Rev. Bras. Hematol. Hemoter. vol.37 número1

rev bras hematol hemoter. 2015;37(1):34–37

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

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

Original

article

Immunohistochemical

evaluation

of

CD20

expression

in

patients

with

multiple

myeloma

Irfan

Yavasoglu,

Gokhan

Sargin

_,

_Gurhan

_Kadikoylu,

_Firuzan

_Kacar

_Doger,

Zahit

Bolaman

AdnanMenderesMedicalSchool,Aydin,Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5July2014 Accepted8August2014

Availableonline26November2014

Keywords:

Multiplemyeloma Laboratorytechniquesand procedures

Immunochemistry

a

b

s

t

r

a

c

t

Objective:CD20 expression was reported at different rates in patients with multiple myeloma.TheimportanceofthisB-cellantigenforplasmacellsisstillunknown.Thisstudy aimedtoinvestigateCD20expressionofmyelomacellsinbonemarrow,andanyrelationship betweenthestageofdisease,isotypeandclinicalfeatures.

Methods:Sixty-onepatientswho wereadmittedto thehematology clinicoftheAdnan MenderesMedicalSchoolwiththediagnosisofmultiplemyelomaaccordingtothe crite-riaofthe“InternationalMyelomaWorkingGroup”wereenrolledinthisstudy.Age,gender, Durie–Salmonstage,historyofautologoushematopoieticstemcelltransplantation,andthe distributionpatternandpositivityofCD20expressiononmultiplemyelomacellsinbone marrowwereevaluated.TheMann–WhitneyUandchi-squaretestswereusedforstatistical analysiswithap-value<0.05beingacceptedasstatisticallysignificant.

Results:Thirtypatients(48.9%)hadpositivescoresforCD20withthedistributionpattern beingmostlikelyinterstitialin55.6%ofthecases.Therewasnostatisticallysignificant dif-ferencebetweenimmunohistochemicalpositivityforCD20expressiononmultiplemyeloma cells,immunoglobulintype,andthestageofdisease.

Conclusion:Thecombinationofimmunohistochemicalstudieswith flowcytometrymay revealtheimportanceofCD20positivityinpatientswithmultiplemyelomamoreclearly.

©2014Associac¸ãoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published byElsevierEditoraLtda.Allrightsreserved.

Introduction

Multiplemyeloma(MM)isaclonalB-cellmalignancy char-acterizedbytheaccumulationofmatureplasmacellsinthe

∗ _{Correspondingauthorat:DepartmentofInternalMedicine,AdnanMenderesUniversityMedicalFaculty,Aytepe,09000Aydin,Turkey.}

E-mailaddress:gokhansargin@hotmail.com(G.Sargin).

bonemarrowandothertissues.ThemyeloidorotherB-cell markerssuchasCD10,CD19,CD20andCD22mayberelated totheprognosisofpatientswithMM.1_{Accordingtostudies,}

CD20expressionmayberelatedtoapoorprognosis,however theprognosticsignificanceinpatientswithMMneedsfurther

http://dx.doi.org/10.1016/j.bjhh.2014.11.013

revbrashematolhemoter.2015;37(1):34–37

35

investigation.1_{CD20isaphosphoprotein,whichplaysarolein}

B-celldevelopment.Therearefewstudiesabout immunohis-tochemicalevaluationsofCD20expressiononmyelomacells inMM.2–4 _{Robillard et al.}2 _{reported 12 (18%)of66 patients}

withCD20expression.TheexpressionofCD20bymyeloma cellsisheterogeneous,andcanbedetectedonlyin13–22% ofpatients.3_{Thesamestudyreportedthatdisease}

stabiliza-tionisseenin50–57%ofCD20+_{patientsoveraperiodof10–27}

months.3 _Mateoetal.5 _{reportedCD20expressionin17% of}

allMMcaseswithmostofthemhavingonlyCD20+_plasma

cells.Inrecentyears,studieshavefocusedonsurface anti-gensduetotheimportanceoftargetedtherapies.Theroleof anti-CD20therapyforMMhasyettobeclearlyestablished.4

ThisstudyaimedtoinvestigateCD20expressiononmyeloma cellsinbonemarrowandanyrelationshipbetweenthestage ofdisease,isotypeandclinicalfeatures.

Methods

Sixty-one patients (31 male, 30 female, mean age 64±11 years) with MM were enrolled in this study. The distribu-tionpatternand positivityofCD20expressiononmyeloma cellsinbonemarrow,age, gender,Durie–Salmonstageand the history of autologous hematopoietic stem cell trans-plantationwereevaluated.ClinicalcharacteristicsandCD20 expressionwereevaluatedatthetimeofdiagnosis. Immuno-histochemicalstainingofCD38,CD138,Kappa/Lambdawere appliedtolysinecoatedslides.CD20expressionwas deter-mined by immunohistochemical staining of bone marrow biopsies.Immunohistochemical analysis wasperformed by theavidin–biotincomplexmethod.Blymphocyteswere evalu-atedcomparingimmunohistochemicalanalysisofCD20with hematoxylin–eosin stain. The patients were evaluated for CD20positivityusingacut-offvalueof10%.Wecouldnot com-pareimmunohistochemistry withflow cytometry todetect CD20inmyelomacells.Statisticalanalysis(Mann–Whitney

U and chi-square tests) was performed using the Statisti-calPackagefortheSocialSciencesandap-value<0.05was acceptedasstatisticallysignificant.

Results

TheimmunoglobulintypewasIgGheavychainfor38(62.3%) patientswithnopatientshavingIgMmyeloma.Forty-three patients(70.5%)werestage3accordingtotheDurie–Salmon staging system and 11 patients had medical histories of autologoushematopoietic stem cell transplantation.Thirty patients(49.2%)hadpositivescoresforCD20withthe distribu-tionpatternbeingmostlikelyinterstitialin55.6%.Although thecut-offvalueforCD20positivitywas10%,CD20 expres-sionwasdetectedin28.7%ofallcasesusingacut-offvalueof 20%.TheclusteredpatternofCD20positivemyelomacellsis showninFigure1.Thecharacteristicfeaturesofpatientswith multiplemyelomaareshowninTable1.Therewasno statis-ticallysignificant differencebetweenimmunohistochemical positivityforCD20ofmyelomacells,immunoglobulintype, andthestageofdisease(p-value>0.05)(Table2).

Figure1–clusteredpatternofCD20-positivemyelomacells inbonemarrow.

Table1–Characteristicfeaturesofpatientswith multiplemyeloma(n=61).

Variable

Gender(M/F) 30/31

Meanage(years) 64±11

Durie–Salmonsystemstage(n)

I 14

II 4

III 43

Immunoglobulintype(n)

IgG 38

IgA 9

Other 6

Lightchain 8

CD20positivitya_{(n) 30}

a _{Cut-offvalueof10%.}

Table2–Associationbetweenimmunohistochemical positivityforCD20ofmyelomacellsand

immunoglobulintypeandthestageofdisease.

Immunohistochemical positivityforCD20a

p-Value

Durie–Salmonsystemstage(n)

I 4

II 1 >0.05

III 25

Immunoglobulintype(n)

IgG 23

IgA 2 >0.05

Other 1

Lightchain 4

36

Discussion

Differentstagesofdifferentiationoftheneoplasticclonemay causeheterogeneityinMM.Cellsurfaceantigensmaybe help-fultodeterminetheantigenicphenotype.Similarresultshave beenreportedfortheheterogeneityofantigenicexpression ofplasma cellsusingtwotechniques (immunofluorescence andimmunohistochemistry)withtheresultsdrawing atten-tiontotheantigenicheterogeneity.6_{Theantigenexpression}

ofmyelomacells isheterogeneous,andimmunophenotype impactsonclinicaloutcomeinMM.7

CD20isatransmembranephosphoproteinthatactsasa calciumionchannelinthecellmembrane,andplaysarole inBlymphocyteactivationanditsdifferentiationtoplasma cells.TheimportanceofthisB-cellantigenforplasmacellsis stillunknown.8

Flowcytometryisaneasy and commonlyused method todeterminecellsurfaceantigens.Apanelincluding CD20 is recommended bythe International Consensus Group to determine plasma cell immunophenotype.9 _{The European}

MyelomaNetwork recommendsa minimalpanelincluding CD19,andCD56butprefersapanelthatincludesCD20,CD117, CD28,CD27todetectabnormalplasmacells.10_{Robillardetal.}2

reportedCD20expressionin12of66patientswithMM.CD10, CD20 and HLA-DR were weakly positive in less than one-thirdofpatientsinastudyconsistingof112untreatedMM patients.6_Ngoetal.7_{evaluated107MMpatientsandreported}

theclinicalimpactofimmunohistochemicalmarkersinbone marrowbiopsysampleswith32%ofthembeingpositivefor CD20.LossofCD20expressionduringthediseasecourse cor-relateswithsignificantworseningbothofoverallsurvivaland event-freesurvivalcomparedtothetimeofdiagnosis.7

Grigo-riadisetal.11_{analyzednewlydiagnosedplasmacellmyeloma}

withanaimofidentifyingclinicopathologicalfeaturesofCD20 inthisdiseaseandfoundthatCD20+_{plasmacellsarenota}

uniquesubsetinmyeloma.CD20positivitywasreportedin 17%ofallmultiplemyelomapatients.5_{Inthecurrentstudy,}

48.9%ofthepatientshadpositivescoresforCD20witha cut-offvalueof10%and28.7%ofthepatientswithacut-offvalue of20%.

CellmorphologymaybeassociatedwithCD20positivity. Histologicaltypeofplasmacellsandprognosiswere evalu-atedin674patientswithMM.Asignificantrelationshipwas reported betweenCD20phenotype and maturecells, more importantlywithsmallplasmacells(lymphoplasmacytic).12

Both morphologies are characterized by low-grade malig-nancy.Acorrelation hasalsobeen reportedbetweensmall matureplasmacells,CD20andt(11.14)inpatientswithMM.2

Inaddition,therewerestatisticallysignificantdifferencesin the expressionofCD20betweenplasmacell leukemia and MM with CD20 displaying higher reactivity in plasma cell leukemia8_{;noneofthepatientsinthecurrentstudyhadthe}

latterdisease.

The CD20 antigen is a suitable target in the treat-ment of lymphoma patients, but the use of monoclonal antibodies to treat MM is still controversial. The differ-encesbetweenthesetwomalignanciesaretheheterogeneity and the weak expression of CD20 on plasma cells. Thus, CD20 positive MM and Non-Hodgkin’s lymphoma with

plasmacytoid differentiation should be treated as separate entities.

The patientsin this study had aM-protein peakin the gammazonebyserumproteinelectrophoresis.Therewasno splenomegalyororganomegaly.Somestudieshavereported thatabouthalftheplasmacellshaveamorphologically lym-phoplasmacytic appearance. Immunohistochemical studies maybehelpfulinthesecases.Cytoplasmicimmunoglobulin isusuallypositive,butnotCD20onplasmacells.13_The

asso-ciation betweenCD20expressionandsmallmatureplasma cellmorphologywasreportedinthestudyofRobillardetal.2

ThesignificanceofCD20expressioninMMpatientsisnotwell understoodyet.

The CD20 antigen is associated with shorter survival, and may be a poor prognosticfactor forMM.4,6,14

Accord-ingtoGrigoriadisetal.,11_CD20+_{plasmacellmyelomacases}

representaheterogeneousdiseaseandnotaunique clinico-pathological entity. However,the prognosticsignificanceof CD20expressioninMMaloneisunclear.Theeffectofother markersofthediseaseisnotknownclearly.4,6

CD20+_{MMcellsoccurredinthreepatternsinthestudyby}

Quinnetal.4_{;thepatternswerereportedasfollows:diffuse}

(63%),interstitial(33%),andclustered(4%).Therelationship betweenpattern,isotypeinbonemarrowandCD20positivity hasbeenreportedintheliterature.4_{Accordingtothisstudy,}

24caseswerepositiveforCD20,62.5%withIgGheavychain andtencaseswithlambdalightchain.Inthecurrentstudy 38(62.2%)patientshadIgGheavychainMMbutnopatients had IgMmyeloma. Moreover there was a higher positivity for CD20 expression compared tothe literature; according tothecut-offvalueof20%,28.7%ofthepatientswere posi-tive.

The limitations of this study were the small number of cases and the use of only one methodology, immuno-histochemical analysis, to evaluate CD20 expression. The prognostic significance of CD20 positivity might be better evaluated bycombining flow cytometryand immunohisto-chemicalanalysisinlargercohorts.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

r

e

f

e

r

e

n

c

e

s

1.Ruiz-ArgüellesGJ,SanMiguelJF.Cellsurfacemarkersin multiplemyeloma.MayoClinProc.1994;69(7):684–90.

2.RobillardN,Avet-LoiseauH,GarandR,MoreauP,PineauD, RappMJ,etal.CD20isassociatedwithasmallmatureplasma cellmorphologyandt(11;14)inmultiplemyeloma.Blood. 2003;102(3):1070–1.

3.KapoorP,GreippPT,MoriceWG,RajkumarSV,WitzigTE, GreippPR.Anti-CD20monoclonalantibodytherapyin multiplemyeloma.BrJHaematol.2008;141(2): 135–48.

revbrashematolhemoter.2015;37(1):34–37

37

5. MateoG,CastellanosM,RasilloA,GutiérrezNC,Montalbán MA,MartínML,etal.Geneticabnormalitiesandpatternsof antigenicexpressioninmultiplemyeloma.ClinCancerRes. 2005;11(10):3661–7.

6. SanMiguelJF,GonzálezM,GascónA,MoroMJ,HernándezJM, OrtegaF,etal.Immunophenotypicheterogeneityofmultiple myeloma:influenceonthebiologyandclinicalcourseof disease.Castellano-Leones(Spain)CooperativeGroupforthe StudyofMonoclonalGammopathies.BrJHaematol. 1991;77(2):185–90.

7. NgoNT,BrodieC,GilesC,HorncastleD,KlammerM,Lampert IA,etal.Thesignificanceoftumourcellimmunophenotype inmyelomaanditsimpactonclinicaloutcome.JClinPathol. 2009;62(11):1009–15.

8. García-SanzR,OrfãoA,GonzálezM,TaberneroMD,BladéJ, MoroMJ,etal.Primaryplasmacellleukemia:clinical, immunophenotypic,DNAploidy,andcytogenetic characteristics.Blood.1999;93(3):1032–7.

9. BraylanRC,OrfaoA,BorowitzMJ,DavisBH.Optimalnumber ofreagentsrequiredtoevaluatehematolymphoidneoplasias: resultsofaninternationalconsensusmeeting.Cytometry. 2001;46(1):23–7.

10.RawstronAC,OrfaoA,BeksacM,BezdickovaL,BrooimansRA, BumbeaH,etal.ReportoftheEuropeanMyelomaNetworkon multiparametricflowcytometryinmultiplemyelomaand relateddisorders.Haematologica.2008;93(3):431–8.

11.GrigoriadisG,GilbertsonM,CameN,WestermanD,FellepaF, JeneN,etal.IsCD20positiveplasmacellmyelomaaunique clinicopathologicalentity?Astudyof40casesandreviewof theliterature.Pathology.2012;44(6):552–6.

12.BartlR,FrischB,Fateh-MoghadamA,KettnerG,JaegerK, SommerfeldW.Histologicclassificationandstagingof multiplemyeloma.Aretrospectiveandprospectivestudyof 674cases.AmJClinPathol.1987;87(3):341–55.

13.HoyerJD,HansonCA,FonsecaR,GreippPR,DewaldGW, KurtinPJ.The(11;14)(q13;q32)translocationinmultiple myeloma.Amorphologicandimmunohistochemicalstudy. AmJClinathol.2000;113(6):831–7.