Original article On-treatment monitoring of liver fibrosis with transient elastography in chronic hepatitis B patients

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1 Antiviral Therapy 2011; 16: (doi: /IMP1726) Original article On-treatment monitoring of liver fibrosis with transient elastography in chronic hepatitis B patients Grace Lai-Hung Wong 1,2, Vincent Wai-Sun Wong 1,2, Paul Cheung-Lung Choi 3, Anthony Wing-Hung Chan 3, Angel Mei-Ling Chim 1,2, Karen Ka-Lam Yiu 1,2, Shirley Hoi-Ting Chu 1,2, Francis Ka-Leung Chan 1,2, Joseph Jao Yao Sung 1,2, Henry Lik-Yuen Chan 1,2 * 1 Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong SAR, China 2 Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong SAR, China 3 Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong SAR, China *Corresponding author hlychan@cuhk.edu.hk Background: The performance of liver stiffness measurement (LSM) to monitor the changes in the severity of liver fibrosis in chronic hepatitis B (CHB) patients on antiviral treatment is uncertain. Methods: We prospectively studied CHB patients undergoing paired liver biopsy and transient elastography before and at week 48 of antiviral treatment. Based on our previously reported LSM algorithm, advanced liver fibrosis (F3 4) could be excluded or confirmed at >90% confidence. Results: A total of 71 CHB patients were studied. The median alanine aminotransferase (ALT) level decreased from 99I U/l to 33I U/l, and the median LSM decreased from 8.8 kpa to 6.6 kpa, respectively, from baseline to week 48. Overall, 17 and 11 patients had regression and progression of histological fibrosis, respectively. Areas under the receiver operating characteristics curves of the LSM algorithm at baseline and week 48 for advanced fibrosis were 0.80 (95% confidence interval [CI] ) and 0.78 (95% CI ), respectively. The sensitivity of LSM algorithm to exclude advanced fibrosis was 100% at baseline and 75% at week 48. The specificity of the LSM algorithm to diagnose advanced fibrosis was 84% at baseline and 91% at week 48. Overall, 11/28 (39%) patients with LSM that decreased by >30%, 28/41 (68%) of patients with LSM that changed within 30% and 1/2 (50%) patients with LSM that increased by >30% had decreased, unchanged and increased histological fibrosis stages, respectively. Conclusions: LSM could predict advanced fibrosis during antiviral therapy according to the ALT-based algorithm. Decrease in absolute LSM value, which could be related to ALT normalization, was unreliable to indicate regression of liver fibrosis. Introduction Chronic hepatitis B (CHB) is a global health problem affecting >350 million people, and causes liver cirrhosis, hepatic decompensation and hepatocellular carcinoma [1]. Liver fibrosis is the important intermediate step in the progression of liver disease, which might eventually result in liver cirrhosis and various cirrhotic complications. Accurate staging of liver fibrosis is of paramount importance in the consideration of antiviral treatment and prediction of prognosis in CHB [2 4]. Effective antiviral therapies leading to potent viral suppression are now available, but not all patients receiving these therapies would have regression of fibrosis [5,6]. Histological response, particularly regression of cirrhosis, is associated with decreased liver-related complications in CHB patients in the long-term [7]; hence, monitoring of changes in liver fibrosis after antiviral therapies provides important information concerning patients prognosis. Liver biopsy has been the gold standard to assess and monitor the stage of liver fibrosis. Repeated biopsy is uncommonly performed outside clinical trial settings because of its invasiveness, risk of complications, patient discomfort and the unavailability of expertise [8,9]. An accurate, non-invasive assessment of liver fibrosis is warranted to monitor the change in liver fibrosis, both in the natural course of disease and during antiviral therapy. Different approaches including ultrasonography, combined clinical laboratory criteria, biochemical and haematological tests, surrogate serum fibrosis markers and serum proteomics or glycomics have been 2011 International Medical Press (print) (online) 165

2 GL-H Wong et al. developed to diagnose significant fibrosis and cirrhosis with accuracies of 51 84% and 79 87%, respectively [10]; however, most of these fibrosis markers have been evaluated only in treatment-naive CHB patients and chronic hepatitis C patients. Among all serum test formulae, Fibrotest was the most extensively studied tool to monitor the change in liver fibrosis in viral hepatitis patients [11]; however, Fibrotest requires the measurement of uncommon and expensive laboratory parameters, and most of the validation studies were conducted by the same group of investigators. Transient elastography (Fibroscan ; Echosens, Paris, France) is a rapid, non-invasive and reproducible method of measuring liver stiffness [12]. Liver stiffness measurement (LSM) has been shown to be a reliable tool to detect early liver cirrhosis in CHB and chronic hepatitis C [6,12 19]. The accuracy of LSM was also unaffected by hepatic steatosis [20]. One logical use of transient elastography is to monitor the change in liver fibrosis during antiviral therapy; however, LSM is increased when alanine aminotransferase (ALT) is increased [6,15], whereas antiviral treatment can reduce ALT level with or without improvement in liver fibrosis. So far, the use of LSM to monitor the change in liver fibrosis has not been properly evaluated with consideration on changes of ALT levels during antiviral therapy. In this study, we aimed to assess the accuracy of LSM to exclude and confirm advanced liver fibrosis in a prospective cohort of CHB patients before and after antiviral treatment. We also aimed to investigate the performance of LSM to monitor the change in liver fibrosis in this cohort. Methods Patients We prospectively recruited consecutive adult patients with CHB undergoing paired liver biopsy and transient elastography examination in our hospital (Prince of Wales Hospital, Hong Kong SAR, China) at baseline and week 48 of antiviral treatment of two multicentre randomized controlled trials (CI-PSI and CI-PSI ) sponsored by Pharmasset, Inc. (Princeton, NJ, USA) from January 2008 to April These trials aimed to compare adefovir dipivoxil 10 mg daily versus clevudine 30 mg daily in hepatitis B e antigen-positive and -negative CHB. The indication of liver biopsy was to assess the severity of liver fibrosis and necroinflammation prior to treatment, as well as to monitor the change in liver fibrosis at week 48 of antiviral treatment. CHB was diagnosed by positive serology tests for serum hepatitis B surface antigen (HBsAg) for 6 months. We excluded patients with evidence of other chronic liver disease by screening with antibody against HCV, anti-nuclear antibody, anti-smoothmuscle antibody, anti-mitochondrial antibody, serum ceruloplasmin, transferrin saturation and ferritin. We interviewed every patient with a standardized questionnaire and excluded men who consumed >30 g of alcohol per week and women who consumed >20 g of alcohol per week. We also excluded patients who had decompensated liver disease, complications of liver cirrhosis, hepatocellular carcinoma, previous liver surgery or liver transplantation. All patients received comprehensive clinical and laboratory assessment at the time of LSM. Serum HBV DNA levels were measured by the Roche Cobas TaqMan Real-Time PCR assay (Roche Molecular Systems, Pleasanton, CA, USA) with a range of detection of copies/ml. The study protocol was approved by the Joint Chinese University of Hong Kong New Territories East Cluster Clinical Research Ethics Committee, and all patients gave written informed consent before enrolled into the study. Histological assessment Percutaneous liver biopsy was performed using the 16G Temno biopsy needle (Cardinal Health, Dublin, OH, USA). Liver histology was assessed by pathologists specialized in liver diseases (PC-LC and AW-HC) without knowledge of the clinical data. A liver sample was considered adequate if it was longer than 15 mm and contained 6 portal tracts. Liver fibrosis and necroinflammatory activity were evaluated semi-quantitatively according to the Metavir scoring system as follows [21]: F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis and few septa; F3, numerous septa without cirrhosis; and F4, cirrhosis. Metavir activity score was defined as: A0, none; A1, mild; A2, moderate; and A3, severe. Advanced fibrosis was defined as Metavir fibrosis score 3 or above (F3 4). Progression or regression of fibrosis was defined as increase or decrease, respectively, in Metavir fibrosis score by 1 stage at week 48 as compared with that at baseline. LSM by transient elastography LSM was performed using transient elastography according to the instructions and training provided by the manufacturer. Details of the technical background and examination procedure have been previously described [12]. The LSM was considered reliable only if 10 successful acquisitions were obtained, with interquartile range (IQR) 30% of LSM and a success rate >60%. The liver stiffness was expressed in kpa. A higher kpa reflected a stiffer liver and more severe liver fibrosis. Exclusion and confirmation of advanced fibrosis with LSM From our previous study validating the performance of LSM against liver histology in a cohort of 156 Chinese International Medical Press

3 Fibroscan as on-treatment monitoring Table 1. Clinical characteristics of the patients Characteristic Baseline Week 48 P-value Patients, n Male gender, n (%) 52 (73) 52 (73) 1.00 Mean age, years (range) 42 (21 65) 43 (22 66) 0.51 Mean albumin, g/l (range) 44 (35 49) 44 (37 49) 0.02 Mean total bilirubin, µmol/l (range) 13 (6 30) 11 (7 42) Mean ALT, IU/l (range) 99 (41 254) 33 (9 153) <0.001 ULN, n (%) 7 (10) 65 (91) >1 5 ULN, n (%) 64 (90) 6 (9) Mean change in ALT from baseline to week 48, IU/l (range) NA -68 ( ) HBeAg 0.13 Positive, n (%) 36 (51) 44 (62) Negative, n (%) 35 (49) 27 (38) HBV DNA levels, log 10 IU/ml 7.5 ( ) 1.8 ( ) <0.001 Length of liver biopsy, mm 21 (15 30) 19 (15 24) <0.001 Number of portal tracts 16 (6 28) 12 (6 22) 0.01 Metavir fibrosis score 2 (0 4) 2 (1 4) 0.39 F0, n (%) 4 (6) 2 (3) F1, n (%) 18 (25) 28 (39) F2, n (%) 29 (41) 25 (35) F3, n (%) 12 (17) 6 (9) F4, n (%) 8 (11) 10 (14) Change of Metavir fibrosis from baseline to week 48 NA 0 (-1 1) Metavir activity score 2 (1 3) 2 (0 3) <0.001 A0, n (%) 0 (0) 0 (0) A1, n (%) 1 (2) 24 (34) A2, n (%) 35 (49) 37 (52) A3, n (%) 35 (49) 10 (14) Change of Metavir activity from baseline to week 48 NA -1 (-3 0) Mean LSM, kpa (range) 8.8 ( ) 6.6 ( ) <0.001 Median LSM ratio, % (IQR) 14 (3 27) 9 (2 21) 0.56 ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; IQR, interquartile range; LSM, liver stiffness measurement; NA, not applicable; ULN, upper limit of normal. CHB patients, an ALT-based algorithm of LSM was derived [16]. For patients with normal ALT levels, advanced (F3 4) fibrosis was excluded at LSM 6.0 kpa and confirmed at LSM>9.0 kpa. For patients with ALT levels >1 5 the upper limit of normal (ULN), advanced fibrosis was excluded at LSM 7.5 kpa and confirmed at LSM>12.0 kpa. In the present study, we prospectively validated these LSM cutoff values in patients with baseline and week 48 liver biopsies performed. Because an IQR>30% was considered significant and the LSM was not reliable, we defined a significant change of LSM after antiviral treatment as any change >30% from the baseline LSM. The absolute change in LSM with respect to change in histological fibrosis was also presented. Statistical analyses Statistical analysis was performed using SPSS version 11.5 (SPSS Inc., Chicago, IL, USA). Continuous variables were expressed in mean ±sd or median (range) as appropriate. Qualitative and quantitative differences between subgroups were analysed by χ 2 or Fisher s exact tests for categorical parameters and Student s t-test or Mann Whitney U tests for continuous parameters, as appropriate. The overall accuracy of LSM in diagnosing advanced fibrosis was calculated using the receiver operating characteristic (ROC) curve and its 95% confidence interval (CI). Areas under the ROC curves were compared by the test developed by DeLong et al. [22]. All statistical tests were two-sided. Statistical significance was defined as P<0.05. Results Patients A total of 71 CHB patients who underwent paired liver biopsy and transient elastography examinations prior to and at week 48 of antiviral treatment were recruited. Overall, 47 patients were treated with clevudine and 24 patients received adefovir dipivoxil. The clinical characteristics of the patients at baseline and week 48 are shown in Table 1. The median baseline ALT level was 99 IU/l (range ), and 64 (90%) patients had Antiviral Therapy

4 GL-H Wong et al. increased ALT levels (that is, >58 IU/l). The median ALT level was decreased to 33 IU/l (range 9 153) at week 48, with 6 (9%) patients having increased ALT levels. The median change of ALT level was -68 IU/l (range ). At baseline, the median Metavir fibrosis score was 2 (range 0 4) and median Metavir activity score was 2 (range 1 3). At week 48, the median Metavir fibrosis score was 2 (range 1 4) and median Metavir activity score was 2 (range 0 3). The median change of Metavir fibrosis score was 0 (range -1 1) and that of the Metavir activity score was -1 (range -3 0; Table 1). A total of 58 (82%) patients had ALT normalization at week 48, whereas 7 and 6 patients had ALT levels that remained normal and increased, respectively. Overall, Figure 1. ROC curves of liver stiffness measurements for advanced fibrosis at baseline and week 48 Sensitivity Specificity Baseline Week 48 Receiver operating characteristic (ROC) curves at baseline (area under the ROC curve =0.80 [95% confidence interval ]) and week 48 (area under ROC curve =0.78 [95% confidence interval ]) visits; DeLong test P= (24%) patients had regression of histological fibrosis, 43 (61%) patients had static fibrosis with no change in Metavir score and 11 (15%) patients had progression of histological fibrosis at week 48. The numbers (%) of patients with complete viral suppression were 11 (69%) for those with regression of fibrosis, 28 (62%) with static fibrosis and 7 (70%) with progression of fibrosis. The virological response to antiviral treatment had no significant relationship with change in fibrosis (P=0.89). There was a weak correlation between ALT level and necroinflammation at baseline (Pearson s correlation coefficient r=0.31; P=0.008) and no statistically significant correlation at 48 weeks (Pearson s correlation coefficient r=-0.007; P=0.95). Performance of the LSM algorithm to exclude and confirm advanced fibrosis Areas under the ROC curves of the LSM algorithm at baseline and week 48 for advanced fibrosis were 0.80 (95% CI ) and 0.78 (95% CI ), respectively (P=0.57; Figure 1). Using the ALT-based LSM algorithm, advanced fibrosis was excluded in 24 patients at baseline and 30 patients at week 48. The sensitivity and negative predictive value of LSM algorithm to exclude advanced fibrosis was 100% and 100% at baseline, and 75% and 87% at week 48, respectively. Among the four false-negative cases at week 48, the median LSM was 5.7 kpa (range ); three patients had F3 fibrosis and one patient had F4 fibrosis. They all had low necroinflammation (A1) and normalized serum ALT levels (median 31 IU/l [range 22 35]). Using the LSM algorithm, 17 patients at baseline and 14 patients at week 48 were diagnosed to have advanced fibrosis. The specificity and positive predictive value of LSM to diagnose advanced fibrosis were 84% and 56% at baseline, and 91% and 64% at week 48, respectively (Table 2). Among the eight false- positive cases at baseline, the median LSM was 12.7 kpa (range ); one patient had F1 fibrosis and seven patients had F2 fibrosis. All but one of them had moderate to severe necroinflammation (A2 A3). Four had mildly increased ALT (>1 2 ULN) and four patients had more markedly increased ALT levels Table 2. Performance of the LSM algorithm to exclude or confirm advanced fibrosis at baseline and week 48 Advanced fibrosis excluded Advanced fibrosis confirmed Parameter Baseline Week 48 Baseline Week 48 Sensitivity, % Specificity, % Positive predictive value, % Negative predictive value, % Positive likelihood ratios Negative likelihood ratios International Medical Press

5 Fibroscan as on-treatment monitoring Table 3. Relationship between change in liver stiffness measurement and change in histological liver fibrosis LSM decreased LSM static LSM increased /necroinflammatory status (change >30%; n=28) (change within 30%; n=41) (change >30%; n=2) Regression of fibrosis, n (%) 11 (39) 6 (15) 0 ALT normalized, n 7 4 ALT remained normal, n 0 0 ALT remained increased, n 4 2 Static fibrosis, n (%) 14 (50) 28 (68) 1 (50) ALT normalized, n ALT remained normal, n ALT remained increased, n Progression of fibrosis, n (%) 3 (11) 7 (17) 1 (50) ALT normalized, n ALT remained normal, n ALT remained increased, n ALT, alanine aminotransferase; LSM, liver stiffness measurement. (>2 5 ULN). Among the five false-positive cases at week 48, the median LSM was 12.6 kpa (range ); two patients had F1 fibrosis and two patients had F2 fibrosis. One of them had mild necroinflammation (A1), whereas three patients had moderate necroinflammation (A2). All patients had normal ALT levels (median 28 IU/l [range IU/l]). Performance of changes in absolute LSM to detect change in liver fibrosis LSM was significantly (that is, change >30% of baseline LSM) decreased and increased in 28 (39%) and 2 (3%) patients, respectively, whereas most of the patients (41/71 [58%]) had static LSM (that is, change within 30% of baseline LSM; Table 3). Among the 28 patients with decreased LSM, only 11 (39%) patients had regression of fibrosis, whereas 14 (50%) and 3 (11%) had unchanged or progression of Metavir staging. Overall, 7 of the 11 patients who had significantly reduced LSM and regression of fibrosis had ALT normalized at week 48, whereas the 4 other patients had persistently increased ALT throughout the treatment period. By contrast, all of the 17 patients who had significantly reduced LSM but static or worsened fibrosis had normalization of ALT at week 48. Among the 41 patients with static LSM, most patients (28/41 [68%]) had static fibrosis, whereas 6 (15%) and 7 (17%) patients had regression and progression of fibrosis, respectively. Four of six patients with static LSM and regression of fibrosis had normalization of ALT at week 48, whereas among two patients ALT persistently increased. By contrast, all of the seven patients of static LSM but progression of fibrosis had ALT normalized at week 48. By comparison, 22 of 28 patients with static LSM and static fibrosis had ALT normalized at week 48, whereas 6 of them had persistently normal ALT throughout the treatment period. Among the two patients who had increased LSM, 1 (50%) of them had progression of histological fibrosis, and the ALT level was increased at baseline and then normalized at week 48. Another patient with increased LSM but static fibrosis had normal ALT levels both at baseline and week 48. The median (IQR) absolute change of LSM was -2.7 kpa ( ) for patients with regression of histological fibrosis, -1.7 kpa (-4.0 0) with static fibrosis and 0.4 kpa ( ) with progression of fibrosis. Figure 2 showed the distribution of the percentage and absolute change in LSM in patients with either regression, static or progression of liver fibrosis. There was a weak correlation between the changes in LSM and changes in histological fibrosis staging (Spearman s rank correlation coefficient r=0.25; P=0.036). Discussion In this study, we demonstrated that LSM with transient elastography (Fibroscan ) could predict the presence of advanced fibrosis before and during antiviral therapy according to an ALT-based LSM algorithm. However, the decrease in absolute LSM value was not able to reflect the change in liver fibrosis according to histological assessment. This finding was probably related to the confounding effect of ALT reduction caused by the antiviral treatment. We previously defined different optimal cutoff values for LSM according to different ALT levels [16], as ALT level has been consistently found to affect LSM independent of liver fibrosis [23,24]. In our previous study, the sensitivities to exclude bridging fibrosis were both high in patients of normal ALT (93%) and ALT>1 5 ULN (96%) [16]; however, in this study, although the sensitivity to exclude advanced fibrosis was high at baseline (100%), it was lower at week 48 (75%). One Antiviral Therapy

6 GL-H Wong et al. of the potential explanations was that ALT did not really provide a good representation of the extent of necroinflammation, as shown in the weak or even lack of correlation between ALT and histological necroinflammation at baseline and 48 week, respectively. Another explanation for the difference compared with results from previous studies was the different patient characteristics regarding ALT levels as well as the severities of liver fibrosis [16]. We found that all the false-negative cases had normalization of ALT levels without resolution of advanced fibrosis at week 48. The reason for the falsely low LSM among these patients was uncertain. As all the false-negative cases had LSM>5.0 kpa, it would be reassuring to exclude advanced fibrosis among patients who have an LSM of <5.0 kpa during antiviral therapy. By contrast, the specificity to diagnose advanced fibrosis was relatively low at baseline (84%) but remained high at week 48 (91%) in the present study. One possible reason for the suboptimal performance might be related to the high prevalence of patients with increased ALT levels at baseline, and some of them had markedly increased ALT levels (>2 5 ULN) that might falsely increase the LSM. Another possible reason for the false-positive results at week 48 would be the underestimation of the severity of hepatic necroinflammation by the relatively low ALT levels in some patients; therefore, an algorithm combining LSM with another serum test formula for liver fibrosis, preferably one independent of serum ALT levels, might be able to improve the performance of LSM to confirm advanced fibrosis in patients with increased ALT levels. This speculation is supported by a recent study showing that a combined LSM Forns algorithm can improve the accuracy to predict advanced liver fibrosis in CHB patients [25]. In addition, changes in LSM in the setting of stable ALT might be more reflective of the changes in histological fibrosis. We defined significant change in LSM as any change >30% of the baseline LSM. It was based on the current evidence that IQR within 30% of LSM would reflect reliable results [12]. In other words, any change of LSM within 30% might be due to variations in measurements instead of change in liver fibrosis. In the current study, up to 60% of patients had insignificant change in LSM, and another 28% of patients had significant decrease in LSM. It was likely due to the fact that most patients had decrease in serum ALT levels and hepatic necroinflammation, which could lead to reduced LSM regardless of the change of liver fibrosis, at week 48. Therefore decrease in absolute LSM value, which could reflect the effect of ALT normalization, was unreliable to indicate regression of liver fibrosis. Indeed, progression of fibrosis could not be totally Figure 2. Box plot of the change in liver stiffness measurements A 60 B 10 Change in liver stiffness measurement, % Change in liver stiffness measurement, kpa regressed static progressed -15 regressed static progressed Liver stiffness measurements are shown for patients with fibrosis that regressed, remained static or progressed in terms of (A) percentage change and (B) absolute stiffness International Medical Press

7 Fibroscan as on-treatment monitoring excluded in patients with reduced or static LSM if ALT was normalized by the antiviral therapy. This study has a few limitations. First, liver biopsy was used as the gold standard despite its limitation of sampling bias. The median length of liver biopsy in the present study was 20 mm. We anticipate the accuracy of fibrosis assessment would be between 65% and 75%, according to the study by Bedossa et al. [26]. Some of the changes in fibrosis staging might be related to sampling bias. Nonetheless, this potential bias should not alter the obvious effect of ALT normalization on the interpretation of LSM changes. By contrast, the Metavir score, which is a made up of five grades only, might not be able to detect modest changes in liver fibrosis. A quantitative analysis of collagen tissue using morphometric analysis [15] might provide additional value in terms of objective and accurate quantitative assessment of fibrosis. Second, the relatively small sample size and even smaller number of patients having change of liver fibrosis might limit the power of this study. As most of our patients had increased ALT levels at baseline and most of them had virological and biochemical responses to antiviral therapy, our cohort represented a relatively homogeneous population indicated for antiviral therapy accordingly to the regional guidelines [2 4]. Third, 48 weeks might be too short to achieve any change in fibrosis in response to viral suppression, which was revealed by the lack of association between complete viral suppression and change in histological fibrosis. Lastly, our study population was only part of the multicentre, randomized trials; therefore, we did not provide any information comparing the efficacy of clevudine versus adefovir dipivoxil, which would be reported together with the results of other centres. In conclusion, LSM with transient elastography could predict advanced fibrosis during antiviral therapy according to the ALT-based algorithm. Decrease in absolute LSM value, which could reflect the effect of ALT normalization, was unreliable to indicate regression of liver fibrosis. Therefore, physicians should not lightly reassure patients on fibrosis resolution just based on the reduction in the absolute LSM results. Unless there is marked improvement of LSM to <5.0 kpa during antiviral therapy, one cannot be certain that advanced fibrosis has resolved. Disclosure statement HL-YC is a member of the advisory boards of Novartis Pharmaceutics, Bristol Myers Squibb, Roche and Pharmasset. VW-SW has received consulting fees from Novartis Pharmaceuticals. GL-HW has served as a speaker for Echosens. All other authors declare no competing interests. References 1. Chan HLY, Sung JJY. Hepatocellular carcinoma and hepatitis B virus. Semin Liver Dis 2006; 26: Liaw YF, Leung N, Kao JH, et al. 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