Rev. Bras. Hematol. Hemoter. vol.39 número2

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rev bras hematol hemoter. 2017;39(2):183–185

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

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Letter

to

the

Editor

Stathmin

1 expression

in

plasma

cell

neoplasms

DearEditor,

Multiplemyeloma(MM)isahematologicalmalignancy char-acterizedby clonalproliferation ofmalignant plasma cells whichaccumulateinthebonemarrow,resultinginrecurrent hypercalcemia,anemia,osteolyticlesions,renalfailure,and increasedriskofinfection.1 _Stathmin _1,_also _named onco-protein18(OP18)orleukemia-associatedphosphoproteinp18 (LAP18), is amicrotubule destabilizer that playsan impor-tant role in cell progression, clonogenicity, differentiation andsurvival.2 _Stathmin₁_{overexpression}_has_been_reported in hematological malignancies, including acute myeloid leukemia, acute lymphoid leukemia, lymphoma, high-risk myelodysplastic syndromes and primary myelofibrosis.3–7 Functionalstudiesindicatethathighstathmin1expressionis abletosustainrapidcelldivisionandproliferationofleukemia cells,whicharesuppressedbystathmin1inhibition.5,8,9_Using themicroarrayapproach,stathmin1hasbeen identifiedas oneof15relevantgenesthatdeterminetheoutcomeinMM patients.10

TheaimofthepresentstudywastoinvestigateStathmin 1 expression in respect to clinical and laboratorial char-acteristics, and outcomes of MM patients. Bone marrow samplescollected from healthy donors(n=21, medianage

A

Stathmin 1 expression in bone marrow cells

MM patients MM patients

5 100 100

80

60

40

20

0 80

60

Low stathmin 1 expression Low stathmin 1 expression High stathmin 1 expression High stathmin 1 expression

p=0.86 p=0.63

40

20

0 10

0 20 30 40 50 60 70 0 10 20 30 40 50 60 70 80 4

3

2

1

0

Healthy donor

n=21 _Months _Months

PCL n=4 MM

n=30

Relative lev

e

l of

stathmin 1

mRNA expression

Progression free survival, %

Overall survival, %

C

B

Figure1–Stathmin1expressioninplasmacellneoplasms.(A)QuantitativepolymerasechainreactionanalysisofStathmin

1mRNAexpressioninbonemarrowcellsfromhealthydonors,andfrompatientswithmultiplemyeloma(MM)orplasma

cellleukemia(PCL).Thehypoxanthinephosphoribosyltransferase1genewasusedasthereferencegeneandahealthydonor

wasusedasacalibratorsample.Horizontallinesindicatemedians.Progressionfreesurvival(B)andoverallsurvival(C)of

MMpatientsstratifiedbymedianStathmin1expression.p-ValueswerecalculatedbytheLong-ranktest.

33 years;range: 18–69),MMpatients(n=30;medianage 64 years;range:45–86)andplasmacellleukemiapatients(PCL;

n=4;medianage72years;range:68–84)were analyzed.All patients included in the study were untreated atthe time of sample collection. Patients’ characteristics are listed in

Table1.Allhealthycontrolsandpatientsprovidedinformed

writtenconsentandthestudywasapprovedbytheResearch EthicsCommitteeoftheUniversidadeEstadualdeCampinas. MM patients received cyclophosphamide, thalidomide and dexamethasone (CTD; n=15), bortezomib, thalidomide and dexamethasone(VTD;n=1),CTD/VTD(n=4),ormelphalan, prednisoloneandthalidomide(MPT;n=9)protocoltreatments orwatchfulwaiting(n=1).TotalRNAwasobtainedfromtotal bonemarrowcellsafterremovaloferythrocytesbyhemolysis usingtheTRIzolreagent(Invitrogen,Carlsbad,CA,USA).The genomicDNAwaseliminatedusingDNAseItreatment (Invit-rogen).ThecDNAwasobtainedfrom1␮gofRNAusingthe

RevertAidTM_First_Strand_cDNA_Synthesis_Kit_(MBI_Fermentas,

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revbrashematolhemoter.2017;39(2):183–185

Table1–TheassociationbetweenclinicalandlaboratorialcharacteristicsofmultiplemyelomapatientsandStathmin1

expression.

Factors n=30 Stathmin1expressiona _p_-Value

Low High

Age(years) 64(45–86)

<60 10 3 7 0.12

≥60 20 12 8

Gender

Male 17 8 9 0.71

Female 13 7 6

Isotype

IgG 20 12 8 0.12

IgA 6 1 5

Lightchain 4 2 2

ISS

I 3 1 2 0.62

II 10 6 4

III 16 7 9

N/A 1 1 0

Albumin(g/L) 36.8(29.9–46.6)

<35 10 4 6 0.52

≥35 19 10 9

N/A 1 1 0

Hemoglobin(g/L) 88(67.8–176)

<100 18 7 11 0.07

≥100 11 8 3

N/A 1 0 1

ˇ2-Microglobulin(pg/mL) 5650(1900–3040)

<3500 3 1 2 0.54

≥3500 25 13 12

N/A 2 1 1

BMplasmacellsrate(%) 29(1–68)

<10 5 3 2 0.92

≥10–30 12 6 6

≥30 12 6 6

N/A 1 0 1

Therapeuticoutcome

VGPR/PR 8/11 5/5 3/6 0.91

SD/PD 6/1 3/0 3/1

Earlydeath 4 2 2

ISS:InternationalStagingSystem;BM:bonemarrow;VGPR:verygoodpartialresponse;PR:partialresponse;SD:stabledisease;PD:progressive disease.

a _Multiple_myeloma_patients_were_divided_into_groups_based_on_Stathmin₁_expression;_Low_Stathmin₁_expression_was_defined_as_lower_median

valuesandhighStathmin1expressionwasdefinedashighermedianvalues.

phosphoribosyltransferase 1 (HPRT1) (FW: GAACGTCTTGCTC-GAGATGTGA; RV: TCCAGCAGGTCAGCAAAGAAT) and the Maxima Sybr green qPCR system (MBI Fermentas). HPRT1

wasusedasthegenereference.ReactionconditionsforRNA extraction,cDNAsynthesisandqPCRwereperformed accord-ingtothemanufactures’instructions.Anegative‘notemplate control’wasincludedforeachprimerpair.Thedissociation protocolwasperformedattheendofeachruntocheckfor nonspecific amplification. Three replicas were run on the sameplateforeachsample.Therelativegeneexpressionwas calculatedusingtheequation,2−CT_.11_Additional_details_of qPCRexperimentswillbeprovideduponrequest.Astatistical analysiswasperformedusingGraphPadPrism5(GraphPad Software, Inc., San Diego, CA, USA). For comparisons, the

Kruskal–WallistestwithDunn’sposttestwasusedfor mea-sured variables with three levels and the chi-squared test wasusedforcategoricalvariables.Coxregressionwasused toestimateoverallsurvival(OS)andprogressionfreesurvival (PFS). OSwasdefinedasthe time(inmonths) betweenthe dateofsamplingandthedateofdeath(fordeceasedpatients) or last follow-up (for censored patients). PFS was defined asthetime(inmonths)betweenthesamplingandthedate of the first event (symptomatic progression based on the InternationalMyelomaWorkingGroupcriteria12_or_death)_or last follow-up (for censored patients). A p-value<0.05 was consideredstatisticallysignificant.

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revbrashematolhemoter.2017;39(2):183–185

185

MM(median:0.86:range: 0.30–2.89) and PCL(median:0.79; range:0.41–4.38)patients(p-value>0.05;Figure1A).To eval-uatethecorrelationofStathmin1expressionwithclinicaland laboratorialfactorsfurther,MMpatientsweredividedintotwo groupsbased on medianStathmin 1levels: high Stathmin 1

expression(median:1.43;range:0.89–2.89)andlowStathmin 1expression(median:0.62;range:0.30–0.83).Inourcohortof MMpatients,noassociationswereobservedbetween Stath-min1expressionand clinicalfactors,laboratorialfactorsor therapyresponseratios(Table1).Stathmin1expressiondid notaffectPFS(lowStathmin1expressionversushigh Stath-min1expression–hazardratio:1.08;95%confidenceinterval: 0.46–2.53;p-value=0.87) nor OS (lowStathmin 1 expression versushigh Stathmin1expression– hazardratio:0.77;95% confidenceinterval:0.28–2.18;p-value=0.68)(Figure1BandC). Insummary,ourfindingssuggestthatStathmin1 expres-sionisnotdifferentlyexpressedinplasmacellneoplasmsand hasnoimpactupon MMoutcomes whenconsideredas an isolatedfactor.TheseresultshighlightthatStathmin1 over-expressionmaybelinkedtohigh proliferatehematological malignanciesasMMischaracterizedbyslowproliferationof malignantplasmacellsinthebonemarrow.13,14_Future_studies usinglargercohortsandfunctionalinvestigationsare neces-sarybeforedisregardingtheinvolvementofStathmin1inthe malignantphenotypeofplasmacellneoplasms.

Conflict

of

interest

Theauthorsdeclarenoconflictsofinterest.

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1. PalumboA,AndersonK.Multiplemyeloma.NEnglJMed.

2011;364(11):1046–60.

2. BellettiB,BaldassarreG.Stathmin:aproteinwithmanytasks.

Newbiomarkerandpotentialtargetincancer.ExpertOpin

TherTargets.2011;15(11):1249–66.

3. HanashSM,StrahlerJR,KuickR,ChuEH,NicholsD.

Identificationofapolypeptideassociatedwiththemalignant

phenotypeinacuteleukemia.JBiolChem.

1988;263(26):12813–5.

4. RoosG,BrattsandG,LandbergG,MarklundU,GullbergM.

Expressionofoncoprotein18inhumanleukemiasand

lymphomas.Leukemia.1993;7(10):1538–46.

5. Machado-NetoJA,deMeloCamposP,FavaroP,LazariniM,

Lorand-MetzeI,CostaFF,etal.Stathmin1isinvolvedinthe

highlyproliferativephenotypeofhigh-riskmyelodysplastic

syndromesandacuteleukemiacells.LeukRes.

2014;38(2):251–7.

6. NylanderK,MarklundU,BrattsandG,GullbergM,RoosG.

Immunohistochemicaldetectionofoncoprotein18(Op18)in

malignantlymphomas.HistochemJ.1995;27(2):155–60.

7. Machado-NetoJA,deMeloCamposP,FavaroP,LazariniM,da

SilvaSantosDuarteA,Lorand-MetzeI,etal.Stathmin1

inhibitionamplifiesruxolitinib-inducedapoptosisin

JAK2V617Fcells.Oncotarget.2015;6(30):29573–84.

8.JehaS,LuoXN,BeranM,KantarjianH,AtwehGF.Antisense

RNAinhibitionofphosphoproteinp18expressionabrogates

thetransformedphenotypeofleukemiccells.CancerRes.

1996;56(6):1445–50.

9.IancuC,MistrySJ,ArkinS,WallensteinS,AtwehGF.Effectsof

stathmininhibitiononthemitoticspindle.JCellSci.

2001;114(Pt5):909–16.

10.DecauxO,LodeL,MagrangeasF,CharbonnelC,GouraudW,

JezequelP,etal.Predictionofsurvivalinmultiplemyeloma

basedongeneexpressionprofilesrevealscellcycleand

chromosomalinstabilitysignaturesinhigh-riskpatientsand

hyperdiploidsignaturesinlow-riskpatients:astudyofthe

IntergroupeFrancophoneduMyelome.JClinOncol.

2008;26(29):4798–805.

11.LivakKJ,SchmittgenTD.Analysisofrelativegeneexpression

datausingreal-timequantitativePCRandthe2(−DeltaDelta

C(T))Method.Methods.2001;25(4):402–8.

12.KyleRA,RajkumarSV.Criteriafordiagnosis,staging,risk

stratificationandresponseassessmentofmultiplemyeloma.

Leukemia.2009;23(1):3–9.

13.PuthierD,Pellat-DeceunynckC,BarilleS,RobillardN,Rapp

MJ,Juge-MorineauN,etal.DifferentialexpressionofBcl-2in

humanplasmacelldisordersaccordingtoproliferationstatus

andmalignancy.Leukemia.1999;13(2):289–94.

14.ScudlaV,OrdeltovaM,BacovskyJ,VytrasovaM,HorakP,

MinarikJ.Therelationshipbetweenproliferationand

apoptosisinpatientswithmonoclonalgammopathyof

undeterminedsignificanceormultiplemyeloma.

Haematologica.2005;90(12):1713–4.

JoãoAgostinhoMachado-Netoa_,_Fernando_Vieira_Pericolea_, FernandoFerreiraCostaa_,_Fabiola_Trainaa,b_,

SaraTeresinhaOlallaSaada,∗

a_Universidade_Estadual_de_{Campinas/Hemocentro}_(UNICAMP),

Campinas,SP,Brazil

b_Universidade_de_São_Paulo_(USP),_Ribeirão_Preto,_SP,_Brazil

∗_{Corresponding}_author_at_:_Hematology_and_Hemotherapy

Cen-ter,University ofCampinas (UNICAMP),RuaCarlosChagas, 480,CEP13083-878,Campinas,SP,Brazil.

E-mails:jamachadoneto@gmail.com(J.A.Machado-Neto),

sara@unicamp.br(S.T.OlallaSaad).

Received21October2016 Accepted8February2017 Availableonline11March2017

1516-8484/

creativecommons.org/licenses/by-nc-nd/4.0/).