| Background:Hepatitis B virus (HBV) infection is a major health problem in the world. There are about 20 million people who have been infected with HBV worldwidely, of which more than 350 million people are chronically infected. HBV infection accounts for annually 1 million deaths worldwide, resulting from cirrhosis, liver failure and hepatocellular carcinoma (HCC). China is a highly endemic area of HBV infection, in which HBsAg-positive rate of the population aged from 1 to 59 years was 7.18%. Chronic HBV infection seriously endangers the health of Chinese. Anti-HBV therapy is the key to the treatment of chronic hepatitis B (CHB) and lamivudine (LAM) is one of commonly used antiviral drugs for Anti-HBV therapy. Its effectiveness has been generally accepted by clinicians as it can prevent the progression of liver diseases and reduce the incidence of HCC. Unfortunately, the inevitable emergence of YMDD mutant (rtM204V/I/S) after long-term lamivudine therapy, which will result in resistance to lamivudine, can largely counteract patients'benefit from lamivunie therapy.Adefovir dipivoxil (ADV) is an oral prodrug of adefovir, a nucleotide analoge that has been shown to have in vitro and in vivo activity against both lamivudine-resistant HBV and wild-type HBV. It has been widely used in the Anti-HBV therapy of patients with lamivudine resistance. PEG interferon a-2a, as a interferferon with long half-time, which can be administrated once a week, can improve the virological response and sustained response rate. It is better than the traditional interferon and is a choice of rescue therapy for patients with resistance to lamivudine.Previous studies have shown that the YMDD mutant will reverse to its wild type after switching to ADV from lamivudine therapy. Few studies have revealed the dynamic changes of YMDD mutant after switching to pegasys from lamivudine therapy.Objective:1. To explore the dynamic changes of YMDD mutant after switching to Pegasys therapy from Lamivudine, and try to find out the differences of dynamic changes of YMDD mutant between patients treated with pegasys and ADV monotherapy.2. To explore the evolutionary pattern of YMDD mutant after withdrawl of lamivudine treatment, try to find out the relationship between Anti-HBV effect and the evolution of YMDD mutant.3. To evaluate the efficacy of ADV monotherapy and in combination with lamivudine in treatment of lamivudine-resistant HBeAg positive CHB. Try to find out some clues to choose appropriate treatment for patients with lamivudine resistance.Methods:1. Reversion of YMDD mutant after cessation of lamivudine therapy.1.1 Source of samples included in present study:All patients included in present study were from one center (Nanfang Hospital) of the clinical trial of ML18376. It was an open label, randomized, multi-center study. All lamivudine-resistant HBeAg positive CHB patients were randomly assigned into two groups by the ratio of 2:1. In our center, twenty five patients of group A (PEG group) received Pegasys treatment (180μg, once a week) for 48 weeks and fifteen patients of group B (ADV group) received ADV treatment for 72 weeks with an initial overlap of 12 weeks lamivudine treatment in all patients.1.2 HBV DNA extraction:Serum samples were collected at week12, week16, week24, week48 and week72. HBV DNA was extracted from serum samples (200μl) by QIAamp DNA Blood Mini Kit.1.3 Amplification of HBV RT region:The RT region of HBV was amplified by nested PCR and the products of the second round were sent to Shanghai Yinjun Company for direct sequencing.1.4 Sequence analysis:Nucleotide sequences available were analyzed by MEGA software, and phylogenetic tree was done with HBV S district for genotyping. HBV sequences of all genotypes loaded from the genbank were used as consensus. Maps of sequencing were analyzed by chromas software.1.5 Statistical analysis:Date analysis was performed with SPSS 13.0 software. Chi-square test was used to compare quantitative data. All P value was two tailed and P<0.05 was considered to be statistically significant.2. Evolution of YMDD mutant after withdrawl of lamivudine therapy.2.1 Source of samples:All of the four patients included in present study were from the ML 18376 clinical trial.2.2 Extraction of HBV DNA and amplification of HBV RT region:HBV DNA was extracted and HBV RT region was amplified as the same as depicted in previous study. Second round PCR products were purified by gel extraction kit for direct sequencing and clone analysis.2.3 Clone analysis of HBV RT region:Purified PCR products were cloned into pMDTM 19-T Vector and then transformed into JM109 competent cells, transformed products were grown on LB plates containing Ampicilin, IPTG and X-Gal. Thirty white clones of each sample were randomly selected and then cultured in LB medium for 6-8 hours at 37℃by shaking at 225 rpm. Positive clones identified by PCR assay were sent to ShangHai Yinjun Company for sequencing. A total of 370 clones from 18 serum samples were sequenced.3. Efficacy of ADV monotherapy and in combination with lamivudine in the treatment of Lamivudine-Resistant HBeAg positive chronic hepatitis B:a comparative study.Fifty two cases of lamivudine-resistant HBeAg positive CHB treated with ADV monotherapy or in combination with lamivudine (24 with monotherapy and 28 with combination therapy) were analyzed retrospectively. Differences between the two groups in HBV DNA, ALT level, rate of HBV DNA undetectability and ALT normalization rate were investigated.Results1. Reversion of YMDD mutant after cessation of lamivudine therapy.1.1Characteristic of HBV gene at the time stopped lamivudine treatment1.1.1 YMDD mutant usually emerges accompanied with secondary mutationsThere were 36 patients infected with YMDD mutant accompanied with secondary mutations such as rtL80V/I, rtL180M or rtL80V/I+rtL180M, which accounts for 90% of all patients.1.1.2 Differences between YVDD and YIDD in choice of secondary mutationsIn present study, there were 17 patients infected with rtL80V/I+rtM204I double mutants and 9 patients with rtL180M+rtM204V double mutants, and the number of patients infected with rtL80V/I+rtM204V and rtL180M+rtM204I double mutants were 8 and 15, respectively. It showed significant differences between YVDD and YIDD in choice of secondary mutations. (χ2=10.059, P=0.002)1.2 YMDD mutant gradually reversed to its wild type after cessation of lamivudine therapyReversion was defined as the replacement of YMDD mutant by wild-type HBV, which was identified by direct sequencing. According to this definition, the reversion rate of YMDD mutant at 4,12,36, and 60 weeks after cessation of LAM therapy were 72.5%(29/40),80%(32/40),87.5%(35/40) and 95%(38/40), respectively.1.3 Effects of treatment regime on the reversion of YMDD mutantAt 4,12,36 and 60 weeks after cessation of lamivudine therapy, reversion rate of YMDD mutant in PEG group were 68.0%(17/25),76.0%(19/25),84.0%(21/25) and 96.0%(24/25), and that of ADV group were 80.0%(12/15),86.7%(13/15),93.3% (14/15) and 93.3%(14/15), respectively. There were no significant difference between the two groups (P value in Chi-Squre test was 0.309,0.685,0.633 and 1.000)1.4 Differences between YVDD and YIDD in the reversion of YMDD mutantReversion rate of YIDD group at 4,12,36 and 60 weeks after cessation of LAM therapy were 66.7%(16/24),79.2%(19/24),83.3%(20/24) and 91.7%(22/24), and that of YVDD group were 81.3%(13/16),81.3%(13/16),93.8%(15/16) and 100% (16/16), respectively. It showed no significant difference between these two groups. (P value in Chi-Squre test was 0.473,1.000,0.631 and 0.508 respectively)1.5 Effects of rtL180M mutant on the reversion of YMDD mutantPatients were divided into rtL180M mutant infected group and rtL180 wild-type HBV infected group by the result of direct sequencing at week 12 (at the time of cessation of lamivudine), and there were 25 patients in the first group and 15 patients in the other group.Revesion rate of YMDD mutant in rtL180M mutant infected group was significantly lower than that of rtL180 wild-type HBV infected group at 4 weeks after cessation of lamivudine treatment.(60.0%(15/25) VS 93.3%(14/15), P=0.03); At 12,36 and 60 weeks after cessation of lamivudine therapy, YMDD mutant reversion rate of rtL180M mutant infected group were lower than that of rtL180 wild-type HBV infected group but without significant difference.(72.0%(18/25),80.0%(20/25), 92.0%(23/25) VS 93.3%(14/15),100%(15/15),100%(15/15), P value in Chi-Squre test was 0.219,0.137and 0.519 respectively.)1.6 Differences between rtL80V/I, rtL180M and rtM204V/I triple mutants group and the other group in the reversion of YMDD mutantPatients were divided into rtL80V/I, rtL180M and rtM204V/I triple mutants infected group and the other group by the result of direct sequencing at week12. There were 14 and 26 patients in these two groups respectively. At 12 and 36 weeks after cessation of lamivudine therapy, YMDD mutant reversion rate of triple mutants infected group were significantly lower than that of the other group (57.1%(8/14), 64.3%(9/14) VS 92.3%(24/26),100%(26/26), P value in Chi-Squre test was 0.014 and 0.003, respectively.) YMDD mutant reversion rate of triple mutants infected group at 4 and 60 weeks after cessation of lamivudine therapy were lower than that of the other group but without significant difference. (57.1%(8/14),85.7%(12/14) VS 80.7%(21/26),100%(26/26), P value in Chi-Squre test was 0.147 and 0.117.)1.7 Simultaneous reversion of YMDD mutant and secondary mutationsSecondary mutations such as rtL80V/I and rtL180M reversed to its wild type with the reversion of YMDD mutant, which was supported by the simultaneous decrease of patients infected with YMDD mutants and secondary mutants.2. Evolution of YMDD mutant after withdrawl of lamivudine therapyPatient 1 received Pegasys treatment (180μg, once a week) for 48 weeks and other three patients received ADV treatment for 72 weeks with an initial overlap of 12 weeks lamivudine treatment in all the four patients.Triple mutant of L80V+L180M+M204I was detected in patient 1 by direct sequencing after emergence of clinical resistance to lamivudine. Clone analysis showed that this mutant accounted for 87.5%(17/20),91%(20/22),95%(18/19), 70%(14/20) and 64%(16/25) in HBV quasispecies at week 12, week 16, week24, week48 and week72, respectively. YMDD-wild HBV, including mutation patterns of L80V/I, L180M, L80V/I+L180M and wild type, accounted for more and more in the viral quasispecies with the increased duration after cessation of lamivudine therapy and reached 30%(6/20) and 32%(8/25) at week 48 and week 72, respectively. In patient 2, mutant of L80I+L180M+M204I was detected and dominated the viral quasispecies all the time, accounted for 47%(8/17),67%(14/21),87.5%(14/16),77% (14/18) and 57%(12/21) in HBV quasispecies at week 12, week 16, week24, week48 and week 72, respectively. Wild type HBV emerges and accounts for 9.5% in the viral quasispecies at week 72.In patient 3, mutant of L80V+L180M+M204I was detected by direct sequencing and revealed by clone analysis to account for 81%(17/21) in the viral quasispecies at week 12. At week 16, wild type HBV became to be the dominant strain and accounted for 94.7%(18/19) in the viral quasispecies. Proportion of wild type HBV in HBV quasispecies were 91%(20/22),67%(14/21) and 69%(15/22) at week 24, week 48 and week 72, respectively. At week 12, the mutant of L80I+L180M+M204I was detected in patient 4, and accounted for 68%(13/19) in the viral quasispecies. Proportion of L80I+L180M+M204I mutant in the viral quasispecies decreased to 33.3%(9/27) because of the emergence of many other mutants, but remained to be the dominant strain in the viral quasispecies at week 16. At week 24, wild type became to be the dominant strain and accounted for 70%(14/20) in the viral quasispecies. At the same time, Proportion of L80I+L180M+M204I mutant in the viral quasispecies decreased to 10%(2/20) at week 24. HBV RT region was not amplified successfully because of the low viral load at week 48 and week 72.3. Efficacy of ADV monotherapy and in combination with lamivudine in the treatment of Lamivudine-Resistant HBeAg positive chronic hepatitis B:a comparative study.There were no significant differences between the two groups in gender, age, pre-treatment HBV DNA and ALT level. ALT level and HBV DNA of the two groups at week 48 were significantly lower than that of pretreatment of the same group, respectively (P<0.05). Significant differences also can be seen between HBV DNA and ALT level at week 72 and that of pretreatment in both of the two groups. At week 48, rate of HBV DNA undetectability of combination group was higher than that of monotherapy group without statistical significance. (50% versus 25%, P>0.05); ALT normalization rate at week 48 was not significantly different between these two groups (67.9% versus 75%, P>0.05). At week 72, HBV DNA undetectability rate of combination group was significantly higher than that of monotherapy group (68% versus 33.3%, P<0.05); Significant difference also can be seen in ALT normalization rate of the two groups (93% versus 70.8%, P<0.05).Conclusions1. Wild-type HBV accounts for more and more in the quasispecies with the extesion of duration after withdrawl of lamivudine therapy and eventually replaces the YMDD mutant to be the dominant strain in the quasispecies.2. Reversion of YMDD mutant to its wild type emerges in 4 weeks after cessation of lamivudine treatment in most cases. Compensatory mutations, such as rtL180M and rtL80V/I, can reduce the reversion rate of YMDD mutant.3. The three mutations rtL80V/I, rtL180M and rtM204V/I usually locate in the same HBV clone and reverse to its wild type simultaneously after cessation of lamivudine therapy.4. ADV monotherapy or in combination with lamivudine is effective in treatment of lamivudine-resistant HBeAg positive CHB. Efficacy of combination therapy is better than that of monothrapy in the long term. 5. It is necessary to combine with LAM in the rescue therapy of patients resistant to LAM, and futher study is needed to explore the efficacy and safety of Pegasys in combination with LAM in treatment of patients resistant to LAM. |
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