Rev. Bras. Hematol. Hemoter. vol.37 número6

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

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Special

article

Proposal

for

the

standardization

of

flow

cytometry

protocols

to

detect

minimal

residual

disease

in

acute

lymphoblastic

leukemia

Maura

Rosane

Valério

Ikoma

,

Miriam

Perlingeiro

Beltrame

,

Silvia

Inês

Alejandra

Cordoba

Pires

Ferreira

_,

_Elizabeth

_Xisto

_Souto

_,

Mariester

Malvezzi

_,

_Mihoko

_Yamamoto

_,

_on

_behalf

_of

_Minimal

_Residual

_Disease

Working

Group

of

the

Brazilian

Society

of

Bone

Marrow

Transplantation

(SBTMO)

a_{Fundac¸ãoAmaralCarvalho(FAC),Jaú,SP,Brazil}

b_{UniversidadeFederaldoParaná(UFPR),Curitiba,PR,Brazil}

c_{CentrodeHematologiaeHemoterapiadeSantaCatarina(HEMOSC),Florianópolis,SC,Brazil} d_{DiagnósticosdaAmérica(DASA),SãoPaulo,SP,Brazil}

e_{UniversidadeFederaldeSãoPaulo(UNIFESP),SãoPaulo,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received3May2015 Accepted27July2015

Availableonline28September2015

Keywords:

Minimalresidualdisease

MRDacutelymphoblasticleukemia Flowcytometry

Immunophenotyping

a

b

s

t

r

a

c

t

Minimalresidualdiseaseisthemostpowerfulpredictorofoutcomeinacuteleukemiaandis usefulintherapeuticstratificationforacutelymphoblasticleukemiaprotocols.Nowadays, themostreliablemethodsforstudyingminimalresidualdiseaseinacutelymphoblastic leukemiaaremultiparametricflowcytometryandpolymerasechainreaction.Bothprovide similarresultsataminimalresidualdiseaselevelof0.01%ofnormalcells,thatis, detec-tionofone leukemiccellinupto10,000normalnucleated cells.Currently, therapeutic protocolsestablishthe minimalresidual diseasethresholdvalueat themost informa-tivetimepointsaccordingtotheappropriatemethodologyemployed.Theexpertiseofthe laboratoryinacancercenteroracooperativegroupcouldbethemostimportantfactor indeterminingwhichmethodshouldbeused.InBrazil,multiparametricflowcytometry laboratoriesareavailableinmostleukemiatreatmentcenters,butmultiparametricflow cytometryprocessesmustbestandardizedforminimalresidualdiseaseinvestigationsin ordertoofferreliableandreproducibleresultsthatensurequalityintheclinicalapplication ofthemethod.TheMinimalResidualDiseaseWorkingGroupoftheBrazilianSocietyofBone MarrowTransplantation(SBTMO)wascreatedwiththataim.Thispaperpresents recom-mendationsforthedetectionofminimalresidualdiseaseinacutelymphoblasticleukemia basedontheliteratureandexpertiseofthelaboratorieswhoparticipatedinthisconsensus, includingpre-analyticalandanalyticalmethods.Thispaperalsorecommendsthatboth

∗ _{Correspondingauthorat:Fundac¸ãoAmaralCarvalho(FAC),RuadasPalmeiras,106,17210-120Jau,SP,Brazil.} E-mailaddress:msikoma@uol.com.br(M.R.V.Ikoma).

1 _{MembersofMRDWorkingGroupofSBTMOarelistedinAppendix.}

http://dx.doi.org/10.1016/j.bjhh.2015.07.012

multiparametricflowcytometryandpolymerasechainreactionarecomplementary meth-ods,andsomorelaboratorieswithexpertiseinimmunoglobulin/Tcellreceptor(Ig/TCR)gene assaysarenecessaryinBrazil.

©2015Associac¸ãoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Publishedby ElsevierEditoraLtda.Allrightsreserved.

Introduction

Minimalresidualdisease(MRD)istodayconsideredthemost powerful predictor ofoutcome inacute leukemias, includ-ing acutelymphoblastic leukemia (ALL). Althoughclassical factorssuchasage,cytogeneticandmolecularfeatures,and leukocytecountaretakenintoaccounttoestablishtheinitial riskgroupsfortherapeuticpurposes,theevaluationof treat-mentresponsebyMRDdetectionallowsclinicianstoidentify relapseriskcategoriesforALLandstratifythechemotherapy accordingtowell-establishedadultor pediatrictherapeutic protocols.1–7_{ResultsofMRDstudiescanalsobeusedtoselect} treatmentintensityandduration,andestimatetheoptimal timingforhematopoieticstemcelltransplantation(HSCT)in childhoodALL.8

Bothmultiparametricflowcytometry(MFC)andthe ampli-fication of immunoglobulin/T cell receptor (Ig/TCR) genes by polymerasechainreaction(PCR)havesimilarresultsinMRD detectionwitha levelof10−4 _{cells.Howeverthe besttime} pointsfordetectionaredifferentbetweenthetwotechniques.

Clinicalsignificanceofminimalresidualdiseaselevels

ThegoalsofMRDstudiesforclinicalpurposesaretoestablish: (i)thelevelsofMRDthatarerelevanttothetherapeutic deci-sion;(ii)themostinformativetimepointsduringtreatment; and(iii)theclinicalrelevanceofinformationthateachmethod providesatthedifferenttimepoints.

Thecut-off valueto define ALL MRDpositivity is0.01% or10−4 _{cells,becausethisrepresentsthe limitofdetection}

byimmunophenotypingandmolecularassays,althoughitis possibleto achieve ahigher sensitivity (betterthan 0.01%) byPCRtechniques.Moreover,withtherecentimprovements intechnology, this thresholdcan nowbe achievedbyflow cytometry.8,9_{Currently,therapeuticprotocolsestablisha} cut-offpointatthemostinformativetimetopredictdangerof relapseaccordingtotheappropriatemethodologyemployed forMRDdetection(Table1).

The identification of the disease relapse risk allows therapeutic stratification and better clinical management, includingrecognitionofpatientswhorequirelessintensive therapyandthoseeligibleforHSCTatfirstremission.5,10

The level of MRD in pediatric patients prior to condi-tioning for allogeneic HSCT has a significant impact on post-transplantoutcomesanditisthemostimportant pre-dictorofrelapseafterHSCT.Patientswithhigh-levelMRDat thetimeoftransplant(>10−3 _{or0.1%malignantcells) have}

significantlypoorer outcomes than those who entered the transplantationwithnegativeMRD(<10−3_cells).11_TheAcute

LymphoblasticLeukemiaBerlin-Frankfurt-MünsterStemCell Transplantation Group (ALL-BFM-SCT) 2003 trial assessed MRDinthe bonemarrow(BM) atDays30,60,90,180, and 365 after HSCT and each time point with a MRD ≥10−4 leukemiccellswasconsistentlycorrelatedwithshorterevent freesurvival.12

Two techniques are available for post-transplant mon-itoring of disease remission: MRD detection and the characterization of post-transplant chimerism. The MRD detection techniquessearchforthe malignantclone,while assessments ofchimerism characterize the origin of post-transplanthematopoiesis.11_{Thesensitivityofinvestigations} ofchimerismvarygreatlydependingonthemethodused.13

PatientswithalowMRDlevelafterHSCT(<10−3_),can

con-vertmixedchimerismtocompletechimerismbypre-emptive immunotherapy,11,14,15 _{whichdemonstrates theimportance} ofMRDmonitoringafterHSCT.Althoughthereisnota well-establishedmanagementscheduleforthesecases,MRDstatus providesarealperspectiveofrationaltherapeuticintervention afterHSCTtopreventrecurrenceofthedisease.14

Methodsofminimalresidualdiseasedetection

ThemostreliablemethodsofevaluatingMRDareMFCanalysis withtheidentificationofleukemia-associated immunophe-notypes(LAIPs)andamplifyingantigen-receptor(Ig/TCR)gene rearrangementsandfusiontranscriptsbyPCR.BothMFCand amplificationofIg/TCRgenesbyPCRprovidesimilarresultsat aMRDlevelof0.01%,5,16_{andbothMFCandPCRhave} advan-tagesand disadvantages. MFCisarapidmethod, usefulin >95%ofALLcasesandismoreinformativethanPCRduring thefirstphaseofinductiontherapy,whilePCRispreferablefor studiesafterHSCTorattheendoftherapybecauseofitshigh sensitivityinthosemoments.10,17_{Duringthefirst2–3weeks} ofremission-inductiontherapy, BMspecimensdo not con-tainlymphoidprogenitors,andsothedetectionofimmature B-cellsbyMFCcanbeanindicationofresidualdisease.17

ThemostimportantcausesofdiscrepancybetweenMFC andPCRassaysare:(i)samplescontainingalimitedcell num-berforMFCassays;(ii)phenotypevariationsofregenerating precursor B-cells(PBC)inBMduringtherapyandrelatedto age; (iii) druginduced antigenic modulation;(iv) qualityof PCRclonalmarkers;(v)amplificationofnonspecificDNAfrom deadcells;and(vi)oligoclonalityandclonalevolution.6,18–20

Table1–ClinicalsignificanceofMRD,cut-offlevelsandtimepointsofdetectionduringinductiontherapyinacute lymphoblasticleukemia.

Reference Therapeutic

protocol

Cut-offlevel(%) Method Timepoint

(inductiontherapy)

Outcomeand therapy

CavéHetal.1998 EORTC >0.1 IgTCR endof

induction

16×higherrelapse

rate

ZhouJetal.2007 Dana-Farber >0.1 IgTCR D30 10,5×higherrelapse

rate

BorowitzMJetal. 2008

COG >0.01 MFC D29 worstEFS

FlohrTetal.2008 AEIOP/BFM2000 ≥0.01 IgTCR D33andD78 highrelapserate

CampanaD,2009 StJude’s ≤0.01

>1

≥0.01 >1

MFC/PCR D15

D15 D42 D42

lowrisk-less intensivetherapy intensiveinduction high-risk

HSCT

GaipaGetal. 2012

AEIOP/BFM2000 ≤0.01 MFCIgTCR D15

D33andD78

EFS

91,6%(5years)

BrüggemannM etal.2012

GMALL <1,0×10−4 _{MFC/IgTCR D71,w16/w30/}

w52

Treatmentreduction high-risk

>1,0×10−4

reconversion

<1yearof treatment

HSCTor experimental therapies

MRD,minimalresidualdisease;MFC,multiparametricflowcytometry;Ig,immunoglobulin;TCR,T-cellreceptor;PCR,polymerasechainreaction; EFS,eventfreesurvival;HSCT,hematopoieticstemcelltransplantation.

PCRtargets21_{;and(iv)therearesubcloneswithdistinctclonal}

Ig/TCR generearrangementsthatareundetectedat diagno-sis and that may become predominant during the course ofthe disease(oligoclonality).22 _{Becauseofthese} disadvan-tages,manylaboratoriestrytouseMFCforMRDdetection,21 althoughnewapproachesofsequencingtechnologiesareof greatpotentialforfacilitatingmolecularMRDstudiesinALL andmaywellsupplantthecurrentmethod.23

Immunophenotyping

Detection ofMRD by MFC consists ofsearching for LAIPs, also called aberrant phenotypes, that are absent in nor-mal,reactiveorregeneratingBMandperipheralbloodcells; this is useful to discriminate neoplastic cells from nor-mal hematopoietic cells. LAIPs include the presence of cross-lineageantigenexpression(e.g.expressionofmyeloid antigensinALLcases),asynchronouspatternsofexpressionof maturationmarkers(e.g.surfaceIgexpressiononCD34+_cells)

andabnormallevelsofexpressionofindividualmarkers(e.g. antigenoverexpressionorunderexpression).LAIPsare identi-fiedinmorethan95%ofALLcases.5,17_{Thus,MFCcanbeused} tomonitor90–95%ofMRDinALLcasesduringtherapeutic management.ImportantlyMRDcanbeusedwithPBsamples inpatientswithTcellALLgivingsimilarresultstotheuseof MRDtestinginBM.HoweverinpatientswithB-lineageALL, MRDisusuallypresentathigherlevelsinBMthaninPB;8_BM ispreferableforMRDdetectioninthissituation.

Thesensitivityofthismethodvariesaccordingtothe num-berofMFCcolorsappliedinMRDassays:10−3_to10−4_MRDcells

with3–4colorsand10−4_to10−5_{with>6colors.}9_Newwaysof samplepreparationsuchasbulklysis24,25_{andimprovements} in analysis strategies can also contribute to increase MFC sensitivity.9_{Althoughtheseimprovementscanbeachievedin} MRDdetection,severalstudiesdemonstratedthatMRD mon-itoringbyfour-colorMFCcanstillbeclinicallyinformative.3,26 Theprincipaladvantagesofimmunophenotypingare:(i) the possibility ofevaluatingthe samplecellularity and the degree of hemodilution; (ii) the maturation of normal BM cells,includinglymphoidrecoveryafterimmunosuppressive therapy;(iii)thespeedofobtainingresults;and(iv)its wide-rangingapplicability.

The limitations ofimmunophenotyping in MRD studies are:(i)thelownumberofcellsavailable;(ii)thesimilarityof theimmunophenotypebetweennormalprecursor cellsand leukemiacellshinderstheidentificationofresidualblastcells; (iii)themodulationofantigenexpressionduringtreatment18_; (iv)thenecessityofhighexpertisetoperformMRDassays;and (v) thelackofinter-laboratorialstandardizationandquality control.

Immunophenotypic modulation, described in ALL patients,5,19,27_{interferesintheabilitytoaccuratelydetermine}

MRD(Table2).SomemodulationsofBcellantigenexpressions

Table2–Commonmodulationofantigenexpression duringacutelymphoblasticleukemiatreatment.

BprecursorcellALL TcellALL

Inductiontherapy(untilDay33) Down-modulationofCD10and CD34

Up-modulationofCD19,CD20, CD45RAandCD11a

Induction therapy Down-modulationof TdT,CD99,CD34 andCD10 AtDay78ofinduction

ReversionofCD10andCD34to initialexpression

therapy, thereis areversionofCD10 and CD34expression

to initial levels and the CD58 expression stablizes.18,19,28 Significantchangesinforward-anglelightscatter(FSC)and side angle light scatter (SSC) signals associatedwith blast cellsatdiagnosisandatDay15arenotobserved.18

Roshaletal.alsodescribeddown-modulationof immatu-ritymarkerssuchasTdT,CD99,CD34andCD10inT-ALLcells duringinductiontherapy.Theydidnotobserveintensity vari-ationsinCD2,CD3,CD4,CD5,CD7,andCD8,andCD45showed aslightgaininmeanfluorescenceintensitycomparedto nor-malTcells.27

Furthermore immunophenotypic changes have been reportedinrelapsedacuteleukemiabyclonalselectionor lin-eageswitch.26_{ThechangesinLAIPscanaffecttheaccuracy} oftheflowcytometricdetectionofMRDrenderingfalse neg-ativeresults.Theuse ofmultiplemarkercombinationscan minimizefalsenegativeresultssuggestingthat thisshould beastrategyforMRDdetectionbyimmunologicalmethods. Thus,theuseofnewmarkersforoverexpressedor underex-pressedPBCALL comparedtonormal PBCincludingCD24, CD44,CD49f, CD69,CD72,CD73,CD79b,CD86,CD97,CD99, CD102,CD123,CD130,CD164,CD200,CD300a,CD304,BCL2, HSPB1,PBX1,CTNNA1, and ITGB7ispromising toimprove sensitivityofimmunophenotypeMRDstudies.29_Inaddition, someofthesemarkersareassociatedtogenetic abnormali-ties,andhavebeenprovedtobestableaftertreatment.29

Detectionofminimalresidualdiseaseinacute lymphoblasticleukemiabymultiparametricflow cytometry–aproposalofstandardization

MFCandamplificationofIg/TCRgenesbyPCRhavesimilar resultsataMRDlevelgreaterthan0.01%cells6,30_butthe intro-ductionofMFCwithsixormorecolors,thestandardization ofinstrumentsettingsandimmunophenotypingprotocols,24 theavailabilityofarobustsinglemulticolorantibody combi-nation,andnovel dataanalysisstrategies,9 _{maycontribute} toimprove thepotentialofMFCinthediagnosisofMRD.21 Thetypeoflaboratoryexpertiseavailabletoacancercenter oracooperativegroupcouldbethemostimportantfactorin determiningwhichmethodshouldbeused.8

InBrazil,asinother countries,thereareflowcytometry laboratoriesinmostleukemiatreatmentcenters,makingthis methodmorereadilyavailable.

TheMRDWorkingGroupoftheSBTMOwascreatedwith theaimofstandardizingMFCforMRDinvestigationsandto

offerreliableandreproducibleresultsthatensurequalityin clinicalapplicationandpatientcare.

Methods

InitiallyallmembersoftheWorkingGroupreviewedthe lit-eraturedata;theyalsoevaluated20list-modeofnormaland regeneratingBMofadultandpediatricsampleswhichwere providedbyeightparticipatingcentersandavailableonline.

TheproposalstostandardizeMFCpresentedinthisarticle arebasedonreporteddataandrelevantinformationcollected fromtheexpertiseoftheparticipatingcenters.Theyinclude: (i) protocols forsamplepreparation and staining, (ii) stan-dardizationofmonoclonalantibodies(MoAbs)andrespective fluorochromes, combined in four-color marker panels, (iii) standardizationofMFCacquisitionprotocolsand(iv)quality controlissues.

According to the Brazilian Group of Flow Cytometry (GBCFLUX)data, mostBrazilian MFClaboratoriesare today equippedwithcytometerswiththreeandfourfluorescence sets.Recently,GBCFLUXpublisheddiagnosticpanelsforacute leukemiarecommendedtomostBrazilianMFClaboratories.31 ThenextstepswillbetotrainWorkingGroupmembers tosupportthe standardizationprocess andevaluate repro-ducibility through online interchange of MFC data files to analyzeMRDcasesasagroup.Dataanalysisisnotthegoal ofthisworkandshouldbeaddressedinafuturepaper.

Minimalresidualdiseaseworkinggrouprecommendations

Generalrecommendations

Cell morphologyshouldbeassessedvisually using conven-tional optical microscopy. Smears of samples collected in tubescontainingethylenediaminetetraaceticacid(EDTA)are alsoanalyzedtoestimatethehemodilutionparameter.The smearsareinternalcontrolsofthelaboratorytoverifythe cel-lularityofthesampletobeprocessed.Nonetheless,ethically, allsamplesmustbeprocessed,includinghemodiluted sam-ples.BM(1–2mL)orPBsamplesmustbecollectedinK3EDTA (7.5%)forbothMFCandacompletebloodcount.Thestorage ofsamplesshouldnotexceed24haftercollection(Table3).

Recommendationforsamplepreparation

Most laboratories of the MRD Working Group that use MFC prepare samples by the conventional stain-and-then-lyse technique or bulk lysis and stain.25 _{One laboratory} uses mononuclearcells separatedusingthe Ficoll-Hypaque method5_{accordingtorequirementsofthespecifictreatment} protocol.

For theconventional technique,freshBMor PBsamples contain106_{cells,10–100␮Lwhite bloodcellsmustbe}

Table3–Summaryoftechnicalproceedings.

Generalrecommendations

Cellmorphologyofcorrespondingsample

Evaluationofsamplecellularity/hemodilutionassessedby smearsfromethylenediaminetetraaceticacid(EDTA)tube Bonemarrow(1–2mL)orperipheralbloodsamplesmustbe collectedinK3EDTA7.5%forbothmultiparametricflow cytometryandcompletebloodcount

Theincubationofsamplesshouldnotexceed24hafter collection

Recommendationforsamplepreparation

Stain-and-then-lysetechniqueorbulkylysisandstain Samplesincubatedfor15minatroomtemperatureinthedark withfluorochromeconjugatedmonoclonalantibodies Lyseofnon-nucleatedredcellswithlysingsolution CentrifugeandwashtwicewithPBSor0.2%PBS–BSAor0.2% PBS+BSAplus0.1%sodiumazide

Re-suspendin300–500␮LofPBSformultiparametricflow

cytometryacquisitionandanalysis

Intracellularstainingmustbeperformedafterstainingforcell surfacemembranemarkers,utilizingpermeabilizingsolutions

Standardoperationalprocedures

Dailycleaningandbeadcalibrationprocedures Compensationmustbedonemonthlyorwhenrequired, accordingtothestabilityofflowcytometerparameters Routinepreventivemaintenancemustbeperformedatleast everysixmonths

PBS:phosphatebuffersolution;BSA:bovineserumalbumin.

Non-nucleatedredcellscanbelysedusingFACSLysing solu-tion(Becton-DickinsonBiosciences-BDBTM_{,SanJose,CA,USA)}

orExcellyseEasy(ExbioTM_{,Vestec,Czech Republic),}

accord-ingtothe manufacturer’sinstructions. Theremainingcells aresequentiallycentrifuged (500g)for5min,washed twice inphosphate bufferedsaline(PBS:pH7.4)or PBSplus0.2% bovineserumalbumin(BSA:pH7.4)orPBSplus0.2%BSAplus

0.1%sodiumazide:pH7.4)andre-suspendedin300–500␮L

inPBSforMFCacquisitionandanalysis.Intracellularstaining forCD3andTdTisperformedafterstainingforcellsurface membranemarkersusing Fix&Permsolution(InvitrogenTM_,

Camarillo, CA, USA or An Der GrubTM_{, Vienna, Austria) or}

theIntraStainkit(DakoTM_{,Carpinteria,CA,USA)asperthe}

instructions ofthe manufacturer.The recommended

solu-tionshavebeentested,appliedandselectedforuseinmost ofthelaboratoriesoftheWorkingGroup.

Standardoperationalprocedures

Daily cleaning and calibration ofbead procedures are

rec-ommendedandcompensationshouldbedoneeverymonth

or when required according to stability of flow cytometer

parameters. Flowcytometerperformancemust be checked

everydayoraccordingtothe manufacturer’s

recommenda-tionstoensurethatthe evaluationofsamplesalwaysuses

the same parameters and enable an accurate analysis of

theunderexpressionandoverexpressionofmarkerswhichis veryimportantforMRDdetection.Routinepreventive main-tenancemustbeperformedperiodically,thatis,atleastevery sixmonths(Table3).

Table4–Fluorochromeconjugatedantibodypanelsused forminimalresidualdiseasedetectioninBprecursor cell-acutelymphoblasticleukemiabymultiparametric flowcytometry.

Mandatorypanel

Tube1–CD20FITC/CD10PE/CD19PerCPorPEcy5.5/CD34APC Tube2–CD45FITC/CD34PE/CD19PerCPorPEcy5.5/CD38APC Tube3–nTdTFITC/CD10PE/CD19PercporPEcy5.5/CD34APC

Recommendedtubesa

Tube4–CD81FITC/CD66cPE/CD19PerCPorPEcy5.5/CD34APC Tube5–CD45FITC/CD123PE/CD19PerCPorPEcy5.5/CD34APC Tube6–CD15FITCandCD65FITC/NG2PE/CD45PerCP/CD19APC

Optionalmarkersa

CD13and/orCD33PE,CD58FITC,CD9FITC/CD25PE,CD22PE

n:nuclearstaining.

a _{Accordingtoantigenexpressionatdiagnosis.}

Fluorochromeselectionforfour-colorpanels

Theselectionofthemostappropriatecombination of fluo-rochromesforfour-colorpanelsshouldallowsimultaneous usageofbackbonemarkersaimedattheidentificationofthe cellpopulationsofinterestandadditionalantibodymarkers devotedtoamoredetailedcharacterizationoftheleukemic cell populationssuchas maturationmarkers, cross-lineage markers,and markersassociatedwithmolecularlesions.A combination of FITC, PE, PerCP or PECy5.5 and APC were selectedbasedontheliteratureandtheexpertiseofthe Work-ingGroup.

ImmunophenotypicabnormalitiesofleukemicBcell popu-lationsareidentifiedbythedeviationfromnormalpatterns ofB-lymphoiddevelopment9_{.PBCorhematogoneshave} mor-phologic and immunophenotypic similarities to neoplastic lymphoblasts.PBCmaybepresentinlargenumbersinmany situations as early as infancy, or in a variety of diseases both in childhood and in adult life, as in some autoim-mune and congenital cytopenias,neoplasms and acquired immunodeficiency syndrome (AIDS).32 _{Hematogones also} maybeparticularlyprominentinregeneratingBMfollowing chemotherapy or HSCT (Figure 1).32–35 _Particularly follow-ing ALLtreatment,hematogonesare oftenexpandedinBM and canpotentiallybemistakenforresidual disease.Some studiesshowedthatthePBCpopulationalwaysexpressesa continuousandcompletematurationspectrumbyfour-color flow cytometry.36 _{In contrast, casesof PBC ALL frequently} show adifferentspectrumtonormalB-lineagematuration. Thesedifferencesinclude:maturationarrest;overexpression, underexpression,and asynchronous expressionofantigens observed in PBC and often the expression of myeloid-associatedantigens.33,37

AccordingtothisMRDWorkingGroupconsensus,residual B-cellALLmustbeinvestigatedinBMandidentifiedbytwo backbonemarkersinafour-colorMoAbpanel,suchasCD19 andCD34.

The mandatory panel for B-cell ALL MRD detection

(Table4)isaddressedtoidentifydeviationsinthematuration

CD 10 PE

CD 20 FITC NG2 PE CD 66 PE _{CD 20 FITC}

A

B

C

D

E

CD 10 PE CD 15+CD 65 FITC CD 58 FITC

1E1

1E1 1

1 1E2

1E2 1E3

1E3 1E4

1E1

–1E2 1E2 1E3 1E4 1E5

–1E2 1E2 1E3 1E4 1E5

1 1E2 1E3 1E4

1E4 1 1E1 1E2 0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5 1E2 1E2

1E3 1E3

1E4 1E4

1E5 1E5

0 0

1E3 1E4

Figure1–MinimalresidualdiseaseinprecursorB-cellacutelymphoblasticleukemiausingmaturationtube: CD20FITC/CD10PE/CD19PerCP/CD34APC.(A)Atdiagnosis(77.6%ofblastcells)and(B)thesamepatientonDay15of

inductiontherapywithpositiveminimalresidualdisease(0.03%).(C)MLLAF4_{precursorB-cellacutelymphoblasticleukemia}

withpositiveminimalresidualdisease(0.28%)onDay40afterhematopoieticstemcelltransplantation.(DandE)BCR-ABL

positiveprecursorB-cellacutelymphoblasticleukemiawithpositiveminimalresidualdisease(0.32%)beforeconditioning treatmentforhematopoieticstemcelltransplantation.GateinCD19+_{cells.Ingreen:matureB-cells(A–C),normalprecursor} andmatureB-cells(DandE).Inred:blastcells.Inblue:normalCD34+_{precursorB-cells.Acquisition:1,000,000oftotal} events.Method:bulklysis.

CD19PerCP or PEcy5.5/CD38APC and Tube 3: nTdT-FITC/CD10PE/CD19PerCPorPEcy5.5/CD34APC.

Recommendedtubesareintendedtorecognize:(i)markers thatarefrequently underexpressedinBcellALL compared to normal B-cells such as CD81; (ii) cross-lineage markers suchasCD15, CD65,CD66c,CD123;and (iii)markers asso-ciatedwithmolecularlesionssuchasCD66c[insomecases presenting the breakpoint cluster region-Abelson murine leukemia(BCR-ABL)fusionprotein]andNG2(associatedwith 11q23alterations);thislatteroneisoftenexpressed concomi-tantlytoCD15and/orCD65(Figure1).

Optionalmarkersare:CD9andCD22thatareexpressedin Bnormalcells andoftenunderexpressedintheir leukemic counterparts; CD58 that has been shown to be overex-pressedinleukemicblastswhencomparedtotheirnormal counterparts,28_{CD13andCD33(myeloidmarkers) as} cross-lineagemarkersandCD25(interleukin2receptor)canalsobe associatedwiththepresenceofBCR-ABLpositivePBCALL.It mustbeemphasizedthatrecommendedandoptionalmarkers shouldbechosenaccordingtotheirexpressionatdiagnosis.

MRDdetectionofT-ALLinthe PBor BMistheoretically easier asmost stagesof normalT lymphocyte maturation occur inthe thymus.Therefore the presenceofan imma-ture T cell subset in the PB or BM should be considered

Table5–Fluorochromeconjugatedantibodypanelsused forminimalresidualdiseasedetectioninTcell-acute lymphoblasticleukemiabymultiparametricflow cytometry.

Mandatorypanel

Tube1–cyCD3FITC/mCD3PE/CD45PerCP/CD7APC Tube2–nTdTFITC/CD2PE/CD5PerCP/CD7APC

Recommendedtube

Tube3–CD1aFITC/CD99PE/mCD3PerCP/CD7APC

Optionalmarkersa

CD10PE,CD13PE,CD33PE,CD34PE,CD44PE,CD117PE

cy: cytoplasmic staining; n: nuclear staining; m: membrane staining.

a _{Accordingtoantigenexpressionatdiagnosis.}

aberrant. For the investigation of MRD in T-cell ALL, CD3 and CD7are recommended asbackbonemarkers(Table5). Mandatoryandrecommendedtubesare addressedto iden-tifymaturation stageofblastcells and includeimmaturity markerssuchasTdT,CD34,CD1a,andCD99(Figure2). Dif-ficultiesinMRDdetectioninT-ALLlargelystemfromtheloss ofimmaturitymarkersontheabnormalblastpopulation fol-lowinginductionchemotherapy.OptionalmarkersforT-ALL

CD 99 PE mCD 3 PE CD 117 PE CD 34 PE

CD 7 APC

CD 45Per CP

–1E2

1E2 0 1E2

1E3 1E3

1E4 1E4

1E5

–1E2 1E2 1E3 1E4 1E5

1E5 1E2 1E3

1E3

1E4 1E4

1E5 1E5

0 0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5

0

1E3 1E4 1E5

0

MRDaimtorecognizecross-lineagemarkerssuchasCD13and CD33;amarkerthatisunderexpressedoroverexpressedin 81%ofT-ALLcasessuchasCD44,29_{andCD117,expressedin} earlyT-ALL.ThesemarkersmaybeusefulforMRDdetection whentheyareexpressedatdiagnosis.

Dataacquisition

Data acquisition must be performed in sequence immedi-atelyaftersamplepreparationiscompleted.Foreachsample aliquot,a minimumof500,000 and maximum of1,000,000 eventsmustbeacquired.Optionally,laboratoriesthatperform bulklysisareabletoacquire5,000,000events.Acriterionfor quantifiableMRDpositivitywasestablishedusingadetection limitofabsoluteMRDcellcountsof10cellsormoreineach tube.38

Conclusion

MFC is a powerful method for MRD investigations of hematology malignancies, but it is fundamental to have good standardization of the pre-analytical, analytical and post-analyticalprocesses. TheMRDWorkingGroup recom-mendationsmeetthisrequirementwiththemaingoalbeing thepatients’safetyandcare.Forthispurpose,italsoshouldbe stressedthattheMFCandPCRtechniquesarecomplementary, andsoitisnecessarythatmoremolecularbiologylaboratories withexpertiseinIg/TCRassaysareavailableinBrazil.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Theauthorsthankthecolleagueswhocontributedtothiswork bysendingdatafrom theirlaboratories:Alex FreireSandes (DivisãodeHematologiadoGrupoFleury,SãoPaulo,SP); Eliz-abeth Delbuono (GRAAC, São Paulo, SP); Leandro S Thiago (InstitutoNacionaldoCancer,RiodeJaneiro,RJ);MariaMirtes Sales(LaboratóriodeCitometriadeFluxodoHospitaldas Clin-icas,FaculdadedeMedicinaUniversidadedeSãoPaulo,SP).

Theauthors especiallythankDrLuciaSilla,presidentof SBTMO,DrNelsonHamerschlakcoordinatorofSBTMO work-inggroupsandDr.VergilioARensiColturato,whoconceived thegroup,forsupportingthedevelopmentofthegroup.

Appendix.

Members

of

MRD

Working

Group

of

SBTMO

AdrianaSeber,AnaPaulaAlegretti,AnaPauladeAzambuja, CintiaGFMachado, ElaineSobralda Costa,ElizabethXisto Souto,IreneGHLorand-Metze,MariaLuizaMenezesCortez, Mariester Malvezzi, Maura Rosane Valério Ikoma, Mihoko Yamamoto,MíriamPerlingeiroBeltrame,NormaLucena-Silva, NydiaStrachmanBacal,SilviaInesAlejandraCórdobaPires Ferreira,VergílioAntonioRensiColturato,VirginiaPires.

r

e

f

e

r

e

n

c

e

s

1.CaveH,vanderWerfftenBoschJ,SuciuS,GuidalC,

WaterkeynC,etal.Clinicalsignificanceofminimalresidual

diseaseinchildhoodacutelymphoblasticleukemia.European

OrganizationforResearchandTreatmentof

Cancer-ChildhoodLeukemiaCooperativeGroup.NEnglJMed.

1998;339(9):591–8.

2.ZhouJ,GoldwasserMA,LiA,DahlbergSE,NeubergD,Wang

H,etal.Quantitativeanalysisofminimalresidualdisease

predictsrelapseinchildrenwithB-lineageacute

lymphoblasticleukemiainDFCIALLConsortiumProtocol

95-01.Blood.2007;110(5):1607–11.

3.BorowitzMJ,DevidasM,HungerSP,BowmanWP,CarrollAJ,

CarrollWL,etal.Clinicalsignificanceofminimalresidual

diseaseinchildhoodacutelymphoblasticleukemiaandits

relationshiptootherprognosticfactors:aChildren’s

OncologyGroupstudy.Blood.2008;111(12):5477–85.

4.FlohrT,SchrauderA,CazzanigaG,Panzer-GrümayerR,van

derVeldenV,FischerS,etal.Minimalresidual

disease-directedriskstratificationusingreal-time

quantitativePCRanalysisofimmunoglobulinandT-cell

receptorgenerearrangementsintheinternational

multicentertrial.AIEOP-BFMALL2000forchildhoodacute

lymphoblasticleukemia.Leukemia.2008;22:771–82.

5.CampanaD.Roleofminimalresidualdiseasemonitoringin

adultandpediatricacutelymphoblastic.LeukemiaHematol

OncolClinNAm.2009;23:1083–98.

6.GaipaG,CazzanigaG,ValsecchiMG,Panzer-GrümayerR,

BuldiniB,SilvestriD,etal.Timepoint-dependent

concordanceofflowcytometryandreal-timequantitative

polymerasechainreactionforminimalresidualdisease

detectioninchildhoodacutelymphoblasticleukemia.

Haematologica.2012;97(10):1582–93.

7.BrüggemannM,SchrauderA,RaffT,PfeiferH,DworzakM,

OttmannOG,etal.StandardizedMRDquantificationin

EuropeanALLtrials:ProceedingsoftheSecondInternational

SymposiumonMRDassessmentinKiel,Germany,18–20

September2008.Leukemia.2010;24(3):521–35.

8.CampanaD.Minimalresidualdiseaseinacutelymphoblastic

leukemia.SeminHematol.2009;46(1):100–6.

9.OrfaoA,Flores-MonteroJ,LopezA,BarrenaS,CiudadJ,

VidrialesB,etal.FlowcytometryforMRDdetection:stateof

theart.In:AbstractBook.MRDTechniques:StateoftheArt.

InternationalSymposiumonMinimalResidualDiseasein

HematologicalMalignancies.ESLHO;2012.p.21–3.

10.ScrhrappeM.Minimalresidualdisease:optimalmethods,

timing,andclinicalrelevanceforanindividualpatient.

HematolAmSocHematolEducProgram.2012;2012:137–42.

11.BaderP,WillaschA,KlingebielT.Monitoringof

post-transplantremissionofchildhoodmalignancies:isthere

astandard?BoneMarrowTransplant.2008;42Suppl2:S31–4.

12.BaderP,KreyenbergH,vonStackelbergA,EckertC,

Salzmann-ManriqueE,MeiselR,etal.Monitoringofminimal

residualdiseaseafterallogeneicstem-celltransplantationin

relapsedchildhoodacutelymphoblasticleukemiaallowsfor

theidentificationofimpendingrelapse:resultsofthe

ALL-BFM-SCT2003trial.JClinOncol.2015;33(11):1275–84.

13.ClarkJR,ScottSD,JackAL,LeeH,MasonJ,CarterGI,etal.

Monitoringofchimerismfollowingallogeneichaematopoietic

stemcelltransplantation(HSCT):technicalrecommendations

fortheuseofShortTandemRepeat(STR)basedtechniques,

onbehalfoftheUnitedKingdomNationalExternalQuality

AssessmentServiceforLeucocyteImmunophenotyping

ChimerismWorkingGroup.BrJHaematol.2015;168(1):26–37.

14.SánchezJ,SerranoJ,GómezP,MartínezF,MartínC,MaderoL,

residualdiseaseinacutelymphoblasticleukaemiaafter

allogeneictransplantation.BrJHaematol.2002;116(3):686–94.

15.SchilhamMW,BalduzziA,BaderP.Istherearoleforminimal

residualdiseaselevelsinthetreatmentofALLpatientswho

receiveallogeneicstemcells?BoneMarrowTransplant.

2005;35Suppl1:S49–52.

16.IrvingJ,JessonJ,VirgoP,CaseM,MintoL,EyreL,etal.

Establishmentandvalidationofastandardprotocolforthe

detectionofminimalresidualdiseaseinBlineagechildhood

acutelymphoblasticleukemiabyflowcytometryina

multi-centersetting.Haematologica.2009;94(6):870–4.

17.GaipaG,BassoG,BiondiA,CampanaD.Detectionofminimal

residualdiseaseinpediatricacutelymphoblasticleukemia.

CytometryBClinCytom.2013;84(6):359–69.

18.GaipaG,BassoG,MagliaO,LeoniV,FainiA,CazzanigaG,

etal.Drug-inducedimmunophenotypicmodulationin

childhoodALL:implicationsforminimalresidualdisease

detection.Leukemia.2005;19(1):49–56.

19.DworzakMN,GaipaG,SchumichA,MagliaO,RateiR,Veltroni

M,etal.ModulationofantigenexpressioninB-cellprecursor

acutelymphoblasticleukemiaduringinductiontherapyis

partlytransient:evidenceforadrug-inducedregulatory

phenomenon.Resultsofthe

AIEOP-BFM-ALLFLOW-MRD-studygroup.CytometryBClin

Cytom.2010;78(3):147–53.

20.SedekL,BulsaJ,SonsalaA,TwardochM,WieczorekM,

MalinowskaI,etal.Theimmunophenotypesofblastscellsin

b-cellprecursoracutelymphoblasticleukemia:howdifferent

aretheyfromtheirnormalcounterparts?CytometryBClin

Cytom.2014;86(5):329–39.

21.vanDongenJJ.MRDdetectionbyIg/TCRtargets:stateofthe

art.In:AbstractBook.MRDTechniques:StateoftheArt.

InternationalSymposiumonMinimalResidualDiseasein

HematologicalMalignancies.ESLHO;2012.p.11–5.

22.vanderVeldenVH,BrüggemannM,HoogeveenPG,deBieM,

HartPG,RaffT,etal.TCRBgenerearrangementsinchildhood

andadultprecursor-BALL:frequency,applicabilityasMRD

PCRtarget,andstabilitybetweendiagnosisandrelapse.

Leukemia.2004;18(12):1971–80.

23.CampanaD.Minimalresidualdiseasemonitoringin

childhoodacutelymphoblasticleukemia.CurrOpinHematol.

2012;19(4):313–8.

24.KalinaT,Flores-MonteroJ,vanderVeldenVH,Martin-Ayuso

M,BöttcherS,RitgenM,etal.EuroFlowstandardizationof

flowcytometerinstrumentsettingsandimmunophenotyping

protocols.Leukemia.2012;26(9):1986–2010.

25.BulkErythrocyteLysingwithAmmoniumChlorideforFlow CytometryImmunophenotyping.Technicalprotocol. Availablefrom:https://www.bdbiosciences.com/documents/

Multicolor-Bulk-Erythrocyte-Lysing-Protocol.pdf[citedJuly

2014].

26.BassoG,VeltroniM,ValsecchiMG,DworzakMN,RateiR,

SilvestriD,etal.Riskofrelapseofchildhoodacute

lymphoblasticleukemiaispredictedbyflowcytometric

measurementofresidualdiseaseonday15bonemarrow.J

ClinOncol.2009;27(31):5168–74.

27.RoshalM,FrommJR,WinterS,DunsmoreK,WoodB.

Immaturityassociatedantigensarelostduringinductionfor

Tcelllymphoblasticleukemia:implicationsforminimal

residualdiseasedetection.CytometryBClinCytom.

2010;78(3):139–46.

28.VeltroniM,ZenL,SanzariMC,MagliaO,DworzakMN,RateiR,

etal.ExpressionofCD58innormal,regeneratingand

leukemicbonemarrowB-cells:implicationsforthedetection

ofminimalresidualdiseaseinacutelymphocyticleukemia.

Haematologica.2003;88(11):1245–52.

29.Coustan-SmithE,SongG,ClarkC,KeyL,LiuP,MehrpooyaM,

etal.Newmarkersforminimalresidualdiseasedetectionin

acutelymphoblasticleukemia.Blood.2011;117(23):6267–77.

30.GarandR,BeldjordK,CavéH,FossatC,ArnouxI,AsnafiV,

etal.FlowcytometryandIG/TCRquantitativePCRfor

minimalresidualdiseasequantitationinacutelymphoblastic

leukemia:aFrenchmulticenterprospectivestudyonbehalfof

theFRALLE,EORTCandGRAALL.Leukemia.2013;27(2):370–6.

31.IkomaMR,SandesAF,ThiagoLS,JuniorGB,Lorand-MetzeIG,

CostaES,etal.Firstproposedpanelsonacuteleukemiafor

four-colorimmunophenotypingbyflowcytometryfromthe

Braziliangroupofflowcytometry-GBCFLUX.CytometryBClin

Cytom.2015;88(3):194–203.

32.DworzakMN,FritschG,FleischerC,PrintzD,FröschlG,

BuchingerP,etal.Multiparameterphenotypemappingof

normalandpost-chemotherapyBlymphopoiesisinpediatric

bonemarrow.Leukemia.1997;11:1266–73.

33.vanWeringER,vanderLinden-SchreverBE,Szczepa ´nskiT,

WillemseMJ,BaarsEA,vanWijngaarde-SchmitzHM,etal.

RegeneratingnormalB-cellprecursorsduringandafter

treatmentofacutelymphoblasticleukaemia:implicationsfor

monitoringofminimalresidualdisease.BrJHaematol.

2000;110(1):139–46.

34.BabusíkováO,ZelezníkováT,KirschnerováG,KankuriE.

Hematogonesinacuteleukemiaduringandaftertherapy.

LeukLymphoma.2008;49(10):1935–44.

35.McKennaRW,AsplundSL,KroftSH.Immunophenotypic

analysisofhematogones(B-lymphocyteprecursors)and

neoplasticlymphoblastsby4-colorflowcytometry.Leuk

Lymphoma.2004;45(2):277–85.

36.vanLochemEG,vanderVeldenVHJ,WindHK,teMarveldeJG,

WesterdaalNAC,vanDongenJJ.Immunophenotypic

differentiationpatternsofnormalhematopoiesisinhuman

bonemarrow:referencepatternsforage-relatedchangesand

disease-inducedshifts.CytometryBClinCytom.

2004;60(1):1–13.

37.McKennaRW,LaBaronTW,AquinoDB,PickerLJ,KroftSH.

Immunophenotypicanalysisofhematogones(B-lymphocyte

precursors)in662consecutivebonemarrowspecimensby

4-colorflowcytometry.Blood.2001;98(8):2498–507.

38.KarawajewL,DworzakM,RateiR,RheinP,GaipaG,BuldiniB,

etal.Minimalresidualdiseaseanalysisbyeight-colorflow

cytometryinrelapsedchildhoodacutelymphoblastic