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

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

Revista

Brasileira

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Hematologia

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Hemoterapia

Brazilian

Journal

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Hematology

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Hemotherapy

Original

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Comparison

of

microRNA

expression

in

high-count

monoclonal

B-cell

lymphocytosis

and

Binet

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chronic

lymphocytic

leukemia

Felipe

Magalhães

Furtado

,

Priscila

Santos

Scheucher

,

Bárbara

Amélia

Santana

,

Dalila

Lucíola

Zanette

_,

_Rodrigo

_do

_Tocantins

_Calado

_,

_Eduardo

_Magalhães

_Rego

_,

Daniel

Mazza

Matos

_,

_Roberto

_Passetto

_Falcão

a_{FaculdadedeMedicinadeRibeirãoPreto(FMRP),RibeirãoPreto,SP,Brazil}

b_{Fundac¸ãoOswaldoCruz(FIOCRUZ),Salvador,BA,Brazil}

c_{HospitalUniversitárioWalterCantídio(HUWC),Fortaleza,CE,Brazil}

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Articlehistory:

Received30November2016 Accepted24March2017 Availableonline22April2017

Keywords:

MonoclonalB-celllymphocytosis Chroniclymphocyticleukemia microRNA

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Background:EvidencesuggeststhatmonoclonalB-celllymphocytosisprecedesallchronic

lymphocyticleukemiacases,althoughthemolecularmechanismsresponsiblefordisease progressionarenotunderstood.AberrantmiRNAexpressionmaycontributetothe patho-genesisofchroniclymphocyticleukemia.TheobjectiveofthisstudywastocomparemiRNA expressionprofilesofpatientswithBinetAchroniclymphocyticleukemiawiththoseof sub-jectswithhigh-countmonoclonalB-celllymphocytosisandhealthyvolunteers(controls).

Methods:Twenty-onechroniclymphocyticleukemiapatients,12subjectswithmonoclonal

B-celllymphocytosisandtenhealthyvolunteerswereenrolledinthisstudy.Flowcytometry CD19+_CD5+_{-basedcellsortingwasperformedforthechroniclymphocyticleukemiaand}

monoclonalB-celllymphocytosisgroupsandCD19+_{cellsweresortedtoanalyzethecontrol}

group.TheexpressionsofmiRNAs(miR-15a,miR-16-1,miR-29b,miR-34a,miR-181a, miR-181bandmiR-155)weredeterminedbyquantitativereversetranscriptasepolymerasechain reaction(qRT-PCR).

Results:Significant differences between the expressions in the chronic lymphocytic

leukemiaandmonoclonalB-celllymphocytosisgroupswererestrictedtotheexpression ofmiR-155,whichwashigherintheformergroup.Acomparisonbetweenhealthy con-trolsandmonoclonalB-celllymphocytosis/chroniclymphocyticleukemiapatientsrevealed highermiR-155andmiR-34alevelsandlowermiR-15a,miR-16-1,miR-181aandmiR-181b inthelattergroup.

∗ _{Correspondingauthorat:LaboratoriodeHematologia–HospitaldasClinicasdeRibeiraoPreto,BandeirantesAvenue,3900,MonteAlegre,}

14051-140RibeirãoPreto,SP,Brazil.

E-mailaddress:felipemf@usp.br(F.M.Furtado). http://dx.doi.org/10.1016/j.bjhh.2017.03.006

Conclusions: OurresultsshowaprogressiveincreaseofmiR-155expressionfromcontrols tomonoclonalB-celllymphocytosistochroniclymphocyticleukemia.TheroleofmiR-155 inthedevelopmentofovertchroniclymphocyticleukemiainindividualswithmonoclonal B-celllymphocytosismustbefurtheranalyzed.

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

Introduction

Chroniclymphocyticleukemia(CLL)isamalignantneoplasm characterizedbyanexcess ofmonoclonalBlymphocytesin peripheralblood,bonemarrow,thespleenandlymphnodes.1 CLListhemostcommonleukemiaintheWesternworld,with anannualincidenceof5.1cases/100,000people.2

Over the last years, advances in multi-parameter flow cytometryallowedtheidentificationofsmallpopulationsof monoclonalBlymphocytesinthebloodofapparentlyhealthy subjects.3_{ThepresenceofmonoclonalB-cellsinthe} periph-eralbloodofupto5×109_{/LisclassifiedasmonoclonalB-cell}

lymphocytosis(MBL)intheabsenceofotherlymphomatous features.4,5 _{This condition is found in 0.6–12% of healthy} individuals.3,6,7

MBL is classified as high-count or low-count MBL. The first is usually diagnosed in asymptomatic subjects with mildlymphocytosis and the latteroccurs inasymptomatic subjectswithnormalbloodcountswho havebeen submit-tedtoflowcytometryscreening.8–10 _{ThemostrecentWorld} HealthOrganization(WHO)classificationdefines low-count MBLasaperipheralbloodCLL countof<0.5×109_/Landno

extramedullarydisease.4_{AsinCLL,MBLismorecommonin} menandinrelativesofCLLpatients11–13 _{withitsfrequency} increasingwithage.11,12,14–17

Virtually,allCLLcasesareprecededbyMBL.18_Eventhough themechanismsunderlyingCLLpathogenesisandMBL pro-gressiontoCLLarestillnotwellunderstood,itisspeculated that the initial genetic lesion in MBL may occur in the immaturebonemarrowBcellcompartmentandafterwards repetitiveantigenicstimulationmayinduceadditionalgenetic lesions,eventuallyleadingtoneoplastictransformation.19

MicroRNAs (miRNA) are 19–25 nucleotide single strand RNAsresponsibleforgeneexpressionandcellularmetabolism regulation.20,21_{ChangesinmiRNAexpressionhavebeen} asso-ciatedwithsolidandhematologictumors.21_{SomemicroRNAs} havebeenshowntobeabnormallyexpressedinCLL.22_Studies addressingthemolecularandgeneticbasisofMBLmayhelp toelucidateinitialstepsofCLLpathogenesisand,therefore, increaseknowledgeabout CLLorigin.Moreover,onlyafew studieshaveinvestigatedmiRNAsinMBL.9,23_Herewe hypoth-esizedthatabnormalitiesinsomemiRNAexpressionsmaybe presentinMBLandmayhavearoleininitialmonoclonalB-cell expansion.

Methods

Cellsamplesfromnormalcontrols,individualswith high-countMBLandCLLpatients

Samples from 12 individuals with high-count MBL and 21 patients withstageACLL accordingtotheBinet classifica-tionwereanalyzed.Allthepatientswerebeingmonitoredin auniversityhospitalinRibeiraoPreto,SaoPaulo,Brazil.Ten healthyindividualswerestudiedasacontrolgroup.Thestudy wasapprovedbytheInstitutionEthicsResearchReviewBoard andwritteninformedconsentwasobtainedfromall partici-pantsinaccordancewiththeDeclarationofHelsinkiandits revisions.

Usingcellsortingbyflowcytometry,CD19+_CD5+

lympho-cyteswereisolatedfromtheperipheralbloodofindividuals inthe twostudy groups, andCD19+ _{lymphocytesfrom the}

peripheral blood ofcontrolsas previouslydescribed.24 _The meanpercentageofthedesiredcellpopulationafterisolation was89.48%(±8.48%).

The clinical characteristics of the enrolled subjects are summarizedinTable1.

RNAisolationandmiRNAexpression

RNAwasextractedfromCD5+ _{Blymphocytesisolated from}

thestudygroupsandBlymphocytesfromthecontrolgroup, usingTrizol(LifeTechnologies,California,USA).Seven miR-NAsknowntohaveabnormalexpressionsinCLLwereselected foranalysis(miR-15a,miR-16-1,miR-29b,miR-34a,miR-181a, miR-181b and miR-155). miRNA expression was quantified by TaqMan miRNAquantitative reverse-transcription poly-merasechainreaction(qRT-PCR)aspreviouslydescribed.25,26 Briefly, 5ng of total RNA were reverse transcribed using themicroRNAreverse-transcriptionkit(AppliedBio-systems, California, USA) with specific stem-loop primers. qRT-PCR analysis was performed using the miRNA-specific Taqman assay(Applied Bio-systems).Expressionlevelsofthe nucle-olar RNAs RNU24, RNU44 and RNU48 were similar for all groupsandthegeometricmeanoftheirexpressionwasused to normalize the expression of the miRNAs, as previously described27_usingthe2−Ct_{formula.Thecoefficientsof}

Table1–Characteristicsoftheenrolledsubjects.

Characteristic Control High-count

monoclonalB-cell lymphocytosis

Chroniclymphocytic leukemia

Numberofsubjects 10 12 21

Mediumage 38 78 70

Range(years) 25–60 57–97 58–81

Gender(n)

Male 2 9 11

Female 8 3 10

Mediumnumberoflymphocytes 2.064 3.933 41.714

Range(L) 1.360–3.300 2.900–5.900 7.500–147.000

%CD5+_{B-Lymphocytesoftotallymphocytes – 34.9 78.02}

Range(%) – 5.97–61.01 58.63–91.09

itsvaluewasconsideredunavailable.AllmiRNAswerestudied induplicate.

Statisticalanalysis

The Kolmogorov–Smirnov normality test was initially per-formedtocomparemiRNAexpressions.Theexpressionsof themiRNAs,miR-15a,miR-16-1,miR-29b,miR-181band miR-155,didnotdeviatefromthenormaldistributionandvariance analysis(ANOVA)wasusedfollowedbytheBonferoniposthoc

test.TheKruskal–Wallisnon-parametrictest(non-parametric ANOVA) was used followed by the Dunn post hoc test for the miRNAs miR-34a and miR-181a that had non-normal distributions.Alltestswithp-values≤0.05 wereconsidered statisticallysignificant.

Results

miRNAexpression

Ofthe sevenmiRNAsdeemed relevantforCLL pathogene-sis,miRNA-155andmiR-34aweredifferentiallyexpressedin theCLL and high-count MBLgroups comparedtothe con-trols(p-value=0.33andp-value=0.003forMBLandcontrols and p-value<0.001 and p-value=0.02 forCLL and controls, respectively)withthehighestvaluesdetectedinCLL(Figure1). miRNA-155expressioninhigh-countMBLhadatendencyto begreater thaninhealthysubjects,but thisdifferencewas notstatisticallysignificant,maybeduetothesmallnumberof samplesstudied(Figure1).miR-34aexpressionwasnot sta-tisticallydifferentbetweenCLLandMBL(Figure1).miR-15a, miR-16-1,miR-181aandmiR-181bweredown-regulatedinCLL andMBL.TheirexpressionwaslowerinCLL patientswhen comparedtocontrols(p-value<0.001forallmiRNAs). High-countMBLsubjectsalsohadlowerexpressionthancontrols formiR-15a,miR-16-1andmiR-181b(p-value=0.003)andfor miR-181a(p-value<0.001)(Figure1).

Expression of miR-29b was similar for all three groups (Figure1).

Discussion

This study with a Binet A CLL group and a high-count MBLgroupdemonstratednewmoleculardifferencesbetween thesetwoentities.

Statisticalsimilaritieshavebeenidentifiedregardingthe frequenciesofimmunoglobulinheavychainvariable(IGHV) genesbetweenhigh-countMBLandtheinitialstagesofCLL, althoughfindingsfromlow-countMBLweredifferentbetween thesegroups.28,29_{Otherauthorsalsofoundbiological} similari-tiesbetweenthesethreeentities.Notonlyhigh-countMBLbut alsolow-countMBLbearcytogeneticabnormalitiescommon inCLL,including13q-,17p-andtrisomy12.6,30–32

Aiming to betterunderstand genetic alterations inCLL, this study comparedtheexpressionsofmiRNAspreviously describedasalteredinthisdiseasewiththeirexpressionin MBL.KnowledgeofmiRNAexpressionindifferentmonoclonal proliferationstagesmayimprovethecomprehensionofCLL pathophysiology.

Severalstudiesusingdifferentmethodologieshave iden-tified overexpressionofmiR-155 inCLL whencomparedto normal controls.23,33–37 _{Our results confirmed the seminal} findingsofFerrajolietal.whoshowedthattheexpressionof miR-155isgreaterinBinetACLLthaninhigh-countMBLand thatitsexpressionintheseindividualsisgreaterthanin nor-malcontrols.23_{Thus,ourfindingssuggestthatmiR-155may} beusedasaprogressionmarkerforMBLindividuals,butthis mustbeconfirmedbyfurtherspecificstudies.

A

miR155

miR15a

miR181a miR181b

miR29b

miR16-1 miR34a

2

–

∆

CT

2

–

∆

CT

X10

3

2

–

∆

CT

X10

2

2

–

∆

CT

X10

3

2

–

∆

CT

X10

4

2

–

∆

CT

X10

2

2

–

∆

CT

X10

4

4 150

100

50

0 3

2

1

0

6 80

60

40

20

0 5

4

3

2

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0

60 1500

1000

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0 40

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Grupos de estudo

Grupos de estudo

Grupos de estudo Grupos de estudo

Grupos de estudo

Grupos de estudo Grupos de estudo

CLL x MBL p=0,002 CLL x Control p<0,001 MBL x Control p=0,33

CLL x MBL p=1,0 CLL x Control p<0,001 MBL x Control p=0,001

CLL x MBL p=1,0 CLL x Control p<0,001 MBL x Control p=0,003

CLL x MBL p=1,0 CLL x Control p<0,001 MBL x Control p<0,001

CLL x MBL p=1,0 CLL x Control p=1,0 MBL x Control p=1,0

CLL x MBL p=1,0 CLL x Control p<0,001 MBL x Control p<0,001 CLL x MBL p=0,96 CLL x Control p=0,02 MBL x Control p=0,003

Control Cont

rol

MBL CLL MBL

Control Control

MBL CLL MBL

Cont rol

Cont rol

MBL CL MBL

L

CLL

Control

MBL CLL

CLL CLL

B

C

D

E

F

G

ThisstudyalsoidentifiedmiR-34atobeoverexpressedin CLLwhencomparedtohealthycontrols.35,40,41 _ThismiRNA hasbeenassociatedtothe regulationofthe tumorprotein p53(TP53) pathway41–45 _{and may berelated tofludarabine} resistanceinCLL.44

The study that first demonstrated down-regulation of miR-15aand miR-16-1inCLL patientswas alsothefirst to demonstrateabnormal expressionsofmiRNAs incancer.46 Subsequent studies using different techniques have con-firmedthisfinding,particularlyinpatientswithdel13q14.47–49 OtherauthorsfoundsimilarexpressionsofthesemiRNAsin CLLandnormalcontrolsusingmicroarrays35_andRT-qPCR.33 Kleinetal.demonstratedthatmousemodelsthathavethe chromosomearea(13q14)responsibleforthetranscriptionof thesemiRNAs deleted developedmonoclonalexpansion of lymphocytesinblood.50

Inthis study,the miRNAsmiR-181aand miR-181b were down-regulatedinCLL whencomparedtonormalcontrols. Other authors found similar results in studies using RT-qPCR,36,48 _{Northern Blot}34 _{and microarrays.}35 _{Visone et al.} demonstratedthat down-regulationofmiR-181b isabetter markerforworseprognosisinCLLthan theIGHVmutation statusandZAP-70expression,andsuggestthatitsexpression shouldbemonitoredinCLLpatients.51

The pattern of miR-29b expression in CLL is not well understood.HeretheresultsforCLLBinetA,high-countMBL andnormalcontrolsweresimilar.Sampathetal.foundthat 70%oftheCLLpatientshaveasimilarexpressiontonormal controls.49_{Fulcietal.alsohadresultssimilartothisstudy.}33 However,Santanam et al. foundthis miRNA tobe overex-pressedinCLL,andZhuetal.foundittobedown-regulated inCLL.48,52 _{All thesestudieshave heterogeneousgroupsof} CLL patientsregarding the stageofthe disease.These dif-ferentresultssuggestthatmiR-29bexpressionmaychange accordingtodiseasestage.However,thishypothesisshould beconfirmedinfuturestudies.

ApartfromthestudyofFerrajolietal.onmiR-155,onlyone othergrouphasevaluatedmiRNAexpressioninhigh-count MBL.23_{Thus,Morabitoetal.usedmicroarraystostudy} indi-vidualspreviouslydiagnosedwithCLLbeforethechangeof diagnosiscriteriaproposedbytheInternationalWorkshopon ChronicLymphocyticLeukemiain2008.9,53_Theyfound miR-130awastheonlyonewithdifferentexpressionsbetweenCLL and high-countMBL.Using another techniqueand a more heterogeneoushigh-countMBLpopulation,thisstudyfound theexpressionsofthemiR-34a,miR-15a,miR-16-1,miR-181a, miR-181bandmiR-29btobesimilarinCLLandhigh-count MBL.

Theresultsreportedhereshouldbeconfirmedbyfurther studies, since the groups were small and more individ-ualsmustbestudiedtoallowrobustconclusions.Moreover, althoughthe CLL and MBL groups were composed mainly of older individuals and the MBL group mainly of males, ourcontrolgroupwascomposedmainlyoffemalesandthe medium age for this group was lower than for the study groups,althoughtherearenodatasuggestingthatthelevels ofthesemiRNAschangewithsexandage.

Inconclusion,thefindingthatsomemiRNAshave abnor-malexpressionsinMBLandthatthisconditionisalsopresent inCLLsuggeststhatthesegeneticchangesmaybepartofthe

initialeventsresponsibleformonoclonalCD5+_B-cell

prolifer-ation.ThedifferentialexpressionofmiR-155inMBLandCLL suggestsitsroleinsignalingpathwaysthatareimportantto thedevelopmentofthedisease.

Author

contributions

FMF,DLZ,RTCSR,EMR,DMMandRPFdesignedtheresearch protocols.FMF,PSSandBSperformedtheresearch.FMF,BS, DMMandRPFanalyzedthedata.FMF,RTCSR,EMR,DMMand RPFwrotethemanuscript.

Conflict

of

interests

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

This research was supported by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico(CNPQ) grant 573.754/2008-0.

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