Fung, JYY

Lai, CL

Wong, DKH

Yuen, RMF

Hong Kong Medical Journal

HKU Scholars Hub

Title Identification of hepatitis B virus DNA reverse transcriptasevariants associated with partial response to entecavirAuthor(s) Wong, DKH; Fung, JYY; Lai, CL; Yuen, RMFCitation Hong Kong Medical Journal, 2015, v. 21 n. 3, Suppl. 4, p. 35-38Issued Date 2015URL http://hdl.handle.net/10722/214352Rights Hong Kong Medical Journal. Copyright © Hong Kong Academyof Medicine Press.35Hong Kong Med J  ⎥  Volume 21 Number 3 (Supplement 4)  ⎥  June 2015  ⎥  www.hkmj.orgK e y  M e s s a g e s 1. Entecavir is a potent antiviral agent against hepatitis B virus (HBV). In a cohort of 370 entecavir-treated chronic hepatitis B patients, 76 (21%) had a partial virological response, defined by detectable HBV DNA at year 1.2. In HBV reverse transcriptase sequence analysis, 20 amino acid variants were identified solely in the partial responders.3. There were 4 reverse transcriptase (rt) amino acid variants that had significantly different distribution between the optimal and partial entecavir responders.4. HBV quasispecies complexity and diversity were Identification of hepatitis B virus DNA reverse transcriptase variants associated with partial response to entecavirIntroductionNucleos(t)ide analogues (NA) are potent antiviral agents against hepatitis B virus (HBV) infection, drastically reducing viral load and improving clinical outcome. Lamivudine, adefovir, entecavir, telbivudine, and tenofovir are licensed in Hong Kong for treatment of chronic hepatitis B. Some patients have high HBV DNA, even at the initial stage.1 One explanation is that some intrinsic viral properties may confer non-responsiveness to a particular NA. As the main target of NAs is HBV reverse transcriptase (rt), some natural polymorphisms in the HBV rt sequences may confer primary non-responsiveness.  HBV in a mixed viral population is known as quasispecies. Viral complexity and diversity change dynamically during the natural history of chronic hepatitis B infection. Studies of their effect on response to antiviral agents are rare. This study aimed to investigate whether there are some pre-treatment HBV rt sequence variations that predict response to entecavir. In addition, the association between quasispecies complexity and diversity and entecavir response were evaluated.MethodsThis study was conducted from May 2009 to November 2011. Between January 2002 and September 2009, 370 chronic hepatitis B patients at Queen Mary Hospital underwent daily entecavir (0.5 mg) therapy and were followed up for >1 year. Hong Kong Med J 2015;21(Suppl 4):S35-8RFCID project number: 08070852DKH Wong *, J Fung, CL Lai, MF YuenDKH Wong *, J Fung, CL Lai, MF YuenDepartment of Medicine, The University of Hong Kong* Principal applicant and corresponding author: danywong@hku.hkNone had previously received NA or interferon/pegylated-interferon therapy. Patients with severe liver disease (chronic hepatitis C and autoimmune and alcoholic liver disease) were excluded.  Patient HBV DNA was measured at baseline and year 1 using the COBAS TaqMan HBV Test (Roche Diagnostics), with a lower limit and upper limit of detection of 60 copies/mL and 6.4 ×108 copies/mL, respectively. Optimal virological response was defined as profound HBV DNA suppression to ≤60 copies/mL at the end of the first year. Partial virological response was defined as a HBV DNA level of >60 copies/mL at year 1.  Baseline HBV rt sequence was amplified using a High Fidelity Taq Polymerase, followed by DNA sequencing. To identify sequence variants present at <20% in the viral population, HBV amplicons of the partial responders and randomly selected optimal responders were subjected to clonal sequencing. Differences in the pretreatment DNA polymorphism and HBV quasispecies complexity and diversity between the optimal and partial responders were noted. ResultsBaseline characteristicsAmong the 370 (129 HBeAg-positive and 241 HBeAg-negative) patients, 76 (21%) exhibited a partial virological response to entecavir at year 1. Of these partial responders, 41% (53/129) were HBeAg-positive and 9.5% (23/241) were HBeAg-negative RESEARCH FUND FOR THE CONTROL OF INFECTIOUS DISEASEShigher in the optimal responders than in the partial responders.5. In multivariate analysis, high baseline HBV DNA, hepatitis Be antigen-positivity, and rt variant rt124N were significantly associated with partial entecavir response.  #  Wong et al #36 Hong Kong Med J  ⎥  Volume 21 Number 3 (Supplement 4)  ⎥  June 2015  ⎥  www.hkmj.org Hong Kong Med J  ⎥  Volume 21 Number 3 (Supplement 4)  ⎥  June 2015  ⎥  www.hkmj.org(P<0.0001). The gender ratio, baseline alanine transaminase (ALT), albumin, and bilirubin levels were comparable between the optimal and partial responders (Table 1). The mean age was higher in the HBeAg-negative optimal responders than partial responders (P=0.026). The partial responders had a higher baseline HBV DNA level than the optimal responders for HBeAg-positive (P<0.001) and HBeAg-negative (P=0.001) patients.HBV rt sequence analysisHBV rt DNA was successfully amplified by PCR in 305 patients (114 HBeAg-positive and 191 HBeAg-negative). Of these, 64 (21%) [47 HBeAg-positive and 17 HBeAg-negative] had a partial response to entecavir at year 1. For the partial responders, the median HBV DNA level at year 1 was 639 copies/mL (range, 72-2.98×107 copies/mL), and the median baseline HBV DNA level was higher than that of the optimal responders (HBeAg-positive patients: 8.81 vs 7.83 log10 copies/mL, P<0.001; HBeAg-negative patients: 8.34 vs 6.62 log10 copies/mL, P=0.001). Of the 344 amino acid residues in the HBV rt region, 217 were conserved among these 305 patients. Major known drug resistance mutations (rtL80I/V, I169T, V173L, L180M, A181V/T, T184G, A194T, S202I, M204I/V, N236T, and M250V) were not detected, except in one patient in whom concomitant rtL80I and rtL180M mutations were detected. Twenty amino acid variations (rt7D, rt7N, rt12Y, rt18K, rt53I, rt80I, rt118H, rt127W, rt128I, rt131S, rt135F, rt139Q, rt153W, rt180M, rt217R, TABLE 1.  Baseline characteristics of the optimal and partial entecavir respondersTABLE 2.  Distribution of reverse transcriptase (rt) polymorphisms found exclusively in partial responders receiving entecavir treatment for hepatitis B virus (HBV)Gender Age at baseline (years)Baseline HBeAg statusHBV genotype Baseline HBV DNA (copies/mL)Year 1 HBV DNA (copies/mL)rt variations exclusively found in partial respondersM 62.9 Positive A >6.4 ×108 5.5 ×105 rt7D + 53I + 139Q + 153W +217RM 62.3 Negative C 2.3 ×108 3.6 ×102 rt7NF 53.8 Positive C >6.4 ×108 4.4 ×103 rt12YM 22.3 Positive B 1.0 ×108 1.6 ×102 rt18KM 34.6 Positive C 1.0 ×108 2.4 ×102 rt18KM 48.2 Positive C 1.1 ×108 2.1 ×102 rt80I + 180MM 37.1 Negative B 2.2 ×108 8.2 ×101 rt118HM 28.7 Positive B >6.4 ×108 1.3 ×103 rt127WM 37.6 Negative B >6.4 ×108 2.7 ×102 rt128IM 45.7 Positive B >6.4 ×108 2.3 ×102 rt131SM 31.4 Positive B 1.0 ×108 1.6 ×102 rt135FM 51.8 Positive C 8.0 ×106 2.1 ×103 rt153WM 54.8 Negative C 1.5 ×105 8.5 ×101 rt219TF 52.5 Negative B >6.4 ×108 4.5 ×102 rt233LF 35.1 Positive C >6.4 ×108 4.5 ×102 rt322IM 22.0 Positive C 1.0 ×108 5.1 ×104 rt329NF 39.9 Positive C >6.4 ×108 1.8 ×103 rt336QCharacteristic HBeAg-positive patients (n=129) HBeAg-negative patients (n=241)Optimal responders (n=76)Partial responders (n=53)P value Optimal responders (n=218)Partial responders (n=23)P valueMean±SD age (years) 42.6±12.3 40.7±10.8 0.36 51.3±9.3 46.8±8.4 0.026No. of males:females 55:21 33:20 0.225 147:71 19:4 0.161Median (range) hepatitis B virus DNA (log10 copies/mL)7.76 (4.40-8.81) 8.81 (5.41-8.81) <0.001 6.43 (3.10-8.81) 7.64 (4.94-8.81) 0.001Median (range) alanine transaminase (IU/L) 92.5 (12-2019) 107 (20-2144) 0.271 71.5 (11-3000) 93 (38-961) 0.133Median (range) albumin (g/L) 42 (16-50) 42 (25-48) 0.657 42 (23-51) 42 (30-48) 0.763Median (range) bilirubin (mmol/L) 14 (6-52) 12 (2-67) 0.162 11 (4-261) 14 (5-216) 0.086Hong Kong Med J  ⎥  Volume 21 Number 3 (Supplement 4)  ⎥  June 2015  ⎥  www.hkmj.org#  HBV DNA reverse transcriptase variants associated with partial response to entecavir  # 37Hong Kong Med J  ⎥  Volume 21 Number 3 (Supplement 4)  ⎥  June 2015  ⎥  www.hkmj.orgrt219T, rt233L, rt322I, rt329N, and rt336Q) were found exclusively in partial responders. These variations were distributed among 17 partial responders (Table 2). Variants rt18K and rt153W were found in two cases, and all other variants were found only once. Due to the rarity, association between these polymorphisms and partial/slow response was not significantly different and needed to be determined by phenotypic assays. The distribution of some rt amino acid variations differed significantly between optimal and partial responders. Specifically, 17 amino acid variants were present at a significantly higher frequency in 64 partial responders than in the 241 optimal responders (Table 3). After controlling for false discovery during multiple comparisons using Bonferroni correction, four rt variants, namely rt 53N, rt118N, rt124N, and rt332S, remained to have a significantly higher frequency in the partial responders than in the optimal responders (Table 3). Multivariate analysis was performed to determine the independent factors associated with partial entecavir response at year 1. All factors with significantly different distribution among the partial and optimal responders were analysed, namely baseline HBeAg status, age, HBV genotype, baseline HBV DNA level, rt53N, rt118N, rt124N, and rt332S (the four rt variants with differential distribution) (Table 3). Multivariate logistic regression analysis revealed that high baseline HBV DNA levels (P<0.0001, OR=2.30, 95% CI=1.60-3.34), HBeAg-positivity (P<0.001, OR=3.68, 95% CI=1.79-7.62), and rt variant rt124N (P=0.001, OR=3.09, 95% CI=1.54-6.18) were independently associated with suboptimal entecavir response at year 1.Minor sequence variations identified by clonal sequencing Clonal sequencing was performed on the 64 partial responders and 34 randomly selected optimal responders. Among the polymorphisms found exclusively in the partial responders (Table 2), the variant rt18K was detected as minor species in five additional cases, rt118H was detected in one additional case, rt128I in two additional cases, rt131S in four additional cases, rt135F in one additional case, and rt153W in one additional case.HBV Quasispecies complexity and diversityQuasispecies complexity and diversity were compared between the optimal and partial responders. The optimal responders had a higher quasispecies complexity (higher normalised Shannon entropy) than the partial responders at the nucleotide level (P=0.036) and at the amino acid level (P=0.087) [Table 4]. Similarly, the optimal responders had a higher quasispecies diversity than the partial responders (all P<0.05, Table 4).DiscussionEntecavir is a potent nucleoside analogue that TABLE 3.  Reverse transcriptase (rt) variants with significantly different distribution among partial responders and optimal respondersrt variant % of variants found in partial responders% of variants found in optimal respondersP value (unadjusted) P value (after Bonferroni correction)rt9H 68.8 53.0 0.025 1rt16T 50.0 29.6 0.002 0.254rt53N 51.6 27.4 0.00024 0.031rt109S 54.7 35.3 0.005 0.635rt118N 48.4 25.3 0.00034 0.043rt121I 51.6 29.5 0.001 0.127rt124N 50.0 23.7 0.000038 0.0048rt127R 46.9 29.0 0.007 0.889rt131N 50.0 28.2 0.001 0.127rt134N 35.9 16.2 0.00048 0.061rt151Y 51.6 30.3 0.001 0.127rt221Y 53.1 37.8 0.026 1rt222A 40.6 21.6 0.002 0.254rt238H 50.0 31.1 0.005 0.635rt271M 48.4 26.1 0.001 0.127rt278V 70.3 54.8 0.025 1rt332S 43.8 20.1 0.00010 0.013  #  Wong et al #38 Hong Kong Med J  ⎥  Volume 21 Number 3 (Supplement 4)  ⎥  June 2015  ⎥  www.hkmj.orgsuppresses HBV DNA replication. The present study showed that 21% of patients still had detectable HBV DNA after 1 year of therapy. HBV rt sequence analysis of 64 partial responders and 241 optimal responders showed that 20 HBV rt variants were present exclusively in partial responders. However, these variants occurred only in one or two cases, and the association was not significant. In addition to the 20 variants found solely in partial responders, four variants were present in a higher proportion in the partial responders than in the optimal responders. Multivariate analysis showed that high baseline HBV DNA, HBeAg-positivity, and the rt variant rt124N were independent factors associated with slow entecavir response. This is consistent with our previous study in which patients with positive HBeAg and HBV DNA of >8 log copies/mL had a lower rate of HBV DNA undetectability at year 1 to 3.1 rt124N variant was associated with partial entecavir response, even though only 50% of the partial responders harboured this variant. Further long-term study is needed to demonstrate the possible role of rt124N and the variants identified solely in the partial responders in susceptibility to entecavir. Two case reports have identified HBV rt variants S219A + Y245H and S246T in entecavir non-responders.2,3 Phenotypic assays determined that the rt variant pattern S219A + Y245H (rather than S246T) are associated with a lower susceptibility to entecavir in vitro. The present study did not identify S219A + Y245H variants. Nevertheless, the findings of rt124N and the 20 ‘unique’ variants may provide a preliminary identification of putative variant pattern of reduced susceptibility to entecavir. In the clonal sequencing data, the optimal responders had a significantly higher quasispecies complexity and diversity than the partial responders. It is possible that in the optimal responders a higher complexity and diversity was present. There may be a ‘less-fit’ subpopulation of viral quasispecies more susceptible to entecavir. In contrast, in the partial responders, the viral population was less diverse and more replication competent, causing a slower entecavir response. Further studies using technology with a greater sequencing depth (such as ultra-deep pyrosequencing or next generation sequencing) are needed to further elucidate the association between quasispecies complexity/diversity and treatment response.ConclusionsWe identified 20 rt variants found exclusively in entecavir partial responders and four rt variants with significantly different distribution among the optimal and partial responders. High baseline HBV DNA levels, HBeAg-positivity, and rt124N were associated with partial entecavir response at year 1. The possible association between rt variants and entecavir response needs to be proved by in vitro phenotypic studies.AcknowledgementsThis study was supported by the Research Fund for the Control of Infectious Diseases, Food and Health Bureau, Hong Kong SAR Government (#08070852). We thank Dr Daniel Fong for his advice on statistical analysis. Results of this study have been published in: Wong DK, Kopaniszen M, Omagari K, et al. Effect of hepatitis B virus reverse transcriptase variations on entecavir treatment response. J Infect Dis 2014;210:701-7.References1. Yuen MF, Seto WK, Fung J, Wong DK, Yuen JC, Lai CL. Three years of continuous entecavir therapy in treatment-naive chronic hepatitis B patients: VIRAL suppression, viral resistance, and clinical safety. Am J Gastroenterol 2011;106:1264-71.2. Karatayli E, Karatayli SC, Cinar K, et al. Molecular characterization of a novel entecavir mutation pattern isolated from a multi-drug refractory patient with chronic hepatitis B infection. J Clin Virol 2012;53:130-4.3. Hu JL, Cui J, Guo JJ, et al. Phenotypic assay of a hepatitis B virus strain carrying an rtS246T variant using a new strategy. J Med Virol 2012;84:34-43.Quasispecies Optimal responders (n=34) Partial responders (n=63) P valueComplexity: normalised Shannon entropyMean (range) nucleotide level 0.9673 (0.6868-1) 0.9316 (0.3324-1) 0.036Mean (range) amino acid level 0.8668 (0.4930-1) 0.7869 (0.2192-1) 0.087DiversityMean (range) genetic distance (nucleotide level) [10-3 substitutions] 8.7083 (3.2747-36.4403) 5.2904 (0.8906-56.1354) 0.019Mean (range) genetic distance (amino acid level) [10-3 substitutions] 14.0147 (3.3561-66.2545) 8.6151 (0.8923-94.822) 0.032Mean (range) No. of synonymous substitutions per site (10-3) 12.4076 (3.8811-42.0139) 7.4706 (0.6533-78.0112) 0.015Mean (range) No. of non-synonymous substitutions per site (10-3) 6.6121 (1.6396-30.8776) 3.9649 (0.4040-43.8353) 0.039TABLE 4.  Quasispecies complexity and diversity of the entecavir optimal and partial responders

Identification of hepatitis B virus DNA reverse transcriptase variants associated with partial response to entecavir

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Identification of hepatitis B virus DNA reverse transcriptase variants associated with partial response to entecavir

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