Rev. Bras. Hematol. Hemoter. vol.39 número3

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w w w . r b h h . o r g

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Original

article

Quantitative

flow

cytometric

evaluation

of

CD200,

CD123,

CD43

and

CD52

as

a

tool

for

the

differential

diagnosis

of

mature

B-cell

neoplasms

Elissandra

Machado

Arlindo

a,∗

,

Natália

Aydos

Marcondes

a,b

,

Flavo

Beno

Fernandes

b

,

Gustavo

Adolpho

Moreira

Faulhaber

a,b

a_Universidade_Federal_do_Rio_Grande_do_Sul_(UFRGS),_Porto_Alegre,_RS,_Brazil b_Laboratório_Zanol,_Porto_Alegre,_RS,_Brazil

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Articlehistory:

Received16February2017 Accepted2May2017 Availableonline29May2017

Keywords:

MatureB-cellneoplasms CD200

CD52 CD43 CD123

a

b

s

t

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t

Background:DistinctionbetweenmatureB-cellneoplasmscanbedifficultdueto overlap-pingofimmunologicfeaturesandclinicalmanifestations.Thisstudyinvestigatedwhether quantifyingmeanfluorescenceintensityoffourmonoclonalantibodiesinaflowcytometry panelisusefulforthedifferentialdiagnosisandcharacterizationofthesedisorders.

Methods:TheexpressionsofCD52,CD200,CD123andCD43wereanalyzedinsamplesfrom 124patientswithmatureB-cellneoplasms.Thequantitativeestimationoftheseantigens wasassessedbymeanfluorescenceintensity.

Results:Thecasesincludedwere78chroniclymphocyticleukemias,threeatypicalchronic lymphocyticleukemias, sixmarginal zone lymphomas,11 splenic marginal zone lym-phomas,ninelymphoplasmacyticlymphomas,sixmantlecelllymphomas,twohairycell leukemias,twohairycellleukemiasvariant,fivefollicularlymphomas,oneBurkitt lym-phomaandonediffuselargeB-celllymphoma.ThemeanfluorescenceintensityofCD200 washigherinatypicalchroniclymphocyticleukemia,chroniclymphocyticleukemiaand hairycellleukemiacases.CD123showedhighermeanfluorescenceintensitiesinhairycell leukemiacells.Chroniclymphocyticleukemia,atypicalchroniclymphocyticleukemiaand mantlecelllymphomahadhigherexpressionofCD43andallfollicularlymphomacases hadverylowmeanfluorescenceintensityvalues.CD52expressionwasconsistentlypositive amongallcases.

Conclusion:Quantitativeevaluationofthesemarkerscanbeausefuladditionaltooltobetter identifysometypesofmatureB-cellneoplasms.

©2017Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published byElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Corresponding}_author_at_:_Rua_Ramiro_Barcelos,_2350,_90035-903_Porto_Alegre,_RS,_Brazil.

E-mailaddress:elissandra.machado@ufrgs.br(E.M.Arlindo).

http://dx.doi.org/10.1016/j.bjhh.2017.05.002

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Introduction

Mature B-cell neoplasms (MBCN) account for around 80% ofall lymphoidneoplasmsandcompriseabroadspectrum of disorders with different morphologies, clinical aspects, geneticsandimmunophenotypes.However,theyhaveamore maturelymphoidprogenitorincommoncomparedto imma-tureneoplasms.AccordingtotheWorldHealthOrganization (WHO),immunophenotypicsimilaritiesofthesecellsata cer-tainstageofmaturationinconjunctionwithmorphological, geneticandclinicalfindingsallowthesediseasestobe classi-fiedanddiagnosed.1

Immunophenotypingbyflowcytometryisafastand cost-effectivetechniquewidelyusedforthediagnosisandfollow upofhematologicaldisorders.Multiparametricflow cytom-etry (MFC) has become more complex in the diagnosis of MBCNduetotheavailabilityofseveralmarkers,inaddition tothe highnumberofentities.Usually,theanalysisis per-formedseparatelyforeachsamplealiquotlabeledwithfourto eightmonoclonalantibodies(MoAbs);anexpertinterpretsthe immunophenotypicprofileofneoplasticB-cellsandspecifies adiagnosis.Preferred markersare those ableto differenti-ateB-cellsfromothercells,definethematurationstageand identifyphenotypic aberrations. Amongthese are the pan B-cellmarkers(CD19andCD20)andthoseusedforthe dif-ferentialdiagnosis (CD10,CD5, CD103,CD43, CD23,CD49d, CD81,CD200,CXCR5,immunoglobulin(Ig)MandKappaand Lambdachains).Adifferentialdiagnosiscanbedifficult,due tooverlappingimmunophenotypicfeaturesandsimilar clin-icalmanifestations,suchascasesofdifferentiationbetween lymphoplasmacyticlymphoma(LPL)andmarginalzone lym-phoma (MZL), or between diffuse large B-cell lymphoma (DLBCL)and follicularlymphoma.For thefinaldiagnosis,it isimportanttointegrateresultsofMFCwithmorphological, clinicalandcytogeneticanalysis.1,2

Somemarkersare knownfortheirexpressioninMBCN, suchas the positivity ofCD52,3 _and _negativity _of_CD43 _in

MZL,4_the _usefulness_of_CD200_in_the_differential _diagnosis

betweenchroniclymphocyticleukemia(CLL)andmantlecell lymphoma(MCL),5_and_the_role_of_CD123_in_the_diagnosis_of

hairycellleukemia(HCL).6_However,_the_quantitative_analysis

ofthesemarkersinMCBNisseldomdiscussed,andits expres-sionmaybeimportantnotonlyforthedifferentialdiagnosis, butalsoforprognosisandpotentialtherapeuticfactors,given theexistenceoftargetdrugsagainstsomeofthesemarkers.

Theaimofthisstudywastoevaluatequantitativelythe expressionofCD200,CD123,CD43andCD52inMBCN.

Methods

All MCBNcases diagnosedina referencelaboratory inthe southofBrazilfromOctober2014toJune2015were consecu-tivelyincludedinthestudy.Specimenssentforreassessment werenotincluded.ThestudywasapprovedbythelocalEthics Committee;writteninformedconsentwasdeemed unneces-saryafterthesignatureofacommitmenttermfortheuseof biologicalmaterialandassociatedinformationbyresearchers.

The panel with the four MoAbs under study was dis-tributedusingthefollowingfluorescences:CD123FITC(clone 7G3),CD52PE(clone4C8),CD43APC(clone1G10)andCD200 PerCP-Cy5.5(cloneMRCOX-104).AllMoAbswerepurchased fromBDBiosciences(SanDiego,USA).About1,000,000cells from whole blood or bonemarrow samplesanticoagulated with EDTAwere incubated for20min inthe dark atroom temperaturewitheachMoAb.Redbloodcellswerelysedby incubation withExcellyse I(EXBIO, Praha, CZ),followed by incubation withdistilled water. Samples were washed and resuspendedinphosphatebufferedsaline(PBS).Allreagents were usedaccordingtothemanufacturer’sinstructions. All sampleswereprocessedwithin48hofcollection.7

Immediatelyafterpreparation,sampleswereanalyzedon aFACSCaliburflowcytometerusingCellQuestTM_Pro_software

(BDBiosciences,SanDiego,CA,USA).About100,000eventsper samplewereobtained.InfinicityTM_Flow_Cytometry_version_1.7

software (Cytognos,SL, ES)wasused fordataanalysis.For thegatingstrategy,debriswereremoved,basedon forward-scatter (FSC) and side-scatter(SSC) distribution. Neoplastic cellswereinitiallyidentifiedbypositivityoftheCD19andCD20 and theninaccordancewiththeexpressionofotherpanel markersforMBCN.Themeanfluorescenceintensities(MFIs) ofneoplasticcellswererecordedinarbitraryunitsfrom0to 104_.

Reproducibility ofthe fluorescence intensities was pre-served bycalibration and daily quality controlprocedures. Calibritebeads(BD Biosciences,SaoDiego, USA)were used inordertoensurestandardization.Alldiagnosiswere estab-lishedinaccordancewiththeWHOcriteria.1

Medians andinterquartile ranges foreach MFIofMoAb werecalculated.TheMann–Whitneytestwasusedto calcu-latethestatisticalsignificanceofdifferencesbetweengroups. BurkittlymphomaandDLBCLwerenotincludedinthe sta-tistical analysis because there was only one case of each disease. Differences were considered significant when the

p-value<0.05.All statisticalanalyseswere performedusing theStatisticalPackagefortheSocialSciencesv.18.0software (SPSS,Chicago,USA).

Results

Immunophenotypicanalysiswasperformedin124samples –67ofperipheralblood(54%)and57ofbonemarrow(46%) –frompatientsdiagnosedwithMBCN.Patientmeanagewas 69.3±12.1yearsoldand51.6%weremen.Thedistributionof differential diagnosesofMBCNisdescribedinTable1.The most common diagnosis, as expected, was CLL due to its highprevalence,andtherarestwereBurkittlymphomaand DLBCL. Therateofneoplastic B-cellsamongtotalcells per samplerangedfrom3.7%to97.1%,withameanof52.3±25.4%. MedianMFIsforeachMoAbofthedifferentgroupsareshown inTable2.

The MFI of CD200 was higher in atypical chronic lym-phocytic leukemias (aCLL),CLL and HCL ascanbe seenin

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aCLL BL CLL DLBCL

CD200 MFI

FL HCL HCLv LPL MCL MZL SMZL

Figure1–BoxplotshowingMFIofCD200in124casesofmatureB-cellneoplasms.aCLL:atypicalchroniclymphocytic

leukemia;BL:Burkittlymphoma;CLL:chroniclymphocyticleukemia;DLBCL:diffuselargeB-celllymphoma;FL:follicular

lymphoma;HCL:hairycellleukemia;HCLv:hairycellleukemiavariant;LPL:lymphoplasmacyticlymphoma;MCL:mantle

celllymphoma;MZL:marginalzonelymphoma;SMZL:splenicmarginalzonelymphoma.

Table1–DistributionofmatureB-cellneoplasms.

Diagnosis Number

ofcases

(%)

Atypicalchroniclymphocyticleukemia 3 2.4

Burkittlymphoma 1 0.8

Chroniclymphocyticleukemia 78 62.9

DiffuselargeB-celllymphoma 1 0.8

Follicularlymphoma 5 4.0

Hairycellleukemia 2 1.6

Hairycellleukemiavariant 2 1.6

Lymphoplasmacyticlymphoma 9 7.3

Mantlecelllymphoma 6 4.8

Marginalzonelymphoma 6 4.8

Splenicmarginalzonelymphoma 11 8.9

comparedtoLPL(p-value<0.001),MCL(p-value<0.001), follicu-larlymphoma(p-value=0.001),MZL(p-value<0.001)andSMZL (p-value<0.001).TheMFIofCD200washigherinLPLcompared

toMCL(p-value=0.003).TheMFIofCD200washigherinHCL comparedtoMCL(p-value=0.046),MZL(p-value=0.046)and SMZL(p-value=0.030).TheMFIofCD200 ofHCL wasabout 6-foldhigherwhencomparedtohairycellleukemiavariant (HCLv),howeverduetothesmallnumberofcasesincluded it wasnot possibleto show statisticaldifference in CD200 expressionbetweenthesetwoneoplasms(p-value=0.121).

CD123hadhigherMFIsinHCLcellsasshowninFigure2. The MFIofCD123 washigher inHCL comparedto CLL (p -value=0.016),LPL(p-value=0.034),MCL(p-value=0.046),MZL (p-value=0.046)andSMZL(p-value=0.030).TheMFIofCD123 washigherinSMZLcomparedtoCLL(p-value=0.003),LPL (p-value=0.037)and follicular lymphoma(p-value=0.050).MFI ofCD123hadatrendtobehigherinHCLcomparedtoHCLv (p-value=0.121).

Figure 3illustratestheexpressionofCD43.All follicular lymphomacaseshadverylowMFIvalues.TheMFIofCD43 was higher in aCLL compared to MZL (p-value=0.034) and

Table2–MedianMFIvaluesofevaluatedmarkersperdiseasecategory.

Diagnosis CD200 CD123 CD43 CD52

Atypicalchroniclymphocyticleukemia 113.7(70.4/122.2) 7.5(5.7/7.6) 133.7(74.5/154.3) 765.2(546.5/814.7)

Burkittlymphoma 4.5(4.5/4.5) 5.9(5.9/5.9) 57.0(57.0/57.0) 143.0(143.0/143.0)

Chroniclymphocyticleukemia 61.1(41.4/89.2) 8.0(5.6/10.7) 179.8(112.8/278.7) 777.9(469.3/1101.0) DiffuselargeB-celllymphoma 3.8(3.8/3.8) 6.1(6.1/6.1) 160.9(160.9/160.9) 549.5(549.5/549.5)

Follicularlymphoma 2.6(1.8/11.4) 6.4(4.5/10.0) 6.8(3.6/13.1) 686.1(455.9/820.2)

Hairycellleukemia 220.3(163.1/277.5) 83.2(61.3/105.0) 79.0(44.4/113.7) 956.4(911.3/1001.6) Hairycellleukemiavariant 36.1(22.1/50.1) 22.0(14.4/29.5) 14.6(11.9/17.2) 990.3(705.9/1274.8) Lymphoplasmacyticlymphoma 14.0(12.30/40.2) 9.0(6.7/11.1) 20.7(6.5/25.3) 697.1(447.7/1150.8) Mantlecelllymphoma 3.5(2.1/4.1) 13.9(8.6/30.9) 90.1(48.6/226.7) 704.5(538.7/1375.5) Marginalzonelymphoma 8.3(4.5/13.2) 11.7(8.1/16.0) 7.8(5.0/23.6) 1326.1(737.5/1847.0) Splenicmarginalzonelymphoma 7.6(4.0/22.9) 12.3(9.4/16.1) 7.1(6.5/8.6) 1297.6(1096.9/1861.8)

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150,0

100,0

50,0

40,0

30,0

20,0

10,0 6,0

3,0

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aCLL BL CLL DLBCL

CD123 MFI

Figure2–BoxplotshowingMFIofCD123in124casesofmatureB-cellneoplasms.aCLL:atypicalchroniclymphocytic

600.0

500.0

400.0

300.0

200.0

100.0

70.0

50.0

30.0

10.0

0

aCLL BL CLL DLBCL

CD43 MFI

Figure3–BoxplotshowingtheMFIofCD43in124casesofmatureB-cellneoplasms.aCLL:atypicalchroniclymphocytic

celllymphoma;MZL:marginalzonelymphoma;SMZL:splenicmarginalzonelymphoma.*Oneoutliercase(MFI=1.1).One

outliercase(MFI=106.3).

SMZL(p-value=0.010). TheMFIofCD43was higher inCLL comparedtoHCLv(p-value=0.021),LPL(p-value<0.001), fol-licularlymphoma(p-value=0.001),MZL(p-value<0.001)and SMZL(p-value=0.003).

The MFI of CD43 was higher in MCL compared to LPL (p-value=0.013), follicular lymphoma (p-value=0.033), MZL (p-value=0.025)andSMZL(p-value=0.001).

CD52expressionwasconsistentlypositiveamongallcases, as shown in Figure 4. The MFI of CD52 was higher in SMZLcomparedtoCLL(p-value=0.001),aCLL(p-value=0.036), LPL(p-value=0.030)andfollicularlymphoma(p-value=0.037).

Despite theBurkittlymphomagrouponlyhavingonecase, theMFIofCD52seemedconsiderablylowercomparedtothe remaininggroups.

Discussion

CD200 isa membraneglycoprotein ofthe immunoglobulin superfamily,expressedindendriticcells,neuronsandT-cell lymphocytes.8_Different_research_groups_have_identified_the

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aCLL BL CLL DLBCL

CD52 MFI

Figure4–BoxplotshowingtheMFIofCD52in124casesofmatureB-cellneoplasms.aCLL:atypicalchroniclymphocytic

versusMCL,includingoneanalyzingaBrazilianpopulation.9

Thecurrentstudyhadsimilarresults,and,althoughoneCLL caseshowedlowexpressionofthismarker,100%ofMCLcases had significantly lower expressionscompared to CLL. In a recentstudy ofCLL patients, caseswith lowMFIofCD200 hadashortertime-to-treatment comparedtopatientswith higherexpressionofthe marker.10 _As_previously_described

intheimmunohistochemicalanalysisbyPillaietal.,CD200 canbeextremelyhelpfulinthedifferentialdiagnosisbetween HCLandHCLv11_;_in_the_present_study,_HCL_cases_showed_a

trendtohavehigherexpressionsthanitsvariantform.There wasnodifferenceinCD200expressionbetweenCLLandaCLL (CD5-negative)cases.

CD200appearstobe usefulin thedifferential diagnosis between CD5-positiveLPL and CLL, as it was lower inthe formercomparedtothelatter.However,ifanalyzed qualita-tively,thisdifferencecouldnothavebeennoted,since30%of casesshowedintermediatepositivity,whichisinagreement withwhatwas describedinapreviousstudyanalyzingthe expressionofthismarkerbyimmunohistochemistry.12_CD200

hasalsobeendescribedasusefultodifferentiatebetweenCLL andMZL;theresultsofthepresentstudyconfirmthefindings describedbyimmunohistochemistrywithalowerexpression ofthemarkerinMZLandSMZL.8

CD123isasubunitoftheinterleukin-3receptor.Ithasbeen described in several hematological neoplasms.13 _Recently,

CD123 expressionwas studied quantitatively using MFI by Garcia-Dabrioetal.whocorrelateditsimplicationasa prog-nosticfactorindenovoacutemyeloidleukemia(AML),14_and

SL-101,ananti-CD123antibody-conjugate,isunderstudyfor the treatment ofCD123-positive AML.15 _The_results _of_the

currentstudyareinagreementwithpreviousstudies investi-gatingthehigherexpressionofthismarkerinHCL,16,17_since

higherMFIvalueswereidentifiedinHCLcasesandatrendof

HCLtohavehigherCD123expressioncomparedtoHCLvcases wasalsoobserved.Thisstudyidentifiedahigherexpressionof CD123inSMZLcomparedtoCLL,LPLandfollicularlymphoma cases.

According toaprevious study,CD43expressionis simi-laramongCD10-positiveMBCN(follicularlymphoma,Burkitt lymphomaandDLBCL),oneofthemostcomplicated differ-entialdiagnoses.18_All_five_cases_of_follicular_lymphoma_had

extremelylowexpressionswhencomparedtoaCLL,CLLand MCL, andthe onlycaseofDLBCLexpressedthis markerat anintermediateintensityasdescribedbypreviousstudies,19

however,analysisofthisgroupwasimpairedbythelow fre-quencyofcases.

DuringtheontogenyofB-cells,CD43isexpressedinearly stagesandislostinintermediatestages,butitisexpressed again in plasma cells and activated mature B-cells.20 _In

agreementwiththeliterature,theseresultsidentifiedhigher expressioninaCLL,CLLandMCL,andlowerexpressioninMZL andSMZL,probablyduetoclonalityintheintermediatestage ofthedisease.DespiteCD43notbeingarelevantmarkerfor thedifferentialdiagnosisofMCLandMZL,cautionshouldbe takenintheinterpretationofthesedatawithrespecttothe differential diagnosisofMCLversusCD5-positiveMZL.One MCLcase(16.0%)hadalowexpressionofthismarkersimilarto MZLandoneMZLcase(5.8%)showedintermediateexpression similartothatseeninMCL.

CD52isacellsurfaceglycoproteinwhosefunctionispoorly understood and is expressed in lymphocytes, monocytes, macrophagesandafewdendriticcells.21 _Its_expression_has

beendescribedinseveralMBCNandpositivityforthismarker involves specific treatments for diseases such as CLL and

LPL.3,22,23_Furthermore,_soluble_CD52_has_been_identified_as_a

markerofdiseaseactivityinCLL.24_This_study_identified_the

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resultsseemtobeincontrastwiththosedescribedbyother studies,suchasRodigetal.whoreportednegativityforsome diseasessuchasBurkittlymphomaand DLBCL,3_the_lowest

MFIsinthe samples ofthe present study were exactly for Burkittlymphomaand DLBCL.Chuang etal. analyzedMCL casesandidentifiedthatonly60%ofcaseswerepositivefor CD52,butthedifferenceintheimmunohistochemical tech-niqueusedmayexplainthedistinctresults.25

Sincefluorescenceintensitymeasuresareimportant deter-minants in the analysis of leukemias and lymphomas,26

reliable methods for measuringMFI are importantfor the correctdatainterpretation,and,consequently,correct classi-ficationofhematologicalmalignancies.Forthisreason,terms suchas“weak”and“strong”areuseful,but,asHendersonetal. suggested,perhapsquantitativevaluescouldbeusedinorder tofurtherexploretheinformationprovided.27

There are numerousvariables involvedin the quantita-tivedeterminationoffluorescenceintensity,somerelatedto thespecificityinthechosenMoAb,sampletype, anticoagu-lantemployed,autofluorescence,typeoffixation,cytometer compensationand unitofmeasurementusedtoreportthe data,amongothers.Even whenallthesevariables arewell controlled,somecautionininterpretingthedatashouldbe taken,butgiventheincreasinguniversalstandardizationin thefield ofimmunophenotyping, thistypeofanalysismay gainground,allowingforcomparativefigureswithgreater reli-abilityovertime.Untilthen,eachcentercandeveloptheuse ofdatainMFIaccordingtotheir caseseries, inaddition to usingthedataavailableintheliterature,aslongasitis crit-icallyinterpretedandthelimitationstothistypeofanalysis areunderstood.

Thisstudyhassomelimitations.Themainoneisthatonly immunophenotypingand biopsy resultsascomplementary testsforthediseaseentitydefinitionwereaccessible.Besides, fewcasesofnon-CLLcases,suchasBurkittlymphomaand DLBCL,wereavailable.

Conclusion

Inconclusion,theresultsofthisstudyshowtheusefulness ofalreadyknownmarkersinMBCNsuchasCD200inthe dif-ferentialdiagnosis ofCLLand MCL.Thesedatasuggestthe usefulnessofmorecomplexanalysesquantifyingMFIinthe differentialdiagnosis ofMBCN, suchasCD52expressionin SMZLversusLPL,andCD43expressioninMZL,LPLandSMZL comparedtoMCLandCLL.Nevertheless,theseresultsshould befurtherexploredinanalysesusingalargersamplesize.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

WeacknowledgetheFundodeIncentivoaPesquisa(FIPE)of HospitaldeClínicasdePortoAlegre(HCPA)forfinancial sup-port.

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