Evolution Of YMDD Mutant And Its Impact On Virologic Response During Rescue Therapy In Chronic Hepatitis B Patients With Lamivudine Resistance

BackgroundAnti-HBV therapy is the key to the treatment of chronic hepatitis B (CHB). and lamivudine (LAM) was the first effective oral hepatitis B virus (HBV) replication suppressive agent approved in the treatment of chronic hepatitis B. It can markedly reduce serum HBV DNA levels, enhance hepatitis B e antigen (HBeAg) seroconversion, and is associated with improvement in liver necroinflammatory activity. It can prevent the progress of liver diseases and reduce the incidence of HCC. It is likely to remain the first choice of therapy for economic reasons, particularly in the developing countries. However, viral resistance to Lamivudine with the emergence of YMDD variants during long-term Lamivudine therapy is a vital problem for optimal therapy. Genotypic resistant mutations have been detected in14-32%of patients after one year of treatment, which increases to38%,49%, and66%after2,3, and4years of treatment, respectively. Continued treatment with LAM in patients who have developed virologic resistance is of no benefit. To avoid clinical relapses that may be severe and occasionally life threatening, an appropriate rescue therapy should be initiated that has the most effective antiviral effect and minimal risk for selection of multidrug resistant (MDR) strains. Unfortunately, the inevitably emergence of YMDD mutant (rtM204V/I/S) after long-term lamivudine therapy, which will result in resistance to lamivudine, can largely eliminate patients’benefit from lamivunie therapy.Adefovir dipivoxil (ADV) is a nucleotide analoge, which has been shown to be effective against both wild-type and LAM-resistant HBV both in vitro and in vivo. LAM resistance patients have been treated with ADV either in monotherapy or in combination with LAM. Peginterferon alfa-2a, as an interferferon with long half-time, administrated once a week, is better than the traditional interferon. Peginterferon has been shown to be highly active against wild-type HBV infection, both HBeAg-positive and HBeAg-negative. Interferon has multiple sites of action in the viral life cycle and previous study confirmed that peginterferon is also active against LAM-resistant HBV. Initial data also suggested that interferon-based therapy (peginterferon alfa-2a) provide a novel therapeutic option for management of lamivudine resistant patients.However, not all patients respond to the rescue therapy. Previous studies revealed that LAM-resistant mutants disappeared and wild-type HBV reappeared during rescue therapy. Evolution of LAM-resistant mutants appears to be important in determining the efficacy of rescue therapy in patients with LAM-resistant mutants. However, to our knowledge, the clinical significance of the reversion is not yet known. Furthermore, few studies have revealed the dynamic changes of YMDD mutant during peginterferon-2a administration in LAM-resistant patients.The aim of this study was to determine the dynamic changes of YMDD mutant and the effect of early reversion of rtM204V/I on virologic and biochemical response of rescue therapy.Objectives1. To determine the dynamic changes of YMDD mutant during peginterferon alfa-2a or adefovir administration. 2. To explore the baseline factors that may affect the reversion of rtM204V/I to wild-type HBV.3. To evaluate the effect of YMDD reversion of rtM204V/I on virologic and biochemical reponse of rescue therapy.Methods1. Study populationPatients in the present study were from a randomized, open-label study, which was carried out in13centers across china (www.controlled-tri als.com. ISRCTN79659320). Male or female patients, aged18-65years, with LAM-resistant (tyrosine-methionine-aspartate-aspartate (YMDD) mutant as determined by the INNO-LipA method) HBeAg-positive CHB who had received lamivudine for at least6months and were still receiving lamivudine at the start of the trial were eligible for inclusion.Patients were randomized to weekly subcutaneous injections of peginterferon alfa-2a180μg for48weeks or once-daily oral adefovir10mg for72weeks by the ratio of2:1. Patients in both treatment groups continued lamivudine100mg/day for the first12weeks.A total of124patients were included in our study based on the availibility of serum.79patients were adefovir-treated, while45patients were peginterferon alfa-2a-treated.2. Extraction of HBV DNASerum samples were collected at baseline, week12, week24, week48and week72. HBV DNA was extracted from serum samples (200μl) by QIAamp DNA Blood Mini Kit.3. Amplification of HBV polymerase geneThe nucleotide sequences of HBV polymerase gene were determined using a nested polymerase chain reaction (PCR) and direct sequencing at baseline, week12, week24, week48and also week72. The nucleotide sequences of the PCR products were determined by Invitrogen Ltd.(ShangHai China).4. Sequence analysesFor comparisons of the nucleotide sequences and deduced amino acid sequence, the reference sequences were got from Genebank and aligned with our sequence data using CLUSTAL-W. Phylogenic analysis was also conducted to determine the HBV genotype using Neighbor-joining algorithm. The reliability of phylogenetic tree was tested by bootstrap analysis with1000replicates. All of these analyses were performed by MEGA4.0software and chromas software.5. Statistical analysesThe independent-samples t-test was used for the quantitative variables. The Chi-square test or Fisher’s exact test was applied for categorical variables. Cumulative YMDD reversion rate were analyzed by Kaplan-Meier method and difference were evaluated by long-rank test. All data was analyzed using the statistical package SPSS (version17.0, SPSS Inc., Chicago, IL). Any p values below0.05were considered statistically significant (Two-tailed).Results1. Evolution of YMDD mutant during rescue therapy1.1Reversion rate of YMDD mutantReversion of YMDD mutant was defined as wild-type HBV dominant in the pool of HBV quasi-species by direct sequencing. According to this definition, reversion of rtM204I/V to wild-type HBV occurred in6%(5/79),38%(30/79),52%(41/79), and57%(45/79) of adefovir-treated patients at12,24,48and72weeks of therapy, respectively. The reversion occurred in7%(3/45),53%(24/45),73%(33/45), and91%(41/45) of peginterferon alfa-2a-treated patients at12,24,48and72weeks of therapy, respectively.1.2Reversion of YMDD mutant and adefovir resistanceThere were four patients developed adefovir resistance during72weeks of therapy in adefovir-treated patients. The rtA181T mutant emerged in three patients and rtA181V mutant emerged in one patients. Interestingly, all four patients developed adefovir resistance after reversion to wild-type HBV.1.3Reversion of compensatory mutants to wild-typeCompensatory mutations such as rtL80V/I and rtL180M also reversed to wild-type accompanying with the reversion of YMDD mutant.2.Baseline factors related with reversion to wild-type HBV2.1Effects of rtL80V/I mutant on the reversion of YMDD mutantPatients were divided into two groups:rt80L group (n=66) and rt80V/I group (n=58). Reversion of rtM204I/V to wild-type HBV occurred in6.1%(4/66),45.5%(30/66),65.2%(43/66) and75.8%(50/66) of patients from rt80L group at12,24,48and72weeks of therapy, respectively. The reversion occurred in6.9%)(4/58),41.4%(24/58),53.4%(31/58), and62.1%(36/58) of patients from rt80V/I group at12,24,48and72weeks of therapy, respectively. There was no significant difference on the reversion rate between two groups (χ2=0.694, P=0.405).2.2Effects of rtL180M mutant on the reversion of YMDD mutantPatients were divided into two groups:rt180L group (n=40) and rt180M group (n=84). Reversion of rtM204I/V to wild-type HBV occurred in7.5%(3/40),50.0%(20/40),67.5%(27/40), and75.0%(30/40) of patients from rt180L group at12,24,48and72weeks of therapy, respectively. The reversion occurred in6.0%(5/84),40.5%(34/84),60.0%(47/84), and66.7%(56/84) of patients from rt180M group at12,24,48and72weeks of therapy, respectively. There was no significant difference on the reversion rate between two groups (χ2=3.098, P=0.078). 2.3Effects of treatment regime on the reversion of YMDD mutantThere were45patients in the peginterferon alfa-2a-treated group, while79patients were adefovir-treated. Reversion to wild-type HBV occurred in7%(3/45),53%(24/45),73%(33/45) and91%(41/45) of peginterferon alfa-2a-treated patients at12,24,48and72weeks of therapy, respectively. The reversion rates from YMDD mutants to wild type infection were6%(5/79),38%(30/79),52%(41/79) and57%(45/79) of adefovir-treated patients at12,24,48and72weeks of therapy, respectively. There was significant difference on the reversion rate between two groups (χ2=3.876, P=0.049).2.4Reversion rate of YIDD mutant group and YVDD mutant groupSequence analysis showed that46patients were YVDD mutant,61patients were YIDD mutant. The rest17patients were YVDD/YIDD mixed mutant. To analyses the effect of YMDD mutant pattern on the reversion rate, these17patients were divided into YVDD mutant or YIDD mutant according to the top wave on the maps of the sequence. Generally,53patients were YVDD mutant, and71patients were YIDD mutant. Reversion to wild-type HBV occurred in7.5%(4/53),50.9%(27/53),71.7%(38/53), and79.2%(42/53) of YVDD mutant patients at12,24,48and72weeks of therapy, respectively. The reversion rates from YMDD mutants to wild type infection were5.6%(4/71),38.0%(27/71),50.7%(36/71),62.0%(44/71) of YIDD mutant patients at12,24,48and72weeks of therapy, respectively. It showed no significant difference between these two groups (χ2=2.509, P=0.113).2.5Effect of the number of compensatory mutants (rtL80V/I, rtL180M) on the reversion of YMDD mutantPatients were divided into three groups according to the number of the compensatory mutants:13patients without compensated mutants were included in group A;81patients with one compensated mutants were included in group B; and30 patients with two compensated mutants were included in group C. Reversion to wild-type HBV occurred in15.4%(2/13),38.5%(5/13),61.5%(8/13),76.9%(10/13) of patients from group A at12.24,48and72weeks of therapy, respectively. The reversion rates from YMDD mutants to wild type infection were3.7%(3/81),46.9%(38/81),61.7%(50/81),71.6%(58/81) of patients from group B at12,24,48and72weeks of therapy, respectively. The reversion rates from YMDD mutants to wild type infection were10.0%(3/30),30.0%(9/30,40.0%(12/30),50.0%(15/30) of patients from group C at12,24,48and72weeks of therapy, respectively. The difference between the three groups was statistically significant (χ2=4.214, P=0.04).2.6Effect of genotype on the YMDD reversionTwo genotypes were found in this study:32patients were genotype B; and92patients were genotype C. Reversion to wild-type HBV occurred in9.4%(3/32),56.3%(18/32),75.0%(24/32),84.3%(27/32) of genotype B patients at12,24,48and72weeks of therapy, respectively. The reversion rates from YMDD mutants to wild type infection were5.4%(5/92),39.1%(36/92),54.3%(50/92),64.1%(59/92) of genotype C patients at12,24,48and72weeks of therapy, respectively. The difference between the two groups was statistically significant (χ2=5.194, P=0.023).2.7Effect of HBV DNA level on the YMDD reversionPatients were divided into three groups according to the baseline HBV DNA level:39patients with HBV DNA<7.0log10IU/mL were included in group A;54patients with7.0log10IU/mL≤HBV DNA<8.0log10IU/mL were included in group B; and31patients with HBV DNA≥8.0log10IU/mL were included in group C. Reversion to wild-type HBV occurred in7.7%(3/39),48.7%(19/39),66.7%(26/39),74.4%(29/39) of patients from group A at12,24,48and72weeks of therapy, respectively. The reversion rates from YMDD mutants to wild type infection were 9.3%(5/54),44.4%(24/54),57.4%(31/54),68.5%(37/54) of patients from group B at12,24,48and72weeks of therapy, respectively. The reversion rates from YMDD mutants to wild type infection were0.0%(0/31),35.5%(11/31),54.8%(17/31),64.5%(20/31) of patients from group C at12,24,48and72weeks of therapy, respectively. The difference between the three groups was statistically significant (χ2=6.295, P=0.043).2.8Bias in selection of compensatory mutations of rtM204V/IIn71patients of YIDD mutant, there were70%(50/71) with L80V/I mutation, and44%(31/71) with L180M mutation. While in the patients of YVDD mutant, there were15%(8/53) with L80V/I mutation, and100%(53/53) with L180M mutation. The rtL80V/I substitutions were more frequent in conjunction with rtM204I than with rtM204V (χ2=37.314, P<0.001), while the rtL180M was in conjunction with rtM204V(χ2=40.078, P<0.001).3.Effect of YMDD reversion on the virologic and biochemical response of rescue therapyPatients were divided into early reversion group and non-early reversion group. Early reversion group was defined as rtM204V/I reversion to wild-type within week24, other patients were classified as non-early reversion group. Finally,110of124patients were included in the analysis,70from adefovir group and40from peginterferon alfa-2a group. The rest14patients were not applicable to the analysis for the PCR negative at week24.3.1Effect of reversion on virological and biochemical response in adefovir-treated patientsBased on the definition above,30patients were classified into early reversion group, another40patients in the non-early reversion group. There was no significant difference between early reversion group and non-early reversion group in terms of baseline HBV DNA, age, gender, genotype, YMDD mutation pattern, frequency of rtL80V/I and rtL180M mutations. The HBV DNA levels over72weeks of therapy were compared between the two groups.The mean HBV DNA reduction at week72showed significant differences between patients with and without early reversion (t=-3.255, P=0.002). The ALT normalization rate showed difference between patients with and without reversion (χ2=3.717, P=0.054). At the end of therapy,10patients in the non-early reversion group achieved complete viral response (HBV DNA level≤60IU/mL), in contrast with only2patients in the early reversion group (χ2=4.057, P=0.044).3.2Effect of reversion on virological and biochemical response in peginterferon alfa-2a-treated patientsIn peginterferon group,24patients were classified into early reversion group, another16patients in the non-early reversion group. There was no significant difference between early reversion group and non-early reversion group in terms of baseline HBV DNA, age, gender, genotype, YMDD mutation pattern, frequency of rtL80V/I and rtL180M mutations. HBV DNA levels over48weeks of therapy and24weeks of follow-up were compared between two groups. The mean HBV DNA reduction at week48showed no significant difference between patients with and without early reversion (t=-0.883, P=0.383). The ALT normalization rate showed no significant difference between patients with and without reversion (χ2=1.778, P=0.182). At the end of therapy, there were5patients in the non-early reversion group achieved complete viral response, while2patients in the early reversion group (χ20.462, P=0.681).Conclusions1. Reversion of rtM204V/I occurred frequently within48weeks both in adefovir-treated patients and in peginterferon alfa-2a-treated patients.2. The incidence of adefovir resistance was higher in lamivudine-resistant patients treated with adefovir than in treatment-naive patients after adefovir therapy. Interestingly, all four patients developed adefovir resistance after reversion to wild-type HBV.3. Compensatory mutations (rtL80V/I or rtL180M) and rtM204V/I usually locate in the same HBV clone and reverse to wild type accompanying with the YMDD reversion.4. Generally, the factors that may affect the reversion were the number of compensatory mutations, genotype, HBV DNA level, and treatment arm. It was difficult for YMDD reversion in patients with more compensatory mutations, patients of genotype C, patients with higher HBV DNA level and patients who were treated with peginterferon alfa-2a.5. In my study, the effect of reversion on virologic response is dependent on the strategy of rescue therapy. Early reversion is associated with suboptimal response to adefovir. Therefore, other treatment strategies should be considered for patients with early reversion. But it has no significant effect on virologic and biochemical response induced by peginterferon alfa-2a. Maybe peginterferon alfa-2a is no a good choice to the rescue therapy in lamivudine resistant patients.6. Our results showed bias in selection of compensatory mutations of rtM204V/I: rtM204I with more rtL80V/I while rtM204V with more rtL180M as compensatory mutations.