The results of the in vitro drug susceptibility assay revealed that this mutation pattern displays more than 1000 fold resistance for 3TC and FTC, and 28.2 fold resistance to ADV. This novel mutation pattern was relatively sensitive to the effect of PMPA and L-FMAU, displaying 5.9 and 5.6 fold resistance, respectively.

 

Conclusion

In conclusion, A181S+M204I mutation pattern is associated with reduction in the susceptibility of all antiviral agents tested. While resistance is very high to 3TC and FTC, it is moderate to ADV and relatively mild to L-FMAU and PMPA This study underlines the potential clinical aid of drug susceptibility assays in the area of multi-resistant viral variants.

 


956.Adefovir Dipivoxil Plus Lamivudine Combination Treatment Is Superior to Adefovir Dipivoxil Monotherapy in Lamivudine-resistant Hepatitis B e Antigen-negative Chronic Hepatitis B Patients

K. Tziomalos; T. Vassiliadis; O. Giouleme; G. Koumerkeridis; C. Koumaras; K. Patsiaoura; A. Mpoumponaris; D. Gkisakis; K. Theodoropoulos; N. Grammatikos; A. Panderi; N. Nikolaidis; N. Evgenidis

 

The aim of this study was to evaluate the long-term efficacy of adefovir dipivoxil (ADV) in patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB) who have developed resistance to lamivudine (LAM) treatment. In addition, we aimed to assess whether LAM should be discontinued in these patients.

 

Methods

Sixty patients with lamivudine-resistant HBeAg-negative CHB were randomized in a 3:1 ratio to receive either ADV + LAM combination treatment (Group A; n = 45) or ADV monotherapy (Group B; n = 15). Baseline characteristics did not differ between groups. After a median follow-up time of 43 months (range, 10 to 63 months), hepatitis B virus (HBV) DNA became undetectable (< 400 copies/ml; virological response) in 37/45 patients in Group A (82.2%) and in 11/15 patients in Group B (73.3%)(p = 0.709). During follow-up, transaminases’ levels normalized in 40/44 patients in Group A (90.9%) and in 8/14 patients in Group B (57.1%)(p = 0.012). ADV-resistant mutations were identified in 2/45 patients in Group A (4.4%) and in 5/15 patients in Group B (33.3%)(p = 0.011).

 

The two patients with ADV-resistance (group A) were suboptimal responders and in one of two (50%) the duplication of ADV dose to 20 mg per day resulted in disappearance of HBV DNA (< 6 IU/ml) six months later. Virological breakthrough (reappearance of HBV DNA after its initial disappearance) did not develop in any of the 37 Group A patients with virological response. In contrast, 3/11 Group B patients with virological response (27.3%) developed virological breakthrough (p=0.011). Treatment was well-tolerated and no adverse effects occurred.

 

Conclusion

In conclusion, ADV + LAM combination treatment is superior to ADV monotherapy in patients with lamivudine-resistant HBeAg-negative CHB, in terms of both efficacy and development of resistance to ADV. Increase of ADV dose to 20mg/day may be effective in suboptimal response patients or even ADV-resistant patients.

 


961. The Effect of Lamivudine and Adefovir Dipivoxil on Preventing Hepatocellular Carcinoma in Hepatitis B Virus-related Liver Cirrhosis

J. Eun; H. Lee; S. Lee; T. Kim; B. Jang; J. Choi; Y. Park; K. Kim; K. Lee; H. Moon; S. Lee

 

Background/Aims

We evaluated the effect of lamivudine and adefovir dipivoxil on preventing HCC in hepatitis B virus (HBV)-related liver cirrhosis.

 

Patients and Methods

We reviewed the medical records of the patients who were diagnosed with HBV-related liver disease at Yeungnam University hospital from January 1983 to December 2005.

 

The number of patients treated with lamivudine over 12 months was 570 and the number of patients treated with adefovir dipivoxil after lamivudine resistance was 284 from March 1997 to December 2005. The number of patients who could not be treated with oral antiviral agent from 1983 to 1999, followed over 12 months, was 1592.

 

In our results using Cox regression model, four factors were related with HCC; gender (male, OR=1.81, p<0.001), age(≥40 years, OR=4.95, p<0.001), platelet count(<150x10^3/mm3, OR=3.48, p<0.001), and ascites (yes, OR=1.69, p=0.009). Using SPSS program, 111 patients of the oral antiviral agent-treated group and the untreated group were randomly selected, respectively, matching an age variable, a gender variable, liver cirrhosis with Child-Pugh class A and positive HBe antigen.

 

Results

The mean follow-up period was 4.4 years in the oral antiviral agent-treated group and 5.4 years in the control group. In oral antiviral agent-treated group, HCC occurred in 5 patients (4.5%) with an annual incidnce rate of 1.02% patients/year, whereas in the control group, HCC occurred in 36 patients (32.4%) with an annual incidence rate of 6.0% patients/year. The cumulative incidence of HCC in the oral antiviral agent-treated group was lower than that in the control group (Log-rank, p=0.003).

 

Conclusion

Oral antiviral agents, such as lamivudine and adefovir dipivoxil may reduce the incidence of HCC in patients with HBV-related liver cirrhosis.

Cumulative Incidence of HCC Between the Treated Group and the Control Group in HBV-Related Child A Liver Cirrhosis with Positive HBe Antigen.

 


964. Mutations of the HBV-polymerase Gene Associated with Adefovir Drug Resistance in Patients Undergoing a First Adefovir Therapy

K. Sauné; F. Abravanel; G. Martin-Blondel; J. Izopet; L. Alric 

 

Background

HBV resistance mutations have been identified for all oral antiviral agents used to treat chronic hepatitis B. Whereas the rtA181V and rtN236T mutations are associated with long-term adefovir dipivoxil (ADV) treatment, the newly discovered mutation I233V is associated with primary resistance to ADV.

 

Aim

To assess the association between existing HBV polymerase mutations and failure of antiviral therapy in patients given ADV for the first time.

 

Patients and methods

86 patients began ADV therapy during 2001 and 2005 for more than 12 months, of these 6 (7%) showed no significant virus suppression (less than 1 log IU/ml) and their ALAT was elevated when therapy was stopped (NR group). The HBV rt amino-acid sequences from these 6 patients were compared to those of 5 patients who had virus sensitive to ADV (R group). The HBV rt gene was sequenced with in-house protocols (ABI 3100) using sera collected at baseline and at the end of treatment.

 

Results

Phylogenetic analysis of the rt gene HBV gave genotypes A (6/11), D (4/11) and E (1/11). The rtA181T/V and rtN236T mutations were never detected at the beginning of ADV therapy. rtM204I/V and/or rtL180M were detected in 5/11 patients (45%) because most of them had been treated with Lamivudine (LMV). Two of them had lost these LMV-resistance-associated mutations at the end of ADV therapy. Only one patient in the NR group developed rtA181T/V and rtN236T mutations (17%) during the ADV treatment. The rtI233V mutation, recently associated with primary resistance to ADV was not detected in any patient and did not exist in patients whose virus was resistant to ADV. But, rtL217R was detected at baseline and at the end of treatment in 3/6 of the NR patients (50%) and in 1/5 R group patients (20%). This mutation, which is more prevalent in HBV strains with genotype A2, was found in 3 patients infected with genotype A.

 

Conclusion

HBV strains that are naturally resistant to adefovir can occur, but are not frequent. The rtI233V mutation cannot explain the failure of ADV therapy in our patients, whereas rtL217R mutation seemed to be more frequent. Further studies are needed to clarify the influence of the rtI233V and the rtL217R mutations on ADV therapy.

 


981. Clinical Covariates Associated with Virological Response and Adefovir Resistance in Chronic Hepatitis B (HbeAg)- negative Lamivudine-resistant Patients Treated with Adefovir Alone or in Combination with Lamivudine for 28 months

A. M. Pellicelli; G. Barbaro; R. Francavilla; M. Romano; G. Barbarini; E. Mazzoni; F. Mecenate; A. Paffetti; A. Barlattani; C. Struglia; R. Villani; L. Nauri; L. Nosotti; O. Armignacco; M. Camporiondo; F. Soccorsi

 

Background

In lamivudine resistance patients (LAM-R) adefovir dipivoxil (ADV) seems to be effective in term of virological (VR) and biochemical response (BR) both in mono or combined therapy with lamivudine (LAM). Clinical covariates of virological response are usually low baseline HBVDNA and alanino amino transferase (ALT) levels. Adefovir associated to LAM in LAM-R patients seems to prevent ADV resistance over time.

 

Aims

To evaluate the efficacy of ADV administered either alone or in combination with LAM, in HbeAg negative patients with chronic hepatitis B or cirrhosis with clinical LAM-R. To evaluate the clinical covariates associated with a sustained virological response (SVR). To determine the occurrence rates of viral resistance to ADV in patients with ADV monotherapy compared to ADV associated to LAM.

 

Methods

Seventy HbeAg-negative patients with clinical LAM-R were treated for a median of 28 months (range: 27 to 36 months) with ADV alone [34 patients (Group A)] or ADV in combination with LAM [36 patients (Group B)]. The study endpoints were: undetectable serum HBV-DNA, normalization of ALT levels, and occurence of resistance to adefovir.

 

Results

By month 3, serum HBV-DNA became undetectable in 30 patients (83%) of Group B and in 26 patients (76%) of Group A and in 5 additional patients (14%) of Group B and in 2 additional patients (6%) of Group A by month 12. One patient of Group B (3%) and 6 patients of Group A (17%) maintained detectable HBV-DNA levels and high ALT levels up to the end of the study period. In Group B and in Group A the rates of ALT normalization were, respectively: 56% and 53% at month 3, 83% and 72% at month 6 and 97% and 82% at month 12, persisting up to the end of the study period. Logistic regression analysis showed that pretreatment levels of HBV-DNA <5 log10 UI/mL, ALT levels >150 U.I./L and a fibrosis score < 2 were the strongest covariates independently associated with a SVR in both groups of patients. A primary non response (basal rtA181V) was identified in 2 patients of the group A who maintained detectable HBVDNA and high ALT levels up to the end of the study period. No adefovir resistance was noted in the remain 5 patients.

 

Conclusions

Adefovir administered either alone or in combination with lamivudine is efficacy in suppressing HBVDNA in HbeAg-negative patients resistant to lamivudine, especially in those with pretreatment low HBV-DNA levels, high ALT levels and low fibrosis score. rtA181V/T resistance to ADV was observed in clinical isolates from 2 patients of group A undergoing long term LAM treatment. In the other 5 patients with detectable HBVDNA no ADV major resistant mutations were disclosed.

 


982. Factors Affecting Response To Adefovir Treatment In Patients With Chronic Hepatitis B And Lamivudine Resistance

S. Pritchett; D. K. Wong; C. Yim; T. Mazzulli; E. Heathcote

 

Background and Aims

Adefovir (ADV) may not always gain control of lamivudine resistant (LAM-R) chronic hepatitis B (CHB). To identify factors that predict adequate ie. undetectable HBV DNA (<60 IU/mL) response to ADV in the treatment of LAM-R CHB.

 

Methods

A retrospective study of CHB with genotypic or phenotypic LAM-R treated with ADV 10mg daily in an Add To or Switch To/Add Back Lam treatment strategy. Adequate response to ADV was defined as undetectable HBV DNA (<200copies/mL or <60IU/mL) at 1 yr. Factors evaluated included eAg status, treatment strategy, pre-treatment (just prior to starting ADV) HBV DNA level (PreTxDNA), whether ADV was added at phenotypic or genotypic resistance, and rapid HBV DNA decline at 6 months, defined as >50% logarithmic decline from PreTxDNA. A multivariate analysis was performed both for all patients and for the subset with cirrhosis.

 

Results

Only 45 of 95 (47%) patients achieved undetectable HBV DNA at 1 yr. In multivariate analysis, only PreTxDNA level and >50% decline in HBV DNA at 6 months predicted achieving undetectable HBV DNA at 1 yr. Though patients with a PreTxDNA >7 log IU/mL were only 12% (3% to 43%) as likely to achieve an undetectable HBV DNA at 1 yr compared to patients with HBV DNA <7 log IU/mL, this effect continued below this cutoff. For each 1 log IU/mL increase in PreTxDNA a patient was only 40% (25% to 64%) as likely to attain an undetectable HBV DNA at 1 yr. Patients who at 6 months had >50% logarithmic decline in HBV DNA compared to pre-treatment were 25.4 (3.1 to 206.1) times more likely to reach undetectability at 1 yr. These same factors significantly predicted response at 1 yr when only patients with cirrhosis were evaluated. Three of 21 patients with a <50% decline in HBV DNA at 6 months developed ADV resistance in 1 yr while on ADV.

 

Conclusions

ADV should be started at the first emergence of LAM-R when HBV DNA is lowest to maximize the probability of achieving an adequate response. Assessing the 6 month rate of decline in HBV DNA may identify those who should have anti-viral therapy changed earlier.

 

Predictive Factors For Effective ADV Therapy In LAM-R CHB (Multivariate Analysis)

Predictive Factor

1 Yr All Patients

OR (95% CI)

1 Yr Cirrhotics

OR (95% CI)

eAg Status

ns

ns

Treatment Strategy

ns

ns

Pheno/Genotypic Resistance

ns

ns

PreTxDNA (>7 vs <7 logIU/mL)

0.12 (0.03-0.43)1

0.23 (0.05-1.0)4

PreTxDNA (per 1 logIU/mL rise)

0.40 (0.25-0.64)2

0.45 (0.26-0.76)1

>50% decline at 6 months

25.4 (3.1-206.1)1

24.4 (2.4-249.3)3

p value: 1<0.005, 20.0001, 3<0.01, 40.05, ns=not significant

 


983. Predictors of Virologic Response and Resistance to Adefovir in Patients With Lamivudine-resistant Chronic Hepatitis B Virus Infection

N. Park; J. Shin; J. Park; S. Jung; I. Jeong; S. Bang; D. Kim

 

Background

Adefovir (ADV) can be used as initial therapy in patients with HBeAg positive or HBeAg-negative chronic hepatitis B or as additional therapy in those with lamivudine (LAM)-resistant HBV. ADV resistance was common in LAM-resistant patients than in treatment-naive patients. We evaluated the virologic response to ADV in patients with LAM-resistant chronic hepatitis B virus infection, and the potential role of primary non-response in prediction of virologic response and resistance.

 

Methods

Liver panel, HBeAg/anti-HBe, HBV DNA PCR (COBAS Amplicor HBV Monitor assay) were measured every 2 or 3 months during ADV treatment. Genotypic mutation was detected using mass spectrometry-based genotyping assay (RFMP). Primary non-response was defined as a decrease in serum HBV DNA by < 2 log10 copies/ml at 24 weeks of therapy. Virologic breakthrough (VBT) was defined as a >1 log10 copies/ml increase in serum HBV DNA from nadir after achieving virologic response. Virologic response was defined as a decrease in serum HBV DNA to undetectable levels by PCR, and loss of HBeAg.

 

Results

146 HBeAg positive CHB patients with LAM resistance were received ADV for at least 12 months. Mean treatment duration was 25 months (range 12-42). Mean pretreatment HBV DNA and ALT levels were 7.7 log10 copies/ml and 357 IU/L, respectively. 115 patients were chronic hepatitis, 31 were liver cirrhosis. Serum HBV DNA became undetectable and ALT normalized in 59 (44%) and 121 (82.9%), respectively. Thirty-four (23.3%) patients represented primary non-response. Virologic response was archived in 34 (23.3%) patients. The cumulative rates of virologic response were 9, 22, and 40% at 12, 24, and 36 months, respectively. In multivariate analysis, the absence of primary non-response (OR, 3.34; 95% CI, 1.017-10.965; P<0.001), higher pretreatment ALT (OR, 1001; 95% CI, 1.000-1.002; P<0.05), and lower pretreatment HBV DNA (OR, 1000; 95% CI, 1.000-1.000; P<0.05) were independently associated with virologic response. VBT and genotypic ADV resistance were observed in 24 (16.9%) and 18 (12.3%), respectively. The rtA181T, rtN236T, and rtA181V and rtN236T were detected in 6, 3, and 9, respectively. The cumulative rates of genotypic ADV resistance at 12, 24, and 36 months were 3, 13, and 26%, respectively. In multivariate analysis, the predictive factor associated with VBT was only the presence of primary non-response (OR, 2.46; 95% CI, 1.089-5.547; P<0.05).

 

Conclusions

Primary non-responder to ADV for LAM rescue therapy showed not only lower rate of virologic response but also higher rate of virologic breakthrough than those of primary responder.

 


996. Impact of Adefovir Dipivoxil on Liver Fibrosis and Activity Assessed with FibroTest-ActiTest in Patients Infected by Hepatitis B Virus

T. Poynard; Y. Ngo; P. Marcellin; S. J. Hadziyannis; V. Ratziu; Y. Benhamou; C. Brosgart

 

Background

The aim was to assess the usefulness FibroTest-ActiTest (FT-AT) as surrogate markers of histological features in patients with chronic hepatitis B based on a correlation analysis with biopsy data in pivotal studies with adefovir dipivoxil.

 

Methods

Patients with chronic hepatitis B randomized in two pivotal trials of adefovir versus placebo, with available paired liver biopsy and FT-AT at baseline and after 48 weeks of treatment were included. Fibrosis and Activity were assessed blindly according to ISHAK scoring system. Diagnostic value of FT-AT was assessed using the area under the receiver operating characteristics curves (AUROCs) for the diagnosis of bridging fibrosis, cirrhosis, and moderate-severe necro-inflammatory activity; sensitivity analyses take into account race, length of biopsy, HBeAg status and sample date. The impact of treatment versus placebo was assessed on liver injury according to baseline stage, and virological response at 48 weeks. For baseline discordant cases, 48 weeks repeated estimates in adefovir virological responders and placebo non-responders permitted to estimates the ratio due to FT-AT or biopsy failures.

 

Findings

We recruited 462 patients, 304 treated with adefovir and 158 received placebo with paired FT-AT and biopsies. The analysis of 924 estimates showed for the diagnosis of bridging fibrosis, cirrhosis and moderate or severe necro-inflammatory activity very significant FT-AT AUROCs: 0.76±0.02, 0.81±0.02 and 0.80±0.01 respectively. Similar impacts on liver fibrosis and activity were observed both with biopsy and FT-AT, with greater efficacy in patients with baseline advanced fibrosis, treated with adefovir and virological responders. The discordances analyses suggested that 66% of discordances were attributable to biopsy failure and 34% to FT-AT failure.

 

Conclusion

In patients with chronic hepatitis B, FibroTest-ActiTest is a simple and non-invasive quantitative estimate of liver fibrosis and necrotico-inflammatory activity, which could be used as a surrogate marker to reduce the need for liver biopsy.

 


998. Factors Affecting Initial Virologic Response and Emergence of Resistant Mutants after Adefovir Treatment in Lamivudine-resistant Chronic Hepatitis B Patients

J. Cheong; J. Cho; S. Cho

 

Background

Adefovir dipivoxil effectively inhibits both wild-type and lamivudine resistant hepatitis B virus (HBV) replication. The development of adefovir resistance is delayed and infrequent compared with lamivudine resistance.

 

Aims

The aim of this study was to characterize the serologic, biochemical and virologic response to adefovir, and to explore the factors affecting initial virologic response (IVR) and adefovir resistance in lamivudine resistant HBV infected patients.

 

Methods

Between March 2004 and December 2006, 76 patients with lamivudine resistance who received adefovir for more than 12 months were included. Assay of adefovir resistant mutant was performed at 6 months and 12 months during adefovir administration. Restriction fragment mass polymorphism analysis was used for YMDD mutant and adefovir mutant detection.

 

Results

After adefovir administration, IVR was observed in 31% patients with lamivudine resistance. Factors associated with IVR were HBeAg negativity(p=0.04), higher pretreatment ALT(p=0.05) / AST (p=0.05) and presence of liver cirrhosis(p=0.04). Age, sex, pretreatment HBV DNA levels, presence of precore mutation and types of YMDD mutants were not related to IVR after adefovir treatment. The prevalence of adefovir resistance was 5% at 6 months and 13% at 12 months after therapy (rtA181T in 6 cases, rtA181V in 3 cases). Mixed infection of precore mutant (TGG+TAG) were risk factors for emergence of adefovir resistance(p=0.01).

 

Conclusion

Patients having HBeAg negativity, high ALT/AST and liver cirrhosis were more likely to achieve initial virologic response after adefovir therapy in lamivudine resistant HBV patients. Adefovir resistance was associated with mixed infection of precore mutant.

 


1000. Adefovir Depivoxil for Decompensated Liver Cirrhosis Patients with Lamivudine-resistant Hepatitis B: Multi-center Long-term Results

H. Woo; J. Choi; S. Yoon; D. Suh; S. Paik; K. Han; S. Um; B. Kim; H. Lee; M. Cho; C. Lee; D. Kim ; J. Hwang

 

Background/Aim

The effect and safety of adefovir dipivoxil for patients with decompensated cirrhosis were not well known. Long-term efficacy and safety of adefovir dipivoxil in decompensated cirrhosis patients with lamivudine-resistant hepatitis B (HBV) were studied.

 

Methods

One hundred nineteen cirrhotic patients who developed hepatic decompensation (any cirrhosis complication or Child-Pugh (CP) score ≥ 7) after lamivudine treatment were enrolled from eleven university hospitals and treated with 10 mg of adefovir dipivoxil for a median 33 months between January 2003 and October 2006. Lamivudine was switched to adefovir dipivoxil with or without overlap period (median 2 months). We evaluated the overall survival rate and the improvement of Child-Pugh score, virologic response (HBV DNA <0.5 pg/ml) (Digene Hybrid Capture II assay, Detection limit 0.5 pg/ml), and virologic breakthrough after long-term adefovir dipivoxil treatment.

 

Results

1) At the baseline, median serum HBV DNA was 7.3 log10 copies/mL, median MELD was 15 (9-37). Child B was 63%, and Child C was 37%. 2) Overall patient survival rate was 90% for a median 33 months. Seven (5.8%) patients died within 6 months. Clinical factor associated with early mortality (≤ 6 months) was initial high MELD score (>25) by multivariate analysis (p=0.020, HR1.270 95% C.I. [1.038-1.553]). 3) The mean change from baseline CP score was -2.9±2.1 points (p<0.001) and 76.4% (84/110) of patients had a decrease of at least 2 points in CP score. 4) The mean change from baseline serum HBV DNA was -2.8 log10 copies/mL. After 48 weeks, serum HBV DNA became undetectable in 71% (78/109). Virologic breakthrough occurred in 33.7% (37/110). 5) Six (5.5%) patients confirmed an increase of at least 0.5 mg/dl in serum creatine concentration.

 

Conclusions

Long-term treatment of adefovir dipivoxil was effective and safe in decompensated cirrhosis associated with lamivudine-resistant HBV. High MELD score at the baseline was associated with early mortality after adefovir dipivoxil treatment.

 


957. Pretreatment Alanine Transaminase Level Might Not Be the Most Important Factor in Predicting HBeAg Loss in Older Patients with More Prolonged HBV Infection

J. Lee; J. Lee; Y. Kim; D. Lee; S. Jeong; H. Park; E. Kim; B. Bang; E. Choi; Y. Paik; K. Lee

 

Background/Aims

Pretreatment ALT level, especially over two times the upper limit of the normal (ULN) reference range, has been accepted as the relevant indicator of initiating the antiviral therapy. Prolonged abnormal ALT level less than 2XULN has been frequently noticed especially in older patients. his analysis was to determine the usefulness of pretreatment ALT level in predicting HBe Ag loss in older patients with lamivudine therapy.

 

Methods

We retrospectively analyzed 820 HBeAg positive chronic hepatitis patients treated with lamivudine. The patients with either hepatocellular carcinoma, or with overt liver cirrhosis recognized by imaging studies were excluded from the analysis. Patients were diagnosed as having persistent HBeAg if they had serum HBeAg positive after three years of antiviral therapy. Patients were determined as having loss of HBeAg when they had HBeAg negative serum within three years of the therapy. Lamivudine was switched into adefovir when lamivudine resistance was detected. 406 patients met the criteria and were recruited for the analysis.

 

Age, sex, ALT, and HBV DNA level were used in multivariate analysis of 406 patients (Group 1). The same analysis was performed in 152 patients with age over 45 (Group 2). Results: Baseline characteristics Group 1 patients were: median age 40 years, 75.1% male, median serum HBV DNA 403 pg/ml and median ALT 4.5 X ULN. Baseline characteristics of Group 2 patients were: median age 50 years, 72.4% male, median serum HBV DNA 470 pg/ml and median ALT 4.0 X ULN. HBe Ag loss was noticed in 47.5% of Group 1, and 45.4% in Group 2. Although HBe Ag loss correlated with increased pretreatment ALT levels in both groups, multivariate modeling indicated that elevated pretreatment ALT levels was a predictor of HBe Ag loss in Group 1 (P<0.05), but not in Group 2. HBV DNA level was not a significant predictor in either groups.

 

Conclusion

Elevated pretreatment ALT levels might not be a powerful predictor of lamivudine-induced HBeAg loss in the patients aged over 45 years. Another marker predicting HBeAg loss might be needed in patients with more prolonged HBV infection.

 

Odds ratios of HBeAg loss for potential prognostic factors; multivariate analysis:

 

Factor

Odd ratio

P value

All Patients (Group1)

ALT level

1.03

<0.05

All Patients (Group1)

HBV DNA level

1.00

0.24

Patients≥45 (Group2)

ALT level

1.03

0.10

Patients≥45 (Group2)

HBV DNA level

1.00

0.30

 


947. Durability of Antiviral Response in Lamivuidne-Treated HBeAg Positive Chronic Hepatitis B Patients Who Did Not Relapse Within a Year of Post-Treatment

J. Kim; M. Joo; S. Lee; S. Suh; Y. Jong; J. Kim; J. Yeon; H. Yim; K. Byun

 

Introduction

On the contrary to Western reports, relapse rate after lamivudine (LMV) discontinuation in HBeAg positive chronic hepatitis B (CHB) patients is higher in Asian patients and most reported relapses occur within one year after LMV discontinuation. The aims of this study to investigate the long-term relapse rate and associated risk factors in HBeAg positive CHB patients who had kept a sustained virologic response (VR) for 1 year after LMV discontinuation.

 

Methods

We enrolled 376 treatment-naïve HBeAg positive CHB patients who took LMV. Among them, 113 patients were achieved VR and then discontinued LMV. During the post treatment follow up, 58 of 113 patients (51.3%) showed relapse within 1 year. For the remaining 55 patients who maintained the VR within a year of treatment discontinuation, cumulative rate and risk factor for long term post treatment relapse were analyzed. VR was defined as loss of HBeAg and undetectable serum DNA in hybridization assay or serum HBV DNA level less than 105 copies/mL. Relapse was defined as reappearance of HBV DNA or increase of HBV DNA more than 105 copies/mL. Delayed relapse was defined as relapse that occurred after sustained VR for 1 year after LMV discontinuation.

 

Results

The baseline characteristics of 55 patients were follows as: Mean age, serum ALT, HBV DNA, duration of LMV treatment, duration of LMV consolidation, and follow up duration after LMV discontinuation was 39.2 years, 374.5 IU/L, 7.5 log10 copies/mL, 22.5 months, 10.5 months (range, 0-28), and 38.8 months (range, 14-95), respectively. During follow up period (mean 26.7 months; range, 2-83), 16 of 55 patients (29%) showed delayed relapse. Cumulative rate of delayed relapse from 1 year after LMV discontinuation was 16%, 29%, 39%, 44% at 1, 2, 3, 4 years, respectively. At relapse, 2 patients showed decompensation and three patients showed flare of hepatitis. In univariate analysis, age, pretreatment albumin, serum HBV DNA > 2000 copies/ml at 3 months after LMV discontinuation predicted the delayed relapse significantly. In multivariate analysis, age (P=0.029) and serum HBV DNA > 2000 copies/mL after 3 months of LMV discontinuation (P=0.047) were significant predictors of delayed relapse.

 

Conclusion

Even in patients who had kept sustained VR for 1 year after LMV discontinuation, delayed relapse are not infrequent. Therefore, a LMV maintenance therapy might be considered in HBeAg positive CHB patients who achieved VR weighing the risk and benefit.

 


948. Analysis of Determinants for Sustained Virologic Response to Lamivudine Monotherapy in Patients with HBeAg Positive Chronic Hepatitis B: A Multi-center Trial

H. Lee; H. Lee; J. Choi; J. Hwang; W. Chung; J. Sohn; T. Kim; J. Jang; K. Han; J. Park; D. Kim; S. Ahn; Y. Paik; C. Lee; K. Lee; C. Chon; Y. Moon; K. Han

 

Backgrounds/Aims

Several reports showed inconsistent results on the durability of virologic response after successful lamivudine monotherapy. Especially, the question remains whether virologic responses can be maintained over extended follow-up period. The aim of our study was to evaluate post-treatment relapse rates for 5 years after virologic responses and to elucidate the predictive factors for sustained virologic response in patients who experienced HBeAg loss to lamivudine monotherapy.

 

Methods

We treated 748 patients (Male : Female = 570 : 178) with HBeAg positive chronic hepatitis B with lamivudine from seven medical institutions in Korea between January 1999 and August 2004. The mean duration of lamivudine monotherapy was 34 months (range, 12-88). Among 178 patients, who discontinued lamivudine monotherapy after complete response (HBeAg loss, undetectable HBV DNA and ALT normalization), 138 patients (77.5%) maintained sustained virologic response. Both host and viral factors were compared between 138 patients with sustained HBeAg response and 40 patients whose response was not sustained.

 

Results

The cumulative relapse rates at 1, 3, and 5 years were 15.9%, 26.4%, and 30.2%, respectively. The mean time to relapse after cessation of lamivudine was 12 months (range, 2-42). Most relapses were occurred within 2 years after discontinuation of lamivudine (35/40, 87.5%). In multivariate analysis, age ≤ 40 years, additional treatment duration over 12 months after HBeAg loss were independent factors for sustained virologic response. In conclusion, only 22.5% of patients who discontinued lamivudine after HBeAg loss experienced a relapse for 5 years follow-up. Age and additional treatment are major predictive factors for sustained HBeAg loss.

 


950. Variability of the HBV Pol Gene Reverse-Transcriptase Domain in Viral Isolates from Untreated and Lamivudine-resistant Chronic Hepatitis B Patients

T. Pollicno; G. Isgrò; R. DiStefano; D. Ferraro; S. Maimone; S. Brancatell1; V. Di Marco; G. Squadrito; A. Craxì; G. Raimondo

 

Different HBV variants carrying point mutations at level of the reverse transcriptase (rt) domain of the viral Pol gene had been detected in patients under treatment with nucleos(t)ide analogues. At present, few information is available about the occurrence of these drug-resistant mutants in untreated patients.

 

Aim

Aim of this study was the genomic characterization of HBV isolates from individuals (1) naive for antiviral therapy or (2) who had developed lamivudine resistance. We analyzed - by amplification, cloning and sequencing procedures - the entire rt-domain in viral isolates from 84 untreated (naive-group) and 49 lamivudine-resistant (Lam-group) consecutive HBsAg carriers with chronic liver disease. All the cases had serum HBV DNA levels > 20,000 UI/mL. Thirty-two of the 84 naive-group cases (38.1%) carried single or multiple mutations potentially predisposing to antiviral resistance.

 

In particular, we found the following mutations: rtV173M in 2 cases (2.4%), rtA181D in 1 case (1.2%), rtV214A/E in 3 cases (3.6%), rtQ215S/P/H in 12 cases (14.3%), rtL217R in 8 cases (9.5%), rtS219S in 4 cases (4.8%), rtF221Y/L in 15 cases (17.8%), rtI233V in 4 cases (4.8%), rtP237T in 3 cases (3.6%), rtN238T/H/D in 4 cases (4.8%).

 

In the lamivudine group – besides the mutations typically inducing lamivudine resistance – 28/49 (57.1%) carried single or multiple mutations conferring resistance to other antivirals. In particular, four cases (8.2%) had either the rtT184S or the rtM250V/L mutations favoring resistance to entecavir, whereas various combined mutations predisposing to adefovir resistance were detected in 26 cases: rtV84M in 1 case (2%), rtA181S/T in 2 cases (4.1%), rtV214A/T in 2 cases (4.1%), rtQ215S/E/P in 12 cases (24.5%), rtL217R in 3 cases (6.1%), rtS219A in 3 cases (6.1%), rtF221Y in 6 cases (12.2%), rtI233V in 1 case (2%), rtP237HT in 2 cases (4.1%), rtN238T/H/D in 12 cases (24.5%).

 

Conclusion

In conclusion, our study shows that HBV mutants predisposing to drug resistance may be present as major infecting population in untreated patients, and that variants emerging during lamivudine treatment may also carry mutations potentially inducing resistance to either adefovir or entecavir. Thus, the genetic characterization of HBVs infecting patients undergoing treatment with nucleos(t)ide analogues may be a helpful tool for tailoring the most proper therapeutic strategy in each individual case.

 


956. Adefovir dipivoxil plus lamivudine combination treatment is superior to adefovir dipivoxil monotherapy in lamivudine-resistant hepatitis B e antigen-negative chronic hepatitis B patients

K. Tziomalos; T. Vassiliadis; O. Giouleme; G. Koumerkeridis; C. Koumaras; K. Patsiaoura; A. Mpoumponaris; D. Gkisakis; K. Theodoropoulos; N. Grammatikos; A. Panderi; N. Nikolaidis; N. Evgenidis

 

The aim of this study was to evaluate the long-term efficacy of adefovir dipivoxil (ADV) in patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB) who have developed resistance to lamivudine (LAM) treatment. In addition, we aimed to assess whether LAM should be discontinued in these patients.

 

Methods

Sixty patients with lamivudine-resistant HBeAg-negative CHB were randomized in a 3:1 ratio to receive either ADV + LAM combination treatment (Group A; n = 45) or ADV monotherapy (Group B; n = 15). Baseline characteristics did not differ between groups. After a median follow-up time of 43 months (range, 10 to 63 months), hepatitis B virus (HBV) DNA became undetectable (< 400 copies/ml; virological response) in 37/45 patients in Group A (82.2%) and in 11/15 patients in Group B (73.3%)(p = 0.709). During follow-up, transaminases’ levels normalized in 40/44 patients in Group A (90.9%) and in 8/14 patients in Group B (57.1%)(p = 0.012). ADV-resistant mutations were identified in 2/45 patients in Group A (4.4%) and in 5/15 patients in Group B (33.3%)(p = 0.011).

 

The two patients with ADV-resistance (group A) were suboptimal responders and in one of two (50%) the duplication of ADV dose to 20 mg per day resulted in disappearance of HBV DNA (< 6 IU/ml) six months later. Virological breakthrough (reappearance of HBV DNA after its initial disappearance) did not develop in any of the 37 Group A patients with virological response. In contrast, 3/11 Group B patients with virological response (27.3%) developed virological breakthrough (p=0.011). Treatment was well-tolerated and no adverse effects occurred.

 

Conclusion

In conclusion, ADV + LAM combination treatment is superior to ADV monotherapy in patients with lamivudine-resistant HBeAg-negative CHB, in terms of both efficacy and development of resistance to ADV. Increase of ADV dose to 20mg/day may be effective in suboptimal response patients or even ADV-resistant patients.

 


957. Pretreatment Alanine Transaminase Level Might Not Be the Most Important Factor in Predicting HBeAg Loss in Older Patients with More Prolonged HBV Infection

J. Lee; J. Lee; Y. Kim; D. Lee; S. Jeong; H. Park; E. Kim; B. Bang; E. Choi; Y. Paik; K. Lee

 

Background/Aims

Pretreatment ALT level, especially over twomes the upper limit of the normal (ULN) reference range, has been accepted as the relevant indicator of initiating the antiviral therapy. Prolonged abnormal ALT level less than 2XULN has been frequently noticed especially in older patients. This analysis was to determine the usefulness of pretreatment ALT level in predicting HBe Ag loss in older patients with lamivudine therapy.

 

Methods

We retrospectively analyzed 820 HBeAg positive chronic hepatitis patients treated with lamivudine. The patients with either hepatocellular carcinoma, or with overt liver cirrhosis recognized by imaging studies were excluded from the analysis. Patients were diagnosed as having persistent HBeAg if they had serum HBeAg positive after three years of antiviral therapy. Patients were determined as having loss of HBeAg when they had HBeAg negative serum within three years of the therapy. Lamivudine was switched into adefovir when lamivudine resistance was detected. 406 patients met the criteria and were recruited for the analysis.

 

Age, sex, ALT, and HBV DNA level were used in multivariate analysis of 406 patients (Group 1). The same analysis was performed in 152 patients with age over 45 (Group 2). Results: Baseline characteristics Group 1 patients were: median age 40 years, 75.1% male, median serum HBV DNA 403 pg/ml and median ALT 4.5 X ULN. Baseline characteristics of Group 2 patients were: median age 50 years, 72.4% male, median serum HBV DNA 470 pg/ml and median ALT 4.0 X ULN. HBe Ag loss was noticed in 47.5% of Group 1, and 45.4% in Group 2. Although HBe Ag loss correlated with increased pretreatment ALT levels in both groups, multivariate modeling indicated that elevated pretreatment ALT levels was a predictor of HBe Ag loss in Group 1 (P<0.05), but not in Group 2. HBV DNA level was not a significant predictor in either groups.

 

Conclusion

Elevated pretreatment ALT levels might not be a powerful predictor of lamivudine-induced HBeAg loss in the patients aged over 45 years. Another marker predicting HBeAg loss might be needed in patients with more prolonged HBV infection.

 

Odds ratios of HBeAg loss for potential prognostic factors; multivariate analysis:

 

Factor

Odd ratio

P value

All Patients (Group1)

ALT level

1.03

<0.05

All Patients (Group1)

HBV DNA level

1.00

0.24

Patients≥45 (Group2)

ALT level

1.03

0.10

Patients≥45 (Group2)

HBV DNA level

1.00

0.30

 


959. Does the Genotoxic Effect of Lamivudine Treatment in Chronic Hepatitis B to the Host DNA?

M. Akdogan; H. Ilgin Ruhi; P. Ozkal; S. Kacar; N. Sasmaz

 

Chronic viral hepatitis B is the main cause of chronic liver disease, cirrhosis and hepatocellular carcinoma in the world. Lamividune, a nucleoside analog, is an inhibitor of viral DNA replication. Patients are treated with lamividune show normalization of serum alanine aminotransferase (ALT) level, and histological improvement. However, discontinuation of therapy often leads to reactivation of HBV so long-term therapy is necessary for many patients with chronic HBV infection.

 

Aim

The aim of this study is to detect the genotoxic effects of the long-term lamividune treatment to the host DNA.

 

Material and Methods

Fourteen chronic hepatitis B (CHB) patients (4 women, 10 men, mean age; 44 ranges; 29-64 yrs) who were treated with lamividune were included in this study. Blood samples were collected before the treatment and 6 months and at least 16 months (mean 27 months) of the treatment. Peripheral blood lymphocytes of these patients were cultured to make cytogenetic evaluation by observing chromosome breakage and cytologic evaluation by micronucleus (MN) test. For each individual 100 metaphase chromosome spreads were analysed. 190-1091 binucleated cells were observed and for each individual MN were scored.

 

Their results were compared with baseline, six months and at least one year of the treatment. Mann-Whitney U test was used for statistical analysis.

 

Results

Our results showed a significantly increase of chromosome breaks after one year of the treatment (p=0,025) Chromosome breaks of the six months of treatment were slightly higher than before the treatment however the difference was not statistically significant (p>0.05) Moreover we did not find any difference at MN scores between each period of the treatment (p>0.05).

 

Conclusion

Based on this data, we concluded that lamividune treatment in patients with chronic HBV may be chromosomal instability and this instability may increases during long-term therapy especially after one year of the treatment.

 


966. Prediction of Long-term Maintenance of Virologic Response During Lamivudine Treatment in HBeAg-Negative Chronic Hepatitis B

A. S. Hadziyannis; P. V. Mitsoula; S. J. Hadziyannis

 

Background and Aims

It has recently been claimed that non-detectability of serum HBV-DNA by PCR after six months of treatment with telbivudine or lamivudine in chronic hepatitis B (CHB) predicts maintenance of the response after two years of treatment with a positive predictive value (PPV) for HBeAg-negative patients of > 80% The aim of the present study was to investigate in such CHB patients the relation between HBV-DNA suppression achieved during the early phases of LAM treatment with the long-term outcome of therapy after 4 and more years.

 

Patients and Methods

The study included 156 patients with 56 of them (37%) in maintained virologic and biochemical responses for more than 4 years. Their virologic data was compared to those of patients who experienced a breakthrough 3 months to 5 years after starting treatment. HBV-DNA data assessed by sensitive real time PCR assays was available every 3 to 6 months and all samples negative or <1000 cp/ml were retested with the COBAS TaqMan Test (Roche Molecular Systems, CA, USA, sensitivity 50 cp/ml)

 

Results

The positive predictive value of negative HBV-DNA after 6 months of treatment was 93% for maintenance of the response for two and 71,5% for maintenance of response for four or years more. Similar percentages were found for the results after 3 months of treatment. On the other hand, a positive result even >10,000 cp/ml at month 6 could not exclude the possibility that LAM therapy would be successful and was associated with a possibility of maintenance of response for 2 years of 36,5% and for 4 or more years of 8%, those percentages being 43,7 and 19,5% for the month three results. Overall, patients who maintained response had lower levels of HBV-DNA both at month 3 and 6 compared to patients who experienced a breakthrough.

 

Conclusion

HBV-DNA non-detectable by sensitive PCR assays during the “early” phases of antiviral treatment of HBeAg(-) CHB are very useful in predicting maintenance of the response up to year 3 of Rx but their positive predictive value decreases to approximately 70% for response maintained longer than 4 years. Moreover their low negative predictive value cannot provide reliable information for deciding treatment discontinuation or modification.

 


972. Long-term Follow-up of Chinese Patients with HBeAg-negative Chronic Hepatitis B Who Discontinued Treatment after Twp Years of Lamivudine Monotherapy

S. K. Fung; F. Wong; A. S. Lok

 

Background

The optimal duration of treatment of HBeAg-negative chronic hepatitis B is unknown. Treatment withdrawal after one year has been associated with a high rate of relapse, while long-term treatment is associated with risks of drug resistance. We previously reported a clinical relapse rate of 18% and 30% at 12 and 18 months, respectively, after withdrawal of a 2-year course of lamivudine.

 

Aims

To determine the long-term durability of response to lamivudine following withdrawal of a 2-year course of lamivudine therapy in HBeAg-negative patients.

 

Methods

Consecutive HBeAg-negative patients attending the Toronto General Hospital Liver Clinic from 07/98- 05/03 were treated with lamivudine 100 mg daily for 2 years. Those who achieved undetectable HBV DNA by PCR assay (COBAS Amplicor, LLOD 200 copies/ml) on 3 consecutive occasions during the second year of therapy were eligible for treatment withdrawal after 2 years of lamivudine. Thereafter, patients were monitored every 6 months. Clinical relapse defined as HBV DNA >3.3 log10 IU/ml [>4 log10 copies/ml] and ALT > 1.5 X ULN [ULN, 40 U/L] and viral relapse (redetection of HBV DNA by PCR) on 2 consecutive occasions were estimated by Kaplan Meier analysis. Antiviral therapy was restarted in those who met criteria for clinical relapse.

 

Results

A total of 50 HBeAg-negative patients, mean age 52+/-11 years, 80% men and 52% cirrhosis were included. Treatment was withdrawn in 45 patients and relapse occurred in 30 with a mean time to relapse of 16 months. The probability of viral and clinical relapse at 24, 36 and 48 months post treatment was 50% and 40%, 62% and 56%, 74% and 60%, respectively. No baseline factor including cirrhosis, gender, age, ALT, or HBV genotype was associated with clinical relapse. During a mean follow-up of 56+/-15 months, no patient underwent reversion to HBeAg-positivity and 3 patients lost HBsAg. All patients who relapsed responded promptly to retreatment using lamivudine with or without adefovir or tenofovir. No patient developed hepatic decompensation or died as a result of clinical relapse.

 

Conclusions

Approximately 60% patients with HBeAg-negative chronic hepatitis had clinical relapse within 4 years of withdrawal of a 2-year course of lamivudine monotherapy. Our data suggest that a longer duration (>2 years) of treatment is needed for patients with HBeAg-negative chronic hepatitis and a long duration of follow-up (>5 years) is needed to determine the post-treatment relapse rate.

 


977. Restoration of HBV-Specific T Cell Responses in Lamivudine Long-term Responder

E. Loggi; F. K. Bihl; C. Fortini; C. Cursaro; E. Grandini; L. Micco; A. Gramenzi; M. Bernardi; C. Brander; P. Andreone

 

Background

Hepatitis B virus (HBV)-specific T cell responses appear to play a pivotal role in infection control and disease outcome. The benefit of Lamivudine (LAM), a nucleoside analogue used for HBV treatment, is compromised by progressively increasing emergence of drug-resistant strains (up to 70% after 5 years). However, 20-30% of patients do not develop resistance despite long-term therapy. While LAM has been shown to induce early but only transient restoration of CD4 and CD8-specific T cell responses, the immunological profile of LAM long-term responders (LAM-LTR) has not yet been analyzed.

 

Aim

To assess the HBV-specific T cell responses in LAM-LTR compared to LAM resistant (LAM-R) and to evaluate if successful long-term LAM therapy restores HBV-specific immunity.

 

Methods

Seven LAM-LTR (male/female: 5/2; median age: 60, range: 42-67), defined by sustained HBV suppression during prolonged LAM administration (median years of treatment: 9, range: 7-11) and 13 LAM-R patients (male/female: 12/1; median age: 52, range: 30-60), defined by occurrence of viral breakthrough, were tested with a panel of overlapping peptides (18-20 mers) spanning the entire HBV sequence. The frequency and magnitude of HBV-specific T cell responses was assessed by an IFN-γ ELISPOT assay after in vitro stimulation with the synthetic HBV peptides.

 

Results

HBV-specific immune responses were detected in all LAM-LTR (100%) and in eight of the 13 subjects with LAM-R (61%). The frequency of HBV-specific T cell responses were significantly higher in LAM-LTR compared to LAM-R (median frequency: 5 vs 1, respectively, p=0.001). Moreover, LAM-LTR presented significantly stronger IFN-γ responses (median: 2’793 vs 1’740 SFC/1,000,000 PBMC, p=0.03). Polymerase-specific immune responses dominated the HBV-specific T cell repertoire in both groups of patients, although LAM-LTR presented multiple responses to all four HBV proteins. Interestingly, two regions, in the core protein and in polymerase, were targeted by 3 LAM-LTR (42%), suggesting two new regions of immunogenicity.

 

Conclusions

LAM-LTR are characterized by a multispecific pattern of HBV specific T cell responses.

 

Moreover, the immune repertoire of responses is stronger in terms of both frequency and magnitude in long-term responders than resistant ones.

These results can contribute to better characterize HBV patients with LTR to antiviral therapy and to help for their clinical management.

 


989. Profiles of HBV DNA, ALT and HBeAg Status After Stopping Lamivudine in Patients with HBeAg Seroconversion

J. Fung; C. Lai; Y. Tanaka; M. Mizokami; J. Yuen; D. Wong; M. Yuen

 

Aim

To determine the virological and biochemical relapse rates in Chinese CHB patients with lamivudine-induced HBeAg seroconversion after stopping lamivudine therapy, and to identify significant viral or biochemical factors predictive of subsequent relapse.

 

Methods

All patients from all Hepatology Clinic with lamivudine-induced HBeAg seroconversion with subsequent cessation of lamivudine therapy were recruited. Liver biochemistry, HBeAg, anti-HBe, and serum HBV DNA by PCR (COBAS Taqman) was determined at the time of stopping lamivudine, at 3 months, 6 months, 1 year, and yearly thereafter.

 

Results

Twenty-two patients were included, of which 16(73%) were male with a median age of 32 years (range, 21-55). The median duration of follow-up was 104 months (range, 31-150). Six (27%) patients had undetectable HBV DNA by PCR at the time of stopping lamivudine treatment. In total, 7(32%) patients had subsequent flare (as defined by ALT >2x upper limit of normal), with associated virological rebound of HBV DNA (as defined by 1 log increase from time of stopping therapy). The cumulative incidence of flare at 5 years was 44%, all occurring before 25 months. There was no significant difference in the cumulative incidence of flares between those patients that continued lamivudine for 6-12 months, 1-2 years and over 2 years after HBeAg seroconversion (p=0.58). In patients who had normal ALT and undetectable HBV DNA, their cumulative incidence of flare was not significantly different to those that had detectable HBV DNA or abnormal ALT at time of stopping lamivudine (p=0.73).

 

Two patients underwent HBeAg seroreversion at 5 and 10 months, with resulting flares. The cumulative incidence of HBeAg seroreversion after stopping lamivudine was 9% at 5 years. Fourteen (64%) patients underwent virological rebound, with a cumulative incidence of 82% at 4 years. There was no significant difference in the cumulative incidence of virological rebound in patients with undetectable HBV DNA at the time of stopping lamivudine therapy compared to those with detectable HBV DNA. Of those with virological rebound, 8(57%) had HBV DNA >105 copies/ml. Neither patient age or gender, genotype, presence of precore or core promoter mutations were significant factors in subsequent flare or virological rebound after stopping lamivudine therapy.

 

Conclusion

Despite the high sustained HBeAg seroconversion (81%) after stopping lamivudine, HBV DNA rebound occurred in 64%, of which 57% had HBV DNA >105 copies/ml and 32% had ALT flares. Patients who had both normal ALT and undetectable HBV DNA by PCR at the time of stopping lamivudine therapy did not have a lower cumulative incidence of flares.

 


999. Prognostic Factors that Associated with Hepatitis B Virus DNA Breakthrough in Chronic Hepatitis B Patients with Lamivudine Treatment

Z. Zeng; G. Tian; D. Tian; J. Cui; H. Lu

 

Objective

To early determine the prognostic factors associated with hepatitis B virus DNA breakthrough in chronic hepatitis B patients with lamivudine treatment.

 

Methods

Clinical data, YMDD genotyping, HBV DNA loads, ALT level, HBeAg titer and lamivudine-resistant HBV mutant ratio, before and during therapy collected from 64 patients throughout lamivudine treatment 48 weeks were retrospectively analyzed by using nonparametric test and logistic regression model test, HBV DNA viral loads were determined by real-time PCR, ALT level were determined by automatic biochemistry analysis system, HBeAg titer were measured by microparticle enzyme immunoassay, and the lamivudine-resistant HBV ratio were determined by Pyrosequencing.

 

Results

The ratio of YMDD and rtL180M mutants were significantly higher in patients who developed HBV DNA breakthrough after lamivudine therapy than before, although they were not significantly associated with HBV DNA breakthrough, for 24 weeks data, multivariate logistic regression analysis revealed that HBeAg titer (24 weeks) was the independent factor for HBV DNA breakthrough (p=0.004, 48w), and was correlated with HBV DNA loads(r=0.493).

 

Conclusions

Pre-existing lamivudine-resistant HBV ratio was not the prognostic factors associated with hepatitis B virus DNA breakthrough, while HBeAg titer after 24 weeks of lamivudine treatment was the independent factor that influenced HBV DNA breakthrough after 48 weeks of lamivudine treatment.

 


949. Multidrug Resistance and Cross-Resistance Pathways in HBV as a Consequence of Treatment Failure

L. Yuen; A. Bartholomeusz; A. Ayres; M. Littlejohn; S. Locarnini

 

Background/Aims

Antiviral resistance is now the single most important factor in treatment failure using nucleos(t)ide analogues (NA). Primary drug resistance mutations refer to amino acid change(s) that result in reduced susceptibility to an antiviral agent. Secondary compensatory mutations restore replication defects associated with primary drug resistance, and may be associated with low level reduced susceptibility. Several evolutionary pathways of drug resistant HBV have been observed in patients treated with NAs. It is possible that the drug resistance mutations selected with one agent may affect the efficacy of other NAs. The aim of this study was to elucidate mutation pathways associated with use of NAs and to determine the potential cross-resistance profiles selected under a particular NA.

 

Methods

The HBV reverse transcriptase (rt) gene was amplified by PCR and sequenced from patients pre- and during-treatment. A software program (SeqHepB) was used to analyse the treatment-associated mutations (Yuen L et al. 2007. AVR;75:64). Associations between drug resistance and rt mutations found in the most recent samples from 159 patients pre- and 215 patients during failing monotherapy with either lamivudine (LMV) or adefovir (ADV) were evaluated using the Fisher’s Exact method and a pattern discovery program Magnum Opus (Rule Quest Research, Australia).

 

Results and Discussion

Therapy with LMV or Telbivudine (LdT) or entecavir (ETV) resulted in the selection of rtM204I/V. Statistical analysis identified several secondary compensatory mutations (rtL80I/V, rtV173L, rtL180M, rtT184G and rtS202I) during LMV monotherapy that can also affect response to ETV or LdT. Therapy with ADV resulted in rtN236T and/or rtA181T/V selection as well as the maintenance of rtL180M and rtM204V in a sub-set of patients with add-on ADV. The loss of rtL180M and/or rtM204I/V was also statistically significant among LMV resistant patients who subsequently switched to ADV monotherapy. The rtA181T/V mutation, which has been implicated with reduced sensitivity to LMV and ADV, could be either common with or separate to the “204 and 236 pathways”.

 

Conclusion

Three major HBV evolutionary NA-resistance pathways (rtM204I/V, rtN236T and rtA181T/V) have been characterised. The rtM204V/I pathway is responsible for resistance to the L-nucleosides such as LMV, LdT and also ETV whilst the rtN236T pathway is responsible for adefovir resistance. Both are associated with clusters of secondary mutations that can affect subsequent treatment with NAs (rtT184G, rtS202I). The third pathway, rtA181T/V, is associated with resistance to LMV and ADV and is a potential multi-drug resistance pathway.

 


976. Drug Resistance Mutation Analysis by Different Methods in a Highly Treatment-experienced Chronic Hepatitis B Patient Population

E. Sablon; J. Doutreloigne; S. K. Fung; T. Mazzulli; G. Moussa; V. Popovic

 

Background

Antiviral drug resistance in patients with chronic hepatitis B has become a major drawback, ultimately leading to antiviral treatment failure, subsequent biochemical breakthrough, and hepatic decompensation. To optimally monitor treatment of HBV patients, a highly sensitive assay is required that can detect resistance mutations early.

 

Patients and Methods

133 adult HBV patients attending the liver clinics of University Health Network (Toronto, Canada) were prospectively monitored for antiviral resistance mutations using INNO-LiPA HBV DR assays (Innogenetics, Belgium) and sequencing. The majority was Asian (78%); HBeAg positive (57%), and had mean HBV DNA upon resistance testing 5.4±2.1 log10 IU/ml. Ninety percent had received lamivudine therapy, 40% adefovir, and a few tenofovir or entecavir. INNO-LiPA HBV DR v2* probes cover mutations at codons 80, 173, 180, 181, 204 and 236; a prototype INNO-LiPA HBV DR v3 strip covers codons 184, 194, 202, 233 and 250. Sequencing and LiPA were performed on the same amplicon. Concordance between the LiPA and sequencing was calculated.

 

Results

Viral loads ranged from 1.2-9.7 log10 IU/ml. All genotypes except F were found, with the majority being genotype C (51.1%).

 

Interpretable LiPA patterns were obtained for 133/133 samples (100%); sequence information for 131/133 samples (98.5%). Thirty-four of the 131 samples (26%) showed no mutations at any one of the 11 codon sites with LiPA or sequencing, whereas 97 samples (74 %) displayed at least one mutation with one or both methods. Of resistance mutations detected by sequencing, concordance with LiPA occurred in 99.2% of cases, while LiPA provided additional information (wild type [wt]/mutant mix) for at least one codon in 66.7% of cases. In more than 1/3 cases, LiPA showed a drug resistance mutation in a wt/mutant mixture versus only wt virus for sequencing. Mutations at codons 180 and 204, associated with lamivudine resistance, were most frequently encountered. Fifty-two patients carried a mutation at codon 180, whereas 76 patients had a known mutation at codon 204 after LiPA analysis. With sequencing, 45 patients (86%) and 70 patients (92%) showed these respective mutations.

 

Conclusions

Both LiPA and sequencing are useful and accurate methods for drug resistance mutation detection. However, LiPA enables detection of minor mutant populations as mixtures earlier than sequencing. Earlier detection of resistance mutations using LiPA followed by prompt salvage therapy could help optimize the management of patients with antiviral-resistant HBV.

* In US: For research use only. Not for use in diagnostic procedures.

 


980. Very High Prevalence of Precore (preC) and/or Basal Core Promoter (BCP) Mutations (Mut) in HBeAg-positive (eAg+) and -negative (eAg-) Chronic Hepatitis B (CHB) Especially Among Older Patients

M. H. Nguyen; H. N. Trinh; R. T. Garcia; J. Phan; G. H. Nguyen; K. Nguyen; H. Nguyen; E. B. Keeffe 

 

Purpose

Relapse is common following anti-HBe seroconversion and discontinuation of therapy for eAg+ CHB. Presence of an eAg- HBV with preC/BCP Mut among patients who “appear” to have eAg+ CHB may be responsible for such treatment failure. Our goal is to examine the prevalence of preC and BCP Mut in eAg+ CHB patients.

 

Methods

We performed a cross-sectional study of 330 CHB patients with genotypic Mut analysis done between 11/05-5/07 at a U.S. clinic. Mut analysis was performed by Quest Diagnostics, San Juan, CA using a direct sequencing test (test code: 10529x) with reported sensitivity of 25%.

 

Results

A total of 330 cases were identified and included. All but 4 were Asians. Mean age=46±14,61%=male, median ALT=35 (range:7-1070 U/L). Treatment history was as follows: naïve=242 (73.3%), adefovir only=27 (8.2%), lamivudine±adefovir=57 (17.3%), and entecavir=4 (1.2%). Mean HBV DNA was lower in patients with preC/BCP Mut (3.6x107±2.7x108 vs. 1.9x108 ±4.2x108 IU/L, p=0.0002). Distribution of HBV genotypes was as follows: A=4%, B=67%, and C=28%. eAg+ was seen in 38% and eAg- in 62%. Overall prevalence of preC/BCP Mut was 77%: 95% in eAg- and 49% in eAg+. Mut prevalences increase with age (Figure). The majority of preC Mut detected were G1698A (87%) with G1896A/G, G1896G/A, 1839, 1841, 1845, 1874, or mixed in the remaining. The majority of BCP Mut detected were A1762T(A)+G1764A(G,A/G)(90%) with the remaining having single Mut. There were no significant differences PreC/BCP Mut prevalence by gender or treatment groups.

 

Conclusions

The prevalence of PreC/BCP Mut is 77% and increases with age. PreC/BCP Mut prevalence among eAg+ patients is 49% which also increases with age. Patients with mixed wild-type and preC-BCP Mut may be at higher risk for relapse after anti-HBe seroconversion. Baseline Mut analysis may help identify pts who would benefit from long-term therapy despite anti-HBe seroconversion.

 

 


986. A Hepatitis B Virus Mutant Implicated In Non-Response To Sequential Treatment With Adefovir, Lamivudine And Entecavir Shows Cross-Resistance To Multiple Antiviral Agents

T. Shaw; T. Sozzi; F. Wong; S. Locarnini

 

Background

Development of safe and effective nucleos(t)ide analogue reverse transcriptase inhibitors (NRTI) has revolutionised chemotherapy for chronic hepatitis B (CHB), but prolonged treatment engenders viral resistance and sequential treatment with different NRTI can promote the emergence of multi-drug resistant mutants, which are refractory to continuing therapy and may be cross-resistant to new drugs. Consequently, control of multi-drug resistant HBV is becoming a major challenge. We describe the phenotype of an HBV mutant isolated from a patient with CHB who responded poorly to sequential treatments with adefovir, lamivudine, and entecavir. Isolates from this patient were genotype D and HBeAg negative due to the presence of the precore [pc] G1896A mutation and encoded multiple substitutions in the polymerase region including rtA181T, rtI233V, rtN236T and rtM250L. Cloning established that these were encoded on a single genome.

 

Aims

To determine antiviral drug resistance phenotype of the putative multi-drug resistant rtA181T/I233V/N236T/M250L mutant, and determine how rtI233V and rtM250L influence phenotype.

 

Methods

Mutant HBV clones encoding rtI233V, rtM250L, rtA181T/N236T/M250L and rtA181T/I233V/N236T/M250L were generated by site-directed mutagenesis from a wild type (WT) genotype D HBV isolate. WT and derived clones harboured the [pc] G1896A mutation, which may increase replication efficiency but does not significantly affect drug resistance. The phenotypes were determined by comparing viral replication in transfected Huh-7 cells. Virus replication and its inhibition were monitored by measuring changes in the amounts of intracellular core-associated viral DNA in cell lysates using a commercial assay kit (Bayer Versant HBV 3.0).

 

Results

The rtA181T/I233V/N236T/M250L substitutions in combination imparted a severe replication defect but conferred high level resistance to all L-nucleosides (lamivudine, telbivudine, clevudine and emtricitabine) as well as high level resistance to the deoxyguanosine analogues diaminopurine, 2’,3’-dideoxy-3’-fluoroguanosine and abacavir, and conferred lower level, but still significant, resistance to adefovir and tenofovir. The rtI233V and M250L substitutions in isolation did not confer significant drug resistance and significantly reduced replication capacity in the absence of selection pressure, but presumably act to compensate for the replication defects associated with acquisition of drug resistance.

 

Conclusions

Sequential treatment with NRTI promotes multi-drug resistance. It is likely that development of multi-drug resistance could be reduced by combination therapy optimized to individual viral phenotypes.

 


987. Cost-effectiveness of New Treatment Paradigms for HBeAg-negative Chronic Hepatitis B.

A. Kamal; L. B. Gerson; A. Ahmed

 

Background

A number of new drugs for chronic hepatitis B have become available in recent years, and several studies show that treatment with either interferon or the oral nucleos(t)ide analogs is cost-effective compared with no treatment. However, the cost-effectiveness of recent treatment algorithms has not been evaluated.

 

Methods

We considered the base case of a forty year old patient with newly-diagnosed, treatment-naïve, e-Ag negative chronic hepatitis B. Using a decision analysis model, we examined seven treatment strategies: (1) no treatment (natural history arm), (2) Adefovir (ADV) with switch to entecavir (ETV) upon emergence of resistance (3) ETV with switch to ADV upon emergence of resistance, (4) ADV with addition of lamivudine (LAM) upon emergence of resistance, (5) ETV with addition of ADV upon emergence of resistance, (6) ADV with addition of ETV upon emergence of resistance, and (7) Peginterferon alfa-2a for one year. We examined the cost-effectiveness of each option from a third-party payor perspective, using a sixty year time horizon and an annual discount rate of 3%. We then separately considered the case of a similar patient with pre-existing LAM resistance. RESULTS: All treatment options yielded more quality-adjusted life years (QALY’s) than the “no treatment” arm. The oral agents demonstrated extended dominance over pegylated interferon. For treatment-naïve patients, the most cost-effective option was initial ADV with switch to ETV upon emergence of resistance, yielding an incremental cost effectiveness ratio (ICER) of $6,697 over the next most cost-effective strategy. Among the “add-on” regimens, initial ETV with addition of ADV was a dominant strategy, giving the highest number of QALY’s at the lowest cost. For patients with pre-existing LAM resistance, adding ADV was preferred over switching to ADV or ETV, with an ICER of $37,118 per QALY gained.

 

Conclusion

New treatment recommendations for the treatment of e-Ag negative chronic hepatitis B are cost-effective. Initial ADV with switch to ETV upon emergence of ADV resistance is the most cost-effective treatment strategy. Initial ETV with ADV add-on is superior to other “add-on” strategies due to a relatively longer allowable period of monotherapy. In patients who have already developed LAM resistance, adding ADV is cost-effective compared with switching to ADV or ETV, due to a lower rate of subsequent resistance.

 


1005. Cross-resistance Characterization of the Main HBV Drug-resistant Mutants

F. Zoulim; S. Villet

 

Introduction

Currently, four nucleoside analogues are approved for the treatment of chronic hepatitis B: lamivudine, adefovir, entecavir and telbivudine. They suppress viral replication through inhibition of HBV DNA polymerase and chain termination. However, prolonged treatments with these analogues result in the emergence of resistant virus harbouring mutations within the HBV polymerase gene which are associated with resistance to treatment.

 

Objectives

We isolated and cloned HBV DNA genomes from patients who developed viral resistance following various therapies. We determined in vitro the antiviral susceptibility of the main resistance mutants to nucleoside analogues approved for chronic HBV treatment, and tested new nucleoside analogues on these mutants.

 

Results

A bank of mutants, constructed by site directed mutagenesis or from patients serum samples were intensively analyzed. We could observed that all the mutants harbouring the rtM204V substitution, except the rtM204V mutation alone, showed a >1000 fold decrease in susceptibility to lamivudine as compared to wild-type (wt) HBV. We also characterized different mutants harbouring the rtA181V mutation and all these mutants were >2.7 fold less sensitive to adefovir than wt HBV. This in vitro decrease in susceptibility to adefovir seems to be sufficient to induce in vivo viral breakthrough. Concerning entecavir, all the mutants harbouring the rtS202G substitution, but only in addition to rtL180M+M204V main lamivudine resistance mutations showed a >200 fold resistance to this drug in comparison with wt HBV. Tenofovir seems to be one of the most efficient drugs on these mutants, since only two of them (rtN236T and rtL180M+M204V+N236T) showed a decrease in susceptibility to tenofovir as compared to wt HBV. We also tested new nucleoside analogues for their capacity to inhibit replication of drug resistant mutants and demonstrated that PMEO, a novel pyrimidine analogue, showed a better activity than adefovir on all the tested mutants except the rtN236T mutant. Phenotypic characteristics of additional complex mutants were also determined.

 

Conclusion

These data on antiviral susceptibility of the main HBV polymerase mutants observed in patients provide useful information to guide anti-HBV therapy.

 


1006. Detection of Hepatitis B Virus YMDD Mutants by Mltiplex-PCR Using Dual Priming Oligonucleotide (DPO)

P. Ji Young; L. Tae Hoon; P. Jeong Hoon; K. Sang Gyune; P. Do Hyun; L. Suck Ho; J. Jae Young; K. Young Seok; C. Il Kwun; K. Hong Soo; P. Sang Heum; K. Sun Joo; K. Seok Hyun; L. Heon Young; K. Chang Jin; J. Dong Jun

 

Background

The mutants of hepatitis B virus (HBV) emerge selectively during long-term lamivudine therapy. Since lamivudine resistance has cross-resistance with other antiviral agents, initial choice of antiviral agents for prevention of multi-drugs resistance is very important. According to recent studies, early detection of HBV mutants at 3 months is useful to predict the long-term outcomes of lamivudine therapy. In addition, predominance of mutants is significantly associated with viral DNA breakthrough. Therefore, early detection and periodical testing of HBV mutants is needed. However current methods of detecting mutants are time-consuming and labor intensive which require multiple steps and expensive instruments.

 

Aims

To determine the effect of recently developed YMDD genetype method using Multiplex-PCR technology, we compared this method with restriction fragment mass polymorphism (RFMP) and direct sequencing method.

 

Patients and Methods:

Total eighty chronic hepatitis B patients treated with lamivudine more than 6 months participated in this study. YMDD mutants were genotyped by RFMP using MALDI-TOF mass spectrometry, sequencing and Multiplex-PCR using dual priming oligonucleotide (DPO).

 

Results

The Multiplex-PCR method with DPO was available for sensitive, specific and simultaneous detection of 4 types of HBV mutants (rtM204V/I/S, rtL180M) by only one time PCR. Additionally, this assay enabled to estimate relative abundance of mutants in variant/variant mixed or variants/wild type mixed serum by agarose gel electrophoresis. The Multiplex-PCR assay detected as few as 1000 copies/ml of HBV genome plasmids, also detected as little as 2% mutant plasmids. The total protocol was carried out easily within 4 hours. The patients with breakthrough (virologic or biochemical), early viral response to lamivudine therapy, HbeAg positivity, HBV-DNA >100,000 copies/ml and elevated ALT (>2 times upper limit of normal) had higher mutation rate. For these patients, Multiplex-PCR with DPO was the most sensitive method for detection of the mutants (especially mixed variants). Interestingly, 18 of 80 patients (22.5%) had single or mixed variants, these mutants only detected by the Multiplex-PCR, but not by RFMP or sequencing. These results indicate that Multiplex-PCR with DPO is more sensitive than RFMP and sequencing to detect variants.

 

Conclusions

Multiplex-PCR with DPO is rapid, sensitive and cost-effective method for detection of the YMDD mutants and relative quantitation as well. Therefore, this method can be useful diagnostic tool for early detection of HBV mutants and estimate their predominance, thereby preventing drug resistance and early rescue therapy.

 


1007. Five-year Retrospective Survey of HBV Genotypic Drug Resistance Patterns in a Database of 381 HBV Reverse Transcriptase Sequences in Southern France.

P. Colson; R. Gerolami; C. Tourres; A. Motte; I. Ravaux; I. Poizot-Martin; J. Moreau; P. Borentain; M. Henry; C. Tamalet

 

Background/Aims

The growing number of HBV nucleos(t)ide inhibitors, the widening of anti-HBV sequential and/or combination therapies, and the viro-clinical impact of drug resistance mutations warrant surveys of HBV genotypic drug resistance. Therefore, we performed a 5-year retrospective survey during the 2002-04/2007 period aiming to analyse the prevalence and dynamic of HBV reverse transcriptase (rt) drug resistance mutations patterns in patients (pts) chronically-infected with HBV followed-up in Marseilles public hospitals.

 

Patients/Methods

381 HBV rt sequences from 312 pts (70% male; mean age, 47) were analysed. 39 pts have been tested twice, and 12 pts >2 times. For each year from 2002 to 2007, only 1 sequence per >18 year-old pt was selected. Serum HBV DNA direct sequencing was performed using in house protocols. 25 amino acid (aa) positions within domains A-E of HBV rt and previously associated with drug resistance were analysed, which included all positions at which association of aa substitution with drug resistance is clearly established.

 

Results

A nearly 1.7-time increase of the number of HBV sequences was observed in 2006 (n=96) as compared to 2002-2003 (55 and 57, respectively). The mean number of aa substitutions per sequence ranged between 2.1 in 2003 and 1.7 in 2007, without significant change. The proportion of non-A HBV genotypes increased from 53-54% in 2002-2003 to >60% in 2006-04/2007 (p=0.24). The proportion of sequences harbouring >3 aa substitutions was significantly higher for genotypes A vs genotypes D or non-A-non-D (23% vs 11 and 9%, respectively; p<0.05). In contrast, the proportion of aa substitutions was significantly higher at positions 80, 181, 215, and 237 for genotypes D vs genotypes A (p<0.02). The most variable aa positions were rt204 and rt180. Substitution rtM204I/V was found in 71 sequences (19%), associated with L180M in all but one case for M204V and with A181V/T in two cases. Substitution rtA181V/T was found in 4.7% of sequences (n=17) and 6.4% in 2007. The proportion of mutated sequences significantly increased between 2002 and 2007 at positions 236, 237, and 238 (p<0.05). Substitution rtN236T was observed in 6 sequences (1.8%), always associated with rt204 wild-type aa, and was significantly associated with rtA181V and rtI169L (in 2 sequences each; p<10-3).

 

Conclusions

Our data underline the changing pattern of HBV drug resistance mutations and the association or exclusion of rt aa mutations in the setting of emerging nucleos(t)ide inhibitors and combination and/or sequential anti-HBV therapies. They also suggest that genotypic drug resistance patterns might differ according to the HBV genotype.

 


939. The Multi-Drug Resistant HBV rtA181T Has a Secretion Defect and Significantly Reduces the Kinetics of Viral Rebound

S. Locarnini; N. Warner

 

Background and Aims

Lamivudine (LMV) [rtM204V/I] or adefovir (ADV) [rtN236T] resistance results in rebound of serum HBV DNA of at least a 1.0 log10 IU/ml increase over 3-6 months (Locarnini et al. 2004. AVT;9:679). The rtA181T mutant is also associated with both LMV and ADV resistance but occurs as a mixed population with wild-type (wt) HBV. The rtA181T also encodes a stop codon in the overlapping surface gene at aa172 (sW172*) resulting in truncation and loss of the last 54 amino acids of the C-terminal hydrophobic region.

 

The aims of this study were to examine the in vitro effects of this mutation on HBV replication, and its effect on HBV DNA levels in patients following its selection

 

Methods

To measure HBV replication, Huh7 cells were transfected with infectious clones encoding wt HBV, rtA181T or a mixture of both. HBV replication products were detected by Western blotting (WB), Southern blotting (SB), Immunohistochemistry (IHC) and electron microscopy (EM). The clinical occurrence and accompanying viral load data from patients with rtA181T was determined using SeqHepB (Yuen et al. 2007. AVR;75:64).

 

Results

The rtA181T change resulted in a severe secretion defect preventing egress of surface proteins and virions from the cell as demonstrated by SB, WB and IHC. WB also revealed that the surface proteins from the rtA181T transfected cells were truncated and displayed differential glycosylation and stability. When rtA181T was transfected and expressed with wt to mimic a mixed infection as found in vivo, secretion of subviral particles was restored, but virion secretion was still greatly impaired. There was also increased intracellular accumulation by IHC, although not as extensive as with rtA181T alone. EM of cell supernatant detected viral-like structures secreted from doubly-infected cells that comprised mainly of spherical particles, with few virions or filaments compared to wt. Thus, rtA181T is dependant on wt HBV for secretion but acts as a dominant negative mutant on HBV virion secretion resulting in its retention within the cell. Examination of sequential HBV DNA levels from the blood of patients where only the rtA181T change was detected by direct PCR-sequencing, revealed that viral load rebound did not occur at all or increased in incremental amounts of less than 1.0 log10 IU/ml over 12-18 months.

 

Conclusion

The HBV rtA181T mutation causes a secretory defect and exerts a dominant negative effect on wt HBV virion secretion. The rtA181T masks viral rebound, which in the clinical situation can result in mis-diagnosis of drug resistance if only viral load is used (> 1.0 log10 IU/ml increase over 3-6 months) as criterion for breakthrough failure.

 


943. An Updated Line Probe Assay (HBV DR v.3) Has High Concordance with Direct Sequencing in Detecting Mutations Associated with Resistance to Lamivudine, Adefovir and Entecavir

A. S. Lok; B. Degertekin; M. Hussain; J. Tan

 

Background and Aims

Early detection of antiviral drug-resistant (DR) mutations enables prompt initiation of rescue therapy. Direct sequencing is necessary in characterizing mutations to new therapies but it is not sensitive in detecting minor variants. Mutations associated with resistance to entecavir (ETV) had been reported at positions: 184, 202 and 250. We compared the concordance and sensitivity of direct sequencing, sequencing of multiple clones, and a line probe assay in the detection of ETV-resistant mutations.

 

Methods

Serial serum samples from patients who received ETV, lamivudine (LAM) or tenofovir (TDF) were tested for DR mutations in the HBV P gene by direct sequencing, sequencing of multiple clones (mean of 20/sample), and an updated line probe assay (Innogenetics, HBV DR LiPA v.2 and v.3) that incorporates probes to detect mutations at 11 positions: 80, 173, 180, 181, 184, 194, 202, 204, 223, 236 and 250 of the reverse transcriptase region of the HBV P gene.

 

Results

We studied 55 samples from 13 patients: 7 received ETV and 6 received TDF, 12 had prior LAM. Results of direct sequencing and LiPA were concordant in 580/605 (96%) positions analyzed. Complete concordance between results of direct sequencing and LiPA was observed in 34 (62%) samples. In 6 samples, direct sequencing did not detect any mutation while LiPA detected 1 (n=5) or 2 (n=1) mutations. In the remaining 15 samples, LiPA identified 1 or 2 more mutations than direct sequencing.

 

Concordance between results of direct sequencing and LiPA was only 36% when mutations were present in <10% of clones and increased to 98% when mutations were present in >10% of clones.

 

LiPA DR v.3 detected ETV-R mutations in 2/13 patients who had received LAM but not ETV: 1 had S202G+M250I and the other had T184A+M250L mutations. In both patients, these mutations were not detected by direct sequencing but were present in 10% of clones sequenced. Three other patients developed virologic breakthrough after switching to ETV for LAM resistance. All 3 patients were confirmed to have ETV-resistant mutations, LiPA DR v.3 detected these mutations earlier than direct sequencing by 6, 9, and 21 months, respectively.

 

Conclusions

The updated version of the line probe assay (LiPA HBV DR v.3) has high concordance with direct sequencing in detecting mutations associated with resistance to LAM and ETV. The improved sensitivity of LiPA permitted earlier detection of genotypic resistance in patients who had virologic breakthrough on ETV. Screening of patients for ETV-resistant mutations using LiPA may help to determine the optimal therapy for patients with LAM resistance.

 


958. In Vitro Anti-HBV Activity of Telbivudine/Tenofovir and Telbivudine/Entecavir Combinations

A. Patty; B. Li; I. Serra; D. N. Standring; M. Seifer

 

Background

Several nucleoside (entecavir, lamivudine and telbivudine) and nucleotide (adefovir) analogs are approved for the treatment of hepatitis B virus (HBV) infection and several others, including tenofovir, are in clinical development. These drugs are effective against HBV but all have been associated with the emergence of drug-resistant mutants. Combinations of antiviral agents with complementary resistance profiles may offer the potential for enhanced suppression of HBV replication and reduced drug resistance. In this study, the L-nucleoside telbivudine was combined with either tenofovir, a nucleotide analog, or entecavir, a nucleoside analog.

 

Methods

Drug combinations were evaluated in stably transfected human liver cells expressing replication-competent HBV nucleocapsids and virions. The anti-HBV efficacy of each drug alone and in combination was determined using in vitro endogenous HBV polymerase assays after 10 days of drug treatment. In vitro cytotoxicity was determined by standard cell viability staining. MacSynergy II, CombiTool, and isobologram plots were used to analyze the data.

 

Results

Telbivudine/tenofovir and telbivudine/entecavir combinations exerted greater anti-HBV in vitro activity than telbivudine, tenofovir or entecavir alone. Bliss independence analysis (MacSynergy) revealed that the interaction volumes of all 2-drug combinations were <25 μM2 suggesting that the antiviral in vitro interactions between the 2 drugs in each combination are additive. Similar results were obtained with CombiTool (Loewe additivity model); two-drug combination points were clustered around the zero-interaction plane consistent with an interpretation of additivity. Telbivudine/tenofovir exhibited an additive interaction in isobolograms, as implied by D values fluctuating around 0, however an average D value of -0.16 was calculated for telbivudine/entecavir suggesting weak but statistically significant synergy (P = 0.0012). There was no evidence of in vitro cytotoxicity with either of these combinations.

 

Conclusions

Telbivudine demonstrated additive to weakly synergistic anti-HBV activity in vitro when combined with tenofovir or entecavir. These data support the investigation of telbivudine as a component in multi-drug therapy of chronic HBV infection, particularly combinations with drugs exhibiting different resistance patterns such as nucleotide analogs.