Autoimmunity and lymphoproliferation markers in naïve HCV-RNA positive patients without clinical evidences of autoimmune/lymphoproliferative disorders

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ContentslistsavailableatScienceDirect

Digestive

and

Liver

Disease

j o ur n a l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / d l d

Liver,

Pancreas

and

Biliary

Tract

Autoimmunity

and

lymphoproliferation

markers

in

naïve

HCV-RNA

positive

patients

without

clinical

evidences

of

autoimmune/lymphoproliferative

disorders

Francesca

Gulli

a,1

_,

_Umberto

_Basile

a,1

_,

_Laura

_Gragnani

b,∗,1

_,

_Elisa

_Fognani

b

_,

Cecilia

Napodano

a

_,

_Luigi

_Colacicco

a

_,

_Luca

_Miele

c

_,

_Nicoletta

_De

_Matthaeis

c

_,

_Paola

_Cattani

d

_,

Anna

Linda

Zignego

b

_,

_Gian

_Ludovico

_Rapaccini

c

a_Department_of_Laboratory_Medicine_of_the_Catholic_University_of_the_Sacred_Heart,_Rome,_Italy

b_Center_for_Systemic_{Manifestations}_of_Hepatitis_Viruses_(MaSVE),_Department_of_Experimental_and_Clinical_Medicine,_University_of_Florence,_Florence,_Italy

c_Institute_of_Internal_Medicine,_Catholic_University_of_the_Sacred_Heart,_Rome,_Italy

d_Institute_of_{Microbiology,}_Catholic_University_of_the_Sacred_Heart,_Rome,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received12January2016

Accepted17May2016

Availableonline24May2016

Keywords:

Antinuclearantibodies(ANA)

Cryoglobulins(CGs)

HepatitisCvirus(HCV)

Rheumatoidfactor(RF)

a

b

s

t

r

a

c

t

Background:HCVcanleadtobothchronicliverdiseaseandB-celllymphoproliferativedisorders.Astrong associationexistsbetweenHCVandmixedcryoglobulinaemia(MC).

Methods:Anti-nuclearantibodies(ANA),rheumatoidfactorIg-G(RF-IgG),freelightchain␬and␭(FLC-␬, FLC-␭)levelsand␬/␭ratiowereevaluatedin50/420subjectsunexpectedlyresultedanti-HCVpositive afterroutinescreeningsfornon-hepathologicalprocedures.

Results:Three/ﬁftypatientshadHCV-RNAundetectableintheserumandwereexcludedfromtheanalysis. Thirty-nine/ﬁftypatientshadlaboratoryevidenceofcirculatingcryoglobulinswithoutliverdiseaseand MC-relatedsymptoms.Amongthem,17resultedANA-positive.

ThemeancryocritwashigherinANA-positivepatients,whilenootherdemographic/clinicaldifferences wereobservedbetweenthegroups.SigniﬁcantlyhigherlevelsofRF-IgGwereobservedinANA-positive vsANA-negativepatients.␬and␭FLCwerehigherinANA-positivepatients.

AROCanalysis,basedonANA-positivityvsANA-negativity,conﬁrmedahighsensitivityandspeciﬁcity ofRF-IgGtest.

Conclusions:PublisheddataconcerningMCcomemostlyfromsymptomaticvasculitis.Weanalyzed HCV-patientswithoutMCsymptoms,foundingcryoglobulinsinthemajorityofthem.

TheincreasedlevelsofFR-IgGandFLCinCGs-ANA-positivepatients,suggestthesetestcouldbeused toidentifyastateofsilentautoimmuneand/orlymphoproliferativeconditionbeforethetransitiontoa frankdiseaseinnaïveHCV-patientswithoutsymptomsofextrahepaticmanifestations.

1. Introduction

Hepatitis C virus (HCV) infects about 200 million people world-wide,leadingtobothchronicliverdiseaseandB-cell lym-phoproliferativedisorders(LPDs).Astrongassociationhasbeen

∗ Corresponding authorat: Center forSystemic Manifestationsof Hepatitis

Viruses(MASVE), Departmentof Experimentaland ClinicalMedicine,

Univer-sityofFlorence,LargoBrambilla3,50134Florence,Italy.Tel.:+390552758087;

fax:+390552758099.

E-mailaddress:laura.gragnani@uniﬁ.it(L.Gragnani).

1 _Contributed_equally_to_this_work.

shownbetweenHCVandmixedcryoglobulinaemia(MC),abenign, butpre-lymphomatouscondition[1,2].

TheactivationofB-lymphocytesisinturnresponsibleforthe productionofimmunecomplexes,includingcryoglobulins(CGs) andvariousautoantibodies[2,3].CGsareimmunoglobulinswhich undergoreversibleprecipitationorgellingwhenexposedto tem-peraturesbelow37◦Candre-dissolveuponrewarming[4].Their cold-insolubilityisstillunclearandmaydependuponvarietyof factors.Lowtemperaturesseemtotriggerareversible cryoprecipi-tation,possiblybyinducingstericmodiﬁcationsoftheIgmolecules

[4].ImmunochemicalcharacterizationofCGshasledtotheir classi-ﬁcationintothreemajorgroupsaccordingtoBrouet’sclassiﬁcation

[5].

http://dx.doi.org/10.1016/j.dld.2016.05.013

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TypeIrepresentsindividualmonoclonalimmunoglobulins, gen-erallyassociatedwithlymphoproliferativediseases;typeIIconsists of mixed immunoglobulins with a monoclonal/oligoclonal IgM componentandisstronglyassociatedwithHCVinfection;typeIIIis constitutedbyamixtureofpolyclonalIgMsandIgGsandis associ-atedwithawiderangeofinfectious(includingHCV),autoimmune andliverdiseases[4,6].TypesIIandIIIarereferredasMC,which impliesthepresenceofcirculatingimmunecomplexescomposed ofpolyclonalIgGs(behavingasauto-antigens)andIgMs(behaving asauto-antibodies),withrheumatoidfactor(RF)activity.

HCVinfectionmaytriggeracollectionofimmunological mech-anismsresponsibleforthedevelopmentofdifferentautoimmune diseases[2,3].Anti-nuclearantibodieswererecentlydetectedin cryoprecipitatesfrom HCV-positive sera [7] and somepatients withchronic HCV infection are anti-liver and kidney microso-malantibodytype1(anti-LKM1)positivewiththeautoantibody titrecorrelatingwithdiseaseseverity[8].Also, thepresence of autoantibodies,RF-IgGandIgG3inthecryoprecipitatesfrom HCV-positivepatientssuggestsastrongactivationoftheimmunesystem

[7].

Cryofibrinogenemiareferstothepresence,inserum,ofa cry-oproteinthatwasfirstidentifiedin1955byKorstandKratochvil. Unlikecryoglobulinaemia,theprecipitateformsonlyinplasmaand notintheserum[9].

Serumfreelightchains(FLCs),␬and␭,havebeenidentifiedas usefulmarkerswhosealterationislinkedtospecificdiseases;the quantitativeassayofKchains,␭chainsand␬/␭ratioisadiagnostic toolinplasmacelldyscrasias,suchasmultiplemyeloma, mono-clonalgammopathyofundeterminedsignificanceandamyloidosis

[10–12].Anabnormal␬/␭FLCratioisinterpretedasasurrogatefor clonalexpansion[13].

ThefastturnoverofFLC,particularlytrueforthe␬chain(less than6hcomparedto20–25daystototalIgG),explainsthatthey areconsideredidealmarkersofresponseinpatientsundergoing rituximabtreatment[14].

Theaimofthisstudywasthereforetoﬁndapanelofanalytic tests,easytoassess,thatcouldidentifyastateofsilent autoim-muneand/orlymphoproliferativeconditionbeforethetransitionto afrankdiseaseinnaïveHCVpatientswithoutsymptomsof extra-hepaticmanifestations.

2. Materialsandmethods

2.1. Patients

NaïvepatientswithHCVinfectionwereconsecutivelyrecruited from January 2014 to December 2014 by the Department of Gastroenterologyof the Instituteof Internal Medicine,Hospital Foundation “A. Gemelli”, Catholic University of Sacred Hearth, RomeItaly. AlltheenrolledsubjectsresultedHCV-RNA positive afterroutine screenings performedbefore undergoingdifferent non-hepatologicalprocedures.Subjectswereincludedinthestudy accordingto thefollowing criteria: noprevious antiviral treat-ments,absenceofsymptomsofhepaticdiseaseandautoimmune and/orLPDsandnormallevelsofC4andC3.Exclusioncriteriawere: presenceofabnormalALTvalues,renalinvolvementorother symp-tomspossiblyrelatedtoMCvasculitis,inﬂammatorydiseaseswith elevatedlevelsofC-reactive protein(CRP)orco-infections(HIV andHBV).Allthepatientssignedawritteninformedconsent,in accordancewiththePrinciplesoftheDeclarationofHelsinki. 2.2. Laboratoryﬁndings

ThequantitativeHCV-RNAdetectionwasassessedbyroutine method and virus genotype was determined for each sample

(Siemens Healthcare, Germany). Briefly, presence and quantifi-cation of HCV-RNA were determined by means of real-time polymerasechainreaction (PCR),transcription-mediated ampli-fication (TMA), and multi-probe reverse hybridization of the 5untranslatedregion(5UTR)oftheHCVgenome.Resultswere reportedin IU/mL.Testing for HCV genotype and subtype was performedthrough reversetranscription and PCR(RT-PCR) and reversehybridizationoftheHCVgenome.Genotypesandsubtypes detectablethroughthistestinclude1a,1b,2a/2c,2b,3a,3b,3c,4a, 4b,4c/4d,4e,4f,4h,5aand6a.

ForCGsdetection,10mLofperipheralbloodwerecollectedand immediatelystoredat37◦Cin pre-warmedtubes without anti-coagulant for 30min toenable complete blood clotting. Serum wastransferredinWintrobetubesandstoredforatleast7days at 4◦C in order to evaluate the presence of precipitates and flocculation.Beforeimmunologictesting,thecryocritpercentage wasassessed,thesupernatantwasremovedandstoredfor sub-sequentanalyses.The remainingcryoprecipitate wasrecovered andwashed3times.Cryoprecipitatewasre-suspendedwithan appropriatevolume of3%PEG 6000solutionand re-solubilized for 30minat 37◦C. CGswere characterizedbyimmunofixation electrophoresis (IFE) with G26 Fully automated system (Inter-lab,Italy)[15].Todetectcryofibrinogenemiaasimilaranalytical procedurehasbeenusedbut peripheralblood wascollectedin tubes containinganticoagulants (oxalate, citrate,or ethylenedi-aminetetraaceticacid).Heparin-coatedtubesshouldnotbeused toavoidfalse-positiveresultsduetotheformationofa cryopre-cipitatecontainingfibronectin,fibrinandfibrinogentogetherwith heparin, knownas “heparin-precipitable fraction” [16]. Antinu-clearantibody(ANA)determinationwascarriedoutby indirect immuno-fluorescence(IIFA)[17].IIFAentailssubstantial techni-calexpertiseanditisconsideredrealgoldstandardassay.Briefly, all serum specimens were titrated in 1:160 in order to avoid false-positivenon-specificlowtitredilution.Dilutedserum was addedtoHepG2cells(ATCC,IL-INOVA,USA)at37◦C.Asecondary polyclonalantibody(anti-humanIgGFITC-conjugatedpolyclonal sheep antiserum) was subsequently added. Slides were fixed withglyceroland thestainingpattern evaluatedbymicroscopy

[18].

Liver-dot,animmunodotblotkit (AXAdiagnosticsSrl,Italy), has been used to detect, in the serum, IgG-autoantibodies directedagainstthefollowingantigens:M2/nPDC,M2/OGDC-E2, M2/BCOADC-E2,M2/PDC-E2, gp210,sp100, LKM1, LC1and SLA

[19].Samplesweretestedat37◦Caccordingtothemanufacturer’s instructionsandserumdilutions,wherenecessary,wereperformed followingtherecommendations.

FLCswereassessedbymeansofTurbidimetricassay(Freelite TMHumanKappaandLambdaFreeKits,TheBindingSite,UK)and performedontheSPAplusinstrument(TheBindingSite,UK). Sam-plesweretestedat37◦Candserumdilutionswereperformedif needed.

Rheumatoidfactor-IgG(RF-IgG)wastestedusingELISAkitsfor RF-IgG(INOVA-diagnostics,USA)andperformedontheELISAand IIFASkyLAB752analyzerautomaticsystem(DASIT-Italy).

ROCcurves(ReceiverOperatingCharacteristic)weregenerated byplottingdataonGraphPadPrism(GraphPadSoftware,Inc.,S. Diego,CA,USA)andtheresultinginformationwasthenre-plotted onMicrosoftExcel(MicrosoftCorporation,Redmond,WA,USA). Data from FLC and RF-IgG serum concentrations were plotted againstANApositivity/negativity,inordertogiveestimatesoftrue positivevalues(sensitivity)andtheproportionoffalsenegatives (speciﬁcity).Plottedvaluesarerepresentedasacurve,andtheArea UndertheCurve(AUC)indicatesdiagnosticaccuracy.AnAUC=1 (100%)denotesfullaccuracyofthetest.Inthisway,itispossibleto identifythebestcut-offvalueforaspeciﬁctestandtodiscriminate normalfromabnormalvalues.

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Table1

Mainbaselinecharacteristicsofthe39HCVpatientswithcirculatingcryoglobulins.

Allpatients ANApositive ANAnegative pvalue

(n=39) (n=17) (n=22)

Meanage(years) 61.74±1.48 60.65±2.13 62.59±2.07 ns

Sex(male/female) 15/24 4/13 11/11 ns HCVgenotype: 1(%) 36 16 20 2(%) 2 – 2 3(%) 1 1 – 4(%) – – –

Viraltitre(IU/mL×105₎ _7.46_±_2.66 _5.61_±_2.11 _8.90_±_4.45 _ns

MeanALT(U/L)a _32.49_±_1.07 _35.01_±_2.44 _30.50_±_2.32 _ns

CharacteristicsofMC: Cryocrit% 2.6±0.2 3.3±0.4 2.2±0.3 0.026 TypeII 18/39(46.2%) 10/17(58.8%) 8/22(36.4%) ns TypeIII 21/39(53.8%) 7/17(41.2%) 14/22(63.6%) RFalteration 8/39(20.51%) 2/17(11.8%) 6/22(27.2%) ns SerumFLCalteration: ␬ 34/39(87.2%) 17/17(100%) 17/22(77.2%) ns ␭ 21/39(53.4%) 11/17(64.7%) 10/22(45.5%) ns FLCratio 13/39(33.3%) 9/17(52.9%) 4/22(18.2%) 0.0392

Valuesareexpressedasmean±standarderrormean(SEM).

ns,notsigniﬁcant;IU,internationalunits;MC,mixedcryoglobulinemia;RF,rheumatoidfactor;FLC,freelightchains.

a_Normal_range_for_ALT:_7–45_U/L.

2.3. Statisticalanalysis

CollecteddatasetswerereportedonMicrosoftExcelTM work-sheets(Microsoft,Redmond,WA,USA)andwascomputedusing StatgraphicsPlus5.1(STATPOINTTECHNOLOGIES,INC.,Warrenton, Virginia,USA)eGraph-PadPrism(GraphPadSoftware,Inc.,S.Diego, CA,USA).Dataareexpressedasmean±standarderrormean(SEM). TheChi-squareandFishertestswereusedtoevaluatecategorical variables.Statisticalsigniﬁcancewassetatavalueofp<0.05.

3. Results

From January 2014 to December 2014, 420 patients were examinedintheDepartmentofGastroenterologyoftheInstitute

of Internal Medicine, Foundation “A. Gemelli” Hospital.Among them,50subjectsresultedanti-HCVpositiveafterroutine screen-ingsperformedbefore undergoingdifferentclinicalprocedures. Three/ﬁftypatientshadHCV-RNAundetectableintheserumand 8 patients of the remained47 had no evidencesof circulating CGs.

Thirty-nine HCV-RNA and CGs positive patients (mean age=61.7yrs, ±1.5,17 males,44%)wereincludedin thestudy. Amongthem,17(44%)resultedpositivetoANAtest.

Themaindemographic,clinicalandvirologicalcharacteristics ofpatientsincludedinthestudyareoutlinedinTable1.

AllthepatientshadnormalALTlevelsandtheMetavirScore, assessed by transient elastography Fibroscan, ranged from F0 toF1.

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Fig.2.RheumatoidfactorIgG(RF-IgG)levelsinHCV-patientswithtypeIIandtype

IIcirculatingcryoglobulins(CGs).

ANApositivepatientsshowedahighermeancryocritandamore frequentalterationofFLCratio,while nootherdemographic or clinicaldifferenceswereobservedbetweenthegroups.

Allsamplesresultednegativetothetestforcryoﬁbrinogenemia andonly6/39patientsshowedpositivesignalinliver-dot, specif-ically5samplesresultedpositiveforLKM1and1sampleforSLA (datanotshown).

ResultsobtainedfromtheanalysesofRF-IgG,FLC␬,FLC␭levels andthe␬/␭ratioarereportedinFig.1.Asigniﬁcantdifferenceof RF-IgGranksmeasuredat37◦CwasobservedbetweenANAnegative andANApositivepatients(p=0.0002,panelA),withhigherlevels shownbythelatestones.Moreover,anincrementofboth␬and ␭FLCwasobservedinANApositivepatientsrespecttothe nega-tiveones(p<0.01,panelB;p<0.03,panelC,respectively),whereas nosigniﬁcantdifferencesbetweenthetwogroupswereobserved concerningthe␬/␭ratio(panelD).

Also,weevaluatedtheincrementofRF-IgGstratifyingfor cryo-globulintype.AsitisshowninFig.2,inbothtypeIIIandtypeII cryoglobulinpatients,ahigheramountofRF-IgGwascorrelatedto ANApositivity.

Moreover,RF-IgG levelsresulteddifferentaccordingto cryo-globulinemia type, speciﬁcally theyappeared lower in type III cryo-patientsrespecttothetypeIIones(Fig.3).

Inordertoverifytheaccuracyoftheperformedtests,we con-ductedaROCanalysisandweelaboratedsensitivityandspeciﬁcity

Fig.3.RheumatoidfactorIgG(RF-IgG)levelsstratiﬁedaccordingtocryoglobuline

type.

ROCcurvesbasedonANApositivevsANAnegativepatients.As showninFig.4,thetestforRF-IgGhasbeenconfirmedtohavevery highsensitivity(83%)andspecificity(76%),aswellasaremarkable AUC(0.88),whiletheothertestsshowlowerpercentages never-thelessmaintainingthestatisticalsignificance.

Toconclude,weperformedalineardiscriminantanalysis.This analysisallowsareclassificationofsubjectsbasedongiven param-eters.WeelaboratedthealgorithmbyusingFLCkappa,RF-IgGand the␬/␭ratiosincetheyresultedthemostsignificantparameters fromROCanalysis,withthehigherAUC.Classificationfunctions coefficientsandrelativealgorithmsareshowninFig.5.

Usingthealgorithmwepropose,91%ofpositiveANApatients and76%ofANAnegativepatientswerecorrectlyreclassiﬁed,with the85%oftotalcasesproperlyidentify.

4. Discussion

HCVrelatedMCisanidealmodeltostudythecomplex interac-tionsbetweenautoimmunityandoncogenesis.

MostofthepreviouslypublishedstudiesanalyzedMC symp-tomaticpatientsreferredtospecializedrheumatology/hepatology centres.Conversely,thepopulationweevaluatedwascomposed ofsubjectswithoutliverdiseaseandwithnosignsorsymptoms oflymphoproliferationandautoimmunityforwhichtheHCV pos-itivitywasdiagnosedthroughroutinetesting,carriedoutbefore undergoingamedicalprocedure. Forthisreason,theoriginality andstrengthofouranalysisliesinhavingtested,forCGs,a pop-ulationwithoutevidencesforsuspectingahiddenautoimmunity withaccuratesamplingandtesting.

Surprisingly, we found CGs in the majority of the patients with HCV infection. The percentage appears high, consider-ing the recruitment of an unselected population without any sign/symptomsofautoimmuneorlymphoproliferativedisease.On theotherhand,theLaboratoryDepartmentoftheGemelli Hospi-talthatperformedallthetests,isrenownedfortheexpertisein detectingthepresenceofCGs[20].

Thisepidemiologicfindingsuggeststheopportunityofamore accurateandfrequenttestingofcirculatingCGsinHCV-positive patientsbecauseitwouldbebeneficialtobringoutnewcaseswith apossibleriskofevolutionintomoreseverediseases.Thisearns evengreatersignificanceinthenewDirectActingAntiviralsera, sincetheguidelinesofallthemajorassociationsforthestudyof theliverindicatethepresenceofHCV-relatedMCvasculitisasone oftherequirementstobetreated,evenintheabsenceofsevere liverdamage[21].

Althoughthemono-oroligo-clonalcomponentofCGswithRF activityisusuallyanIgM,ourresultsshowedahighpresenceof

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Fig.4. SensitivityandspeciﬁcityROCcurvesbasedonanti-nuclearantibodies(ANA)positivevsANAnegativepatients.

RF-IgGinCGspatients.Tothebestofourknowledge,thisisthe ﬁrststudytotestthelevelofRF-IgGinpatientswithHCVinfection and CGs.Despite theinterest raisedbythis ﬁnding,we cannot explainitsmeaningandweareplanningtoperformfurtherstudies inordertounderstandtheroleofRF-IgGinHCV-relatedMC.

FLC serum levels were altered in the majority of HCV-CGs patientsandmorethan30%showedanabnormallyelevated␬/␭ ratio,completelyconﬁrmingthevalueoftheseserumproteinsas markersofHCV-relatedMC,eveninpatientswithoutsymptoms andwithanearly-stagedisease.Morededicatedstudiesareneeded toclarifythepathogeneticroleofthesemolecules:despiteFLCs havebeenconsideredas“extra-products”ofIgsynthesisinthepast, itisnowrecognizedthattheyareveryactiveinmanypathwaysof naturalandacquiredimmunity,interactingwithspeciﬁcimmune mediators,surfacemembranereceptors,andvariouscells ofthe immunesystem[22].

Here,weevaluatedthepresenceofANAinCGsHCV-patients. ThedetectionofANAinHCV-infectedindividualshasbeen pre-viouslydescribedbyseveralauthorswithpercentageslowerthan 44%[23–26].

Althoughconﬂictingresultshavebeenreportedonthistopic, themajorityofauthorsstatedthattheANApositivityismore com-moninHCV-relatedadvancedliverdamage[23–25]andinfemale gender[24].

Interestingly,symptomaticMCisaconditionmorefrequently affectingwomen and thatis usually characterizedbyadvanced stagesofliverdisease.

Ourresultsconfirmthesepreviousfindingssince62%of CG-positive and 76% of ANA-positive patients were females (the differencewasnotstatisticallysignificant).

ThedeterminationofcirculatingCGshasnotbeenperformedin thesepreviousworks,thereforethereisnoinformationavailable ontheANA-CGsrelationshipinHCV-infectedindividuals.

SinceouranalysisconsideredonlythesubgroupofCGs-positive HCV-patients,it is conceivable thatwe studieda special popu-lationenriched inANA-positive patients:thiscouldexplain the higherpercentageofANApositivitywefound,comparedto pre-viousreports.Also,ourdatasuggestthattheproductionofANA seemsstrictlyrelatedtoCGsproduction.

Considering the stratiﬁcation of CGs-HCV-patients in ANA-positiveandANA-negativepatients,weshowedthatRF-IgGand FLClevelsaresigniﬁcantlyhigherintheANA-positivepopulation. Furthermore,thelineardiscriminantanalysisshowsthepossibility offormulatinganalgorithmbasedonthreeparameters(RF-IgG,FLC ␬and_␬␭ratio)whichcanpredictANApositivity/negativityinHCV patientswithcirculatingCGs.Thisisaclear,furtherproofthatall thesedifferentbiohumoralparametersarecloselycorrelatedand complementeachother.

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Fig.5.Lineardiscriminantanalysisandrelatedalgorithm.

Theimmunologicaltolerancerepresentsthelossoftheimmune systemabilitytorespondtoaparticularantigen.Themolecular mimicrybetweenviralandself-antigenscancausecross-reaction againstself-antigensthemselves.HCV-patientsshowawiderange ofautoantibodiesusuallyatverylowtitres.Amongthem, ANA-positivitywas foundin 10–33%of HCV-patients[27].Although an interpretationof the mechanism responsible for ANA onset andtheirclinicalmeaningarenot clear,themolecularmimicry could,atleastinpart,explainthisphenomenon.Infact,asequence homology hasbeen reported betweensome HCV antigens and nuclearcomponentsofhostcells. Thereactivityagainstspeciﬁc viralproteinscouldalsojustifythepresenceofcirculatingCGsthat representtheﬁrststepofB-cellcloneactivationandarethesignof alatent,sub-clinicalandnotyetsymptomaticauto-reactivity.

Thepersistenceoftheantigenicstimulusleadstothesecretion ofIgGsthat,insomecases,showRFactivity.Thebiochemicalnature oftheselatterIgGscouldbeIgG3subtype[7].Itisconceivablethat, afterthis ﬁrstphasecharacterizedbyasymptomaticIgGRF,the nextstepwouldbetheappearanceofIgM-RFpossiblydetermining aclinicallyevidentdisease.Inotherwords,thisstudyopentheway forfurtheranalysesabletoclarifythemechanismsinvolved.

Indeed,itisconceivablethatthemoreconsistentinvolvement oftheimmunesystem,assuggestedbytheincreasedlevelsof FR-IgGandFLCobservedinCGs-ANA-positivepatients,corresponds toa highpropensitytoevolve towards symptomatic vasculitis.

Prospectivededicatedstudiesbasedonalong-termfollowupofthis specialpopulation,willhopefullyhelpinascertainthishypothesis. Inthefuture, itwillbeinteresting toevaluatethese param-eters afteran effectiveantiviral treatment.In fact,the patients described in this study did not undergone any antiviral ther-apy,since, accordingtotheAIFA(AgenziaItalianadelFarmaco; Italian Medicines Agency) current indications, they were not eligibletotreatmentwithDAAs(http://www.agenziafarmaco.gov. it/it/content/aggiornato-l%E2%80%99algoritmo-la-scelta-della-terapia-l%E2%80%99epatite-c-cronica). In conclusion, our pre-liminary results outline the importance of RF-IgG and FLCs in asymptomaticCGsandHCV-positivepatients,suggestingtheiruse asbiomarkersoftheevolutionofanunderlyingautoimmune-LPD. Also,thepossibilityofidentifyingrisksubpopulationsmayopen newscenariostotargetedtreatmentstrategiesofHCVpatientsin extremelyearlyphases(sub-clinical)fordiseasescharacterizedby unfavourableprogressioninanunpredictabletime–course.

Conﬂictofinterest

Nonedeclared.

Funding

LGwassupportedbya“FondazioneUmbertoVeronesi” fellow-ship;EFwassupportedbyanA.I.R.C.fellowship;theworkofthe

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CenterforSystemicManifestationsofHepatitisViruses(MaSVE)of theUniversityofFlorencewassupportedbygrantsfromthe “Asso-ciazioneItalianaperlaRicercasulCancro”(AIRC),IG2015Id.17391, “IstitutoToscanoTumori” (ITT)and“EnteCassa diRisparmiodi Firenze”.

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