Department of Internal Medicine, Konkuk University School of Medicine, Konkuk University Hospital, Seoul, South Korea 2

Antiviral Therapy 9 1:95 993 (doi: 1.351/IMP117) Original article Evolution of hepatitis B virus mutation during entecavir rescue therapy in patients with antiviral resistance to lamivudine and adefovir Won Hyeok Choe 1,, Sun Pyo Hong 3, Byung Kook Kim 1, Soon Young Ko 1, Young Kul Jung, Ji Hoon Kim, Jong Eun Yeon, Kwan Soo Byun, Kyun-Hwan Kim,5, Seung Il Ji 3, Soo-Ok Kim 3, Chang Hong Lee 1, and So Young Kwon 1, * 1 Department of Internal Medicine, Konkuk University School of Medicine, Konkuk University Hospital, Seoul, South Korea Center for Cancer Research and Diagnostic Medicine, Institute of Biomedical Science and Technology, Konkuk University, Seoul, South Korea 3 Research and Development Center, GeneMatrix, Inc., Yongin, South Korea Department of Internal Medicine, Korea University College of Medicine, Guro Hospital, Seoul, South Korea 5 Department of Pharmacology, Konkuk University School of Medicine, Seoul, South Korea *Corresponding author: e-mail: sykwonmd@kuh.ac.kr These authors made an equal contribution to this work Background: The efficacy of entecavir (ETV) monotherapy in treatment-experienced patients with chronic hepatitis B (CHB) is debatable. Methods: A total of hepatitis B e antigen (HBeAg)- positive CHB patients who had shown viral breakthrough or suboptimal response with lamivudine () and adefovir disoproxil (ADV) therapy were treated with 1. mg of ETV. Clinical and virological parameters were monitored every 3 months. Restriction fragment mass polymorphism assays were used to detect antiviral resistance. Results: During and ADV therapy, 11 patients had rtmv/i mutations, had rta11v/t or rtn3t, 7 had both and had no - or ADV-related mutations. After switching to ETV monotherapy, the median change in serum hepatitis B virus (HBV) DNA level was -.1 log 1. Virological response (HBV DNA<3 ) was achieved in 1 of 1 patients with pre-existing rt mutations, whereas it was achieved in all patients without pre-existing rt mutations regardless of the presence of rt11 or rt3 mutations. Changes in mutational patterns during ETV therapy showed that rt mutations persisted or re-emerged. Relative abundances of rtmv/i mutations in total viral populations gradually increased under ETV rescue, whereas those with rta11v/t and rtn3t mutations decreased. ETV resistance mutations (rtl1m+rtt1i/l[rtsg]+rtmv) were detected in five patients with pre-existing rt mutations. Conclusions: ETV monotherapy resulted in a limited virological response in patients who had previously failed and ADV rescue therapy. The limited efficacy might be associated with residual or reselected rtmv/i mutations leading to ETV resistance. Combination treatment including potent antiviral agents should be recommended for patients with pre-existing rtmv/i mutations. Introduction Hepatitis B virus (HBV) replication has a key role in the disease progression and development of hepatocellular carcinoma (HCC) in chronic hepatitis B (CHB) patients [1 3]; therefore, the major goal of anti-hbv therapy is the sustained suppression of HBV replication without the emergence of drug resistance mutations and the prevention of hepatic decompensation and HCC []. Lamivudine () was the first oral agent approved for the treatment of CHB patients and was initially successful in suppressing HBV DNA [5]; however, the wide use of monotherapy during the past 1 years has led to a progressive increase in the number of patients with resistance because of its poor resistance profile []. Adefovir dipivoxil (ADV) was the second oral anti-hbv agent approved for first-line therapy or rescue therapy for resistance; however, long-term treatment with ADV has led to the emergence of ADV resistance [7]. Moreover, a higher rate of ADV resistance has been reported in patients with pre-existing - resistant mutations compared with treatment- 9 International Medical Press 1359-535 (print) -5 (online) 95

WH Choe et al. naive patients, especially in those treated with ADV monotherapy []. Recently, there has been increasing evidence indicating that the addition of ADV to continued therapy is associated with a lower rate of emergence of ADV resistance [9,1], but does not result in significantly improved viral suppression [9]. Entecavir (ETV) is a recently approved oral agent for treatment-naive or treatment-experienced CHB patients. ETV has an excellent potency and a high genetic barrier to resistance in treatment-naive patients [11,1]. The high genetic barrier to resistance is attributable to the requirement for three amino acid substitution mutations for resistance to occur, including two of the mutations associated with resistance (MV/I and L1M) plus an additional ETV-associated mutation at codons rt19, rt1, rt or rt5 [13]. Despite the great efficacy of ETV in treatment-naive patients, there is controversy about the antiviral efficacy of ETV in treatment-experienced CHB patients [1 1]. The aim of this study was to evaluate the antiviral efficacy of ETV in patients who had failed to achieve viral response during and ADV rescue therapy. We also investigated the evolution of antiviral resistance mutations and the emergence of HBV mutations associated with ETV resistance during ETV monotherapy by using restriction fragment mass polymorphism (RFMP) assays. Methods Patients Between September and September 7, CHB patients who had viral breakthrough or a suboptimal response to and ADV rescue therapy were included. The patients were treated with ETV monotherapy at a daily dose of 1. mg for 1 months. There was no time gap when the drugs were switched. They were followed-up every 3 months during ETV monotherapy for clinical assessments of tolerability, physical examination and blood sampling to measure laboratory parameters and HBV status. Serum HBV DNA levels were assessed using the COBAS Amplicor PCR assay (Roche Molecular Systems, Branchburg, NJ, USA) with a lower limit of detection of 3 (.5 log 1 ). This study was approved by the Institutional Review Board of the Konkuk University Hospital (Seoul, South Korea). Definitions Virological response was defined as a decrease in serum HBV DNA to an undetectable level (<.5 log 1 ) by PCR assay [19]. Viral breakthrough was defined as a 1 log 1 increase in HBV DNA from nadir after an initial decrease in HBV DNA by > log 1 [19]. Suboptimal response was arbitrarily defined as HBV DNA> log 1 after months of antiviral therapy []. Detection of antiviral-resistant mutations Serial samples were collected from each patient at the time of initiation of each antiviral agent, once every 3 months during treatment and at the time of viral breakthrough, and they were stored frozen at - C. For surveillance of HBV antiviral resistance mutations, the serum samples taken at baseline and at the time of viral breakthrough or suboptimal response to the prior or ADV treatment were tested. After switch to ETV monotherapy, mutational analysis of HBV polymerase in each patient was performed using the individual serial samples at 3-month intervals. Analysis of the mutations associated with (rt1 and rt) or ADV (rt11 and rt3) were performed with a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MS)-based genotyping assay, termed RFMP, as previously described [1 ]. The RFMP assay system had a low detection limit of.5 log 1 of HBV DNA in serum [1] and could detect antiviral resistance mutations that constituted as little as 1% of the total viral population [19,]. In addition, its feasible range for measuring relative abundance ratio of mutant versus wild-type virus was from 5% to 95% when the total viral load was log 1 [3]. The predominance of mutations was arbitrarily defined as a twofold or greater increase of antiviral resistance mutants than wild type in this study. RFMP analysis of mutations associated with ETV resistance was established with following primer pairs: 5 -GGATGCCTGGGCTTTCGCAAG-3 (nucleotides 1 33) and 5 -GGATGCACTCCCA TAGG-3 (nucleotides 37 ) for codon rt19, 5 -GGATGTCCTGGCTCAGTTT-3 (nucleotides 7) and 5 -GGATGACAAATGGCACT-3 (nucleotides 93) for codon rt1, 5 -GGATG TGTTTGGCTTTC-3 (nucleotides 71 73) and 5 -TTGGCCCCCAGGATG TACCACATGATC-3 (nucleotides 7 7) for codon rt, 5 -GGATGT GTTTGGCTTTCAGTTAT-3 (nucleotides 71 73) and 5 -TTGGCCCCCAGGATGTACC ACATGATC-3 (nucleotides 7 7) for codon rt, and 5 -GGATGTCCCTTAACTTC-3 (nucleotide 5 7) and 5 -GGATGAGCTTCCAATTACATATCC-3 (nucleotides 9) for codon rt5. The underlined sequence in each primer introduced new FokI sites into the PCR products. The presence of mutations was analysed by MS of the 9-mer, 1-mer, 7-mer, 11-mer and 1-mer fragments for rti19t, rtsg, rtt1s, rtmv and rtm5v, respectively, followed by comparison to expected mass patterns summarized in Additional file 1. The RFMP assay results were validated by 1% concordance with those 9 9 International Medical Press

Entecavir rescue therapy for antiviral-resistant HBV obtained by repetitive direct sequencing. Analytical sensitivities were estimated to be.5 log 1 and lower limits of detection for minority viral populations of RFMP in detecting the ETV-resistant strains was 1% and was similar to those obtained in - or ADV-resistance-related mutant strains [1,]. Statistical analyses Results are reported as median (range). HBV DNA levels were logarithmically transformed for analysis. Continuous variables were compared with the twotailed Student s t-test and categorical variables with the two-tailed χ test or Fisher s exact test. P-values <.5 were considered as statistically significant. Results Patient characteristics prior to ETV monotherapy Baseline characteristics of patients prior to ETV monotherapy are summarized in Table 1. A total of patients were included. All patients were hepatitis B e antigen (HBeAg)-positive and negative for hepatitis B e antibodies. None of the patients were coinfected with HIV or hepatitis C virus. The patients had compensated liver disease and seven of them had liver cirrhosis. All patients had been treated with (1 mg/day) as the first-line therapy for a median of 15 months. During monotherapy, they experienced viral breakthrough or a suboptimal response to, and 1 patients among them were confirmed to have rtmv/i mutations. After viral breakthrough or suboptimal response to, was switched to ADV (1 mg/day); 1 patients were treated with ADV monotherapy and 1 patients were treated by adding on ADV while continuing. During ADV rescue therapy, all patients experienced viral breakthrough or a suboptimal response. Of the 1 patients who experienced viral breakthrough during ADV therapy, were confirmed to have rta11v/t and/or rtn3t mutations. Of the 1 patients who were suboptimal responders to ADV, 3 were confirmed to have rta11v/t mutations. Antiviral responses to ETV monotherapy At the start of ETV rescue therapy, the median HBV DNA level was. log 1 (range 5.9.). The median decreases of HBV DNA level were -1. and -. log 1 at 3 and months of ETV therapy, respectively. At the end of the observation period (median 13.5 months), the median decrease of HBV DNA level was -.1 log 1. Virological response was achieved in 5 (3%) patients after a median duration of 9. months (range. 1.) of ETV treatment. In the remaining 17 patients, 13 patients were suboptimal responders to ETV and patients experienced viral breakthrough. All four Table 1. Patient characteristics Baseline variable Value, n= Mean age, years (range) 5 (1 59) Sex Male, n 1 Female, n Hepatitis B e antigen status Positive, n Negative, n Liver disease Chronic hepatitis B, n 15 Cirrhosis, n 7 monotherapy prior to ADV therapy Median duration of treatment, months (range) 15. (7. 3.) Response to treatment Viral breakthrough, n 1 Suboptimal response, n MV/I, n 1 Wild type, n a ADV therapy prior to ETV therapy Median duration of ADV treatment, months (range) 15. (. 9.) ADV monotherapy, n 1 ADV+ combination, n 1 Response to ADV treatment Viral breakthrough, n 1 Suboptimal, n 1 MV/I, n 1 A11V/T, n 7 b A11V/T+N3T, n Wild type, n a 3 a Wild type at codons rt19, rt1, rt11, rt1, rt, rt, rt3 and rt5. b One patient had mutations rta11t and rtmi. ADV, adefovir dipivoxil; ETV, entecavir;, lamivudine. patients without pre-existing rtmv/i mutations (regardless of the presence or absence of pre-existing rta11v/t or rtn3t mutations) achieved virological response to ETV rescue, whereas only 1 of 1 patients with pre-existing rtmv/i mutations achieved virological response. The mean reduction in HBV DNA was significantly lower in patients with pre-existing rtmv/i mutations compared with patients without rtmv/i mutations at ETV treatment months 3,, 9 and 1, respectively (Figure 1). Univariate analyses showed that virological response was only significantly associated with an absence of pre-existing rtmv/i mutations (P<.1) among clinical and virological parameters, such as age, sex, liver cirrhosis, pre- existing - or ADV-resistant mutations, HBV DNA or alanine aminotransferase (ALT) levels at the start of ETV therapy and treatment duration. At the start of ETV rescue therapy, the median ALT level was IU/l (range 3 ) and of patients had an ALT level above the upper limit of normal (ULN; IU/l). ALT normalization Antiviral Therapy 1.7 97

WH Choe et al. was achieved in 11 of (55%) patients during ETV monotherapy and HBeAg seroconversion occurred in one patient. Treatment outcomes of the patients during ETV therapy are summarized in Table. Mutational analysis during ETV treatment At the initiation of ETV rescue therapy, rtmv/i mutations were detected in 1 patients and A11V/T and/or N3T mutations were detected in patients. One patient had both -resistant and ADV-resistant mutations, and three patients had wild type at multiple sites of HBV mutations associated with antiviral resistance. During ETV rescue therapy, MV/I mutations re-emerged or persisted, and their relative abundances in total viral populations gradually increased in the 15 of 1 patients with pre-existing -resistant mutations (Table 3). In five of those patients, ETV- resistant mutations (rtl1m+rtt1l/i+rtmv in two patients and rtl1m+rtsg+rtmv in three patients) were detected at a median of 1 months of ETV therapy (range 9 1 months; Figure ), and of 5 patients with ETV resistance mutations experienced viral breakthrough (patients 5 and 13). Representative RFMP analyses of mutations associated with ETV resistance in patient 9 and patient 13 are shown in Figure 3. Mutations at codons rt19 or rt5 were not detected in any patients. Changes in mutational patterns demonstrated that the relative abundances of ADVresistant mutations in total viral populations decreased under ETV rescue; however, 3 of 9 patients (patients 15, 1 and 1) still had ADV-resistant mutations at the last follow-up period (Table 3). Discussion This study demonstrated that ETV monotherapy is not effective in patients who failed and ADV as a rescue treatment, especially in patients with pre-existing rtmv/i mutations. We observed that pre- existing MV/I mutations persisted or re-emerged during ETV rescue therapy in those patients. ETV exhibits potent activity against HBV and the occurrence of ETV resistance mutations is extremely low [5,], with a cumulative genotypic resistance rate of 1.% at 5 years in treatment-naive patients [7]. However, antiviral efficacy of ETV against -resistant HBV is still unsatisfactory [1 1]. Clinical studies showed that ETV resulted in a limited virological response and ETV-resistant mutations emerged more frequently in -experienced patients compared with treatment-naive patients [,13]. Antiviral activity of ETV against ADV-resistant HBV is another controversial issue. Theoretically, rescue therapy with ETV was suggested to be beneficial for ADVresistant patients because of the different resistance profiles between ETV and ADV [,], and in vitro studies demonstrated that ADV-resistant mutations were susceptible to ETV [9,3]. However, recent clinical reports have offered conflicting results regarding the efficacy of ETV in CHB patients with ADV resistance [17,1]. Recently, Reijnders et al. [1] reported that ETV monotherapy dosed at 1 mg resulted in a limited virological response in patients with a persistently high level of viral replication during ADV treatment. However, in their study, 75% of the - and ADV- Figure 1. Viral decreases according to the pre-existing lamivudine resistance mutation rtmv/i Decrease of HBV DNA, log 1 - - - 3 ETV monotherapy Months P<.1 9 1 Patients with pre-existing -r mutations (n=1) Patients without pre-existing -r mutations (n=) ETV, entecavir; HBV, hepatitis B virus;, lamivudine; -r, lamivudine resistance. Table. Treatment outcomes of entecavir rescue therapy Baseline variable Value Median HBV DNA at initiation of ETV, log 1 (range). (5.9.) Median duration of ETV, months (range) 13.5 (1. 1.) Median HBV DNA decrease at months, log 1 (range) -. (.1 -.) Median HBV DNA decrease at last follow-up, log 1 (range) -.1 (1. -.9) Virological response to ETV, n (%) 5 (3) HBeAg seroconversion or loss of HBeAg, n (%) 1 (5) ETV, entecavir; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus. 9 9 International Medical Press

Entecavir rescue therapy for antiviral-resistant HBV Table 3. Changes in HBV DNA levels and mutational patterns during ETV rescue therapy ADV ± ETV treatment at the start ETV treatment at months ETV treatment at last follow-up Patient treatment treatment HBV DNA, Relative abundance a HBV DNA, Relative abundance a Duration of HBV DNA, Antiviral resistance number (rt) (rt11) log 1 rt rt11 log 1 rt rt11 ETV treatment, log 1 mutations months 1 I A 7. I A I A 1 5.3 rtmi, rtl1m I A.5 I<M A 5. V/I>M A 1. rtmi 3 V A.7 V/I>M A 5. V A 15 5 rtmv, rtl1m V A.9 M A. NA b NA b 1 UD UD 5 I A. I>M A.9 I A 1 5.9 rtsg, rtmv, rtl1m V/I A.3 V/I=M A 5. V/I A 1 5.5 rtmv/i, rtl1m 7 V/I A.5 V A. V A 1 3.9 rtmv, rtl1m V A. V<M A. V A 1. rtmv, rtl1m 9 V/I A.7 V/I A.1 V/I A 15.3 rtt1i/l, rtmv, rtl1m 1 V A 7. V A.9 V A 1. rtmv, rtl1m 11 I A. I A.9 I A 15. rtsg, rtmv, rtl1m 1 I T/A I=M T=A. I A 15.5 rtmi 13 V/I T c 7. M T c.7 V A 1 7.5 rtsg, rtmv, rtl1m 1 I V/T/A 7. M V/T>A.7 I<M T=A 1.9 rtmi 15 V T/A 7. M T>A 5.7 M T<A 1.1 rta11t 1 V/I V 7. M V.1 M V=A 1 3. rta11v 17 I V 5.9 M V 5. I A 15. rtt1i, rtmv/i, rtl1m 1 I T M T.1 I<M T=A 1.9 rtmi, rta11t 19 M T/A. M T=A. NA b NA b 1 UD UD M V c 7.1 M V c UD NA b NA b 1 UD UD 1 M A.7 M A 3. NA b NA b 15 UD UD M A 7. M A 3.3 NA b NA b 15 UD UD The ± symbol indicates with or without treatment. The > and < symbols indicate which type is predominant, mutant or wild type. The = symbol indicates that the proportions of mutant type and wild type in total viral population are similar. a Restriction fragment mass polymorphism assays showed wild type at the codons rt19, rt1, rt and rt5. b The relative abundances were not applicable (NA) because total viral loads were < log 1 in samples at months after entecavir (ETV) treatment. c Patient 13 had rta11t with rtn3t and patient had rta11v with rtn3t. There were no patients who had only rtn3t. ADV, adefovir dipivoxil; HBV, hepatitis B virus; UD, undetectable (<.5 log 1 );, lamivudine. Antiviral Therapy 1.7 99

WH Choe et al. Figure. Changes in HBV DNA and ALT levels during antiviral therapy in five patients who had entecavir resistance mutations A rtm I I>M I V/I V rtl1 L L L M<L M rta11 A A A A A rtn3 N N N N N rts S S S S G>S C HBV DNA, log 1-1 - 1 1 rtm I I I V V rtl1 M M M M M rta11 A A A A A rtn3 N N N N N rtt1 T T T I=T T rts S S S S G HBV DNA, log 1 1 ADV ETV -1-1 15 E ADV rtm I M M I V/I rtl1 L L L L M=L rta11 A V V A A rtn3 N N N N N rtt1 T T T T I=T ADV ETV ADV+ ALT, IU/ ULN ALT, IU/ ULN ETV B D rtm V/I V/I V/I V/I V V rtl1 M M M M M M rta11 A A A A A A rtn3 N N N N N rtt1 T T T T T I/L HBV DNA, log 1 ADV ADV+ ETV -1-1 15 rtm V/I M M V V V rtl1 M<L L L M M M rta11 A T T A A A rtn3 N N T>N N N N rts S S S S G G HBV DNA, log 1 ADV ADV+LMV ETV - -1-1 - 1 HBV DNA ALT level 1 ALT IU/l ULN ALT IU/l ULN HBV DNA, log 1-1 -1-1 ALT, IU/ ULN (A) Patient 5. (B) Patient 9. (C) Patient 11. (D) Patient 13. (E) Patient 17. All patient numbers correspond to those in Table 3. Hepatitis B virus (HBV) mutations associated with antiviral resistance are shown above each graph. The solid bar above each graph represents the duration of antiviral therapy. The vertical dotted line represents the start of adefovir or entecavir treatment. The horizontal dotted lines represent the lower limit of detection of HBV DNA (.5 log 1 ). The upper limit of normal (ULN) for alanine aminotranferase (ALT) is IU/l. ADV, adefovir dipivoxil; ETV, entecavir;, lamivudine; rt, reverse transcriptase. 99 9 International Medical Press

Entecavir rescue therapy for antiviral-resistant HBV Figure 3. Representative mass spectra showing mutations that are associated with entecavir resistance A Patient 9 Relative peak intensity,1.,1.7,19.,193. rt1i/l Relative peak intensity 3,39.3 3,17.3 rtv,1,15,15,175,,5,5,75,3 Mass to charge ratio 3,5 3,3 3,35 3, 3,5 Mass to charge ratio B Patient 13 Relative peak intensity 3,75.1 3,113.1 3,39. rtg Relative peak intensity 3,17.5 rtv 3,5 3,1 3,15 3, 3,5 Mass to charge ratio 3,5 3,3 3,35 3, 3,5 Mass to charge ratio (A) Mutations rt1i/l (,19. Da/,1. Da and,1. Da/,193. Da) and rtv (3,17. Da/3,39. Da) detected in patient 9. (B) Mutations rtg (3,113. Da/3,75. Da) and rtv (3,17. Da/3,39. Da) detected in patient 13. experienced patients had previously developed resistance; therefore, it is difficult to suggest that prior experience of ADV therapy or ADV resistance mutations might have affected the limited overall efficacy of ETV in those patients. In this study, patients who experienced viral breakthrough or suboptimal response during and ADV as a rescue therapy were treated with ETV monotherapy sequentially. Of these patients, 1 (%) patients had previously developed resistance, 15 (%) had showed suboptimal responses to ETV and (9%) patients experienced viral breakthrough during a median observation of 13.5 months with ETV monotherapy. Also, ETV-resistant mutations were detected in 5 (3%) patients at a median of 1 months of ETV therapy, and it appeared earlier and more frequently than was reported in previous studies on treatmentnaive patients or refractory patients [11,1]. The limited efficacy of ETV might be related to preexisting -resistant mutations. Our data showed that only 1 of the 1 patients with pre-existing resistant mutations achieved virological response and the presence of pre-existing -resistant mutation significantly reduced the virological response. Mutational analyses also demonstrated that -resistant mutations re-emerged or persisted and their relative abundances in total viral populations gradually increased under ETV monotherapy in most patients with pre-existing -resistant mutations. In addition, ETV-resistant mutations occurred among these patients. These results also suggest that pre-existing - resistant mutations could continue to be selected by ETV therapy. If we compare the five patients who had ETV- resistant mutations in Figure, their HBV DNA levels decreased transiently under ETV therapy, only when a proportion of wild-type at codon rtm was present at the start of ETV therapy. Subsequently, the response to ETV was blunted and remained relatively flat when the transition to MV/I mutations predominance took place for several months; thus, allowing the selection of the additional ETV-resistant substitutions required to overcome drug suppression and resulting in a viral breakthrough. This trend of association of evolution of MV/I mutations with Antiviral Therapy 1.7 991

WH Choe et al. clinical response to ETV is consistent with a previous in vitro study [31] indicating that MV/I mutations exhibited a higher relative level of phenotypic resistance to ETV; therefore, ETV monotherapy does not seem to be a good choice as a rescue for treatment of patients with pre-existing -resistant mutations. By contrast, ETV still has some importance in treatment-experienced patients, especially in suboptimal responders to or ADV without antiviralresistant mutations. In this study, all four patients who had not experienced -resistant mutations achieved virological response regardless of the presence or absence of pre-existing ADV-resistant mutations. Changes in mutational patterns under ETV therapy also demonstrated that the relative abundances of ADV-resistant mutations were slowly decreased; however, ADV- resistant mutations still remained in three of nine patients at the last follow-up. Although N3T and A11V/T mutants are susceptible to ETV in vitro, the potential presence of cross-resistance or other compensatory mutations in HBV polymerase might contribute to the persistence of ADV-resistant mutants. Further investigations are needed to clarify the persistence of ADV-resistant mutation during ETV monotherapy. Approval of multiple nucleoside/nucleotide analogues to clinical practice and their sequential or combinational use increase the chance of selection of HBV variants with complex mutational patterns; thus, there is a need to simultaneously analyse several mutational hotspots associated with antiviral resistance and for optimal management of CHB. In this study, we introduced an advanced RFMP-based assay that exploited differential lengths of oligonucleotides (7 1-mer) depending upon each codon associated with ETV resistance, which would not necessitate repetitive PCR and MS analyses for multiple mutations. The RFMP method sensitively and reproducibly detected known point mutations of amino acids in the reverse transcriptase domains of HBV polymerase. Analytical sensitivity and a lower limit of detection for the minority mutant population as low as.5 log 1 and 1% of RFMP assays for ETV mutation should enable early detection of treatment failure, subsequent intervention and prevention of drug resistance as it develops [1,]. However, there is a possibility that mutations in other codons that were not covered by the RFMP assay might contribute to the individualized drug response to a certain extent during sequential treatment; therefore, clonal identification of HBV quasispecies during longitudinal sampling and subsequent in vitro susceptibility testing of potential variants with or without known primary mutations against antiviral agents should be supplemented to elucidate the viral evolution and acquisition of multiple antiviral drug resistance mutations. Our patients were treated with as the first-line anti-hbv treatment and ADV as a rescue therapy. Recent guidelines for treatment of CHB patients no longer recommend as a first-line agent or sequential monotherapy [3,33]. In many Asian countries with HBV endemic areas, including Korea, many patients have already been treated with and ADV sequential therapy because of a relatively low cost and the drug-by-drug approval process of anti- HBV agents. However, the effective rescue therapy for patients who have suffered from treatment failure with the sequential therapy is still controversial. This study clearly demonstrated that ETV is not an ideal option for the rescue therapy for those who failed sequential treatment with and ADV. Recent guidelines recommend tenofovir disoproxil fumarate (TDF)-based therapy with or without a combination of nucleoside/ nucleotide analogues as a rescue for the treatment of these patients [3,33]. We have reported that TDF and combination markedly suppressed HBV replication in ADV-resistant patients with pre-existing resistance [3]. Combination treatment that includes TDF could be a useful therapeutic option for patients with and/or ADV resistance In conclusion, ETV monotherapy showed a limited antiviral efficacy in CHB patients who had previously failed and ADV rescue therapy. The suboptimal response to ETV rescue therapy could predispose HBV to emergence of ETV resistance mutations. The limited efficacy of ETV in those patients might be primarily related to persistence or reselection of pre-existing -resistant mutations. Sequential monotherapy with an anti-hbv agent should be avoided to prevent emergence of multidrug resistance of HBV. Further investigations for efficacy of ETV combined with potent antiviral agents are needed to provide the therapeutic strategies for antiviral-resistant patients. Acknowledgements This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A). Disclosure statement The authors declare no competing interests. Additional file Additional file 1: A supplementary file of an interpretation table for mass to genotype conversion in the RFMP assay can be found at http://www.intmedpress.com/ uploads/documents/15_kwon_additional_file.pdf 99 9 International Medical Press

Entecavir rescue therapy for antiviral-resistant HBV References 1. Iloeje UH, Yang HI, Su J, et al. Predicting cirrhosis risk based on the level of circulating hepatitis B viral load. Gastroenterology ; 13:7.. Chen CJ, Yang HI, Su J, et al. Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA ; 95:5 73. 3. Yu MW, Yeh SH, Chen PJ, et al. Hepatitis B virus genotype and DNA level and hepatocellular carcinoma: a prospective study in men. J Natl Cancer Inst 5; 97:5 7.. Ayoub WS, Keeffe EB. Review article: current antiviral therapy of chronic hepatitis B. Aliment Pharmacol Ther ; :17 177. 5. Lok AS. The maze of treatments for hepatitis B. N Engl J Med 5; 35:73 7.. Locarnini S. Molecular virology and the development of resistant mutants: implications for therapy. Semin Liver Dis 5; 5 Suppl 1:9 19. 7. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, et al. Longterm therapy with adefovir dipivoxil for HBeAg-negative chronic hepatitis B for up to 5 years. Gastroenterology ; 131:173 1751.. Lee YS, Suh DJ, Lim YS, et al. Increased risk of adefovir resistance in patients with lamivudine-resistant chronic hepatitis B after weeks of adefovir dipivoxil monotherapy. Hepatology ; 3:135 1391. 9. Rapti I, Dimou E, Mitsoula P, Hadziyannis SJ. Adding-on versus switching-to adefovir therapy in lamivudine-resistant HBeAg-negative chronic hepatitis B. Hepatology 7; 5:37 313. 1. Lampertico P, Vigano M, Manenti E, et al. Low resistance to adefovir combined with lamivudine: a 3-year study of 15 lamivudine-resistant hepatitis B patients. Gastroenterology 7; 133:15 151. 11. Gish RG, Lok AS, Chang TT, et al. Entecavir therapy for up to 9 weeks in patients with HBeAg-positive chronic hepatitis B. Gastroenterology 7; 133:137 1. 1. Colonno RJ, Rose R, Baldick CJ, et al. Entecavir resistance is rare in nucleoside naive patients with hepatitis B. Hepatology ; :15 15. 13. Tenney DJ, Levine SM, Rose RE, et al. Clinical emergence of entecavir-resistant hepatitis B virus requires additional substitutions in virus already resistant to lamivudine. Antimicrob Agents Chemother ; :39 357. 1. Sherman M, Yurdaydin C, Simsek H, et al. Entecavir therapy for lamivudine-refractory chronic hepatitis B: improved virologic, biochemical, and serology outcomes through 9 weeks. Hepatology ; :99 1. 15. Suzuki F, Toyoda J, Katano Y, et al. Efficacy and safety of entecavir in lamivudine-refractory patients with chronic hepatitis B: randomized controlled trial in Japanese patients. J Gastroenterol Hepatol ; 3:13 13. 1. Su CW, Wu JC, Lee SD. Is entecavir ideal for the treatment of lamivudine-refractory chronic hepatitis B? Hepatology ; :17 177. 17. Kamar N, Milioto O, Alric L, et al. Entecavir therapy for adefovir-resistant hepatitis B virus infection in kidney and liver allograft recipients. Transplantation ; :11 1. 1. Reijnders JG, Pas SD, Schutten M, de Man RA, Janssen HL. Entecavir shows limited efficacy in HBeAg-positive hepatitis B patients with a partial virologic response to adefovir therapy. J Hepatol 9; 5:7 3. 19. Lok AS, Zoulim F, Locarnini S, et al. Antiviral drugresistant HBV: standardization of nomenclature and assays and recommendations for management. Hepatology 7; :5 5.. Zoulim F, Buti M, Lok AS. Antiviral-resistant hepatitis B virus: can we prevent this monster from growing? J Viral Hepat 7; 1 Suppl 1:9 3. 1. Hong SP, Kim NK, Hwang SG, et al. Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments. J Hepatol ; :37.. Kim HS, Han KH, Ahn SH, et al. Evaluation of methods for monitoring drug resistance in chronic hepatitis B patients during lamivudine therapy based on mass spectrometry and reverse hybridization. Antivir Ther 5; 1:1 9. 3. Lee CH, Kim SO, Byun KS, et al. Predominance of hepatitis B virus YMDD mutants is prognostic of viral DNA breakthrough. Gastroenterology ; 13:11 115.. Yeon JE, Yoo W, Hong SP, et al. Resistance to adefovir dipivoxil in lamivudine resistant chronic hepatitis B patients treated with adefovir dipivoxil. Gut ; 55:1 195. 5. Chang TT, Gish RG, de Man R, et al. A comparison of entecavir and lamivudine for HBeAg-positive chronic hepatitis B. N Engl J Med ; 35:11 11.. Lai CL, Shouval D, Lok AS, et al. Entecavir versus lamivudine for patients with HBeAg-negative chronic hepatitis B. N Engl J Med ; 35:111 1. 7. Tenney DJ, Rose RE, Baldick CJ, et al. Long-term monitoring shows hepatitis B virus resistance to entecavir in nucleoside-naive patients is rare through 5 years of therapy. Hepatology 9; 9:153 15.. Locarnini S, Hatzakis A, Heathcote J, et al. Management of antiviral resistance in patients with chronic hepatitis B. Antivir Ther ; 9:79 93. 9. Villet S, Pichoud C, Billioud G, et al. Impact of hepatitis B virus rta11v/t mutants on hepatitis B treatment failure. J Hepatol ; :77 755. 3. Qi X, Xiong S, Yang H, et al. susceptibility of adefovirassociated hepatitis B virus polymerase mutations to other antiviral agents. Antivir Ther 7; 1:355 3. 31. Yang H, Qi X, Sabogal A, et al. Cross-resistance testing of next-generation nucleoside and nucleotide analogues against lamivudine-resistant HBV. Antivir Ther 5; 1:5 33. 3. Keeffe EB, Dieterich DT, Han SH, et al. A treatment algorithm for the management of chronic hepatitis B virus infection in the United States: an update. Clin Gastroenterol Hepatol ; :93 9. 33. Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology 7; 5:57 539. 3. Choe WH, Kwon SY, Kim BK, et al. Tenofovir plus lamivudine as rescue therapy for adefovir-resistant chronic hepatitis B in hepatitis B e antigen-positive patients with liver cirrhosis. Liver Int ; :1. Accepted for publication 13 June 9 Antiviral Therapy 1.7 993