This is an author version of the contribution published on:
Questa è la versione dell’autore dell’opera:
[Hepatology Research, DOI: 10.1111/hepr.12811]
ovvero [Gian Paolo Caviglia, Maria Lorena Abate, Daniele Noviello, Antonella Olivero,
Chiara Rosso, Giulia Troshina, Alessia Ciancio, Mario Rizzetto, Giorgio Maria Saracco
and Antonina Smedile, John Wiley & Sons, 2016, pagg.1-8]
The definitive version is available at:
La versione definitiva è disponibile alla URL:
Hepatitis B core-related antigen kinetics in chronic HBV-genotype-D infected
patients treated with nucleos(t)ide analogues or pegylated-interferon-α
Caviglia GP1,*, Abate ML1,*, Noviello D1, Olivero A1, Rosso C1, Troshina G2, Ciancio A1,2, Rizzetto
M1,2, Saracco GM2,3, Smedile A1,2
1Department of Medical Sciences, University of Turin, Turin, Italy
2Department of Gastroenterology, Città della Salute e della Scienza Hospital, Turin, Italy 3Department of Oncology, University of Turin, Turin, Italy
Correspondence: Gian Paolo Caviglia, Department of Medical Sciences, University of Turin, Via San
Massimo 24, Turin 10100, Italy. Tel: +39 (0)11 6333922; Fax: +39 (0)11 6333976; e-mail: caviglia.gi-ampi@libero.it
*These authors contributed equally to this work
Short title: HBcrAg kinetics in CHB therapy
Conflict of interest: None to declare
Abstract
Aim. To evaluate Hepatitis B core-related antigen (HBcrAg) correlation with HBV DNA and hepatitis
B surface antigen (HBsAg) levels and to investigate HBcrAg kinetic during nucleos(t)ide analogues (NAs) or pegylated-interferon (PEG-IFN)-α treatment in a cohort of chronic hepatitis B (CHB)-geno-type-D patients.
Methods. One-hundred-thirty-eight sequential serum samples were collected from 28 Hepatitis B e
antigen (HBeAg)-negative CHB-genotype-D patients (20M/8F; median age 54 [47-58] years), who underwent NAs (n=20) or PEG-IFN-α (n=8) treatment. Serum HBcrAg levels were determined by chemiluminescent enzyme immunoassay. Longitudinal analysis was performed at 6, 12, 24 and 36 months after NAs treatment initiation and at 6, 12, 18 months and at follow-up month-6 after PEG-IFN-α administration.
Results. Basal HBcrAg levels were 4.7±1.8 LogU/mL and 3.3±1.6 LogU/mL in NAs and PEG-IFN-α
treated patients, respectively. HBcrAg showed a moderate correlation with HBV DNA (r=0.498, p<0.0001) and no correlation with HBsAg (r=0.192, p=0.0669). In serial serum samples, a significant HBcrAg reduction was observed only in patients receiving NAs (p=0.019). In these patients, we observed a group (n=12) with an early HBcrAg decline to undetectable levels between months 6-12, while the other group (n=8) had still detectable HBcrAg at month-36 (4.4±0.6 LogU/mL),
independently from HBV DNA and HBsAg kinetics.
Conclusions. Serum HBcrAg correlates with HBV DNA levels, most likely expression of viral
replication activity. We observed two different HBcrAg kinetics in NAs treated patients that may reflect distinct intrahepatic virological status. Further studies on larger patients cohorts will elucidate if HBcrAg may have a role for safely discontinuing NAs in CHB-genotype-D patients.
Key words: hepatitis B core-related antigen, hepatitis B surface antigen, hepatitis B virus DNA,
INTRODUCTION
Hepatitis B virus (HBV) is the second greatest cause of chronic viral hepatitis worldwide. Despite decades of vaccination, the estimated number of chronic HBV surface antigen (HBsAg) carriers in the world is approximately 350–400 million.1 In Western Countries, chronic hepatitis B (CHB) is mainly
the result of an acute, unresolved infection, that overtime may lead to cirrhosis and its complications such as liver failure and hepatocellular carcinoma (HCC).2 Currently, there are several drugs approved
for CHB treatment including recombinant interferon (IFN), an immune-modulatory agent, and nucleos(t)ide analogues (NAs),oral drugs that directly inhibit viral replication.3
The major goal in CHB treatment is to prevent disease progression suppressing viral replication and maintaining a virological remission. Then, inflammation reduction prevents fibrosis progression, decreasing the risk of cirrhosis and theoretically of HCC. The final endpoint is HBsAg loss and eventually seroconversion to anti-HBs.4 Several reliable serological and molecular parameters are
available to clinicians for an optimal management of CHB infected patients. According to international guidelines, indication for treatment initiation depends on well-known parameters such as HBV DNA levels, alanine aminotransferase (ALT) and severity of liver disease.1,5,6 In addition, HBV DNA levels
and quantitative HBsAg gained increasing attention as potential predictors of hepatitis reactivation, disease progression and HCC development.7-9
Beside conventional serological markers for hepatitis B, a novel 22 KDa precore/core protein (p22cr), named hepatitis B core-related antigen (HBcrAg) has been recently described as the main capsid protein in viral DNA-negative Dane particles. In serum, p22cr containing-particles are more abundant than those containing HBcAg. In addition, because the precore non-sense mutation abolishes precore protein expression, the precore mutation must influence the HBcAg/HBcrAg ratios and the ratios of particle-forming p22cr to soluble HBeAg ratios in serum.10 Furthermore, due to its correlation
proposed as a useful tool for monitoring intrahepatic HBV status.11,12 Low HBcrAg levels were
associated with favorable entecavir (ETV) treatment outcome, response to NAs/IFN-α sequential therapy and with lower risk of hepatitis reactivation following discontinuation of lamivudine (LAM). 13-15 Conversely, elevated serum HBcrAg levels have been associated with higher risk of either HCC
development or HCC recurrence after curative therapy.16,17 In addition, it has been shown that the
different phases of CHB infection can be distinguished according to HBcrAg serum levels.18,19
Since current available data on HBcrAg are mainly limited to Asian CHB patients infected with HBV genotypes B and C, the aim of the present study is to evaluate HBcrAg kinetics during NAs and pegylated (PEG)-IFN-α treatment and its correlation with HBV DNA and HBsAg levels in an Italian cohort of HBeAg-negative patients chronically infected with HBV-genotype-D.
METHODS Patients
A total of 138 sequential serum samples were collected from 28 HBeAg-negative patients (20 men and 8 women; median age 56 [39 - 68] years), chronically infected with HBV-genotype-D who underwent NAs (n = 20) or PEG-IFN-α (n = 8) treatment between 2001 and 2015 at the Department of Gastroen-terology, Città della Salute e della Scienza - Molinette Hospital, Turin, Italy. In detail, of the 20 pa-tients treated with NAs, 2 papa-tients received LAM 100 mg/day, 5 received LAM 100 mg/day + adefovir dipivoxil (ADV) 10mg/day, 11 received ETV 0.5 mg/day and 2 received tenofovir (TDF) 300 mg/day. Patients treated with PEG-IFN-α received a dose of 180 µg/week for a 18 months course of treatment. Virological and biochemical response were defined as sustained decrease of HBV DNA to < 20 IU/mL and sustained ALT normalization (< 40 IU/L), respectively. A pre-treatment serum sample was avail-able for all included patients. Patients included in the study were either treatment naïve (n = 16) or ex-perienced (n = 12).
All patients gave their written informed consent prior to recruitment. The study protocol con-formed to the principles of the Declaration of Helsinki and it was approved by the Institutional Ethics Committee.
Laboratory testing
The two-step fully automated chemiluminescent microparticle immunoassay ARCHITECT-QT (Abbott Diagnostics, Abbott Park, IL, USA) was used for quantitative determination of human serum HBsAg concentrations using acridium-labeled anti-HBs conjugate as previously described.20 The assay has a
dynamic range of 0.05 - 250.00 IU/mL with onboard dilution for results out of the range. HBeAg qualitative determination was performed with ARCHITECT HBeAg assay. Results are expressed as Sample/Cut Off relative light units (S/CO). Specimens with S/CO values < 1.000 are considered nonreactive. HBV DNA was detected and quantified in patients serum samples by a fully automated real-time polymerase chain reaction system, the COBAS/AmpliPrepCOBAS TaqMan HBV test, version 2.0 (Roche Molecular Diagnostics, Branchburg, NJ, USA) whose detection limit is 20 IU/mL.21
HBV genotypes were determined based on reverse hybridization line probe assay (INNO-LiPA ® HBV
Genotyping, Fujirebio Europe, Gent, Belgium) designed to identify HBV genotypes A to H by detection of type specific sequences in the HBV polymerase gene domain B and C.
Serum HBcrAg levels were determined using a chemiluminescent enzyme immunoassay kit Lu-mipulse G HBcrAg (Fujirebio Europe, Gent, Belgium) on a fully automated system, LuLu-mipulse ® G600 II analyzer (Fujirebio Europe) as previously described.11 The assay is measuring HBeAg, HBcAg
and HBcrAg and the concentration is calculated by comparison with A standard curve generated using recombinant pro-HBeAg. The immunoreactivity of pro-HBeAg at 10 fg/mL is defined as 1 U/mL. HBcrAg values are expressed as Log U/mL with an analytic measurement range between 2.0 - 7.0 Log U/mL.
Statistical analysis
Continuous variables are expressed as mean ± standard deviation (SD) or median (range). The t-Stu-dent or Kruskal-Wallis test were used to analyze continuous variables according to whether distribution was normal or not. Test for normal distribution was performed by D'Agostino-Pearson normality test. Fisher’s exact test was used to compare categorical variables. Pearson’s correlation coefficient (r) was used to measure the degree of association between two continuous variables. Comparison of correlation coefficients was performed by Fisher’s z test.
Repeated measures ANOVA was performedto analyze kinetics of HBV markers. Longitudinal analysis was carried out on patients sera at baseline, months 6, 12, 24 and 36 of NAs therapy, and at baseline, months 6, 12, 18 (end of treatment) and months 6 of post-therapy follow-up (FU) of PEG-IFN-α treatment. For all analyses, a p < 0.05 was considered statistically significant. All statistical analyses were performed using MedCalc software version 12.7.0.0 (MedCalc, Ostend, Belgium).
RESULTS
Patients’ characteristics
Demographic, virological and clinical baseline characteristics of the study cohort are reported in Table 1. We found no significant differences between patients that underwent NAs of PEG-IFN-α treatment regarding age, gender and pre-treatment levels of HBV DNA, HBsAg, HBcrAg and ALT. The only significant difference was found in naïve/experienced ratio to previous treatment (p = 0.0084). Regard-ing treatment response, all patients that underwent NAs had a virological and biochemical sustained re-sponse, whereas among patients that underwent PEG-IFN-α, 4 patients did not respond to therapy and 4 patients relapsed within 6 months post-treatment (HBV DNA > 2000 IU/mL and ALT > 40 IU/L). Baseline HBcrAg levels were significantly higher in non-responder patients compared to relapse
pa-tients (4.4 ± 2.4 Log U/mL vs. 2.4 ± 0.8 Log U/mL, respectively; p = 0.0458). No differences were found in HBV DNA (5.4 ± 1.6 LogIU/mL vs. 3.8 ± 1.8 Log IU/mL; p = 0.2375) and HBsAg (3.6 ± 0.6 Log IU/mL vs. 2.7 ± 1.0 Log IU/mL; p = 0.2099) basal levels according to treatment outcome.
Correlation analysis
The correlation between HBcrAg, HBV DNA and/or HBsAg in the 138 serum samples of the 28 en-rolled patients is depicted in Figure 1. Overall, HBcrAg showed a moderate correlation with HBV DNA levels (r = 0.4981, 95% confidence interval [CI] 0.3599 - 0.6149; p < 0.0001) (Fig. 1A) but no correlation with HBsAg (r = 0.1919, 95%CI -0.01344 - 0.3817; p = 0.0669) (Fig. 1B). A weaker corre-lation was observed between HBV DNA and HBsAg (r = 0.2678, 95%CI 0.06427 - 0.4499; p = 0.0107) (Fig. 1C). Next, HBcrAg concentration was correlated to HBV DNA levels according to ther-apy. In patients undergoing PEG-IFN-α treatment it was found a better correlation compared to those who received NAs treatment (r = 0.7865, 95%CI 0.6292 - 0.8820; p < 0.0001 vs. r = 0.4960, 95%CI 0.3281 - 0.6335; p < 0.0001, respectively; z-statistics 2.6661, p = 0.0077). Regarding the correlation between HBcrAg and HBsAg, no statistically significant correlation was found in patients that under-went either PEG-IFN-α or NAs treatment (r = 0.2830, 95%CI -0.1463 - 0.6226; p = 0.1908 vs. r = 0.1555, 95%CI -0.0843 - 0.3782; p = 0.2021, respectively). In contrast, HBV DNA and HBsAg were correlated in both groups of patients without a statistically significant difference between patients treated with PEG-IFN-α and those treated with NAs (r = 0.4190, 95%CI 0.0082 - 0.7088; p = 0.0466 vs. r = 0.0327, 95%CI 0.0225 – 0.4719; p = 0.0327, respectively; z-statistics 0.6987, p = 0.4848).
Longitudinal analysis
Kinetics of HBcrAg, HBV DNA and HBsAg mean levels in patients treated with NAs and those treated with PEG-IFN-α are depicted in Figure 2. In serial serum samples, a significant HBcrAg reduction was
observed only in patients receiving NAs (p = 0.019). No significant variation was observed in PEG-IFN-treated patients (p = 0.172). A statistically significant longitudinal variation resulted for HBV DNA levels either in patients treated with NAs and in those treated with PEG-IFN-α (p < 0.001 and p = 0.001, respectively). Almost no changes occurred in HBsAg kinetics either in NAs treated patients or in PEG-IFN-α group (p = 0.077 and p = 0.190, respectively). Of interest, among patients treated with NAs we observed a subgroup (n = 12) that experienced an early HBcrAg decline reaching undetectable val-ues between months 6 and 12 (< 2 Log U/mL) whereas another group (n = 8) maintained sustained HBcrAg values at last FU (4.4 ± 0.6 Log U/mL at month 36) irrespectively from HBV DNA and HB-sAg kinetics (Fig. 3). Moreover, the rapid decliner group showed HBcrAg kinetic similar to HBV DNA (p = 0.243) and significantly different from HBsAg (p = 0.021). Contrarily, patients with HBcrAg-posi-tive values at last FU showed HBcrAg kinetic significantly different from HBV DNA (p = 0.006) and similar to HBsAg (p = 0.216). Furthermore, basal HBcrAg levels in patients with early decline were significantly lower compared to patients with still detectable HBcrAg at month 36 (3.8 ± 1.7 Log U/mL vs. 5.9 ± 1.3 Log U/mL, p = 0.0109).
DISCUSSION
Primary markers for treatment monitoring in CHB patients include HBeAg for seroconversion of HBeAg-positive patients, ALT for biochemical response and HBV DNA for virological response.22
HBsAg quantitation became another important marker to predict HBsAg-negativization and
seroconversion to anti-HBs. Several studies investigated the role of such markers in therapy outcome prediction.23 In particular, it has been shown that in IFN-based therapy HBV DNA is not a reliable
predictor of sustained response because of similar kinetics between non-responder patients and those who relapse, whereas in NAs treated patients, HBV DNA becomes rapidly undetectable without ensuring that viral replication will not reactivate in case of therapy cessation.15,24 In the last years, basal
serum HBsAg quantification and on-treatment kinetics have been proposed as surrogate markers for treatment outcome prediction in patients treated with PEG-IFN.25-27 Interestingly, guidelines for
avoiding risks resulting from discontinuation of NAs have been recently proposed and incorporated in the official Japan Society of Hepatology guidelines for the management of HBV infection.28,29 Such
recommendations are based on a score that includes HBsAg and HBcrAg levels for NAs therapy cessation in CHB patients with at least two years of NAs administration, with undetectable HBV DNA and negative serum HBeAg.29 Accordingly, patients with HBsAg load <1.9 Log IU/mL and HBcrAg
<3.0 Log U/mL have a low risk of relapse (predicted success rate 80-90%) whereas patients with HBsAg >2.9 Log IU/mL and HBcrAg >4.0 Log U/mL must continue treatment (predicted success rate 10-20%).29
In the present study investigating e-minus patients, we found that HBcrAg levels were moderately correlated with HBV DNA in both NAs and PEG-IFN-α treated patients. However, no correlation was found between HBcrAg and HBsAg which is in contrast to a recent study by
Maasoumy and colleagues performed on 249 blood samples of European untreated HBsAg-positive patients chronically infected with HBV-genotype A/D.19 The smaller study cohort and the impact of
therapy could explain the different results. In our study, we found that treatment affected more HBcrAg than HBsAg kinetics, probably explaining the weak correlation between these two markers.
Among patients treated with PEG-IFN-α, we found higher baseline HBcrAg levels in non-responder patients compared to relapsers (p = 0.0458) in spite of no difference in HBV DNA and HBsAg pre-treatment levels. Because of the low number of patients treated with PEG-IFN-α and the absence of patients who achieved a sustained response, it was not possible to perform any statistical analysis to investigate the role of basal HBcrAg levels in such patients. To note, recent reports suggest that a rapid on-treatment HBcrAg level decline predicts response to IFN-based therapy in both HBeAg-positive and -negative patients.30,31
To our knowledge, no studies evaluated HBcrAg kinetics in comparison to HBV DNA and HBsAg in a cohort of HBeAg-negative CHB-genotype-D patients treated with NAs or PEG-IFN-α. Interestingly, in NAs treated patients we identified a subgroup with lower basal HBcrAg levels (3.8 ± 1.7 Log U/mL) who experienced a rapid decline to < 2 Log U/mL within the first year of treatment, and a subgroup with higher basal HBcrAg levels (5.9 ± 1.3 Log U/mL) with still detectable HBcrAg after 3 years of treatment. The two subgroups were similar for HBV DNA and HBsAg basal levels and
kinetics. Recently, Jung and colleagues showed that HBcrAg levels could be a useful marker to identify HBeAg-negative patients who are at risk of hepatitis reactivation after LAM or ETV therapy
cessation.32 Indeed, authors found that age > 40 years (odds ratio [OR] = 6.690; 95%CI 1.314 - 34.057;
p = 0.022) and end-of-treatment HBcrAg level > 3.7 Log U/mL (OR = 3.751; 95%CI 1.187 - 11.856; p = 0.024) were significantly and independently associated with virological relapse.32 Similarly,
Matsumoto and colleagues previously reported that HBcrAg levels were significantly higher in patients who experienced hepatitis reactivation after LAM discontinuation (4.9 [4.7 - 4.9] Log U/mL vs. 3.2 [< 3.0 - 4.5] Log U/mL, p = 0.009).32 Beside, it has been reported that HBcrAg is able to reflect viral
replication and much more importantly intrahepatic HBV cccDNA levels.11 This feature is particularly
relevant in patients undergoing NAs therapy since HBV proteins production depends on the
transcription of mRNA from cccDNA that is unaffected by NAs treatment. In addition, HBeAg and core protein production requires the entire HBV cccDNA whereas HBsAg can be synthetized either from cccDNA or HBV DNA fragments integrated into the host genome.33 Therefore, we can cautiously
speculate that HBcrAg measurement may represent a reliable surrogate marker for intrahepatic cccDNA evaluation.
The major limitations of our research are represented by the retrospective nature of the study and the low number of included patients. Scientific reports for longitudinal data comes mainly from studies involving Asian patients infected with HBV-genotypes B/C. Here, we provide data on HBV
markers correlation and kinetics on a cohort of HBeAg-negative CHB-genotype-D patients treated either with NAs or PEG-IFN-α.
In conclusion, we found a positive correlation between serum HBcrAg and HBV DNA levels, particularly in PEG-IFN-α treated patients, most likely expression of viral replication activity. In NAs treated patients, we observed two distinct patterns of HBcrAg kinetic. Since consensus criteria for stopping NAs treatment are still lacking particularly in CHB-genotype-D patients, further studies are needed to elucidate if these two patterns are consistent and reflect a different intrahepatic virological status and, accordingly, if HBcrAg may represent a marker for safe discontinuation of NAs therapy in such patients.
ACKNOWLEDGMENTS
This study was supported by local research grant of the Department of Medical Sciences, Università degli Studi di Torino, Turin, Italy.
Authors thank Fujirebio Italia S.r.l. for supply of HBcrAg test reagents. Fujirebio had no role in study design, data collection, analysis and interpretation, in manuscript preparation and decision to publish.
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Figure 1 Correlation between hepatitis B core-related antigen (HBcrAg) and hepatitis B virus (HBV) DNA (A), HBcrAg and hepatitis B surface antigen (HBsAg) (B) and HBV DNA and HBsAg (C) in serum samples of the 28 enrolled patients.
Figure 2 Kinetics of hepatitis B core-related antigen (HBcrAg), hepatitis B virus (HBV) DNA and hepatitis B surface antigen (HBsAg) in patients treated with NAs (A) and in patients treated with
Figure 3 Subgroup of 12 patients that experienced an early hepatitis B core-related antigen (HBcrAg) decline (A) and subgroup of 8 patients with still detectable HBcrAg at last follow up. The bottom grey area represents HBcrAg assay lower limit of detection (LLOD).
Table 1 Baseline demographic, clinical and virological characteristics of the 28 HBeAg-negative CHB-genotype D patients.
Characteristics Total NAs PEG-IFN-α p†
No. of patients 28 20 8
Age (years), mean ± SD 56 (39 - 68) 57 (39 - 68) 54 (47 - 58) 0.1608
Gender (M/F) 20/8 15/5 5/3 0.6508
Naïve/experienced 16/12 8/12 8/0 0.0084
HBcrAg (Log U/mL), mean ± SD 4.3 ± 1.8 4.7 ± 1.8 3.3 ± 1.6 0.0775
HBV DNA (LogIU/mL), mean ± SD 5.5 ± 1.9 5.7 ± 1.9 4.7 ± 1.8 0.2584
HBsAg (Log IU/mL), mean ± SD 3.8 ± 0.5 3.7 ± 0.5 3.9 ± 0.2 0.4792
ALT (IU/mL), median (range) 73 (18 - 490) 73 (18 - 490) 63 (21 - 200) 0.5428
†Analysis of normally distributed continuous variables performed by t-Student test; analysis of non-normally distributed continuous variables performed by Kruskal-Wallis test; analysis of dichotomous variables performed by Fisher’s exact test.
ALT, alanine aminotransferase; HBcrAg, hepatitis B core-related antigen; HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; NAs, nucleos(t)ide analogues; PEG-IFN-α, pegylated-interferon-α; SD, standard deviation.
