Antiviral Therapy 14:

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1 Antiviral Therapy 14: Original article Combination of baseline parameters and on-treatment hepatitis B virus DNA levels to start and continue patients with lamivudine therapy Man-Fung Yuen 1 *, James Fung 1, Wai-Kay Seto 1, Danny Ka-Ho Wong 1, John Chi-Hang Yuen 1 and Ching Lung Lai 1 1 Department of Medicine, University of Hong Kong, Queen Mary Hospital, Hong Kong *Corresponding author: mfyuen@hkucc.hku.hk Background: This study aimed to identify the baseline hepatitis B virus (HBV) DNA, alanine aminotransferase (ALT) levels and on-treatment HBV DNA levels for favourable outcome in patients receiving 5-year lamivudine. Methods: Virological, serological and biochemical parameters were assessed in 74 hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients at year 5 of therapy. Results: Patients with baseline HBV DNA levels <9 log 10 and ALT 2 the upper limit of normal (ULN) had a significantly higher chance of HBV DNA suppression to <4 log 10 (76.5%) and HBeAg seroconversion (82.4%), and a lower chance of YMDD mutations (35.3%) compared with patients with and ALT<2 ULN and patients with HBV DNA 9 log 10 (all P<0.05). All patients with these two baseline parameters plus week 4 HBV DNA<4 log 10 achieved HBV DNA<35, HBeAg seroconversion and ALT normalization without YMDD mutations at year 5. When these two baseline parameters were combined with week 24 HBV DNA<3 log 10, 60%, 80% and 90% of patients had HBV DNA<35, <3 log 10 and <4 log 10, respectively at year 5. Overall, 90% of patients had HBeAg seroconversion and only 10% had YMDD mutations. Conclusions: For HBeAg-positive patients with baseline and ALT 2 ULN, lamivudine could be initiated. For those with HBV DNA<4 log 10 at week 4 or <3 log 10 at week 24, continuation of lamivudine treatment would be more likely to result in a good long-term response. Introduction Lamivudine is the first nucleoside analogue approved for the treatment of chronic hepatitis B (CHB) infection. According to the registration trial comparing lamivudine with placebo for 1 year [1], lamivudine treatment is associated with a higher rate of histological improvement, hepatitis B virus (HBV) DNA suppression, hepatitis B e antigen (HBeAg) seroconversion and normalization of alanine aminotransferase (ALT) levels. Lamivudine is also the only drug examined in controlled trials demonstrating that long-term viral suppression is translatable to a reduced chance of developing cirrhosis and hepatocellular carcinoma for both patients with early or advanced diseases [2 5]. However, the major drawback of lamivudine is the increased chance of developing lamivudine- resistant HBV, the YMDD mutants, upon long-term therapy. The resistance rate is as high as 70% after 5 years of treatment [3]. Despite this, lamivudine is still commonly used in many countries because of its established long-term safety and its relatively lower cost compared with other antiviral agents for CHB. There are several baseline factors that are known to be associated with better viral suppression, a higher chance of loss of HBeAg and a lower chance of YMDD mutations. These include lower HBV DNA levels, higher ALT levels and higher histological activity at baseline [6 10]. We have shown that a greater HBV DNA suppression at 24 weeks of lamivudine treatment is associated with a lower chance of YMDD mutations [11]. In particular, only 13% of patients with HBV DNA levels 3 log 10 (200 IU/ml) at 24 weeks of treatment had YMDD mutations compared with 63.2% of patients with HBV DNA levels >3 log 10 (P<0.001) at a median follow-up of 29 months International Medical Press (print) (online) 679

2 M-F Yuen et al. These findings are the basis for the proposal of the roadmap model to guide plans for antiviral treatment for CHB [12]. In addition, a more recent study from our centre also found that all HBeAg-positive patients with HBV DNA levels <4 log 10 (2,000 IU/ ml) at week 4 or <3.6 log 10 (800 IU/ml) at week 16 of lamivudine treatment achieved HBV DNA levels <2,000 (400 IU/ml), HBeAg seroconversion and ALT normalization with no YMDD mutations at 5 years of lamivudine [13]. To further improve the selection of patients, studies are required to identify patients who might have a good outcome by combining both the favourable predictive parameters at baseline and during treatment. In the present study, we aimed firstly to determine the virological and biochemical outcome as well as the chance of YMDD mutations at 5 years of lamivudine treatment using baseline HBV DNA and ALT levels. If these baseline parameters were shown to be associated with a favourable outcome, we then proceeded to determine whether combining these baseline factors with the on-treatment HBV DNA response at defined time points could further improve treatment outcome. Methods Patient population and follow-up The present study recruited the same cohort of patients (n=74) reported in our previous study [13]. All 74 patients received lamivudine 100 mg daily under the trials (NUCB 3009, NUCB 3018 [a rollover trial for NUCB 3009] and NUCB 4003) conducted with Glaxo- SmithKline Research Laboratories (Glaxo Wellcome Research and Development, Greenford, UK) during the period between June 1994 and August These trials were approved by the institutional review board of the University of Hong Kong, Queen Mary Hospital, Hong Kong. After the first 3 years of lamivudine treatment given under the trials, patients were maintained on lamivudine for at least 2 years by our own initiatives. Therefore, all patients had received 5 years of lamivudine without treatment interruption and alteration (adefovir was only available in Hong Kong in October 2003). In a previous study [13], the aim was to determine the best early time point and the best cutoff value for HBV DNA levels to predict good response during a prolonged period of lamivudine treatment. We found that patients with HBV DNA levels <4 log 10 at week 4 or <3.6 log 10 at week 16 could predict excellent viral response with no YMDD mutation after 5 years of lamivudine treatment. HBV DNA levels were determined by branched DNA assay (Versant HBV DNA 3.0 assay (Bayer HealthCare Diagnostic Division, Tarrytown, NY, USA) with a lower limit of detection of 2,000 ) and YMDD mutations were determined by the Trugene HBV genotyping assay kit, (Bayer HealthCare Diagnostic Division). Patients were positive for hepatitis B surface antigen (HBsAg) for at least 6 months and were positive for HBeAg at baseline. These were tested by microparticle enzyme immunoassay (Abbott Laboratories, Chicago, IL, USA). All patients were negative for antibodies against hepatitis C and D, as well as HIV. Patients consuming >20 g per day of alcohol on a regular basis were excluded. Patients were followed up bi-weekly for the first 4 weeks, every 4 weeks until year 1, every 8 weeks until year 2 and every 16 weeks until year 5. Sera were taken during every follow-up and stored at -20 C. Measurements at baseline Liver biochemistry, α-fetoprotein (AFP), HBsAg, HBeAg, antibodies against HBeAg (anti-hbe) and HBV DNA levels were determined at baseline. HBV genotypes were determined by an ELISA as described previously [14]. Patients (n=41) recruited in the trials NUCB 3009 and NUCB 3018 had undergone protocol liver biopsies. The liver biopsies were graded according to the necroinflammatory score (score 0 18) and fibrosis score (Ishak scoring system 0 6) [15,16]. Measurements during follow-up The liver biochemistry, AFP, HBsAg, HBeAg and anti- HBe were determined every 3 4 months. Patients with increased AFP levels (>20 ng/ml) were required to undergo ultrasonography of the liver. HBeAg seroconversion was defined as loss of HBeAg with the development of anti-hbe for at least two consecutive follow-ups and maintained as this status till 5 years of follow-up. HBV DNA levels were determined at week 4, week 24, and every year from year 1 to year 5. These were measured by the COBAS TaqMan HBV test (Roche Molecular Systems, Inc., Branchburg, NJ, USA) with a lower limit of detection of 35 (6.0 IU/ ml). The line probe assay (INNO-LiPA HBV DNA; Innogenetics NV, Gent, Belgium) was used to survey the presence of YMDD mutations at baseline and every year until year 5. Statistical analyses All statistical analyses were performed using SPSS version 16 for Windows (SPSS, Inc., Chicago, IL, USA). Continuous variables with skew distributions were expressed as median (range). The Mann Whitney U test was used to compare these continuous variables between different groups. The χ 2 test with Yates correction factor or Fisher s exact test were applied for categorical variables for two groups. The χ 2 for trend (Mantel Haenszel trend test) was used for categorical variables for three groups. The Kaplan Meier method adopting the log-rank test was used to compare groups for cumulative events. HBV International Medical Press

3 Baseline and on-treatment HBV DNA levels to predict response to lamivudine Table 1. Demographics for three groups of patients classified by baseline HBV DNA and ALT levels and their outcome at 5 years Variable Group 1, n=17 Group 2, n=22 Group 3, n=35 Demographics Male:female sex, n 14:3 19:3 26:9 Age, years 31.1 ( ) 33.9 ( ) 32.6 ( ) Genotype B:C, n 4:13 6:17 8:26 HBV DNA, log ( ) a 7.7 ( ) b 9.4 (9 11.7) ab ALT, U/l 143 (83 506) cd 53 (27 97) c 57 (14 19) d Albumin, g/l 46 (40 50) 46 (42 52) 47 (42 54) Bilirubin, µmol/l 12 (5 19) 9 (3 19) 10 (5 27) Outcome at 5 years ALT normalization, n (%) 15/17 (88.2) e 6/11 (54.6) e 13/20 (65) e HBV DNA <4 log 10, n (%) 13/17(76.5) 5/22 (22.7) 4/35 (11.4) HBV DNA<35, n (%) 7/17 (41.2) 4/22 (18.2) 2/35 (5.7) HBeAg seroconversion, n (%) 14/17 (82.4) 5/22 (22.7) 5/35 (14.3) Group 1 had hepatitis B virus (HBV) DNA levels <9 log 10 and alanine aminotransferase (ALT) 2 the upper limit of normal (ULN), Group 2 had HBV DNA levels <9 log 10 and ALT<2 ULN, and Group 3 had HBV DNA levels 9 log 10. Continuous variables are expressed in median (range). a P< b P< c P< d P< e Number of patients out of the total number of patients with increased ALT levels at baseline. DNA levels below the lower detection limit (<35 copies/ ml [6 IU/ml]) were recorded as 35 for statistical calculations. Results Demographics The median (range) age was 32.2 years ( ) for the 74 patients (male to female ratio 59:15). A total of 18 (24.3%) patients had genotype B and 56 (75.7%) patients had genotype C infection. The median (range) HBV DNA level was 8.6 log 10 ( ). The median (range) albumin, bilirubin and ALT levels were 47 g/l (40 54), 10 µmol/l (3 27) and 56 U/l (14 506), respectively. Of the 41 patients with baseline liver biopsies, the median (range) necroinflammatory and Ishak fibrosis scores were 7 (2 14) and 1 (0 4), respectively. Only two (4.9%) patients had advanced fibrosis with an Ishak fibrosis score 4. Patients were divided into three groups according to their baseline HBV DNA and ALT levels: Group 1 (n=17) included patients with baseline HBV DNA<9 log 10 and ALT 2 the upper limit of normal (ULN), Group 2 (n=22) included patients with baseline and ALT<2 ULN, and Group 3 (n=35) included patients with baseline HBV DNA 9 log 10. The baseline demographics of these three groups of patients are listed in Table 1. There were no significant differences in the demographic parameters between the three groups except for the median HBV DNA and ALT levels. ALT normalization and HBV DNA suppression at year 5 A total of 11 out of 22 Group 2 patients and 15 out of 35 Group 3 patients had normal ALT levels at baseline. ALT normalization was observed in 15 out of 17 (88.2%) Group 1 patients, 6 out of 11 (54.6%) Group 2 patients and 13 out of 20 (65%) Group 3 patients with baseline increased ALT levels. Although Group 1 had a higher percentage of patients achieving ALT normalization compared with Group 2 and Group 3, the differences did not reach statistical significance. This is probably related to the relatively small number of patients in each group. There was a significant decrease in the chance of achieving HBV DNA<4 log 10 at year 5 from Group 1 (13 out of 17 [76.5%] patients) to Group 2 (5 out of 22 [22.7%] patients) to Group 3 (4 out of 35 [11.4%] patients) with a P-value < Similarly, there was also a significant decrease in the chance of achieving HBV DNA<35 (lower limit of detection) from Group 1 (7 out of 17 [41.2%] patients) to Group 2 (4 out of 22 [18.2%] patients) to Group 3 (2 out of 35 [5.7%] patients) with a P-value of Cumulative HBeAg seroconversion and YMDD mutations The cumulative chance of HBeAg seroconversion is depicted in Figure 1. Group 1 patients had a significantly higher chance of HBeAg seroconversion compared with Group 2 patients (P<0.001) and Group 3 patients (P<0.001). A total of 82.4%, 22.7% and 14.3% of Groups 1, 2 and 3 patients, respectively, had HBeAg seroconversion at year 5. There was no difference observed between Group 2 and Group 3 (P=0.44). A total of 49 patients developed YMDD mutations at year 5 (3 more were detected to have the YMDD mutations compared with our previous study [13] by using the line probe assay). The cumulative chance of YMDD mutations is depicted in Figure 2. There was a significant increasing trend in the chance of YMDD Antiviral Therapy

4 M-F Yuen et al. Figure 1. Cumulative chances of HBeAg seroconversion in patients with different baseline HBV DNA and ALT levels 100 Group 1 Group 2 Group 3 Patients with HBeAg seroconversion, % Months Patients at risk, n Group 1 Group Group Group 1 had hepatitis B virus (HBV) DNA levels <9 log 10 and alanine aminotransferase (ALT) 2 the upper limit of normal (ULN), Group 2 had HBV DNA levels <9 log 10 and ALT<2 ULN, and Group 3 had HBV DNA levels 9 log 10. HBeAg, hepatitis B e antigen. mutations from year 2 onward until year 5 from Group 1 to Group 2 to Group 3 patients. The chance of YMDD mutations increased from 35.3% (6 out 17) of Group 1 patients to 63.6% (14 out of 22) of Group 2 patients and finally to 82.9% (29 out of 35) of Group 3 patients at year 5 (P=0.003). YMDD with virological breakthrough, defined as HBV DNA level >1 log 10 increase from the nadir, was observed in 23.5% (4 out of 17) Group 1 patients, 50% (11 out of 22) of Group 2 patients and 60% (21 out of 35) of Group 3 patients at year 5. The differences were statistically significant between Groups 1 and 3 (odds ratio 5.3, 95% confidence interval ; P=0.023). In summary, Group 1 patients had more favourable 5-year responses in terms of higher chances of normalization of ALT level (88.2%), HBV DNA suppression to <4 log 10 (76.5%; 41.2% of patients had undetectable HBV DNA<35 ), HBeAg seroconversion (82.4%) and lower chances of YMDD mutations (35.3%) compared with Group 2 and Group 3 patients. Combining favourable baseline parameters with on-treatment HBV DNA response In order to improve the selection criteria for continuation of lamivudine treatment, the baseline HBV DNA and ALT levels combined with the early on-treatment HBV DNA response were assessed. Of the 17 patients (Group 1) with baseline ( IU/ml) and ALT 2 ULN, 5 (29.4%) patients met the favourable earliest time point assessment at week 4 with HBV DNA levels <4 log 10 defined by our previous study [13]. All 5 patients had undetectable HBV DNA (<35 ), ALT normalization and HBeAg seroconversion without YMDD mutations at year 5. Although these patients had excellent outcomes, the percentage of patients (29.4%) achieving HBV DNA levels <4 log 10 was small. Another commonly used early on-treatment time point assessment is at 24 weeks. Combining the favourable baseline factors ( copies/ ml and ALT 2 ULN with HBV DNA<3 log 10 copies/ ml at week 24 (a cutoff level shown to have favourable outcome for both lamivudine- and telbivudinetreated patients) [11,17,18], 10 out of the 17 (58.8%) patients were under this category. Of these 10 patients, 6 patients had undetectable HBV DNA. The remaining 4 patients had HBV DNA levels of 48.4, 254, 4,370 and at year 5. Therefore, 60% of patients had HBV DNA<35, 80% had HBV DNA<3 log 10 and 90% had HBV DNA<4 log 10 at year 5. In total, 90% (9 out of 10) of patients had HBeAg seroconversion without YMDD mutations at year 5. One patient remained HBeAg-positive and had YMDD mutation. All patients had normal ALT levels at year International Medical Press

5 Baseline and on-treatment HBV DNA levels to predict response to lamivudine Figure 2. Cumulative chances of YMDD mutations in patients with different baseline HBV DNA and ALT levels Patients with YMDD mutations, % Group 1 Group 2 Group 3 P= P= P= P= P= Year 1 Year 2 Year 3 Year 4 Year 5 Year of treatment Group 1 had hepatitis B virus (HBV) DNA levels <9 log 10 and alanine aminotransferase (ALT) 2 the upper limit of normal (ULN), Group 2 had HBV DNA levels <9 log 10 and ALT<2 ULN, and Group 3 had HBV DNA levels 9 log 10. Discussion Although lamivudine has the lowest cost and best established safety profile among all the antiviral agents for CHB, its use is largely hampered by the high chance of emergence of YMDD mutations and the relatively modest degree of HBV DNA suppression. However, there exists a group of patients who can have good outcomes with lamivudine treatment [3]. This observation is also seen in short-term telbivudine treatment. Recently, subgroup analyses of the GLOBE study revealed that for HBeAg-positive patients receiving telbivudine with baseline HBV DNA levels <9 log 10 copies/ ml and ALT 2 ULN, 71% achieved undetectable HBV DNA at week 24. Of these, there was an 89% chance of undetectable HBV DNA, a 52% chance of HBeAg seroconversion and an 81% chance of ALT normalization at year 2 [19]. Drug resistance only occurred at 1.8% of these patients. Whether the favourable shortterm outcomes in this selected group of patients can be maintained on a longer term of telbivudine remains to be studied. In the present 5-year study of lamivudine treatment, we showed that patients with baseline and ALT 2 ULN had a very favourable longterm outcome (88.2% chance of ALT normalization, 82.4% chance of HBeAg seroconversion, and 76.5% and 41.2% chances of HBV DNA suppression to the levels <4 log 10 and <35, respectively, at year 5) compared with those with baseline HBV DNA<9 log 10 and ALT<2 ULN, and those with HBV DNA 9 log 10. Achieving HBV DNA levels <4 log 10 (occurring in 76.5% of this selected population receiving lamivudine) is already associated with a significantly lower risk of development of hepatocellular carcinoma and cirrhosis, according to two large-scale studies conducted in Taiwan [20,21]. There are, however, two concerns of using only baseline HBV DNA and ALT levels to determine whether lamivudine should be given continuously. Firstly, it has been shown that the predictive power for favourable response is less optimal by using baseline HBV DNA levels compared with using the achievement of a fixed HBV DNA response at 24 weeks of treatment [22]. Secondly, even with the criteria of and ALT<2 ULN at baseline, 35.3% and 23.5% of patients would develop YMDD mutations and virological breakthroughs, respectively, at year 5. The early prediction of good response could be improved by the additional step of combining these two favourable baseline factors with the early HBV DNA response at week 4. We were able to show that for patients with baseline and ALT 2 ULN, who additionally had HBV DNA reduction to <4 log 10 at week 4, would all achieve ALT normalization, undetectable HBV DNA levels and Antiviral Therapy

6 M-F Yuen et al. HBeAg seroconversion without emergence of YMDD mutations at year 5. Therefore, patients with baseline and ALT 2 ULN might be considered for lamivudine treatment, and for those with HBV DNA<4 log 10 at week 4, continuation of lamivudine for long-term treatment would result in an excellent outcome. However, only 29.4% of patients with baseline HBV DNA<9 log 10 and ALT 2 ULN could achieve this early HBV DNA target and, hence, long-term benefits. We therefore also assessed the predictive value using the HBV DNA response at week 24, a time point consistently shown to have good prediction for outcome in several studies [11,17,18]. For patients achieving HBV DNA levels <3 log 10 at week 24 (achievable in 58.8% of patients with baseline and ALT 2 ULN), 90% and 60% of patients had HBV DNA<4 log 10 and <35, respectively, at year 5. All patients had normal ALT levels at year 5. Only 10% remained HBeAg positive with YMDD mutations at year 5. According to our previous study, all patients with HBV DNA<4 log 10 at week 4 will achieve excellent virological, biochemical and serological responses without YMDD mutations at year 5 [13]. Without adding the baseline parameters of and ALT 2 ULN as selection criteria, only 6 out of 74 (8.1%) patients achieved this early HBV DNA target (data not shown in our previous study). By combining these two baseline parameters in the present study, a better selection of patients in terms of a higher chance of achieving this early HBV DNA target (5 out of 17 [29.4%]) at week 4 was achieved. In the present study, the outcome using the combination of baseline HBV DNA and ALT levels together with 4-week treatment HBV DNA response was better than that of using 24-week treatment HBV DNA response. We therefore recommend using the former combination in an ideal situation. However, if frequent HBV DNA measurement is not easily available (for example, because of its high cost) using 24-week treatment HBV DNA response as the combination parameter with the baseline parameters is a practical alternative. Using these combined criteria, we were able to select more patients who would continue to respond to longterm lamivudine. For those with parameters outside the defined values either at the baseline or during treatment, the addition of more potent antiviral agents should be considered to lower the chance of emergence of drug resistance. It remains to be determined whether these lamivudine-experienced patients with 24-week exposure to lamivudine would have a higher chance of entecavir resistance if their therapy is switched to entecavir. A viable alternative is to add on tenofovir to the regimen. One of the limitations for the present study is the restriction to HBeAg-positive patients, this being one of the entry criteria for the previous trials. The selection of baseline HBV DNA levels and ALT levels might be different for HBeAg-positive and -negative patients. One study had shown that HBeAg-negative patients receiving lamivudine with baseline HBV DNA>6 log 10 ( IU/ml) had a significantly higher chance of virological breakthroughs with YMDD mutations [23]. Similarly, in the subgroup analysis of the GLOBE study [19] for HBeAg-negative patients receiving telbivudine with baseline HBV DNA levels <7 log 10 ( IU/ml), 93% had undetectable HBV DNA levels by PCR assay, 83% had ALT normalization and only 2% had drug resistance at year 2. The present study might also serve as an example of using the baseline parameters and on-treatment response to antiviral agents in predicting drug resistance. Baseline ALT and HBV DNA levels are proven to be an important parameter to predict response to adefovir used for lamivudine-resistant patients [24 27]. Apart from the baseline parameters, it has been shown that lamivudine-resistant patients with adefovir resistance had higher HBV DNA levels at week 24 of adefovir treatment than those without adefovir resistance [28]. This is confirmed by a recent study showing that 49% of lamivudine-resistant patients receiving adefovir with HBV DNA>3 log 10 at week 48 developed adefovir resistance at 192 weeks compared with only 6% of patients with HBV DNA<3 log 10 at week 24 [29]. For entecavir in lamivudine-resistant CHB, it has been shown that lamivudine- resistant CHB patients with HBV DNA<7 log 10 at baseline or <4 log 10 at week 24 had better viral suppression and lower chance of entecavir resistance after 2 years of treatment [30]. The selection criteria might be specific for different antiviral agents and patient populations, such as treatment- naive patients or patients with drug-resistant CHB. The present study provided an evidence-based approach to selecting patients for the most cost- effective way to achieve a favourable long-term outcome with the relatively low-cost lamivudine therapy. This might be of particular importance for developing countries where CHB is of high prevalence. In conclusion, HBeAg-positive patients with baseline and ALT 2 ULN, whose HBV DNA levels could be reduced with lamivudine treatment to <4 log 10 at week 4 or <3 log 10 at week 24, continued treatment with lamivudine was associated with good viral suppression and a very high chance of HBeAg seroconversion and ALT normalization, with a minimal chance of YMDD mutations International Medical Press

7 Baseline and on-treatment HBV DNA levels to predict response to lamivudine Disclosure statement The authors declare no competing interests. References 1. Lai CL, Chien RN, Leung NW, et al. A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N Engl J Med 1998; 339: Liaw YF, Sung JJ, Chow WC, et al. Lamivudine for patients with chronic hepatitis B and advanced liver disease. N Engl J Med 2004; 351: Yuen MF, Seto WK, Chow DH, et al. Long-term lamivudine therapy reduces the risk of long-term complications of chronic hepatitis B infection even in patients without advanced disease. Antivir Ther 2007; 12: Matsumoto A, Tanaka E, Rokuhara A, et al. Efficacy of lamivudine for preventing hepatocellular carcinoma in chronic hepatitis B: A multicenter retrospective study of 2795 patients. Hepatol Res 2005; 32: Eun JR, Jang BIK, Kim TN, Lee HJ, Lee KS. 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