Proc.Natl.Acad.Sci.USAVol.81,pp.593-597,January1984MedicalSciencesImmu - PDF document

Proc.Natl.Acad.Sci.USAVol.81,pp.593-597,January1984MedicalSciencesImmunoglobulingenerearrangementasadiagnosticcriterionofB-celllymphoma(Southernblothybridizationtechnique/cancer/DNAprobe/clonalanalysis/immunogenotyping)MICHAELL.CLEARY,JAMESCHAO,ROGERWARNKE,ANDJEFFREYSKLARDepartmentofPathology,StanfordUniversity,Stanford,CA94305CommunicatedbyPaulBerg,September26,1983ABSTRACTWedescribetheuseoftheSouthernblothy-bridizationtechniquetodiagnoseB-celllymphomabydetect-ingclonalimmunoglobulingenerearrangementsinlymphnodeandotherbiopsytissues.DNAwasisolatedfromawidevarietyofneoplasticandnon-neoplasticspecimensandana-lyzedforthepresenceofrearrangedimmunoglobulingenesusingradiolabeledDNAprobesspecificfortheheavy-andlight-chainimmunoglobulinconstantregiongenes.Amongthespecimensexamined,clonalimmunoglobulingenerearrange-mentswerefoundonlyinbiopsysamplesofB-celllymphomaandnotinsamplescontainingreactivelymphoidprocessesornon-B-cellcancers.Inlymphomas,thepresenceofrearrange-mentsforeithertheKorAlight-chaingenecorrelatedwithexpressionofoneortheotherofthesechainswhencellularimmunoglobulinscouldbedetectedbyfrozen-sectionimmuno-phenotypingtechniques.Theanalysisofimmunoglobulingenerearrangementsoffersseveraladvantagesoverconventionaldiagnosticmethodsforlymphomas,includingimprovedsensi-tivityindetectingminorpopulationsofneoplasticlymphocytescomposingaslittleas1%ofthetotalcellpopulation.Inaddi-tion,clonalimmunoglobulingenerearrangementsaredemon-strableinasubsetoflymphomasthatlackdetectablesurfaceorcytoplasmicimmunoglobulin,thusofferingpositiveevi-denceforbothmalignancyandtheB-celloriginofthesetu-mors.Ourstudiesindicatethatdetectionofimmunoglobulingenerearrangementsisavaluablemethodfordiagnosisandclassificationofvariouslymphoproliferativedisordersthataredifficulttoevaluatehistologicallyorthatlackdistinctiveanti-genicmarkers.Diagnosisofmalignantlymphomadependsonhistologicevaluationoftissuebiopsies.However,distinguishingbe-tweenmalignantandbenigndisordersinlymphnodesandotherlymphoidtissuesbylightmicroscopyremainsoneofthepathologist'smostdifficulttasks.Althoughthemajorityofsuchbiopsiesareunambiguous,asignificantminorityposeseriousproblemsforeventhemostexperthistopathol-ogist.Inlargepart,thebiologicalcauseforthisdifficultyisthatantigenicallystimulatedlymphocytesmaymorphologi-callyresembleneoplasticlymphocytes.Conversely,so-calledwell-differentiatedneoplasticlymphocytesmaybecy-tologicallyindistinguishablefromnormalunstimulatedlym-phocytes.Therearealsocasesinwhichreactiveconditionscoexistwithandobscuremalignancy,furthercomplicatinghistologicinterpretation.Occasionally,poorlydifferentiatedmetastaticcarcinomaormelanomamaybemistakenforlym-phoma.Animportantmethoddevisedtodealwiththeseproblemsistheanalysisofimmunologicmarkersonthesurfaceorinthecytoplasmoflymphoidcellsintissuesectionsorincellsuspensionsoflymphnodebiopsies(1,2).Thismethodtakesadvantageoftheclonalnatureofmalignancy,suchthatlargenumbersofcellswithinahistologicsectionorcellsuspensionbearthesameantigenicmarkersiftheprolifera-tionisneoplastic.Forinstance,homogeneousneoplasticproliferationofBlymphocytesthatsynthesizedetectableimmunoglobulinwillshowonlyasingleimmunoglobulinlightchain,KorX,whenanalyzedforthesetwopolypep-tides.Althoughthisimmunophenotypingtechniqueisrapidandhasprovedtobehelpfulinevaluatingcertainbiopsyspecimens,itsuffersfromseveraldisadvantages.Onefre-quentproblemisthatmalignantB-cellproliferationwithinlymphnodesisoftenintermixedwithvariousamountsofnormalBcells,inwhichcasethistechniquemaydependondetectingsmalldeviationsfromthe2:1ratioofK-toA-bear-ingBcellsfoundinnormalhumanlymphoidtissue.Otherproblemsincludeartifactsassociatedwithsuboptimalhan-dlingorfixationoftissues,therequirementforgoodanti-bodyreagentsdirectedagainstantigenicmarkers,andtheabsenceofmarkersincertainlymphoidtumors.RecentlywehaveexploredanalternativeapproachtothediagnosisofB-celllymphoma.Thisapproachreliesonde-tectinguniformrearrangementsofimmunoglobulingeneswithinclonalpopulationsofBlymphocytes,asdetectedpre-viouslyinhumanB-cellleukemias(3-5).OurworkisbasedonthefactthatBlymphocytesmustundergoaseriesofDNArearrangementspriortoimmunoglobulinproduction(6,7).Ingerm-linecellsthevariableandconstantdomainsofeachtypeofimmunoglobulinchain(oneheavychainandtwolightchains,KandX)areencodedinseparatediscontinu-ousregionsofspecificchromosomes.DuringB-lymphocytematuration,aninitialeventinimmunoglobulinsynthesisisthesomaticrecombinationoftheseparatedvariableandcon-stantgenesegments.Thisresultsintheremovalofinterven-ingDNAandthecloseappositionofspecificvariableandconstantDNAsequencestoformanactiveimmunoglobulingene(seeFig.1A).ThehighdegreeofvariabilitywithwhichimmunoglobulingenesegmentsarerearrangedandthefactthateachindividualBcelliscapableofexpressingonlyasingleantibodyidiotypemaketheconfigurationofrear-rangedimmunoglobulingenesegmentsanentirelyspecificmarkerforagivenBcellandforanyclonethatmayarisefromthatBcell.Inthisreport,weshowthatdetectionofimmunoglobulingenerearrangementsinbiopsytissuebytheSouthernblothybridizationprocedureaffordsanaccurateandhighlysen-sitivemeansofidentifyingclonallymphoidproliferationsinawidevarietyofB-cellmalignancies.Inaddition,benignre-activeprocessesandnon-B-cellmalignanciesaredistin-guishedbytheabsenceofdetectableimmunoglobulingenerearrangements.Thistechnique,therefore,providesavalu-ableadjuncttocurrentlyavailablemethodsfordiagnosingB-celllymphoma.Furthermore,thistechniqueavoidsmanyoftheproblemsassociatedwithimmunologicmarkerstudiesandconventionalmorphologicdiagnosis.Abbreviations:kb,kilobase(s);CandJ,constantandjoiningregionsofimmunoglobulinchains.593Thepublicationcostsofthisarticleweredefrayedinpartbypagechargepayment.Thisarticlemustthereforebeherebymarked"advertisement"inaccordancewith18U.S.C.§1734solelytoindicatethisfact. 594MedicalSciences:Clearyetal.A44$12345VK1VK2VK3VIK4VK5VK6JKDNAREARRANGEMENTRearrangedff3CKProbetCKLymphoma9VAU'l-.k1F.iAllelevvVKVK4JK3CKIAlleleK1K21C3Po4bCalProbeGermlineMucGeneBJH123456GermlineKappaGeneCli1234lkbh~CDiProbeJKCKt123451kbGermlineLambdaGene7IICKProbeCAK-eOFCAMcgCXKOe0CXProbeiBanH1#EcoRI2k2kbFIG.1.(A)Hypotheticalgenerearrangementforthelight-chainKlocus.DuringB-cellmaturationpriortoimmunoglobulinproduction,oneofthemultipleKvariable-regiongenes(V44)undergoessomaticrecombinationwithoneofseveralseparateJsegments(1K3)thatliedirectlyupstreamfromasingleKconstant-regiongene(CK).DNArearrangementleavesthedownstreamBamHIrestrictionsite(downwardarrowtotherightofCK)unalteredwhiletheBamHIsiteupstreamandnearesttotheCgeneischangedintherearrangedlymphomaallele.Thisallowsdistinctionbetweengerm-lineandrearrangedKgenes,becausetheC,,probewilldetectdifferent-sizedBamHIDNAfragments(bracketedlinesattopandbottom)bytheSouthernblothybridizationprocedure.[FigurebasedonCossmanetal.(18).](B)Chromosomalmapofthegerm-lineconfigurationfortheheavy-chain,K,andXCgenes.Theprobesusedinthisworkareindicatedbyhatchedboxes.Theheavy-chainJ-regionprobeconsistedofa6.5-kilobase(kb)BamHI/HindIIIDNAfragment.AprobespecificfortheC,.geneconsistedofa1.4-kbEcoRIfragment,whichincludesthefirst,second,andpartofthethirdexonsofthehumanC,.gene.TheJprobeispreferabletotheC,.probeforshowingheavy-chainrearrangementsbecausetheC,.genesegmentmaybedeletedduringheavy-chainclassswitching.Thelight-chainCKprobecontaineda2.5-kbEcoRIfragmentspanningtheentirehumanC,.gene.TheCAlocusconsistsofafamilyofatleastsixcloselylinkedrelatedgenes-e.g.,Mcg,Ke7Oz-,andKe-Oz+(15).AcombinedC,probewasusedconsistingofa3.5-kbEcoRI/HindllIfragmentcontainingthe�Ke-Oz-C,geneanda2.5-kbEcoRI/HindIIIfragmentcontainingtheMcgCAgene.METHODSBiopsytissueswereroutinelycollectedfromtheoperatingroom,frozeninairtightplasticcapsulesbyimmersioninadryice/isopentanebath,andstoredat-70'Cforuptofiveyears(8).Normalcontroltissuesfortwocasesstudiedcon-sistedofperipheralbloodgranulocytesandautopsylivertis-sue.DNAwasextractedfromlymphnodebiopsiesandothertissuesandpurifiedaccordingtostandardprocedures(9).Thestartingmaterialrepresented10-25mg(wetweight)oftissue.Afterpurification,highmolecularweightDNAwasdigest-edwithappropriaterestrictionenzymesaccordingtocondi-tionsrecommendedbythesupplier(BethesdaResearchLaboratories).Digestionproductswereelectrophoresedovernightinan0.8%agarosegel,asdescribed(10).AfternickingoftheDNAbyultravioletlighttodecreasetheaver-agechainlength,DNAfragmentsweretransferredoutoftheagarosegelsontonitrocellulosefiltersasdescribedbySouthern(11).Thefilterswerethenhybridizedwithradiola-beledpBR322plasmidDNAcarryingimmunoglobulinDNAfragments.Hybridizationreactionswerecarriedoutunderconditionsdescribedelsewhere(12),using50%formamideat420C.Afterextensivewashinganddryingoffilters,auto-radiographywascarriedoutat-70'Cagainstasingleinten-sifyingscreenfor12-72hr.HumangenomicDNAfragmentsspecificfortheK(13,14)and(15)constantregions(C)andtheheavy-chainjoining(J)and,uregions(16)wereisolatedfromrecombinantbacte-riophagekindlyprovidedbyP.Leder(HarvardMedicalSchool).ThesefragmentsweresubclonedintotheEsche-richiacoliplasmidpBR322usingstandardprocedures(17).ThepositionsoftheDNAprobefragmentswithrespecttotheimmunoglobulingenesareshowninFig.1B.PlasmidscontainingimmunoglobulinDNAwereisolatedfromE.coliandnick-translatedinvitrowith[a-32P]dNTPsasdescribedelsewhere(19),toaspecificactivityof3-5x108cpm/pgg.RadiolabeleddNTPswereobtainedfromAmersham.Alllymphomaspecimenswerecategorizedhistopathologi-callyasdescribed(20).Analysisofimmunologicsurfaceandcytoplasmicmarkerswascarriedoutinfrozensectionsasdescribed(8).RESULTSImmunoglobulinGeneRearrangementsArePresentinLymphomasbutNotinNonlymphoidControlTissuefromtheSamePatient.Fig.2Ashowsthedataobtainedfromalymphnodebiopsydiagnosedhistologicallyasadiffuselargecelllymphoma.Theheavy-chainJprobedetectedtwobandsinthelymphomaDNA,oneofwhichcomigratedwiththesin-JKAWI'aFIG.2.Autoradiogramsfrom_lymphomaandcontrolDNAhy-bridizedwithimmunoglobulingeneprobes.(A)Case1.(B)Case2.Analysesforheavy-chainandKlight-chaingenerearrangementswerecarriedoutonDNAdigested*withtheBamHIrestrictionen-zyme;theXlight-chainlocuswasanalyzedwithDNAdigestedwith.JAtheEcoRIrestrictionenzyme.i8rLanes1,controlDNA;lanes2,lymphomaDNA.Dasheshavebeenplacedalongsidegerm-lineJobands,arrowsarebesiderear-rangedbands.BasedonmarkerDNAfragmentscoelectropho-resedwithDNAdigestsintheseblotsbutnotshowninthefigure,thegerm-linebandsareofexpect-edsize:about19.3kb(heavychain);12kb(Klightchain);and16,14,and8kb(Xlightchain).GermlineGeneAlleleftProc.NatLAcadSci.USA81(1984)f Proc.Natl.Acad.Sci.USA81(1984)595Table1.Correlationofimmunoglobulingenerearrangements(immunogenotype)withimmunoglobulinantigens(immunophenotype)invariouslymphoproliferativeconditionsImmunogenotypeImmunophenotypeCaseHistologicdiagnosisKA/KX1DiffuselargecellMLRRG++2DiffuselargecellMLRRG++3ReactivefollicularhyperplasiaGGGNDNDND4LargecellimmunoblasticMLRRG---5DiffusesmallcleavedcellMLRRG++6SmallnoncleavedcellML,non-BurkittRRG++7SmalllymphocyticMLRRG++8UnclassifiedMLRRG++9LymphocyticML,intermediatedifferentiationRRR+-+10DiffuselargecellMLRRG11LymphoblasticMLtGGG---ML,malignantlymphoma;R,rearrangedimmunoglobulingene;G,germ-line;ND,notdetermined.*Rearrangementsoftheheavy-chainlocusdetectedwithheavy-chainJ-specificprobewereconfirmedwiththeC,probe.Ineachcase,theC,JprobehybridizedtoatleastonerearrangedbanddetectedwiththeJprobe.tT-celldifferentiationdeterminedbyanalysisofsurfacemarkers(Leu-1+,Leu-2a',Leu-3a+,Leu-4',la-).glegerm-linebandobtainedfromperipheralgranulocyteDNA.Thesecondband,whichmigratedinapositionbelowthatofthegerm-lineband,representsclonalrearrangementofoneheavy-chainimmunoglobulinallele.SeparateblotspreparedfromthesameDNAandhybridizedwithaprobefortheKlight-chaingenealsorevealedasingleclonallyrear-rangedbandthatmigratedslightlyaheadofthepositionofthegerm-linebandaswellasaweakerbandthatcomigratedwiththegerm-linebandfromgranulocytecontrolDNA.AutoradiogramsobtainedfromblotshybridizedwiththeXlight-chainprobeshowedidenticalpatternsforthecontrolandlymphomaDNAs.Theresultsobtainedinthiscaseareconsistentwithaclonalrearrangementofatleastonealleleoftheheavy-chainandK-chainlociandcorrelatewithimmu-nologicphenotypingoffrozensectionsofthispatient'slym-phoma,whichrevealeduandKsurfaceimmunoglobulin(Ta-ble1).Asimilaranalysiswascarriedout(Fig.2B)foradiffuselargecelllymphomathatdevelopedinapatientwithWis-kott-Aldrichsyndrome.NonlymphomacontrolDNAwasextractedfromautopsyliver,whichwasgrosslyandmicro-scopicallyfreeoftumor.Whentheheavy-chainJprobewasusedinthehybridization,threebandswereseen,twoofwhichmigratedfasterthantheunrearrangedgerm-linebandpresentinthecontrolDNA.Thisfindingcanbeexplainedbyrearrangementofbothheavy-chainallelesinthelymphomacells.TheKlight-chainprobeshowedasinglerearrangedbandrepresentingafragmentlargerthanthegerm-linefrag-mentinadditiontoabandthatcomigratedwiththecontrolDNAband.HybridizationofDNAfromcontrolandlympho-matissuewiththeXlight-chainprobeproducedidenticalpat-terns.AsinFig.2A,thesedataareconsistentwithacloneofmalignantcellsthatexpressedA-Kimmunoglobulin,inagree-mentwithsurfacemarkeranalyses,asshowninTable1.Differentamountsofgerm-linebandwerepresentinanal-ysesofbothlymphomaspecimens.Thesebandsmayhaveresultedfromeitheranunrearrangedimmunoglobulinallelewithintumorcellsorfromcontaminationofthetumortissuewithnormalpolyclonallymphocytes,bloodcells,fibroustis-sue,andbloodvessels.ImmunoglobulinGeneRearrangementsAreDetectableinVariousHistologicSubtypesofB-CellLymphoma.Fig.3showsresultsobtainedwhenDNAisolatedfromlymphnodebiopsiesrepresentingavarietyofconditionswasanalyzedwithheavy-chainJ-regionDNAprobes.RearrangementswereseeninallcasesinvolvinghistologicsubtypesofB-celllymphoma,assummarizedinTable1.Light-chainrear-rangementswerealsoseeninallcasesofB-celllymphoma(datanotshown).Whenpresent,thesurfaceorcytoplasmiclightchainidentifiedinfrozensectionscorrespondedtothelight-chainclassthatshowedgenerearrangement(Table1).BothKandXgenerearrangementswerefoundinasinglespecimencontainingonlyXsurfaceimmunoglobulin(case9),butnoXgenerearrangementwasfoundinlymphomaswithKsurfaceimmunoglobulin.Thisobservationconformstotheproposedhierarchyofimmunoglobulingenerearrangement,suchthatrearrangementsoftheXgeneoccuronlyafterde-fectiveornonproductiverearrangementsofbothKgeneal-leles(4,21).ImmunoglobulinGeneRearrangementsAreDetectableinLymphomasLackingCellularImmunoglobulin.Twoofthecasesexamineddidnotshowstainingforsurfaceorcyto-plasmicimmunoglobulinbutcontainedimmunoglobulingenerearrangements.Bothcases10and4(Table1)arediffuselargecelllymphomasthatlackdistinctivemarkersexceptforB1(22)andIaantigens.Despitetheabsenceofimmunoglob-ulinproductioninbothcases,clonalheavy-chainimmuno-globulingenerearrangements(Fig.3)supporttheB-celllin-eageofthesetumors.Moreover,light-chainKgenerear-rangementswerefoundineachinstance,whiletheXgeneswereinagerm-lineconfiguration.34567891011FIG.3.AutoradiogramsobtainedwithDNApreparedfromlymphnodeswithvariousproliferativedisordersandhybridizedwithaheavy-chainJ-specificprobe.Analysesforheavy-chaingenerearrangementswerecarriedoutusingBamHI-digestedDNAex-tractedfromlymphnodebiopsies.ThenumberscoincidewiththecasenumbersinTable1.Dashesindicatethegerm-line19.3-kbband;arrowsshowrearrangedheavychainJ-specificbands.Inmostoftheautoradiograms,thereisafaint12-kbbandofunknownoriginthathybridizesweaklywiththeJ-specificprobeunderthecondi-tionsused.Thisbandisalsodetectedinblotspreparedfromnon-lymphoidDNA.MedicalSciences:ClearyetaL 596MedicalSciences:Clearyetal.)0(')-'Cv'0)0FIG.4.SensitivityoftheSouthernblothybridizationtechniquefordetectingimmunoglobulingenerearrangements.Mixturescon-tainingatotalof10jugofDNAwerepreparedwithlymphomaDNAandvariousamountsofnonmalignantlymphnodeDNA.TheDNAwasdigestedwithBamHIrestrictionenzyme.Afterelectrophoresisandtransfer,thesampleswerehybridizedwitharadiolabeledCK-specificprobe,washed,andautoradiographedfor48hr.Theper-centageoflymphomaDNAwithineachmixtureisindicatedabovethelanesoftheautoradiogram.Dashindicatesthepositionofthegerm-lineband;arrowsindicatethepositionoftworearrangedKbandsinthelymphomaDNA.ImmunoglobulinGeneRearrangementsAreNotSeeninTissueSpecimensOtherThanB-CellLymphomas.WehaveexaminedfourT-celllymphomas-forexample,alympho-blasticlymphoma(Fig.3,case11).Aflshowedonlygerm-linebandsandnodetectableimmunoglobulingenerear-rangements.WehavealsoexaminedDNAfromlymphnodescontainingawidevarietyofbenignandreactivecon-ditions,includingreactivefollicularhyperplasia(case3),an-gioimmunoblasticlymphadenopathy,rheumatoidarthritis,andsarcoidosis.Othernon-B-cellneoplasticprocesseswereexamined;theseincludedacutemyelocyticleukemia,acutemonocyticleukemia,malignanthistiocytosis,Hodgkindis-ease,metastaticcarcinomasandsarcomas,thymoma,andWarthin'stumorofthesalivarygland.DNAsfromnoneofthesespecimensshowedbandsotherthanthosecomigratingwithunrearrangedgerm-linebands.HighSensitivityoftheSouthernBlottingTechniqueforDe-tectingClonalImmunoglobulinGeneRearrangements.TotestthesensitivityoftheSouthernblottingtechniqueforde-tectingminorsubpopulationsofmalignantlymphocyteswithinbiopsies,blotswerepreparedfromknownlymphomaDNAmixedwithvariousamountsofnonmalignantlymphnodeDNA.AfterhybridizingwithaK-specificprobe,rear-rangedallelescouldbeconfidentlydetecteddowntoalevelof1-2.5%lymphomarelativetononlymphomaDNA(Fig.4).DISCUSSIONClinicallyapparentmalignantlymphomaresultsfromtheclonalproliferationofneoplasticlymphocytes(1).Clonalityisnot,however,theequivalentofmalignancy.Benignmono-clonalgammopathyandexpansionofanisolatedB-cellcloneinresponsetoanantigenicstimulusareexamplesofclonalprocessesthatarenotmalignant.Benignmonoclonalgam-mopathyseldomifeverinvolveslymphnodes,andneithermonoclonalnoroligoclonalimmuneresponsehaseverbeendocumentedasacauseofclinicallymphadenopathy.Never-theless,theexistenceorpotentialexistenceofsuchprocess-esindicatesthatclonalB-cellproliferationinabiopsyshouldberegardedasstronglycorrelatedwith,butnotanabsolutecriterionof,malignancy.TheresultsdescribedinthisreportshowthatanalysisofimmunoglobulingenerearrangementisanaccurateandpracticalmethodofdetectingB-lymphocyteclones.Usedintheproperclinicalcontextandinconjunc-tionwithavailablemorphologicinformation,thistechniqueprovidesvaluableevidenceforB-celllymphomainbiopsyspecimens.Inthisreportwepresentdatafromcasesofunequivocallymphomatoshowtheapplicabilityandvalidityofthistech-nique.Ineachcase,rearrangementswerefoundinwhichadiagnosisoflymphomawasmadebymorphologiccriteriaalone.Surfaceandcytoplasmicimmunoglobulinwasana-lyzedinthesecasesaswell.Whenpresent,immunoglobulinmarkerscorrelatedwiththepatternofimmunoglobulingenerearrangements,sothatanytumorexpressingeitherKorXlightchainsalwaysshowedarearrangementinatleastonealleleforthecorrespondinggene.Normalandreactivelymphnodes,avarietyofnon-B-celllymphomas,andseveralnonlymphoidcancershavebeentestedforimmunoglobulingenerearrangements.Thesehavebeenconsistentlynegativeforbothheavy-andlight-chaingenerearrangements.AsshowninFig.3(case3),normalorreactivelymphnodesshowonlygerm-linebands.Therearepresumablyinnumerablerearrangedimmunoglobulingenesinsuchtissues;however,apparentlynosinglerearrangedDNAfragmentissufficientlyabundanttobedetectablebytheSouthernblottechnique.Onthebasisofthesefindings,weconcludethatdetectionofarearrangedimmunoglobulinbandinaSouthernblotanalysisoflymphoidDNAisareli-abletestforclonalproliferationand,withinthelimitsdis-cussedabove,forB-celllymphoma.OurfailuretodetectimmunoglobulingenerearrangementsinhumanT-celllymphomascontrastswithstudiesonsmallnumbersofmouseT-celltissueculturelines,inwhichocca-sionalheavy-chaingenerearrangementswerefoundintheabsenceoflight-chainrearrangements(23,24).In11of12casesofhumanT-cellacutelymphoblasticleukemia(ALL),Korsmeyeretal.(3)foundgerm-lineconfigurationsofimmunoglobulingenes.AsingleTALL-derivedcelllineinthisseriescontainedanonproductiveheavy-chaingenerear-rangementbutretainedgerm-lineconfigurationsoftheKandXgenes.Thesignificanceofrearrangementsincelllinesisunclear,however,becauserearrangementsmayhaveoc-curredinthesecellsatsomepointaftertheywereputintoculture.Atthepresenttime,availableinformation,summa-rizedabove,suggeststhatifimmunoglobulingenerearrange-mentsoccurinhumanT-celltumorstheyarerareanddonotinvolvelight-chainloci.Therefore,analysisofimmunoglob-ulingenerearrangementsdoesnotseemapplicabletodiag-nosisofT-celllymphoma.AsadiagnosticmethodforB-celllymphoma,analysisofimmunoglobulingenerearrangementssuffersfromthetimenecessarytocompletethetest(5-10days)andtheuseofradioactiveprobesrequiredbythepresentformoftheSouthernblottechnique.Ontheotherhand,analysisofimmunoglobulingenerearrangementspossessesseveralad-vantagesoverconventionaldiagnosticmethods.Aboveallitoffersamethodthatisnotdependentonsubjectivecriteriaandtheexperienceoftheobserver.Inaddition,thetech-niquerequiressmallamountsoftissue(aslittleas1mgperimmunoglobulinchainanalysis)thatneedsnospecialhan-dlingorpreparation.Infact,wehaveobtainedhighqualityautoradiogramswithDNAextractedfromautopsytissueseveraldaysafterdeath.Thiscontrastswiththedifficultyexperiencedinmicroscopicexaminationoflymphnodebi-opsies,bothmorphologicallyandformarkerstudies,iftis-sueiseithernotproperlyfixedornotfrozenimmediatelyonremovalfromthepatient.Also,DNAimmunoglobulinprobesarestableandeasyreagentstouse.Theycanbepre-paredinlargequantitiesandstoredindefinitelyinthefreez-er.Thereisverylittlevariationinqualityfrompreparationtopreparation,aproblemsometimesencounteredwithanti-serausedinimmunologicmarkerstudies.Apossibledifficultyassociatedwithanalysisofimmuno-globulingenerearrangementsinlymphomaisthedetectionofspuriousrearrangementsthatareactuallyduetoinheritedpolymorphismsinimmunoglobulingeneDNA.Anabsolutecontrolagainstsuchoccurrencesisparallelanalysisofnon-lymphocyticDNAfromthesamepatient,asshownfortwocasesinthisreport.Inpractice,suchpolymorphicmutationsseVD0Proc.Natl.AcadSci.USA81(1984) Proc.NatL.Acad.Sci.USA81(1984)597appeartoberarefortheheavy-chainandKloci,nonehavingbeenencounteredbyusfortherestrictionenzymesusedinthisreportinspecimensofnormalornon-B-celltissuesfromover40patients.Othershavereportedsimilarexperiences(3,4).However,theXlight-chainlocusdoesshowoccasion-alfragmentpolymorphismsthatinvolvethesmallestofthethreeEcoRIrestrictionfragmentsdetectedinthegerm-lineDNAofmostpatients(Figs.1and2).Thesepolymorphismsconsistofacquisitionorlossofmultiplesofa5-kbDNAse-quenceresultinginasmallsetofpredictablevariantbands(15).Thiscomplicatesbutusuallydoesnotpreventinterpre-tationwithoutparallelgerm-linecontrols.ClonalimmunoglobulingenerearrangementswerefoundineveryhistologicsubtypeofB-celllymphomatested.Anal-ysisofimmunoglobulingenerearrangementsfailstodistin-guishbetweenthesesubtypesand,therefore,providesnoinformationaboutexpectedbiologicbehavior(e.g.,clinicalaggressiveness),ascanbeobtainedfromthemorphologiccharacteristicsofthetumor.Nevertheless,itmaybepossi-bletopredictfeaturesofthebiologicalbehavioroftumorsbasedontheextentofimmunoglobulingenerearrange-ments.Forexample,atumorshowingrearrangementsofonlyheavy-chaingenesandnorearrangementsoflight-chaingenesmayhaveabiologicbehaviorandresponsetotherapydifferentfromatumorshowingrearrangementsofbothheavy-andlight-chaingenes.Althoughanalysisofimmunoglobulingenerearrange-mentsdoesnotpermithistologicsubtypingofB-celltumors,thistechniqueishelpfulindistinguishingB-cellneoplasmsamonglymphoidtumorsthatfailtoshowthedefinitiveim-munologicmarkersforeitherT-orB-celldifferentiation(nonT/nonBor"nullcell"lymphomas).TheworkofWarnkeetal.(25)indicatesthatthesetumorsmayrepresentupto25%oflargecelllymphomasor10-15%ofallnon-Hodgkinlym-phomas.InextensivestudiesofhumanB-cellleukemias,Korsmeyerandcolleagues(3,4)haveshownthatheavy-chainimmunoglobulingenerearrangementsoccurearlyinhumanB-celldifferentiationbeforetheacquisitionofallknownB-cellmarkersexceptTa.However,theirstudiesdidnotrevealIaantigenonanyB-cellprecursorslackingheavy-chainrearrangements.Furthermore,thisantigenisnoten-tirelyspecificforBcells,beingpresentonsomeactivatedTcellsandoncellsofcertainothertissues.Theearlyappear-anceofimmunoglobulingenerearrangementsinB-cellde-velopmentmeansthatthisgeneticmarkermaybepresentincellslackingdefinitiveimmunologicB-cellmarkers.Thissit-uationisshownincases4and10ofthisreport.Thepresenceofheavy-andlight-chainimmunoglobulingenerearrange-mentsinthesecasesrevealstheB-celloriginofbothtumors.Thesecasesalsoshowthatanalysisofimmunoglobulingenerearrangementsprovidesforthefirsttimeameansofdocu-mentingclonalityforatleastsomenull-celllymphomas.Analysisofimmunoglobulingenerearrangementsisanex-tremelysensitivetestforthepresenceofaminorclonalpop-ulationoflymphocytes.Thisfeaturemaybeimportantinthediagnosisofabiopsyinwhichthereisasmallclusterofma-lignantcellsthatmayescapecarefulexaminationunderthemicroscopeorevenmissbeingsectioned.BecauseDNAanalysisofbiopsytissuescreensathree-dimensionalfrag-mentoftissueandcaneasilybescaleduptoseveralgramsofstartingmaterial,thelikelihoodoffailingtodetectaclonalrearrangementinasmallportionofabiopsyislowaslongastheclonalfractionofcellswithinthebiopsyexceedsthethresholdofsensitivityofthemethod.Alternatively,diffuseadmixturesofmalignantcloneswithnonclonalbenignlym-phocyteswithinatissuesection,ararebutoccasionallyen-counteredsituation,aredifficulttoevaluatebyconventionaldiagnosticmethods.Thissituationcreatesnospecialprob-lemforimmunoglobulingeneanalysis.Sensitivitymaybeevenmorecriticalinstagingoflymphomaormonitoringpa-tientsaftertreatment.Forinstance,smallnumbersofmalig-nantcellsmaybedetectedinlymphnodesorbloodinrelaps-ingpatientsorinthosewithpersistentdisease.Wehavesuc-cessfullyisolatedDNAfrombonemarrowneedlebiopsiesandanalysisofsuchsamplescouldbeofvalueinfollowingtreatedlymphomapatientsaswellasthosewhohaveunder-gonebonemarrowtransplantation.ThisworkwassupportedbyGrantsNP-376fromtheAmericanCancerSocietyandCA34233fromtheNationalInstitutesofHealth.M.L.C.isaPostdoctoralFellowoftheJaneCoffinChildsMemorialFundforMedicalResearch.J.S.isaFellowoftheJohnA.HartfordFoundation.1.Levy,R.,Warnke,R.,Dorfman,R.F.&Haimovich,J.(1977)J.Exp.Med.145,1014-1028.2.Aisenberg,A.C.,Wilkes,B.M.,Long,J.C.&Harris,N.L.(1980)Am.J.Med.68,206-213.3.Korsmeyer,S.J.,Arnold,A.,Bakshi,A.,Ravetch,J.V.,Sie-benlist,U.,Hieter,P.A.,Sharrow,S.O.,LeBien,T.W.,Kersey,J.H.,Poplack,D.G.,Leder,P.&Waldmann,T.A.(1983)J.Clin.Invest.71,301-313.4.Korsmeyer,S.J.,Hieter,P.A.,Ravetch,J.V.,Poplack,D.G.,Waldmann,T.A.&Leder,P.(1981)Proc.Natl.Acad.Sci.USA78,7096-7100.5.Hieter,P.A.,Korsmeyer,S.J.,Waldmann,T.A.&Leder,P.(1981)Nature(London)290,368-372.6.Hozumi,N.&Tonegawa,S.(1976)Proc.Natl.Acad.Sci.USA73,3628-3632.7.Leder,P.,Max,E.E.&Seidman,J.G.(1980)inImmunology80,eds.Fougereau,M.&Dausset,J.(Academic,London),pp.34-50.8.Wood,G.S.&Warnke,R.(1981)J.Histochem.Cytochem.29,1196-1204.9.Pettersson,V.&Sambrook,J.(1973)J.Mol.Biol.73,125-130.10.Robbins,J.,Rosteck,P.,Jr.,Haynes,J.R.,Freyer,G.,Cleary,M.L.,Kalter,H.D.,Smith,K.&Lingrel,J.B.(1979)J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