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Journal of Clinical Microbiology, September 2004, p. 3932-3936, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.3932-3936.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Long-Term Follow-Up Study of Chinese Patients with YMDD Mutations: Significance of Hepatitis B Virus Genotypes and Characteristics of Biochemical Flares

Man-Fung Yuen,1* He-Jun Yuan,1 Erwin Sablon,2 Danny Ka-Ho Wong,1 Annie On-On Chan,1 Benjamin Chun-Yu Wong,1 and Ching-Lung Lai1*

Division of Gastroenterology and Hepatology, Department of Medicine, The University of Hong Kong, and Queen Mary Hospital, Hong Kong,1 Innogenetics N.V., Ghent, Belgium2

Received 23 December 2003/ Returned for modification 29 April 2004/ Accepted 1 June 2004


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ABSTRACT
 
We sought to examine the role of hepatitis B virus (HBV) genotypes in virological breakthroughs and biochemical flares in patients with YMDD mutations during lamivudine therapy. Virologic breakthroughs (i.e., the reappearance of HBV DNA as determined by bDNA assay) and biochemical flares (mild flares = alanine aminotransferase [ALT] between 2 and 10 times the upper limit of normal [ULN]; severe flares = ALT >10 times ULN) were monitored in 154 hepatitis B e antigen-positive patients receiving long-term lamivudine. The HBV genotypes and YMDD mutations were determined. Forty-three patients had virological breakthroughs with YMDD mutations (median follow-up of 29.6 months [range, 22.3 to 61.4]). Twenty patients (47%) patients had mild biochemical flares; seven (16%) had severe flares. Two patients showed an elevation of bilirubin level that is >2 times the ULN. All patients recovered spontaneously. The cumulative risks for biochemical flares were 28, 47, and 58% for the first 3 years, respectively. Patients with biochemical flares compared to those without flares had a significantly higher median pretreatment ALT level (61 U/liter versus 34.5 U/liter [P = 0.012]). There were no differences in the cumulative risk of virological breakthroughs, risk, and severity of biochemical flares between patients with genotypes B (n = 11) and C (n = 32). There was an increase in the percentage of patients with single YMDD mutant at last follow-up compared to that at the time of virological breakthroughs (74% [n = 32] versus 47% [n = 20], respectively; P = 0.015). The chances of YMDD mutations with virological breakthroughs and biochemical flares were similar in patients with genotypes B and C. Biochemical flares were common, with 16% being severe in nature. High pretreatment ALT levels were associated with a higher chance of biochemical flares.


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INTRODUCTION
 
The efficacy of lamivudine is limited by mutation of the YMDD (tyrosine, methionine, aspartate, aspartate) motif at the region of the reverse transcriptase (rt204), where M is substituted either by valine (YVDD) or isoleucine (YIDD). The chances of YMDD mutations are 14, 38, 57, and 67% by the first, second, third, and fourth years, respectively, according to long-term follow-up studies conducted in Asia (9, 12, 14). HBV DNA levels and alanine aminotransferase (ALT) levels after the emergence of YMDD mutants are usually lower than pretreatment levels (5, 8, 10, 13). In spite of the fact that YMDD mutants are comparatively less replication competent than wild-type YMDD (4, 15, 17, 19), biochemical flares resulting in hepatic decompensation may occasionally occur (7, 12, 14).

The frequency, severity, and predictive factors for ALT flares have not been fully investigated. Another aspect that has not been fully studied is the effect of HBV genotypes on the development of YMDD mutations. One study shows that, compared to patients with genotype D, patients with genotype A are more likely to have YMDD mutations (24). Another study with a relatively short period of follow-up shows that there is no difference in the chance of having YMDD mutations between patients with genotypes B and C (6).

We sought to examine the role of HBV genotypes in the development of virological breakthroughs with YMDD mutations and the frequency, nature, and factors associated with subsequent biochemical flares.


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MATERIALS AND METHODS
 
A total of 154 patients were recruited for the present study. These patients were originally recruited into three clinical trials NUCB3009, which was continued as trial NUCB3018. The third trial was NUCB4003. The entry criteria for trial NUCB3009/3018 were that patients (i) were ≥16 years old, (ii) were positive for hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) for at least 6 months, and (iii) had HBV DNA levels of ≥1.4 x 106 copies/ml as determined by solution-hybridization assay (Abbott Diagnostics, Chicago, Ill.) and ALT levels of ≤10 times the upper limit of normal (ULN) (8). For entry into trial NUCB4003, patients had to (i) be ≥16 years old; (ii) be positive HBsAg and HBeAg for at least 6 and 3 months, respectively; and (iii) have detectable HBV DNA levels as determined by branched DNA assay (Bayer Corp., Tarrytown, N.Y.; lower limit of detection = 0.7 x 106 copies/ml) and ALT levels of between 1.3 and 10 times the ULN (9). All trials were sponsored by GlaxoSmithKline Research Laboratories and were approved by the Ethics Committee, The University of Hong Kong, Hong Kong.

Of the 154 patients, 83 patients were given 100 mg of lamivudine daily. Twenty-seven patients were given 25 mg of lamivudine daily for 1 to 3 years, followed by 100 mg of lamivudine daily at the end of the third year. Forty-four patients were given 500 mg of famciclovir three times daily for 12 weeks, followed by 100 mg of lamivudine daily. All patients were maintained on lamivudine up to the time of writing. A total of 43 of 154 patients (28%) developed virological breakthroughs with YMDD mutations, with a median follow-up duration of 29.6 months (range, 22.3 to 61.4 months). Virological breakthrough is defined as the reappearance of HBV DNA (as determined by the branched DNA assay [see below]) for at least two consecutive follow-ups (the follow-up schedule is described below) and the presence of YMDD mutants. Of these patients, 22 received 100 mg of lamivudine daily; 18 received 25 mg of lamivudine daily for 1 to 3 years, followed by 100 mg of lamivudine daily; and 3 received 500 mg of famciclovir three times a day for 12 weeks, followed by 100 mg of lamivudine daily.

To study the characteristics and factors for biochemical flares of long-term lamivudine therapy, the 43 patients with documented virological breakthroughs with YMDD mutations had continuous regular follow-up after the time of virological breakthroughs (median follow-up time after virological breakthrough of 51.7 months [range, 28.7 to 71.5]). This was scheduled as every 2 weeks for the first 4 weeks, every 4 weeks until week 24, every 8 weeks until week 52, and every 16 weeks until the last follow-up. Patients without virological breakthrough were followed up at 3- to 6-month intervals. Liver biochemistry and prothrombin time were monitored during every visit. HBV serological analyses, including HBsAg, HBeAg, and antibody to HBeAg (anti-HBe), were performed at each follow-up visit. The HBV DNA levels before treatment and during every subsequent follow-up were measured by branched DNA assay (lower limit of detection of 700,000 copies/ml).

Biochemical flares in patients with YMDD mutations are defined as increases in ALT levels of >2 times the ULN from the normal ALT level in the preceding follow-up. Mild and severe biochemical flares are defined as elevated ALT levels between 2 and 10 times the ULN and >10 times the ULN, respectively. Decompensated biochemical flares are defined as elevated bilirubin levels of >2 times ULN, prothrombin times of controls of more than 3 s, and/or the development of ascites or encephalopathy.

YMDD mutations were checked by a line probe assay (INNO-LIPA HBV DR; Innogenetics N.V., Ghent, Belgium) (21) at the time of the virological breakthroughs. The YMDD mutations were also checked in the 43 patients with virological breakthroughs at the last follow-up. Serum samples before treatment were taken for the detection of HBV genotypes. This analysis was performed by using another line probe assay (INNO-LiPA HBV Genotyping) that is described in a previous study (23).

Statistical analysis. The Mann-Whitney test for continuous ordinal data, the chi-square test with Yates' correction, and the Fisher exact test for categorical data were used for statistical analyses. The differences in nonparametric paired samples were tested by Wilcoxon signed rank test. The relationship of two continuous variables was tested by using Kendall's rank correlation test. Kaplan-Meier analysis with the log rank test was used to compare the cumulative risk of virological breakthroughs with YMDD mutations between different HBV genotypes. Cox regression analysis was used to compare the cumulative risk of virological breakthroughs with YMDD mutations between different HBV genotypes with known confounding factors.


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RESULTS
 
HBV genotypes and virological breakthroughs with YMDD mutations. The demographic data, ALT levels, HBV DNA levels, and HBV genotypes of the 154 patients are listed in Table 1. There were no differences in the demographic parameters between patients with or without virological breakthroughs with YMDD mutations. The cumulative risk of the virological breakthroughs with YMDD mutations was examined in 153 patients with either genotypes B or C alone (one patient with a coinfection with genotypes B and C was excluded from analysis). There was no significant difference in the cumulative risk of the development of virological breakthroughs with YMDD mutations between patients with genotypes B and C (P = 0.87) with a median follow-up of 29.6 months (range, 22.3 to 61.4 months). Cox regression analysis was performed by including the variables of HBV genotypes, HBV DNA, and ALT levels on presentation. There was still no significant difference in the cumulative risk of virological breakthroughs with YMDD mutations between patients with genotypes B and C (P = 0.95). In order to eliminate the effect of a suboptimal dose of lamivudine (25 mg daily) and preceding famciclovir treatment, the cumulative risk of the development of virological breakthroughs with YMDD mutations was calculated for patients who received 100 mg of lamivudine daily throughout. There was again no significant difference in the cumulative risk of the development of virological breakthroughs with YMDD mutations between patients with genotypes B and C (P = 0.19).


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  		TABLE 1. Demographic data for patients with or without virological breakthroughs with YMDD mutations

Long-term follow-up of 43 patients with virological breakthroughs. (i) Changing patterns of YMDD wild types and mutants during follow-up. Assessment of the presence of YMDD wild types and mutants was done at the time of virological breakthroughs and at the last follow-up with all of the patients maintained on lamivudine after the emergence of YMDD mutants. Two patients (5%), one with YMDD wild-type/YIDD and one with YIDD/YVDD, had the YMDD mutants disappearing and the wild-type YMDD taking over. Both patients had normal ALT levels and low HBV DNA levels (2.85 and <0.7 x 106 copies/ml) at the last follow-up at 43.2 and 71.1 months from the time of the virological breakthroughs, respectively. Twenty-five patients (58%) had the same YMDD mutants at the time of virological breakthrough and at the time of the last follow-up (17 [68%] had single YMDD mutants of either YIDD or YVDD). Thirteen patients had an elimination of one of the mutants at the last follow-up. The remaining three patients had either an addition of one more mutant or a switch from one mutant to another. In summary, a single YMDD mutant of either YIDD or YVDD was found in 32 patients (74%) at the last follow-up compared to 20 patients (47%, P = 0.015) at the time of the virological breakthroughs.

For the concomitant mutations at rt180 in which leucine is substituted by methionine (rtL180M), 39 (91%) of the 43 patients at the time of virological breakthroughs with YMDD mutations had rtL180M mutations. At the last follow-up, a substantial proportion of patients (33% [14 of 43]) had wild-type rt180L only, whereas the presence of rtL180M was still found in 29 patients (67%).

(ii) Biochemical flares of 43 patients with virological breakthroughs. Of the 43 patients with virological breakthroughs, 8 (19%) had continually normal ALT levels after virological breakthroughs with YMDD mutations until last follow-up. Another eight patients (19%) had at least one abnormal ALT level of between 1 and 2 times ULN. The remaining 27 patients (63%) had at least one episode of biochemical flares with ALT levels of >2 times the ULN. The median time of biochemical flares from the time of virological breakthroughs was 13.8 months (range, 0 to 60). The cumulative risk of biochemical flares is plotted in Fig. 1. The majority of these flares (20 of 27 patients [74%]) were mild and transient. (The severe flares will be described below.) The cumulative risks of biochemical flare by the end of the first, second, and third year of follow-up after virological breakthroughs with YMDD mutations were 28% (n = 12), 47% (n = 20), and 58% (n = 22), respectively. Over the 2-year period of follow-up after emergence of YMDD mutants, only one patient (2%) had more than three episodes, nine patients (21%) had two episodes, and ten patients (23%) had only one episode of biochemical flares.



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  		FIG. 1. The cumulative risk of biochemical flares after virologic breakthroughs with YMDD mutations. All of the biochemical flares resolved spontaneously.

The median follow-up after virological breakthrough was comparable between patients with genotypes B and C (54.6 months [range, 29.0 to 67.0] versus 50.4 months [range, 28.7 to 71.5], respectively [P = 0.57]; Table 1). There was no significant difference in the cumulative risk of biochemical flares between patients with genotypes B and C (P = 0.66).

There were no differences in the median HBV DNA levels before treatment and at the time of virological breakthroughs with YMDD mutations between patients with or without subsequent biochemical flares (HBV DNA levels before treatment of 1,100 x 106 copies/ml [range, 15 to 11,000] versus 1,289.32 x 106 copies/ml [range, 24.67 to 11,000], respectively, P = 0.26; HBV DNA levels at breakthroughs of 8.2 x 106 copies/ml [range, 1.71 to 7,900] versus 11.69 x 106 copies/ml [range, 2.08 to 390], P = 0.65, respectively).

Patients with elevated ALT levels (>1 times the ULN) after virological breakthroughs with YMDD mutations had a significantly higher median pretreatment ALT level compared to that of patients with continually normal ALT levels (60 U/liter [range, 9 to 269] versus 27 U/liter [range, 14 to 41], respectively; P = 0.002). Patients with biochemical flares (ALT levels of >2 times the ULN) had a significantly higher median pretreatment ALT level (61 U/liter [range, 9 to 269]) than that of patients without biochemical flares(34.5 U/liter [range, 14 to 122]; P = 0.012). For biochemical flares, there was no significant difference in the median peak ALT level between patients with genotypes B and C (ALT level of 481 U/liter [range, 88 to 854] versus 417 U/liter [range, 71 to 1,906], respectively; P = 0.78).

Seven patients (16%) had severe biochemical flares (ALT level of >10 times the ULN) after the virological breakthroughs with YMDD mutations. The characteristics, liver biochemistry, and HBV DNA levels of these seven patients are listed in Table 2. Two patients had elevated peak bilirubin level of >2 times the ULN during the severe biochemical flares. No patient had prolongation of prothrombin time, and none developed ascites or encephalopathy. None of the patients required adefovir dipivoxil. All patients had full spontaneous recovery with normalization of ALT afterward. Two patients had HBeAg seroconversion at 13.8 and 32.6 months after the severe biochemical flares.


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  		TABLE 2. Features of seven patients with severe biochemical flares

(iii) HBV DNA levels. The median HBV DNA level at the time of virological breakthroughs with YMDD mutations (8.9 x 106 copies/ml [range, 1.7 x 106 to 7.9 x 109]) and at the time of last follow-up (2.1 x 107 copies/ml [range, <0.7 x 106 to 9.7 x 108]) was significantly lower than that of baseline value (1.2 x 109 copies/ml [range, 1.5 x 107 to 1.1 x 1010]; both P < 0.0001)]. The median peak HBV DNA level was 1.1 x 109 copies/ml (range, 1.6 x 108 to 2 x 1010). There were no correlations between HBV DNA levels at baseline, at the virological breakthroughs with YMDD mutations, and at the last follow-up and for the peak HBV DNA levels during follow-up. Five patients (12%) (three had HBeAg seroconversions) had undetectable HBV DNA levels (<0.7 x 106 copies/ml) at the last follow-up.

(iv) HBeAg seroconversion. One patient had an HBeAg seroconversion before the virological breakthrough with YMDD mutations. The remaining 42 patients with virological breakthroughs with YMDD mutations were HBeAg positive. HBeAg seroconversion occurred in five patients (12%) during subsequent follow-up. These five patients had continually normal ALT levels without biochemical flares after HBeAg seroconversion. There were no differences in the percentages of patients with genotypes B and C, the pretreatment ALT levels, the peak ALT levels after virological breakthroughs, the pretreatment HBV DNA levels, the HBV DNA levels at the time of virological breakthroughs, and the peak HBV DNA levels between patients with or without HBeAg seroconversion.


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DISCUSSION
 
High pretreatment ALT level and HBV DNA level are associated with a higher chance of YMDD mutations (21). The role of HBV genotypes on the development of YMDD mutations is less clear. The present study showed that there was no difference in the cumulative risk of development of virological breakthroughs with YMDD mutations between the two most common genotypes prevailing in Asia, i.e., genotypes B and C. This finding supports the preliminary Taiwan study involving 31 patients conducted by Kao et al., who demonstrated that both genotypes have a similar risk in developing YMDD mutations after a mean follow-up of 9 months of lamivudine treatment (6). In addition, Akuta et al. recently show that there is no difference in the chance of emergence of YMDD mutations between patients with genotypes B and C in long-term follow-up (1). However, another recent study comparing HBV serotype adw (genotype A) with ayw (genotype D) shows that patients with serotype adw have a higher chance of development of YMDD mutations compared to patients with serotype ayw (24). This finding is important since the YMDD mutation rate is apparently higher in Caucasian patients (32%) than in Asian patients (14%), as demonstrated in the 1-year lamivudine trials conducted in the United States and Asia, where genotype A and genotypes B/C, respectively, are commonly found (3, 18). Whether this difference is due to the high prevalence of genotypes A in Caucasians needs more studies to confirm. Although the present study demonstrated no difference in the chance and severity of biochemical flares between patients with genotypes B and C, there was an increase in the proportion of patients with YMDD mutations as the sole viral strain at the last follow-up. It is likely that the increased chance of biochemical flares with time was related to the mutant becoming the dominant strain in the viral population. This finding is in agreement with findings from other studies that the increase in viral load and the occurrence of biochemical flares are the result of gradual increase in the concentration of particular mutants (16, 18, 20).

In addition, patients with higher pretreatment ALT levels had a higher chance of biochemical flares. Therefore, patients with high baseline ALT levels, although they have a higher chance of undergoing HBeAg seroconversion induced by lamivudine (2), they are also more prone to virological breakthroughs with YMDD mutations (as shown in our previous study) (21), with a higher chance of experiencing biochemical flares afterward. Patients with high pretreatment ALT levels should be monitored more closely once YMDD mutations develop.

Severe biochemical flares leading to hepatic decompensation have been observed in the present study and in previous studies (7, 12, 14, 22). The majority of the patients had normalization of ALT levels within 6 months after the onset of severe biochemical flares, although it might take as long as 21 months, as observed in one patient (Table 2). This finding is in accord with the study conducted by Lau et al., who reported that the ALT levels of patients with YMDD resistance after virological breakthroughs may increase to a level above the baseline values for 1 to 6 months only and then fluctuate at values at or below baseline levels (10). HBeAg seroconversion occurred in two of our patients. Therefore, although severe biochemical flare is not uncommon, the majority of patients would have an uneventful course, and some patients may undergo HBeAg seroconversion afterward. In fact, it has been suggested that hepatitis flares due to YMDD mutations have a high rate of HBeAg seroconversion (12).

Finally, similar to other studies (5, 8, 10, 14), the median HBV DNA level of patients with YMDD mutations at the last follow-up was significantly lower than the median pretreatment HBV DNA level. Even with longer periods of follow-up, the viral load of the patients with YMDD mutations was still lower than that at the baseline.

In conclusion, there was no difference in the risk of virological breakthroughs with YMDD mutations between patients with genotypes B and C. Biochemical flares occur in more than half of the patients by the end of the third year after virological breakthroughs with YMDD mutations. Patients with high pretreatment ALT levels would have a higher chance of biochemical flares. Severe biochemical flares occurred in 16% patients. These patients usually recover spontaneously. The HBV DNA levels of patients with YMDD mutations were still lower than at the baseline after a prolonged period of follow-up.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Rd., Hong Kong, Peoples Republic of China. Phone: (852) 28553111. Fax: (852) 28162863. E-mail for M.-F. Yuen: yuenmf{at}netvigator.com. E-mail for C.-L. Lai: hrmelcl{at}hkucc.hku.hk. Back


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Journal of Clinical Microbiology, September 2004, p. 3932-3936, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.3932-3936.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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