Viral evolutionary changes during tenofovir treatment in a chronic hepatitis B patient with sequential nucleos(t)ide therapy

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Case Report

Viral evolutionary changes during tenofovir treatment in a chronic hepatitis B patient with sequential nucleos(t)ide therapy Hyun Woong Lee, Hye Young Chang, Suh Yoon Yang, Hyung Joon Kim ∗ Department of Internal Medicine, Chung-Ang University College of Medicine, Seoul, Republic of Korea

a r t i c l e

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Article history: Received 6 January 2014 Received in revised form 27 March 2014 Accepted 29 March 2014 Keywords: Tenofovir Chronic hepatitis B Resistance Mutation

a b s t r a c t A 54-year-old man diagnosed with HBeAg-positive chronic hepatitis B (CHB) was treated with entecavir (ETV) 1 mg/day following an initial unsuccessful lamivudine (LAM) treatment (rtL180M, rtM204V/I). Subsequently, virological breakthrough with ETV mutation (rtT184A/L) developed. The LAM and adefovir combination therapy was followed by virological breakthrough. The therapy had been switched to TDF monotherapy. However, this patient experienced virological breakthrough under TDF with a HBV strain bearing rtL80M, rtL180M, rtM204V/I, rtA200V, rtF221Y, rtS223A, rtT184A/L, rtR153Q, and rtV191I combined mutations without rtA194T mutation. TDF resistance may emerge due to multi-site polymerase mutations rather than single-site polymerase mutation. © 2014 Elsevier B.V. All rights reserved.

1. Why this cases is important? Tenofovir (TDF) has shown promising antiviral efficacy in patients with lamivudine (LAM) resistance and in patients with incomplete response to adefovir (ADV) without genotypic resistance [1,2]. To date, the development of TDF resistance has been identified only very rarely, in two hepatitis B virus (HBV) – human immunodeficiency virus (HIV) co-infected patients with the rtA194T nucleotide exchange in the HBV polymerase [3]. The rtA194T mutation was reported to confer a reduced susceptibility to TDF in the presence of LAM-associated mutation in vitro [3]. In addition, precore (PC) and basal core promoter (BCP) mutations reportedly enhance the reduced replicative capacity of rtA194T mutants [4]. However, in another report, this mutation did not confer in vitro resistance to TDF as a single mutation or in LAM-resistant viral background [5]. It is very difficult to explain this discrepancy. We experienced one case of virological and biochemical breakthrough during TDF treatment of a chronic hepatitis B (CHB) patient who received sequential nucleos(t)ide therapy. Interestingly, the

Abbreviations: ADV, adefovir; ALT, alanine aminotrasferase; Anti-HBe, hepatitis B virus e antibody; AST, aspartate aminotrasferase; CHB, chronic hepatitis B; CT, computed tomography; ETV, entecavir; HBsAg, hepatitis B virus surface antigen; HBV, hepatitis B virus; LAM, lamivudine; NA, nucleos(t)ide analog; U/S, ultrasonography. ∗ Corresponding author at: Department of Internal Medicine, Chung-Ang University College of Medicine, 224-1 Heuk Seok-Dong, Dongjak-Ku, Seoul 156-755, Republic of Korea. Tel.: +82 2 6299 1417; fax: +82 2 6299 1137. E-mail address: [email protected] (H.J. Kim).

evolution of multi-drug resistant mutation may be associated with the virological and biochemical breakthrough, despite the lack of known rtA194T mutation. In this report, we describe this case in detail. 2. Case report The patient was a 54-year-old Korean man who first visited Chung-Ang University in July 2007. At a local clinic, LAM therapy was initiated when HBV viral load rapidly rose to 8.0 log10 copies/mL and alanine aminotransferase (ALT) was elevated to 82 IU/L (normal level ≤40 IU/L) at age 52. After 1 year, the HBV DNA level was lower than 5.1 log10 copies/mL as determined using the Hybrid Capture II HBV DNA assay (Digene Diagnostics, Bestivelle, MD, USA) and the ALT level was normal. However, after 2 years of continuous treatment, HBV DNA was detectable (5.7 log10 copies/mL) and ALT was slightly elevated to 46 IU/L. The patient was referred to our hospital for further evaluation. The laboratory data at the first visit were ALT 88 IU/L. The tests for HBeAg were positive. The serum HBV DNA level determined by the AmplicorTM Monitor PCR assay (lower limit of detection, 140 copies/mL; Roche Diagnostics, Basel, Switzerland) was 8.2 log10 copies/mL. HBV genotype was determined by INNOLiPA HBV Genotyping assay (Innogenetics, Belgium). He was infected with HBV genotype C. Because of the development of LAM mutation (rtL180M, rtM204V/I), the LAM regimen was replaced by entecavir (ETV) 1.0 mg/day monotherapy. However, virological breakthrough with ETV mutation (rtT184A/L) developed after 120 weeks. The therapy had been switched to LAM and ADV for 24 weeks. This led

http://dx.doi.org/10.1016/j.jcv.2014.03.018 1386-6532/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Lee HW, et al. Viral evolutionary changes during tenofovir treatment in a chronic hepatitis B patient with sequential nucleos(t)ide therapy. J Clin Virol (2014), http://dx.doi.org/10.1016/j.jcv.2014.03.018

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Fig. 1. Clinical course of a 54-year-old man with nucleos(t)ide experienced chronic hepatitis B during tenofovir rescue therapy. The therapy had been switched to TDF 300 mg/day monotherapy with adequate adherence for 1 year. However, the levels of HBV DNA and ALT rebounded from 3.8 log10 copies/mL and 23 IU/mL, respectively, at week 24–7.0 log10 copies/mL and 357 IU/mL, respectively, at week 48. HBV DNA level in solid line and ALT levels in dashed line are plotted against time in weeks from the start of each antiviral drugs. Direct DNA sequence analysis of amplified PCR products from serum samples obtained at 14 different weeks of sequential therapy was performed. Abbreviations: ADV, adefovir; ALT, alanine aminotransferase; ETV, entecavir; HBV, hepatitis B virus; LAM-R, lamivudine resistance; TDF, tenofovir.

only to a temporary HBV DNA decline, which was soon followed by virological breakthrough, despite the lack of known ADV resistance mutations. The therapy had been switched to TDF 300 mg/day monotherapy for 1 year. He underwent regular follow-ups and performed with good compliance. However, the levels of HBV DNA and ALT rebounded from 3.8 log10 copies/mL and 23 IU/mL, respectively, at week 24 to 7.0 log10 copies/mL and 357 IU/mL, respectively, at week 48. The compliance was also confirmed during clinical breakthrough. Virological and biochemical breakthrough developed in spite of the lack of known rtA194T mutation (Fig. 1). Direct DNA sequence analysis of amplified PCR products from serum samples obtained at 14 different weeks of sequential therapy revealed the mutations shown in Table 1. Sequence analysis showed that rtL180M, rtM204V/I, rtA200V, rtF221Y, rtS223A with a BCP mutation were detected continuously. The rtT184A/L mutation was detected intermittently. A longitudinal clonal analysis of

HBV genomes was performed by evaluating a total of 50 clones from five different serum samples (Table 2). Samples 7, 10, and 12 were taken at development of virological and biochemical breakthrough of ETV, LAM + ADV, and TDF treatment, respectively. Sample 13 and 14 were taken at 3 and 9 months after ETV + TDF treatment. The dominant viral strain continuously revealed rtL80M, rtL180M, rtM204V/I, rtA200V, rtF221Y, rtS223A, rtT184A/L, rtR153Q, and rtV191I mutations (Table 3). The new dominant viral strain was not detected in sample 12 at the development of TDF resistance. Its proportion in the quasispecies pool was slightly increased in sample 12 compared to in sample 7 and 10. In sample 14, the proportion of variants significantly decreased but variants did not disappear in spite of the combination treatment of ETV + TDF (Table 1). Although clinical data are lacking, European Association for the study of the Liver guideline recommend that in patients with multi-drug resistance, a combination of a nucleoside and a nucleotide (preferably

Table 1 Direct DNA sequence analysis of the HBV polymerase gene from serum samples obtained at 14 different weeks of sequential therapy. Amino acid residuesa

RT region LAM resistant mutation L180 A200 M204 F221 S223 ETV resistant mutation T184 S202 M250 ADV resistant mutation A181 N236 TDF resistant mutation A194 BCP/Pre C region A1762 G1764 G1896 a

Sample number 1

2

3

4

5

6

7

8

9

10

11

12

13

14

M V V/I Y A

M V V/I Y A

M V V/I Y A

M V V/I Y A

M V V/I Y A

M V V/I Y A

M V V/I Y A

M V V/I Y A

M V V/I Y A

M V V/I Y A

M V V/I Y A

M V V/I Y A

M V V/I Y A

L/M V M/V/I F/Y A

L S M

T/L S M

T S M

T S M

T S M

T/L S M

T/A/L S M

L S M

T/L S M

T/L S M

T/L S M

T/L S M

T/L S M

T S M

A N

A N

A N

A N

A N

A N

A N

A N

A N

A N

A N

A N

A N

A N

A

A

A

A

A

A

A

A

A

A

A

A

A

A

T A G

T A G

T A G

T A G

T A G

T A G

T A G

T A G

T A G

T A G

T A G

T A G

T A G

T A G

The reference gene of HBV genotype C was NCBI GenBank Nos. DQ683578, AY123041, which was considered the wild type.

Please cite this article in press as: Lee HW, et al. Viral evolutionary changes during tenofovir treatment in a chronic hepatitis B patient with sequential nucleos(t)ide therapy. J Clin Virol (2014), http://dx.doi.org/10.1016/j.jcv.2014.03.018

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Table 2 Hepatitis B virus polymerase gene variants characterized by clonal analysis of 50 clones from five different serum samples. Sample number (clones)

Amino acid residues (DQ683578, AY123041)

L80 L84 I91 D134 F151 R153 H160 F166 L180 T184 V191 A200 M204 F221 S223 N238 N248 M250 V253 S256 L269 7 (n = 10) Pattern Number 10 (n = 10) Pattern Number 12 (n = 10) Pattern Number 13 (n = 10) Pattern Number 14 (n = 10) Pattern Number

– –

M 4

F 1

– –

Y 4

Q 2

R 1

L 1

M 10

A/L 4/3

I 2

V 3

V/I 8/2

Y 10

A 10

–

H 1

I 1

A 1

G 1

I 1

– –

M 1

F 1

A 1

Y 7

Q 1

– –

– –

M 10

A/L 1/7

I 1

V 7

V/I 8/2

Y 10

A 10

– –

H 1

I 1

– –

– –

I 10

– –

M 5

F 5

A 5

Y 1

Q 5

– –

– –

M 10

A/L 2/3

I 4

V 2

V/I 8/2

Y 10

A 10

H 1

– –

– –

– –

– –

– –

– –

M 3

F 3

A 3

Y 3

Q 1

– –

– –

M 10

L 3

I 1

V 3

V/I 9/1

Y 10

A 10

– –

– –

– –

– –

– –

– –

– –

– –

– –

– –

– –

– –

– –

– –

M 5

– –

I 1

V 1

V/I 4/1

Y 5

A 10

– –

– –

– –

– –

– –

– –

tenofovir) should be used [6,7]. At 48 weeks, ETV 1.0 mg was added to TDF to decrease the risk of a hepatitis flare and to reduce the risk of subsequent TDF resistance. The serum HBV DNA level declined to 2.6 log10 copies/mL at week 60, and the serum ALT level remained normal.

with HIV and HBV co-infection showed genotypic resistance to TDF [3]. Nevertheless, this mutation was not verified in another study [5].

3. Other similar and contrasting cases in the literature

We experienced the first case of virological and biochemical breakthrough without rtA194T mutation during TDF treatment of a CHB patient who received sequential nucleos(t)ide therapy. Serum samples obtained at different months of sequential therapy were analyzed to investigate new TDF resistant mutations of HBV reverse transcriptase region using direct DNA sequencing assay. Although the rtL180M, rtM204V/I, rtA200V, rtF221Y, rtS223A with BCP mutation were continuously detected in serial samples, new dominant viral strain was not detected at the development of TDF resistance. Three mutations (rtA200V, rtF221Y, rtS223A) were apparent and might be compensatory mutations for replication fitness [9].

Although ETV has a high genetic barrier to resistance, three mutations (rtL180M, rtM204V/I, and a single mutation at rtT184, rtS202, or rtM250) are needed for resistance [8]. Studies have demonstrated the mutations of adefovir resistance (A181T/V, N236T) to be associated with reduced sensitivity to TDF [1,2]. However, the genetic barrier of TDF is not yet fully clarified because resistance mutations have not been identified through 4 years of treatment. In vitro, the combination of rtA194T, rtM204V, and precore mutation induced the partial resistance to TDF. Two patients

4. Discussion

Table 3 Evolution of multi-drug resistant mutations: clonal analyses of serial samples. Sample number (50 clones)

Course of treatment

Mutations of clonal analysis

Number

7 (10 clones)

Time at breakthrough during ETV 1 mg

L80M + L180M + M204I + F221Y + S223A L80M + L180M + M204I + F221Y + S223A + T184A L80M + L180M + M204I + A200V + F221Y + S223A + T184A + F151Y + N248H L80M + L180M + M204I + A200V + F221Y + S223A + T184A + F151Y + S256G L80M + L180M + M204I + A200V + F221Y + S223A + V84M + T184A + F151Y L80M + L180M + M204I + A200V + F221Y + S223A + V84M + R153Q + V191I L80M + L180M + M204I + F221Y + S223A + V84M + T184A + M250I + V253A + S256G L80M + L180M + M204I + F221Y + S223A + V84M + I91F + F151Y + R153Q + F166L + V191I

1 3 1 1 1 1 1 1

10 (10 clones)

Time at breakthrough during LAM + ADV

L80M + L180M + M204I + F221Y + S223A + T184L + L269I L80M + L180M + M204I + F221Y + S223A + M250I + F151Y + L269I L80M + L180M + M204I + A200V + F221Y + S223A + T184L + F151Y + L269I L80M + L180M + M204I + A200V + F221Y + S223A + T184L + F151Y + N248H + L269I L80M + L180M + M204I + F221Y + S223A + V84M + I91F + D134A + R153Q + V191I + L269I

2 1 5 1 1

12 (10 clones)

Time at breakthrough during TDF

L80M + L180M + M204V + F221Y + S223A + L269I L80M + L180M + M204V + F221Y + S223A + T184A + L269I L80M + L180M + M204V + F221Y + S223A + L269 + V84M + I91F + D134A + R153Q + V191I L80M + L180M + M204V + A200V + F221Y + S223 + V84M + I91F + D134A + F151Y + R153Q + L269I L80M + L180M + M204V + A200V + F221Y + S223A + V84M + T184A + I91F + D134A + R153Q + V191I + N238H + L269I

1 4 3 1 1

13 (10 clones)

Time at during ETV 1 mg + TDF

L80M + L180M + M204V + F221Y + S223A L80M + L180M + M204V + F221Y + S223A + T184L L80M + L180M + M204V + A200V + F221Y + S223A + T184L + F151Y L80M + L180M + M204V + F221Y + S223A + V84M + I91F + D134A + R153Q + V191I L80M + L180M + M204V + A200V + F221Y + S223A + V84M + I91F + D134A + F151Y L80M + L180M + M204V + A200V + F221Y + S223A + V84M + T184L + I91F + D134A + F151Y

5 1 1 1 1 1

ETV, entecavir; LAM, lamivudine; ADV, adefovir; TDF, tenofovir.

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Especially, the mutations with ADV resistance have been reported to be associated with mild decrease in TDF susceptibility. However, these mutations also did not exist. A longitudinal clonal analysis of HBV genomes was performed to find a new HBV genome pattern displaying resistance to TDF. Unfortunately, new dominant mutation did not show up in the clones sequenced until the last serum sample of the patient where TDF resistance was documented. International guidelines do not recommend sequential therapy with nucleos(t)ide analog (NA) when resistance is suspected. It is recommended to add a second NA with a non-overlapping resistance profile. We suggest that patients with persistent HBV DNA after prolonged, sequential antiviral treatment should be closely evaluated for the development of TDF resistance variants. High rate of mutations coupled with the rapid production of HBV can result in daily generation of all single substitutions at each site of the HBV genome or even all possible double substitutions [10]. Therefore, potential TDF resistance substitutions may readily emerge during the course of HBV infection in patients who have been exposed to sequential therapy. We experienced one case who developed virological breakthrough under TDF with a HBV strain bearing rtL80M, rtL180M, rtM204V/I, rtA200V, rtF221Y, rtS223A, rtT184A/L, rtR153Q, and rtV191I combined mutations without previous reported rtA194T mutation. Despite TDF’s potent antiviral activity and high genetic barrier to resistance, patients with high pretreatment viral load and sustained detectable HBV DNA while treating with several HBV polymerase inhibitors sequentially should be carefully monitored for TDF resistance. In addition, the patient’s compliance needs to be assured before resistance testing with TDF treatment. Contributors Hyun Woong Lee analyzed the data and wrote the paper; Hye Young Chang performed experiments. Hyung Joon Kim and Suh Yoon Yang analyzed the data and edited the manuscript.

Funding None. Competing interests The authors declare no competing interests related to this manuscript. Ethical approval The patient gave consent to the publication of the case. References [1] Patterson SJ, George J, Strasser SI, Lee AU, Sievert W, Nicoll AJ, et al. Tenofovir disoproxil fumarate rescue therapy following failure of both lamivudine and adefovir dipivoxil in chronic hepatitis B. Gut 2011;60(2):247–54. [2] van Bommel F, de Man RA, Wedemeyer H, Deterding K, Petersen J, Buggisch P, et al. Long-term efficacy of tenofovir monotherapy for hepatitis B virus-monoinfected patients after failure of nucleoside/nucleotide analogues. Hepatology 2010;51(1):73–80. [3] Sheldon J, Camino N, Rodes B, Bartholomeusz A, Kuiper M, Tacke F, et al. Selection of hepatitis B virus polymerase mutations in HIV-coinfected patients treated with tenofovir. Antivir Ther 2005;10(6):727–34. [4] Amini-Bavil-Olyaee S, Herbers U, Sheldon J, Luedde T, Trautwein C, Tacke F. The rtA194T polymerase mutation impacts viral replication and susceptibility to tenofovir in hepatitis B e antigen-positive and hepatitis B e antigen-negative hepatitis B virus strains. Hepatology 2009;49(4):1158–65. [5] Delaney 4th WE, Ray AS, Yang H, Qi X, Xiong S, Zhu Y, et al. Intracellular metabolism and in vitro activity of tenofovir against hepatitis B virus. Antimicrob Agents Chemother 2006;50(7):2471–7. [6] EASL clinical practice guidelines: management of chronic hepatitis B virus infection. J Hepatol 2012;57(1):167–85. [7] KASL Clinical Practice Guidelines: management of chronic hepatitis B. Clin Mol Hepatol 2012;18(2):109–62. [8] Gish R, Jia JD, Locarnini S, Zoulim F. Selection of chronic hepatitis B therapy with high barrier to resistance. Lancet Infect Dis 2012;12(4):341–53. [9] Rhee SY, Margeridon-Thermet S, Nguyen MH, Liu TF, Kagan RM, Beggel B, et al. Hepatitis B virus reverse transcriptase sequence variant database for sequence analysis and mutation discovery. Antiviral Res 2010;88(3):269–75. [10] Thai H, Campo DS, Lara J, Dimitrova Z, Ramachandran S, Xia G, et al. Convergence and coevolution of hepatitis B virus drug resistance. Nat Commun 2012;3:789.

Please cite this article in press as: Lee HW, et al. Viral evolutionary changes during tenofovir treatment in a chronic hepatitis B patient with sequential nucleos(t)ide therapy. J Clin Virol (2014), http://dx.doi.org/10.1016/j.jcv.2014.03.018