Journal of Antimicrobial Chemotherapy Advance Access published April 25, 2013

Transcription

1 Journal of Antimicrobial Chemotherapy Advance Access published April 25, 213 J Antimicrob Chemother doi:1.193/jac/dkt147 Virological response to entecavir reduces the risk of liver disease progression in nucleos(t)ide analogue-experienced HBV-infected patients with prior resistant mutants Shih-Cheng Yang, Chuan-Mo Lee, Tsung-Hui Hu, Jing-Houng Wang, Sheng-Nan Lu, Chao-Hung Hung, Chi-Sin Changchien and Chien-Hung Chen* Division of Hepatogastroenterology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan *Corresponding author. Division of Hepatogastroenterology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, 123 Ta Pei Road, Kaohsiung, Taiwan. Tel: ; Fax: ; e5836@ms31.hinet.net Received 7 December 212; returned 7 January 213; revised 6 February 213; accepted 25 March 213 Objectives: This study investigated the influence of virological response () to entecavir on clinical liver disease progression in nucleos(t)ide analogue (NA)-naive and -experienced patients. Methods: We investigated 487 chronic hepatitis B patients (323 NA-naive, 164 NA-experienced) treated with entecavir monotherapy for at least 12 months. was defined as hepatitis B virus DNA level,3 copies/ml during entecavir therapy. Clinical events were defined as hepatic decompensation, hepatocellular carcinoma (HCC), death and liver transplantation. Results: Of the 487 patients, 49 developed clinical events during entecavir treatment. Of those, 36 developed HCC. For all patients, Cox regression analysis showed that age, baseline cirrhosis, alanine aminotransferase level 2 U/L, albumin level and no during entecavir treatment were independent predictors for clinical events and HCC development. However, the benefit of to entecavir was significant for clinical events and HCC only in NA-experienced patients, but not in NA-naive patients. For the further analysis of the different subgroups of NAexperienced patients, the benefit of to entecavir was significant for clinical events or HCC only in patients with prior lamivudine- or adefovir-resistant mutants, but not in NA-experienced patients who had never developed lamivudine- or adefovir-resistant mutants. at month 12 (but not early at month 6) remained a significant predictor associated with the development of clinical events and HCC in NA-experienced patients. Conclusions: to entecavir was associated with a reduced risk of clinical events and HCC in NA-experienced patients, particularly in those who had prior lamivudine- or adefovir-resistant mutants. Keywords: hepatitis B virus, lamivudine, hepatocellular carcinoma Introduction Hepatitis B virus (HBV) infection is a major health problem, with an estimated prevalence of 35 million carriers worldwide. Each year HBV infection is responsible for more than 1 million deaths from cirrhosis and hepatocellular carcinoma (HCC). 1 The risk of progression to cirrhosis and HCC, and liver-related mortality, are strongly correlated with serum HBV DNA levels, and a reduction in HBV DNA to undetectable levels has been adopted as an important endpoint for antiviral efficacy in patients with chronic hepatitis B (CHB). 2 6 Entecavir is a potent antiviral agent superior to lamivudine and adefovir for hepatitis B virological suppression. 7 9 Suppression of HBV DNA replication to undetectable levels was observed in 92% of treatment-naive patients positive for hepatitis B e antigen (HBeAg), and genotypic resistance to entecavir is rare in nucleos(t)ide analogue (NA)-naive patients throughout 5 years of treatment Moreover, long-term entecavir therapy has been shown to improve fibrosis in patients with CHB However, limited data are available on the effect of virological response () to entecavir on the incidence of major clinical events such as HCC, decompensation and death in patients undergoing long-term entecavir therapy. A recent study demonstrated that to entecavir is associated with a lower probability of disease progression in patients with cirrhosis. 14 However, this study did not investigate the role of to entecavir in reducing the risk of disease progression separately according to NA-naive or NA-experienced patients. 14 The role Downloaded from at Pennsylvania State University on September 17, 216 # The Author 213. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oup.com 1of1

2 Yang et al. of in reducing the risk of liver disease progression may differ according to NA experience. Furthermore, a previous review study demonstrated that achievement of did not seem to significantly reduce the HCC risk in patients with lamivudine resistance treated with adefovir with or without continued lamivudine. 15 However, the benefit of for clinical events or HCC in patients with prior lamivudine- or adefovir-resistant mutants treated with entecavir monotherapy was not reported. The aim of this study was to investigate whether the role of to entecavir in predicting clinical liver disease progression or HCC differs between NA-naive and NA-experienced patients, particularly in those who have had prior NA-resistant mutants during entecavir therapy. Patients and methods Patients From August 26 to May 21, 487 consecutive CHB patients who received entecavir monotherapy were analysed retrospectively. Inclusion criteria consisted of seropositivity for hepatitis B surface antigen (HBsAg) for at least 6 months, a viral load of at least 2 IU/mL at the initiation of entecavir and duration of entecavir therapy of at least 12 months. Patients were excluded if they exhibited any evidence of autoimmune hepatitis, alcoholic liver disease or viral coinfections [hepatitis C virus (HCV), hepatitis D virus (HDV) or HIV]. Patients were excluded if they had HCC at baseline or if HCC occurred during the first 6 months of entecavir therapy. Of the 487 subjects, 323 were NA-naive and 164 had received prior lamivudine or adefovir treatment. A liver ultrasound scan was performed every 3 6 months and an ultrasound scoring system for liver surface, parenchyma, vascular structure and spleen size was used to describe the severity of hepatic parenchymal damage. 16,17 Cirrhosis was diagnosed by: (i) liver biopsy histology or (ii) combined repeated ultrasound findings suggestive of cirrhosis from at least two examinations performed a minimum of 6 months apart with clinical features such as splenomegaly, thrombocytopenia, oesophageal varices, ascites or hepatic encephalopathy. The diagnosis of HCC was based on: (i) histopathological findings of tumour tissues; or (ii) one typical HCC feature on a dynamic image or serum a-fetoprotein (AFP) levels.2 ng/ml if the nodule was.2 cm in cirrhotic liver; or (iii) two typical HCC features on dynamic images if the nodule was between 1 and 2 cm in a cirrhotic liver. 18 This study was approved by the Ethics Committee of Chang Gung Memorial Hospital and each patient provided informed consent. Methods All patients were followed up every 12 weeks throughout therapy. Supplementary weekly or twice-weekly visits were scheduled if alanine aminotransferase (ALT) levels were increasing more than five times the upper limit of the normal range (ULN; 4 U/L). Follow-up studies consisted of clinical assessment, conventional liver biochemical tests and measures of serological hepatitis B marker levels (including HBeAg and anti-hbe). Each patient was assessed for HBV DNA quantitatively prior to therapy and every 6 months during treatment or at the time of biochemical breakthrough. Genotypic analysis was performed: (i) in NA-experienced patients using serum stored at the end of all prior NA treatments if serum HBV DNA was 3 copies/ml; (ii) in NA-naive subjects using serum stored at the entecavir baseline if entecavir-resistant mutations were detected during entecavir treatment; or (iii) for all patients in case of virological breakthrough during entecavir monotherapy or if serum HBV DNA 3 copies/ml was observed at the end of follow-up. Definitions Virological response was defined as a serum HBV DNA level,3 copies/ml during entecavir treatment and -12 were defined as achieved at month 6 and 12, respectively. For non-cirrhotic and compensated cirrhotic patients at baseline, clinical events were defined as a composite of development of hepatic decompensation [jaundice (serum bilirubin level 3 mg/dl), new development of variceal bleeding, ascites or encephalopathy], new HCC, death or liver transplantation. 14 For patients with decompensated cirrhosis at baseline, clinical events were defined as recurrent hepatic decompensation (new or recurrent development of jaundice, varices bleeding, ascites or encephalopathy after clinical remission during entecavir treatment), new HCC, death or liver transplantation. Biochemical breakthrough was defined as an ALT level of more than twice the ULN during continued treatment in patients who initially had normalization of ALT. Virological breakthrough was defined as an increase in serum HBV DNA levels of 1 log 1 copies/ml from nadir. Serology The presence of HBsAg, HBeAg, anti-hcv antibodies and anti-hdv antibodies was determined using commercial assay kits [HBsAg enzyme immunoassay (EIA; Abbott, North Chicago, IL, USA); HBeAg EIA (Abbott); anti-hcv EIA 3. (Abbott); anti-hdv radioimmunoassay (Abbott)]. Serum HBV DNA levels were determined using a quantitative real-time PCR assay, the COBAS AmpliPrep-COBAS TaqMan HBV test (CAP-CTM; Roche Molecular Systems Inc., Branchburg, NJ, USA), with a lower detection limit of 12 IU/mL (7 copies/ml). Detection of HBV-resistant mutants in HBV polymerase genes The viral mutational analysis was performed by LiPA assay (Innogenetics NV, Gent, Belgium). LiPA DR version 2 and version 3 were used to identify the amino acids at codons rt173, rt18, rt24 and rt184, rt22, rt25, respectively. HBV genotyping The HBV genotypes in sera were determined using restriction fragment length polymorphism analysis of the S-gene sequence, amplified by PCR with nested primers, as previously described. 19,2 Data analysis Data are displayed as means+standard deviation (SD), proportions or medians (range). On comparing the values between two groups, the x 2 test was used to analyse categorical variables, and Student s t-test and the Mann Whitney U-test were applied for continuous variables with normal and skewed distributions, respectively. Cumulative incidences of, clinical events and HCC were determined using the Kaplan Meier method using a log-rank test. Univariate and multivariate analyses were performed to identify factors associated with clinical events and HCC development, including baseline characteristics and during entecavir therapy, using Cox proportional hazards regression models. All statistical tests were two-sided, and a P value of,.5 was considered statistically significant. Downloaded from at Pennsylvania State University on September 17, 216 2of1

3 Virological response to entecavir and liver disease JAC Results Baseline characteristics of the study population Baseline characteristics of the study population are shown in Table 1. NA-experienced patients were more often men and had lower serum total bilirubin levels and prothrombin time at baseline compared with NA-naive patients. Sixty-two (37.8%) and 25 (15.2%) NA-experienced patients had a history of genotypic resistance to lamivudine and adefovir, respectively. The study population consisted of 314 patients with CHB without cirrhosis, 152 patients with compensated cirrhosis, and 21 patients with decompensated cirrhosis. Overall median follow-up duration for the study population was 36 (12 73) months. Predictive factors for developing clinical events and HCC for all patients Forty-nine patients in the study population developed clinical events during entecavir therapy. Of these, 1 developed an episode of hepatic decompensation, 36 developed HCC and 3 died (2 from hepatic failure and 1 from oesophageal variceal bleeding). The cumulative probabilities of developing clinical events at months 12, 24, 36 and 48 were.3%, 1%, 1.8% and 5.1% for non-cirrhotic patients, respectively; 1.4%, 9.2%, 15.4% and 22.3% for compensated cirrhotic patients, respectively; Table 1. Baseline characteristics of the study population Characteristics Naive (n¼323) NA-experienced (n¼164) P value Age (years) Male 256 (79.3%) 144 (87.8%).2 Genotype C 121 (37.5%) 62 (37.8%).92 Cirrhosis 121 (37.5%) 52 (31.7%).21 Decompensated 15 (4.6%) 6 (3.7%).61 cirrhosis Child Pugh score a ALT (U/L) Total bilirubin (mg/dl) Prothrombin time (s) Albumin (g/l) Platelets ( 1/mL) HBV DNA (log copies/ ml) HBeAg positive 118 (36.5%) 69 (42.1%).24 Prior interferon 2 (6.2%) 15 (9.1%).23 experience Prior 3TC experience 155 (94.5%) Prior ADV experience 45 (27.4%) Prior 3TC resistance 62 (37.8%) Prior ADV resistance 25 (15.2%) Achieved during entecavir treatment 313 (96.9%) 132 (8.5%).1 3TC, lamivudine; ADV, adefovir dipivoxil; ALT, alanine aminotransferase. a Child Pugh scores were analysed only in cirrhotic patients. and 23.8%, 38.5% and 49.7% (no subject had follow-up for 4 years) for decompensated cirrhotic patients, respectively (P,.1, Figure 1a). The cumulative probabilities of developing HCC at months 12, 24, 36 and 48 were %,.7%, 1.9% and 3.8% for non-cirrhotic patients, respectively;.7%, 5.7%, 11.2% and 19.1% for compensated cirrhotic patients, respectively; and 14.3%, 28.9%, 34.4% and 34.4% for decompensated cirrhotic patients, respectively (P,.1, Figure 1b). Patients with decompensated cirrhosis had the highest incidence of developing clinical events and HCC during entecavir therapy. The cumulative probabilities of developing clinical events and HCC at months 12, 24, 36 and 48 were.5% and %, 1% and.5%, 1% and.5%, and 2.8% and.5% for naive non-cirrhotic patients, respectively. The cumulative probabilities of developing clinical events and HCC at months 12, 24, 36 and 48 were 1.7% and.8%, 1% and 6.7%, 27.4% and 13.1%, and 26.3% and 23.2% for naive cirrhotic patients, respectively. (a) 1 Cumulative incidence of clinical events A B C (b) 1 Cumulative incidence of HCC A B C P < P <.1 A: decompensated cirrhosis B: compensated cirrhosis C: non-cirrhosis A B C A: decompensated cirrhosis B: compensated cirrhosis C: non-cirrhosis A B C Figure 1. The cumulative incidence of developing (a) clinical events and (b) HCC in non-cirrhotic, compensated cirrhotic and decompensated cirrhotic patients. Downloaded from at Pennsylvania State University on September 17, 216 3of1

4 Yang et al. Table 2. Univariate and multivariate analysis of factors predictive of clinical events for all patients Univariate analysis Multivariate analysis a Variable Comparison hazard ratio (95% CI) P value hazard ratio (95% CI) P value Age (years) increase per 1 year 1.5 ( ), ( ).31 Sex male versus female.84 ( ).62 HBV genotype C versus B 1.4 ( ).89 Cirrhosis yes versus no 1.6 ( ), ( ).9 HBeAg yes versus no.4 (.2.83).13 HBV DNA increase per 1 log copies/ml.76 (.63.91).3 ALT U/L.2 versus 2.25 (.11.59).1.32 (.13.75).9 Total bilirubin increase per 1 mg/dl 1.4 ( ).23 Prothrombin time increase per 1 s 1.19 ( ),.1 Albumin increase per 1 g/l.24 (.16.37),.1.29 (.16.5),.1 Platelets increase per 1/mL.98 (.98.99), ( ).5 Naive yes versus no.78 ( ).39 Prior interferon experience yes versus no.22 (.3 1.6).14 Prior 3TC experience yes versus no 1.3 ( ).38 Prior ADV experience yes versus no 1.92 (.9 4.1).92 Prior LAM resistance yes versus no 1.59 ( ).23 Prior ADV resistance yes versus no 3. ( ).12 ETV resistance yes versus no 2.34 ( ).11 b yes versus no.29 (.13.62).1.16 (.7.36),.1-6 c yes versus no 1.11 (.6 2.7) c yes versus no.49 ( ).54 3TC, lamivudine; ADV, adefovir; ALT, alanine aminotransferase; ETV, entecavir. a Variables for the multivariate analysis included all factors of the univariate analysis. b during entecavir therapy. c -6 and -12, at month 6 and month 12 of entecavir therapy, respectively. The baseline factors and the achievement of during entecavir treatment were analysed for association with clinical events (Table 2) and HCC (Table 3). Cox regression analysis showed that age, baseline cirrhosis, pre-treatment ALT level 2 U/L, albumin level, platelet count and non-achievement of during entecavir treatment were independent predictors for clinical events (Table 2). Age, baseline cirrhosis, pre-treatment ALT level 2 U/L, albumin level and non-achievement of were predictive of HCC (Table 3). during entecavir treatment remained an independent predictor for clinical events and HCC after excluding decompensated cirrhotic patients. Of the 487 patients, 25 (1 naive, 24 NA-experienced) developed entecavir resistance during entecavir therapy. There was no significant difference in terms of developing clinical events and HCC between patients with and without occurrence of entecavir resistance (Tables 2 and 3). Predictive factors for developing clinical events and HCC for NA-experienced patients The baseline factors and the achievement of during entecavir treatment for NA-experienced patients were analysed for association with clinical events (Table 4) and HCC (Table 5). Cox regression analysis showed that baseline cirrhosis, HBeAg-negative status, pre-treatment HBV DNA level, prothrombin time and nonachievement of during entecavir treatment were independent predictors for clinical events (Table 4). Age, baseline cirrhosis and non-achievement of were independent predictors for HCC (Table 5). Effect of on clinical events or HCC in NA-naive and NA-experienced patients The cumulative probabilities of achieving a at months 12, 24, 36 and 48 were 74%, 95.2%, 98% and 98% in NA-naive patients, respectively, and 55.5%, 79.9%, 83.3% and 83.3% in NA-experienced patients, respectively (P,.1). NA-experienced patients had a lower rate of than NA-naive patients [hazard ratio (HR) 1.47; 95% CI ; P,.1] after adjusting for age, ALT levels, HBeAg status and HBV DNA at baseline. For all patients, the cumulative incidences of clinical events and HCC development according to during entecavir therapy are shown in Figure 2. In non-cirrhotic patients, the incidence of clinical events (HR.15, 95% CI.3.8) and HCC development (HR.8, 95% CI.1.5) was lower in patients with than in those without. In cirrhotic patients, the incidence of clinical events (HR.15, 95% CI.3.8) and HCC development (HR.21, 95% CI.7.6) was lower in patients with compared with in those without. Because the rate of was different between NA-naive and NA-experienced patients, we compared the influence of on the risk of developing clinical events and HCC in both groups. Downloaded from at Pennsylvania State University on September 17, 216 4of1

5 Virological response to entecavir and liver disease JAC Table 3. Univariate and multivariate analysis of factors predictive of HCC development for all patients Univariate analysis Multivariate analysis a Variable Comparison hazard ratio (95% CI) P value hazard ratio (95% CI) P value Age (years) increase per 1 year 1.6 ( ), ( ).2 Sex male versus female.75 ( ).47 HBV genotype C versus B 1.6 ( ).86 Cirrhosis yes versus no 1.9 ( ), ( ),.1 HBeAg yes versus no.43 (.19.99).48 HBV DNA increase per 1 log copies/ml.73 (.59.91).5 ALT U/L.2 versus 2.16 (.5.53).3.25 (.8.84).24 Total bilirubin increase per 1 mg/dl.97 ( ).61 Prothrombin time increase per 1 s 1.14 ( ).49 Albumin increase per 1 g/l.32 (.18.56),.1.4 (.2.78).7 Platelets increase per 1/mL.99 (.98.99),.1 Naive yes versus no.87 ( ).68 Prior interferon experience yes versus no.4 (. 6.69).22 Prior 3TC experience yes versus no 1.12 ( ).75 Prior ADV experience yes versus no 2.1 ( ).12 Prior 3TC resistance yes versus no 2.6 ( ).86 Prior ADV resistance yes versus no 2.73 ( ).59 ETV resistance yes versus no 2.55 ( ).12 b yes versus no.27 (.11.65).4.19 (.8.48),.1-6 c yes versus no.98 (.48 2.) c yes versus no.44 ( ).55 3TC, lamivudine; ADV, adefovir; ALT, alanine aminotransferase; ETV, entecavir. a Variables for the multivariate analysis included all factors of the univariate analysis. b during entecavir therapy. c -6 and -12, at month 6 and month 12 of entecavir therapy, respectively. In NA-naive patients, no significant difference was noted between patients with and without for clinical events and HCC development in non-cirrhotic and cirrhotic patients. All 3 NA-naive patients who developed clinical events (including 23 cases of HCC) did so after achieving. Furthermore, early at month 6 or 12 was not a significant predictor for developing clinical events and HCC in NA-naive patients either with or without cirrhosis. In NA-experienced patients, the cumulative incidences of clinical events and HCC development according to during entecavir therapy are shown in Figure 3. There was a significant difference between patients with and without for clinical events and HCC development in non-cirrhotic patients (clinical events and HCC: HR.1, 95% CI.1.77) and cirrhotic patients (clinical events: HR.15, 95% CI.5.44; HCC: HR.13, 95% CI.3.49). In addition, at month 12 of entecavir therapy (-12) was also significantly predictive for clinical events (P¼.35 and P¼.7 by log-rank test in non-cirrhotic and cirrhotic patients, respectively) and HCC (P¼.35 and P¼.5 in non-cirrhotic and cirrhotic patients, respectively) in NA-experienced patients. Furthermore, -12 was also an independent predictor for clinical events (HR.15, 95% CI.6.4, P,.1) and HCC (HR.17, 95% CI.5.54, P¼.3) after adjusting for other baseline factors (except during entecavir therapy) in NA-experienced patients. By contrast, at month 6 was not a significant predictor for developing clinical events and HCC in NA-experienced patients either with or without cirrhosis. All risk factors included in Table 2 were analysed. In non-cirrhotic patients, multivariate analysis showed that lower pre-treatment HBV DNA levels (increase per 1 log copies/ml; HR.48, 95% CI.25.93, P¼.29) and (HR.34, 95% CI.3.474, P¼.12) were independent predictors for clinical events and HCC development. In cirrhotic patients, only during entecavir therapy was an independent predictor for developing clinical events (HR.15, 95% CI.5.44, P¼.1) and HCC (HR.13, 95% CI.3.49, P¼.3). Effect of on clinical events or HCC in different subgroups of NA-experienced patients Of the 164 NA-experienced patients, 92 had no prior lamivudine or adefovir resistance, 62 had prior lamivudine-resistant mutants and 25 had prior adefovir-resistant mutants (15 had adefovir-resistant mutations after switching to adefovir sequential monotherapy for lamivudine-resistant CHB). The rates of, clinical events and HCC development in the three subgroups are presented in Figure 4. Compared with naive patients, rates were not decreased by NA experience in those who never developed lamivudine- or adefovir-resistant mutants (P¼.33 by log-rank test). By contrast, patients with prior lamivudine- or adefovir-resistant mutants were significantly associated with reduced rates (P,.1). In patients who had never developed lamivudine- or adefovir-resistant mutants, to Downloaded from at Pennsylvania State University on September 17, 216 5of1

6 Yang et al. Table 4. Univariate and multivariate analysis of factors predictive of clinical events for NA-experienced patients Univariate analysis Multivariate analysis a Variable Comparison hazard ratio (95% CI) P value hazard ratio (95% CI) P value Age (years) Increase per 1 year 1.5 ( ).22 Sex male versus female.73 ( ).616 HBV genotype C versus B.41 ( ).117 Cirrhosis yes versus no 7.45 ( ), ( ).1 HBeAg yes versus no.19 (.4.84) (.4.83).29 HBV DNA increase per 1 log copies/ml.82 ( ) (.56.96).28 ALT U/L.2 versus 2.11 (.1.82).31 Total bilirubin increase per 1 mg/dl 1.11 ( ).324 Prothrombin time increase per 1 s 1.4 ( ) ( ).8 Albumin increase per 1 g/l.36 (.18.72).4 Platelets increase per 1/mL.98 (.97.99),.1 Prior interferon experience yes versus no.42 ( ).41 Prior 3TC experience yes versus no 1.17 ( ).878 Prior ADV experience yes versus no 1.87 ( ).179 Prior 3TC resistance yes versus no 1.46 ( ).421 Prior ADV resistance yes versus no 2.88 ( ).33 ETV resistance yes versus no 2.9 ( ).195 b yes versus no.18 (.7.47),.1.7 (.2.22),.1-6 c yes versus no.6 ( ) c yes versus no.23 (.9.58).2 ALT, alanine aminotransferase; ETV, entecavir; 3TC, lamivudine; ADV, adefovir. a Variables for the multivariate analysis included all factors of the univariate analysis. b during entecavir therapy. c -6 and -12, at month 6 and month 12 of entecavir therapy, respectively. entecavir was not a significant predictor for the development of clinical events and HCC. By contrast, in patients with lamivudine-resistant mutants, lack of to entecavir was an independent predictor for the development of clinical events and HCC after adjusting for other baseline factors. In patients with adefovir-resistant mutants, lack of to entecavir was an independent predictor for the development of clinical events after adjusting for other baseline factors, but not for HCC (Figure 4). Discussion In the large randomized trial described by Liaw et al., 21 in which CHB patients with advanced fibrosis or cirrhosis were included, long-term lamivudine monotherapy was shown to reduce the risk of liver disease progression and HCC in the absence of genotypic resistance compared with placebo. Another study showed that in HBeAg-negative patients, long-term lamivudine treatment significantly improved survival and reduced the risk of major complications compared with interferon treatment in patients without sustained response or untreated patients. 22 There was a trend for better major-event-free survival in patients remaining in than in those with virological breakthrough or no response to lamivudine. 22 Thus, CHB patients receiving NA therapy had a significantly lower incidence of liver-related complications and HCC than untreated patients. Furthermore, maintenance of was important because patients developing lamivudine resistance were at increased risk of developing liverrelated complications and HCC. 15,21 23 A recent study reported significant beneficial effects of to entecavir in preventing liver disease progression in CHB patients with cirrhosis. 14 However, the study did not investigate the role of in reducing the risk of liver disease progression separately according to NA-naive or NA-experienced patients. 14 In the present study, our data showed the beneficial effects of to entecavir in preventing liver disease progression in those both with and without cirrhosis. Furthermore, to entecavir reduced the probability of developing clinical events and HCC in NA-experienced patients but not in NA-naive patients. This difference is probably caused by the relatively small number of NA-naive patients who did not achieve (3.4%, 11/323), making it impossible to perform statistically robust comparisons. In addition, most of the NA-naive patients without were positive for HBeAg and had high HBV DNA levels at baseline. These patients were younger and did not have cirrhosis, and had a relatively low risk of developing clinical events and HCC. In our study, all 3 NA-naive patients who developed clinical events (including 23 cases of HCC) did so after achieving. Thus, achieving early during entecavir treatment did not completely eliminate the risk of clinical events and HCC in NA-naive patient, and such patients should be continuously monitored for clinical events and HCC, especially those with cirrhosis. However, we found that was significantly associated with a lower probability of clinical events and HCC in both Downloaded from at Pennsylvania State University on September 17, 216 6of1

7 Virological response to entecavir and liver disease JAC Table 5. Univariate and multivariate analysis of factors predictive of HCC development for NA-experienced patients Univariate analysis Multivariate analysis a Variable Comparison hazard ratio (95% CI) P value hazard ratio (95% CI) P value Age (years) increase per 1 year 1.9 ( ) ( ).33 Sex male versus female.44 ( ).212 HBV genotype C versus B.48 ( ).269 Cirrhosis yes versus no 4.35 ( ) ( ).15 HBeAg yes versus no.3 ( ).122 HBV DNA increase per 1 log copies/ml.84 ( ).36 ALT U/L.2 versus 2.3 (. 3.28).139 Total bilirubin increase per 1 mg/dl 1.5 ( ).759 Prothrombin time increase per 1 s 1.35 ( ).65 Albumin increase per 1 g/l.38 (.16.9).28 Platelets increase per 1/mL.99 (.98.99).4 Prior interferon experience yes versus no.4 ( ).41 Prior 3TC experience yes versus no.782 (.1 6.2).813 Prior ADV experience yes versus no 2.23 ( ).15 Prior 3TC resistance yes versus no 2.41 ( ).116 Prior ADV resistance yes versus no 2.72 ( ).97 ETV resistance yes versus no 2.44 ( ).182 b yes versus no.16 (,.5.48).1.18 (.5.57).4-6 c yes versus no.41 ( ) c yes versus no.18 (.6.54).2 3TC, lamivudine; ADV, adefovir; ALT, alanine aminotransferase; ETV, entecavir. a Variables for the multivariate analysis included all factors of the univariate analysis. b during entecavir therapy. c -6 and -12, at month 6 and month 12 of entecavir therapy, respectively. non-cirrhotic and cirrhotic NA-experienced CHB patients. Previous studies have shown that the antiviral efficacy of entecavir is significantly decreased in patients with lamivudine-resistant mutations. 24 In our study, 37.8% (62/164) of NA-experienced patients had a prior history of lamivudine resistance. Of these, only 59.7% (37/62) achieved during entecavir therapy. Our study showed that to entecavir was the most important independent predictor for clinical disease progression and HCC in NA-experienced patients after adjusting for other baseline factors, particularly in patients with pre-existing cirrhosis. We further investigated the effect of on clinical outcome in the different subgroups of NA-experienced patients. We did not show a significant effect of as a predictor for clinical outcome in NA-experienced patients who had not developed lamivudine- or adefovir-resistant mutants. However, patients without during entecavir therapy were at increased risk of developing liver-related complications and HCC if they were NA-experienced patients with lamivudine- or adefovir-resistant mutants. A previous review study demonstrated that achievement of did not seem to significantly reduce the HCC risk in patients with lamivudine resistance treated with adefovir with or without continued lamivudine. 15 Our result is not compatible with previous studies. The benefit of for reducing the liver disease progression or HCC risk seems to be different in patients with lamivudine resistance who received adefovir with or without lamivudine or entecavir rescue therapy. A previous study suggested that early monitoring of to NA therapy in NA-naive patients is essential to identify cases of suboptimal response at week 24 or 48 in order to modify the subsequent therapy accordingly. 6,25 However, it remained unclear whether early to entecavir at week 24 or 48 could predict clinical outcomes. In our study, we found that achieving a at month 12 (but not at month 6) was an effective predictor for clinical disease progression and HCC in NA-experienced patients. By contrast, at month 6 or 12 was not a significant predictor for clinical disease progression and HCC in NA-naive patients. Early to entecavir at month 6 was therefore not a useful predictor in NA-naive or -experienced patients. However, multivariate analysis showed that achieving a during entecavir therapy (but not -12) was an independent predictor for clinical events and HCC development (Tables 2 and 3). Considering these results, it is important to note that a delayed to entecavir over 12 months is still useful for reducing the risk of clinical disease progression and HCC in NA-experienced patients. We suggest that adding or shifting to other appropriate antiviral agents to achieve in NA-experienced patients who did not achieve a at or beyond month 12 under entecavir treatment is important in reducing the risk of clinical disease progression and HCC. It is interesting to note that in our study, a higher pretreatment ALT level (.2 IU/L) was an independent protective factor against clinical events and HCC. The rate of pre-treatment ALT ( 2 U/L) was observed more frequently in cirrhotic patients than non-cirrhotic patients (42.4% versus 23.7%, Downloaded from at Pennsylvania State University on September 17, 216 7of1

8 Yang et al. (a) 1 (a) 1 Cumulative incidence of clinical events (HCC) no (b) Cumulative incidence of clinical events no no (c) 1 Cumulative incidence of HCC P = no P < P = no no 63 3 Figure 2. The cumulative incidence of developing clinical events and HCC during entecavir therapy according to : (a) clinical events (all HCC) in non-cirrhotic patients; (b) clinical events in cirrhotic patients; and (c) HCC in cirrhotic patients. P.1). This might partially explain how a higher ALT at baseline would be protective against clinical events. However, it remains unclear what mechanism lies behind the association Cumulative incidence of clinical events (HCC) no (b) (c) Cumulative incidence of clinical events P =.7 no no Cumulative incidence of clinical events no P <.1 P < no no Figure 3. The cumulative incidence of developing clinical events and HCC during entecavir therapy in NA-experienced patients according to : (a) clinical events (all HCC) in non-cirrhotic patients; (b) clinical events in cirrhotic patients; and (c) HCC in cirrhotic patients. between a higher ALT and the decreased risk of clinical events, even after adjusting for other factors. In conclusion, our study suggests that during entecavir therapy was associated with a reduced risk of clinical liver Downloaded from at Pennsylvania State University on September 17, 216 8of1

9 Virological response to entecavir and liver disease JAC No 3TC or ADV resistance n = 92 Prior 3TC-resistant mutants n = 62 Prior ADV-resistant mutants n = 25 Yes No Yes No Yes No Events 7/87 HCC 3/87 disease progression and HCC only in NA-experienced patients who had prior lamivudine- or adefovir-resistant mutants, but not in NA-naive patients or NA-experienced patients who had never developed lamivudine- or adefovir-resistant mutants. However, only a relatively small number of NA-naive patients and NA-experienced patients without lamivudine- or adefovirresistant mutants did not achieve, which limited the power of the statistical analysis among both subgroups. to entecavir at month 12 (but not early at month 6) remained a significant predictor associated with the development of clinical events and HCC in NA-experienced patients. Funding This study was supported by grants CMRPG from Chang Gung Memorial Hospital, Taiwan. Transparency declarations None to declare. 1/5 1/5 Cox regression Events HR: % CI: HCC HR:.1. 95% CI: /37 2/37 References 1 Lee WM. Hepatitis B virus infection. N Engl J Med 1997; 337: Iloeje UH, Yang HI, Su J et al. Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer-In HBV (the REVEAL-HBV) Study Group. Predicting cirrhosis risk based on the level of circulating hepatitis B viral load. Gastroenterology 26; 13: Chen CJ, Yang HI, Su J et al. Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA 26; 295: Liaw YF, Kao JH, Piratvisuth T et al. Asian-Pacific consensus statement on the management of chronic hepatitis B: a 212 update. Hepatol Int 212; 6: Lok AS, McMahon BJ. Chronic hepatitis B: update 29. Hepatology 29; 5: /25 5/25 HR:.9. 95% CI:.2.48 HR: % CI: /17 2/17 4/8 2/8 HR: % CI:.3.79 HR:.3. 95% CI: Figure 4. Incidence of clinical events and HCC during entecavir therapy according to in the different subgroups of NA-experienced patients. 3TC, lamivudine; ADV, adefovir; HR, hazard ratio. 6 European Association for the Study of the Liver. EASL Clinical Practice Guidelines: Management of chronic hepatitis B virus infection. J Hepatol 212; 57: 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 26; 354: Lai CL, Shouval D, Lok AS et al. Entecavir versus lamivudine for patients with HBeAg-negative chronic hepatitis B. N Engl J Med 26; 354: Leung N, Peng CY, Hann HW et al. Early hepatitis B virus DNA reduction in hepatitis B e antigen-positive patients with chronic hepatitis B: a randomized international study of entecavir versus adefovir. Hepatology 29; 49: 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 29; 49: Gish RG, Lok AS, Chang TT et al. Entecavir therapy for up to 96 weeks in patients with HBeAg-positive chronic hepatitis B. Gastroenterology 27; 33: Chang TT, Lai CL, Kew YS et al. Entecavir treatment for up to 5 years in patients with hepatitis B e antigen-positive chronic hepatitis B. Hepatology 21; 51: Chang TT, Liaw YF, Wu SS et al. Long-term entecavir therapy results in the reversal of fibrosis/cirrhosis and continued histological improvement in patients with chronic hepatitis B. Hepatology 21; 52: Zoutendijk R, Reijnders JG, Zoulim F et al. for the VIRGIL Surveillance Study Group. Virological response to entecavir is associated with a better clinical outcome in chronic hepatitis B patients with cirrhosis. Gut 213; 62: Papatheodoridis GV, Lampertico P, Manolakopoulos S et al. Incidence of hepatocellular carcinoma in chronic hepatitis B patients receiving nucleos(t)ide therapy: a systematic review. J Hepatol 21; 53: Lin DY, Sheen IS, Chiu CT et al. Ultrasonographic changes of early liver cirrhosis in chronic hepatitis B: a longitudinal study. J Clin Ultrasound 1993; 21: Hung CH, Lu SN, Wang JH et al. Correlation between ultrasonographic and pathologic diagnoses of hepatitis B and C virus-related cirrhosis. J Gastroenterol 23; 38: Downloaded from at Pennsylvania State University on September 17, 216 9of1

10 Yang et al. 18 Bruix J, Sherman M, Practice Guidelines Committee, American Association for the Study of Liver Diseases. Management of hepatocellular carcinoma. Hepatology 25; 42: Mizokami M, Nakano T, Orito E et al. Hepatitis B virus genotype assignment using restriction fragment length polymorphism patterns. FEBS Lett 1999; 45: Lee CM, Chen CH, Lu SN et al. Prevalence and clinical implications of hepatitis B virus genotypes in southern Taiwan. Scand J Gastroenterol 23; 1: Liaw YF, Sung JJ, Chow WC et al. Lamivudine for patients with chronic hepatitis B and advanced liver disease. N Engl J Med 24; 351: Papatheodoridis GV, Dimou E, Dimakopoulos K et al. Outcome of hepatitis B e antigen-negative chronic hepatitis B on long-term nucleos(t)ide analog therapy starting with lamivudine. Hepatology. 25; 42: Sung JJ, Tsoi KK, Wong VW et al. Meta-analysis: treatment of hepatitis B infection reduces risk of hepatocellular carcinoma. Aliment Pharmacol Ther 28; 28: Reijnders JG, Deterding K, Petersen J et al. for the VIRGIL Surveillance Study Group. Antiviral effect of entecavir in chronic hepatitis B: influence of prior exposure to nucleos(t)ide analogues. J Hepatol 21; 52: Keeffe EB, Zeuzem S, Koff RS et al. Report of an international workshop: roadmap for management of patients receiving oral therapy of chronic hepatitis B. Clin Gastroenterol Hepatol 27; 5: Downloaded from at Pennsylvania State University on September 17, of 1