her 2 assessment

3/22/2015 www.medscape.com/viewarticle/839097_print

http://www.medscape.com/viewarticle/839097_print 1/12

www.medscape.com

AbstractandIntroductionAbstract

Humanepidermalgrowthfactorreceptor2(HER2)overexpressionispresentinapproximately15%ofearlyinvasivebreastcancers,andisanimportantpredictiveandprognosticmarker.ThesubstantialbenefitsachievedwithantiHER2targetedtherapiesinpatientswithHER2positivebreastcancerhaveemphasisedtheneedforaccurateassessmentofHER2status.CurrentdataindicatethatHER2testaccuracyimprovedfollowingpreviouspublicationofguidelinesandtheimplementationofanexternalqualityassessmentschemewithadeclineinfalsepositiveandfalsenegativerates.ThispaperprovidesanupdateoftheguidelinesforHER2testingintheUK.TheaimistofurtherimprovetheanalyticalvalidityandclinicalutilityofHER2testingbyprovidingguidelinesoftestperformanceparameters,andrecommendationsonthepostanalyticalinterpretationoftestresults.HER2statusshouldbedeterminedinallnewlydiagnosedandrecurrentbreastcancers.Testinginvolvesimmunohistochemistrywith>10%completestrongmembranestainingdefiningapositivestatus.Insituhybridisation,eitherfluorescentorbrightfieldchromogenic,isusedeitherupfrontorinimmunohistochemistryborderlinecasestodetectthepresenceofHER2geneamplification.SituationswhererepeatHER2testingisadvisedareoutlinedandtheimpactofgeneticheterogeneityisdiscussed.Strictqualitycontrolandexternalqualityassuranceofvalidatedassaysareessential.Testinglaboratoriesshouldperformongoingcompetencyassessmentandproficiencytestsandensurethereliabilityandaccuracyoftheassay.Pathologists,oncologistsandsurgeonsinvolvedintestinterpretationandclinicaluseshouldadheretopublishedguidelinesandmaintainaccurateperformanceandconsistentinterpretationoftestresults.

Introduction

Overexpressionofthehumanepidermalgrowthfactorreceptor2(HER2)protein,mainlyduetoHER2geneamplification,inbreastcancerisassociatedwithaggressivehistologicalfeaturesandpoorprognosis.[1,2]SeveralrandomisedclinicaltrialshavedemonstratedsubstantialsurvivalbenefitsinpatientswithHER2positivebreastcancertreatedwithantiHER2targetedtherapy,suchastrastuzumab[35]andthetyrosinekinaseinhibitorlapatinib[68]butnotinHER2negativepatients.[9]This,inadditiontopotentialsideeffectsofthesecostlydrugsandevidenceofhigherresponseratestoneoadjuvantchemotherapyinHER2positivetumours,[10]hasemphasisedtheneedforaccurateassessmentofHER2statusinpatientswithallinvasivebreastcancer.Earlystudies,withrelativelysmallnumbersofcases,suggestedthatasmanyas30%ofbreastcancershadHER2overexpression,withafalsepositiverateupto19%andafalsenegativerateof510%.[1113]However,followingpublicationofguidelinerecommendations[11,1418]andrefinementoftestperformanceparametersincludingthestandardisationoftissuehandling,assaymethodologyandadoptingqualityassurancemeasures,recentdataindicatethatthefrequencyofHER2positivityisbetween13%and20%.[11,12,1921]Thefalsepositiverateisreducedtolessthan6%,thefalsenegativerateismuchlower(



multipleipsilateraltumoursiftheyarehistologicallysimilarandcolocatedinthesamequadrant/regionofthebreast.Thereisnoconsensusontestingresidualinvasivetumourfollowingneoadjuvanttherapy,althoughsomerecommendthisapproach.RetestingnonrespondingstableorprogressiveHER2negativetumoursparticularlyhighgradetumoursorthosewithalongtimeperiodbetweenpreoperativebiopsyandexcisionmaybeconsideredbutcannotberecommendedroutinelyinviewofthelackofevidence.

Excellentconcordancebetweencorebiopsyandsurgicalspecimenshasbeenshownusingimmunohistochemistry(IHC)andinsituhybridisation(ISH).[20,22,23]InthemajorityofUKcentres,HER2testingisperformedonthediagnosticneedlecorebiopsyspecimens,mainlytoensuretimelyavailabilityofresultsatthetimeofpostoperativemultidisciplinaryteam(MDT)treatmentplanningdiscussionandalsotoenableconsiderationforneoadjuvanttreatmentusewhichisincreasinglyusedforoperablecases.AlthoughassessmentofHER2statusonneedlecorebiopsyisrecommendedandnorepeatonexcisionspecimensisneededifthetestisclearlypositiveornegative,performing/repeatingtheassayonincisionalorexcisionalsurgicalspecimensshouldbeconsideredif:

(1)thecorebiopsyisnotavailable(ie,thereisonlyacytologysample)or(2)thereisapossibilitythattheHER2testonthecorebiopsyisunreliableorunrepresentativeofthetumouridentifiedintheresectionspecimenasfollows:

1. HER2assessmentisuninterpretableonthecoreduetotechnicalartefacts(ie,suboptimalprocessingorstaining)orthereisdoubtaboutthecorebiopsyhandling.

2. ThecorebiopsyHER2statusremainsintheequivocalcategoryafterIHCandISHforexample,repeatassessmentisadvisedifthecorebiopsywasscoredas2+onHER2IHCwithborderlinenegativeISH(ratioofnumberofHER2tochromosome17centromerecopiesof1.81.99orHER2genecopynumberis46).

3. Invasivetumouronthecoreistoosmallforreliableassessment,orifinvasivediseaseisintimatelyadmixedwithinsitucarcinoma,oronlyidentifiedintheexcisionspecimen.Thereisinsufficientdatatodefinetheamountofinvasivetumourtissueincorebiopsysufficientforanalysishoweverthiscanbelefttothereportingpathologist'sdiscretion.

4. Ifthetumourintheresectionspecimensismorphologicallydistinctfromthatinthecorebiopsy,forexampleofaclearlydifferenthistologicaltypeorhistologicalgrade(eg,lowgradeonthecoreandhighgradeontheexcision,butnotjustreflectingminordifferenceinthemitoticcountorproportionofsolidareas).[24]Arepeatmayalsobeundertakenonconcurrentmetastaticnodaldiseaseifitismorphologicallydistinctfromtheprimarybreasttumour.

5. IfthecorebiopsystainingisheterogeneousandshowsafocusofstrongHER2positivityin



morethan72hCentresusingrapidfixationandprocessingmustvalidatetheirmethodologyforHER2assessment.

Sectionsshouldbestainedwithin12daysofcuttinganddrying.Excessivesectiondryingtimehasalsobeenshowntocausea

lossofHER2expressionanditisthereforerecommendedthatfreshlycutsectionsareeitherdriedat60Cfor1hor37C

overnight[29]

(http://www.ukneqasicc.ucl.ac.uk/neqasicc.shtml).

AlgorithmsforHER2Testing

IHCfordetectionofproteinoverexpressionandISHfordetectionofgeneamplificationstatusarethetechniquesrecommended

fordeterminingHER2status.HighconcordancebetweenIHCandgeneamplificationstatusisreported.[16,30,31]

ThecurrentUK

recommendationsforHER2testingareforatwotiersystemusingIHCwithreflexISHtestingifrequired,usingthemodelshown

infigure1,oraonetierISHstrategy.IngeneraltestingisperformedusingIHCwithanalysisofequivocalcasesbyISH,butthis

doesnotprecludelaboratoriesfromusingprimaryHER2ISHtesting,particularlyifthequalityoftissuefixationisquestionable.[32]

ISHhasusuallybeenconductedusingafluorescenceISH(FISH)technique.BrightfieldISH,whichcanbeusedtoassessHER2

statuswitharegularlightmicroscope,isnowacceptedasanalternativetoFISH.[33]

ThemostcommonbrightfieldISHusesa

DNAprobecoupledtoachromogenicISHorsilverISHdetectionsystem,oracombinationofboth.[33]

ISHcanbeconducted

usingasingleprobetoenumerateHER2copiespernucleusorasadualprobetechniquewhichallowsdeterminationofthe

HER2:CEP17ratioandHER2genecopynumber.Forthisreasontheinclusionofachromosome17probeisstronglyadvocated.

Currently,otheravailableHER2testingtechniques(PCR,ELISA,Southernblotting,mRNAassaysandDNAmicroarray)should

beusedforresearchonly.Similarly,HER2resultsobtainedfromanonISHtechniqueaspartofaprognosticpanelcannotbe

regardedasdiagnosticandshouldnotreplacestandardassaymethodsdetailedabove.

Figure1.



RecommendedHER2scoringalgorithmforimmunohistochemistry(IHC)andinsituhybridisation(ISH).*Insufficientdataisavailabletocommentonmoderatecompletemembranestainingin10%oftumourcellsorstrongincompletemembranestainingin>10%oftumourcells.Arepeatonanotherspecimen/tissueblockisadvisable.**Membranestainingmustbeintenseanduniformandresemblechickenwire.Ignoreincompleteorpalemembranestaininginthepercentageestimation.

ScoringImmunohistochemistry

OnlymembranestainingoftheinvasivetumourshouldbeconsideredwhenscoringHER2.Cytoplasmicstainingandstainingofinsitudiseaseshouldnotbescored,andnormalepitheliumshouldbenegative.TheHER2IHCscoringmethodisasemiquantitativesystembasedontheintensityofreactionproductandpercentageofmembranepositivecells,givingascorerangeof03+(figure1).Samplesscoring3+areregardedasunequivocallypositive,andthosescoring0/1+asnegative.Borderlinescores(2+)areregardedasequivocalandmandatefurtherassessmentusingISH(figure1).Appropriatecontrolsfeaturingdifferentscores(3+,2+and1+/0)shouldbeincludedineverytestrun.Somecentresalsoincludeanonslidepositivecontrolsection.TheHER2testshouldbereportedasindeterminate,andrepeatedwherepossible,iftechnicalissuespreventoneorbothtests(IHCandISH)frombeingreportedaspositive,negativeorequivocal.Examplesinclude,inadequatespecimenhandling,artefacts(eg,crushormarkededgeartefacts)thatmakeinterpretationdifficult,analyticaltestingfailureorifcontrolsarenotasexpected(ie,sampleshowsstrongmembranestainingofnormalbreasttissue).Insuchacase,analternativetest,oranotherspecimenifavailable,shouldbeusedtodetermineHER2status.TheseguidelinesreverttothepreviouslyusedIHCcriterionof>10%cellsstainingforHER2[14,15]insteadofthe>30%cutoffusedinthepreviousguidelines.[11,16]

ScoringISH

HER2ISHtesting,whichusesadualprobemethod,isinitiallyexpressedastheratioofHER2signaltochromosome17centromericenumerationprobe(CEP)signal.SubsequentlytheaverageHER2genecopynumberreportinghasbeenusedinsomecountrieswhenusingdualprobeandsingleHER2geneprobemethodology.TheUKrecommendationistousedualprobeISHandreporttheHER2/CEP17signalratioandHER2copynumber.Tumoursshowingaratio2.0and/orameanHER2genecopynumber6areconsideredtobepositive.AssigningcasesaspositivebasedonaHER2genecopynumber6wheretheHER2/CEP17ratiois



tomanageheterogeneousHER2geneamplificationinbreastcancerandisrecommendedintheseguidelines:[37]

InallcaseswhereISHisperformedtheentireslideshouldbescannedbeforecounting,areasofapparentheterogeneityshould

beidentifiedduringthisscanand/orbyreferencetoanIHCstainedslide.Thenumberofchromosome17(CEP17)andHER2

signalsshouldbecountedin2060nonoverlappinginvasivecancercellnuclei,usingatleastthreedistincttumourfields.Ifthere

isevidenceofheterogeneitybetweenfields(orlessfrequentlywithinfields)additionalcells(atleast20perfield)and/orfields(up

to6)shouldbecounted.TheHER2/CEP17ratioshouldbecalculatedforeachfieldindividually.WherethemeanHER2/CEP17

ratioinanyfieldis2.00orgreater,thetumourshouldberegardedasamplified.Forallcaseswheretheratioisbetween1.80and

2.20resultsshouldbebasedoncountingatleast60tumourcells,andincaseswhereheterogeneityissuspectedthisshouldbe

60cellsperassessedfield.Inrarecaseswhereamplifiedandnonamplifiedtumourcellsareintermingledinasinglefield,

interpretationisdifficultandevidenceislacking.Wesuggestthatforsuchcasesonlythepresenceofclearlyamplifiedcells,with

multipleHER2signals,isconsideredevidenceofheterogeneity,againevidenceislackinginthisarea.Currentevidencedoesnot

supportusingtheexistenceofsmallnumbersofapparentlyamplifiedcellswithinanindividualtumourfieldtoidentify

heterogeneousamplification.[36,38]

Inborderlinecases,thatis,thosewithaHER2/CEP17ratioof1.802.20,additionalcellsshouldbecountedwhenpossible

(optimallyaminimumof60percase),ideallythisshouldincludeadualcount(fromasecondobservereitherinternallyorina

secondcentre).Theoptimalapproachtoimprovingaccuracyinthisrangeistoincreasethenumberofcellscountedto60120,

and/orrepeatthetest.Aratioof1.801.99,aftercountingfurthercellsand/orrepeatingthetest,shouldbereportedasborderline

butnotamplifiedandincludeaclearstatementthatthecarcinomaisregardedasHER2negative(takingthemeanHER2copy

numberintoconsideration(mean10%oftheinvasivetumourareausingIHCisusedtodefinepositivity,casesshowingcompleteintense

membranestainingin



differentiateinvasivefrominsitudiseaseintheindextumourblocksubmittedforISH,IHCmarkersformyoepithelialcellscanbeused.

ImageanalysissystemsmayprovidealternativestomanualscoringforHER2IHCandISH.However,atpresent,insufficient

evidenceisavailabletorecommendtheirroutineuseinthediagnosticsetting.

QualityAssuranceMeasures

Controls

Theinclusionofcontrols,ideallyincludingonslidecontrol(s),andtheirdetailedscrutinyareessentialtoensuretest

accuracy.ControlswhoseHER2statushasbeenvalidatedandproducingresultsclosetoimportantdecisionmaking

pointsarerecommended.Tissuebasedcontrols,frombreastcancers,shouldalsobeusedinallassayruns,ideally

showing3+,2+and1+/0patterns.Controlmaterialshouldbesimilarlyfixedandprocessedtothetesttissue.Control

sectionsshouldideallybecutatthesametimeasthetestmaterial.Longtermstorageofprecutcontrolsectionsisstrongly

discouraged.Thereisnoevidencethatstorageofblocksleadstodeteriorationofsignal.

CelllinepreparationscontainingmultiplesamplesofknownHER2statuscharacterisedbyFISHandIHCandinclusionofa

tumourtissuefromIHC3+caseoneachslideareusefulasadditionalcontrols.

Excessiveantigenretrievalshouldbemonitoredbyevaluatingnormalbreastepithelialcellsasaninternalcontrol.Should

membranestainingbeidentifiedinthenormalcellpopulation,excessiveantigenretrievalmayhaveoccurredandretestingofthe

entirerunshouldbeconsidered.Anysuchtestsshouldcertainlybeinterpretedwithgreatcareitisreasonabletoscorea0or1+

tumourasnegative,but2+or3+tumoursshouldhavestainingrepeated.Ifthereisdoubtbetweena1+/2+resultora2+/3+

result,eithertheIHCshouldberepeatedoramplificationstatusshouldbeassessedusingISH.Ifmembranestainingofnormal

epithelialcellsisseeninanumberofcasesfromthesamestainingrunconsiderationshouldbemadetorepeatstainingofthe

wholerun.

Crushingandedgeartefact,particularlyaffectcorebiopsies.ISH,orrepeatIHConthesurgicalspecimen,maybeneeded.

Thepotentialgradienteffectsofsuboptimalfixation,particularlyinlargersurgicalspecimens,mustalsobeconsideredin

interpretationofstaining.

Itisessentialthatassayproceduresbestandardisedsothatstainingisreliable.Astherecanbevariationbetweenbatches

ofreagents,itisvitalthatcontrolsareassessedcriticallyforeveryrun.Newbatchesofantibodyshouldalsobetested

beforecommencingroutineapplication.Useofstandardisedoperatingprocedures,includingroutineuseofcontrol

materials,isrecommended.

AppropriateLaboratoryAssayMethods

ForIHCandISHbasedHER2testing,comprehensivestandardisationofmethodology,includingmonitoringofscoringprocedures

andtheinclusionofvalidatedcontrols,ismandatory.IntheUK,participationandsatisfactoryperformanceintheUKNational

ExternalQualityAssessmentSchemeforImmunocytochemistryandInSituHybridisation(UKNEQASICC&ISH)HER2IHCandISHmodulesisarequirement(http://www.ukneqasicc.ucl.ac.uk).

StandardisationofHER2IHCstainingisbestachievedbyusingacommercialkit/assay.Inhouse'homebrew'(laboratory

validated)methodsarenotrecommendedbut,ifused,strictprotocolsneedtobefollowed,includingchoiceofantibody,antibody

dilutionandretrievalmethod,eachofwhichcancausevariabilityinstainingresults.Ifacommercialkit/assayisused,itis

recommendedthatlaboratoriesadherestrictlytothekit/assayprotocolandscoringmethodology.Localmodificationsof

techniquescanleadtofalsepositiveandnegativeresults.Therefore,itisimportanttocheckandauditcontrolscarefullyinorder

toensuretestaccuracy.LaboratoriesusingbrightfieldISHshouldperformaninitialvalidationagainstFISH.

InterobservervariationintheassessmentofIHCstainingcanleadtomisclassificationofHER2status.Eachindividualassessor

shouldstandardisescoringagainstknownpositive,negativeandborderlinecases.Itisalsopreferabletoassesscomparabilityof

scoringwithacolleagueonaregularbasis.BeforeundertakingevaluationofHER2,assessorsshouldreceiverelevanttraining.



PublisheddatasuggestthatinterobservervariationissignificantlylowerforFISHthanforIHC.However,especiallywhen

developinganewservice,careneedstobetaken.Therecommendationisthatlaboratoriesshouldperformvalidationstudiesby

dualobserverscoringwhentrainingnewstaffuntilthereisconcordanceof95%.ForISHvalidationpurposes,eachstaffmember

shouldperformaminimumof100ISHtestsinparallelwithanexperiencedISHscorertoattainaminimumconcordanceof95%

ondiagnosticresults(amplifiedandnonamplifiedstatus)andnumericalresults(forHER2andCEP17).Continuedmonitoringof

scoringoffersadvantagesinqualitycontrolandtraining,butisnotarequirement.

ValidationofStandardisedAssayMethodology

Testconditionsshouldbeoptimisedsothatdistinctmoderateorstrongmembranestainingshows>90%concordancewithHER2

ISHpositivesamples.Thiscanbeachievedby:

1. DualHER2IHCandISHassayofacontemporaryseriesofbreastcarcinomas(minimum100cases).Useoftumourtissue

arrayblocksforthispurposemayreducecosts.HER2ISHassaycanbeconfinedtothosecasesdemonstrating3+,2+

and1+membranereactivity.

2. Alternatively,aseriesofcarcinomasthathavealreadybeenscoredforHER2IHCandISH,fromareferencelaboratory,

canbeused.

Laboratoriesnotabletostandardiseinhousemethodologyshouldalsoconsiderusingacommercialvalidatedkitassaysystem.

ISHforHER2GeneEvaluation

ISHtestingforHER2shouldmeetthefollowingcriteria:

1. Comprehensivestandardisationofmethodology

2. Validatedcontrols:theinclusionofachromosome17probetoallowforcorrectionoftheHER2signalnumberfor

chromosome17aneusomy(seenin~30%ofcasesandreportedlymorecommonintumoursthatshowdiscrepantHER2

expressionandintumourswithdiscordantHER2proteinandgenecopynumbermeasurements)isrecommended.

CaseLoad

Laboratoriesprovidingatestingserviceshouldbecarryingoutaminimumof250assaysperyearfor

immunohistochemicaldetectionofHER2.Thistargetlevelhasbeensettoensurehigherconsistencyofassayqualityand

continuingexpertiseofassayproviders.

Centreswithlownumbersofcases(



Itisrecommendedthatcommerciallyavailablevalidatedprobesareused.ThereareanumberofcommercialkitsforHER2ISHusingfluorescenceandchromogenbaseddetectionsystemsandwhichareallacceptable,onceproperlyvalidated.

ShortturnaroundtimesforHER2testingthatdonotdelaythemanagementofpatientsarerecommended.Turnaroundtimeisrecognisedtobevariablebetweendifferentcentres,andcanbeaddressedatthelevelofcancernetworksandlocalservices(figure2).TheNationalInstituteforCareExcellencerecommendsthatHER2statusofthetumourbeassessedandtheresultsmadeavailablewithin2weekstoallowplanningofsystemictreatmentbytheMDTandthatlocalarrangementsandwrittenclinicalprotocolsareinplacetoensureHER2statusresultsareavailablewithinthistime(http://publications.nice.org.uk/breastcancerqualitystandardqs12/qualitystatement5pathologyerandher2status#qualitymeasure5).Itisalsoimportanttoemphasisetheroleofimprovedcommunicationbetweenpathologists/laboratoriesperformingthetestandclinicianstoensureproperhandlingofspecimens(ie,prefixationtimeandfixationtype),shortturnaroundtimeandproperinterpretationofthetestresults.

Figure2.

PathwayforHER2testing.

Audit

Regularandongoingauditshouldbeundertaken.LaboratoriesshouldaudittheiroverallpositiverateforHER2usingacombinationofIHCandISH.Itisimportanttoensurethatthesamplesizeisadequate.Ofnote,theaverageproportionofinvasivebreastcancercasesrecordedasHER2positiveis14.5%(UKNEQASICC&ISHcombined5yearnationalauditdata),with14.3%ofprimarycarcinomasand18%ofmetastaticcasesbeingHER2positive().Ofthesecasesapproximately22%casesare



reportedasborderline(2+)onIHCofwhich1516%arereportedasHER2ISHamplified.[16]TheproportionofHER2positivebreastcancersfoundinscreendetectedbreastcancercasesisrecognisedtobelowerthaninsymptomaticpractice.AuditofHER2assayturnaroundtimeisalsoimportantasitiscriticaltopatientpathway.

Table1.ProportionofHER2positiveprimaryandmetastaticbreastcancers*

0 1+ 2+ 3+ ISH+ OverallHER2positive

Overall(%) 32.8 33.1 21.8 11.6 14.7 14.5

Primarycarcinoma(%) 32.6 33.7 21.8 11.5 14.6 14.3

Metastaticlesion(%) 36.7 27.2 21.1 14.9 15.8 18.0*

*UKNEQASICC&ISHcombined5yearnationalauditdata(unpublisheddata).

ISH,insituhybridisationISH+,proportionof2+carcinomasthatareamplifiedUKNEQASICC&ISH,UKNationalExternalQualityAssessmentSchemeforImmunocytochemistryandInSituHybridisation.

QualityAssuranceforHER2ReceptorEvaluationintheUK

AllUKclinicallaboratoriesusingIHCorISHtoassessHER2statusasapredictivemarkermustparticipateinanappropriateexternalqualityassuranceprogramme,suchasthatrunbytheUKNEQASICC&ISH.

Communication

IntheeraofpersonalisedmedicineandthecommonplaceroutinepracticeofMDTmeetingfordiscussionofdiagnosisandmanagementofallpatientswithcancerintheUK,improvedcommunicationwithintheteamisconsideredofparamountimportance.AlthoughformanyyearstherehasbeencollaborationbetweenpathologistsandpatientfacingcliniciansintheUK,thisguidelinefurtheremphasisestheimportanceofthiscollaboration.Closecommunicationwithsurgeonsandradiologistsisthereforeadvisedinordertoimprovecontroloversamplesprefixationtimeandfixationtype,andwithoncologiststoimproveunderstandingofinterpretationoftheresults.ThisisalsoexpectedtofacilitatecontroloverHER2testturnaroundtime.

Inconclusion,thisupdatecontainsrecommendationssupportedbyasufficientlevelofevidenceonkeypointsrelatedtoHER2testingmethodology,testingalgorithm,interpretationoftheresultsandthepotentialneedforretesting.LaboratoriesofferingaHER2testingserviceandpathologists,oncologistsandsurgeonsinvolvedintestinterpretationandclinicaluseshoulddotheirbesttoadheretopublishedguidelinesandensureaccurateperformanceandinterpretationofsuchtests.

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JClinPathol.201568(2):9399.2015BMJPublishingGroupLtd&AssociationofClinicalPathologists

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her 2 assessment