Overview to Diagnosis of Acute leukemia

Diagnosis of acute leukemia according to WHO classification

1. Overview to diagnosis of Acute
Leukemia
By:Ahmed MakboulAhmed
M.B.B.Ch, M.Sc
Assistant Lecturer, Clinical Pathology Department, South Egypt Cancer Institute

2. INTRODUCTION:
Allhematopoieticandlymphoidneoplasmscanbedescribedaccordingto
threemajorcharacteristics:
•Aggressiveness:AcuteversusChronic.
•Lineage:LymphoidversusMyeloid.
•PredominantSiteofInvolvement:BloodandBoneMarrowversusTissue.

3. 1.Aggressiveness:ACUTEvs.CHRONIC
•Hematologicneoplasmscanbedividedintoacuteandchronictypesbasedontwocharacteristics:survivalandmaturation:
SURVIVAL:
•Acute:Survivalmeasuredinweeksorafewmonths(withouteffectivetherapy).
•Chronic:Survivalmeasuredinyears.
Witheffectivetherapyformanyoftheacuteneoplasms,thesurvivaldifferencebetweenacuteandchronicneoplasmshasbeengreatlydiminishedoreliminated.
MATURATION:
•Acute:Predominanceofimmaturecells(blasts)
•Chronic:Predominanceofmaturecells

4. 2. Lineage: Lymphoidvs. Myeloid
•Thisdivisionrelatestothefirststepindifferentiationofthehematopoieticstem
cellintotheCFU-L(colony-formingunit-lymphoid)andtheCFU-GEMM(colony-
formingunit-granulocyte-erythrocyte-megakaryocyte-macrophage).
•NeoplasmsderivedfromtheCFU-Laredesignatedlymphoid;thosederivedfrom
theCFU-GEMMaremyeloid.

5. 3. Predominant Site of Involvement: Bloodand Bone
Marrowvs. Tissue
•Neoplasmswithpredominantbloodandbonemarrowinvolvementare
calledleukemia.
•Neoplasmswithpredominanttissueinvolvementarecalledlymphomasif
composedoflymphocytesandgranulocyticsarcomas(sometimescalled
chloromasorextramedullarymyeloidtumors)ifcomposedpredominantlyof
myeloidcells(nowcalledmyeloidsarcoma).

6. Definition:
•AcuteLeukemiaisamalignantdiseasecharacterizedbyclonalexpansionofhematopoieticprecursors
(Blasts)inthebonemarrowBM,peripheralbloodPBorothertissues.Themostimportantfactoristhe
presenceof≥20%blastsintheperipheralbloodorbonemarrow.
•Accordingtoage,acuteleukemiacanbedividedintochildhoodacuteleukemiainpatients<15years,
adultacuteleukemiainpatients>15yearsandacuteleukemiaatelderlyageinpatients>60years.
Inthelattergrouptheresponsetotherapyisinferior.
•Accordingtocelltype,acuteleukemiaisdividedinto2maingroups,acutemyeloidleukemiaAML
forming80%ofadultcasesandacutelymphoblasticleukemiaALLforming80%ofchildhoodcases.

7. Prerequisite laboratory techniques for diagnosis of
Acute Leukemia:
Theyinclude:
1.Morphology.
2.Cytochemistry.
3.Immunophenotyping.
4.Geneticstudies:Cytogeneticanalysis&Moleculargeneticanalysis.

8. 1. Role of Morphology:
I.Differentialcount:
•Manualdifferentialcountsofbloodfilmsandbonemarrowfilmsshouldbeperformedinall
patients.
•TheWHOexpertgroupadvisesa200–celldifferentialcountonabloodfilmanda500–cell
differentialcountonthebonemarrowfilminanareaasclosetoaparticleandasundilutedwith
bloodaspossible.Performinga500–celldifferentialcountonthebonemarrowisparticularly
importantifthepercentageofblastcellsisatthelevelof20%whichisthecriticallevelforthe
diagnosisofacuteleukemia.
•Theblast%derivedfromthebonemarrowaspiratecorrelateswithanestimateoftheblast%in
thetrephinebiopsy,althoughfocalclustersorsheetsofblastsareregardedasdisease
progression.

9. II.Identificationofblasts/blastequivalents:
1.Myeloblasts:
Size:Theyarelargecells.
Nucleus:
-Blastscommonlyhavemorethan2nucleoli.
-Nuclearfoldingwithlippingismorein
myeloblastswhilenuclearcleftingismorein
lymphoblasts.
-Prominentnucleoli.
Cytoplasm:
-Thecytoplasmissmooth,abundant,
homogeneousandusuallypaleattheperiphery.It
maycontainAzurophilicgranulesorAuerrods.The
presenceofAuerrodsshouldbementioned.

10. 2.MonoblastsvsPromonocytes:
CriteriaMonoblastPromonocyte
Size:Large. Intermediatetolarge.
Nucleus:oRoundnuclearcontours.
oFinechromatin.
oProminentnucleolus.
oSmaller,variablyprominentnucleoli.
oGentlylobulatednucleiwithdelicate
nuclearfolding.
oMoreeven/dispersedchromatin
patternthantypicalmonocyte.
Cytoplasm:oAbundantvariablybasophilic
cytoplasm.
oFinecytoplasmicazurophilic
granules.
oNoAuerrods.
oMayseecytoplasmicvacuoles.
oAbundant,lightlybasophilic
cytoplasm.

11. MonoblastPromonocyte

12. 3.Abnormalpromyelocytes:
•Blast equivalent in acute promyelocytic leukemia.
•Has 2 variants:
a). Typical (Hypergranular) variant:
oAbnormalpromyelocyteswithirregularandoften
bilobednuclei.
oNumerouslargeintracytoplasmicgranulesand
granulescoveringnuclei.
oAbnormalcellswithnumerousAuerrods(faggot
cells)canbeidentifiedinmajorityofcases.
Hypergranular APL

13. b).Microgranular(Hypogranular)variant:
oAbsentorscantcytoplasmicgranulesbylight
microscopy.
oPresenceofabundantsubmicroscopic
granuleshighlightedbystrong
myeloperoxidasereactivity.
oFrequentbilobednuclei(slidingplates).
oRarefaggotcellspresentinmostcases.
Microgranular APL

14. 4.Erythroblasts:
•Blast equivalent in pure erythroid leukemia.
•Size: Variably sized, small to large.
•Nucleus:
oRound nucleus.
oFine/immature chromatin.
oProminent nucleolus.
•Cytoplasm:
oDeeply basophilic cytoplasm.
oCytoplasmic vacuoles.

15. 5.Megakaryoblasts:
•Size:Theyareofmediumtolargesize
•Nucleus:Nucleusisround,slightlyirregularorindentedwithfinereticularchromatinandonetothreenucleoli.
•Cytoplasm:Thecytoplasmisbasophilic,agranularandmayshowdistinctblebsorpseudopodformation.
•InsomecasesblastsarepredominantlysmallwithhighnuclearcytoplasmicratioresemblingL1lymphoblasts.Large&smallblastsmaybepresentinthesamepatient.

16. Lymphoblasts:
CriteriaL1 blastsL2 blasts
Size:Small blasts.Largeblasts.
Cytoplasm:Scant cytoplasm.Moderatecytoplasmandmayhave
vacuoles.
Nucleus:oCondensed chromatin.
oIndistinctive nucleoli.
oDispersedchromatin.
oVariablenucleoli.

17. L1 BlastsL2 Blasts

18. L3 Blasts

19. 2.Cytochemistry:
i.Myeloperoxidase(MPO):
•Myeloperoxidaseisanenzymelocatedintheazurophil(primary)granulesofmyeloidcells.
•MPOpositivityappearsascolouredgranulesinthecytoplasmofcellsmainlyatthesiteofenzymeactivity(Golgizone).
•AllthestagesofneutrophilseriesshowMPOpositivity.
•InmonocyteseriesazurophilgranulesaresmallerandMPOactivitystainslessstronglyandappearslateduringmaturation.
•MPOisneverseeninlymphoblasts.
•ThemainuseofMPOistodistinguishAMLfromALL.
•MPOispositiveinAMLsubtypesM1,M2,M3(strongpositivity),andM4

20. ii.SudanblackB(SBB):
•PhospholipidsinthemembraneofneutrophilgranulesarestainedbySBB.SBBpositivityparallelsthatofMPOinneutrophilseries.
iii.Estrases:
1.Chloroacetateesterase(CAE)(a.k.a.specificesterase):
•Thereactionispresentinallcellsofneutrophilseries(thoughlesssensitivethanMPOandSBB)andisnegativeinmonocyteseries.
•Itiscommonlyusedincombinationwithnon-specificesterase(NSE)fordiagnosisofleukemiawithbothmyeloidandmonocytecomponents(AMLM4).Bothesterases(CAEformyeloidandNSEformonocyticcomponents)canbedemonstratedinthesamebloodfilm;thisiscalledascombinedordoubleesterasereaction.

21. 2.Non-specificesterase(NSE)reaction
(usuallydemonstratedbyANAEorANBE):
•α-naphthylacetateesteraseisanenzymethatis
presentinlargequantitiesinmonocyticcells.
•Itispresentinsmallamountsinmyeloidandlymphoid
cells.
•Thenon-specificesterasereactionisintenselyand
diffuselypositiveinmonocyteseriesandissensitiveto
sodiumfluoride.
•ANAE/NASDAgivesacharacteristicallystrongdiffuse
reactioninmonocyticleukemias(M4&M5)andgivesa
localizedpatterninM6&M7.
•Esterasereactionissensitivetoinhibitionbysodium
fluorideinmonocytes.ItispartiallyinhibitedinM4and
totallyinhibitedinM5.

22. iv.Periodicacidschiff’sreaction(PAS):
•Periodicacidisanoxidisingagentthattransformsglycolsandrelatedcompoundstoaldehydes.
•ThealdehydegroupsthenalongwithSchiff’sreagentformaninsolublered-ormagenta-colouredcompound.
•Inhematopoieticcells,positivereactionisduetotheabundanceofglycogenincytoplasm.
•InALL-Blineage,PASstainshowscharacteristicpatternofblockpositivity.
•InTcell-ALLandinL3subtypeofALL,PASreactionisnegative.
•PASpositivityisalsoseeninmonoblasts(inAMLM5)andinerythroblasts(inAML-M6);however,inthesecellssmallblocksofpositivematerialarepresentagainstadiffuselypositivecytoplasmicbackground.

23. v.Acidphosphatasestain:
•ItisusedforrecognitionofGolgizonein
T-lineageALL.
•BothPAS&acidphosphatasestainsare
nolongerindicatedinsuspectedALL
unlessthereisnoaccessto
immunophenotyping.

24. 3.FlowcytometricImmunophenotyping:
Roleofimmunophenotypinginacuteleukemia:
1.IPTisnecessaryforlineageassignmentandtodetectmixedphenotypeacute
leukemia.
2.IPTisalsoimportantindistinguishingbetween:
•MinimallydifferentiatedAMLandALL.
•MyeloidblastphaseandlymphoidblastphaseinCML.
3.AsecondaryroleforIPTisinmonitoringminimalresidualdisease.Thisisachievedby
definingaleukemiarelatedphenotypeinanindividualpatient.Forthispurpose,an
extendedantibodypanelisneeded.
4.Ifthereisapossibilityofamonoclonalantibodybeingusedintherapye.g.Myelotarg;
antiCD33inAML,thenitshouldbeincludedinthediagnosticpanel.

25. 4. Genetic studies:
I.Cytogeneticanalysis:
-Whenresourcespermit,itshouldbecarriedoutinallpatientsforthefollowingreasons:
1.AMLcanbeassignedtocertainprognosticgroupssotreatmentcanbemodifiedaccordingly.e.g.
goodriskcytogeneticgroupswhichinclude:
◦t(8;21)(q22;q22):itoccurspredominantlyinAMLM2andsomecasesofAMLM4.
◦Inv16(p13;q22)ort(16;16)(p13;q22):itoccursinAMLM4withnotableassociationwithabnormal
eosinophilia.
◦t(15;17)(q24;q21):itisspecificforAMLM3&M3variant.TheresponsetoallretenoicacidATRAis
remarkable.
2.Itfacilitatestherecognitionofsecondaryleukemiae.g.therapyrelatedALandthedistinctionbetween
thealkylatingagentrelatedleukemiainvolvingchromosomes5&7andthetopoisomeraseIIinteractive
drugrelatedleukemiainvolvingchromosome11q23e.g.t(4;11)(q21;q23).
3.CytogeneticanalysisisessentialiftheWHOclassificationofAMListobeused.

26. A case of AML with normal karyotype (46,XY)

27. -CytogeneticanalysisinALLisdifficultduetothelowmitoticindexofthe
leukemicblastsandthepoorchromosomemorphology.Thiscanbe
overcomebyusingmoleculargeneticanalysis.

28. II.Moleculargeneticanalysis:
•Moleculargeneticanalysisonlypermitsthedetectionofabnormalitiesthatarespecifically
soughtwhereasconventionalcytogeneticanalysiscanassessallchromosomes.Somolecular
geneticanalysissupplementscytogeneticanalysisanddoesnotreplaceit.
•TechniquesusedinmoleculargeneticanalysisincludeFluorescenceinsituhybridization
(FISH)&RT-PCR.
•ApotentialroleformoleculargeneticsismonitoringofMRDe.g.monitoringthetranscription
offusiongenee.gPML-RARAfusiongene.
•Forprognosis,identificationofabnormalgeneexpressione.g.FLT3expression,FLT3-ITD
mutationsareassociatedwithanadverseoutcome.SomecasesofAMLwithanapparently
normalkaryotypecarrymutationsinnucleophosmin(NPM)gene,haveafavorableprognosis.

29. Leftpanel:2redand2green(2R2G)signalpatterncorrespondtonormalPML(red)andRARA
(green).
Rightpanel:FISHanalysisusingprobesdirectedtoPML(green)andRARA(red),demonstrate
intactPMLandRARAsignals(redandgreen)and2fusionsignals(yellow).

30. ExampleofquantitativeRT-PCRusingTaqManprobesandprimersdesignedtoamplify3
PML-RARAfusiontranscriptsdemonstratehighcopynumbersofPML-RARAshortform
transcript(Blue).TheamplificationplotinblackrepresentsABL1internalcontrolgene.

31. AcuteMyeloidLeukemiaGeneticRiskClassification(EuropeanLeukemiaNET2017risk
stratification):
RiskStatusCytogeneticMolecular Abnormalities
Favorable:oCorebindingfactorAML:AMLwith
t(8;21),AMLwithinv(16)/t(16;16).
oAcutepromyelocyticleukemiawithPML-
RARA.
Normalkaryotypewith:
oNPM1mutationandabsenceofFLT3-ITD
mutation.
oBiallelicCEBPAmutation.
Intermediate:oAMLwitht(9;11).
oNormalkaryotype.
oOthernon-defined.
oCorebindingfactorAMLwithKITmutation.
oMutatedNPM1withFLT3-ITD.
Unfavorable:oComplex(>3clonalabnormalities).
oAMLwithinv(3)/t(3;3)(GATA2-MECOM).
ot(11q23-KMT2Agene)otherthant(9;11).
oAMLwitht(6;9)(DEK-NUP214).
oAMLwitht(9;22).
oMonosomalkaryotype.
oMonosomy5,del(5q).
oMonosomy7,del(7q).
Normalkaryotypewith:
oFLT3-ITDmutation.
oTP53mutation.
oMutatedRUNX1.
oMutatedASXL1.
oWildtypeNPM1withFLT3-ITD.

32. FavorableIntermediateUnfavorable
Hyperdiploidy(>50chromosomes,
especiallywithtrisomy4,10,17).
t(5;14)(IL3-IGH).Hypodiploidy(<45chromosomes).
t(12;21)(ETV6-RUN1).t(1;19) (TCF3-PBX1)t(9;22)(BCR-ABL-1).
Normalkaryotype.KMT2Arearrangement;t(4;11)
Anyotherabnormalitiesnotin
favorableorunfavorable
categories.
B-ALLwithiAMP21
BCR-ABL1-likeB-ALL
Complexabnormalities

33. Moleculargeneticanalysishasthefollowingadvantagesoverconventional
cytogeneticanalysis:
1.Itcanconfirmthepresenceofspecificfusiongeneinpatientswithvariant
ratherthanclassicaltranslocation.
2.Itcanyieldresultswhencytogeneticanalysisfailsorgivesnormal
metaphases.
3.Itcandetectrelevantabnormalitiesthatarenotdetectedbycytogenetic
analysis(i.e.cryptic)e.g.ETV6-RUNX1(TEL-AML1)fusiongeneinB-lineageALL
associatedwithcryptict(12;21).
4.Itcandetectsubmicroscopicabnormalitiese.g.GATA1mutationsinAML-M7in
DownsyndromeorinternaltandemduplicationofFLT3inmultiplemorphological
subtypesofAML.
5.Itgivesrapidresultssoclinicaldecisionscanbemade.