J Viral Hepat. 2019;26:697–709. wileyonlinelibrary.com/journal/jvh  | 697© 2019 John Wiley & Sons Ltd

Received:23October2018  |  Accepted:9January2019DOI: 10.1111/jvh.13068

O R I G I N A L A R T I C L E

Immunological biomarkers as indicators for outcome after discontinuation of nucleos(t)ide analogue therapy in patients with HBeAg- negative chronic hepatitis B

Hariklia Kranidioti1,2 | Spilios Manolakopoulos2,3 | George Kontos2 | Michael S. Breen1 |  Anastasia Kourikou2 | Melanie Deutsch2 | Maria Ester Quesada-Del-Bosque1 |  Rocio T. Martinez-Nunez1 | Mohammed M. Naiyer1 | Christopher H. Woelk1 |  Tilman Sanchez-Elsner1 | Emilia Hadziyannis3 | George Papatheodoridis3  |  Salim I. Khakoo1

Abbreviations:AUC,areasunderthecurve;CHB,chronichepatitisB;EOT,endoftreatment;FDR,falsediscoveryrate;GAPDH,glyceraldehydephosphatedehydrogenase;HBV,hepatitisBvirus;HCC,hepatocellularcarcinoma;HCV,hepatitisCvirus;HDV,hepatitisDvirus;KEGG,KyotoEncyclopaediaofGenes;NA,nucleos(t)ideanalogues;PBMC,peripheralbloodmono-nuclearcell;PCA,Principalcomponentanalysis;ROC,Receiveroperatingcharacteristic;ULN,upperlimitofnormal.

1DepartmentofClinicalandExperimentalSciences,FacultyofMedicine,UniversityofSouthampton,Southampton,UK22ndAcademicDepartmentofInternalMedicine,HippokrationGeneralHospitalofAthens,Athens,Greece3AcademicDepartmentofGastroenterology,LaikoGeneralHospitalofAthens,Athens,Greece

CorrespondenceSalimKhakoo,DepartmentofClinicalandExperimentalSciences,FacultyofMedicine,UniversityofSouthampton,Southampton,UK.Email:s.i.khakoo@soton.ac.uk

Funding informationBristol-MyersSquibb;EASL

SummaryTheoptimaldurationof treatmentwithnucleos(t)ideanalogues (NAs) forpatientswithHBeAg-negativechronichepatitisB (CHB) isunknown.Theaimofthisstudywastoidentifyanimmunesignatureassociatedwithoff-treatmentremissiontoNAtherapy.We performedmicroarray analysis of peripheral bloodmononuclear cell(PBMCs)fromsixpatientswithchronichepatitisBwhostoppedNAtherapy(threewithoff-treatmentremission,threewithrelapse)andfivepatientswithchronicHBVinfection(previouslytermed‘inactivecarriers’)servedascontrols.Resultswerevali-datedusingqRT-PCRonasecondgroupof21individuals(17patientswhostoppedtreatmentandfourcontrols).PBMCsfrom38patientsonlong-termNAtreatmentwereanalysedforpotentialtostoptreatment.Microarrayanalysisindicatedthatpa-tientswithoff-treatmentremissionsegregatedasadistinctout-group.Twenty-onegeneswereselectedforsubsequentvalidation.Tenofthesewereexpressedatsig-nificantlylowerlevelsinthepatientswithoff-treatmentremissioncomparedtothepatientswith relapse and predicted remissionwith AUC of 0.78-0.92. IFNγ, IL-8,FASLGandCCL4werethemostsignificantbylogisticregression.Twelve(31.6%)of38patientsonlong-termNAtherapyhadexpressionlevelsofallthesefourgenesbelowcut-offvaluesandhencewerecandidatesforstoppingtreatment.OurdatasuggestthatpatientswithHBeAg-negativeCHBwhoremaininoff-treatmentremis-sion3years afterNAcessationhave adistinct immune signature and thatPBMCRNA levels of IFNγ, IL-8, FASLGandCCL4may serve as potential biomarkers forstoppingNAtherapy.

698  |     KRANIDIOTI eT Al.

1  | INTRODUCTION

ChronicinfectionwithhepatitisBvirus(HBV)remainsaseriouspub-lic health problemworldwidewith a highmorbidity andmortalityrate.1Patientswithactiveviralreplicationandnecro-inflammationwhoremainuntreatedareatparticularriskforcirrhosisandhepa-tocellularcarcinoma(HCC).2,3Theinfectioncanbecontrolledeitherwithalimitedcourseofinterferon-αorlong-termnucleos(t)ideana-logue(NA)therapy.4,5Potentfirstlinenucleos(t)ideanalogues(NAs)havebeenwidelyusedandfoundtobeassociatedwithvirologicalresponse,biochemicalremission,reversioninliverfibrosisandsig-nificantreductioninmorbidityandmortality.5-8However,long-termadministration of NAs has raised several safety and economic is-sues,makingthequestionoftreatmentdiscontinuationanemergingissueespecially inpatientswithHBeAg-negative chronichepatitisB(CHB).Severalsmallstudieshaveshownthataproportionofpa-tientsremainedinremissionafterNAdiscontinuation.9-13Therefore,theAsianPacificAssociation14andmorerecentlyEuropeanguide-lines5 recommended that NAs could be discontinued in selected,noncirrhoticHBeAg-negativepatientswhohaveachievedvirologi-calsuppressionforatleast3yearsandareunderclosemonitoring.5 However,as55%-70%ofpatientsrelapsewithin24monthsafterNAdiscontinuation,andsincenoacceptableendpointmarkerexiststosafely predict long-term off-treatment remission, NA discontinua-tioninHBeAg-negativepatientsstillremainsadebatableissue.9-13

ControlofHBV infection involvesthehost immuneresponsetocontrolviralreplication,whilstHBVhasevolvedmechanismstoevadebothinnateandadaptiveimmuneresponsesinordertoestablishper-sistentinfection.15Long-termtherapywithNAsseemstofacilitatetherestorationofthehostimmunesystemfollowingreductionofserumHBVDNA.16BiochemicalreboundinpatientsafterNAdiscontinua-tion,followedbyHBsAgclearanceinsomeofthem,mightbeexplainedbytherestorationofimmunesystemfunctionsatthetimeofNAdis-continuation.Patientswhocancontrolvirologicalreboundandremaininremissionordevelopoff-treatmentHBsAgclearancemayhavedif-ferent immunological characteristics compared to individuals in dif-ferentstagesofHBVinfection.Inthisstudy,weinvestigatedwhetherperipheralbloodmononuclearcell(PBMC)RNAlevelsareassociatedwith off-therapy remission acting therefore as potential immune-relatedbiomarkerstoidentifyindividualswithHBeAg-negativeCHBonNAtherapywhocanbenefitfromstoppingtreatment.

2  | PATIENTS AND METHODS

2.1 | Patients

InJuly2013,weinitiatedaprospectivemulticentrestudyexaminingthepotentialofNAdiscontinuationinpatientswithHBeAg-negative

CHBwhowerereceivingNAtherapyforatleast5yearsandhadnoevidenceofcirrhosisbeforeNAinitiation.Thecurrentstudyrepre-sentsagroupofpatientswithlong-term(3years)offollow-upaftertreatmentdiscontinuationthatwererecruitedfromtheoutpatientclinic of the 2nd Academic Department of Internal Medicine at‘Hippocration’GeneralHospitalofAthens,Greece.

Twenty-threeadultpatientsundermaintenancetherapystoppedNAtreatment.Themaininclusioncriteriaforparticipationinthedis-continuation studywere as follows: documentedHBeAg-negativechronichepatitisBbeforeNAtherapyinitiation,absenceofcirrhosisdefinedbyliverstiffness<10kPaassessedbytransientelastography(Echosens)or Ishakscore<4at liverbiopsy;treatmentwithNAoracombinationofNAsforat least5yearsandundetectableserumHBVDNAforat least4years.Patientswereexcluded if theyhadco-infectionwith hepatitisD virus (HDV), hepatitis C virus (HCV)or human immunodeficiency virus (HIV); any additional cause forliver injury; history of liver decompensation,malignancy includinghepatocellular carcinoma (HCC)orhistoryof liver transplantation.ThestudywasapprovedbyanindependentethicscommitteeoftheHippocrationGeneralHospital andwasconductedaccordingwiththeprinciplesoftheDeclarationofHelsinski.Allpatientssignedaninformedconsentbeforescreeningfortheirparticipation.

NineuntreatedpatientswithHBeAg-negativechronicHBV in-fection(previouslytermed‘inactivecarriers’)servedascontrolsforthe present study.All of themwereHBsAgpositive,HBeAgneg-ative/anti-HBe positivewithALT values <40IU/L on at least fouroccasionsforthelast12months,serumHBVDNAlevels<2000IU/mLandliverstiffness<7kPaattransientelastography.

A second matched population of 38 noncirrhotic HBeAg-negativeCHBpatientsundermaintenancetherapywithoutstoppingNAswererecruitedtoassessthepotentialforusingcut-offvaluesofidentifiedgenestostoptreatment.

2.2 | Study design—definitions

Thestudywasdividedintotwoparts.Firstly,aninvestigativestudyto identify determinants associated with off-treatment remissionand secondly a cross-sectional analysis of patients on long-termtreatment(Figure1).ThissecondpartwasconductedtoinvestigateifdeterminantsidentifiedinthefirstpartofthestudycouldbeusedtosubdividepatientsonNAtherapyintotwodiscretepopulations,oneofwhichcouldbetargetedforcessationoftherapy.

Peripheral blood mononuclear cell samples for gene expres-sion analysiswere collectedat theendof treatment (EOT) andat6months after NA discontinuation for three of the patients. Allpatients were followed up for at least 36months after stoppingtreatment.Accordingtotheprotocol,allpatientshadmonthlyvisitsduringthefirst3monthsandevery2-3monthsthereafter.Clinical

K E Y WO RD S

chronichepatitisB,nucleos(t)ideanalogues,treatmentdiscontinuation

     |  699KRANIDIOTI eT Al.

examinationand liverfunctiontestswereperformedateachvisit.SerumHBVDNAlevelsweremeasuredevery3monthsforthefirstyear and 6 monthly thereafter. Serum HBsAg quantification wasperformedattheEOTandevery6months.Theupperlimitofnor-mal(ULN)forALTandASTwas40IU/L,andserumHBVDNAlevelswereassessedbyapolymerasechainreactionassaywithsensitivityof13IU/mL.

Inthepresentstudy,thedefinitionofremissionwasbasedonthecombination of biochemical and virological profile; remission wasdefinedasALT<40IU/LandserumHBVDNAlevels<20000IU/mLorALT>40IU/Linonlyone-measurementandserumHBVDNAlev-els<20000IU/mLforatleast36monthsaftertreatmentcessation.

3  | METHODS

3.1 | Isolation of PBMCs and mRNA purification

Blood sampleswere collected from 23 patientswho stoppedNAtreatmentattheEndOfTreatment(EOT)andthreeofthepatientswith off-treatment remission at 6months post-therapy cessation.Blood sampleswere also collected from38patientson long-termNAtherapyandfromninecontrols.PBMCswereisolatedfrom10to 20mL blood using Ficoll Histopaque 1077-1 (SIGMA, St Louis,MO,USA)densitycentrifugation.CellpelletswerelysedwithTRIzol(Invitrogen, LifeTechnologies,Grand Island,NY,USA) andkept at

−80°C until mRNA extraction. mRNAwas extracted and purifiedusingtheRNeasyMinikit (QIAGEN,CA,USA).ANanoDrop1000Spectrophotometer(ThermoScientific,Wilmington,Delaware)wasusedtodeterminethequalityandquantityofthemRNA.

3.2 | Agilent Whole Human Genome Oligo Microarrays

AgilentWholeHumanGenomeOligoMicroarrays(MiltenyiBiotec,GmbH)wereusedtoanalysePBMCs:takenattheEOTfromthreepatients with off-treatment relapse; from three patients withoff-treatment remission taken at the EOT and at 6months post-treatment; and from five controls. Microarray image files wereprocessedusingAgilentFeatureExtractionsoftware (FES,AgilentTechnologies, Inc). For determination of differential gene expres-sion,FES-derivedoutputdatafileswerefurtheranalysedusingtheRosettaResolver®Gene expression data analysis system (RosettaInpharmaticsLLC).Thesignalintensitieswerenormalizedbydivid-ingtheintensityvaluesbytheirmedian.Thesignificancethresholdwas anominalP-value<0.05.Principal component analysis (PCA)wasperformedusingtherglpackageinR.17Resultswerealsodis-played as a Heatmap usingMultiExperiment Viewer.18 To assesstherelationshipbetweenthegenes,KyotoEncyclopaediaofGenes(KEGG) and Genomes pathway analysis (DAVID v6.7)19 softwarewereapplied.

F IGURE  1 Flowchartdiagramillustratingstudydesign.Patients:NoncirrhoticHBeAg-negativechronichepatitisB(CHB)patientsonlong-termtreatmentwithnucleos(t)idesanalogues(NAs);sub-cohorts:patientswhostoppedNAtherapy(DevelopmentandValidationcohort)andhadatleast36monthsoffollow-up(n=23);patientsonNAswithoutcessationoftherapy(Cross-sectionalcohort,n=38);Controls(C):nineuntreatedpatientswithHBeAg-negativechronicHBVinfection(previouslytermed‘inactivecarriers’);off-treatmentremissionwasdefinedasALT<40IU/LandserumHBVDNAlevels<20000IU/mLorALT>40IU/Linonlyone-measurementandserumHBVDNAlevels<20000IU/mLforatleast36monthsaftertreatmentcessation

700  |     KRANIDIOTI eT Al.

3.3 | Reverse transcription—qRT- PCR

mRNAsamplesof10patientswithoff-treatmentrelapse(attheEOT),fromsevenpatientswithoff-treatmentremission(attheEOT),fromfour controls and from38patients onNA therapy,wereused forqRT-PCR.cDNAwasreversetranscribedusingAppliedBiosystemsHighCapacitycDNAKit(USA).qPCRwasperformedusingTaqman® LowDensity Array System (Life Technologies, USA). The TaqManarraywasdesignedforcustomconfigurationwith21pre-dispensedprimer assays for the target genes and three for the endogenouscontrols (Housekeeping genes: GAPDH, B2-microglobulin and B-actin).RelativelevelsofgeneexpressionwerecalculatedusingΔCt(thresholdcycle)method.TheGAPDHhousekeepinggenewasusedto normalize the RNA amounts. Results are expressed relative toGAPDHusing theequation:Relativequantity (Foldchange):2−ΔCt,where ΔCt=Cttargetgene–CtGAPDH

3.4 | Statistical analysis

DatawereanalysedusingGraphPadPrism6.0(GraphPadSoftware,Inc., SanDiego,CA) and SPSS (version22; SPSS Inc,Chicago, IL).Patient characteristics were compared using Mann-Whitney testfor continuousvariables andchi-squareorFisher's exact tests forcategoricalvariables.TheMann-Whitneytestwasusedtocomparegene expression levels between the clinical groups andWilcoxonmatched-pairs signed-rank test tocomparegeneexpression levelsbetweendifferenttimepointsofthesameindividuals.Receiverop-eratingcharacteristic(ROC)curvesfortheaccuracyofpredictionofoff-treatmentremissionorrelapsewerederived,andthresholdsofgeneexpression levelswerecalculated[withareasundertheROCcurves(AUCROC)andtheir95%confidenceintervals(CI)].Geneex-pressionlevelswerelog-transformedtoachievenormalityandtobeincludedintheunivariablelogisticregressionanalysis.Multivariablelogisticregressionanalysiswasappliedtothepredictors,whichwere

significant at the univariable analysis. Correlations were analysedusingSpearman'sranktest (rho).The levelofP < 0.05wasconsid-eredtobestatisticallysignificant.

4  | RESULTS

4.1 | Population characteristics and outcome

ThemaincharacteristicsofthepatientsareshowninTable1.FortheindividualsthatstoppedNAtherapy,themeanagewas59±9yearsand15ofthe23patientsweremales.ThemediandurationofNAtreatmentwas8(5-14)yearsandthemedianoff-treatmentfollow-upperiod4.6(3.2-5.2)years.Off-treatmentremissionwasobservedin10ofthe23patients(43%);sevenoutoftenpatientswithremissionachievednondetectableserumHBVDNAlevelsat30(±20)monthsaftertreatmentdiscontinuation.TwopatientshadALT>40IU/mLinonlyonemeasurementduringthefollow-up.

According to the study design, six of the 23 patients andfiveof the controlswereused toperformwhole genomegeneexpressionanalysis(‘developmentcohort’).Threeoutofsixpa-tientsrelapsedafterameanof2.6(±0.6)months,andthreehadoff-treatmentremissionwithmedianserumHBVDNAlevels172(0-9300) IU/mL, 2000 (0-4000) IU/mL and 2470 (0-4410) IU/mLat12,24and36months, respectively.Seventeenof the23patients and four of the controlswere studied as a ‘validationcohort’performingtheqRT-PCRgeneexpressionanalysis.Tenofthesehadoff-treatmentrelapseafterameanof5.2±4months,and seven had remission with median serum HBV DNA levels3084(0-9200)IU/mL,170(0-6650)IU/mLand63(0-10907)IU/mL at 12, 24 and 36months, respectively. HBVDNA levels ofthe patientswith off-treatment remissionduring the follow-upperiodareshowninTableS1.Nosignificantdifferenceinbase-linecharacteristicswasobservedbetweenthedevelopmentandvalidationgroups(Table1).

TABLE  1 BaselinecharacteristicsofthechronichepatitisB(CHB)patientsandinactivecarriers(controls)

Baseline characteristics

Development cohort (N = 11) Validation cohort (N = 21) Cross- sectional cohort

CHB patientsInactive carriers (Controls) CHB patients

Inactive carriers (Controls) CHB patients

Number(N) 6 5 17 4 38

Gender(M/F) 3/3 2/2 12/5 2/3 26/12

Age(years)† 57±8.4 44±19 59.8±11 37.5±10.4 53.9±12

ALT(IU/mL)a 23.2±5.6 23±3.4 22.4±6.2 21±5.4 22±7.6

HBVDNA(IU/mL)a Undetectable 864±1019 Undetectable 1091±425 Undetectable

HBsAg(log10IU/mL)a 3.4±3.4 N/A 3±3.1 N/A N/A

DurationofNAtreatment(years)b 8(6-14) 0 8(5-13) 0 8(5-13)

Durationoffollow-up(months)b 65(61-68) - 61(38-68) - -

Nosignificantdifferenceswereobservedbetweenthepatientsorcontrols.NA,nucleos(t)ideanalogue;N/A,notavailable.aMean±SD.bMedian(range).

     |  701KRANIDIOTI eT Al.

4.2 | Patients with off- treatment remission have a distinct gene expression profile

Microarray analysis was performed in ‘the development sub-cohort’: three patients with off-treatment relapse at the end of

treatment (EOT); three patients with off-treatment remission atthe EOT and 6months following cessation of therapy; and fivecontrols. This demonstrated that there were 1649 differentiallyexpressedgenes(P<0.05afterBonferonicorrection)betweenpa-tientswithoff-treatmentremissionandrelapse,2231differentially

F IGURE  2 A,Hierarchicalclusteringanalysisforallthegenesfromthemicroarrayanalsyisshowingthatpatientswithoff-treatmentremission(REM-EOT)clusterasanout-group;B,principalcomponentanalysisforallthegenes.C,HeatMaprepresentingthemicroarrayscomparingthedifferentiallyexpressedgenesbetweenpatientswithoff-treatmentremissionandrelapseattheendoftreatment(EOT).Geneswhicharerelativelyup-regulatedarecolouredredandthosedown-regulatedarecolouredgreen.R-EOT(N=3):patientswithoff-treatmentrelapse(EndOfTreatment),REM-EOT(N=3):patientswithoff-treatmentremission(EndOfTreatment),REM-6months(N=3):patientswithoff-treatmentremission(6monthsoff-treatmentforthesamepatientsasREM-EOT),Control(N=5):untreatedinactivecarriers

–2 –1 00

50

100

150

Colour Key and Histogram

1 2

Row Z-Score

Cou

nt

PC1PC2

PC3

Height0.00 0.01 0.02 0.03 0.04 0.05

REM1-EOT

REM2-EOT

REM3-EOT

Carrier 1

Carrier 2

Carrier 3

Carrier 4

REM3-6M

REM2-6M

REM1-6M

R1-EOT

R2-EOT

0

50

–50 0 50 100 60 40 20 0 –20 –40

Carrier 5

R3-EOT

CarrierRelapse EOTRemission EOTRemission 6M

Carrier

Relapse EOT

Remission EOT

Remission 6M

(A)

(C)

(B)

702  |     KRANIDIOTI eT Al.

expressed genes between patients with off-treatment remissionandcontrols,and730differentiallyexpressedgenesbetweenpa-tientswith off-treatment remission at the EOT and at 6monthspost-treatment. One hundred and sixty differentially expressedgeneshadagreaterthantwo-foldincreaseand201agreaterthantwo-fold decrease between patients with off-treatment remis-sionandrelapse.Unsupervisedhierarchicalclusteringanalysisoftheoligo-microarraydata suggested that the threepatientswithoff-treatment remission at theEOTclusteredas adistinct groupcompared to the other groups (relapsers, controls and patientswithremissionat6monthsaftertreatmentcessation)(Figure2A),which was also illustrated by the principal component analysis(PCA)(Figure2B).

The heatmap,whichwas created using only the differentiallyexpressedgenesbetweenpatientswith remissionat theEOTandrelapse,againdemonstratedthatthegroupwithremissionclusteredasanout-grouptotheothers(Figure2C).Thecytokine-cytokinere-ceptor interactionpathwaywas themost significantly different inthe off-treatment remission group compared to the other groups[P=3.14×10−4,falsediscoveryrate(FDR)0.002]asdeterminedbyKyotoEncyclopediaofGenes(KEGG)andGenomespathwayanaly-sis(DAVIDv6.7).

4.3 | Comparison of expression levels for the target genes between patients with off- treatment remission and relapse

Weselected21differentiallyexpressedkeygenesbasedonthefoldchangedifferencebetween thepatientswithoff-treatment remis-sionandrelapse,theirparticipationinthecytokine-cytokinerecep-torinteractionpathwayandtheirpotentialimmunologicalrelevanceforcontrollingHBVinfection.AllhadPvaluesoflessthan0.05afterBonferonicorrection.The levelsofexpression for these21 targetgenesweredeterminedbyqRT-PCRonthe‘validationsub-cohort’.Theresultsindicatedthat10ofthe21geneswereexpressedatsig-nificantly lower levels inthepatientswithoff-treatmentremissionascomparedtothepatientswithrelapse(Figure3).Specifically,pa-tientswith remissionhad lower expressionofCCL20 (14 fold de-crease(fd),P=0.03),CCL4(5.9fd,P=0.02),CXCL2(18fd,P=0.02),CXCL3(17.6fd,P=0.01),IFNγ(5.3fd,P=0.01),IL-8(5.7fd,P=0.01),IL-1A(61fd,P=0.03),IL-1B(8.6fd,P=0.05),FASLG(2fd,P=0.01)andTNFRSF9(2.9fd,P=0.05)(TableS2).Additionally,ROCcurveanalysisofthe10targetgenesshowedthatexpressionlevelsofthegeneswere a potentially usefulmarker for discriminatingpatientswhowill haveoff-treatment remissionwithAUROCsof0.78-0.92(95%CI:0.56-1.00,P<0.05)(Figure4).ThebestperforminggeneswereCCL4(AUROC:0.929),IFNγandIL-8(AUROC:0.871),CXCL3andFASLG(AUROC:0.857).

4.4 | Predictive model of the off- treatment remission or relapse

Univariable logisticregressionanalysisoftheexpressionofthe10targetgenesindicatedthatlowexpressionofthetargetgeneswasassociatedwithoff-treatmentremissionafterNAtherapycessation(Table2).Specifically,weobservedsignificantassociationforFASLG(P=0.030),IFNγ(P=0.032),IL-8(P=0.048)afterlog-transformationandatrendfortheCCL4(P=0.053).Multivariatelogisticregressionanalysis revealed thatnoneof the fourgenesassociatedwithoff-treatmentremissiononunivariateanalysisremainedindependentlysignificant;atrendtowardsassociationwithoff-treatmentremissionwasobservedforIFNγ(P=0.073).

4.5 | Expression levels of the target genes and HBsAg clearance

Fourofthe23patients(17.4%)whostoppedNAtherapyachievedHBsAglossafterameanof30(±23)months.Thesepatientshadameanage71±4years,ALT levels20±3IU/mLand liver stiffness5.4±1kPa.ThedurationofNAtreatmentwas6±0.5years,threeweretreatedwithTDFandonewithETV.Thedurationoffollow-upfor themwas54±12months,andatEOT,allpatientshadHBsAg<1000IU/mL with two having levels <100IU/mL. These four pa-tientshadsignificantlylowerexpressionlevelsofFASLG(P=0.04),IL-8(P=0.02),CCL4(P=0.008)andatrendforIFNγ(P=0.06),com-paredtothosewhodidnotachieveseroclearanceduringfollow-up.

4.6 | Stratifying patients on NA treatment using levels of immune response genes

ExpressionofCCL4,IFNγ,IL-8andFASLG,thefourgenesthatweresignificantlyassociatedwithoff-treatmentremission inthe logisticregressionanalysis,wasdeterminedbyqRT-PCRinthe38patientsonlong-termNAtherapytodetermineifadiscretesubgroupofin-dividualscouldbeidentifiedwithasimilarimmunesignaturetothesustainedresponders.Theseindividualscouldthenbepotentialcan-didatesforstoppingtreatment.WeusedROCcurveanalysesofthevalidationsub-cohorttoderivethecut-offvaluesingeneexpressionlevels,whichdiscriminatepatientsintotwosubgroups.Patientswithgeneexpression levels lower than thecut-offvalueswillhavepo-tentialoff-treatmentremissionandhigherpotentialrelapse.Weap-pliedthesecut-offvaluesonthegeneexpressionlevelsofthe38ontreatmentpatients.PatientswithCCL4expressionlevelslowerthan0.098,IFNγexpressionlevelslowerthan0.0057,IL-8expressionlev-els lowerthan0.12orFASLGexpression levels lowerthan0.0038couldhaveapotentialoff-treatmentremissionwithasensitivityofprediction of 71.4%-85.7% and specificity 80%-90% (Figure5A).

F IGURE  3 RelativemRNAexpressionofthe10targetgenes,whicharesignificantlydifferentiallyexpressedbetweenthepatientswithoff-treatmentremission(REM-EOT,n=7),relapseoff-treatment(REL-EOT,n=10)anduntreatedcarrierswithinactivedisease(n=4).mRNAwasquantifiedbyqRT-PCR,expressionlevelsnormalizedtoglyceraldehydephosphatedehydrogenase(GAPDH)andtheresultexpressedasfoldexpression.Shownarethemean±SDexpressionlevels.*P<0.05,**P≤0.01

     |  703KRANIDIOTI eT Al.

704  |     KRANIDIOTI eT Al.

F IGURE  4 Receiveroperatorcharacteristic(ROC)forpredictionofoff-treatmentrelapseusingtheexpressionlevelsofthe10significantlydifferentiallyexpressedgenes.TheareasundertheROCcurve(AUC)areshown

     |  705KRANIDIOTI eT Al.

Twelve(31.6%)ofthe38patientshadexpressionlevelslowerthanthecut-offvaluesforallfourgenesandthereforecouldbeconsid-eredforstoppingtreatment(Figure5B).

5  | DISCUSSION

Nucleos(t)ideanaloguesarewidelyusedbythemajorityofthephy-siciansinthetreatmentofpatientswithCHB,andtheyhavebeenprovedveryeffectiveandsafe.4,5Long-termmaintenance therapywithNAsisassociatedwithsustainedviralsuppressionandanim-provement inhistologyandoverall outcomes.6-8However, it doesnot affect serumHBsAg levels andHBsAg loss rarely is achievedparticularlyinHBeAg-negativepatients.20,21Recently,anumberofsmall studies have shown thatNA therapy can be stopped in pa-tients with HBeAg-negative CHB who have long-term viral sup-pressionandtheevaluationofNAtreatmentwithdrawalhasshownhigherHBsAglossratesthaninstudieswithlong-termNA.Thishasresultedininclusionofpossibletreatmentwithdrawalasanoptionin recentEuropeanguidelines for the treatmentofHBV infection,basedonthedurationofHBVDNAnegativity.5Thehost immuneresponseplays a crucial role inHBVclearanceand is expected toparticipate in themaintenance of an off-treatment remission.22-24 This is thefirststudy investigatingPBMCRNA levelsasanacces-sibleindicatorofimmuneactivityinpatientswithHBeAg-negativeCHBwhostopped long-termNAtherapy.Our resultshaveshownthatlowgeneexpressionofCCL4,IFNγ,IL-8andFASLGisassociatedwithoff-treatmentremission.

Ourstudywasbasedonprospectivelycollecteddatafromaclin-icalmulticentercohortofHBeAg-negativenoncirrhoticpatients.Weusedtwodifferentcohorts.Inthedevelopmentcohort,weanalysedPBMCsusingthewholegenomemicroarraymethodology.Wefoundthatpatientswithoff-treatmentremissionhadadistinctimmunolog-icalpattern,whichwasconfirmedwhenweperformedqRT-PCRinthevalidationcohort.Tenoutof21genesweresignificantlydown-regulatedinthepatientswithoff-treatmentremissionascomparedto the patients who relapsed during the follow-up. These genesbelong to thecytokine-cytokine receptor interactionpathway,arepleiotropicandsecretedbybothadaptiveandinnateimmunecells.Using robust statistical analysis with the areas under the curve(AUC)andlogisticregression,wefoundthatfourgenes(IFNγ,IL-8,FASLGandCCL4)couldserveaspredictivefactorsforoff-treatmentremissionwithAUC:0.78-0.92(95%confidenceinterval(CI)0.56-1,P<0.05).Noneofthesefactorsareindependentlyassociatedwithoff-treatmentremission,whichisnotsurprisingastheimmunesys-tem is complex with multiple interactions and individual immunecomponentsdonotfunctioninisolation.Thesamefourgenes,whichpredictoff-treatmentremission,aredown-regulatedinpatientswithsuccessfulseroclearanceduringfollow-up,thusfurthersupportingthese immunologicalmarkers forsustainedHBVsuppressionafterNAdiscontinuation.

We hypothesized that gene expression patterns within thePBMCwouldindicate iftherewasabeneficial immunestatusthat

waspredictiveofremission.Overall,thisappearstoshowthatlowerlevelsofmarkersassociatedwithimmunecellactivationwerefoundinpatientswithoff-treatment remission, suggesting that their im-munesystemhadbeen ‘turneddown’.Patientswithoff-treatmentremissionhadadistinctimmunologicalpatternattheEOTcomparedto6monthspost-treatmentprobablyduetoasubsequentincreaseinviral loadwithanassociated immuneresponse.Onehypothesisis that the turningdownof the immunesystemmaysubsequentlyresult in ‘re-priming’ of the immune system if the virus rebounds,and resulting in better subsequent viral control, as observed byHönerzuSiederdissenetal.,25whocorrelatedreboundinHBVDNAwith subsequent HBsAg loss. Further longitudinal studies wouldhelp toascertain if these individuals relapse in the longrunand ifthis is associatedwithanup-regulationof these immunemarkers.IFNγ is awell-knowneffectorcytokine, an immunemarker,whichcan ‘turn on’ the cellular antiviralmechanism by inducing the ex-pression of several antiviral proteins modulating both innate andadaptiveimmuneresponses.26,27FasLandIL-8havebothbeenpre-viously associatedwith activeCHB. Inparticular, theHBxproteincantransactivatetheFasL28,29andtheIL-8promoters,30therebyupregulatingtheexpressionofthesegenes.Also,HBV-specificTcellscanproduceIL-8duringactiveCHB,andflaresofliverinflammation(eitherspontaneousor inducedbyantiviralwithdrawal)havebeenshowntobeprecededbyaparallelincreaseofIL-8productionandserumHBVDNAlevels.31-34SpecificinhibitionofIL-8increasedthepotencyofIFNαagainstHBVinvitroandtheadditionofrecombi-nanthumanIL-8almosttotallyrescuedHBVreplicationduringIFNchallenge,stronglysuggestingthatIL-8expressioninducedbyHBVcanimpairtheabilityofendogenousIFNαtoinhibitearlystagesofviralreplication,thusfavouringviralpersistence.35InhibitionofIL-8activitycouldbeinstrumentaltothesuppressionofviralactivityandtotheimprovementofIFNα antiviral action.

PreviousworkfromRivinoetal36andRinkeretal37hasanalysedT-cell responses following cessation of NA therapy. Both studiesused in vitro stimulationofT cellswithHBVpeptides in order tospecificallyamplifylow-frequencyT-cellresponses,withcontrastingresults.InthestudyofRivinoetal,expressionofPD-1wasassoci-atedwithbeneficialresponsesasdefinedbyanabsenceofaflareinALT,whereasRinkeretaldescribedanabsenceofPD-1onTcellsasbeingbeneficial intermsofHBsAgloss.Thesedataareconsistentwithdiscretemechanismsoperating inthesetwodifferentscenar-ios.Forprolongedviralsuppression,thepresenceofPD-1positiveT cells, whichmay represent T-cell memory populations with im-provedsurvivalcharacteristics,38appears important.Whereas fol-lowingavirologicalrelapseT-cellprimingmayberequired,inwhichcasePD-1-negativeTcellsmaybemoreimportant.Ourdatastudypatientswith a response pattern similar to thework of Rivino, inwhichmemoryresponsesmaybepresentandsolowerlevelsofT-cellhelparerequiredtomaintainimmuneresponse,andcontrolofviral replication.Thus, the immuneresponseappearstobeturneddown.Furthermore,low-frequencyT-cellresponses,whichrequireantigen-specificstimulationinvitroinordertobecomedetectable,areunlikelytobeidentifiedusingourunbiasedapproach.

706  |     KRANIDIOTI eT Al.

Thepercentageof our patientswith off-treatment remissionandHBsAg loss is in accordancewith the results of other clini-cal studies.9,10,13,39-41 Although several studies tried to explorethe factors that may predict the post-cessation outcome, nonerevealed a strong or reliable indicator.10,13,25,39,42,43 The signif-icance of serumHBsAg levels at the time of discontinuation as

an indicatorof remissionhasbeendebatablewithcontradictoryresultsamongthestudies13,41,44-48whilstnovelHBVmarkerssuchas core-related antigen (HBcrAg)49,50 andHBVRNA51,52 need to be assessed in larger cohorts prospectively. The data regardingthecourseofpatientsbeforelosingHBsAgafterNAdiscontinua-tionareconflicting.11 Interestingly,HönerzuSiederdissenetal25

Variables B SE P value Odds ratio

95% CI of OR

Lower Upper

CCL20 0.653 0.344 0.057 1.922 0.980 3.769

CCL4* 3.317 1.911 0.053 27.568 0.652 1165.958

CXCL2 0.954 0.535 0.074 2.596 0.910 7.401

CXCL3 1.257 0.663 0.058 3.514 0.958 12.894

FASLG* 3.394 1.568 0.030 29.783 1.379 643.079

IFNγ* 1.242 0.580 0.032 3.463 1.112 10.788

IL-1A 0.618 0.332 0.063 1.855 0.968 3.555

IL-1B 0.858 0.484 0.076 2.358 0.914 6.083

IL-8* 1.090 0.550 0.048 2.973 1.012 8.737

TNFSF9 1.188 0.625 0.057 3.280 0.963 11.166

*P ≤ 0.05.

TABLE  2 Univariablelogisticregressionanalysisoftheexpressionofthe10targetgeneswiththeoff-treatmentremissionandrelapse

F IGURE  5 A,CCL4,IFNγ,IL-8andFASLGexpressionlevelsofthepatientsontreatment(n=38).Thecut-offvaluesasdeterminedbytheROCanalysesweresuedtodividethepopulationintotwosubgroups,onewithpotentialoff-treatmentremission(potentialREM)andonewithpotentialoff-treatmentrelapse(potentialREL).Barsrepresentmean±SD.****P-value<0.0001.B,Thepercentageofpatientsontreatmentwithone,two,threeorfourgenesbelowthecut-offvalues

(A)

(B)

     |  707KRANIDIOTI eT Al.

reportedthatpatientswithHBsAglosshadsignificantincreasesinserumHBVDNAaftertreatmentdiscontinuation.Theseauthorsalso reported the presence of antigen-specific PD-1-negative Tcells correlatingwith this.37Asnotedabove, thispopulationap-pears to be immunologically distinct from individuals who haveprolongedviralsuppression.Howevergiventheconflictingresultsin this area, we consider that an approach which predicts pro-longedviralsuppressionwouldcurrentlybemoreclinicallyappli-cable,thanonewhichreliesonalaterassociationwithHBsAgloss.Further studies in thisareawillbe importantboth in identifyingadditionalmarkers,butalsoindeterminingwhichshorttermout-comesarebestpredictiveoflong-termbenefit.

Thestudyhassomelimitations.Applyingimmunologicalpredic-tivefactorsinclinicalpracticeismorecumbersomethanserologicalanalysis;howevergivencurrenttechnologicaladvancesandthead-ventofpersonalizedmedicine, costs are likely to fall.Additionally,furtherinvestigationisneededtodefinethethresholdsofgeneex-pression levelsgivingthehighestAUC,whichmaypredict theoff-treatmentremission.ThesmallnumberofpatientswhostoppedNAtreatmentisanotherdrawbackbuttheriskofpost-treatmentrelapseand the need for close follow-up after treatment cessation madetheidentificationofthesepatientschallenging.Thirdly,weincludednoncirrhotic Caucasians with HBV genotype D infection patients,and therefore,our resultsmaynotbegeneralizable toallpatients.Finally,itmightbearguedthatoff-treatmentremissionisadynamicconditionandremissionorrelapseratesmaychangeduringthefol-low-upwiththepercentagesofrelapsevaryingconsiderablyamongstudies.10However,weshouldemphasizethatwehaveprospectivelycollecteddata,weusedstrictcriteriaforremission,andallpatientshavebeenfollowedfor36months,whichstrengthenourdata.

Inconclusion,ourresultsdemonstratedthattheHBeAg-negativeCHBpatientsoneffectivelong-termtherapywithNAswhostoppedNAtreatmentandhadoff-treatmentremissionseemtoshareadis-tinctimmuneprofilefromthosepatientswithanoff-treatmentre-lapse. Expression levels of theCCL4, IL-8, IFNγ andFASLGgenescould be used as potential biomarkers of off-treatment remissionandcouldstratifythosepatientsonNAtreatmentforconsiderationoftreatmentcessation.Ourdatashowedforfirsttimethepredictivesignificanceof immunologicalbiomarkers inpatientswithHBeAg-negativeCHBwhostoppedNAsandunderlinedtheneedforfurtherresearchwithahighernumberofpatientsconfirmingourresults.

ACKNOWLEDGEMENTS

ThisworkwassupportedbyEuropeanAssociationfortheStudyofthe Liver (EASL Physician Scientist Fellowship) and Bristol-MyersSquibb. We would like to acknowledge the contribution of DrBorislavDmitrovtothiswork.

CONFLICT OF INTEREST

HK: Research support from Bristol-Myers Squibb. SM: Researchgrants, lectures and advisory boards forGilead, Abbvie,Novartis,

GlaxoSmithKline, Janssen, Merck Sharp & Dohme, and Bristol-MyersSquibb.MD:Advisoryboard forGilead, lecturer forGileadand BMS. EH: Research grant from Gilead. GP: advisor/lecturerfor Abbvie, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead,GlaxoSmithKline,Janssen,MerckSharp&Dohme,Novartis,Roche;researchgrantsfromAbbvie,Bristol-MyersSquibb,Gilead,Janssen,Roche, Data SafetyManagement Board for Gilead. SIK: AdvisoryboardforBristol-MyersSquibb,researchgrantsfromBristol-MyersSquibbandGilead.GK,BD,MSB,AK,MEQDB,RTMN,MMN,CHW,TSE,EH:Noconflict.

AUTHOR CONTRIBUTIONS

ConceptionandDesign:H.K.,S.M.,S.I.K.,Datacollection:H.K.,S.M,G.K.,A.K.,M.D.,G.P.;Experimentaldesign:S.I.K.,S.M.,H.K.,T.S.E.,M.E.Q.B., R.T.M.N., M.M.N., C.H.W., E.H.; Experimentation: H.K.;Data Analysis: H.K., M.S.B., B.D.; Manuscript writing: H.K., S.M.,S.I.K.

ORCID

George Papatheodoridis https://orcid.org/0000-0002-3518-4060

Salim I. Khakoo https://orcid.org/0000-0002-4057-9091

REFERENCES

1. Trepo C, Chan HL, Lok A. Hepatitis B virus infection. Lancet. 2014;384(9959):2053-2063.

2. ChanSL,WongVW,QinS,etal.Infectionandcancer:thecaseofhepatitisB.J Clin Oncol.2016;34(1):83-90.

3. El-SeragHB.Epidemiologyofviralhepatitisandhepatocellularcar-cinoma. Gastroenterology.2012;142(6):1264-1273e1.

4. TerraultNA,BzowejNH,ChangKM, et al.AASLDguidelines fortreatmentofchronichepatitisB.Hepatology.2016;63(1):261-283.

5. European Association for the Study of the Liver. Electronic ad-dresseee,EuropeanAssociationfortheStudyoftheL.EASL2017ClinicalPracticeGuidelinesonthemanagementofhepatitisBvirusinfection.J Hepatol.2017;67(2):370-398.

6. Manolakopoulos S, Karatapanis S, Elefsiniotis J, et al. Clinicalcourse of lamivudine monotherapy in patients with decompen-satedcirrhosisduetoHBeAgnegativechronicHBVinfection.Am J Gastroenterol.2004;99(1):57-63.

7. Liaw YF, Sung JJ, ChowWC, et al. Lamivudine for patientswithchronic hepatitis B and advanced liver disease. N Engl J Med. 2004;351(15):1521-1531.

8. Marcellin P, Gane E, Buti M, et al. Regression of cirrho-sis during treatment with tenofovir disoproxil fumarate forchronic hepatitis B: a 5-year open-label follow-up study. Lancet. 2013;381(9865):468-475.

9. KranidiotiH,ManolakopoulosS,KhakooSI.Outcomeafterdiscon-tinuationofnucleot(s)ideanaloguesinchronichepatitisB:relapserateandassociatedfactors.Ann Gastroenterol.2015;28(2):173-181.

10. Papatheodoridis G, Vlachogiannakos I, Cholongitas E, et al.DiscontinuationoforalantiviralsinchronichepatitisB:asystematicreview. Hepatology.2016;63(5):1481-1492.

11. Papatheodoridis GV, Manolakopoulos S, Su TH, et al.Significanceofdefinitionsof relapseafterdiscontinuationoforal

708  |     KRANIDIOTI eT Al.

antivirals in HBeAg-negative chronic hepatitis B. Hepatology. 2018;68(2):415-424.

12. vanBommelF,BergT.Stoppinglong-termtreatmentwithnucleos(t)ideanaloguesisafavourableoptionforselectedpatientswithHBeAg-negativechronichepatitisB.Liver Int.2018;38(Suppl1):90-96.

13. BergT,SimonKG,MaussS,etal.Long-termresponseafterstoppingtenofovirdisoproxilfumarateinnon-cirrhoticHBeAg-negativepa-tients-FINITEstudy.J Hepatol.2017;67(5):918-924.

14. Sarin SK, KumarM, Lau GK, et al. Asian-Pacific clinical practiceguidelines on the management of hepatitis B: a 2015 update.Hepatol Int.2016;10(1):1-98.

15. BertolettiA,FerrariC. Innateandadaptive immune responses inchronichepatitisBvirusinfections:towardsrestorationofimmunecontrolofviralinfection.Gut.2012;61(12):1754-1764.

16. BoniC,LaccabueD,LamperticoP,etal.RestoredfunctionofHBV-specificTcellsafterlong-termeffectivetherapywithnucleos(t)ideanalogues.Gastroenterology.2012;143(4):963-973e9.

17. LangfelderP,HorvathS.WGCNA:anRpackageforweightedcor-relationnetworkanalysis.BMC Bioinformatics.2008;9:559.

18. SaeedAI,SharovV,WhiteJ,etal.TM4:a free,open-sourcesys-tem formicroarraydatamanagement andanalysis.Biotechniques. 2003;34(2):374-378.

19. HuangDW,ShermanBT,TanQ,etal.TheDAVIDGeneFunctionalClassificationTool:anovelbiologicalmodule-centricalgorithmtofunctionallyanalyzelargegenelists.Genome Biol.2007;8(9):R183.

20. ButiM,TsaiN,PetersenJ,etal.Seven-yearefficacyandsafetyoftreatmentwithtenofovirdisoproxilfumarateforchronichepatitisBvirusinfection.Dig Dis Sci.2015;60(5):1457-1464.

21. StrikiA,ManolakopoulosS,DeutschM,etal.HepatitisBsantigenkineticsduringtreatmentwithnucleos(t)idesanaloguesinpatientswith hepatitis B e antigen-negative chronic hepatitis B. Liver Int. 2017;37(11):1642-1650.

22. FerrariC.HBVandtheimmuneresponse.Liver Int.2015;35(Suppl1):121-128.

23. MainiMK, SchurichA. Themolecular basis of the failed immuneresponse in chronic HBV: therapeutic implications. J Hepatol. 2010;52(4):616-619.

24. Yuan T, Jiang Y, Li M, et al. Chronic hepatitis B surface an-tigen seroclearance-related immune factors. Hepatol Res. 2017;47(1):49-59.

25. Honer Zu Siederdissen C, Rinker F, Maasoumy B, et al. Viraland host responses after stopping long-term nucleos(t)ide ana-loguetherapy inHBeAg-negativechronichepatitisB.J Infect Dis. 2016;214(10):1492-1497.

26. LinFC,YoungHA.Interferons:successinanti-viralimmunotherapy.Cytokine Growth Factor Rev.2014;25(4):369-376.

27. Schroder K, Hertzog PJ, Ravasi T, et al. Interferon-gamma: anoverview of signals, mechanisms and functions. J Leukoc Biol. 2004;75(2):163-189.

28. KimSY,KimJK,KimHJ,etal.HepatitisBvirusXproteinsensitizesUV-inducedapoptosisby transcriptional transactivationofFas li-gandgeneexpression.IUBMB Life.2005;57(9):651-658.

29. ZhangSJ,ChenHY,ChenZX,etal.Possiblemechanism forhep-atitisBvirusXgene to induceapoptosisofhepatocytes.World J Gastroenterol.2005;11(28):4351-4356.

30. MaheY,MukaidaN,KunoK,etal.HepatitisBvirusXproteintrans-activateshumaninterleukin-8genethroughactingonnuclearfac-tor kB and CCAAT/enhancer-binding protein-like cis-elements. J Biol Chem.1991;266(21):13759-13763.

31. GehringAJ,KohS,ChiaA,etal.Licensingvirus-specificTcellstosecretetheneutrophilattractingchemokineCXCL-8duringhepati-tisBvirusinfection.PLoS ONE.2011;6(8):e23330.

32. DunnC,BrunettoM,ReynoldsG,etal.CytokinesinducedduringchronichepatitisBvirusinfectionpromoteapathwayforNKcell-mediatedliverdamage.J Exp Med.2007;204(3):667-680.

33. ZimmermannHW,SeidlerS,GasslerN,etal.Interleukin-8isacti-vatedinpatientswithchronicliverdiseasesandassociatedwithhe-paticmacrophageaccumulationinhumanliverfibrosis.PLoS ONE. 2011;6(6):e21381.

34. TanAT,KohS,GohW,etal.A longitudinalanalysisof innateandadaptiveimmuneprofileduringhepaticflaresinchronichepatitisB.J Hepatol.2010;52(3):330-339.

35. PollicinoT,BellinghieriL,RestucciaA,etal.HepatitisBvirus(HBV)induces the expression of interleukin-8 that in turn reducesHBV sensitivity to interferon-alpha. Virology. 2013;444(1–2): 317-328.

36. RivinoL,LeBertN,GillUS,etal.HepatitisBvirus-specificTcellsassociate with viral control upon nucleos(t)ide-analogue therapydiscontinuation.J Clin Invest.2018;128(2):668-681.

37. Rinker F, Zimmer CL, Siederdissen CHZ, et al. Hepatitis B virus-specific T cell responses after stopping nucleos(t)ide ana-logue therapy in HBeAg negative chronic hepatitis B. J Hepatol. 2018;69(3):584-593.

38. OdorizziPM,PaukenKE,PaleyMA,etal.GeneticabsenceofPD-1promotes accumulation of terminally differentiated exhaustedCD8+Tcells.J Exp Med.2015;212(7):1125-1137.

39. Liu F,Wang L, Li XY, et al. Poor durability of lamivudine effec-tivenessdespitestringentcessationcriteria:aprospectiveclinicalstudyinhepatitisBeantigen-negativechronichepatitisBpatients.J Gastroenterol Hepatol.2011;26(3):456-460.

40. HeD,GuoS,ChenW,etal.Long-termoutcomesafternucleos(t)ideanaloguesdiscontinuation inchronichepatitisBpatientswithHBeAg-negative.BMC Infect Dis.2013;13:458.

41. Papatheodoridis GV, Rigopoulou EI, Papatheodoridi M, et al.DARING-B: discontinuation of effective entecavir or tenofovirdisoproxil fumarate long-termtherapybeforeHBsAg loss innon-cirrhoticHBeAg-negativechronichepatitisB.Antivir Ther.2018;(inpress).https://doi.org/10.385/3256.

42. HadziyannisSJ,SevastianosV,Rapti I, et al. Sustained responsesandlossofHBsAginHBeAg-negativepatientswithchronichepati-tisBwhostoplong-termtreatmentwithadefovir.Gastroenterology. 2012;143(3):629-636e1.

43. SohnHR,MinBY, Song JC, et al.Off-treatment virologic relapseandoutcomesofre-treatmentinchronichepatitisBpatientswhoachieved complete viral suppression with oral nucleos(t)ide ana-logs.BMC Infect Dis.2014;14:439.

44. ChanHL,WongGL, ChimAM, et al. Prediction of off-treatmentresponsetolamivudinebyserumhepatitisBsurfaceantigenquan-tification in hepatitis B e antigen-negative patients. Antivir Ther. 2011;16(8):1249-1257.

45. LiangY,JiangJ,SuM,etal.PredictorsofrelapseinchronichepatitisBafterdiscontinuationofanti-viraltherapy.Aliment Pharmacol Ther. 2011;34(3):344-352.

46. Lee HA, Seo YS, Park SW, et al. Hepatitis B surface antigentiter is a good indicator of durable viral response after ente-cavir off-treatment for chronic hepatitis B. Clin Mol Hepatol. 2016;22(3):382-389.

47. JengWJ, Sheen IS, Chen YC, et al. Off-therapy durability of re-sponse to entecavir therapy in hepatitis B e antigen-negativechronichepatitisBpatients.Hepatology.2013;58(6):1888-1896.

48. SetoWK,HuiAJ,WongVW,et al. Treatment cessationof en-tecavir in Asian patients with hepatitis B e antigen negativechronic hepatitis B: a multicentre prospective study. Gut. 2015;64(4):667-672.

49. MatsumotoA,YatsuhashiH,NagaokaS,etal.Factorsassociatedwiththeeffectof interferon-alphasequential therapy inorder todiscontinue nucleoside/nucleotide analog treatment in patientswithchronichepatitisB.Hepatol Res.2015;45(12):1195-1202.

50. Matsumoto A, Tanaka E, Minami M, et al. Low serum level ofhepatitis B core-related antigen indicates unlikely reactivation

     |  709KRANIDIOTI eT Al.

of hepatitis after cessation of lamivudine therapy. Hepatol Res. 2007;37(8):661-666.

51. WangJ,ShenT,HuangX,etal.SerumhepatitisBvirusRNAisencap-sidatedpregenomeRNAthatmaybeassociatedwithpersistenceofviralinfectionandrebound.J Hepatol.2016;65(4):700-710.

52. YuY,WangJ,LiG,etal.Nucleos(t)ideanalogueinterruption:alter-nativeapproachtointrahepaticsetpointforspontaneouscontrolofHBVreplication?J Hepatol.2018;68(3):609-610.

SUPPORTING INFORMATION

Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle.

How to cite this article:KranidiotiH,ManolakopoulosS,KontosG,etal.Immunologicalbiomarkersasindicatorsforoutcomeafterdiscontinuationofnucleos(t)ideanaloguetherapyinpatientswithHBeAg-negativechronichepatitisB.J Viral Hepat. 2019;26:697–709. https://doi.org/10.1111/jvh.13068