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Clinical response to long-term tenofovir monotherapy in Korean chronic hepatitis B patients
Clinica Chimica Acta 471 (2017) 308–313
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Clinical response to long-term tenofovir monotherapy in Korean chronic hepatitis B patients
MARK
Eun-Hyung Yoo, Hyun-Jung Cho⁎ Department of Laboratory Medicine, Konyang University School of Medicine, Daejeon, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords: Chronic hepatitis B Tenofovir HBV DNA Complete virologic response Quantitative hepatitis B surface antigen
Background: Tenofovir disoproxil fumarate (TDF) is a potent nucleotide analogue recommended as first-line monotherapy for chronic hepatitis B (CHB). We investigated the clinical response to TDF monotherapy in Korean CHB patients. Methods: A total of 90 CHB patients [55 hepatitis B e antigen (HBeAg)-positive and 35 HBeAg-negative] who received TDF monotherapy for > 2 year, were enrolled. Quantitative hepatitis B surface antigen (qHBsAg) levels, serum alanine aminotransferase (ALT), HBeAg, anti-HBe and HBV DNA levels were measured during treatment. Virologic response (VR) was defined as undetectable HBV DNA level. Results: The cumulative incidences of complete virologic response (CVR) were 75.6% and 89.9% at months 12 and 24, respectively. The cumulative CVR rates were significantly higher in HBeAg-negative than HBeAg-positive group (P < 0.001). HBeAg loss/seroconversion was observed in 21 (38.2%) out of 55 HBeAg-positive patients. One HBeAg-positive and 1 HBeAg-negative patients (2.2%) achieved HBsAg loss at months 6 and 8 of TDF therapy, respectively. Baseline HBV DNA level and qHBsAg were significant predictive factors for a CVR (P = 0.001 and P < 0.001, respectively). Conclusions: Virologic, serologic, biochemical responses were achieved in both HBeAg-positive and HBeAg-negative patients under 24-month TDF therapy. Monitoring using baseline HBV DNA and qHBsAg levels would be helpful to predict CVR.
1. Introduction Chronic hepatitis B (CHB) is a worldwide health problem and a major cause of liver cirrhosis (LC), liver failure and hepatocellular carcinoma (HCC) [1]. Patients with CHB infection are at a 100-fold higher risk for developing HCC compared to healthy individuals while hepatitis B virus (HBV) cirrhotic patients are at an even higher risk [2,3]. Therefore, effective long-term treatment and regular follow-up monitoring are necessary. Seven antiviral agents including interferon-alpha, pegylated interferon-alpha, 3 nucleoside analogues (lamivudine, entecavir and telbivudine), and 2 nucleotide analogues (adefovir and tenofovir) have been approved for the treatment of CHB. Among them, oral administration of potent anti-HBV analogues has become the most popular treatment strategy worldwide given the excellent efficacy and safety of thirdgeneration nucleos(t)ide analogues (NA) such as entecavir and tenofovir. Tenofovir disoproxil fumarate (TDF) is a potent nucleotide
analogue of adenosine 50-monophosphate, suppresses viral genomereplication by HBV DNA polymerase activity via competition with the natural substrate. It has excellent safety profile and potent anti-HBV efficacy [4,5]. It is approved for the treatment of CHB by Korean Food and Drug Administration in December 2012. Owing to its potent activity against HBV and high genetic barrier to the development of resistance for up to 8 years [5], TDF is recommended as a first-line monotherapy for CHB patients [6]. Several surrogate markers including alanine aminotransferase (ALT) normalization, undetectable HBV DNA, and hepatitis B e antigen (HBeAg) loss/seroconversion have been developed for the end point of treatment [7]. Baseline and on-treatment response of HBV DNA loads are well known predictors of treatment outcomes for patients on antiviral therapy. As the serum HBV DNA level increases, the risk of LC and HCC and the mortality due to liver disease increase [8,9]. Therefore, the long-term suppression of HBV replication is an important goal in the effective treatment of CHB [10,11].
Abbreviations: Ab, antibody; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCC, covalently closed circular; CHB, chronic hepatitis B; CVR, complete virologic response; HBeAg, hepatitis B e antigen; HCC, hepatocellular carcinoma; LC, liver cirrhosis; PVR, partial virologic response; qHBsAg, quantitative hepatitis B surface antigen; VB, virologic breakthrough; VR, virologic response; TDF, tenofovir disoproxil fumarate ⁎ Corresponding author at: Department of Laboratory Medicine, Konyang University Hospital, #158, Gwangeodong-Ro, Seo-gu, Daejeon 35365, Republic of Korea. E-mail address: [email protected] (H.-J. Cho). http://dx.doi.org/10.1016/j.cca.2017.06.019 Received 13 March 2017; Received in revised form 22 June 2017; Accepted 27 June 2017 Available online 04 July 2017 0009-8981/ © 2017 Published by Elsevier B.V.
Clinica Chimica Acta 471 (2017) 308–313
E.-H. Yoo, H.-J. Cho
Recently, quantitative hepatitis B surface antigen (qHBsAg) has been developed as a marker for a predictor of antiviral treatment response. The qHBsAg is claimed to reflect not only the extent of viral replication but also the state of HBV infected hepatocytes, as it closely correlates with covalently closed circular (CCC) DNA, the key intrahepatic HBV replicative intermediate [12]. However, currently there is limited data on the role of qHBsAg levels in CHB patients treated with oral antiviral therapy. Although several studies have evaluated the efficacy of TDF and it is one of the most effective and safe antiviral agents, long term follow-up data are still lacking, especially in Asia. The aim of this study was to investigate the efficacy of TDF monotherapy for at least 24 months and to analyze the predictive factors for therapy response in Korean CHB patients.
Table 1 Baseline characteristics of the Korean CHB patients in the present study.
2. Materials and methods 2.1. Patients A total of 208 CHB patients started TDF monotherapy at 300 mg daily in Konyang university hospital, Daejeon, Korea from February 2013 to December 2014. Among them, patients with younger than 18 years old, prior antiviral treatment history, duration of TDF therapy < 24 months, concurrent hepatitis C and human immunodeficiency virus infection, prior diagnosis of HCC or insufficient laboratory data were excluded. As a result, a total of 90 CHB patients were enrolled in the present study. This study was approved by the Institutional Review Board of Konyang university hospital.
Characteristics
All
HBeAg-positive
HBeAg-negative
P-valuea
Number Age, year Male sex Duration of F/ U, months LC ALT, IU/mL AST, IU/mL HBV DNA level (log10 IU/ mL) qHBsAgb (log10 IU/ mL) Disease progression LC HCC
90 50.5 (18–74) 51 (56.7%) 36.5 (24–46)
55 47 (18–72) 32 (58.2%) 36 (24–45)
35 52 (29–74) 19 (54.3%) 37 (24–46)
0.065 0.828 0.667
40 (44.4%) 92 (13–2760) 74 (17–2130) 6.40 ± 1.64
22 (40%) 94 (13–2760) 77 (17–2130) 6.88 ± 1.35
18 (51.4%) 91 (15–740) 70 (20–510) 5.66 ± 1.80
0.384 0.582 0.313 < 0.001
3.63 ± 0.78
3.82 ± 0.51
3.33 ± 1.02
0.029
8 (16%, 8/50) 3 (3.3%, 3/90)
4 (12.1%, 4/33) 2 (3.6%, 2/55)
4 (23.5%, 4/17) 1 (2.9%, 1/35)
0.419 1.000
Values are expressed as median (range), mean ± SD or number (frequency, %). Abbreviations: HBeAg, hepatitis B envelop antigen; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LC, liver cirrhosis; qHBsAg, quantitative hepatitis B surface antigen level. a Comparison was between patients with HBeAg-positive and HBeAg-negative. b Results from 59 patients. Table 2 Virologic responses between HBeAg-positive and HBeAg-negative patients.
2.2. Laboratory analyses
Virologic responses
Serologic markers including HBsAg, HBeAg, anti-HBs antibody (Ab), anti-HBe Ab, anti-HBc Ab were tested with Architect (Abbott laboratories, Abbott park, IL, USA). HBV DNA was tested at baseline and every 3 to 6 months with real-time PCR using COBAS TaqMan HBV test (Roche diagnostics, Branchburg, NJ, USA; lower limit of detection: 20 IU/mL; upper limit of detection: 1.7 × 108 IU/mL). The qHBsAg levels were measured with chemiluminescent microparticle immunoassay using Architect HBsAg QT (Abbott laboratories, Abbott Park, IL, USA). Serum aspartate aminotransferase (AST) and ALT were also measured.
At month 12 Virologic response, n (%) Partial virologic response, n (%) Virologic breakthrough, n (%) At month 24 Virologic response, n (%) Partial virologic response, n (%) Virologic breakthrough, n (%)
HBeAg-positive N = 55
HBeAg-negative N = 33
P value
35 (63.6) 20 (36.3) 0 (0)
28 (84.8) 5 (15.2) 0 (0)
0.05
40 (72.7) 11 (20.0) 4 (7.3)
31 (93.9) 0 (0) 2 (6.1)
0.01
Values are expressed as number (frequency). Abbreviations: HBeAg, hepatitis B envelop antigen.
2.3. Definitions factor affecting CVR and HBeAg loss/seroconversion. Data analysis was performed with SPSS Statistics 24.0 software (IBM corporation, Armonk, NY, USA). A P-value of < 0.05 was considered statistically significant.
Complete virologic response (CVR) was defined as undetectable HBV DNA level (< 20 IU/mL, lower limit of detection) by a quantitative PCR assay during the treatment follow-up period. Partial virologic response (PVR) was a decrease in HBV DNA of > 1 log10 IU/mL but detectable HBV DNA after at least 12 months of therapy. Virologic breakthrough (VB) was confirmed increase in HBV DNA level of > 1 log10 IU/mL compared to the lowest HBV DNA level during continued treatment. Serological response is HBe loss/seroconversion or HBsAg decline > 1 log10 IU/mL from baseline. Biochemical response was defined as ALT normalization (≤ 40 IU/L).
3. Results 3.1. Baseline characteristics Table 1 showed baseline characteristics of the 90 Korean CHB patients. The median age was 50.5 years (range, 18 to 74) and 56.7% of patients were male. At the time of treatment initiation, 55 (61.1%) patients were HBeAg-positive and the other 35 (38.9%) patients were HBeAg-negative. The mean HBV DNA level was 6.41 ± 1.64 log10 IU/ mL and the mean qHBsAg in available 59 patients was 3.63 ± 0.78 log10 IU/mL. The median duration of TDF treatment was 36.5 (range, 24 to 46) months. Baseline qHBsAg and HBV DNA levels were significantly higher in the HBeAg-positive compared to those in the HBeAg-negative group (P = 0.029 and P < 0.001, respectively). ALT and AST levels did not differ significantly between the two groups.
2.4. Statistical analyses Microsoft Excel 2010 software (Microsoft, Seattle, WA, USA) was used for the database. Quantitative variables were expressed as median (range) or mean ± SD and categorical variables as absolute and relative frequencies. HBV DNA and qHBsAg levels were logarithmically transformed for analysis. Groups of quantitative and qualitative variables were compared using the Mann-Whitney and the chi-square or Fisher's exact tests, respectively. The cumulative incidence of therapy responses was estimated by Kaplan-Meier method and analyzed using the log-lank test. The cox regression analyses were used to identify risk 309
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Fig. 1. HBV DNA changes through TDF therapy.
Fig. 2. Cumulative incidence of complete virologic response according to HBeAg status (A) and baseline HBV DNA level (B).
Fig. 3. Cumulative probability of HBeAg loss/seroconversion during TDF therapy.
Fig. 4. Quantitative HBsAg changes through TDF therapy.
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12 and 19 patients were HBeAg-positive. Among 24 PVR patients, 14 patients achieved CVR at month 24 with continuous TDF therapy. Four HBeAg-positive and 2 HBeAg-negative patients showed VB at month 24.
Table 3 Univariate and multivariate Cox regression analyses to identify predictive factors for a complete virologic response. Variables
Univariate
Multivariate
3.3. Serological responses Age, year Male sex LC HBeAg (–) ALT, IU/mL AST, IU/mL HBV DNA level (log10 IU/mL) qHBsAg (log10 IU/ mL)
HR (95% CI)
P value
1.009 (0.989–1.030) 0.937 (0.610–1.441) 1.230 (0.801–1.890) 0.443 (0.282–0.697) 1.001 (1.000–1.001) 1.001 (1.000–1.002) 0.694 (0.610–0.790) 0.479 (0.341–0.671)
0.358
HR (95% CI)
P value
The proportion of patients achieving HBeAg loss or seroconversion was observed in 21 (38.2%) out of 55 HBeAg-positive patients during follow-up period (Fig. 3). The mean time to HBeAg loss or seroconversion was 17 months (range, 1 to 29 months). The cumulative incidences were not different between patients with baseline HBV DNA level < 6 log10 IU/mL and those with baseline HBV DNA ≥ 6 log10 IU/mL (P = 0.263). One HBeAg-positive and 1 HBeAg-negative patients (2.2%, 2/90) achieved HBsAg loss at months 6 and 8 of TDF therapy, respectively. Among 40 patients with available qHBsAg data at baseline and month 12, only one patient showed HBsAg decline > 1 log10 IU/mL from baseline level. The qHBsAg levels were not significantly changed through 12-month therapy (Fig. 4.). The qHBsAg changes from baseline at months 6 and 12 were not different according to HBeAg status (P = 0.109 and P = 0.900, respectively). Baseline qHBsAg levels did not differ between patients with CVR and with PVR or VB at month 24 (P = 0.073).
0.768 0.344 < 0.001 0.003 0.020 < 0.001 < 0.001
0.702 (0.383–1.288) 1.006 (1.000–1.012) 0.994 (0.984–1.005) 0.730 (0.608–0.877) 0.466 (0.312–0.697)
0.253 0.057 0.293 0.001 < 0.001
Abbreviations: HR, hazard ratio; CI, confidence interval; LC, liver cirrhosis; HBeAg, hepatitis B envelop antigen; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HBV, hepatitis B virus; qHBsAg, quantitative hepatitis B surface antigen level.
3.4. Biochemical responses
Table 4 Univariate and multivariate Cox regression analyses to identify predictive factors for HBeAg loss/seroconversion. Variables
Age, year Male sex LC ALT, IU/mL AST, IU/mL HBV DNA level (log10 IU/mL) qHBsAg (log10 IU/ mL)
Univariate
The cumulative biochemical response rates were 79.3% and 94.3% at months 12 and 24, respectively. Forty-two [76.4%; median (range), 31 (11 −133) IU/mL] and 44 [80%; median (range), 28 (13–86) IU/ mL] patients in HBeAg-positive group had normalized ALT at months 12 and 24, respectively. In HBeAg-negative group, 22 [62.9%; median (range), 32 (16–96) IU/mL] and 27 [77.1%; median (range), 27 (17–141) IU/mL] patients showed biochemical response at months 12 and 24, respectively. The proportion of ALT normalization was comparable between HBeAg-positive and HBeAg-negative patients during therapy (P = 0.860).
Multivariate
HR (95% CI)
P value
0.991 (0.954–1.028) 1.484 (0.598–3.682) 0.837 (0.347–2.020) 1.002 (1.001–1.003) 1.003 (1.002–1.005) 0.779 (0.600–1.012) 0.353 (0.100–1.241)
0.622
HR (95% CI)
P value
1.001 (0.992–1.010) 1.001 (0.985–1.017) 0.783 (0.542–1.133) 0.615 (0.181–2.089)
0.841
0.394 0.692 < 0.001 < 0.001 0.062 0.104
3.5. Predictive factors for treatment response to TDF therapy
0.902 0.195
In univariate Cox regression analysis, HBeAg negativity, baseline ALT, AST, HBV DNA level and qHBsAg level showed significant association with a CVR (Table 3). Baseline HBV DNA level and qHBsAg were significant predictive factors for a CVR in multivariate analyses. No variables were significantly associated with HBeAg loss/seroconversion in multivariate analysis with HBeAg-positive patients (Table 4).
0.436
Abbreviations: HR, hazard ratio; CI, confidence interval; LC, liver cirrhosis; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HBV, hepatitis B virus; qHBsAg, quantitative hepatitis B surface antigen level.
4. Discussion
3.2. Virologic responses
We investigated therapeutic efficacy of a long-term TDF monotherapy and prediction factors of therapeutic responses. The present study showed that TDF as an initial therapy was effective for Korean CHB patients and associated with favorable virologic, serologic and biochemical responses. The main intermediate-term purpose of CHB treatment is HBV DNA suppression as profound and sustained as possible to prevent the longterm complications of CHB [13]. The long-term effective suppression of HBV allows the regression of fibrosis and LC and delays the development of HCC as well as its progression [14]. In addition, HBV DNA determination represents the only validated tool to monitor on-treatment efficacy in HBeAg-negative patients [15]. Therefore, close monitoring of the virologic response is needed. In the current study, the cumulative CVR rate at month 24 was 89.9% and significantly different according to HBV DNA levels and HBeAg status. The cumulative CVR rates were 83.6% in HBeAg-positive group and 97.0% in HBeAg-negative patients at months 24, which was comparable to those in previous studies. Ahn et al. reported that CVR was achieved in 91.5% of patients
Virologic responses for TDF monotherapy were summarized in Table 2 and Fig. 1 showed overall HBV DNA changes through TDF therapy. The cumulative incidences of CVR in Korean CHB patients were 75.6% and 89.9% at months 12 and 24, respectively. The cumulative CVR rates in HBeAg-positive group were 65.5% and 83.6% at months 12 and 24, respectively. Two HBeAg-negative patients had no viremia at initiation of therapy. The cumulative CVR rates in 33 HBeAgnegative patients were 90.9% and 97.0% at months 12 and 24, respectively. The cumulative CVR rates were significantly higher in HBeAg-negative than HBeAg-positive group (P < 0.001) (Fig. 2A). The mean declines from baseline in serum DNA level were 4.74 and 4.96 log10 IU/mL at months 6 and 12, respectively. Reduction of HBV DNA was significantly higher in HBeAg-positive than HBeAg-negative at months 6 and 12 (P = 0.016 and P < 0.001, respectively). Patients with baseline HBV DNA level < 6 log10 IU/mL achieved significantly higher CVR (%) than those with baseline HBV DNA ≥ 6 log10 IU/mL (P < 0.001) (Fig. 2B). PVR occurred in 24 (26.7%) patients at month 311
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monitoring of qHBsAg level would be helpful to predict clinical response and allow individualization of therapy.
at month 24 (85.2% of HBeAg-positive and 95.5% of HBeAg-negative patients) [16]. The CVR rate was 87% at month 24 and 100% at month 36 in 55 Australian CHB patients [17]. Compared to 12-month therapy, CVR rate was increased in both groups at month 24. Prolonged TDF therapy might be more effective for virologic response. Buti et al. showed that CVR was observed in 99.3% of patients (99.4% of HBeAgpositive and 99.3% of HBeAg-negative patients) who remained on study at 7-year therapy [18]. The baseline HBV DNA and qHBsAg levels were confirmed as independent predicting factors for CVR in a multivariate analysis. The baseline qHBsAg level was significantly associated with HBV DNA level (P < 0.001). In the previous studies, HBV DNA levels and/or HBeAgnegativity have been confirmed as major predicting factors for CVR [17,19,20]. However, HBeAg negativity, baseline ALT and AST were significantly associated with CVR in only univariate regression analysis in the present study. HBeAg seroconversion is an established therapeutic end point for the management of HBeAg-positive CHB and is associated with reduced morbidity and mortality [21]. The relative risk of HCC was > 6-fold higher in HBeAg-positive patients than HBeAg-negative [22]. Patients who undergo HBeAg seroconversion are more likely to experience improved long-term outcomes, including disease remission, a lower incidence of cirrhosis and HCC, increased rates of survival, and the possibility of HBsAg loss or seroconversion [23–25]. There were 21 (38.2%) patients of HBeAg loss or seroconversion in HBeAg-positive group at month 24 in this study. HBe seroconversion occurred in 7–18% of patients in European cohort [26] and 17.6% in Korean group [16]. On study at 7-year therapy, among 154 observed HBeAg-positive patients, 84 (54.5%) experienced HBeAg loss and 61 (39.6%) experienced seroconversion [18]. Long-term use of TDF monotherapy might increase serological response rate. HBsAg seroconversion is the ultimate laboratory marker of successful therapy for patients with CHB. However, this seldom occurs. Serum loss of HBsAg is reported to be 0.5% to 3% every year during the course of natural history [27–29]. It is reported that the rate of HBsAg loss increases from 3% to 8% when the treatment period is longer with strong antiviral agents such as entecavir or tenofovir [30,31]. In the present study, 1 HBeAg-positive and 1 HBeAg-negative patients (2.2%, 2/90) achieved HBsAg loss at months 6 and 8, respectively. In the past 10 years, there has been a lot of enthusiasm surrounding the use of serum HBsAg quantification to predict disease activity and monitor treatment response in CHB [32]. Baseline qHBsAg levels did not differ between patients with CVR and with PVR or VB at month 24, therefore, TDF had little effect on qHBsAg levels in this study. HBsAg decline during NA therapy is much slower and less pronounced compared to pegylated interferon treatment, although NA are much more potent on HBV DNA suppression [33,34], because NAs do not target the cccDNA directly. The present study was limited in its small sample size and retrospective nature. Long-term follow-up data from well-designed trials will allow physicians to select the best therapeutic options for their patients with CHB. Furthermore, although 95.6% to 100% of CHB patients in Korea have genotype C, which has been associated with a poor clinical outcome and response to antiviral therapy [35,36], the baseline genotype was not measured. There were a few patients showed high HBV DNA level after 24-month TDF therapy. Although there is no known resistance mutation for TDF treatment [5], additional mutation analyses for these patients with inappropriate virologic response would be helpful. In summary, the present study provides 24-month data on the efficacy of TDF in naïve CHB patients in Korea. Favorable virologic, serologic, biochemical responses were achieved in both HBeAg-positive and HBeAg-negative patients. In addition, baseline HBV DNA and qHBsAg levels were predictive factors for virologic response under TDF therapy. Although the role of qHBsAg titers in predicting clinical response for NA therapy is still yet to be defined, long term on-treatment
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398–411. [33] M.R. Brunetto, F. Moriconi, F. Bonino, G.K. Lau, P. Farci, C. Yurdaydin, et al., Hepatitis B virus surface antigen levels: a guide to sustained response to peginterferon alfa-2a in HBeAg-negative chronic hepatitis B, Hepatology 49 (2009) 1141–1150. [34] J.G. Reijnders, V. Rijckborst, M.J. Sonneveld, S.M. Scherbeijn, C.A. Boucher, B.E. Hansen, et al., Kinetics of hepatitis B surface antigen differ between treatment with peginterferon and entecavir, J. Hepatol. 54 (2011) 449–454. [35] B.C. Song, X.J. Cui, H. Kim, Hepatitis B virus genotypes in Korea: an endemic area of hepatitis B virus infection, Intervirology 48 (2005) 133–137. [36] E. Orito, M. Mizokami, H. Sakugawa, K. Michitaka, K. Ishikawa, T. Ichida, et al., A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Japan HBV Genotype Research Group, Hepatology 33 (2001) 218–223.
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Clinical response to long-term tenofovir monotherapy in Korean chronic hepatitis B patients
MARK
Eun-Hyung Yoo, Hyun-Jung Cho⁎ Department of Laboratory Medicine, Konyang University School of Medicine, Daejeon, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords: Chronic hepatitis B Tenofovir HBV DNA Complete virologic response Quantitative hepatitis B surface antigen
Background: Tenofovir disoproxil fumarate (TDF) is a potent nucleotide analogue recommended as first-line monotherapy for chronic hepatitis B (CHB). We investigated the clinical response to TDF monotherapy in Korean CHB patients. Methods: A total of 90 CHB patients [55 hepatitis B e antigen (HBeAg)-positive and 35 HBeAg-negative] who received TDF monotherapy for > 2 year, were enrolled. Quantitative hepatitis B surface antigen (qHBsAg) levels, serum alanine aminotransferase (ALT), HBeAg, anti-HBe and HBV DNA levels were measured during treatment. Virologic response (VR) was defined as undetectable HBV DNA level. Results: The cumulative incidences of complete virologic response (CVR) were 75.6% and 89.9% at months 12 and 24, respectively. The cumulative CVR rates were significantly higher in HBeAg-negative than HBeAg-positive group (P < 0.001). HBeAg loss/seroconversion was observed in 21 (38.2%) out of 55 HBeAg-positive patients. One HBeAg-positive and 1 HBeAg-negative patients (2.2%) achieved HBsAg loss at months 6 and 8 of TDF therapy, respectively. Baseline HBV DNA level and qHBsAg were significant predictive factors for a CVR (P = 0.001 and P < 0.001, respectively). Conclusions: Virologic, serologic, biochemical responses were achieved in both HBeAg-positive and HBeAg-negative patients under 24-month TDF therapy. Monitoring using baseline HBV DNA and qHBsAg levels would be helpful to predict CVR.
1. Introduction Chronic hepatitis B (CHB) is a worldwide health problem and a major cause of liver cirrhosis (LC), liver failure and hepatocellular carcinoma (HCC) [1]. Patients with CHB infection are at a 100-fold higher risk for developing HCC compared to healthy individuals while hepatitis B virus (HBV) cirrhotic patients are at an even higher risk [2,3]. Therefore, effective long-term treatment and regular follow-up monitoring are necessary. Seven antiviral agents including interferon-alpha, pegylated interferon-alpha, 3 nucleoside analogues (lamivudine, entecavir and telbivudine), and 2 nucleotide analogues (adefovir and tenofovir) have been approved for the treatment of CHB. Among them, oral administration of potent anti-HBV analogues has become the most popular treatment strategy worldwide given the excellent efficacy and safety of thirdgeneration nucleos(t)ide analogues (NA) such as entecavir and tenofovir. Tenofovir disoproxil fumarate (TDF) is a potent nucleotide
analogue of adenosine 50-monophosphate, suppresses viral genomereplication by HBV DNA polymerase activity via competition with the natural substrate. It has excellent safety profile and potent anti-HBV efficacy [4,5]. It is approved for the treatment of CHB by Korean Food and Drug Administration in December 2012. Owing to its potent activity against HBV and high genetic barrier to the development of resistance for up to 8 years [5], TDF is recommended as a first-line monotherapy for CHB patients [6]. Several surrogate markers including alanine aminotransferase (ALT) normalization, undetectable HBV DNA, and hepatitis B e antigen (HBeAg) loss/seroconversion have been developed for the end point of treatment [7]. Baseline and on-treatment response of HBV DNA loads are well known predictors of treatment outcomes for patients on antiviral therapy. As the serum HBV DNA level increases, the risk of LC and HCC and the mortality due to liver disease increase [8,9]. Therefore, the long-term suppression of HBV replication is an important goal in the effective treatment of CHB [10,11].
Abbreviations: Ab, antibody; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCC, covalently closed circular; CHB, chronic hepatitis B; CVR, complete virologic response; HBeAg, hepatitis B e antigen; HCC, hepatocellular carcinoma; LC, liver cirrhosis; PVR, partial virologic response; qHBsAg, quantitative hepatitis B surface antigen; VB, virologic breakthrough; VR, virologic response; TDF, tenofovir disoproxil fumarate ⁎ Corresponding author at: Department of Laboratory Medicine, Konyang University Hospital, #158, Gwangeodong-Ro, Seo-gu, Daejeon 35365, Republic of Korea. E-mail address: [email protected] (H.-J. Cho). http://dx.doi.org/10.1016/j.cca.2017.06.019 Received 13 March 2017; Received in revised form 22 June 2017; Accepted 27 June 2017 Available online 04 July 2017 0009-8981/ © 2017 Published by Elsevier B.V.
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Recently, quantitative hepatitis B surface antigen (qHBsAg) has been developed as a marker for a predictor of antiviral treatment response. The qHBsAg is claimed to reflect not only the extent of viral replication but also the state of HBV infected hepatocytes, as it closely correlates with covalently closed circular (CCC) DNA, the key intrahepatic HBV replicative intermediate [12]. However, currently there is limited data on the role of qHBsAg levels in CHB patients treated with oral antiviral therapy. Although several studies have evaluated the efficacy of TDF and it is one of the most effective and safe antiviral agents, long term follow-up data are still lacking, especially in Asia. The aim of this study was to investigate the efficacy of TDF monotherapy for at least 24 months and to analyze the predictive factors for therapy response in Korean CHB patients.
Table 1 Baseline characteristics of the Korean CHB patients in the present study.
2. Materials and methods 2.1. Patients A total of 208 CHB patients started TDF monotherapy at 300 mg daily in Konyang university hospital, Daejeon, Korea from February 2013 to December 2014. Among them, patients with younger than 18 years old, prior antiviral treatment history, duration of TDF therapy < 24 months, concurrent hepatitis C and human immunodeficiency virus infection, prior diagnosis of HCC or insufficient laboratory data were excluded. As a result, a total of 90 CHB patients were enrolled in the present study. This study was approved by the Institutional Review Board of Konyang university hospital.
Characteristics
All
HBeAg-positive
HBeAg-negative
P-valuea
Number Age, year Male sex Duration of F/ U, months LC ALT, IU/mL AST, IU/mL HBV DNA level (log10 IU/ mL) qHBsAgb (log10 IU/ mL) Disease progression LC HCC
90 50.5 (18–74) 51 (56.7%) 36.5 (24–46)
55 47 (18–72) 32 (58.2%) 36 (24–45)
35 52 (29–74) 19 (54.3%) 37 (24–46)
0.065 0.828 0.667
40 (44.4%) 92 (13–2760) 74 (17–2130) 6.40 ± 1.64
22 (40%) 94 (13–2760) 77 (17–2130) 6.88 ± 1.35
18 (51.4%) 91 (15–740) 70 (20–510) 5.66 ± 1.80
0.384 0.582 0.313 < 0.001
3.63 ± 0.78
3.82 ± 0.51
3.33 ± 1.02
0.029
8 (16%, 8/50) 3 (3.3%, 3/90)
4 (12.1%, 4/33) 2 (3.6%, 2/55)
4 (23.5%, 4/17) 1 (2.9%, 1/35)
0.419 1.000
Values are expressed as median (range), mean ± SD or number (frequency, %). Abbreviations: HBeAg, hepatitis B envelop antigen; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LC, liver cirrhosis; qHBsAg, quantitative hepatitis B surface antigen level. a Comparison was between patients with HBeAg-positive and HBeAg-negative. b Results from 59 patients. Table 2 Virologic responses between HBeAg-positive and HBeAg-negative patients.
2.2. Laboratory analyses
Virologic responses
Serologic markers including HBsAg, HBeAg, anti-HBs antibody (Ab), anti-HBe Ab, anti-HBc Ab were tested with Architect (Abbott laboratories, Abbott park, IL, USA). HBV DNA was tested at baseline and every 3 to 6 months with real-time PCR using COBAS TaqMan HBV test (Roche diagnostics, Branchburg, NJ, USA; lower limit of detection: 20 IU/mL; upper limit of detection: 1.7 × 108 IU/mL). The qHBsAg levels were measured with chemiluminescent microparticle immunoassay using Architect HBsAg QT (Abbott laboratories, Abbott Park, IL, USA). Serum aspartate aminotransferase (AST) and ALT were also measured.
At month 12 Virologic response, n (%) Partial virologic response, n (%) Virologic breakthrough, n (%) At month 24 Virologic response, n (%) Partial virologic response, n (%) Virologic breakthrough, n (%)
HBeAg-positive N = 55
HBeAg-negative N = 33
P value
35 (63.6) 20 (36.3) 0 (0)
28 (84.8) 5 (15.2) 0 (0)
0.05
40 (72.7) 11 (20.0) 4 (7.3)
31 (93.9) 0 (0) 2 (6.1)
0.01
Values are expressed as number (frequency). Abbreviations: HBeAg, hepatitis B envelop antigen.
2.3. Definitions factor affecting CVR and HBeAg loss/seroconversion. Data analysis was performed with SPSS Statistics 24.0 software (IBM corporation, Armonk, NY, USA). A P-value of < 0.05 was considered statistically significant.
Complete virologic response (CVR) was defined as undetectable HBV DNA level (< 20 IU/mL, lower limit of detection) by a quantitative PCR assay during the treatment follow-up period. Partial virologic response (PVR) was a decrease in HBV DNA of > 1 log10 IU/mL but detectable HBV DNA after at least 12 months of therapy. Virologic breakthrough (VB) was confirmed increase in HBV DNA level of > 1 log10 IU/mL compared to the lowest HBV DNA level during continued treatment. Serological response is HBe loss/seroconversion or HBsAg decline > 1 log10 IU/mL from baseline. Biochemical response was defined as ALT normalization (≤ 40 IU/L).
3. Results 3.1. Baseline characteristics Table 1 showed baseline characteristics of the 90 Korean CHB patients. The median age was 50.5 years (range, 18 to 74) and 56.7% of patients were male. At the time of treatment initiation, 55 (61.1%) patients were HBeAg-positive and the other 35 (38.9%) patients were HBeAg-negative. The mean HBV DNA level was 6.41 ± 1.64 log10 IU/ mL and the mean qHBsAg in available 59 patients was 3.63 ± 0.78 log10 IU/mL. The median duration of TDF treatment was 36.5 (range, 24 to 46) months. Baseline qHBsAg and HBV DNA levels were significantly higher in the HBeAg-positive compared to those in the HBeAg-negative group (P = 0.029 and P < 0.001, respectively). ALT and AST levels did not differ significantly between the two groups.
2.4. Statistical analyses Microsoft Excel 2010 software (Microsoft, Seattle, WA, USA) was used for the database. Quantitative variables were expressed as median (range) or mean ± SD and categorical variables as absolute and relative frequencies. HBV DNA and qHBsAg levels were logarithmically transformed for analysis. Groups of quantitative and qualitative variables were compared using the Mann-Whitney and the chi-square or Fisher's exact tests, respectively. The cumulative incidence of therapy responses was estimated by Kaplan-Meier method and analyzed using the log-lank test. The cox regression analyses were used to identify risk 309
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Fig. 1. HBV DNA changes through TDF therapy.
Fig. 2. Cumulative incidence of complete virologic response according to HBeAg status (A) and baseline HBV DNA level (B).
Fig. 3. Cumulative probability of HBeAg loss/seroconversion during TDF therapy.
Fig. 4. Quantitative HBsAg changes through TDF therapy.
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12 and 19 patients were HBeAg-positive. Among 24 PVR patients, 14 patients achieved CVR at month 24 with continuous TDF therapy. Four HBeAg-positive and 2 HBeAg-negative patients showed VB at month 24.
Table 3 Univariate and multivariate Cox regression analyses to identify predictive factors for a complete virologic response. Variables
Univariate
Multivariate
3.3. Serological responses Age, year Male sex LC HBeAg (–) ALT, IU/mL AST, IU/mL HBV DNA level (log10 IU/mL) qHBsAg (log10 IU/ mL)
HR (95% CI)
P value
1.009 (0.989–1.030) 0.937 (0.610–1.441) 1.230 (0.801–1.890) 0.443 (0.282–0.697) 1.001 (1.000–1.001) 1.001 (1.000–1.002) 0.694 (0.610–0.790) 0.479 (0.341–0.671)
0.358
HR (95% CI)
P value
The proportion of patients achieving HBeAg loss or seroconversion was observed in 21 (38.2%) out of 55 HBeAg-positive patients during follow-up period (Fig. 3). The mean time to HBeAg loss or seroconversion was 17 months (range, 1 to 29 months). The cumulative incidences were not different between patients with baseline HBV DNA level < 6 log10 IU/mL and those with baseline HBV DNA ≥ 6 log10 IU/mL (P = 0.263). One HBeAg-positive and 1 HBeAg-negative patients (2.2%, 2/90) achieved HBsAg loss at months 6 and 8 of TDF therapy, respectively. Among 40 patients with available qHBsAg data at baseline and month 12, only one patient showed HBsAg decline > 1 log10 IU/mL from baseline level. The qHBsAg levels were not significantly changed through 12-month therapy (Fig. 4.). The qHBsAg changes from baseline at months 6 and 12 were not different according to HBeAg status (P = 0.109 and P = 0.900, respectively). Baseline qHBsAg levels did not differ between patients with CVR and with PVR or VB at month 24 (P = 0.073).
0.768 0.344 < 0.001 0.003 0.020 < 0.001 < 0.001
0.702 (0.383–1.288) 1.006 (1.000–1.012) 0.994 (0.984–1.005) 0.730 (0.608–0.877) 0.466 (0.312–0.697)
0.253 0.057 0.293 0.001 < 0.001
Abbreviations: HR, hazard ratio; CI, confidence interval; LC, liver cirrhosis; HBeAg, hepatitis B envelop antigen; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HBV, hepatitis B virus; qHBsAg, quantitative hepatitis B surface antigen level.
3.4. Biochemical responses
Table 4 Univariate and multivariate Cox regression analyses to identify predictive factors for HBeAg loss/seroconversion. Variables
Age, year Male sex LC ALT, IU/mL AST, IU/mL HBV DNA level (log10 IU/mL) qHBsAg (log10 IU/ mL)
Univariate
The cumulative biochemical response rates were 79.3% and 94.3% at months 12 and 24, respectively. Forty-two [76.4%; median (range), 31 (11 −133) IU/mL] and 44 [80%; median (range), 28 (13–86) IU/ mL] patients in HBeAg-positive group had normalized ALT at months 12 and 24, respectively. In HBeAg-negative group, 22 [62.9%; median (range), 32 (16–96) IU/mL] and 27 [77.1%; median (range), 27 (17–141) IU/mL] patients showed biochemical response at months 12 and 24, respectively. The proportion of ALT normalization was comparable between HBeAg-positive and HBeAg-negative patients during therapy (P = 0.860).
Multivariate
HR (95% CI)
P value
0.991 (0.954–1.028) 1.484 (0.598–3.682) 0.837 (0.347–2.020) 1.002 (1.001–1.003) 1.003 (1.002–1.005) 0.779 (0.600–1.012) 0.353 (0.100–1.241)
0.622
HR (95% CI)
P value
1.001 (0.992–1.010) 1.001 (0.985–1.017) 0.783 (0.542–1.133) 0.615 (0.181–2.089)
0.841
0.394 0.692 < 0.001 < 0.001 0.062 0.104
3.5. Predictive factors for treatment response to TDF therapy
0.902 0.195
In univariate Cox regression analysis, HBeAg negativity, baseline ALT, AST, HBV DNA level and qHBsAg level showed significant association with a CVR (Table 3). Baseline HBV DNA level and qHBsAg were significant predictive factors for a CVR in multivariate analyses. No variables were significantly associated with HBeAg loss/seroconversion in multivariate analysis with HBeAg-positive patients (Table 4).
0.436
Abbreviations: HR, hazard ratio; CI, confidence interval; LC, liver cirrhosis; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HBV, hepatitis B virus; qHBsAg, quantitative hepatitis B surface antigen level.
4. Discussion
3.2. Virologic responses
We investigated therapeutic efficacy of a long-term TDF monotherapy and prediction factors of therapeutic responses. The present study showed that TDF as an initial therapy was effective for Korean CHB patients and associated with favorable virologic, serologic and biochemical responses. The main intermediate-term purpose of CHB treatment is HBV DNA suppression as profound and sustained as possible to prevent the longterm complications of CHB [13]. The long-term effective suppression of HBV allows the regression of fibrosis and LC and delays the development of HCC as well as its progression [14]. In addition, HBV DNA determination represents the only validated tool to monitor on-treatment efficacy in HBeAg-negative patients [15]. Therefore, close monitoring of the virologic response is needed. In the current study, the cumulative CVR rate at month 24 was 89.9% and significantly different according to HBV DNA levels and HBeAg status. The cumulative CVR rates were 83.6% in HBeAg-positive group and 97.0% in HBeAg-negative patients at months 24, which was comparable to those in previous studies. Ahn et al. reported that CVR was achieved in 91.5% of patients
Virologic responses for TDF monotherapy were summarized in Table 2 and Fig. 1 showed overall HBV DNA changes through TDF therapy. The cumulative incidences of CVR in Korean CHB patients were 75.6% and 89.9% at months 12 and 24, respectively. The cumulative CVR rates in HBeAg-positive group were 65.5% and 83.6% at months 12 and 24, respectively. Two HBeAg-negative patients had no viremia at initiation of therapy. The cumulative CVR rates in 33 HBeAgnegative patients were 90.9% and 97.0% at months 12 and 24, respectively. The cumulative CVR rates were significantly higher in HBeAg-negative than HBeAg-positive group (P < 0.001) (Fig. 2A). The mean declines from baseline in serum DNA level were 4.74 and 4.96 log10 IU/mL at months 6 and 12, respectively. Reduction of HBV DNA was significantly higher in HBeAg-positive than HBeAg-negative at months 6 and 12 (P = 0.016 and P < 0.001, respectively). Patients with baseline HBV DNA level < 6 log10 IU/mL achieved significantly higher CVR (%) than those with baseline HBV DNA ≥ 6 log10 IU/mL (P < 0.001) (Fig. 2B). PVR occurred in 24 (26.7%) patients at month 311
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monitoring of qHBsAg level would be helpful to predict clinical response and allow individualization of therapy.
at month 24 (85.2% of HBeAg-positive and 95.5% of HBeAg-negative patients) [16]. The CVR rate was 87% at month 24 and 100% at month 36 in 55 Australian CHB patients [17]. Compared to 12-month therapy, CVR rate was increased in both groups at month 24. Prolonged TDF therapy might be more effective for virologic response. Buti et al. showed that CVR was observed in 99.3% of patients (99.4% of HBeAgpositive and 99.3% of HBeAg-negative patients) who remained on study at 7-year therapy [18]. The baseline HBV DNA and qHBsAg levels were confirmed as independent predicting factors for CVR in a multivariate analysis. The baseline qHBsAg level was significantly associated with HBV DNA level (P < 0.001). In the previous studies, HBV DNA levels and/or HBeAgnegativity have been confirmed as major predicting factors for CVR [17,19,20]. However, HBeAg negativity, baseline ALT and AST were significantly associated with CVR in only univariate regression analysis in the present study. HBeAg seroconversion is an established therapeutic end point for the management of HBeAg-positive CHB and is associated with reduced morbidity and mortality [21]. The relative risk of HCC was > 6-fold higher in HBeAg-positive patients than HBeAg-negative [22]. Patients who undergo HBeAg seroconversion are more likely to experience improved long-term outcomes, including disease remission, a lower incidence of cirrhosis and HCC, increased rates of survival, and the possibility of HBsAg loss or seroconversion [23–25]. There were 21 (38.2%) patients of HBeAg loss or seroconversion in HBeAg-positive group at month 24 in this study. HBe seroconversion occurred in 7–18% of patients in European cohort [26] and 17.6% in Korean group [16]. On study at 7-year therapy, among 154 observed HBeAg-positive patients, 84 (54.5%) experienced HBeAg loss and 61 (39.6%) experienced seroconversion [18]. Long-term use of TDF monotherapy might increase serological response rate. HBsAg seroconversion is the ultimate laboratory marker of successful therapy for patients with CHB. However, this seldom occurs. Serum loss of HBsAg is reported to be 0.5% to 3% every year during the course of natural history [27–29]. It is reported that the rate of HBsAg loss increases from 3% to 8% when the treatment period is longer with strong antiviral agents such as entecavir or tenofovir [30,31]. In the present study, 1 HBeAg-positive and 1 HBeAg-negative patients (2.2%, 2/90) achieved HBsAg loss at months 6 and 8, respectively. In the past 10 years, there has been a lot of enthusiasm surrounding the use of serum HBsAg quantification to predict disease activity and monitor treatment response in CHB [32]. Baseline qHBsAg levels did not differ between patients with CVR and with PVR or VB at month 24, therefore, TDF had little effect on qHBsAg levels in this study. HBsAg decline during NA therapy is much slower and less pronounced compared to pegylated interferon treatment, although NA are much more potent on HBV DNA suppression [33,34], because NAs do not target the cccDNA directly. The present study was limited in its small sample size and retrospective nature. Long-term follow-up data from well-designed trials will allow physicians to select the best therapeutic options for their patients with CHB. Furthermore, although 95.6% to 100% of CHB patients in Korea have genotype C, which has been associated with a poor clinical outcome and response to antiviral therapy [35,36], the baseline genotype was not measured. There were a few patients showed high HBV DNA level after 24-month TDF therapy. Although there is no known resistance mutation for TDF treatment [5], additional mutation analyses for these patients with inappropriate virologic response would be helpful. In summary, the present study provides 24-month data on the efficacy of TDF in naïve CHB patients in Korea. Favorable virologic, serologic, biochemical responses were achieved in both HBeAg-positive and HBeAg-negative patients. In addition, baseline HBV DNA and qHBsAg levels were predictive factors for virologic response under TDF therapy. Although the role of qHBsAg titers in predicting clinical response for NA therapy is still yet to be defined, long term on-treatment
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