Elevated Expression Of Serum Soluble ST2 In Clinical Relapse After Stopping Long-term Nucleos(t)ide Analogue Therapy For Chronic Hepatitis B

BackgroundHepatitis B virus(HBV)infection causes acute and chronic hepatitis and is a threat to public health across the world.Globally,an estimated 240 million people suffer HBV infection with a varying prevalence geographically.Every year,HBV-associated end-stage liver disease results in approximately 1 million deaths.Current clinical guidelines from the European Association for the Study of the Liver(EASL)and American Association for the Study of the Liver Disease(AASLD)recommend nucleos(t)ide analogue(NA)therapy in chronic hepatitis B(CHB)patients.Effective suppression of HBV DNA using NA has been shown to delay disease progression in patients with CHB.However,discontinuation of NA therapy is often associated with HBV rebound and recurrence of active hepatitis after anti-HBe seroconversion.It has been suggested that patients who achieve hepatitis B surface antigen(HBsAg)seroclearance can discontinue NA treatment,but HBsAg seroclearance is rare,even after long-term NA treatment.Serum soluble ST2(sST2)is a member of the Toll-like/interleukin-1 receptor superfamily.It has two isoforms:transmembrane ST2 expressed on the cell surface and soluble ST2 in the serum.sST2 is reported to act as a binding decoy for IL-33 and therefore modulates IL-33 activity during inflammatory responses.sST2 expression is elevated in several inflammatory diseases.These include atopic individuals with allergic symptoms or exacerbation of asthma,atopic dermatitis,rheumatoid arthritis,ulcerative colitis,Crohn's disease,and systemic lupus erythematosus.Shu et al.found a positive correlation between sST2 and alanine aminotransferase(ALT)in chronic hepatitis patients.Shao et al.found that sST2 is a promising prognostic biomarker in HBV-related acute-on-chronic liver failure.Currently,many studies have demonstrated that patients have very high rates of relapse after NA treatment cessation.Related studies have shown that the factors used to predict relapse after treatment discontinuation are still limited to clinical indicators such as patient age,serum HBsAg,consolidation treatment time,residual HBV DNA levels,hepatitis B virus core-related antigen(HBcrAg)levels,and intrahepatic HBV covalently closed circular DNA(cccDNA)levels at the end of therapy.There is still a lack of reliable laboratory tests to predict early relapse after stopping long-term NA therapy.AimsPrevious studies from our group demonstrated that after NA treatment discontinuation,a lower relapse rate was observed in younger patients and in those with low end-of treatment HBsAg levels.The level and persistence of off-treatment elevated HBV DNA levels were useful in the prediction of a subsequent biochemical relapse and may be used to guide off-treatment management.Serum levels of Anti-HBc might be used to select patients suitable for NA treatment discontinuation However,the role of sST2 in patients after discontinuation of long-term NA therapy has yet to be explored.This prospective study investigated the expression and clinical significance of sST2 in predicting relapse after cessation of long-term NA therapy in CHB.Materials and methodsStudy subjectsThis study was a prospective,single-center,observational study.The enrolled patients were Asian patients with CHB(positive for HBsA>6 months).Patients provided informed consent and voluntarily entered the study cohort.Patients were recruited from Nanfang Hospital(Guangzhou,China)from November 2012 until May 2018.This analysis includes follow-up data collected until May 2018,when all patients had completed at least 6 months of follow-up.Patients underwent cessation of NA therapy in accordance with the 2012 The Asian Pacific Association for the Study of the Liver(APASL)guidelines.Patients who were HBeAg-positive at the start of treatment underwent NA treatment until they met the criterion of discontinuation NA therapy:HBeAg seroconversion,undetectable HBV DNA,and normalized ALT level,and consolidated treatment for at least 12 months.Patients who were HBeAg-negetive at NA initiation were eligible if they fulfilled the criterion of discontinuation NA therapy:HBeAg seroconversion,undetectable HBV DNA,and normalized ALT level,and consolidated treatment for at least 18 months.Additional eligibility criteria were age?18 years,HBsAg positivity,undetectable HBV DNA at the moment of NA discontinuation.Exclusion criteria are as follows:1.Coinfection with hepatitis C virus,hepatitis D virus,or HIV virus.2.Clinical manifestations of decompensated liver disease before the previous treatment,including but not limited to:total bilirubin(TBIL)?normal upper limit(ULN),prothrombin time>3 seconds greater than ULN,serum albumin<32 g/L,with liver dysfunction,compensatory medical history(such as ascites,variceal bleeding,or hepatic encephalopathy);3.Biopsy-confirmed cirrhosis or a liver stiffness of>9 kPa measured using Fibroscan(Echosens,Paris,France);4.ultrasound or radiological examination of suspicious hepatocellular carcinoma(HCC)or alpha-fetoprotein(AFP)>20 ng/mL;5.liver transplantation or plan to have a liver transplant;6.other liver diseases.Follow-upPatients enrolled in the study were followed up once a month within first 3 months.Thereafter,the patients were followed every 3 months.After two years,the patients were checked every 6 months.Biochemical(ALT levels)and virological(quantitative measurement of HBsAg,HBV DNA)tests were performed.End points,retreatment,and definitionsThe main clinical endpoint of this clinical trial was clinical relapse,defined as:HBV DNA>2000 IU/mL,combined with ALT>2ULN.The patients who experienced clinical relapse were withdrawn from the follow-up study and retreated using NA.The rest of the patients were defined as the non-clinical relapse group.The non-clinical relapse group include the virological relapse group and the sustained response group.The virological relapse defined as:HBV DNA>2000 IU/mL.Laboratory testsAn Olympus AU5400 automatic biochemical analyzer was used for biochemical detection.The ULN of the ALT level was 40 U/L for males and 35 U/L for females.Quantitative analysis of HBV DNA was performed using the Cobas HBV-specific TaqMan polymerase chain reaction assay with a lower limit of detection(LLOD)of 20 IU/mL(Roche Diagnostics,Basel,Switzerland).Serum HBsAg detection(LLOD,0.05 IU/mL)qualitative HBeAg were performed using an Architect Assay(Abbott Laboratories,Chicago,IL).Quantitative total serum Anti-HBc(IgG and IgM)was detected with a double-sandwich immunoassay(Wantai,Beijing,China,LLOD,0.1IU/mL).Enzyme-linked immunosorbent assay(ELISA)Serum was stored at-80? after centrifugation until use.The concentration of sST2 was quantitated using a commercial human sST2 ELISA kit(R&D Systems,Human ST2/IL-33 R DuoSet ELISA Kit,USA,DY523B-05)in accordance with the manufacturer's instructions.Statistical analysisContinuous data are expressed as either the median(minimum-maximum)or the mean ± SEM.An independent samples t test,the Mann-Whitney U test or the Chi square test were used in group comparisons.To examine the dynamic change of sST2 after cessation of long-term NA therapy,the paired T test and the repeated measures ANOVA with Bonfreroni test were used to compare the baseline sST2 levels with other different timepoints,and the Mann-Whitney U test was used to compare the sST2 between the CR group and the NCR group at different timepoints.Finally,the Cox proportional hazards regression models to identify factors associated with clinical relapse after stopping long-term NA therapy.All statistical analyses were based on two-tailed hypothesis tests with a significance level of P<0.05.All statistical analyses were conducted using SPSS 25.0 or GraphPad Prism 8.0 software.ResultsDemographic data and clinical characteristicIn total,91 non-cirrhotic Asian patients with CHB who stopped NA therapy according to international guidelines were prospectively followed.All patients met the withdrawal criteria and signed a withdrawal agreement.The withdrawal time was at least 6 months.The demographic data and clinical characteristics are presented in Table 1.Among the 91 patients,75 were male and 16 were female.A total of 39 patients underwent first-line treatment.The average age of the patients was 35.7±7.9 years.The median duration of antiviral treatment was 56.7±26.2 months.The consolidated treatment time was 28 months(maximum-minimum,18-46 months)The level of ALT was 0.5×ULN and HBV DNA was undetectable at baseline.At the start of treatment,61 patients were HBeAg positive,and 30 patients were HBeAg negative.Clinical relapse and virological relapse after stopping long-term NA therapyAll 91 patients who met the international withdrawal criteria were followed up to 240 weeks.The ratio of clinical relapse,virological relapse,and sustained response at each time point is shown in Fig.1A.The cumulative virological relapse rate was 80.16%(Fig.1B),and the cumulative clinical relapse rate was 48.86%(Fig.1C),which was consistent with previous findings.Moreover,it could be found that the majority of virological relapse(61/91,67.03%)and clinical relapse(26/91,28.57%)occurred within 48 weeks.Dynamic change of serum sST2 concentrations after cessation of long-term NA therapy.Then,we examined the concentration of serum sST2 by ELISA in patients after discontinuation of NA therapy for 48 weeks.The CHB patients were divided into CR and NCR group depending on their ALT and HBV DNA levels before week 48.Patients in the CR group were older than those in the NCR group(p=0.039)and had relatively higher end-of-treatment HBsAg levels(p=0.071).Pre-treatment HBeAg status,ALT,and HBV DNA levels no significant differences between CR and NCR group.After stop of treatment,the level of the HBV DNA was increased for 24 weeks and slightly decreased during 24-48 weeks in CR group.In the NCR group,HBV DNA levels have the same trend,but is lower than the CR group at each timepoint.ALT levels were significant higher at week 24 and week 48 in CR group than NCR group.These results indicated that most patients develop clinical relapse after discontinuation of cessation of NA treatment at earlier time.We further compared serum sST2 levels in the CR group and NCR group at week 0,week 4,week 12,week 24,and week 48 after discontinuation of NA therapy.There was no significant difference between the two groups at baseline and week 4,and the CR group expressed higher sST2 levels then NCR group at week 12,week 24 and week 48.By using repeated measures ANOVA analysis,we only observed the significant difference of serum sST2 levels in the CR group between week 48 and week 12(p=0.004),week 48 and week 0(p=0.007),respectively.Further compared with the baseline at the end of treatment,sST2 expression was increased at clinical relapse timepoint in CR group(p<0.001),while there was no significant difference between baseline and 48W sST2 in the NCR group.Serum sST2 expression is positively correlated with HBsAg,ALT,HBV DNA and Anti-HBc levelsPearson analysis was used to evaluate the association between serum sST2 with HBsAg,ALT,HBV DNA and Anti-HBc levels in patients after discontinuation of NA treatment.As shown,there were positive correlations between serum sST2 levels and HBsAg(r=0.2055,p<0.0001),ALT(r=0.1953,p<0.0001),HBV DNA(r=0.2562,p<0.0001)and Anti-HBc(r=0.2068,p<0.0001).These data further suggested that sST2 expression might be associated with liver inflammation caused by virus replication rebound during clinical relapse.Baseline sST2 expression could not predict clinical relapse after stopping of NA treatmentTo elucidate whether serum sST2 levels at the end of treatment were associated with clinical relapse follow-up to 240 weeks,univariate and multivariate COX regression analyses were conducted.As shown in Table 3,the age>35 y(p=0.006;hazard ratio[HR]:2.647,95%confidence interval[CI]:1.324-5.291),HBsAg>200 IU/ml(vs?200 IU/ml)(p=0.006;HR:3.697,95%CI:1.447-9.444),HBV DNA level elevation>20000 IU/ml(vs?20000 IU/ml)(p=0.017;HR:2.531,95%CI:1.179-5.435)and the levels of sST2 at week 12(p<0.001;HR:4.655,95%CI:2.345-9.243)were found to be independent predictors for clinical relapse in off-treatment CHB patients.The baseline serum sST2 expression was not statistically significant to be an independent predictor of clinical relapse.However,we could observe a HR of 1.718 in the factor of sST2,indicating that the risk of clinical relapse is 1.718 times higher in patients with elevated sST2 than in patients with non-elevated sST2.Conclusions1.We demonstrated that the cumulative rate of virological relapse and clinical relapse was high,and the cumulative rate of virological relapse was higher than the cumulative rate of clinical relapse,consistent with previous reports.In a total of 91 patients,57 patients suffered virological relapse and 26 patients suffered clinical relapse.The cumulative rate of virological relapse was 80.16%and the cumulative rate of clinical relapse was 48.86%.We found that clinical relapse predominantly occurred within 48W.2.We analyzed the differences between the CR group and the NCR group.We found that patients in the CR group were older than those in the NCR group.Basline ALT,HBsAg,Anti-HBc levels have no significant differences between CR and NCR group.Pre-treatment HBeAg status,ALT,and HBV DNA levels have no significant differences between CR and NCR group.3.We analyzed the dynamic changes of serum sST2,HBV DNA,and ALT after stopping long-term NA treatment.There was an increase in HBV DNA in the CR group from 0 to 24 weeks,and a slight downward trend in 24-48 weeks.The NCR group had a similar trend,but the amplitude was smaller than that in the CR group.This indicates that HBV DNA increased after stopping the NA treatment,and the CR group was higher than the NCR.4.In current study,serum sST2 levels were measured after stopping long-term NA therapy,and the correlation between serum sST2 levels and HBsAg,HBV DNA,ALT and Anti-HBc levels was analyzed.It was found that serum sST2 was positively correlated with HBsAg,HBV DNA,ALT and Anti-HBc.5.COX regression analyses were conducted that the age>35 y,HBsAg>200 IU/ml(vs?200 IU/ml),HBV DNA level elevation>20000 IU/ml(vs?20000 IU/ml)and serum sST2 levels of week 12 were found to be independent predictors for clinical relapse in off-treatment CHB patients.Unfortunately,the baseline serum sST2 expression was not statistically significant to be an independent predictor of clinical relapse.6.This suggests that sST2 in the serum may be an inflammatory cytokine that isactivated following an immune response,and which plays a role in immune regulation after the cessation of long-term NA therapy.However,the specific mechanism underlying this phenomenon remains to be explored in the future.