Virus Genes (2014) 48:32–37 DOI 10.1007/s11262-013-1004-1

Two rescue therapies in lamivudine-resistant patients with chronic hepatitis B in the central China: adefovir monotherapy and adefovir plus lamivudine Ming Wang • Leyong Yuan • Bin Qiao Yan Li

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Received: 17 August 2013 / Accepted: 21 October 2013 / Published online: 8 November 2013 Ó Springer Science+Business Media New York 2013

Abstract The emergence of mutations that confer drug resistance in patients with chronic hepatitis B (CHB) is increasing in China. We aimed to compare the cumulative efficacy and resistance of adefovir (ADV) monotherapy and ADV add-on lamivudine (LAM) (ADV?LAM) therapy in LAM-resistant patients. One-hundred adult CHB patients with LAM-resistance mutations were identified. Of these 100, 52 patients were treated with ADV monotherapy and 48 were treated with ADV?LAM combination therapy for at least 24 months. After 2-year treatment, the cumulative rates of serum alanine aminotransferase normalization were, respectively, 73.1 and 83.3 % in the ADV monotherapy and ADV?LAM therapy groups (P = 0.216). Additionally, 36 patients receiving ADV plus LAM had hepatitis B e antigen loss/seroconversion, as compared with 30 in patients (P = 0.068). More patients who received LAM plus ADV than those who received ADV alone had HBV DNA levels below 1,000 international unit/milliliters (83.3 vs. 50 %, P \ 0.001). Viral breakthrough and genotypic mutations were detected in 19 (36.5 %) and 9 (18.8 %) patients in the ADV monotherapy and ADV?LAM therapy groups, respectively (P = 0.048). ADV?LAM combination therapy demonstrated faster and significantly greater suppression of HBV DNA compared with ADV therapy alone for patients with LAM-resistance mutations. ADV?LAM was superior to ADV monotherapy in achieving the initial viral breakthrough and genotypic mutations. ADV?LAM combination therapy was rational for most of LAM-resistant Chinese patients with chronic hepatitis B.

M. Wang  L. Yuan  B. Qiao  Y. Li (&) Department of Clinical Laboratory, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuhan 430060, People’s Republic of China e-mail: [email protected]

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Keywords Adefovir  Lamivudine  Hepatitis B e antigen  Chronic hepatitis B

Introduction Chronic hepatitis B (CHB) affects over 400 million people worldwide, constituting an important health burden in areas where hepatitis B virus (HBV) infection is endemic. Up to 40 % of CHB patients may develop cirrhosis and its complications, including hepatocellular carcinoma [1]. Nucleos(t)ide analogs directly suppress HBV replication and retard disease progression [2, 3]. However, nucleos(t)ide analog therapy requires long-term or, in many cases, indefinite treatment, which unfortunately often results in the emergence of mutations that confer drug resistance as HBV appear mutations under the drug selective pressure [4]. Lamivudine (LAM), which was the first nucleos(t)ide analog approved for the treatment of HBV infection, had been used extensively in China due to its low cost. However, the LAM-resistance rate is as high as 23 % after 1 year of treatment and 70–80 % after 4–5 years and the most important mutation involves substitution of methionine for valine or isoleucine (rtM204V/I) in the highly conserved tyrosine–methionine–aspartic acid– aspartic acid (YMDD) motif of the reverse transcriptase domain of HBV polymerase [4]. As rescue therapies, switching to adefovir (ADV) or entecavir (ETV) monotherapy and adding on ADV were once suggested against LAM-resistant HBV [5]. Additionally, incidence of drug resistance after initiation of rescue therapies varied markedly between studies [6–8]. The aim of this article is to investigate various rescue strategies in LAM-resistant patients with CHB in real-world clinical practice in China and compare the incidence of drug resistance among them.

Virus Genes (2014) 48:32–37

Materials and methods

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HBV reverse transcriptase (RT) gene amplification and sequencing

Patients We retrospectively collected clinical data between January 2007 and March 2013 on 100 chronic HBV-infected patients who had developed LAM resistance under monotherapy with LAM and subsequently took nucleos(t)ide analogs as rescue strategy for at least 24 months in our hospital. The rescue therapy regimens for all the patients were chosen based on attending physicians’ own discretion and then were divided into two rescue treatment groups. Of these, 52 patients were treated with ADV monotherapy (ADV 10 mg/day) and 48 were treated with ADV add-on LAM therapy (LAM 100 mg/day and ADV 10 mg/day). All patients had compensated CHB with serum hepatitis B surface antigen (HBsAg) detectable for at least 6 months and serum HBV DNA levels [4 log10 international unit/ milliliters (IU/mL), regardless of hepatitis B e antigen (HBeAg) status. All had received LAM therapy for at least 6 months and LAM-resistance mutations accompanied by virologic breakthrough ([1 log10 increase in serum HBV DNA level from the nadir in a patient who had an initial virological response, [9]). Patients with hepatic decompensation, past or current hepatocellular carcinomas, or liver transplantation were excluded. Other criteria for exclusion were infection with hepatitis A, C, D, E, or human immunodeficiency virus (HIV), or the presence of other forms of liver diseases such as autoimmune or alcoholic liver disease, drug hepatitis, or Wilson’s disease. All patients were followed-up every 2–3 months by clinical examination and biochemical and virologic assessments. Adherence to treatment was assessed (checked with pill count) during each visit to the clinic. Informed written consent for the analysis was obtained from each patient. The study was approved by the ethics committee of Renmin Hospital of Wuhan University.

Biochemical and serological markers and quantification of HBV DNA Serum HBV DNA, HBsAg, HBeAg, anti-HBe, alanine aminotransferase (ALT), total bilirubin concentrations, etc., were routinely detected in the Central Clinical Laboratory of Renmin Hospital of Wuhan University. Among these, HBV DNA level was determined by a popular real-time quantitative PCR (qPCR) kit (Abbott Laboratories, Chicago, IL, USA), according to the manufacturer’s instructions, with a linear dynamic detection range of 1.5 9 101 to 1 9 109 IU/mL.

HBV gene fragment (nucleotides (nt) 54–1,278) encompassing the complete RT gene (nt 130–1,161) was amplified by nested PCR. Primer set 1 50 -AGTCAGGAA GACAGCCTACTCC-30 (sense, nt 3,146–3,167)/50 -AGG TGAAGCGAAGTGCACAC-30 (antisense, nt 1,577–1,596) and primer set 2 50 -TTCCTGCTGGTGGCTCCAGTTC-30 (sense, nt 54–75)/50 -TTCCGCAGTATGGATCGGCAG-30 (antisense, nt 1,258–1,278) were used for first-round PCR and second-round PCR, respectively. The first-round PCR consisted of 10 cycles of 94 °C for 35 s, 59 °C for 35 s (decreasing by 2 °C every other cycle), and 72 °C for 70 s; and 30 cycles of 94 °C for 35 s, 56 °C for 35 s, and 72 °C for 70 s. The second-round PCR (conducted in the same tube) consisted of 35 cycles of 94 °C for 25 s, 56 °C for 25 s, and 72 °C for 50 s. PCR products were purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Sequencing was performed using an ABI 3100 DNA Analyzers (Applied Biosystems, Foster City, CA). Analysis and assembly of sequencing data were performed with the Vector NTI Suite software package (Informax, Frederick, MD, USA).

HBV genotype analysis The genotyping was based on S-gene sequences encompassing the RT domain of HBV, which was amplified as the above. HBV genotype was determined by molecular evolutionary analysis of the viral sequences using the MEGA4 software, which based on the phylogenetic analysis of the S-gene sequence. Standard reference sequences were acquired from the online hepatitis virus database (http:// www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi) as previously reported [10].

Statistical analysis Quantitative variables were expressed as mean, and categorical variables were presented as counts and percentages, and HBV DNA levels were presented as log transformation. Comparisons between groups of quantitative and qualitative variables were performed using the Student’s t test and Chi square test (or Fisher’s exact test), respectively. Data were analyzed using SPSS (version 17.0; SPSS, Inc., Chicago, IL, USA). P values less than 0.05 were considered significant. Flow chart for study methods are shown in Fig. 1.

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Virus Genes (2014) 48:32–37

Fig. 1 Flow chart for study methods

Results

Biochemical response

Baseline characteristics

Serum ALT levels declined in both treatment groups, After 6/12/24 months of rescue treatment, ALT normalization was observed in 65.4 %/70.8 %, 71.2 %/79.2 %, and 73.1 %/83.3 % of patients who received ADV monotherapy and add-on LAM therapy, respectively; and showed no significant difference (Fig. 2a; Table 2; P [ 0.05).

The baseline demographic and disease characteristics of the two rescue treatment groups of ADV monotherapy and ADV add-on LAM therapy were well balanced (Table 1). All patients had genotype C. The mean baseline characteristics of HBV DNA, serum total bilirubin, serum albumin, and serum ALT for the three study groups were comparable. Additionally, the rate of HBeAg positivity, the pattern of the LAM-resistance mutation, age, gender, and the duration of prior LAM treatment were also comparable between the two study groups.

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Virological response The reduction in serum HBV DNA levels from baseline of retreatment was greater in LAM plus ADV than in ADV retreatment alone (Fig. 2b; Table 2). At 6 months, the

Virus Genes (2014) 48:32–37

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Table 1 Baseline characteristics of LAM-resistant patients with CHB before receiving different rescue strategies Characteristics

Age (mean ± SD, years) Gender (male/female, N)

ADV monotherapy group (n = 52) 43.0 ± 10.4 40/12

Serum ALT (mean ± SD, IU/mL)

99.2 ± 39.8

Serum total bilirubin (mean ± SD, lmol/L)

35.8 ± 9.1

ADV add-on LAM group (n = 48) 42.6 ± 9.9 35/13 101.4 ± 37.1 37.4 ± 11.2

Table 2 Virologic, biochemical, and HBeAg responses ADV monotherapy group (n = 52)

P value

ADV add-on LAM group (n = 48)

P value

Proportion of virological response, n (%)

0.839 0.644 0.777 0.435

Month 6

17 (32.7 %)

28 (58.3 %)

0.010

Month 12

23 (44.2 %)

37 (77.1 %)

0.001

Month 24

26 (50.0 %)

40 (83.3 %)

0.001

Normalization of ALT, n (%) Month 6

34 (65.4 %)

34 (70.8 %)

0.560

Month 12 Month 24

37 (71.2 %) 38 (73.1 %)

38 (79.2 %) 40 (83.3 %)

0.355 0.216

HBeAg positivity

43 (82.7 %)

41 (85.4 %)

0.710

Genotype (C/B, N)

37/15

33/15

0.793

Month 6

18 (34.6 %)

24 (50.0 %)

0.119

Serum HBV DNA (mean ± SD, log10 IU/mL)

5.14 ± 1.10

5.05 ± 1.14

0.700

Month 12

25 (48.1 %)

32 (66.7 %)

0.061

Month 24

30 (57.5 %)

36 (75.0 %)

0.068

LAM-resistant mutations rtM204I/V rtM204I/V?(rtL80I/M and/or rtL180 M and/or rtV173L)

20 (38.5 %) 32 (61.5 %)

17 (35.4 %) 31 (64.6 %)

Age, serum ALT, serum total bilirubin, and serum HBV DNA using the Student’s t test; Gender, HBeAg positivity, genotype, and LAMresistant mutations using Chi square test ADV adefovir, LAM lamivudine, ALT alanine aminotransferase, HBeAg hepatitis B e antigen, HBV hepatitis B virus, SD standard deviation

HBeAg loss/seroconversion n (%)

Proportion of virological response, Normalization of ALT and HBeAg loss/seroconversion using Chi square test

receiving LAM plus ADV as compared to 44.2 % (23/52) of patients receiving LAM alone. At 24 months, the proportion of patients with virological response was achieved in 83.3 % (40/48) of patients receiving LAM plus ADV as compared to 50.0 % (26/52) of patients receiving LAM alone. The difference in virological response between two groups was statistic significant at 6, 12, and 24 months, respectively (P = 0.010 for month 6, P = 0.001 for month 12, and P = 0.001 for month 24). HBeAg response

proportion of patients with virological response (HBV DNA \3 log10 IU/ml) was achieved in 58.3 % (28/48) of patients receiving LAM plus ADV retreatment as compared to 32.7 % (17/52) of patients receiving ADV alone. At 12 months, the proportion of patients with virological response was achieved in 77.1 % (37/48) of patients

After 6/12/24 months of rescue treatment, the rate of patients who achieved HBeAg loss/seroconversion was 34.6 %/50.0 %, 48.1/66.7, and 57.5.0 %/75.0 % in patients treated with ADV monotherapy and ADV add-on LAM therapy, respectively (Fig. 2c; Table 2; P [ 0.05).

Fig. 2 a Biochemical response rates, b Virological response rates and c HBeAg loss and seroconversion rates in patients receiving ADV alone or ADV?LAM during the 2 years of retreatment

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Table 3 Viral breakthrough and genotypic mutations ADV monotherapy group (n = 52)

ADV add-on LAM group (n = 48)

P value

Pattern of LAM-resistant mutations (24 months) rtM204I/V

7 (13.5 %)

2 (4.2 %)

rtM204I/V?(rtL80I/M and/or rtL180 M and/or rtV173L)

12 (23.1 %)

7 (14.6)

Total mutation

19 (36.5 %)

9 (18.8 %)

0.048

Pattern of ADV-resistant mutation (24 months) rtA181T

2 (3.8 %)

1 (2.1 %)

rtA181V

8 (15.4 %)

4 (8.3 %)

rtN236T

4 (7.7 %)

2 (4.2 %)

rtA181T?rtN236T

3 (5.8 %)

2 (4.2 %)

rtA181V?rtN236T Total mutation

2 (3.8 %) 19 (36.5 %)

0 9 (18.8 %)

0.048

Pattern of LAM/ADV-resistant mutations using Chi square test

Viral breakthrough and genotypic mutations Viral breakthrough and genotypic mutations at 24 months after the initiation of each antiviral treatment were as follows. In ADV monotherapy group, the most frequently detected mutations of LAM-resistant mutations or ADV resistance were rtM204I/V?(rtL80I/M and/or rtL180 M and/or rtV173L) (12/19), rtA181V (8/19), respectively. In ADV add-on LAM therapy group, rtM204I/V?(rtL80I/M and/or rtL180 M and/or rtV173L), rtA181V were more frequently detected (7/9, 4/9), respectively (see Table 3).

Discussion Current treatment of CHB with NA rarely eradicates HBV, necessitating long-term therapy to avoid viral reactivation and the progression of liver decompensation. However, such long-term treatment has been associated with the emergence of drug-resistant viral strains [11]. For mutants resistant to one NA, switching to another NA may exert a selective pressure on the viral quasi-species, allowing additional mutations to occur in preselected mutants, resulting in multidrug resistance [12]. A new and emerging concept in the management of antiviral resistance is the superiority of add-on therapy rather than switching therapy, in preventing subsequent multidrug resistance [11]. This concept came from the studies of patients with LAMresistant HBV who were managed by switching to ADV monotherapy or by adding adefovir to ongoing lamivudine [13, 14]. In those studies, the rates of viral breakthrough

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and genotypic resistance to ADV were significantly higher in the group which was switched to ADV than in the addon group, thus underscoring the benefit of combination therapy in preventing the development of multi-drug resistance. A similar study about rescue therapy for lamivudineresistant patients with chronic hepatitis B used risk factors indentified on multivariable analyses, a simple index for future GR (antiviral resistance prediction index, ARPI) was developed with the rescue therapy regimens, HBV DNA reduction at 12 weeks, and the initial HBV DNA level [5]. However, in this study, we explored and compared retreatment options for chronic hepatitis B patients with viral relapse after cessation of LAM. After 2-year retreatment, the proportion of ALT normalization was achieved in 83.3 % of patients receiving LAM plus ADV as compared to 73.1 % of patients receiving ADV alone and there was no significant difference between them. Moreover, the proportion of HBeAg loss or seroconversion was also reached 75.0 % in LAM plus ADV as compared to 57.5 % of patients receiving ADV alone and there was no significant difference between them. Several other studies also had reported that the combination of LAM and ADV could lead to effective viral suppression in most cases after development of viral breakthrough due to ADV monotherapy [15, 16]; and patients receiving LAM plus ADV combination therapy have a lower risk of developing genotypic resistance to ADV [16–19]. In our study, we found that incidence of viral break-through and genotypic mutations in the group of switching to ADV monotherapy was much higher than that in the group of adding on ADV (respectively, 36.5 and 18.8 %) at 24 months after the initiation of each antiviral treatment. Additionally, the proportion of virological response was achieved in 83.3 % of patients receiving LAM plus ADV as compared to 50.0 % of patients receiving ADV alone. Those findings gave us another hint that the combination of LAM plus ADV was associated with lower antiviral resistance compared with ADV alone. In fact, for some special populations with chronic hepatitis B, the combination of agents without cross-resistance had been clearly put forward by authoritative guidelines [20, 21], and data from many clinical observational trials also suggested combination therapy would bring more benefits to nucleosides or nucleotides-refractory CHB patients. In conclusion, ADV?LAM combination therapy has demonstrated faster and significantly greater suppression of HBV DNA compared with ADV monotherapy for patients with LAM-resistance mutation. ADV?LAM is superior to ADV monotherapy in achieving the initial virological response, viral breakthrough, and genotypic mutations. ADV?LAM combination therapy was rational for most of LAM-resistant Chinese patients with chronic hepatitis B.

Virus Genes (2014) 48:32–37 Acknowledgments This work was supported by a Grant from the National Clinical Key Specialty Construction Projects to the Department of Clinical Laboratory of Renmin Hospital of Wuhan University.

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9. 10.

Conflict of interest interest exists.

The authors have declared that no competing 11. 12.

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