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MolecularMedicalCytogenomicsFluorescenceinsituHybridizationFISH-aprocesswhichvividlypaintschromosomesorportionsofchromosomeswithfluorescentmoleculesOpeningpicture-HumanM-phasespreadusingDAPIstainIdentifieschromosomalabnormalitiesAidsingenemapping,toxicologicalstudies,analysisofchromosomestructuralaberrations,andploidydeterminationUsedtoidentifythepresenceandlocationofaregionofDNAorRNAwithinmorphologicallypreservedchromosomepreparations,fixedcellsortissuesectionsHistoryofInsituhybridization

1）Isotopicinsituhybridization

——1970s

2）Non-isotopicinsituhybridization

——1986,1994

（1）ILASA

（2）Fluorescenceinsituhybridization,FISH

DisadvantageofISH：

1）unstable；

2）highbackground；

3）longertime；

4）statistics5）isotopetreatment

PrinciplesofFISHSchematicdiagramforFISHtechnique(ReprintedwithpermissionfromO'Connor,2008.Fluorescenceinsituhybridization(FISH).NatureEducation1(1).FISHtechniquesFISHProcedureDenaturethechromosomesDenaturetheprobeHybridizationFluorescencestainingExamineslidesorstoreinthedarkEachfluorescentlylabeledprobethathybridizestoacellnucleusinthetissueofinterestwillappearasadistinctfluorescentdotDiploidnucleiwillhavetwodotsIfthereisduplicationintheregionofinterest,thegainwillresultinmorethantwodotsIfthereisalossintheregionofinterest,oneorzerodotwillresultIfmorethan4dots,indicatinggeneamplificationInterpretationofFISHFISH:AdvantagesPossiblecorrelationbetweenFISHresultandtumormorphologywithconventionallightmicroscopyCanbeusedinformalin-fixed,paraffinembeddedsectionsorfreshfrozentissueAllowssimultaneousinterrogationofmultiplecytogeneticsignaturesCanbeusedeitherinbulkytumorsortumorswherethemalignantcomponentcontributestoasmallproportionoftheoverallcellularpopulationlesslabor-intensivemethodforconfirmingthepresenceofaDNAsegmentwithinanentiregenomethanotherconventionalmethodslikeSouthernblottingFISH:LimitationsProbedesignrequiresknowledgeofspecificchromosomalabnormalitiestobestudiedCutoffsignalsmaybedifferentamonglaboratoriesProcessingerrors,imperfecthybridization,non-specificbinding,photobleaching,interobservervariability,andfalsepositiveandnegativeresultsarepossible

FISH

Probes

1.Singlecopyprobe：

（1）YAC，BAC，Cosmid，Plasmid，cDNA

fragment

（2）applications

（a）positionofDNA

fragmentandmosaicclone；

（b）microdeletionandduplication；

（c）chromosomebreakageaffirmation

（d）interphasechromosomeabbrations14Centromerspecificprobes●chromosome7centromerspecificsignal●chromosome8centromerspecificsignal２）simplerepetitiveprobes

——heterochromaticcentromereprobes

alphasatellite/satelliteIIIDNA

advantage：verystrongsignals

applications：(a)

markerchromosomes

(b)chromosomenumberdisorders

(c)interphase

17DiGeorgeSyndrome(microdeletionon22)●22q13specificcontrolsignal●22q11.2specificDiGeorgeprobeSRYlocusspecificprobe.SRY/Xprobewasused.●Xchromosomecentromerspecificprobe●SRYprobe Locusspecificprobes

chromosomepaintingprobes

Wholechromosomeorpartials

Proberesources：

（1）human-rodenthybridmaterials

（2）fluorescence-activatedcellsorter,FACS,thenPCR

（3）microsection，PCR；

Applications（1）numberandstructuredisorders

（2）comparation；

（3）tumorstudies

24WholechromosomepaintingprobesChromosome8paintingprobeFISHandTelomeresTelomericprobesdefinetheterminalboundariesofchromosomes(5’and3’ends)UsedinresearchofchromosomalrearrangementsanddeletionsrelatedtocellagingorothergeneticabnormalitiesSpecialtelomericprobesspecifictoindividualchromosomeshavebeendesignedProbeisbasedontheTTAGGGrepeatpresentonallhumantelomeresApplicationincytogenetics-candetectsubmicroscopicdeletionsandcryptictranslocationsofgenesassociatedwithunexplainedmentalretardationandmiscarriages27

TELOMERICPROBES29FiberFISHMulti-colorFISH

多色复合染色体FISH探针(multiplexFISH,M-FISH

probes)

#两种以上不同的非同位素标记，不同的荧光检测系统，通过不同的滤光片组合或极少数几种非同位素标记探针后，按照不同的比例混合，可以显示多种颜色。31SpectralKaryotyping(SKY)Figure6.ImagescapturedfortheM-FISHkaryotypinganalysis:DAPI(counterstain)aloneandcombinedwithSpectrumAqua™,SpectrumGreen™,SpectrumGold™,SpectrumRed™,andSpectrumFarRed™.Distinctchromosomesfluoresceindistinctcolorsduetothecombinatorialprobe-labelingscheme.33MultiplexFISH

Metaphasespreadofalungcancercelllineafterhybridizationofthe

5-fluorochromeM-FISHmixshownin"true-color"(a)and"classification-color"representation(b).Thekaryotypeisshownin(c).

34Cobra-FISHOverviewofthe12-colorimageobtainedusingahumanCOBRA–FISHprobeset.Onlythreefluorochromeswereusedforratiolabeling(DEAC,Cy3andCy5).Thewhitearrowindicatesatranslocationthatwasnotdetectedbyconventionalcytogenetictesting./.../v1/n1/full/nprot.2006.41.htmlFISHUsesDetectionofhighconcentrationsofbasepairsEg:MousemetaphasepreparationstainedwithDAPI(anon-specificDNAbindingdyewithhighaffinityforA-Tbonds)Centromereregionsstainedbrighter-meanstheyarerichinA-TbondsAlsousedingermcellorprenataldiagnosisofconditionssuchasaneuploidiesFigure5AcomparisonofSNParrayresultsforchromosome7usingDNAfromfreshandfixedcells.A.AcomparisonofSNParrayresultsfromfresh(left)andfixed(right)U937cellline.Thebracketsontheleftshow(a)a38Mbdeletionand(b)a70Mbdeletionof7qwhichencompasses(a).B.Cellswiththesmaller(top)andlarger(bottom)7qdeletionvalidatedbyFISHwiththeVysisLSID7S486SpectrumOrange(7q31,red)andCEP7SpectrumGreen(centromere,green)probesinmetaphasespreads.Thedeletedchromosome7isarrowed.TheIlluminaCytoSNP12arrayprobeshaveamedianspacingof6.1kb.FISH检测微小缺失Figure1Asupernumeraryringmarkeroriginatingfromchromosome16.Thesampletestedwasamnioticfluid.Bandedmetaphase(left)andspectralkaryotype(right)areshown.MarkeroriginwasconfirmedbyFISHusingachromosome16centromericprobe(notshown).Figure3Aninsertionofgeneticmaterialfromchromosome9intochromosome6.Thesampletestedwasperipheralbloodfroma1-year-oldboy.Bandedmetaphase(left)andspectralkaryotype(right)areshown.TheinsertionwasconfirmedbyFISHusingpaintingprobesspecificforchromosomes6and9(notshown).Figure4Spectralkaryotypeanalysisidentifiedoriginsofmultiple(3-6)markerspresentina3-year-oldgirl.Thesamplewasperipheralblood.Bandedmetaphase(left)andspectralkaryotype(right)areshown.Spectralkaryotypeanalysisindicatedthatthemarkerswererespectivelyderivedfromchromosomes12,19,21,22,andX.MARILEILAVARELLA-GARCIAFigure7.Dual-colorFISHassayinbreastadenocarcinomaswithsequencesfortheHER-2genelabeledinred(SpectrumOrange™)andthechromosome17centromerelabeledinSpectrumGreen™.MARILEILAVARELLA-GARCIAFigure8.EvaluationofHER-2proteinstainingbyimmunohistochemistryandgenestatusbyFISHinlungtumors[38].Normalepitheliumshowingapicalstaining(A),well-differentiatedtumorshowingbasolateralstaining(B),moderatelydifferentiatedtumorscoredas2+(C),andpoorlydifferentiatedtumorscoredas3+(D).FISHassaysshowedthreemajorpatterns:balanceddisomyforHER-2geneandchromosome17(E),balancedgainforHER-2geneandchromosome17(F),andHER-2geneamplificationwithagene/chromosomeratio>2(G).Figure10.Multitargetprobeset(LAVysion™;Vysis)fordetectionofaneusomyinlungtumors.A)Fourchromosomaltargetsareaddressed:5p15(labeledinSpectrumGreen™),chromosome6centromere(inSpectrumAqua™),EGFRsequencesat7p12(inpectrumRed™),andMYCsequencesat8q24(inSpectrumGold™).B)Normalbronchialepithelialnucleishowingtwocopiesofeachprobe.C)HighlyaneusomicnucleusfromasquamouscelllungcarcinomashowingadditionalcopiesforallfourDNAtargets.Figure9.AhighlyaneusomiccellfoundinvoidedurineusingtheUroVysion™bladdercancer.Thetoppanelsegmentedimagesforeachofthethreechromosomeenumerationprobes(CEP)includingcentromericsequencesforchromosomes3(CEP3labeledinSpectrumRed™),7(CEP7labeledinSpectrumGreen™),and17(CEP17inlabeledpectrumAqua™)andthelocusspecificindicator(LSI)probeforp16(9p21labeledinSpectrumGold).ThebottompanelshowsnucleistainedwithDAPIandthemulticolorimage.Thelargenucleusatthetopleftineachimageofthetoppanelshows,respectively,6,11,10,and6copiesoftheDNAtargets.Figure4AcomparisonofSNParrayresultsforchromosome2usingDNAfromfreshandfixedcells.A.AcomparisonofSNParrayresultsfromfresh(left)andfixed(right)U937cellline.Gainofasectionofthelongarm(bracketsa,b)isdenotedbyasingleverticalgreenbarforthefixedspecimen(FoundReg=FoundRegion)(b)whereasinthefreshspecimen(a)thisisdividedintotwoseparatesections.ThisisrepresentativeoftheminorboundarydifferencesdeterminedbytheKaryostudiosoftware,betweenthetwoexperiments.B.TheM-BANDpatternforchromosome2.Theidiogramsbelowthebandedchromosomesshowthesectionofchromosome2presentineachchromosome.Thegreenbarrepresentsthehomologfromoneparentandthebluebarsrepresentthehomologfromtheotherparent(inferredfromBallelefrequencies,seeMethods).TheIlluminaCytoSNP12arrayprobeshaveamedianspacingof6.1kb.50

ComparativeGenomicHybridization(CGH):51DNAfromNormalTissue(CONTROL)DNAfromDiseaseTissue(TEST)LabelIncorporating(REDFluorescent)

LabelIncorporating(GREENFluorescent)HybridizetoNormalMetaphaseChromosome-202(log2ratio)DeletionAmplificationComparativeGenomicHybridization(CGH)52ComparativeGenomicHybridization(CGH)53ComparativeGenomicHybridization(CGH)54ComparativeGenomicHybridization(CGH)55ComparativeGenomicHybridization(CGH)56

Microarraytechnique

aCGHProbesBACsorP1(PAC)clones:DNAintheformofbacterialartificialchromosomes(sizeof75–200kb),CosmidsandFosmids:smallerinsertclonessizeof30–40kbandsizeof40–50kbOligonucleotides:(25–85mers).ThegenomicresolutionofthedifferentaCGHplatformsisdeterminedbyspacingandlengthoftheDNAprobes.Figure2.EvolutionofaConstitutionalCMADesignEarlyversionsofarray-basedComparativeGenomicHybridization(aCGH)platformsusedforconstitutionalcytogenetictestingtargetedthesubtelomericandericentromericregionsanddefinedmicrodeletionandmicroduplicationsyndromes.61,62(B)Later,moreextensivecoveragewasaddedatthesubtelomericandericentromericregionsandincludedadditionalprobesoutsidethetargetedregions;thisisso-called‘‘backbone’’coverage.(C)Higher-densitybackbonecoverageorhigh-densitygenomewidearraysprovideessentiallywhole-genomecoverage,yieldingevenhigherdetectionratesIndicationsforOrdering

Individualswithanunexplainedabnormalphenotype,suchas:◦Autism/autismspectrumdisorder(ASD)/pervasivedevelopmentaldisorder(PDD)◦Developmentaldelay/intellectualdisability,withorwithoutdysmorphicfeatures◦Multiplecongenitalanomalies◦Heartdefects&Epilepsy/seizuresScreeningformicrodeletionsandmicroduplicationsassociatedwithknownsyndromes/clinicalphenotypesScreeningforuniquemicrodeletionsandmicroduplicationsnotassociatedwithknownsyndromesIdentificationofLCSHthatmaybesuggestiveofUPDorincreasedriskofarecessivedisorderFurthercharacterizationofachromosomalabnormality,includingmarkerchromosomes,ringchromosomes,apparentterminaldeletions,unbalancedtranslocations,orsubsequentanalysisofanapparentlybalanceddenovorearrangementseeninpatientswithabnormalphenotypesARUPVariabilityinpreparingchromosomesKaryotypingvariationasresultofoperatorskillsResolutioncanrangefrom5Mbto10MbdependingonpreparationMicroarraysofferunbiasedmeansofdetectingchromosomalaberrations.Thesamechromosomeprepared6differenttimes!SMS:del(17)(p11.2)PurposeofCMADetectionofDNAcopynumbergainsandlossesassociatedwithunbalancedchromosomalaberrations.Regionswithanabsenceofheterozygosity(AOH),alsoreferredtoaslossofheterozygosity,regions/runsofhomozygosity,orlongcontinuousstretchesofhomozygosity,mayalsobedetectedbyplatformsWithsingle-nucleotidepolymorphism(SNP)-detectingprobes.SomeregionswithAOHmaybeindicativeofuniparentalisodi-somyorregionsofthegenomeidenticalbydescentTheutilityofthistechnologyfordetectionofgainsandlossesinpatientswithintellectualdisabilities,autism,and/orcongenitalanomalieshasbeenwelldocumented,andCMAisnowrecommendedasafirst-tiertestfortheseindicationsAdvantagesofCMAs

ThebenefitsfromtheuseofCMAsfordetectionofgainsandlossesofgenomicDNAinclude:1.AbilitytoanalyzeDNAfromnearlyanytissue,includingarchivedtissueortissuethatcannotbecultured.2.DetectionofabnormalitiesthatarecytogeneticallycrypticbystandardG-bandedchromosomeanalysis.3.Abilitytocustomizetheplatformtoconcentrateprobesinareasofinterest.4.Betterdefinitionandcharacterizationofabnormalitiesdetectedbyastandardchromosomestudy.5.Interpretationofobjectivedata,ratherthanasubjectivevisualassessmentofbandintensities.6.AbilitytodetectcopyneutralAOHwithplatformsincorporatingSNPprobes.7.Areadyinterfaceofthedatawithgenomebrowsersanddatabases.LimitationsofCMAsMostplatformscan’tdetectgeneticeventsthatdonotaffecttherelativecopynumberofDNAsequences.2.Low-levelmosaicismforunbalancedrearrangementsandaneuploidymaynotbedetectedbyCMAs.3.Thechromosomalmechanismofageneticimbalancemaynotbeelucidated.4.Tetraploidyorotherploidylevelsmaynotbedetectedormaybedifficulttodetect.5.Copynumbervariations(CNVs)ofgenomicregionsnotrepresentedontheplatformwillnotbedetected.6.CurrentCMAtechnologiesarenotdesignedtodetectduplicationsanddeletionsbelowthelevelofdetectionaccordingtoprobecoverageandperformance,pointmutations,geneexpression,andmethylationanomaliesthatmaycontributetothepatient’sphenotype.7.Nomicroarrayplatformwilldetectallmutationsassociatedwithagivensyndrome.Failuretodetectacopynumberabnormalatanylocusdoesnotexcludethediagnosisofadisorderassociatedwiththatlocus.

CarrierdetectionPreimplantationgeneticdiagnosisPrenataldiagnosisNewbornscreeningPostnataldiagnostictestingGermlinemutationsSomaticmutationPredictive,presymptomatictestingandPersonalizedMedicinePrevention,Diagnosis,andTreatmentbasedonArrayCGHCurrentOpinioninBiotechnology2008,19:36–40Copynumber+SNParrayCopy#AllelepeaksSNParraysmoresensitivefordetectionofmosaicismNon-mosaicdeletionMosaicdeletionAAABBBDeletionNormalDuplicationNormalAAABBBAAAABBBBAABABBBBAAAABBABNormalDeletionBBAAAABBBAAADuplicationABBABABBABChromosome2SmalldeletionLongcontinuousstretchesofhomozygosity(LCSH)withnormalcopynumberSNPsandconsanguinityorUPDFigure3.

Detectionof

UPD

andIBDby

SNP

Arrayanalysis.

SNP

Arrayanalysisshowingevidenceofuniparentaldisomy(UPD)andidentitybydescent(IBD).(a)

UPD

fortheentirechromosome15isindicatedbyabsenceofheterozygosity(toppanel-lackofsignalat0.5Ballelefrequency(BAF)whichrepresentsgenotypeA/B)andnochangeincopynumber(bottompanel-allsignalsareat0Logratio).(b)Blocksofabsenceofheterozygosity(AOH)oftheproximalregionsofchromosome9pand9qasdemonstratedbylackofsignalsatthe0.5BAF.WithintheblockofAOHat9p(redoval)isthegeneforgalactosaemia,

GALT.Thepatientisaffectedwithgalactosaemiaduetoahomozygousmutationinthe

GALT

gene.Theparentsareconsanguineous,whichisconsistentwiththemultipleblocksofAOH.芯片检出9p和9q部分纯合缺失，导致乳糖血症，父母系近亲结婚。ComparativeGenomicHybridizationintheStudyofHumanDisease,SauWaiCheung,

2011Figure2.

ModelofthemechanismofUPDincancer.

(a)

Bothmaternalandpaternalallelesarepresentinthenormalcell.

(b)

Mutatedcell(*).Duringmitosis,incompletesegregationofthechromosomecanoccuraseither

(c)

atrisomiccell(bynon-disjunction)or

(d)

amonosomiccell(byanaphaselag),and

(e)

theremainingalleleisduplicatedbymitoticnon-disjunction.Alternatively,

(f)

(i–iv)oneormoremitoticrecombinationeventscanoccurduringmitosis,resultinginsegmentalUPD.

(g)

(i)Lossofachromosomearmand(ii)reduplicationbynon-disjunctionmightfollow.Thewhitesegmentindicatesdeletion.Thebottompanelillustratesrepresentativelog2plotfiguresfromAsCNARshowingUPDmechanisms.Iftheredlineisover1andthecorrespondingregiongreenlineisbelow1,itrepresentsUPD.Ifjusttheredlineisover1,itrepresentsgain,andifjustthegreenlineisbelow1,itrepresentsloss.ThisfigurewasmodifiedfromRef.

[25],©theAmericanSocietyofHematologyNormalallelehomozygosityWholechromosomeisodisomyHomozygousblocksof1-3MbAABBCopynumber=2Figure3:

MultidimensionalomicsdataintegrationusingSIGMA2software.Combinedgenetic,epigeneticandgeneexpressionanalysisofcancersamplesfacilitatesidentificationofoncogenesandtumorsuppressorgeneswhichareconcertedlydisrupted.(a)Examplesofannotationtracks.(b)CopynumberprofilefromarrayCGHexperiment—afocalDNAamplificationofaregionon17q12ishighlightedinyellow.(c)Allelicstatus(SNParray).Thisregionisalsoencompassedinalargestretchofallelicimbalance.Bluehorizontalbarsindicatelocithatbecomehomozygous(lossofheterozygosity)inthetumorsample.(d)DNAmethylationanalysis(MeDIP-microarray)showsaconcurrentlossofmethylation,asindicatedbyapeakshiftedtoleftofthecenterline.(e)Heatmapsummaryofgeneexpressionprofileintheregionofinterest.Thegeneboxedinblueontheheatmapis

ERBB2.Itshowsthehighestlevelofdifferentialexpressionbetweenthetumorlineandapanelofnormaltissuesamples.(f)Thehistogramdisplaystherelativeexpressionofthetumorsampleascomparedwiththenormalsamplesfor

ERBB2(i.e.,expandedviewfromheatmap).ThisSNParraykaryotypefromacolorectalcarcinomademonstratesallele-specificanalysis(redandgreenplot)generatedusingtheOscaralgorithm(YamamotoG,etal.AmJHumGenet.2007Jul;81(1):114-26).Theallele-specificplotprovidestherelativecopynumberofeachalleleseparately.Inanormaldiploidstate,thereisonecopyoftheredalleleandonecopyofthegreenallele(onefromeachparent),foranoverallcopynumberoftwo(e.g.,chromosome2inthistumor).WhenthereiscopyneutralLOH/acquiredUPD,bothcopiesarecomingfromthesameparent,sotherearetwocopiesoftheredalleleandzerocopiesofthegreenallele,foranoverallcopynumberoftwo(e.g.,5qinthistumor).Inatrisomy,therearetwocopiesoftheredalleleandonecopyofthegreenallele,foranoverallcopynumberof3(e.g.,chromosome7inthistumor).Adeletionisshownfor11qinthistumor.Thereisonecopyoftheredalleleandzerocopiesofthegreenalleleforanoverallcopynumberof1.Note:Thetumor-initiatingeventinthiscasewascopyneutralLOHoftheAPCgeneonchromosome5q.Inotherwords,the2ndhitforthispatientwasviaacquireduniparentaldisomyratherthandeletion.

iKaryosandiKaryosDiagnosticsOverviewofallgeneticaberrationsfoundwithSNParrayin45adultandadolescentALLcases.©2008byNationalAcademyofSciencesOverviewofallgeneticaberrationsfoundwithSNParrayin45adultandadolescentALLcases.Minimallyinvolvedregionsareshowntotherightofeachchromosome.Foreachtypeofaberration,eachlinerepresentsadifferentcase.Bluelinesareregionsofuniparentaldisomy,lightgreenlinesarehemizygousdeletions,darkgreenlinesarehomozygousdeletions,andredlinesarecopy-numbergains.Notethehighfrequencyofdeletionsinvolvingchromosomes9p21.3,9p13.2,7p12.2,12p13.2,and13q14.2correspondingtotheCDKN2A,PAX5,IKZF1,ETV6,andRB1loci,respectively.IdentificationofthenovelKPfusioninaglioblastomatumor.OzawaTetal.GenesDev.2010;24:2205-2218©2010byColdSpringHarborLaboratoryPressProgressionofmyelodysplasticsyndromeA58-year-oldmalepatientpresentedwithreportsoffatigue,dyspnea,andpalpitations.Hisinitialbloodworkupidentifiedanemia.Subsequentpathologyconsultationidentified5%blastsinbonemarrow,resultinginaclassificationofrefractoryanemiawithexcessblasts-1(RAEB-1).Knowingthatcytogeneticstudiescanbetterclassifyprognosisforpatientswithmyelodysplasticsyndromes(MDS),thepathologistorderedakaryotypeandtheOncoChip™|CopyNumberEvaluation.3

TheOncoChip™|CopyNumberEvaluationresults,reportedonthe5thdayafterspecimenreceiptinthelaboratory,identifiedthreeclinicallysignificantaberrations,includingdeletion5q,trisomy21,anda380kilobasedeletionoftheRUNX1gene.Karyotypingresults,completedfivedaysaftertheOncoChip™|CopyNumberEvaluation,showedthelargeaberrationsresultinginakaryotypeof46,XY,del(5)(q13q33)[11]/47,idem,+21[13].BecausethechromosomeresolutionwasnotsufficienttoidentifyasmalldeletionandFISHforRUNX1wasnotperformedonthespecimen,thedeletionwasnotidentified.PatientswithMDSwhoareyoungerthan60atdiagnosis,haveanemiabutnoothercytopenias,andpresentwithRAEB-1intheabsenceofcytogeneticinformationhavealow-tointermediate-riskdiseasestate.3Additionally,5q-isconsideredagoodprognosticmarkerwhenseeninisolation,althoughwhenseenincombinationwithothercytogeneticabnormalitiestheprognosisbecomespoorerasthenumberofabnormalitiesincrease.3Presenceof2cytogeneticabnormalitiesincluding5q-classifiesthediseaseashavinganintermediateprognosis.RUNX1deletion,whichwasnotdetectedbykaryotypeorFISHinthispatient,hasbeenreportedinMDSpatientsatthetimeoftransformationtoacutemyeloidleukemia(AML).4

Thepresenceofthisdeletionchangedthispatient’sprognosisfromanintermediate-levelprognosistoidentificationofimmediatediseaseevolutionandthereforeindicatedthatmoreintensivetherapy,suchasstem-celltransplant,shouldbeconsidered.2,3Furthermore,somepatientswithRUNX1deletionshaveafamilyhistoryoffamilialplateletdisorderwithpropensitytoAML,andthereforefamilyhistoryofthispatientshouldbeconsideredtoevaluateriskforotherfamilymembers.1Figure1.OncoChip™|CopyNumberEvaluationshowingtrisomy21andconcurrentRUNX1deletion.Thetopshowsanideogramofchromosome21withtheproximalq-armtotheleftandthedistalq-armtotheright.ThesectionofchromosomeindicatedinredisexpandedtoshowtheplotofarrayCGHdatafortheentireq-arm.Thepinkhighlightedareawithintheplotindicatesagainoftheentireq-arm,consistentwithtrisomy21.ThebluehighlightedareawithintheplotshowsasubmicroscopicdeletioninvolvingRUNX1.AcutemyeloidleukemiawithnormalkaryotypeA35-year-oldmalepatientpresentedwithfatigueandreportedbruisingwithouttrauma.Workupincludedsendingbonemarrowaspirateforcytogenetics,flowcytometry,andpathologyreview.Pathologyresultsindicatedadiagnosisconsistentwithacutemyeloidleukemia,unspecifiedsubtype.Karyotypeshowedanormalmaleresultwithanoteindicatingthatchromosomemorphologywaspoorandthatonlylargeabnormalitiescouldbeexcluded.Giventhepathologyresults,aFISHpanelincludinganMLLbreak-apartprobewaschoseninanefforttodetectaberrationsinAMLthatmaybemissedbyapoorresolutionkaryotype.ResultssuggestedthepresenceofanMLLtranslocation,with50%ofcellsindicatingpotentialrearrangement.MetaphaseswerenotavailableforFISHanalysis,sotheMLLpartnerwasnotclarifiedfurtherbyFISH.KnowingthatidentificationofthespecificMLLpartnercanbecriticalforestablishingprognosisandhelpfulindeterminingtreatment,thecytogeneticslaborderedtheOncoChip™|HemEssential™panel.3TheychosethispanelbecauseitspecificallytargetsthreeofthemostcommonMLLtranslocationsandhasthepotentialtoidentifytranslocationswithupto40differentMLLpartners.1TheOncoChip™|HemEssential™testingidentifiedaberrationsintheMLLandMLLT3genes,suggestingat(9;11)(p22;q23)creatingafusionbetweenMLLandMLLT3.ThistranslocationisthemostcommonMLLrearrangementinAML,particularlyinAML-M5,whichareviewofpathologycharacteristicsconfirmedthatthepathologywasconsistentwiththispatient’sfindings.1-3WhiletypicallyMLLtranslocationsareassociatedwithpoorprognosis,theMLLT3translocationistypicallyassociatedwithabetteroutcomeinpatientswithdenovoAML,particularlywhenpatientsaretreatedwithintensivepost-remissiontherapy.2,3Figure1.OncoChip™|HemEssential™plotsshowingMLLT3/MLLtranslocation.Thetoppanelshowsanideogramofchromosome9withthep-armtotheleftandq-armtotheright.ThesectionofchromosomeindicatedinredisexpandedtoshowtheplotofarrayCGHdataforthe9p21.3sub-bandincludingMLLT3.ThepinkhighlightedareawithintheplotindicatesapeakdelineatingthetranslocationbreakpointwithinMLLT3.Thebottompanelsimilarlyshowsanideogramofchromosome11withtheregionbelowexpandedtoshowtheplotofarrayCGHdataforthe11q23.3sub-bandincludingMLL.Thepinkhighlightedareaindicatesap