Original article Partial virological response to entecavir in treatment-naive patients with chronic hepatitis B

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1 Antiviral Therapy 2011; 16: (doi: /IMP1772) Original article Partial virological response to entecavir in treatment-naive patients with chronic hepatitis B Young Eun Chon 1, Seung Up Kim 1, Chun Kyon Lee 2, Jeong Heo 3, Ja Kyung Kim 1,4, Ki Tae Yoon 3, Mong Cho 3, Kwan Sik Lee 1,4, Dong Hwan Kim 2, Eun Hee Choi 5, Jun Yong Park 1,4, Do Young Kim 1,4, Chae Yoon Chon 1,4, Kwang-Hyub Han 1,4,6, Sang Hoon Ahn 1,4,6 * 1 Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea 2 Department of Internal Medicine, National Health Insurance Corporation Ilsan Hospital, Koyang, Korea 3 Department of Internal Medicine, Pusan National University School of Medicine, Busan, Korea 4 Liver Cirrhosis Clinical Research Center, Seoul, Korea 5 Department of Biostatistics, Yonsei University College of Medicine, Seoul, Korea 6 Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea These authors made an equal contribution to this work. *Corresponding author ahnsh@yuhs.ac Background: The proposed definition of a partial virological response (PVR) to nucleos(t)ide analogue therapy in the 2009 European Association for the Study of the Liver (EASL) guidelines is based on limited evidence, especially in terms of the cutoff HBV DNA level and the time point at which to judge it. This study assessed optimal PVR criteria for predicting virological response (VR) at week 96 in treatment-naive patients with chronic hepatitis B (CHB) receiving entecavir (ETV). Methods: A total of 175 patients (126 men, 49 women) who completed 96 weeks of first-line ETV therapy were prospectively recruited. For predicting VR at week 96, the area under the receiver operating characteristic curve (AUC) was used to find the optimal time point and the Youden index was used to calculate the optimal cutoff HBV DNA level. Results: After 96 weeks of ETV therapy, 139 (79.4%) patients achieved VR. The AUC at week 48 was significantly better than that at week 24 for predicting VR at week 96 (P=0.023). The optimal cutoff HBV DNA level at week 48 was 35 IU/ml. Forty-one (23.4%) patients met this PVR criteria of ETV (HBV DNA level >35 IU/ml at week 48). Conclusions: An HBV DNA level >35 IU/ml at week 48 is the optimal PVR criteria for predicting non-vr at week 96 in treatment-naive patients with CHB who are receiving ETV. This study supports the proposed EASL PVR for ETV based on scientific evidence. Introduction Approximately 400 million people are chronically infected with hepatitis B worldwide and about 1 million suffer from complications related to HBV, such as cirrhosis and hepatocellular carcinoma (HCC) [1,2]. Because the risk of developing HCC or liver cirrhosis increases with an increase in HBV DNA level [3,4], suppressing HBV replication and maintaining a sustained reduction of HBV DNA level is important to stop or reverse the progression of liver diseases by reducing the hepatic necroinflammatory response [5 7]. Hence, the main aim of chronic hepatitis B (CHB) treatment is a complete and persistent suppression of viral replication [8]. Recently, the European Association for the Study of the Liver (EASL) HBV Clinical Practice Guidelines proposed a definition for the response to antiviral therapy [9]. According to these guidelines, partial virological response (PVR) to nucleos(t)ide analogue (NUC) in CHB was defined as a more than 1 log decline of viraemia compared with baseline but still detectable serum levels of HBV DNA by real-time PCR assay (>10 15 IU/ml) at week 24 or 48, depending on the genetic barrier of the anti-hbv drugs. However, this PVR definition did not suggest clear evidence for the proposed cutoff HBV DNA level or for the time point at which to judge it. With first- (lamivudine) and second- (telbivudine and adefovir) generation anti-hbv drugs, it has been clearly demonstrated that patients who show PVR to 2011 International Medical Press (print) (online) 469

2 YE Chon et al. these NUCs are at high risk for developing resistance to antiviral therapy [10 14]. Therefore, early detection of patients with PVR and appropriate intervention (changing to or adding other potent drugs) for achieving sustained viral suppression have been emphasised. However, the relationship between the residual viral load and the treatment response has not been fully evaluated for entecavir (ETV), a third-generation NUC with potent activity against HBV [15 17] and a low risk of resistance [18]. Recent data have revealed that 10 33% of ETV-treated patients show >300 HBV DNA copies/ml at week 48 [19]. Moreover, ETV-resistant strains were detected in two NUC-naive patients with incomplete suppression [20]. Therefore, it is necessary to establish PVR criteria and subsequent management guidelines for treatmentnaive patients with CHB who are receiving ETV. This study aimed to determine the optimal PVR criteria for predicting virological response (VR) at week 96 of ETV therapy in treatment-naive patients with CHB. Methods Study population Between March 2007 and December 2009, 420 treatment- naive patients with CHB starting antiviral therapy with 0.5 mg ETV at Sinchon Severance Hospital (Yonsei University College of Medicine, Seoul, Korea), Gangnam Severance Hospital (Yonsei University College of Medicine, Seoul, Korea), National Health Insurance Corporation Ilsan Hospital (Koyang, Korea) and Pusan National University Hospital (Pusan University College of Medicine, Busan, Korea) were prospectively enrolled. Inclusion criteria for this study were age >16 years, persistent serum hepatitis B surface antigen (HBsAg) levels for >6 months and HBV genotype C, the prevalence of which reaches nearly 98~100% of HBV in Korea [21 24]. Exclusion criteria included decompensated liver cirrhosis (evidence of ascites, hepatic encephalopathy, varices, serum total bilirubin >2.5 mg/dl, serum albumin <3 mg/dl or prothrombin time >3 s longer than normal), previous antiviral treatment for CHB (either interferon or NUC), current use of corticosteroid or immunomodulatory drugs, co-infection with hepatitis C or D virus or HIV, serious concurrent medical illness, evidence of HCC or prior organ transplantation [25]. Written informed consent was obtained from all patients. Our study protocol was consistent with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the independent institutional review boards of each institute. Study design and definitions ETV (0.5 mg) was given once daily according to the clinician s judgment to initiate antiviral therapy. Hepatitis B e antigen (HBeAg) and the antibody to HBeAg (anti-hbe) were checked at baseline and every 6 months. HBV DNA and alanine aminotransferase (ALT) levels were checked at baseline and every 3 months during follow-up. VR was defined as undetectable HBV DNA by PCR (<12 IU/ml) at any time point during ETV therapy [9]. Patients with a decrease in HBV DNA level <1 log 10 IU/ml from baseline at week 12 (3 months) were defined as primary non-responders [9]. Virological breakthrough (VB) was defined as an increase in HBV DNA of >1 log 10 IU/ml compared with the nadir HBV DNA level during ETV therapy [9]. Biochemical response (BR) was defined as normalization of serum ALT levels from abnormal baseline levels; a threshold of 40 IU/l was used as the upper normal range [26 28]. Biochemical breakthrough (BB) was defined as an increase in serum ALT levels above the upper limit of normal after achieving normalization during ETV therapy [29]. HBeAg seroclearance was defined as a persistent absence of antigenaemia on at least two consecutive follow-ups [30]. HBeAg seroconversion was defined as the loss of HBeAg with the development of anti-hbe on at least two consecutive follow-ups [31]. Serological response was defined as HBeAg seroclearance with or without HBeAg seroconversion in HBeAgpositive patients. Genotypic resistance was defined as the appearance of viral populations bearing amino acid substitutions in the reverse transcriptase region of the HBV genome that conferred resistance to antiviral drugs in patients with VB [29]. Liver cirrhosis was defined as follows: (1) liver cirrhosis confirmed by biopsy, (2) either current or past evidence of cirrhosis-related complications (for example, ascites, varices or hepatic encephalopathy) or (3) a platelet count <100,000/mm 3 with ultrasonographic findings suggestive of cirrhosis, including blunted or nodular liver edge accompanied by splenomegaly (>12 cm) [32,33]. Compliance with therapy was assessed when patients visited the outpatient clinic and phone calls were made to those who did not make their scheduled appointment. Laboratory assay HBV DNA levels were measured by quantitative PCR assay (Amplicor HBV Monitor Test, Roche Diagnostics, Basel, Switzerland; detection limit approximately 12 IU/ml). HBV serological tests for HBsAg, HBeAg and anti-hbe were conducted with enzyme-linked immunoassays (Dade Behring, Marburg, Germany). Biochemical data including levels of ALT, aspartate aminotransferase, total bilirubin, albumin, creatinine and prothrombin time were analysed using a sequential multiple auto-analyser (Hitachi Ltd, Tokyo, Japan). For genotypic analysis, HBV DNA was extracted from serum samples using commercial kits (Qiagen, Velencia, CA, USA) and the HBV polymerase reverse transcriptase domain (encoding International Medical Press

3 Partial virological response to entecavir Table 1. Baseline characteristics Characteristic Total population Patients with VR a Patients without VR a P-value Patient number, n (%) 175 (100) 139 (79.4) 36 (20.6) Mean age, years ±sd 45 ±11 46 ±11 42 ± Male gender, n (%) 126 (72.0) 100 (71.9) 26 (72.2) HBeAg positivity, n (%) 121 (69.1) 89 (64.0) 32 (88.9) Cirrhosis, n (%) 61 (34.9) 52 (37.4) 9 (25.0) Mean baseline ALT, IU/l ±sd 166 ± ± ± Mean baseline HBV DNA, log 10 copies/ml ±sd 7.03 ± ± ± a Virological response (VR) indicates undetectable HBV DNA (<12 IU/ml). ALT, alanine aminotransferase. HBeAg, hepatitis B e antigen. amino acids 1 344) was amplified by PCR methods and sequenced directly. End point The primary end point was to investigate the optimal PVR criteria in terms of cutoff HBV DNA level and time point to predict VR at week 96 in treatment-naive patients with CHB who were receiving ETV. Statistical analysis Baseline characteristics of the patients with and without VR were compared by Student s t-test (or Mann Whitney U-test) for continuous variables and χ 2 test (or Fisher s exact test) for categorical variables. To define the optimal time point for PVR, receiver- operating characteristic (ROC) curves and the area under the ROC curve (AUC) using HBV DNA levels at weeks 24 and 48 were calculated, and their overall accuracies were compared. After the optimal time point was established, the optimal cutoff HBV DNA level for PVR was subsequently calculated using the Youden index (defined as sensitivity + specificity -1) [34]. Statistical analysis was performed using SAS software version (SAS, Cary, NC, USA). Two-sided P-values <0.05 were considered statistically significant. Results Patient baseline characteristics A total of 420 patients with CHB from four institutes were consecutively treated with ETV as first-line therapy during the study period. Of these, 22 (5.2%) patients were lost to follow-up, 20 (4.8%) showed poor compliance, 8 (1.9%) were primary non-responders and 195 (46.4%) were followed for <2 years. Finally, 175 patients who completed 2 years of ETV therapy were enrolled for analysis. Table 1 shows the baseline characteristics of the patients. The mean age was 45 years (range 34 56) and male gender was predominant (n=126, 72.0%). HBeAg was positive in 121 (69.1%) patients and accompanied by cirrhosis in 61 (34.9%). The baseline median serum ALT and HBV DNA levels were 166 IU/l (range ) and 7.03 log 10 copies/ml (range ), respectively. VR at week 96 was identified in 139 (79.4%) patients. Age, gender and the presence of liver cirrhosis were not significantly different between patients with VR at week 96 and those without (all P>0.05). HBeAg positivity and baseline HBV DNA level were significantly higher in patients without VR (P=0.004 and P=0.001, respectively), whereas baseline ALT level was significantly higher in patients with VR at week 96 (P=0.008). Treatment outcomes Table 2 shows the treatment outcomes following ETV therapy. After 2 years of ETV therapy, 139 (79.4%) patients achieved VR (89 [73.6%] HBeAg-positive and 50 [92.6%] HBeAg-negative). BR was achieved in 164 (93.7%) patients after 2 years of ETV therapy (113 [93.4%] HBeAg-positive and 51 [94.4%] HBeAg-negative). The VR and BR rate were always higher in HBeAg-negative patients than in HBeAgpositive patients at the same time point. The 24, 48 and 96 week cumulative serological response rates in HBeAg-positive patients (n=121) were 7.4%, 13.2% and 18.2%, respectively. The cumulative occurrence of VB was two (1.1%) patients at week 48, four (2.3%) at week 72 and eight (4.6%) at week 96. All of these eight patients underwent genotypic resistance tests, but none showed genotypic resistance. BB occurred in three (1.7%) patients at week 48 and in six (3.4%) at week 96. Of the six patients with BB, three showed VB. HBV DNA level versus HBV DNA reduction to define PVR The ROC curves of the HBV DNA level or the HBV DNA reduction from baseline to predict VR at week 96 and their corresponding AUC values are shown in Figure 1. The AUC values of the HBV DNA level at weeks 24 and 48 were consistently higher than those of HBV DNA reduction from baseline (AUC versus at week 24; AUC versus at week 48). Therefore, HBV DNA level was adopted to define Antiviral Therapy

4 YE Chon et al. PVR. Subsequent comparative analysis showed that the AUC for the HBV DNA level at week 48 was significantly higher than that at week 24 (AUC versus 0.824; P=0.023). The optimal cutoff HBV DNA level to define PVR Because HBV DNA level at week 48 was more useful to predict VR at week 96, we investigated the optimal cutoff HBV DNA level at week 48 using the Youden index Table 2. Treatment outcomes during 2 years of entecavir therapy Entecavir therapy duration Outcomes 24 Weeks 48 Weeks 96 Weeks Treatment response, n (%) Virological response Total (n=175) 68 (38.9) 120 (68.6) 139 (79.4) HBeAg-positive patients (n=121) 36 (29.8) 74 (61.2) 89 (73.6) HBeAg-negative patients (n=54) 32 (59.3) 46 (85.2) 50 (92.6) Biochemical response Total (n=175) 140 (80.0) 150 (85.7) 164 (93.7) HBeAg-positive patients (n=121) 94 (77.7) 101 (83.5) 113 (93.4) HBeAg-negative patients (n=54) 46 (85.2) 49 (90.7) 51 (94.4) Serological response in HBeAg-positive patients 9 (7.4) 16 (13.2) 22 (18.2) Seroclearance 7 (5.8) 11 (9.1) 14 (11.6) Seroconversion 2 (1.6) 5 (4.1) 8 (6.6) Antiviral resistance, n (%) Virological breakthrough 0 (0.0) 2 (1.1) 8 (4.6) Biochemical breakthrough 0 (0.0) 3 (1.7) 6 (3.4) Genotypic resistance 0 (0.0) 0 (0.0) 0 (0.0) Virological response is defined as undetectable HBV DNA (<12 IU/ml), biochemical response as normalization of serum alanine aminotransferase (ALT) levels (<40 IU/l), serological response as hepatitis B e antigen (HBeAg) seroclearance with or without HBeAg seroconversion in HBeAg-positive patients, virological breakthrough as an increase in HBV DNA >1 log 10 IU/ml compared with the nadir HBV DNA, biochemical breakthrough as an increase in serum ALT levels above the upper limit of normal after achieving normalization and genotypic resistance as the appearance of viral populations bearing amino acid substitutions in the reverse transcriptase region of the HBV genome in patients with virological breakthrough. Figure 1. Defining partial virological response: HBV DNA level versus HBV DNA reduction A 1.0 B Sensitivity HBV DNA level AUC Week Week P=0.023 Sensitivity HBV DNA reduction AUC Week Week P= Specificity Specificity Receiver-operating characteristic curve analysis using (A) HBV DNA level and (B) HBV DNA reduction from the baseline at week 24 and week 48 to predict virological response at week 96. AUC, area under the curve International Medical Press

5 Partial virological response to entecavir (sensitivity + specificity -1). According to the maximal Youden index, a HBV DNA level of 35 IU/ml (2.24 log 10 copies/ml, 174 copies/ml) was the optimal cutoff to predict VR at week 96 with a sensitivity of 92.8%, a specificity of 86.1%, a positive predictive value of 96.3% and a negative predictive value of 75.6% (Table 3). The flow of patients according to treatment response According to the cutoff HBV DNA level at week 48 (35 IU/ml), 41 (23.4%) patients met the PVR criteria (HBV DNA level >35 IU/ml at week 48), whereas 134 (76.6%) exhibited a HBV DNA level <35 IU/ml at week 48 (defined as favourable VR in this study). Figure 2 shows the treatment response during 2 years of ETV therapy. Of 41 patients with PVR, 31 (75.6%) still Table 3. Distribution of patients at week 96 according to optimal partial virological response Patients with Patients without Optimal cutoff HBV DNA a VR, n VR, n >35 IU/ml IU/ml Sensitivity 92.8%, specificity 86.1%, positive predictive value 96.3% and negative predictive value 75.6%. a Optimal cutoff HBV DNA was determined by the maximal Youden index (sensitivity + specificity -1) [34]. Virological response (VR) is defined as undetectable HBV DNA (<12 IU/ml). Figure 2. The flow of patients according to treatment response Proportion of patients, % n=175 (100%) Baseline n=41 (23.4%) n=134 (76.6%) Week 48 n=36 (20.6%) n=139 (79.4%) Total patients (baseline) PVR: HBV DNA>35 IU/ml at week 48 Favourable VR: HBV DNA<35 IU/ml at week 48 VR: HBV DNA negative (<12 IU/ml) at week 96 Non-VR: HBV DNA positive (>12 IU/ml) at week Week 96 The circled number denote the number of patients. PVR, partial virological response; VR, virological response. had detectable HBV DNA levels at week 96. Of 134 patients with a favourable VR, 129 (96.3%) achieved VR at week 96. Finally, 139 patients (79.4%) achieved VR at week 96. Patients with PVR at week 48 showed a significantly higher risk for detectable HBV DNA levels at week 96 than those with favourable VR at week 48 (odds ratio 79.9). Factors predicting VR at week 96 in patients with PVR Although 41 (23.4%) patients were PVR at week 48, 10 (24.4%) achieved undetectable HBV DNA levels at week 96 (Figure 2). We conducted further analysis to identify which of these PVR patients would finally achieve VR at week 96 (Table 4). When we compared baseline and on-treatment variables between patients with VR and those without at week 96, none of the variables differed except for the baseline HBV DNA level. The baseline HBV DNA level was significantly lower in patients who later achieved VR at week 96 (6.88 ±1.20 log 10 copies/ ml in patients with VR versus 7.88 ±1.35 log 10 copies/ ml in patients without VR; P=0.045). In patients with PVR at week 48, the best cutoff to predict VR at week 96 was a baseline HBV DNA 1.77E+08 IU/ml (7.85 log 10 copies/ml, 8.84E+08 copies/ml), according to the maximal Youden index (a sensitivity of 93.6%, a specificity of 83.5%, a positive predictive value of 94.2% and a negative predictive value of 89.8%) [34]. Discussion Suppressing HBV replication is a principal goal of CHB therapy [6]. According to the EASL HBV Clinical Practice Guidelines, the end point of CHB therapy is to reduce the HBV DNA level to as low a level as possible to ensure virological suppression and biochemical remission, ultimately resulting in histological improvement and prevention of two major complications including cirrhosis and HCC [9]. Several strategies can be considered in view of viral replication suppression, as reflected by the HBV DNA level. The first strategy is to continue current NUC until complete viral suppression is achieved; however, this strategy can raise issues of high cost and appearance of resistance [35]. The second strategy is to predict when, and under what conditions, complete viral suppression might be attained in the future. If this is possible, then time and costs on ineffective NUC would not be wasted. Considering these issues, the EASL guidelines recently suggested a new PVR criteria concept for the use of NUCs, which was defined as more than 1 log decline of viraemia compared with baseline but still detectable serum levels of HBV DNA by real-time PCR assay (>10 15 IU/ml) at week 24 or 48, depending on the genetic barrier of the anti-hbv drug. However, the cutoff HBV DNA level and the time point of this PVR Antiviral Therapy

6 YE Chon et al. Table 4. Factors predicting virological response at week 96 in patients with PVR Factors Total PVR Patients with VR Patients without VR P-value Patient number, n (%) 41 (100) 10 (24.4) 31 (75.6) Baseline variables Mean age, years ±sd 45 ±12 51 ±14 43 ± Male gender, n (%) 31 (75.6) 8 (80.0) 23 (74.3) HBeAg positivity, n (%) 35 (85.4) 7 (70.0) 28 (90.3) Cirrhosis, n (%) 9 (22.0) 3 (40.0) 6 (19.4) Mean baseline ALT, IU/l ±sd 120 ± ± ± Mean baseline HBV DNA, log 10 copies/ml ±sd 7.64 ± ± ± On-treatment variables Mean ALT level at week 48, IU/l ±sd 31 ±13 32 ±14 31 ± Biochemical response at week 48, n (%) 32 (78.0) 8 (80.0) 24 (77.4) Mean HBV DNA level at week 48, log 10 copies/ml ±sd 4.02 ± ± ± HBV DNA reduction at week 48, log 10 copies/ml ±sd ± ± ± Partial virological response (PVR) is defined as HBV DNA >35 IU/ml at week 48, virological response (VR) as undetectable HBV DNA (<12 IU/ml) and biochemical response as normalization of serum alanine aminotransferase (ALT) levels. HBeAg, hepatitis B e antigen. criterion were not based on scientific evidence, but on experts perceptions. Several investigations have focused on the issue of PVR in patients treated with lamivudine or adefovir. However, although ETV is thought to be a potent NUC with a high genetic barrier, viral resistance to ETV has emerged. Yao et al. [36] reported that three patients during 96 weeks and an additional two patients during the following 48 weeks developed ETV resistance among 160 patients who received first-line ETV therapy for 144 weeks. Suzuki et al. [20] reported an ETV-resistant strain in an NUCnaive patient with genotype H. In the same context, PVR in ETV-treated patients has been demonstrated by several investigators. Chang et al. [16] reported 33% PVR (HBV DNA level <300 copies/ml at week 48) in HBeAg-positive patients and Lai et al. [15] reported 10% PVR in HBeAgnegative patients. To prevent treatment failure and the emergence of ETV-resistant strains, it is crucial to define optimal PVR criteria and change treatment strategies. Therefore, this study investigated the cutoff HBV DNA level and the time point of optimal PVR to predict VR at week 96 in treatment-naive patients with CHB who were receiving ETV by scientific method To establish optimal PVR criteria, we determined whether the HBV DNA level or the HBV DNA reduction from baseline was more useful for predicting VR at week 96 during ETV therapy. On the basis of our data, the AUCs of the HBV DNA level were consistently superior to those of HBV DNA reduction from the baseline both at week 24 and 48. In addition, the AUC of the HBV DNA level at week 48 was more predictable than that at week 24 with statistical significance. Furthermore, the AUC of the HBV DNA at week 48 was significantly higher than that of the baseline HBV DNA (0.908 versus 0.713; P=0.036). This observation implies that the response to ETV therapy might not be totally determined by baseline HBV DNA level and viruses that show an excellent response to ETV might exist even in a patient with a high baseline viral load. Therefore, a whole genomic analysis of the virus, such as revealing the frequency of core promoter mutations, should be the focus of future studies to explain this intrinsically different responsiveness. Finally, our PVR criteria for ETV therapy were defined as an HBV DNA level exceeding 35 IU/ml (2.24 log 10 copies/ml, 174 copies/ml) at week 48. That is, if patients achieve HBV DNA levels of less than 35 IU/ml at week 48 (favourable VR), ETV should be continued because the majority of patients (96.3%) could achieve VR at week 96 with high positive predictive value. On the contrary, if patients show an HBV DNA level of >35 IU/ml at week 48 (PVR), more than half these patients (75.6%) might not achieve VR at week 96. However, decisions on switching or add-on strategies for patients with PVR at week 48 should be considered cautiously, because 24.4% of patients with PVR at week 48 finally achieved VR after 2 years of ETV therapy. Considering that the kinetics of viral clearance are different between HBeAg-positive and HBeAg-negative HBV, we stratified the study population according to the presence or absence of HBeAg positivity. The results from 121 HBeAg-positive patients were consistent with those from all patients. AUC of the HBV DNA level at week 24 and week 48 were consistently higher than those of HBV DNA reduction from baseline (AUC versus at week 24; AUC versus at week 48). Moreover, AUC of the HBV DNA level at week 48 was significantly higher than that at week 24 (AUC versus 0.812; P=0.019). By contrast, statistically significant time point and cutoff HBV DNA levels were not International Medical Press

7 Partial virological response to entecavir Figure 3. A practical management guideline in treatment-naive patients with chronic hepatitis B receiving entecavir Week 12 Response assessment Responder (>1 log 10 decline in HBV DNA) Primary non-responder (<1 log 10 decline in HBV DNA) Week 48 Response assessment Compliant Identify HBV resistant mutations Add or switch to potent drug Non-compliant Counsel on adherence Favourable virological response (HBV DNA<35 IU/ml) Partial virological response (HBV DNA>35 IU/ml) Continue ETV therapy with regular monitoring Compliant Non-compliant Counsel on adherence Baseline low viral load (Baseline HBV DNA<1.77E+08 IU/ml) Baseline high viral load (Baseline HBV DNA>1.77E+08 IU/ml) Continue ETV therapy with regular monitoring Add or switch to potent drug ETV, entecavir. identified in 54 HBeAg-negative patients (all P-values for AUC >0.05, data not shown). As these unsatisfactory results might be due to the small number of HBeAg-negative patients (n=54), future studies with larger number of HBeAg-negative patients will overcome this statistical problem. Among the patients with PVR, we performed a subgroup analysis to identify those who would achieve VR at week 96. Only baseline HBV DNA level was significantly lower in patients who later achieved VR at week 96 (6.88 ±1.20 log 10 copies/ml versus 7.88 ±1.35 log 10 copies/ml; P=0.045). This finding indicates that in patients with PVR, baseline HBV DNA level can be used as another predictor for VR at week 96. In patients who show high baseline viral load (HBV DNA >1.77E+08 IU/ml) and PVR at week 48, the treatment strategy should be changed because these patients will not benefit from further use of ETV. By contrast, patients who show low baseline viral load (HBV DNA<1.77E+08 IU/ml) and PVR at week 48 might need more time to achieve VR. Thus, modification of treatment strategies should be delayed in these patients, because there is a chance for them to achieve VR if ETV is continued for up to 2 years. However, the role of baseline HBV DNA level measurements in patients with PVR should be validated in the future. Additionally, we analysed the influence of the pattern of HBV DNA level between week 48 and week 72 on the prediction of VR at week 96 for 41 patients with PVR. Of these, 35 (85.4%) patients exhibited a decreasing pattern of HBV DNA from week 48 to week 72, whereas the other 6 (14.6%) patients had an increasing pattern. Although all patients who showed PVR at week 48 and VR at week 96 (n=10) had a decreasing pattern of HBV DNA from week 48 to week 72, the patterns and differences of HBV DNA level from week 48 to week 72 were not selected as a predictor for VR at week 96 (all P>0.05). Antiviral Therapy

8 YE Chon et al. It is a great challenge for physicians to manage PVR, which forewarns treatment failure in the use of third-generation NUCs such as ETV. Pan and Hu [37] reported that among 236 CHB patients receiving ETV therapy for 68 weeks, 3% had a suboptimal response (<1 log 10 copies/ml of HBV DNA reduction at week 24). After switching to tenofovir monotherapy, all patients achieved HBV DNA <160 copies/ml within 24 weeks. However, there is risk of resistance with a switch-to strategy when maintaining an alternative monotherapy for a long period. By contrast, for add-on strategies, not only uncertain safety data for the concurrent use of ETV and tenofovir but also increased cost can be a problem. So far, there are conflicting opinions among experts on this issue and more investigations should be conducted. To the best of our knowledge, this is the first attempt to establish optimal PVR criteria for treatment of CHB using the scientific method. We propose a practical management guideline based on our PVR criteria (Figure 3). An initial response assessment should be performed at week 12. Responders should continue ETV and primary non-responders should be checked for compliance. The treatment response should be assessed again at week 48. Patients with a favourable response (<35 IU/ml of HBV DNA at week 48) should continue ETV, whereas patients with PVR (>35 IU/ml of HBV DNA at week 48) should be managed differently according to their baseline HBV DNA level, if they are compliant. Our study still leaves several issues unresolved. Firstly, the PVR criteria in our study should be validated in a cohort with different ethnicities and settings to test its extended use and corresponding management flow. Secondly, we could not predict the long-term VR beyond 2 years of ETV therapy, because few patients used ETV for more than 96 weeks and even fewer for more than 144 weeks. In addition, 195 patients were initially excluded only because they received ETV therapy for less than 96 weeks. Therefore, long-term large population-based studies should be followed to deduce comprehensive conclusions. Lastly, although we demonstrated that measuring HBV DNA level at week 48 is the best way to predict VR at week 96 among available variables, this criterion could not perfectly discriminate patients who would benefit from continued use of ETV for up to 2 years (23.4% of patients with PVR). Thus, if future studies identify new factors that are better predictors than those currently available, such as differences in viral sequence, our PVR criteria can be modified using these newly identified predictors. In conclusion, an HBV DNA level exceeding 35 IU/ ml (2.24 log 10 copies/ml, 174 copies/ml) at week 48 was the optimal criterion for treatment-naive patients with CHB receiving ETV. The results of this study support the PVR criteria suggested by the EASL guidelines for treatment-naive patients with CHB receiving ETV. Acknowledgements This work was supported by a grant of the Bilateral International Collaborative R&D Program from the Ministry of Knowledge Economy and by the Good Health R&D Project from the Ministry for Health, Welfare and Family Affairs, Republic of Korea (A050021). The authors are grateful to Dong-Su Jang, (Medical Illustrator, Medical Research Support Section, Yonsei Universtity College of Medicine, Seoul, Korea) for his help with the figures. The English in this document has been checked by at least two professional editors from Textcheck, both native speakers of English. A certificate can be produced on request. Disclosure statement The authors declare no competing interests. References 1. Hepatitis B vaccines: WHO position paper recommendations. Vaccine 2010; 28: Lee WM. Hepatitis B virus infection. 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