InvestigationofBcell malignancies

Dr.JoannaSheldon ProteinReferenceUnit St.George sHospital St.George sHospital

TheBcell
progressionfrom Pluripotentstemcell Lymphoidcommittedstemcell Blineagecommitted Heavychainrearrangement Lightchainrearrangement Encountersantigeninlymphoidtissue Affinitymaturation Classswithching Enddifferentiatedplasmacell

TheBcell
Manyopportunitiesforanerrortooccur Someerrorsresultinamalignant transformationoftheBcells Someerrorsresultinaclonalexpansion ofBcellsandBcellmalignancies Monoclonalproteinsareoneofthe featuresofBcellmalignancies

Beta gammaregionofEP Beta

Normal(polyclonal)
(kappa>lambdaapprox.2:1)

Polyclonalraised
(kappa>lambdaapprox.2:1)

Oligoclonalbanding Monoclonalprotien protien

Historyofthelab
1847BenceJonesproteindescribed 1937electrophoresisseparatesplasmaprotiens 1940thetermparaproteinintroducedbyApitz 1959 theimmunoglobulinintroducedbyHeremans 1965 measurementofimmunoglobulinconcentrationby radialimmunodiffusiondescribedbyMancini 1966measurementofproteinsbyrocketelectrophoreiss describedbyLaurell 1976automatedimmunonephelometry forprotein measurementintroduced

Settingthestandards
ALLlaboratorymethodsshouldbe: ACCURATEwherepossible,becalibratedagainstanIRP PRECISE
showCVsof<10%realistic(<5%preferable)withinbatch showCVsof<20%realistic(<10%preferable)betweenbatch

CONSISTENTBETWEENUSERS
showCVsof<20%realistic(<10%preferable)inEQA

CLINICALLYSPECIFIC
showalownumberoffalsepositives

CLINICALLYSENSITIVE
showalownumberoffalseNEGATIVES

CLINICALLYSENSITIVE
showalownumberoffalsePOSITIVES

VALUEFORMONEY
alloftheaboveandcosteffective

Whatisnormal?
SERUM
polyclonalgammaregiononelectrophresis
Adultconcentrations

IgG616g/L,IgA0.84.0g/L,IgM0.52.0g/L IgGhalflife~21daysanddependentonconcentration IgAandIgMhalflife~5daysindependentofconcentration URINE Totalprotein<0.1g/L atraceofalbuminshouldbedetectableineveryurine normalurine(adequatelyconcentrated)willalsoshowsomeother proteine.g.transferrinandsomepolyclonalfreelightchains thesefreelightchainsareanormalresultofBcelldevelopment

Monoclonalproteins
developmentofamonoclonaldoesnothappen overnight
willstartasasmallband maydevelopquicklyorveryslowly mayincreaseinconcentrationasclonegrows mayremainatalowandstableconcentration maydisappearovertime maysuppressbackgroundBcellpopulation

thesameimmunoglobulinconcentrationmayrelateto polyclonal,oligoclonalormonoclonalpopulations thereisNOantibodythatiscapableofdistinguishing amonoclonalproteinfromapolyclonalprotein

Thingstoremember
monoclonalproteinsarenot(usually) normalproteins(intermsofstructure) monoclonalproteinsdonotbehavelike polyclonalproteins presenceofamonoclonaldoesnot meanmalignancy absenceofamonoclonaldoesnot excludemalignancy

Whatarethestages?
Detection Typing Quantification Monitoring

Detectionofmonoclonalproteins
ALWAYScheckserumandurine
approx.20%ofmyelomaonlymakeBJP BJPissmall~22kDa(butcanpolymerise) BJPcanpasseasilythroughtheglomerulus

Serumimmunoglobulinsshouldalwaysbe donewithserumproteinelectrophoresis InternationalGuidelinesforBJPanalysis nd voidofthedayfordetection recommends2

Detectionofmonoclonalproteins
Highqualityelectrophoresis
agarose,celluloseacetateorcapillary serum(prefereabletoplasma) urineconcentratedorsensitivestain(atleast atraceofalbuminMUSTbeseeninallurines)

monoclonalproteinscanappearanywhere fromthealpha1tothepostgammaareas lowthresholdforimmunofixation

IMMUNOFIXATION
usehighqualityantiserum
antitotal(freeandbound)lightchainantiserumisbetter thanantifreelightchainantiserum antilightchainantiserumoftenshowsgreaterbindingto freelightchainsthantoboundlightchains

oneantiserumwillnotdetectALLmonoclonals immunofixationdoesincreasesensitivityover electrophoresis(by1020x) goodinterpretationincreasesspecificity immunofixationisnotquantitative

Typingofmonoclonalproteins
Immunofixationistheonlyreliablewaytotypemonoclonalproteins itcan confirmtheclonalityofbanddetectedbyelectrophoresis testfor a, g and m heavychainsand k and l lightchains testforthe d and e heavychainswhereaserumshowsmonoclonal lightchainswithoutacorresponding a, g or m heavychain excludelowconcentrationmonoclonalcomponentsevenwhereno bandisapparentonelectrophoresisbutwithclinicalindicationse.g. ALamyloidosis

Typingofmonoclonalproteins
Immunofixationistheonlyreliablewaytotypemonoclonal proteinsitcan excludethepresenceofmonoclonalIgAorIgMiftheyare showingraisedconcentrationswithoutincreasedstaininginthe betagammaregionoftheelectrophoresis positivelyidentifyotherproteinsthatmaybemistakenfor monoclonalimmunoglobulinse.g.fibrinogen,Creactive protein,beta2microglobulinandcomplementcomponents detectminimalresidualdiseaseorcompleteremissionpost stemcelltransplantationwhennomonoclonalcomponentis seenontheelectrophoreticseparation.

Glomerularproteinuria
Examplesofglomerularproteinuria

Overflowproteinuria
Examplesofoverflowproteinuria

Proteinuria Mixedproteinuria
glomerular,tubularandoverflow canalloccurtogether patternshardtoclassify

Proteinuria Mixedproteinuria

Lightchains
polyclonalBcellsproduceaslightexcessoflight chainsaspartoftheirnormalprocesses thesefreelightchainsarriveatthekidneysandare filteredbytheglomerulus(mwtapprox.25kDa) inflammatoryresponsescanincreasetheamountof polyclonalfreelightchainsproduced kidneysareimportantsitesoflightchaincatabolism lightchaincatabolism(plusdehydration,acidosisetc) cancauseaggregationofexcesslightchainsand tubulardamage

BenceJonesprotein
MONOCLONALfreelightchains firstdescribedin1846! importantmarkerofBcellmalignancy rarelyseeninbenignconditions canformamyloidormyelomacasts kidneysareimportantsitesoflightchaincatabolism lightchaincatabolism(plusdehydration,acidosisetc) cancauseaggregationofexcesslightchainsand tubulardamage thereisNOantiserumavailableANYWHEREthat candistinguishmonoclonalfrompolyclonallightchains

BenceJonesprotein
FreelightchainsnotnecessarilyBJP BJPismonoclonalfreelightchains reliabledetectionofBJPcanonlybe donebygoodqualityelectrophoresisand immunofixation findingandtypingBJPisprobablythe hardestthingwedoinproteinlabs..

BenceJonesprotein

Dontforget..
intactmonoclonalIgalsoappearsinthe urine(withorwithoutBJP) willusuallyhavedifferentmobilityBJP b2microglobulincanalsobealarge bandonurineEP(especiallyifpatientis onalphainterferon) patientswithamyloidmayhaveheavy glomerularortubularproteinuriaandonly asmallamountofBJP

Why?
patientswithinfectionandinflammatoryconditions showincreasedfreelightchainexcretionnotBJP patientswithBcellmalignancieswithBJPcanhave glomerular,tubular,overflowormixedproteinuria elderlypatientsoftenhavesometubularproteinuria tubularcatabolismcanmakelightchainsfragments thataggregate tubularcatabolismcanmakelightchainsfragments thataggregateandhavesimilarcharge degradedurinesshowveryfuzzypatterns highresolutionelectrophoresispicksuptinyamounts ofprotein

Whatcanwedo?
useanelectrophoretictechniquethatis sensitiveto10mg/LBJP seealbuminineveryurine confirmwithimmunofixationincreases sensitivityandspecificity dontbeafraidtoaskforafreshsampleifthe urineisdegraded,smellyorshowsanindistinct pattern positiveidentificationimportantifthereisa band,whatisit(BJP,Hb, b2M,lysozymeetc.)

Quantificationbestofabad job!
electrophoresis,scanningdensitometryandtotal protein NOTideal totalproteinmethodsarepoor EPseparationcanhaveahighbackground dueto proteinfragments tubularproteins crud limitationofurinevolumetimed,24hour, random withinapatient,urinepatternsaresurprisinglystable

MultipleMyeloma
Summary amalignantdiseaseoftheplasmacellsinthebonemarrow. Proliferationofonecloneofplasmacells(monoclonal proliferation)resultsinproductionofamonoclonal immunoglobulin(Ig)moleculewhichcanbedetectedintheserum ortheurine. frequentlyassociatedwithbonepain,anaemia,andrenalfailure. accountsforabout1%ofallcancers2,500newcasesof myelomaeachyearinUK. incidenceincreaseswithagemostpatientsareovertheageof60 years thecauseisunknowninmostpatientsradiationexposureis knowntoincreasetherisk. incurableinmostpatients,averagesurvival34years.

Monoclonal Immunoglobulins
therearenormallymanydifferentplasmacell clones,producingmanydifferentIg molecules Inresponsetoinfectionorinflammation,there isproliferationofanumberofdifferent plasmacellclonesleadingtoanincreased numberofplasmacellsinthemarrow(a reactiveincrease)andapolyclonal increaseinIgs,whichappearasabroad bandinthegammaregiononserum electrophoresis.

Monoclonal Immunoglobulins
inmyelomaonecloneovergrowsandproducesoneparticular immunoglobulinmoleculeamonoclonalimmunoglobulin, alsotermedamonoclonalprotein(Mprotein)or paraprotein whichappearsasadensenarrowbandon electrophoresis.Thereisnearlyalwaysareductioninnormal polyclonalIgs. theabnormalplasmacellsmayalsoproducefreelightchains, whicharesmallenoughtopassintotheurine(Bence JonesproteinorBJP).Freelightchainscanalsobe measuredintheserumusingthenewFreeliteassay. Individualpatientsmayhaveplasmacellswhichsecretea wholeimmunoglobulinalone,BJPalone,orboth.

TheESRinMyeloma
ahighlevelofIgsinthebloodcausesaraisedESR (erythrocytesedimentationrate).thisismeasured asmmin1hourthedegreeofseparationofred cellsfromtheplasmathathastakenplacein1 hour. theESRisthereforeraisedinmostpatientswith myeloma,butisalsoraisedinconditionswhere thereisapolyclonalincreaseinIgs(infectionand inflammation) inpatientswithmyelomawhoproduceonlyfreelight chainsandnoserumparaprotein,theESRis normal

ClinicalFeaturesof Myeloma
Bonepain.Thisaffectsabout60%ofpatients. Xraysmayshowlyticlesions(punchedoutholes). Generalisedosteoporosis(thinningofthebone texture)isalsocommonandtheremaybe compressionfracturesofthevertebrae,leadingto backpainandoccasionallytocompressionofthe spinalcordandneurologicalsymptoms.Thebone destructionisduetotheactivationofosteoclasts resultingfrominteractionbetweenmyelomacells andthebonemarrowstromalcells.

Clinical Featuresof Myeloma

Clinical Featuresof Myeloma

ClinicalFeaturesof Myeloma
Hypercalcaemiaresultingfrom bonedestruction.Causes dehydration,drowsiness,confusion, constipationandrenaldamage.

ClinicalFeaturesof Myeloma
Renalfailure.About30%ofpatients havesomedegreeofrenal impairmentandabout5%have severerenalfailure.Thisismost commonlyduetoBJPwhichdamages thetubules.Otherfactorswhichcan contributeincludehypercalcaemia, infection,dehydrationanddrugs.

ClinicalFeaturesof Myeloma
Anaemia.Thisiscommonin myelomapatients.

ClinicalFeaturesof Myeloma
Increasedsusceptibilitytoinfection, bothbacterialandviral.

ClinicalFeaturesof Myeloma
Amyloid.About10%ofpatientswith myelomadeveloplightchainamyloidosis (ALamyloid),inwhichthelightchainsare depositedinthetissuesintheformof amyloidcausesenlargementandstiffness ofthetissue.(Amyloid=starchlike)The kidneyisusuallyaffected,withglomerular damageleadingtolossofalbumen,low serumalbumenlevelandconsequent oedema(nephroticsyndrome)

ClinicalFeaturesof Myeloma
Asymptomaticpatientsmaybe pickedupbythefindingofaraised ESRorabnormalprotein electrophoresis.

DiagnosisofMyeloma
Bonepain.Thisaffectsabout60%of patients.Confirmationofthediagnosisat least2ofthefollowing3featuresmustbe present: aparaproteininserumorinurine(no specificlevel) >10%plasmacellsinthebonemarrow lyticlesionsonXray.

CriteriaforMGUS
<30g/lparaprotein <10%plasmacellsinmarrow nobonedisease normalhaemoglobinandrenalfunction

TreatmentSpecific Aspects
Chemotherapyisthemainstayof treatmentinmyeloma.Oralor intravenousdrugsmaybeused,often combinedwithsteroids. Radiotherapyisusedforlocalareasof disease,e.g.forpainorcord compression

TreatmentSpecific Aspects
Older/lessfitpatientsaretreatedwithoral chemotherapy.Responseismonitoredbythe paraproteinlevel,andsometimesbybonemarrow tests.Generallyaplateauphaseisreachedafter whichtheleveldoesnotdeclineanyfurtherand treatmentisstopped.Itisunusualforthe paraproteintodisappear(completeremission). Soonerorlatertheparaproteinstartstoriseagain indicatingrelapse.Furthertreatmentmayinducea secondresponsebutthisisusuallyshorterthan thefirst.

TreatmentSpecific Aspects
youngerpatientsaretreatedwithinitial intravenouschemotherapytoinducea remissionfollowedbyhighdose chemotherapyandautologousstemcell transplantation.Allogeneictransplantation (fromahealthydonor)isanoptionfora minorityofpatientswhoareunder55 yearsandhaveamatcheddonor

FutureTreatment
Theaveragesurvivalofpatientstreatedwith conventionalchemotherapyisaround3years, althoughsomepatientsmaysurvivemuchlonger Anumberoffactorspredictprognosis,ofwhichmost importantistheserumbeta2microglobulinlevel. Othersincluderenalfunction,serumalbumin,calcium andhaemoglobin Highdosetherapyappearstoprolongsurvivalbutis notdefinitelyabletocurethedisease.

Monitoringresponse
responseismonitoredbythelevelof paraproteinintheserumorurine(24 hrexcretion) repeatbonemarrowsorXraysmaybe neededoccasionally

Monitoringresponse
responseismonitoredbythelevelof paraproteinintheserumorurine(24 hrexcretion) repeatbonemarrowsorXraysmaybe neededoccasionally

Monitoringresponse
responseisgradedasfollows(simplifiedcriteria) nochange: <25%decreasein paraprotein minimalresponse: 2549%decreasein paraprotein partialresponse: 5099%decreasein paraprotein completeresponse: undetectable paraproteinbyEPorbyimmunofixation andbonemarrowshowing<5%plasmacells

Monitoringresponse
inpatientswithminimalorpartialresponse,a 25%increaseinparaproteinisconsidered progression(relapse) inpatientsincompleteremissionreappearance oftheparaproteinisconsideredrelapse. relapsecanalsooccurwithoutincreasein paraprotein,e.g.withnebonelesions.

Whatisbest?
highqualityelectrophoresis lowthresholdforfixation skilledinterpretation quantificationby%BJPand TP