Update on developments in the diagnosis and prognostic evaluation of patients with myelodysplastic syndromes (MDS): Consensus statements and report from an expert workshop.

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ContentslistsavailableatSciVerseScienceDirect

Leukemia

Research

j o ur n a l ho m e p age : w w w . e l s e v i e r . c o m / l o c a t e / l e u k r e s

Meeting

report

Update

on

developments

in

the

diagnosis

and

prognostic

evaluation

of

patients

with

myelodysplastic

syndromes

(MDS):

Consensus

statements

and

report

from

an

expert

workshop

Uwe

Platzbecker

a,∗

_,

_Valeria

_Santini

b

_,

_Ghulam

_J.

_Mufti

c

_,

_Claudia

_Haferlach

d

_,

_Jaroslaw

_P.

_Maciejewski

e

_,

Sophie

Park

f

_,

_Francesc

_Solé

g

_,

_Arjan

_A.

_van

_de

_Loosdrecht

h

_,

_Detlef

_Haase

i a_{Universitätsklinikum}_{Carl-Gustav-Carus,}_Medizinische_Klinik_I,_Dresden_01307,_Germany

b_AOU_Careggi,_University_of_Florence,_Italy

c_Kings_College_London_and_Kings_College_Hospital,_Department_of_{Haematological}_Medicine,_London,_UK d_MLL_Munich_Leukemia_Laboratory,_Munich,_Germany

e_Taussig_Cancer_Institute,_Cleveland_Clinic,_Cleveland,_OH,_USA

f_Hôpital_Cochin,_Université,_Paris_V,_Assistance_Publique_des_Hopitaux_de_Paris,_Paris,_France g_Hospital_del_Mar-Parc_de_Salut_Mar,_Barcelona,_Spain

h_Department_of_Hematology,_VU_University_Medical_Center,_Amsterdam,_The_Netherlands i_Department_of_Hematology_and_Oncology,_{Georg-August-University,}_Göttingen,_Germany

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15September2011

Receivedinrevisedform7November2011 Accepted8November2011

Available online 3 December 2011 Keywords:

MDS Diagnosis Prognosis

a

b

s

t

r

a

c

t

Severalnewtreatmentsformyelodysplasticsyndromes(MDS)haverecentlybecomeavailable,orare indevelopment.Patientswhocouldbenefitfromactivetreatmentmustbeeffectivelyidentifiedand followedup.Therefore,guidelinesforthediagnosisandprognosticevaluationofMDSneedtobekept uptodatewithtechnologicalandscientificadvances.Anexpertworkshopwasconvenedtoreview currentlyavailableandemergingdiagnostictechnologiesanddevelopmentsinprognosticclassification systems,toensureappropriatemanagementofindividualpatients.Thepanelalsoprovidedsuggestions toensureadherencetoguidelinesandhighlightedthemandatoryrequirementforcytogeneticevaluation inpatientswithMDS.

1. Introduction

Myelodysplasticsyndromes(MDS)constituteaheterogeneous group of clonal hematopoietic disorders characterized by bone marrow(BM)failure,dysplasiaandanincreasedriskof develop-ingacutemyeloidleukemia(AML)[1].ClinicalvariabilityofMDS, diversity of cytomorphological phenotypes and genetic hetero-geneitycan make thediagnosisand assignment ofprognosis a challenge[2].Anumberofparameters,bothclinical(e.g. cytope-nias,ageandperformancestatus)andcytological(e.g.blastcount, morphologyandkaryotype)mustbetakenintoaccount[3,4].Given therecentdevelopmentof newtreatmentsthat canpotentially changethecourseofthedisease[5–8],itisimperativethatMDS iscorrectlydiagnosedandclassifiedtoensureappropriate selec-tionandapplicationoftreatment.Thisis particularlyimportant given that inadequate diagnosiscan leadto delayed identifica-tion, or incorrect classification of, MDS which could result in

∗ Correspondingauthor.Tel.:+493514584190;fax:+493514585362. E-mailaddress:Uwe.Platzbecker@uniklinikum-dresden.de(U.Platzbecker).

inappropriatetreatmentdecisions[1].However, manymethods appliedtopatientswithsuspectedMDS,particularlycytogenetics andmorphologicalassessment,aresubjectiveanddependon indi-vidualexperience.Therefore,strictstandardizationandassurance ofanadequatequalityoftechniquesisrequired.

Aschromosomalaberrationshaveahugeimpactonboththe diagnosisandprognosisofpatientswithMDS,itisessentialthat accuratecytogeneticanalysisisundertakeninpatientswithMDS. Chromosomal aberrations are present in approximately 50% of patientswithMDS[9],thoughdetectionratesseemtovaryfrom institution toinstitution. This mayreﬂect differences in cohort selectionbetweencenters,asthefrequencyofaberrationsishigher inpatientswithadvancedMDSthanpatientswithlessadvanced disease,andvariabilityinthequalityofsampleprocessingbetween individuallaboratories.Theimpactofcytogeneticstatuson prog-nosis,andrateoftransformationtoAML,iswellknownandthe impact of speciﬁc abnormalities is becoming increasingly well understood[10–12].Asanintegralcomponentofprognostic scor-ingsystems, includingthewidelyusedInternationalPrognostic ScoringSystem(IPSS)[13],accuratecytogeneticanalysiscanbe instrumentalindeterminingappropriatetreatmentoptionsfrom 0145-2126/$–seefrontmatter © 2011 Elsevier Ltd. All rights reserved.

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bestsupportivecare,immunomodulatorydrugs,hypomethylating agents,chemotherapyorhematopoieticstemcelltransplantation. Moreover, theselection of certain therapeutic options may be drivenby theexistence ofspeciﬁc chromosomal abnormalities. Forexample,lenalidomideisparticularlyeffectiveinpatientswith low-risk,transfusion-dependentMDSwithadeletionof chromo-some5q[7].

GiventheimportanceofaccuratediagnosisofMDS,diagnostic criterianeedtobeperiodicallyreviewedandupdatedinorderto incorporatemodificationsinclassificationcriteria,rapid techno-logicaldevelopmentsindiagnosticmethodologies,andadvances intheunderstandingofthepathophysiologyofthedisease. Con-sequently,an expert panelworkshop wasconvened toreview: (i)currentguidelinesonminimaldiagnosticcriteriaforMDSand obstaclesto theirimplementation; (ii) the role of fluorescence insituhybridization(FISH),flowcytometryandmicroarray tech-nologiesinimprovingdiagnosis,aswellasmonitoringofdisease progressionand assessing response totherapy inpatientswith MDS;and(iii)howemergingdata,particularlycytogenetics,may impacttheprognosticclassificationsystemsIPSSandWorldHealth Organization (WHO)-based Prognostic Scoring System [WPSS]. Thepanelalsoformulatedconsensusrecommendationstoensure inter-laboratoryadherencetooptimaldiagnosticandprognostic procedures.

2. Currentguidelinesonminimaldiagnosticcriteriafor MDSandobstaclestotheirimplementation

Severalguidelinesregardingtheminimaldiagnosticcriteriafor MDShaverecentlybeenpublished.

Followingaworking conferencein 2006inVienna, minimal diagnosticcriteria[12]wereproposedwiththefollowing recom-mendations: two prerequisitecriteria:(i) marked and constant cytopenia(_≥6months)inatleastonecelllineageand(ii)exclusion of another clonal or non-clonal hematopoietic diseaseor non-hematopoieticdiseaseastheprimaryreasonforcytopenia.Atleast oneout ofthree additionalMDS-relatedcriteria isrequired:(i) morphologicaldysplasiain≥10%oferythroidcells(or>15%ringed sideroblasts),neutrophilsandtheirprecursorsormegakaryocytes inBMsmears;(ii)typicalcytogeneticabnormalityor(iii)aconstant blastcountof5–19%.Theauthorsalsorecommendedco-criteria inpatientswhofulﬁllbothprerequisitecriteriaandshowtypical clinicalfeaturesofMDS,butdonotdemonstrateanyofthethree additionalcriteria.Theseco-criteriainclude:(i)anabnormalBM phenotypecompatiblewithadiagnosisofMDSaccordingto Euro-peanLeukemiaNetworkcriteriabasedonﬂowcytometryand(ii) clearmolecularsignsofamonoclonalcellpopulationbasedona humanandrogenreceptorassay,genechipanalysis,ormutation analysis(e.g.EZH2mutations).

Otherrecentlypublishedguidelines,suchastheItalianSociety ofHematology’spracticeguidelines,highlighttheimportanceof thoroughcharacterizationofpatientswithsuspectedMDSfor accu-ratediagnosisandtoguidetreatmentdecisions[14].Theguidelines recommendthatpatientsshouldreceiveacompletebloodcount,a peripheralblood(PB)smearwithdifferentialleukocytecount,aBM aspirationforcytogeneticandmorphologicevaluation,aBMbiopsy toassessmarrowarchitecture,cellularity,ﬁbrosis,blastpercentage anddysmegakaryopoiesis[15].

Clearly,therefore,guidelines stipulatethat cytogenetic anal-ysis of BM should be undertaken in all cases of suspected MDS; at least 20 metaphases should be analyzed [16] and if <20 metaphases are scored, then the results should be con-sidered invalid, provided the karyotype appears normal. It is also recommended that patients undergo regular BM analysis to monitor disease progression and response to therapy. FISH

analysis,withavalidatedpanelofprobes[17],isrecommended incertaininstances,e.g.toclarifycomplexaberrations,todetect speciﬁcanomalies,e.g.monosomy 7ordel(5q),orwhen onlya lownumberofmetaphasesareavailableforstandardG-banding [18,19].

Sufﬁcient cytogenetic evaluation is not always undertaken, or is undertaken long after initial presentation, in patients withMDS.One reasonfor this couldbeinsufﬁcientknowledge amongsomehematologistsand healthcareprofessionals (HCPs) regarding the importance of cytogenetic analysis for accurate diagnosis,prognosis,andtreatmentdecisions.Importantly,there may also be a lack of inter-laboratory uniformity in cytoge-netic methodology. Other reasons for sub-optimal cytogenetic evaluation in patients with suspected MDS include logistical problems, i.e. the time elapsed between BM biopsy/aspirate and analysis and cost issues. Furthermore, BM samples may not always be available. For example, BM aspiration is not always undertaken in elderly patients, or patients with co-morbidities,becausetheyaredeemedtoofrailorunsuitablefor activetreatmentoptions.Althoughconventionalcytogenetic anal-ysisis possible on PBsamples,it is frequentlyunsuccessful or incomplete[20].Moreover, ifit is successful but shows a nor-mal karyotype, the existence of an aberrant clone cannot be excludedbecausethesecansometimesonly bedetectedinBM samples.

2.1. Recommendationsoftheexpertpanel

The technical challenges of cytogenetic analysis necessitate specialistexperienceinorderfordatatobemeaningful,and there-fore it is recommended that cytogenetic testing be performed by a cytogenetic laboratoryexperienced in MDS. However,the majority of patientswith suspected MDS, especially with mild cytopeniasat diagnosis,are seen in community settings which maynothaveaccesstoanexperiencedcytogeneticslaboratory. Thesepatientsshouldbereferred toahematologist inorderto performthediagnosticwork-up.Inorder toimprove karyotyp-ingdata acrossEuropethepanelsuggested:(i)standardization ofthemethodology;(ii)increasedapplicationofinter-laboratory qualityassurancemeasureswhichhaveprovedsuccessfulinsome countries. For example, in the UK a quality assurance system is in place whereby every 3 months test samples are sent to allcytogenetics laboratories;ifresultsare below anacceptable thresholdthelaboratoryisinformedandmaybeinspectedbya memberofthequalityassuranceteam;and(iii)introductionof a certiﬁcation process to ensurehomogeneity of results across Europe.

Theconsensusviewofthepanelwasthat underdiagnosisis a problemlargely due to a poorunderstanding and awareness of MDS among HCPs. The panel suggested that further edu-cation of HCPs and hematologists is required to: (i) improve awareness of MDS, particularlywith regard tothe importance of undertakingcytogenetic analysis toensurecorrect diagnosis andtoguideappropriatetreatmentdecisions;(ii)improve knowl-edgeofnewtreatmentoptions;and (iii)clarifytherisksofnot undertakingcytogeneticanalysis.ItisimportantthatHCPs under-standthatthecostsinvolvedinundertakingcytogeneticanalysis are far surmounted by therapeutic costs; therefore, avoidance of cytogenetic analysis on cost grounds may be a false econ-omyifitleadstoinappropriatetherapeuticdecisions.Withthese factorsinmind, simpleguidelineshave beendevelopedto out-line criteria required for HCPs to refer cases to hematologists, and to guide hematologists as to what the minimal require-mentsarefromcytogeneticanalysistoensureaccuratediagnosis (Table1).

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Table1

RecommendationstoensureeffectivecytogeneticanalysisofpatientswithMDS.

(A)Criteriaforhealthcareprofessionalstorefercasestohematologists •Unexplainedcytopeniaforoveratleast6months

(B)Guidancetohematologistsforminimalrequirementsfromcytogeneticanalysistoensureaccuratediagnosis •AlwaysperformconventionalcytogeneticsfrombonemarrowinMDSorsuspectedMDSpatients

•Ifkaryotypeshowednoabnormalities,evaluatethereliabilityofcytogeneticresultsbycheckingwhetheratleast20metaphaseswereanalyzed •Undertakedel(5q)FISHwhen:(i)adel(5q)anomalyissuspectedbutisabsentaccordingtoconventionalcytogenetics(bytypicalmorphology);and(ii)in patientswithseeminglymonosomy5

•Inpatientswith<20normalmetaphases,orwithnomitosisconsider:(i)undertakingFISHwithprobesto5q,7q,centromericregionofchromosome8 and20q.Additionalmeaningfulprobesarefor12p,21qandtheYchromosome(inmales);and(ii)undertakeSNP/CGHarrayanalysis,ifavailable FISH,ﬂuorescenceinsituhybridization;MDS,myelodysplasticsyndromes.

3. TheroleofFISH,ﬂowcytometryandmicroarray technologiesinimprovingdiagnosis,monitoringofdisease progressionandassessingresponsetotherapyinpatients withMDS

3.1. FISH

AsFISHanalysisdoesnotoffergenome-widecoverageandcan

onlybeusedtoevaluateselectedchromosomalregions,itisnot

normallyrecommendedforscreeningforcytogenetic

abnormali-tiesintheﬁrstinstance.However,inconjunctionwithconventional

cytogenetics FISH couldpotentially bea powerful approach to

improve identiﬁcation of commonchromosomal aberrations in

patientswithMDS.For example,ina recent study,Malloetal.

analyzed716patientswhohadpreviouslybeenassessedby

stan-dardcytogeneticswitha5q31FISHprobe[19].Interestingly,38

(5.9%)of637patientswhodidnothaveadetectabledel(5q) aber-rationaccordingtostandardcytogeneticshadadel(5q)deletion thatwasdetectedbyFISHonly.Inthese38patients,30%hadnoor insufﬁcient(<20)metaphasesavailableforstandardcytogenetics.

Inanotherstudyof28patientswithMDSorAML,24(85.7%) patientsidentifiedashavingmonosomy5withconventional cyto-geneticswereshowntohavedel(5q)aberrationsbyFISH,aspart ofcomplexaberrations[18].ThesefindingsdemonstratethatFISH inadditiontoconventionalcytogeneticsmayidentifyadditional patientswhocouldbenefitfromlenalidomide.Consequently,the authorsofthesestudiesconsideritmandatorytoapply5q31FISH toallMDSpatientswithanabnormalkaryotypeinvolving chromo-some5,patientswithinsufficientmetaphases,andpatientswitha suspecteddiagnosisofMDSwithisolated5q–whereconventional cytogeneticsdidnotrevealdel(5q)[18,19].

Inadditiontoprovidingincreasedsensitivityforthedetection ofchromosomal abnormalities,preliminarystudiessuggestthat FISHwithapanelofprobescouldbeausefultooltomonitor dis-easeprogressionandresponsetotherapy,particularlyifitcouldbe appliedtoPBsamples.InarecentstudywhereFISHwasundertaken onparallelBMandPBsamplestakenfrom77patientswithMDS, concordantresultswereobtainedin92%ofcases,indicatingthat PBsamplesmaybeinformativewhenfollowinguppatients[20]. Braulkeetal.recentlydescribedanovelmethodbywhich circulat-ingCD34+(hematopoieticprogenitor)cellsfromPBsampleswere enrichedandutilizedtomonitorresponsetotherapyin16patients treatedwithazacitidine[17].AnalysisofenrichedCD34+cellsfrom PBbetterreﬂects theclonal situationin thebone marrowthan non-enrichedPB.ThetechniqueislessinvasivethanBMaspiration which facilitatesfrequentmonitoring,enabling CD34+enriched cellstobeassessedbyFISHonceamonthimmediatelypriorto each cycleof azacitidine.Cytogeneticresponse wasdetectedin 67%ofpatientsusingthistechnique;ineachcasethecytogenetic responseprecededahematologicalresponseby1–3cycles.Despite theseencouragingdata,anotherrecentstudyconcludedthatFISH fordel(5q)alonemaybeinadequateformonitoringdisease pro-gressionandresponse totherapy,becauseitmaymiss casesof cytogeneticprogression/clonalevolution[21].

FISHanalysiscouldpotentiallypredictdiseaseprogressionin patientswithactivetherapy.Braulkeetal.demonstratedthatina patientwithIPSShigh-riskMDSwithacomplexaberrantkaryotype atdiagnosis,thecomplexkaryotypewaseliminatedfollowingeight cyclesofazacitidine(onlyanisolateddel[5q]aberrationremained). Azacitidinetherapywasinterruptedbetweenweek32andweek 52;cytogeneticprogressionwasdetectedatweek52which pre-cededlossofhematologicalimprovementthatoccurredatweek 57.Moreextensivestudiesareongoingtoinvestigateutilization ofthetechniquetoscreenandmonitorpatientswithMDSunder differenttreatmentmodalities(FISHanalysiswithastandard [7 probes]or‘super’panel[12probes]undertakenevery2–3months, andannually,respectively).Preliminarydatahaveindicatedhigher than expected occurrences of certain anomalies, e.g. del(12p), and 12% karyotypic evolution ratewithin a very short median follow up period of 8 months[22].FISH could alsopotentially beusedtomonitorlower-riskpatientsreceivingsupportivecare only.The identiﬁcationofcytogenetic progressioncould poten-tially prompt early treatment interventions (e.g. azacitidine or hematopoieticstem celltransplantation) priortohematological progressiontherebyimprovinglong-termoutcomes.

3.1.1. Recommendationsoftheexpertpanel

FISHshouldplayasupplementaryroletocytogenetics; how-ever,FISHwillnotsupersedeconventionalchromosomalbanding analysisforinitialdiagnosisofMDSbecauseitmaynotdetectall chromosomalaberrationsinapatient[21].Thepossibilityof fre-quentFISHanalysisonPBsamplesfromthesamepatienttomonitor diseaseprogressionandresponsetotherapyisexcitingthough fur-therprospectivedataarerequiredinorderforthistechniquetobe integratedintostandardclinicalpractice.

3.2. Flowcytometry

Cytomorphologicalanalysisisthebackboneofthediagnosisand classificationofMDS.However,accurateassessment ofthetype anddegreeofdysplasiaandpercentageofblastscanbedifficult, witha degree of subjectivityand variability between laborato-ries.Biologicalmarkersareoftenrequiredtoimprovediagnosis andclassificationinordertooptimizetreatmentdecisions.Asthe patternofcellsurfaceantigenexpressionduringhematopoiesisis highlyconserved,aberrantcells canbeeasilyidentifiedbyflow cytometry.Anumberofaberrantantigenshavebeenidentifiedas beingofmajorprognosticimportanceinpatientswithMDS.For examplethepresenceofCD7onCD34+myeloidprogenitorcells hasbeenshowntobeassociatedwithrapidevolutiontoAMLand poorsurvival[23].

Standardizedcombinationsofantibodies,aswellastechnical recommendations, have beenput forwardin order tofacilitate theuseofflow cytometryinthediagnosisandprognostic eval-uation of MDS[24].A standardized scoring systemtoquantify immunophenotypicaberrations in the myelomonocyticlineage, theflowcytometricscoringsystem,hasbeendevelopedand vali-datedtofacilitatetheuseofflowcytometryintheclinic[23,25].

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Emerging clinicalevidence suggeststhat flow cytometry could have prognostic value in patients withMDS in terms of help-ingtopredictpost-transplantoutcomes[26],aswellasassessing therisk of transfusion dependence and progression to AML in patientswithlow-/int-1-riskMDS[23].Flowcytometrymayalso bearobustapproachtoidentifyingpatientswithrefractory ane-miawithunilineage dysplasia(RCUD) and refractory cytopenia withmultilineagedysplasia(RCMD)accordingtotheWHO2008. TheseWHOsubgroupsareparticularlydifficulttodistinguishby morphologybuttheydohavedifferentprognosticoutcomes[16]. However,accordingtocurrentEuropeanLeukemiaNetwork guide-lines, the role of flow cytometry is not yet established in the standarddiagnosticwork-upofMDS,butguidelinesareduetobe updatedin2011.

Flow cytometry could be useful for predicting response to therapyinpatientswithMDS.Astudyof46patientswith low-/int-1-riskMDS, demonstratedthatincorporation offlow cytometry into a previously validated model to predict response to ery-thropoietindefinedthreepatientsubgroupswith94,17and11% probabilitiesofresponse,respectively[27].Preliminarydata sug-gestthatflowcytometrymayidentifypatientswhocouldbenefit from azacitidine therapy and could also identify patients who wouldbenefitfromcontinuedprolongedtreatmentwith azaciti-dinehavingachievedstabledisease[28].

Atpresent,flowcytometrycannotsubstituteformorphology, butmayaddsignificantlytothediagnosisandprognostic assess-mentofpatientswithMDSandshouldbeintegratedintoclinical practicewhenpossibleandappropriate.Furtherprospective eval-uationisrequiredtoassesstheroleofflowcytometryintreatment decisions.Thereisaneedforstandardizationofflowcytometric techniquesin MDSand steps shouldbeputin place toensure inter-laboratoryreproducibility,whichisamajorfocuswithinthe EuropeanLeukemiaNetwork.

3.3. Microarraytechnologies

Developmentswithmicroarraytechnologyhaveprovidedtools tofacilitate high-resolution genome-wide analysiswhich could potentiallysupplement traditional methodologies suchas stan-dardcytogenetics.Suchtoolsincludecommerciallyavailablesingle nucleotide polymorphism (SNP) arrays. With this technology genomicDNAisdigestedwithrestrictionendonucleases,ampliﬁed, labeledandhybridizedtoachip-basedplatformthatcontainsDNA probesencompassingthousandsofSNPsspreadacrosstheentire genome(50K-,250K-,500K-and1000K-SNPchipsareavailable). Followinghybridizationareadoutisgeneratedthatprovides geno-typeinformationforeachSNPandcopynumberinformationacross thegenome[29].

SNP arrays offer a number of advantages over traditional techniques: (i) higher resolution versus standard cytogenetics (small‘cryptic’deletionsand duplicationscanbedetected);(ii) no dependency on the availability of live dividing cells and, therefore, they can provide results when routine cytogenet-ics are not available; and (iii) regions of uniparental disomy (UPD;chromosomalregionswherebothcopiesarederivedfrom the same parent) can be detected. The disadvantages of SNP arrays include an inability to detect balanced rearrangements (i.e. translocations and inversions), an inability to distinguish multipleclones and an inabilitytodetectsmalleraberrant cell clones (<15–20%of a cell population). However,as cytogenetic abnormalitiesare relativelyfrequent and mostly unbalanced in patientswithMDS,thediseaseisconducivetoSNParray analy-sis.

SNParrayscandetectlargenumbersofchromosomal aberra-tionsinpatientswithMDSthat arenot detectableby standard cytogenetics.UPDwasdetectedin46% ofpatients, deletionsin 10% of patients and ampliﬁcations in 8% of cases in a cohort of 119 patients with low-risk MDS who had either a normal karyotype or del(5q) by standard cytogenetics [30]. In another studyof 175patientswithMDS,MDS/myeloproliferative disor-derorsecondaryAML,chromosomallesionsweredetectedinup to80%ofpatients[31].Patientswithanormalkaryotypebased onstandardcytogeneticshad reducedsurvival ifcryptic abnor-malitieswerepresent(16versus39months;P=0.002)indicating thatcrypticabnormalitiesmayhavesomeprognosticsigniﬁcance [31].

Arraytechnologyhasbeeninstrumentalinidentifyinggenetic lesions that could both improve knowledge of the underlying pathogenesisof MDSandhave prognosticsignificance.The dis-coveryofacommonregionofUPDspanningchromosome4q24 inMDSpatientsledtotheidentificationofmutationsintheTET2 gene[32].Around19–26%ofpatientswithMDS,andapproximately 50%withchronicmyelomonocyticleukemia(CMML),have muta-tionsintheTET2gene,althoughtheprognosticsignificanceofthese mutationsremainuncertain[33,34].Discoveryofmicrodeletions byarraytechnologyhasledtotheidentificationofmutationsin genesinvolvedinhistonemethylation,suchasEZH2on chromo-some7q36.1andASXL-1onchromosome20q11[35,36].Mutations inASXL-1arebelievedtonegativelyaffectprognosisinpatients withMDS[37].

Geneticmutationsmaypredictresponsetotherapy.For exam-ple,inacohortof63patientstreatedwithazacitidine,mutations inTP53wereidentiﬁedasanindependentprognosticvariable neg-ativelyinﬂuencingoverallsurvival[38].Inpatientswithdel(5q) MDS,mutationsinTP53arethoughttobeassociatedwithpoor prognosis,lossofresponsetolenalidomide,anddisease progres-sion [39]. In another recent study of 86 patients treated with azacitidine,mutationsinTET2predictedresponsetotherapy inde-pendentlyofkaryotype[40].

Theadoptionofgene mutationanalysisintoclinicalpractice couldbefacilitatedby‘next-generation’technologythathasseveral advantages over traditionalsequencing, including:(i) improved throughputwhichallowsanalysisforalargenumberofpatients. Forexample,TET2hasrecentlybeenfullysequencedin355patients withMDSandCMML[41];(ii)improvedsensitivitythatenables detectionofmutationsthatarepresentinonly∼1%cells;and(iii) potentialusetomonitorclonal evolutionduringtreatment.Ina recentstudyofsevenpatientstreatedwithazacitidine,sequence analysissuggestedthatchangesinclonesizecorrelatedwith treat-mentresponse[38].Geneexpressionarrayscouldalsoplayarole inthefuturemanagementofpatientswithMDS.Recentdatahave suggestedthatgeneexpressionarrayscanbeusedtodeﬁnegene ‘signatures’thatidentifypatientswithhigh/intermediate/lowrisk ofprogressingtoAML[42].

Technological advances have led to rapid evolution in the understandingof molecularmechanismsunderlying the patho-genesisand malignant transformation ofMDS. However,atthe moment genomic technologies (SNP arrays, expression arrays, next-generationsequencing) cannotbeintegratedintostandard clinicalpracticeduetolackofexpertiseandprohibitivecosts.For thetechnologiestoentertheclinicinthefuture,groupsneedto cooperatetostandardizetheassays andreadout,manufacturers needtomakethetechnologiesmore user-friendlyand cheaper, andfurtherprospectivedataarerequiredtoevaluatetheeffectof particulargenemutationsondiagnosis,treatmentoutcomeand prognosis.

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4. Impactofemergingdataonprognosticclassiﬁcation systemsforpatientswithMDS

Variousprognosticsystemshavebeenproposed,withtheaimof improvingtheabilitytopredictsurvivalandprogressioninpatients withMDS;theseincludetheIPSSandWPSS[13,43].

IPSSremainsthegoldstandardforriskstratificationinpatients withMDSatdiagnosis.However,givenrecentprogressinthe dis-sectionofthebiologyofMDS,andtheidentificationofadditional prognosticrisk factors,it islikely that IPSSmay underestimate riskinitscurrentincarnation.Forexample,IPSSint-1patientscan sometimesbereclassifiedashighriskaccordingtotheWPSS,a time-dependentregression model, basedon WHOclassification criteria.UnlikeIPSS,WPSSprovidesdynamicprognostic informa-tionandincorporatestransfusiondependenceasanadditionalrisk factor.However,thedefinitionoftransfusiondependencyisvery subjectiveacrosscountriesandamongclinicians.Designationof redbloodcelltransfusiondependencyreliesheavilyonthe opin-ion of theclinician, who has totakeinto account theage and cardiaccomorbiditiesofthepatient.Thereisalsogreat discrep-ancybetween clinicians onwhen to transfuse; someclinicians willtransfusepatientswithhemoglobinlevels<10g/dLwhile oth-erswilltransfuseata8g/dLthreshold[44].Forthesereasons,it hasrecentlybeenproposedthattheWPSSismodifiedusing sex-specifichemoglobinthresholds(8g/dLforwomenand9g/dLfor men),insteadoftransfusionrequirement[45].Analysisofatotal of1344MDSpatientswiththemodifiedWPSSwasabletoidentify fivedistinctriskgroupswithclearlydifferentsurvivals[45].

BothIPSSandWPSSalsorequireupdatingbasedon improve-ments in knowledge concerning prognostic factors that have occurredsincetheintroductionofIPSSin1997[13],particularly theimpactofdistinctcytogeneticabnormalities.Itisalsoimportant toacknowledgethatWHOclassificationcriteriaofMDShavebeen updatedsincetheWPSSwasdeveloped[46].Importantchanges include:(i)theadditionofRCUDasadistinctcategory;(ii)patients withrefractorycytopenia(s)but lackingdiagnostic morphologi-calfeaturescannowbeconsideredashavingMDSiftheyhave specificMDS-relatedcytogeneticabnormalities(i.e.unclassifiable MDS[MDS-U]);(iii)thepreviouslydefinedRCMDwith≥15%ringed sideroblastscategory(RCMD-RS)isnolongerconsideredaseparate categoryduetoclinicalinsignificanceandisnowincorporatedinto RCMD;and(iv)patientswith2–4%blastsinPBand<5%blastsin BMarenowdiagnosedasrefractoryanemiawithexcess blasts-1 (RAEB-1)ifother clinicaland laboratoryfindings of MDSare present.Itishopedthatincorporationofnewprognostic informa-tionmayassistselectionofappropriatetreatmentoptionsandwill facilitatepredictionofresponsetotherapy.

ManyclinicaltrialsusetheIPSSfordifferentiationof ‘lower-’versus ‘higher-risk’ disease.However,somepatients classiﬁed as ‘lower-risk’, particularly those in the IPSS int-1 group, may havehigh-riskfeatures,e.g.patientswithRBC-transfusion depen-dentRCMDwithintermediatekaryotype.Consequently,arevised IPSS (IPSS-R) has been proposed which incorporates several changes includingan updated systemfor assessing cytogenetic risk and the consideration of the severity of cytopenias (with thresholds of hemoglobin: 10g/dL; absolute neutrophil count: 0.8×109_L−1_;_platelets:₁₀₀_×₁₀9_L−1_)._In_a_{retrospective}_analysis

of4417patientswithMDS,TheIPSS-Rdefinedfiveratherthanfour majorprognosticgroupsforclinicaloutcomes[47].Theupdated cytogeneticscoringsystemusedintheproposedIPSS-Risbased ondatafromaninternationalstudythatassessed the prognos-tic impactof 20 cytogeneticsubgroupsin ∼3000 patientswith MDSwhoweretreatedwithsupportivecareonly[10]. Abnormal-itieswere groupedasnormal, single(one abnormality),double (twoabnormalitiesstratifiedintothreesubgroups:double includ-ing del(5q);double including– 7/7q –; anyother double) and

complex(threeormoreabnormalitiesstratifiedintotwogroups: threeabnormalities;morethanthreeabnormalities).Basedonthis classification,fiveprognosticgroupswereidentifiedwith signifi-cantdiscriminationofoverallsurvival(OS)andriskforleukemic transformationacrossthegroups.Recently,thenewcytogenetic classificationsystemhasbeenindependentlyvalidatedinacohort of631consecutivepatientswithdenovoMDS[48].Applicationof thesamecytogeneticscoringsystemintheGroupeFrancophone desMyelodysplasiesregister(acombinationoftreated,untreated andcensoredpatients)resultedindiscriminationofOSbetween groupsbutdidnotconferthesamelevelofpredictivityasinother patientcohorts.

4.1. Recommendationsoftheexpertpanel

BoththeIPSSandWPSSarerobustandclinicallymeaningful toolstoestimatetheprognosisofagivenMDSpatient.Asmost patientsarenowtreatedwithactivetherapythatcanalterthe nat-uralhistoryofMDS,andimprovesurvival,thepanelemphasized theimportanceofhavingscoringsystemsthatpredictbothrisk andresponsetotreatment.Itisessentialthatfactorsare identi-ﬁedthatcanpredictresponsetoparticulartherapiesinpatients withdifferentprognosticriskscores.Emergingtoolssuchasﬂow cytometryandSNParrayswillprobablybeimportantinthisregard. Nevertheless,atthistime, pre-diagnosisassessmentofpatients’ riskbasedonphysiologicalageandcytogeneticdatawillcontinue tounderpinthemanagementofthedisease.Therefore,new scor-ingsystemsneedtobevalidatedincohortsofbothuntreatedand treatedpatients.Thismaybechallenging,asfewuntreatedpatients arenowavailabletovalidateriskassessment.

5. Concludingremarksandfutureperspectives

TheemergenceofnewtherapiesthatcanalterthecourseofMDS hasincreasedtheoptionsavailableforthemanagementofthe dis-ease.However,itisessentialthatpatientswhocouldbeneﬁtfrom thesetreatmentsareaccuratelydiagnosedassoonaspossible fol-lowinginitialpresentation.Forthistobecomeareality,itwould bedesirableforphysicianstohaveaccesstoscreeningtoolsthat wouldallowthemtomonitordiseaseprogressioninpatients dur-ingitsearlystagesandhelpthemtoassesswhenactivetreatment isappropriate,aswellasmonitoringtheresponsetotherapy.

Moving forward, morphological assessment and standard metaphasecytogenetics willremain centralto thediagnosisof MDS.However,FISHcouldplayanimportantsupplementaryrole, particularlyindetectingspecificabnormalities(e.g.del[5q])when insufficientmetaphases are available for standard cytogenetics. Recent data have demonstrated that a multifaceted diagnostic approachcanhelpimprovediagnosticprecisioninpatientswith suspectedMDS[2,49].Rapidprogresshasalreadybeenmadein thedevelopmentofmethodologiestomonitordiseaseprogression andresponsetotherapy.Forexample,FISHanalysisofPBsamples isbeingintensivelystudiedandhasshownsignificantpromise.In addition,flowcytometrymayprovetobea valuabletoolinthe diagnosis,prognosticevaluation,and monitoringofresponse to therapyinpatientswithMDSandmaybeintegratedintostandard clinicalpracticeinthefuture.Developmentsinmolecular technolo-gieshaveledtohugestridesintheunderstandingofthemolecular pathogenesisofMDS.Asourknowledgeofgenesinvolvedinthe pathogenesisandprogressionofMDSincreases,thisshouldhave animpactonprognosticscoringsystemsandallowforbetter strat-ificationofpatientsfordifferenttypesoftreatment.Asdiagnostic andprognosticguidelinesdevelopoverthecomingyears,amajor challengewillbetoensureadherencetoguidelinesacrosscenters indifferentcountries.

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Roleoffundingsource

Developmentofthismanuscriptwassupportedbyfundingfrom CelgeneInternational.

Conﬂictofintereststatement

UP,DH,VSandCHhavereceivedhonorariafromCelgeneand Novartis.JM hasreceivedresearch supportfromCelgene, Eisai, AlexionandhonorariafromCelgeneandAlexion.SPhasreceived honorariafromCelgene,AmgenandJanssen-Cilag.Otherauthors disclose no conﬂicts of interest relevant to the contentof this manuscript.

Acknowledgement

The authors would like to acknowledge writing assistance of Lynn Pritchard of FireKite during the development of this manuscript.

Contributions.Allpersonslistedasco-authorsactively partic-ipated in the Celgene-convened expert workshop (Munich, 27 October2010)andcontributedequallytothedevelopmentofpanel recommendations.Eachco-authorprovidedessentialinputintothe developmentofthemanuscriptandapprovedtheﬁnalversionof thedocument.

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