A - Fetoprotein Level-Dependent Early Hepatitis B Surface Antigen Decline During Entecavir Therapy in Chronic Hepatitis B With Hepatitis Flare
A - Fetoprotein Level-Dependent Early Hepatitis B Surface Antigen Decline During Entecavir Therapy in Chronic Hepatitis B With Hepatitis Flare
A - Fetoprotein Level-Dependent Early Hepatitis B Surface Antigen Decline During Entecavir Therapy in Chronic Hepatitis B With Hepatitis Flare
Liver Research Unit, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan
Received 31 July 2015; returned 16 October 2015; revised 11 January 2016; accepted 15 January 2016
Objectives: Hepatitis B surface antigen (HBsAg) reduction during nucleos(t)ide analogue therapy is related to ALT
level. ALT reflects hepatocytolysis while a-fetoprotein (AFP) ≥100 ng/mL during hepatitis flare reflects more
extensive hepatocytolysis (bridging hepatic necrosis). The impact of AFP levels on early HBsAg kinetics during
entecavir therapy was investigated.
Methods: HBsAg level was measured at baseline and months 6 and 12 of entecavir therapy in 149 chronic
hepatitis B patients with hepatitis flare, defined as ALT ≥5× upper limit of normal (ULN), and 58 patients with
ALT ,5× ULN.
Results: There was a significantly greater HBsAg reduction in an ALT (,5, 5– 10, 10 –20 and ≥20× ULN, P ¼ 0.001)
and AFP (,20, 20–99 and ≥100 ng/mL, P ¼0.000) level-dependent manner. In hepatitis flares with a peak AFP
level ≥20 ng/mL, the differences in HBsAg reduction across all ALT levels became non-significant. HBsAg reduc-
tion was greater in genotype B- than genotype C-infected patients with baseline ALT ≥20× ULN, but the differ-
ence became non-significant in those with peak AFP ≥100 ng/mL. Multivariate linear regression analysis showed
that AFP level ≥100 ng/mL, baseline HBsAg level and genotype B were independent significant factors for greater
HBsAg decline at month 6 of entecavir therapy.
Conclusions: During entecavir therapy, early HBsAg reduction increased in an AFP and ALT level-dependent
manner, suggesting the impact of hepatocytolysis rather than nucleos(t)ide analogue per se. Notably, a higher
AFP level during hepatitis flare, reflecting more extensive hepatic necrosis, was a more powerful factor than ALT
and genotype for greater HBsAg decline.
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46% of those with AFP 21 –100 ng/mL and only 9% of those with method. Statistical analysis was performed with the x2 test or Fisher’s
AFP ≤20 ng/mL.9,10 Given these findings, together with a high exact test and independent Student’s t-test or Mann–Whitney U-test for
degree of AFP-producing progenitor oval cell activation in patients the categorical and continuous variables, respectively, among patients
with BHN, a recent review suggests that AFP .100 ng/mL during with hepatitis B flares with different ALT (5 – 10×, 10 – 20× and .20×
hepatitis B flare can be used as a surrogate marker of BHN.11 ULN) and AFP (,20, 20–99 and ≥100 ng/mL) levels.9 – 11 Continuous vari-
ables are shown as the median (range). Multiple linear regression analysis
Asian-Pacific HBV guidelines recommend that patients with ALT
was performed to find the predictor of HBsAg log reduction. Variables that
.5× ULN should be monitored closely with weekly or biweekly
were previously identified as significant factors in univariate analysis
serum ALT, bilirubin and prothrombin time measurement.12 In (P, 0.05) and factors reported to be significant in earlier studies were
addition to these tests, we have included serial AFP assays to included in multivariate analyses.
detect the peak level of AFP since the relation of AFP and BHN dur-
ing hepatitis flare was reported three decades ago.9 With these
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AFP-dependent rapid HBsAg decline JAC
Table 1. Baseline demographic features and HBsAg reduction during entecavir therapy in patients with and those without hepatitis B flare
Hepatitis B flare
HBsAg kinetics in relation to pretherapy ALT and peak AFP ULN. The differences were not significant across all categories of
level in patients with hepatitis B flare ALT flares with AFP ≥20 ng/mL (Table 2).
Patients with higher pretherapy ALT, especially those with higher
AFP levels, showed greater HBsAg reduction in an ALT and AFP level- HBsAg kinetics in relation to HBV genotypes
dependent manner (Table 2). Patients with higher ALT (≥10×versus HBV genotype data were available in 149 patients with hepatitis B
5–10×ULN) also had more frequent ‘rapid HBsAg decline’ at month flare (genotype B¼110, genotype C¼38 and not typeable¼1) and
6 and month 12 of entecavir therapy, being greatest in patients 58 patients without flare (genotype B¼45 and genotype C¼13).
with ALT ≥10× (ALT 5 –10×¼ 30.8%, 10– 20×¼ 63.5% and ≥20× Taking all patients together, HBsAg decline at month 6 of entecavir
ULN ¼ 71.1%, P ¼ 0.000) and AFP ≥20 ng/mL (,20 ¼ 41.2%, therapy was greater in genotype B-infected patients (20.27 versus
20 – 99 ¼ 60% and ≥100 ¼ 79.5% in patients with ALT ≥5× ULN, 20.16 log10 IU/mL, P¼0.097). In patients without hepatitis flare,
P ¼ 0.000). The clinical courses of two representative patients there was similar HBsAg reduction between genotypes B and C
with hepatitis flare and a patient with pretherapy ,5× ULN are (20.06 versus 20.08 log10 IU/mL, P¼0.867) while greater HBsAg
shown in Figure 2. HBsAg reduction during the first year of entecavir reduction was observed in genotype B patients with hepatitis
therapy occurred primarily by month 6. Comparisons of data at flare (20.715 versus 20.295 log10 IU/mL in genotype C,
month 6 showed greater reduction in patients with ALT levels P¼0.028), which was significantly greater in both HBeAg-positive
≥10× ULN than in those with ALT 5 – 10× ULN if peak AFP was and -negative patients. However, breakdown data showed that
,20 ng/mL (P ¼ 0.012), but the differences were non-significant the difference was significant only in patients with ALT ≥20× ULN
in those with peak AFP ≥20 ng/mL. Compared with patients with (genotype B : genotype C ¼ 20.965 versus 0.04 log10 IU/mL,
peak AFP ,20 ng/mL, patients with peak AFP ≥20 ng/mL showed P¼0.002). Of the patients with ALT ≥20× ULN, the different impact
significantly greater HBsAg decline at month 6 of entecavir therapy of genotypes B and C was observed only in patients with
in patients with ALT ≥10× ULN, but not in those with ALT 5–10× AFP ≥20 ng/mL (genotype B : genotype C ¼ 21.19 versus 0.07
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Jeng et al.
ALT and
4.00 AFP
category
1
2
3
3.75 4
5
Median HBsAg (log10 IU/mL)
3.50
Group 2
3.00
Group 1
Group 5
2.75 Group 3
Group 4
Figure 1. Reduction of serum HBsAg from baseline to month 6 was highly significant, but the reduction from month 6 to 12 of entecavir therapy was not
significant in patients regardless of ALT and AFP levels. Greater reduction of HBsAg was noted in those with ALT ≥10× and AFP ≥20 ng/mL (21.09 log
reduction at month 6). vs, versus.
log10 IU/mL, P ¼ 0.008), among whom the difference became AFP ≥100 ng/mL were factors for HBsAg reduction in the univariate
non-significant in patients with AFP ≥100 ng/mL. Among patients analysis. In the multivariate analysis, genotype B [adjusted b (95%
with flare who had AFP ,100 ng/mL, median HBsAg reduction CI): 0.164 (0.079–0.517), P¼0.008], higher baseline HBsAg level
in genotype B was greater than in genotype C (20.545 versus [adjusted b (95% CI): 20.474 (20.642 to 20.335), P¼0.000] and
20.240 log10 IU/mL, P ¼ 0.042). In contrast, the difference was AFP ≥100 ng/mL [adjusted b (95% CI): 20.254 (20.705 to 20.215),
not statistically significant in patients with AFP ≥100 ng/mL (geno- P¼0.000] were significant factors while ALT ≥10× ULN [adjusted b
type B:genotype C¼21.34 versus 20.92 log10 IU/mL, P¼0.399). (95% CI): 20.123 (20.41720.009), P¼0.060] was of borderline sig-
nificance for HBsAg reduction at month 6 of therapy (Table 3).
Factors predicting HBsAg decline at month 6 of
Of these independent factors for HBsAg reduction at month 6,
entecavir therapy higher baseline HBsAg level [adjusted b 20.709 (20.809 to
Since there was only a small and non-significant decline between 20.444), P ¼ 0.000] and ALT ≥10× ULN [adjusted b 20.287
months 6 and 12 of entecavir therapy, only factors predicting (20.496 to 20.147), P ¼0.000] were factors for HBsAg reduction
‘rapid HBsAg decline’ at month 6 were analysed. By multiple linear in patients with hepatitis flare whose AFP was ,20 ng/mL, while
regression analysis, genotype B, higher baseline HBsAg level, higher baseline HBsAg level was the only factor in those with AFP
higher baseline HBV DNA level, pretherapy ALT ≥10× ULN and ≥20 ng/mL [adjusted b 20.414 (20.712 to 20.168), P ¼ 0.002].
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AFP-dependent rapid HBsAg decline JAC
Table 2. HBsAg reduction at month 6 of entecavir therapy in relation to pretherapy ALT and peak AFP levels in patients with hepatitis B flare
ALT (ULN)
5 –10× (n¼52) 20.18 (22.8– 1.05) 16 (30.8)
P¼0.000
≥10× (n ¼97) 20.80 (22.94 – 1.28) 65 (67)
AFPa (ng/mL)
5 –10×b 20.18 (21.66 to +1.05)d 20.1 (21.32 to +0.14)e 21.34 (22.8 to 20.45)f
≥10×c 20.57 (21.82 to +1.28)g 20.76 (22.51 to +1.27)h 21.19 (22.94 to +0.61)i
P 0.012 0.169 0.806
Long-term outcome in patients with ‘rapid HBsAg decline’ (APASL) stopping rule.12 The end-of-treatment HBsAg level in
patients with and without ‘rapid HBsAg decline’ was similar
Of the 60 HBeAg-positive patients in this study, 35 (68.6%) of
(29.6 – 7515 versus 14 – 2086 IU/mL, P ¼ 0.716); however, more
the 51 patients with hepatitis flare at baseline achieved HBeAg
achieved an HBsAg level ,100 IU/mL, though the difference
seroconversion, much higher than 2 (22.2%) of the 9 with baseline
was non-significant (15% versus 8%, P¼ 0.654).
ALT ,5× ULN (P ¼ 0.021). Of these HBeAg-positive patients,
the 3 year cumulative HBeAg seroconversion rate was 83% in
patients with ‘rapid HBsAg decline’ and 45% in those without
rapid decline (P¼ 0.03).
Discussion
HBsAg loss was documented in 3 (2%) of the 149 patients with The results of the present study showed that patients with hepatitis
baseline hepatitis flare and 2 (3.4%) of the patients without flare, B flare had a steep and significantly greater HBsAg reduction and
at 1303 – 2015 (median 1590) days after the start of therapy more frequent ‘rapid HBsAg decline’, in an ALT level-dependent
(P ¼ 0.616). The median time from baseline to HBsAg loss was manner, and patients with ‘rapid HBsAg decline’ during the early
also similar between these two groups (with flare¼1590 days ver- phase of entecavir therapy had a significantly higher HBeAg sero-
sus without flare¼1641 days). Of the five patients with HBsAg loss, conversion rate. Previous studies showed: a ‘rapid HBsAg decline’ at
none had prior IFN therapy and one had liver cirrhosis at baseline. week 24 (≥0.5 log10 IU/mL decline) and year 1 (≥1.0 log10 IU/mL
Of the 149 patients with baseline hepatitis flare, HBsAg decline) during telbivudine therapy in 33% and 20%, respectively,
seroclearance was observed in 3 (3.7%) of the 81 patients of 162 HBeAg-positive patients with a mean pretherapy ALT of
with ‘rapid HBsAg decline’ and none of the patients without 4 – 5×ULN;14 a ‘rapid HBsAg decline’ at month 6 during lamivudine
(P ¼ 0.251). In addition, two of the three patients with HBsAg therapy in 30% of 86 HBeAg-positive patients with a mean
loss had peak AFP .100 ng/mL. The average annual HBsAg reduc- pretherapy ALT of 4.58× ULN;15 and a ‘rapid HBsAg decline’ at
tion in patients with ‘rapid HBsAg decline’ was greater than in month 6 of Nuc therapy in 51% of 74 HBeAg-negative patients
those without it, in both HBeAg-positive and -negative patients with severe reactivation (ALT ≥10× ULN).7 The present study has
(20.439 versus 20.041 log10 IU/mL, P ¼ 0.000 and 20.379 versus further shown that the proportion of patients with ‘rapid HBsAg
20.099 log10 IU/mL, P ¼0.000, respectively). Average reduction of decline’ increases significantly along with the increasing ALT
HBsAg from baseline to the end of treatment was greater in those level, being 6.9% in patients with baseline ALT ,5× ULN
with ‘rapid HBsAg decline’ than in those without (median: 21.092 (Table 1), 30.8% in those with ALT of 5 – 10× ULN, 63.5% in
versus 20.183 log10 IU/mL, P ¼ 0.000). those with 10– 20× ULN and 71.1% in those with ALT ≥20× ULN
Of the 53 HBeAg-negative patients with hepatitis flare at base- at month 6. Although previous studies demonstrated that higher
line who had stopped entecavir therapy, 45 patients had stopped pretherapy ALT level is a factor for greater HBsAg decline during
therapy by the Asian Pacific Association for the Study of the Liver Nuc therapy,4 – 7,16,17 the data in the present study may be the
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Jeng et al.
HBV DNA
ALT (U/L)
HBsAg
102
400 lowed by HBeAg seroclearance, in contrast to ,20% in flares
with AFP ,100 ng/mL, including those with ALT .25× ULN.11,13
101 102 Another study also reported AFP elevation of 52 –1636 ng/mL in
200
seven patients with an ALT level .5× ULN (217 – 480 IU/L), of
103
HBV DNA is probably related to a greater extent of hepatocytolysis and sub-
HBsAg
102 104 cccDNA.8 If AFP levels were also properly measured in patients
400 with baseline ≥5× ULN in the above-mentioned earlier studies,
perhaps we might explain why the early and ‘rapid HBsAg decline’
101 102 rates differ among different studies and being much higher in the
200
present study as compared with a lower rate in patients with ALT
0 0 .10× ULN in the Hong Kong study.7
0 8 16 24 32 40 48 56 60 There are variable findings on HBsAg reduction during Nuc
(c) 1000 108 therapy among patients infected with different HBV genotypes.
Therapy 104
Greater HBsAg decline was observed in patients with genotype
A than in patients with genotypes B, C and D HBV infec-
AFP
800
103 106 tion.5,14,19,20 Between genotypes B and C HBV-infected patients,
HBV DNA
HBsAg
600 greater HBsAg decline in the first year of entecavir therapy was
documented in patients with genotype B HBV infection,5,19,21,22
ALT
102 104
400 but there was no difference in patients treated with tenofovir.20
The results of the present study showed significantly greater
101 102 HBsAg decline in genotype B HBV-infected patients with hepatitis
200
flare as a whole group, but the significant difference was actually
limited to those with ALT ≥20× ULN. In addition, the difference
0 0
–8 0 8 16 24 32 40 48 56 60 became non-significant in patients with AFP ≥100 ng/mL during
Weeks of therapy hepatitis flare regardless of ALT levels, although genotype B
remained a significant independent factor for HBsAg reduction
Figure 2. Clinical course during antiviral therapy in three representative at month 6 of therapy in the multivariate analysis. These findings
patients. (a) ‘Rapid HBsAg decline’ in a patient with high ALT and peak suggest that the impact of HBV genotype in HBsAg decline is over-
AFP .100 ng/mL. (b) Slow HBsAg decline in a patient with high ALT, but powered by the more extensive hepatocytolysis, as reflected in an
peak AFP ,20 ng/mL. (c) Slow HBsAg decline in a patient with ALT ,5× AFP level ≥100 ng/mL.9,10 Perhaps the different effects of HBV
ULN and AFP ,20 ng/mL. genotype in patient populations with different ALT and/or AFP
levels may explain the different findings related to HBV genotype
first showing the influence of different ALT levels ≥5× ULN. Since reported in previous studies.5,14,19 – 21
ALT elevation in chronic hepatitis B has been considered to be the The current study has several limitations. First, only a small
result of endogenous immune response against HBV and the ALT number of patients had liver biopsy and an even smaller number
levels reflect the strength of the immune response,10,11 the of patients with AFP ≥20 ng/mL showed evidence of BHN (3 of 5)
above-mentioned results showing HBsAg reduction in an ALT and none of those with AFP ,20 ng/mL had BHN (0 of 12).
level-dependent manner are consistent with the concept that Therefore, the greater HBsAg decline could only be speculated
greater HBsAg decline during entecavir therapy is linked to stron- to be the result of more extensive hepatocytolysis based on higher
ger pre-existing immune activity.4,7 This is also in line with the AFP levels. However, previous studies have provided strong
finding of association between greater HBsAg reduction and evidence that elevated AFP, especially ≥100 ng/mL, during hepa-
stronger immune modulation reflected in higher IFN-g-inducible titis B flare is closely associated with biopsy findings of BHN9,10 and
protein of 10 kDa (IP-10).6,16 AFP ≥100 ng/mL can be considered a surrogate marker of BHN.11
Perhaps the most important new finding of the present study Second, the study aimed to examine the early HBsAg kinetics and
is that the peak AFP level is a more powerful baseline factor than therefore did not include long-term HBsAg kinetics. However, earl-
ALT level and HBV genotype for HBsAg decline during the early ier studies have shown that HBsAg declines mainly during HBV
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AFP-dependent rapid HBsAg decline JAC
Table 3. Linear regression analysis of baseline factors in 149 patients with hepatitis B flare for HBsAg reduction at month 6 of entecavir therapy
Univariate Multivariate
Variable regression coefficient (b) (95% CI) P regression coefficient (b) (95% CI) P
Gender
female 1.0
male 20.005 (20.312– 0.295) 0.956
Genotype (n¼148)
B 1.0 1.0
C 0.177 (0.029–0.613) 0.031 0.164 (0.079– 0.517) 0.008
HBsAg (log10 IU/mL) 20.586 (20.74 to 20.467) 0.000 20.474 (20.642 to 20.335) 0.000
HBV DNA (log10 IU/mL) 20.418 (20.372 to 20.178) 0.000 20.127 (20.181– 0.015) 0.095
ALT
5 –10× 1.0 1.0
≥10× 20.276 (20.717 to 20.198) 0.001 20.123 (20.417– 0.009) 0.060
a
AFP
,20 1.0 1.0
20– 99 20.133 (20.607– 0.044) 0.090 0.037 (20.197– 0.354) 0.576
≥100 20.437 (21.062 to 20.509) 0.000 20.254 (20.705 to 20.215) 0.000
a
Peak level during flare episode.
DNA declining phase, which is mostly in the first year of Nuc ther- more powerful factor that overwhelms ALT and genotype. This
apy. Third, the observation period was not long enough to show AFP factor may reflect the effects of more extensive immune-
differences in long-term efficacy. Nevertheless, the current mediated hepatocyte killing as HBsAg decline was greatest in flares
study showed that the HBeAg seroconversion rate was much with AFP ≥100 mg/mL, which is considered to be a surrogate
higher in patients with hepatitis flare than in those without flare marker of BHN.11 Therefore, both ALT and AFP factors should be
(68.6% versus 22.2%, P ¼ 0.021). For patients with ‘rapid HBsAg taken into account when comparing HBsAg reduction between dif-
decline’, the 3-year cumulative HBeAg seroconversion rate was ferent studies or different patient populations with high ALT levels.
83%, significantly higher than 45% (P ¼ 0.03) in patients without Measurement of AFP during hepatitis flare has long been neglected
‘rapid HBsAg decline’. Their HBsAg loss rate was two times higher since it was reported 30 years ago.9 Perhaps proper measurement
(3.5% versus 1.7%; P ¼ 0.332) though the difference was not sig- of AFP levels during hepatitis B flare deserves more attention in clin-
nificant statistically. Previous studies also suggest that ‘rapid ical practice in the era of HBsAg quantification during natural course
HBsAg decline’ may predict long-term outcomes of Nuc therapy and antiviral therapy.
such as HBsAg seroclearance.7,14,16,20,23 Given a very low rate of
HBsAg seroclearance (,1%) in Asian patients (with genotype B
or C HBV infection) in previous studies with entecavir therapy over Acknowledgements
3 – 5 years,24,25 longer follow-up is needed to see if these initial
We thank Ms Li-Hua Lu for laboratory work, Ms Shin-Huei Yang for
dynamics translate into clinically meaningful events. Fourth, the database construction, Ms Su-Chiung Chu for manuscript preparation
study patients were exclusively infected with genotype B or C HBV. and Dr Hwai-I Yang for statistical consultation.
Perhaps similar findings are also applicable in patients with predom-
inant HBV genotypes other than B and C, especially genotypes A and
D. However, further studies are required to confirm this.
In conclusion, HBsAg kinetics during Nuc therapy is a complex Funding
issue because it involves multiple factors. The small HBsAg This study was supported by grants from the Chang Gung Medical
reduction of 0.09 log10 IU/mL at month 12 of entecavir therapy Research Fund (SMRPG1005, OMRPG380061 and CMRPG3A0901-3) and
the Prosperous Foundation, Taipei, Taiwan.
in patients with baseline ALT ,5× ULN in the current study
(Table 1) implies that Nuc per se has limited impact on HBsAg
decline during the early phase of therapy. Notably, there was a
greater HBsAg reduction in an ALT and AFP level-dependent Transparency declarations
manner during the early phase of entecavir therapy in patients Y.-F. L. has been involved with clinical trials and served as a global advisory
with pretherapy ALT ≥5× ULN. Further, AFP ≥100 ng/mL is a board member of Roche. All other authors: none to declare.
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Jeng et al.
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