Incidence And Predictors of Relapse After Stopping Antiviral Therapy In Pediatric Chronic Hepatitis B

doi:10.21203/rs.3.rs-1150647/v1

Objective: The objective was to evaluate the incidence of relapse after stopping antiviral therapy and to identify the predictors of relapse.

Methods: All HBsAg positive children with who had been on antivirals for at least 2 years with undetectable HBV-DNA and normal alanine-aminotransferase (ALT) on three consecutive occasions over last 12 months were included. Antivirals were stopped if liver biopsy showed histological activity index <5 and fibrosis (metavir) <3. Children were monitored for virological relapse (elevation of HBV-DNA >2000 IU/mL) and biochemical relapse (ALT levels >2 × upper limit of normal (ULN)). Those having biochemical relapse were started on pegylated interferon alpha-2b based sequential therapy.

Results: Antivirals were stopped in 31 HBsAg positive children. Virological and biochemical relapse was seen in 12 (38.7%) and 5 (16.1%) children within 12 months of stopping antiviral treatment. Majority of virological relapse occurred within a month and biochemical relapses within 6 months of stopping therapy. HBeAg positive status at the time of stopping antiviral therapy (HR: 7.206, p =0.005) and longer time taken for HBV-DNA to become undetectable while on antivirals (HR: 1.030, p=0.037) were found to be the 2 independent predictors of relapse after stopping antiviral treatment.

Conclusion: Discontinuation of antiviral treatment in children with CHB resulted in relapse in one third of the patients. Relapse was more common in those with HBeAg positivity at the time of stopping therapy and in those with longer time taken for HBV-DNA to become undetectable on antivirals.

Gastroenterology & Hepatology

ccc-DNA

biochemical relapse

virological relapse

HBeAg

pediatric hepatitis B

pegylated interferon

stopping rule

repeat liver biopsy

antiviral therapy

The goal of antiviral therapy in chronic hepatitis B (CHB) patients is to improve survival by preventing the progression of liver damage to cirrhosis, end-stage liver disease, hepatocellular carcinoma (HCC) and death [1-2]. There are clear guidelines for stopping antiviral therapy in adults [3-6]. However, there is no study to evaluate the natural course and risk of relapse after stopping therapy in children with chronic hepatitis B. There is no ambiguity in stopping antiviral therapy in children who achieve hepatitis B surface antigen (HBsAg) seroclearance. However, only 1.7 to 8.3% of children treated with antivirals achieve HBsAg seroclearance [7-10]. There is no consensus about the duration of antiviral therapy in the vast majority who continue to remain HBsAg reactive. Initiation of antivirals at a very young age in children coupled with lack of definite endpoint for stopping therapy implies extremely long cumulative duration of exposure to antivirals, thereby increasing the risk of adverse events and drug resistance [7-10]. The incidence and predictors of relapse after stopping antiviral treatment in children with CHB has never been studied previously. Therefore, we planned this pilot research to determine the incidence and identify risk factors of relapse on stopping antiviral therapy in children with CHB.

It was a prospective interventional study conducted at a tertiary care centre over a period of 2 years (January 2019 to December 2020). Approval was obtained from the institutional ethical committee vide letter no IEC/2019/67/MA-06. The study was conducted in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments. Children between the ages of 2 and 18 years with CHB who had received antivirals for at least 24 months with (i) alanine aminotransferase (ALT) levels persistently < 1.5 times the upper limit of normal (ULN) (3 consecutive readings done 6 months apart in last 12 months); (ii) hepatitis B virus – deoxyribonucleic acid (HBV DNA) not detected on 3 consecutive occasions 6 months apart in last 12 months and (iii) whose parents/ guardians gave consent for the study, were included. Normal ALT levels were defined as per the cut-offs obtained from SAFETY study [11]. Children with family history of cirrhosis, HCC or co-infection with hepatitis C virus and human immuno-deficiency virus were excluded. Liver Biopsy was done to evaluate the histological activity index (HAI) and fibrosis as per Ishak staging [12]. Children with HAI < 5 and fibrosis < 3 in liver histology were offered the option of stopping antiviral therapy. Additionally, quantitative estimation of covalently closed circular DNA (ccc-DNA) was also done in the liver tissue. ccc-DNA levels were measured by real-time polymerase chain reaction (PCR) based in-house assay on lightcycler 480 (LC480) (Roche Diagnostics, Netherlands) instrument using following primers: Forward primer: 5’-CTCCCCGTCTGTGCCTTCT-3’; Reverse Primer: 5’-GCCCCAAAGCCACCCAAG-3’. This is a qualitative real-time PCR based assay using sybergreen probes for the target detection [13]. HBV-DNA was measured in the plasma samples by using automated real-time PCR kit, COBAS® AmpliPrep/COBAS® TaqMan®, v2.0 (Roche Diagnostics, GmbH, Mannheim, Germany). This is an automated hepatitis B viral load quantitative assay needing 650 µL of serum or plasma, with the linear range of quantification being 1.3-8.23 log₁₀ IU/ml in all of them. HBsAg quantification was done by chemiluminiscent immunoassay (CLIA) method (Abbott Laboratories, Chicago, IL, USA) [14].

After stopping the antivirals, the patient was followed up at 1 month, 3 months and every 3 months thereafter. Workup at each follow up included: ALT, quantitative HBV-DNA (qHBV-DNA), quantitative HBsAg (qHBsAg), HBeAg, alfa-fetoprotein (AFP) and ultrasonography of the abdomen. Virological relapse was defined as any elevation of qHBV-DNA > 2000 IU/mL. Biochemical relapse was defined as elevation of ALT > 2 times ULN. Primary outcome of the study was the incidence of relapse after stopping antiviral therapy. Secondary outcome was identification of the predictors of relapse after stopping antiviral therapy. The predictors studied included clinical parameters, type of therapy, HBeAg seroconversion status, time taken for HBV-DNA to become undetected on therapy, HBV genotype, ccc-DNA levels, duration of normal ALT before stopping therapy and qHBsAg at the time of stopping antivirals. Those with only VR were continued on regular follow up whereas those with VR as well as BR were treated with combination therapy: 8 weeks of antivirals (entecavir in those younger than 12 years; tenofovir in those older than 12 years), followed by combination of peg-IFN alpha 2b (60 mcg/m²/week) for 48 weeks. The study design is depicted in figure 1.

Statistical analysis: All analyses were performed with SPSS 22.0 (SPSS, Chicago, IL). As this was a pilot study, all the children fulfilling the inclusion criteria during the study period were enrolled. Continuous variables were expressed as median and inter-quartile range (IQR), while categorical variables were expressed as proportions. Both categorical and continuous variables were analyzed using cox regression analysis. Cox regression model was used for univariate and multivariate analyses for predictors of relapse. Receiver operating characteristic curves were used to calculate the area under the receiver operating characteristic curve (AUROC).

A total of 114 CHB children were screened during the study period, out of which 39 (34.2%) children satisfied the criteria for inclusion in the study. Eight of them were HBsAg negative; whose treatment was stopped without a repeat liver biopsy. Parents of all remaining 31 HBsAg reactive children gave consent for inclusion in the study, liver biopsy and consideration of stopping antiviral treatment. All the 31 children had HAI < 5 and fibrosis < 3 in liver biopsy and all their parents agreed to stop antiviral treatment with close follow-up. Table 1 depicts the baseline clinical, biochemical and virological characteristics of these children at the time of enrolment in the study. The cohort predominantly consisted of children with genotype D (75%). All of these children had been HBeAg reactive chronic hepatitis B at the time of initiation of their antiviral therapy. Majority were males (96.8%) with median age of 15 years (IQR: 11.5-17 years). Thirteen (41.9%) were persistent HBeAg reactive (no HBe seroconversion) at the time of enrolment. The median ccc-DNA in the liver histology was 4 log₁₀ copies/ml (IQR: 3.1 log₁₀– 5.4 log₁₀). None had advanced fibrosis either on histology or on transient elastography (TE) (median TE: 5 kPa (4.4 – 5.4)). These children had received oral antivirals for a median duration of 60 months (IQR: 36-78 months). They had a normal ALT on therapy for a median on 30 months.

Relapse after stopping antivirals: All 31 HBsAg positive children enrolled in the study were followed up for a minimum of 12 months. Twelve of these 31 children (38.7%) developed virological relapse. Five children (16.1%) additionally had a biochemical relapse. Most (58.3%) of the virological relapse occurred within a month of antiviral stoppage (figure 2). Biochemical relapse was more common in those developing virological relapse at 3 months or beyond after stopping antivirals. In 4 children, virological as well as biochemical relapse were detected at the same time-point during follow up (Figure 2). Of the 7 children who developed only virological relapse, 6 (85.7%) developed it within 1^stmonth after stopping antiviral treatment. None of the 8 children with HBsAg negative status showed virological or biochemical relapse till 12 months of follow up.

Risk factors for development of relapse: On univariate analysis, HBeAg positive status at the time of stopping therapy (OR: 7.058, 95%CI: 1.825 – 27.29, p = 0.005) and longer time taken for qHBV-DNA to become undetectable on antiviral treatment (OR: 1.024, 95%CI: 1.003-1.046, p = 0.028) were the significant predictors of virological relapse after stopping antiviral therapy (Table 2). All the variables with p value less than 0.1 on univariate analysis [(i) HBeAg positive at the time of stopping therapy; (ii) time taken for qHBV-DNA to become undetectable; and (iii) duration of normal ALT on antivirals] were entered in the Cox regression model. On Cox regression analysis, HBeAg positive status at the time of stopping therapy (HR: 7.206, 95%CI: 1.81-28.74, p =0.005) and time taken for qHBV-DNA to become undetectable (HR: 1.030, 95%CI: 1.002-1.058, p=0.037) were independent predictors of relapse (Table 2). This model predicted occurrence of relapse with an AUROC of 0.855. A longer time taken (more than 22.5 months) for qHBV-DNA to become undetectable predicted virological relapse with a sensitivity of 83.3% and specificity of 47.4% (AUROC: 0.737, 95% CI 0.56-0.913, p=0.029) (Figure 3). Children who were HBeAg positive at the time of stopping therapy were at significantly higher risk of relapse as compared to those who were HBeAg negative (log rank 10.705, p = 0.001) (Figure 4). In the present study, 69.2% HBeAg positive children relapsed within 12 months as against 16.7% of the HBeAg negative children (OR: 7.058, 95%CI: 1.825 – 27.29, p = 0.005). ccc-DNA in liver tissue although higher in those with relapse (4.19 log₁₀ copies/ml (IQR: 3.5-6.99) than in those without relapse 3.2 log₁₀ copies/ml (IQR: 2.5 -5.1), was not statistically different between the 2 groups.

Treatment of children with relapse: Four (80%) of the 5 children with biochemical relapse were treated with sequential therapy. The fifth child only had a transient biochemical relapse; hence, he was treated with antivirals alone. Of the 4 children who were treated with sequential therapy, 3 were HBeAg positive while 1 was HBeAg negative. None of the 3 HBeAg positive children, showed HBeAg seroconversion at 1 year of therapy. Two of them achieved virological clearance (qHBV-DNA undetected). All 4 achieved normalization of their ALT levels at 1 year of therapy. The rest of the patients with only virological relapse are being continued on strict 3 monthly follow up for timely identification of biochemical relapse and possible re-treatment.

Virological relapse was seen in 38.7% and biochemical relapse in 16.1% of HBsAg reactive children whose antivirals were stopped as per the protocol. Most of the relapse occurred within a month of stopping the therapy. None of the HBsAg negative children developed relapse on follow up. HBeAg positive status at the time of stopping therapy (HR: 7.206, p =0.005) and longer time taken for qHBV-DNA to become undetectable while on therapy (HR: 1.030, p=0.037) were independent predictors of relapse. While 69.2% of HBeAg positive children developed virological relapse; only 16.7% of those with HBeAg negative status relapsed.

Most of the guidelines in adult hepatitis B recommend stopping antivirals after 12-36 months of HBe seroconversion with undetectable HBV-DNA and persistently normal ALT levels [4-6]. There are no pediatric studies to evaluate the timing and criteria for stopping antiviral therapy in children. Very low HBsAg seroconversion (1.7 – 8.3%) and HBeAg seroconversion (21 – 31.3%) rates observed in children on antivirals implies longer, often indefinite antiviral therapy [7-10]. Longer duration of therapy increases the cumulative risk of adverse events as well as risk of development of antiviral resistance [15]. Present study provides some insights into patient selection for stopping antivirals in children who fail to achieve HBsAg seroconversion. Virological relapse rate of 38.7% and biochemical relapse rate of 16.1% observed in children in our study is consistent with relapse rates observed in adults after antiviral discontinuation. The virological relapse rates reported have varied between 42% at 3 months to 68% at 6 years of follow up, while biochemical relapse rates have varied between 21.7% at 3 months to 63% at 6 years of follow up after antiviral discontinuation [16-17].

HBeAg positive status at the time of stopping therapy and longer time taken for qHBV-DNA to become undetectable were the predictors of relapse in the present study. The optimal end-point for stopping therapy would be the loss of HBsAg, often termed ‘functional cure’, but it is only rarely achievable in children [5,9-10]. Short of it, HBeAg seroconversion, coupled with HBV-DNA loss and ALT normalization characterizes a state of good immune control with low replicative phase of disease [5]. The high rates of relapse (69.2%) in HBeAg positive children in the present study suggests that treatment withdrawal should not be attempted in absence of HBeAg seroconversion. The important predictors of relapse after stopping antivirals in adults include (i) higher qHBsAg level at the time of stopping the therapy [18-21]; (ii) shorter duration of consolidation therapy [19-20,22-24]; (iii) slower HBV-DNA decline while on therapy [25]; (iv) baseline pre-treatment HBV-DNA [26-27]; (v) hepatitis B core-related antigen (HBcrAg) [28-29]; and (vi) hepatitis B virus- ribonucleic acid (HBV-RNA [29]. The emphasis is maximum on qHBsAg levels at the time of stopping therapy and recently Asia Pacific Association for Study of Liver Diseases (APASL) has suggested qHBsAg less than 100 IU/ml as a safe endpoint for stopping long term antivirals [18]. qHBsAg levels was not significantly higher in the relapse in the present study. HBcrAg and HBV-RNA are among the newer serological markers predicting relapse either singly or in combination [28-29]. We evaluated the role of liver tissue ccc-DNA which although higher in relapse was not statistically different. This could be due to the smaller sample size and would need evaluation in larger multicentric studies. Recently, Lai et al investigated the effect of antiviral withdrawal in patients with undetectable ccc-DNA in the hepatocytes in adults [30]. All 13 patients with undetectable ccc-DNA randomized to stop therapy had virological relapse.

The main strength of our study is the pilot effort in determining the relapse rate and it’s risk factors in children with CHB. The limitation is a smaller sample size due to single centre study and a short follow up of 12 months. However, we continue to closely follow-up these children. To conclude, the virological and biochemical relapse rates were 38.7% and 16.1% respectively with most relapses occurring within a month of stopping therapy. HBeAg positive status and longer duration to achieve virological remission while on therapy were independent predictors of relapse.

Conflict of interest: Piyush Upadhyay, Bikrant Bihari Lal, Vikrant Sood, Rajeev Khanna, Ekta Gupta, Archana Rastogi and Seema Alam have no potential conflict of interest to declare.

Ethical Approval: All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. The study was approved by the Institutional Ethics Committee of Institute of Liver and Biliary Sciences, New Delhi, Indiavide letter no IEC 2019/67/MA 06.

Consent to participate: Informed, written consent to participate was obtained from the parents of the children included in the study.

Funding: The authors did not receive support from any organization for the submitted work.

Author Contributions: All authors contributed to the study conception and design. Data collection was done by PU, BBL and VS. All the virological and serological studies were performed by EG. Histological evaluation and inputs were provided by AR. Analysis of the data and writing of the first draft of the manuscript was done by PU, SA and BBL. All authors commeneted on previous versions of manuscript, provided inputs and approved the final manuscript.

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Table 1: Baseline clinical and laboratory profile of the 31 HBsAg positive at the time of stopping antiviral treatment

Parameter
Age [median (IQR)]	15 years (11.5-17)
Gender Male Female	30 (96.8%) 1 (3.2%)
HBeAg status at the time of enrolment Reactive Non reactive	13 (41.9%) 18 (58.1%)
ALT at the time of enrolment	30 IU/L (23-35)
Liver histology findings Histological activity index 0-1 2-3 >3 Fibrosis 0 1 2 >2 ccc-DNA (Log₁₀in copies/ml) [median(IQR)]	20 (64.5%) 11 (35.5%) 0 14 (45.2%) 13 (41.9%) 4 (12.9%) 0 4 (3.1 – 5.4)
Liver stiffness by transient elastography	5 kPa (4.4 – 5.4)
Genotype (n = 24) Genotype D Genotype A Genotype C	18 (75%) 4 (16.7%) 2 (8.3%)
Duration of antiviral therapy [median(IQR)]	60 months (36-78)
Previous sequential therapy with peg-IFN	13 (41.9%)
Duration of antiviral treatment post sequential therapy [median(IQR)]	36 months (27-54)
Time taken for qHBV-DNA to become undetected during antiviral therapy [median(IQR)]	32 months (16-49)
Duration of normal ALT on antivirals [median(IQR)]	30 months (15.8-31)

*Effect size described as odds ratio, #Effect size described as mean difference

Abbreviations: ALT: Alanine aminotransferase; HBeAg: Hepatitis B e antigen; HBsAg: Hepatitis B surface antigen; IQR: Inter Quartile Range; qHBV-DNA: Quantitative HBV-DNA; Peg-IFN: Pegylated interferon

Table 2: Cox univariate and regression analyses of risk factors for development of virological relapse after stopping antiviral treatment in children with chronic hepatitis B

Univariate Analysis
Variable		Relapse (n=12)			No Relapse (n= 19)			Hazard ratio (95% CI)		p Value
Age at enrolment (years) [median (IQR)]		16 (13.5-17)			13 (10-17)			1.129 (0.96 – 1.326)		0.140
qHBsAg (Log₁₀) [median (IQR)]		3.84 (3.15 – 4.41)			3.71 (2.92 – 4.19)			1.465 (0.806 – 2.66)		0.211
HBeAg positive status n (%)		9 (75%)			4 (21%)			7.058 (1.825 – 27.29)		0.005
Duration of antiviral treatment (months) [median (IQR)]		60 (36-78)			48 (32-72)			0.960 (0.905 – 1.019)		0.136
History of sequential treatment, n (%)		5 (41.6)			8( 42.1)			0.982 (0.227 - 4.251)		1.000
Duration of normal ALT on antivirals (months) [median (IQR)]		16 (13.5-33)			32 (19.5-48)			0.979 (0.932 – 1.029)		0.07
Time taken for qHBV-DNA to become undetectable (months), [median (IQR)]		46 (23-64)			24 (11-39)			1.024 (1.003-1.046)		0.028
Duration since HBeAg seroconversion (months) [median (IQR)]		24 (12-28)			24 (12-36)			1.103 (0.417 – 2.919)		0.843
ccc-DNA in liver (log₁₀) copies/ml, [median (IQR)]		4.19 (3.5-6.99)			3.2 (2.5 -5.1)			1.176 (0.893-1.550)		0.248
Cox Regression Analysis
Variables in the Equation	B		Wald	Significance		Exp(B)	95% CI for Exp (B)
							Lower		Upper
HBeAg positive status	1.975		7.829	0.005		7.206	1.807		28.743
Time taken for qHBV-DNA to become undetected (months)	0.029		4.356	0.037		1.030	1.002		1.058

Abbreviations: ALT:Alanine aminotransferase; CI: Confidence interval; Exp (B): Exponential B; IQR : Inter Quartile Range; HBeAg: Hepatitis B e antigen; qHBsAg: Quantitative HBsAg; qHBV-DNA: Quantitative Hepatitis B virus deoxyribonucleic acid; ccc DNA: Circular covalently closed circular deoxyribonucleic acid;

Incidence And Predictors of Relapse After Stopping Antiviral Therapy In Pediatric Chronic Hepatitis B

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