Her2 immunohistochemical evaluation by traditional microscopy and by digital analysis, and the consequences for FISH testing

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ContentslistsavailableatScienceDirect

Pathology

–

Research

and

Practice

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / p r p

Original

article

Her2

immunohistochemical

evaluation

by

traditional

microscopy

and

by

digital

analysis,

and

the

consequences

for

FISH

testing

Thomas

Marcuzzo

a

_,

_Fabiola

_Giudici

a,∗

_,

_Elisa

_Ober

b

_,

_Clara

_Rizzardi

a,b

_,

_Cristina

_Bottin

a

_,

Fabrizio

Zanconati

a,b

a_Department_of_Medical,_Surgical_and_Health_Science_–_University_of_Trieste,_Strada_di_Fiume_447,₃₄₁₄₉_Trieste,_Italy

b_UCO_di_Anatomia_e_Istologia_Patologica_–_Azienda_Sanitaria_{Universitaria}_Integrata_di_Trieste,_Strada_di_Fiume_447,₃₄₁₄₉_Trieste,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received30June2015 Keywords: Breastcancer Her2 Digitalanalysis Immunohistochemistry Fluorescence

a

b

s

t

r

a

c

t

Aim:Her2proteinisthekeymarkerdeterminingthechoiceofHerceptintherapyafteradiagnosisof breastcancer.Itsevaluationismadeinmostlaboratoriesbyimmunohistochemistry,andinterpretedby apathologistusinganopticalmicroscope,aprocesssubjecttointer-observervariability,particularlyfor samplesscoredasequivocal(2+).Softwareanalysisproductshavebeenintroduced,seekingtoreducethis variability.Inthisstudy,wecomparedtheresultsofbothtraditionalevaluationandaspeciﬁcsoftware package(VISIAImaging)tothosefromﬂuorescentinsituhybridization(FISH).

Materialsandmethods:Weselected176casesofinvasivebreastcancersampledduring2012–2014that wereclassiﬁedasequivocalafterevaluationofHer2immunohistochemistry,andthatwerealsoevaluated byFISH.EachtissueslidewasscannedwithadigitalD-SightFluo2.0microscopeandanalysedwithVISIA ImagingS.r.l.software.Theﬁnalresultswerecategorisedasfollows:negative(0–1+),equivocal(2+),or positive(3+).TheneachresultwascomparedtothatobtainedbyFISH.

Result:Thedigitalmethodconfirmed85samples(48.3%)asequivocal(2+),while23(15.1%)were reclas-sifiedasnegative(1+)and44(28.9%)aspositive(3+).Ofthe176cases,24(13.6%)werenotsuitable fordigitalanalysis(inadequate).Of67reclassifiedcases(1+or3+),62wereinagreementwithFISH results(concordancerate92.5%).Thesensitivityandspecificityofthedigitalmethodwere100%and82%, respectively.

Conclusion:Theapplicationofthisanalysissoftwareledtoanimprovementintheinterpretationofcases classiﬁedasequivocal,decreasingtheneedforFISHandincreasingdiagnosticcertainty.

1. Introduction

Theexpressionofthehumanepidermalgrowthfactor2(Her2)

membraneproteinisakeymarkerintheprognosticandpredictive

assessmentofbreastcancer[1].Itsevaluationisgenerallymade

usingimmunohistochemicaltestsinterpretedbyapathologist,and

usinginsitu hybridisationtesting foruncertaincases[2].

How-ever,apathologist’sevaluationofopticalmicroscopyisasubjective

parameter,opentointer-observervariability[3].Manyeffortshave

beenmadetoﬁndamoreobjectiveprocedure,includingdigital

analysis[4–8].Inthisstudy,adigitalanalysissoftwareproductfor

Her2immunohistochemicalevaluationwastested.Thesoftware

usesslideimagespreviouslyacquiredwithadigitalmicroscopeand

∗ Correspondingauthor.

E-mailaddress:fgiudici@units.it(F.Giudici).

semiautomaticanalysisofthevirtualslide,classifyingthesample

aspositive(3+),negative(0–1+),orequivocal(2+).Weevaluated

whethertheapplicationofthissoftwareanalysiscouldleadtoan

improvementintheinterpretationofequivocalcases,decreasing

theuseofFISH.Weexaminedtheconcordanceratebetweenthe

resultsobtainedbydigitalanalysisandbyFISH.

2. Materialsandmethods

We selected 176 cases of invasive breast cancer diagnosed

during2012–2014,classiﬁedasequivocalafterHer2

immunohisto-chemicalslideevaluation.AconﬁrmingFISHtestwasmadeonlyon

equivocalcases[1].Allslideswereselectedfromthearchiveofthe

InstituteofPathologicalAnatomyandHistologyofthe

Academic-Hospitalpublic-servicecorporation“OspedaliRiuniti”ofTrieste,

Italy.Immunohistochemistrywasperformedafterprevioustissue

ﬁxation(10%neutralbufferedformalin)andparafﬁnembedding.

http://dx.doi.org/10.1016/j.prp.2016.07.016

(2)

Table1

Immunohistochemicalprocedure.

√

Antigenretrieval:applicationofaheatprocedurewithPT-Link instrument(Bioptica)andW-CAPEDTABufferpH8.0(Bioptica): Preheat:heatingofthebuffer,itstemperatureincreasesfromroom

temperatureto65◦_C_in₂₀_min_(min).

Retrieval:thetemperatureincreasesto95◦_C_and_becomes_stable_for

20min;

Cool:coolingofthebufferfrom95◦Cto65◦Cin20min; √

Endogenousperoxidaseinhibition:H2O2at3%(Bioptica)for10min;

√

Primaryantibodyincubation:cloneMIB-1(Bioptica),1:200dilution for20min;

√

Secondarysystem:UltravisionQuantolargevolumedetectionkit-HRP (ThermoScientiﬁc):

Enhancerfor10min; Polymerfor10min; √

Chromogen:3,3_{diaminobenzidine}_(DAB_Quanto)_for₅_min

(ThermoScientiﬁc); √

Counterstaining:Mayerhaematoxylinfor3min; √

Dehydration:arisingsequenceofalcohol; √

Clariﬁcationwithxylene;

√

Assembling.

Allimmunohistochemicaltestswereperformedusinganautomatic

stainer(Lab Vision,ThermoScientiﬁc),following theprocedure

showninTable1.Eachstepwasfollowedbywashingwith

Tris-bufferedsalinecontainingTween20(ThermoScientiﬁc).AfterJuly

2014,anotherwashingstepwithrunningwaterwasaddedafter

counterstaining,toimprovethehaematoxylinresults.Everyslide

had been evaluated by a pathologist onan optical microscope

withalowmagniﬁcationandclassiﬁedasequivocal,inagreement

withthemostrecentguidelinesoftheAmericanSocietyof

Clini-calOncologyandtheCollegeofAmericanPathologists(ASCO-CAP

2013).Cases evaluatedbeforethe2013 guidelinescut-off date,

which had been classiﬁed using the previousset of guidelines

[9],wererevaluated,andallwereconﬁrmedasequivocal.Each

slideunderwentdigitalacquisition,usingaD-SightFluo2.0

dig-italmicroscope(A.MenariniDiagnostics,Firenze,Italy)andwas

analysedwithVISIAImagings.r.l.software(version2.5.0.1,San

Gio-vanniValdarno,Italy).Thedigitizingofasliderequiresacquisition

withalow-magniﬁcationlens(4×)tocreateaslidepreview,on

whichtheareaofinteresttobescannedwithahigh-magniﬁcation

lens(20×)isselected.Withthisprocedure,adigitalequivalentof

theslideisobtained, andit canbedisplayedat any

magniﬁca-tionfrom0to20×,maintaininganexcellentimagequality.The

importanceofusingahigh-magniﬁcationlens isthatthe

analy-sissoftwarerequiresahigh-resolutionimage(notlessthan20×

magniﬁcation).EveryequivocalcaseunderwentaFISHconﬁrming

test(PathVysionHer2DNAProbeKitII,AbbotMolecular)evaluated

using a ﬂuorescent microscope,following international criteria

(ASCO-CAP2013).Resultscollectedbeforetheadoptionofthese

guidelineswere reevaluated, and no changes wereseen in the

ﬁnaldiagnosisforanycase.AfterJuly2014,ﬂuorescentslideswere

scannedwithaD-SightFluo2.0digitalmicroscope,andanalysed

withspeciﬁcsoftware.

Digitalslidesweredividedintocategoriesbasedon:

• Typeofspecimen:Surgicalsampleorbiopsy.

Thedifferenceinthequantityoftumoursamplethatcanbe

foundinthetwodifferenttypesofspecimenisimportant,asisthe

subsequenthandlingofsamplesduringallthestepspreceedingthe

immunohistochemicalstainingtoachieveaperfectreactionquality

[10].

• Suitabilityfordigitalanalysis:Inadequateoradequate.

Table2

Parametersfordigitalanalysis.

Parameter Range Function

Onlycellswithnucleus ON/OFF Countsonlythemembrane associatedwithadetected nucleus.

Threshold 0–255 Detachmentbetween backgroundandnucleionthe haematoxylinchannel. Minimumarea 0–6000 Minimumareaunderwhich

theobjectisidentiﬁedas nucleus.

Crumbling 0–255 Settlesthesensibilityonthe membraneseparation. Threshold+vs− 0–255 Settlesthethresholdof

membranecompletenessto classifycellsasnegative(0)or positive(1+,2+,3+).

Threshold2+vs3+ 0–255 Settlestheaverageintensityof completemembrane classiﬁcationasstrong(3+)or moderate(2+).

Someoftheslidesinourcollectiondidnotmeetthe

require-mentsoftheanalysissoftware,mostlyduetopoorqualityofthe

immunohistochemicalsignal.Thesecaseswereclassiﬁedas

inad-equate(technicalissues).

• Proﬁle:A,B,C,D,E,F,G.

Theanalysissoftwareusesasequenceofparametersthatallow

evaluationofstainingintensityandofthepercentageofthecancer

cellmembranethatisstained(Table2).Anaccurateanalysisofthe

sliderequiresthattheseparametersbecarefullychosentoavoid

misclassiﬁcationofcells.Thisisdirectlyaffectedbythequalityof

theimmunohistochemicalreaction,andbytheintensityofboththe

haematoxylinstainingofthenucleiandthe3,3diaminobenzidine

(DAB) staining and its distribution.Speciﬁc proﬁles have been

developedbasedonslideswiththesamestainingquality,with

ref-erencetoarepresentativeslide,fromAtoG.Everyscannedslide

wasevaluatedatlowmagniﬁcationandenteredinthe

appropri-ateanalysiscategory.Aspeciﬁcproﬁle(F)wasdevelopedonlyfor

biopsy,evenifthestainingoftheslideisidenticaltothesurgical

specimenthatunderwentBproﬁleanalysis.Thisisduetothe

differ-entnatureofthetwosamples,biopsieshavefewertissuequantity

anddifferenthistologicaltissuetreatment(ﬁxationand

process-ing)comparedtosurgicalspecimens,causingstainingdifferences

thattheanalysissoftwarecandetect,astheyarenotvisibletothe

nakedeye.Itisreallyimportanttoseparatethetwoproﬁlestoavoid

misclassiﬁcationoftheslide.Overall,sevendifferentproﬁleswere

deﬁned(Table3),eachwithitsrepresentativeslide(Image1),to

maketheanalysisreproducibleandreliable.

• Stainingdistribution:Homogeneous,heterogeneous.

DABstainingintensitydistributionisanessentialparameterin

thechoiceoftheanalysisarea.Eachslidewasvisualizedatlow

magniﬁcation(max4×)andthestainingintensitywasevaluated.

Ifthedistributionwashomogeneous,theanalysisareawaschosen

randomlyontheslide.Ifthedistributionwasheterogeneous,two

separateareaswerechosen,oneinthemostintenseregion(hot

spot),anotherintheweakest,containingapproximatelyan

equiv-alentnumberofcells.Theseareaswereanalysedseparatelyanda

ﬁnalaverageresultfrombothwasconsidered.

Whentheseparameterswereset,theimageanalysiswas

per-formedusinga20_{×magniﬁcation.}Itwasdecidedtolimitthearea

ofanalysisto20mm2₍₄_×_ﬁeld),_due_to_the_{time-consuming}_nature

(3)

Table3

Proﬁleforthedigitalanalysis.

Proﬁle Parameters Value

A

Greynuclei,weak intensity Representativeslide B13-1404I

Onlycellswithnucleus Threshold Minimumarea Crumbling Threshold+vs− Threshold2+vs3+ OFF 1 10 25 137 77 B

Intensebluenuclei Representativeslide B14-8580/1B

Onlycellswithnucleus Threshold Minimumarea Crumbling Threshold+vs− Threshold2+vs3+ OFF 40 120 25 167 87 C

Faintslide,softDAB Representativeslide B12-772E

Onlycellswithnucleus Threshold Minimumarea Crumbling Threshold+vs− Threshold2+vs3+ OFF 1 10 25 172 102 D Faintslide, well-deﬁnedDAB Representativeslide B12-9154G

Onlycellswithnucleus Threshold Minimumarea Crumbling Threshold+vs− Threshold2+vs3+ OFF 1 20 10 187 117 E Grey/bluenuclei, cytoplasmicor intenseDAB Representativeslide B12-13575/2C

Onlycellswithnucleus Threshold Minimumarea Crumbling Threshold+vs− Threshold2+vs3+ OFF 1 10 25 140 65 F

Biopsy,Intenseblue nuclei

Representativeslide B14-9699

Onlycellswithnucleus Threshold Minimumarea Crumbling Threshold+vs− Threshold2+vs3+ OFF 40 10 10 140 60 G

Strongintensitymix slide(A/E) Representativeslide B12-15122/2o B14-1761

Onlycellswithnucleus Threshold Minimumarea Crumbling Threshold+vs− Threshold2+vs3+ OFF 1 10 25 97 35

havetakenanexcessiveamountoftime,andthenumberofcellsin

anareaof20mm2_was_expected_to_be_high_enough_to_be

represen-tativeofthewholesection.Thiswasdemonstratedtobethecase

(seeResultsbelow).Theanalysiswasbasedontheidentiﬁcation

ofDAB-positivecellmembranesandonthepercentageofthecell

membranestained.Sampleswereclassiﬁedintofourcategories,as

follows:

• 0Negative:nopresenceofmembranestaining,theanalysisﬁnds

onlythehaematoxylinstainingofthenuclei.

• 1+Negative:weakandincompletestaining.

• 2+Equivocal:moderateorincompletemembranestaining.

• 3+Positive:strongandcompletemembranestaining.

Thisclassiﬁcationis equivalenttothat recommended inthe

most recent published guidelines [1]. Every cell is categorized

accordingtothesecriteriaandthesoftwarereportsthe

percent-ageof cellsineachcategory fromthetotalnumber ofanalysed

cells.Theﬁnalresultisscoredbasedonapositivitycut-offsetat

10%ofthetotal cellnumber,asnegative(0–1+),equivocal(2+),

orpositive(3+).Resultsfromeachcasewerecomparedtothose

obtainedbyearlierFISHtests,whichareconsideredthediagnostic

goldstandard.

• Numberofcells:Adequate,inadequate.

Whenthetotalnumberofanalysedcellswaslessthan10,000,

thewholeanalysiswasconsideredunreliable.Weobservedthat

with fewer cells the positive/negativepercentage wasstrongly

inﬂuencedbythenumberofcellsanalysed,withsmalldifferences

leadingtolargechanges.Becausethisvariationstartedtostabilize

above10,000cells,weusedthis numberasareliability

thresh-old.Ifasingleareadidnotcontainenoughcells,theanalysiswas

extendeduntiltheminimumthresholdwasreached.Ifinsufﬁcient

cellswerepresentwithinthewholesection,theslidewas

consid-eredinadequate(<10,000).Totestthesuitabilityoftheselected

threshold,weexaminedthetotalnumberofcellsanalysedforall

areasofeachslide.Alloftheanalysedareaswereincludedinthis

examination,thusoneforeveryhomogeneouscase(129)andtwo

foreachheterogeneouscase(23).Furthermore,wealsoexamined

cellnumbersonslidesscoredasinadequate(<10,000),foraﬁnal

totalof191analysedareas.

3. FISH

Aspreviouslydescribed,theconﬁrmingFISHtestneedsits

anal-ysiswithaﬂuorescentmicroscope.Theanalysisstartedwithan

observationoftheslidebythepathologistwithaDAPIﬁlter.This

allowedhimtoevaluatethenucleimorphologyandeventualnuclei

overlapping,choosingthesuitableone.ThentheDAPIﬁltermust

beswitchedwiththeSpectrumOrangetodetectHer2/neusignals

andcountthem,losingthenuclearmorphologicaldetailthatthe

observermustkeepinmind.Thentheﬁlterisswitchedagainto

theSpectrumGreen,tocountCep17signals.Boththecountswere

signedandtheobservermovedonanothernucleus.Thisprocess

continueduntiltheachievementoftheminimalnucleinumber,not

lessthan20nuclei[1].AfterJuly2014,theevaluationoftheFISH

resultswasmadeusingthedigitalequivalentoftheslide:weused

theD-SightFluo2.0digitalmicroscopetocreatedigitalﬂuorescent

slide,andthenanalysedthemwiththespeciﬁcanalysissoftware

madebyVISIA.Thissoftwareisanupgradeofthesameusedina

recentarticle[11]aboutstandardizationofcompletelyautomated

FISHanalysis.Theanalysiswasmadeonselectedareasafter

exam-inationofallﬂuorescentslides.Thesoftwarescannedtheselected

ﬁeldsat 60× magniﬁcation.A mercurylamp poweredthelight

directedonthesampleandaDAPI

(4,6-diamidino-2-phenylindol)-stainedimagewasacquiredtoidentifynuclei,usinganautomated

perfectfocussystem.Thesoftwareproceededtoscansignalsfrom

probesforbothHer2/neu(SpectrumOrange)andCEP

(Spectrum-Green),usingappropriateﬁlters,andcollectingdatafromagreater

numberofZ-levelscomparedtotheDAPIimages.Levelnumber

isadjustable,butforthesesamplesitwasﬁxedat13,separated

fromeachotherby1␮m.Thesoftwarecompressedthescanned

imagesintoonethattheusercanscrollthrough,removing

back-groundfrombothprobes.Thepathologistgainedtheadvantagesof

observingtheslideonascreen,andnotdirectlyontheﬂuorescent

microscope,withahighimagequalityincludingallﬁltersand

with-outneedoffocusing[12].Thesoftwareautomaticallyhighlighted

therecognizablenucleiandtheredandgreenprobes.The

pathol-ogisthadtocheckeverynucleusandeverysignaltocontrolthe

suitabilityoftheanalysis.Ifneeded,hecoulderase,modifyoradd

nucleiorprobessignalswithadedicatedmanualcontrolsystem.

Thesoftwarecontinuouslyupdateditsﬁnalresultsbasedoneach

probeandnucleusacceptedbythepathologist.Thisallowedafast,

effective,andreproducibleanalysisoftheselectednuclei.Itwas

alsopossibletostorehigh-qualityimagesofeachsample,avoiding

lossofsampleinformationduetoﬂuorescencedecay.Furthermore,

digital analysisallowedtheobservationofa greater numberof

nucleicomparedtodirectopticalﬂuorescentmicroscopy

evalua-tion.Themostrecentguidelines[1]changedtheminimumnumber

ofnucleithata pathologistmustcounttogivea reliableresult,

(4)

Image1. Representativeslides.

thisreduction.Eachofthe49Her2FISHdigitisedcaseswascounted

multipletimes,startingfromaminimumof20andincreasingthe

numberevaluateduntilthemaximumanalysablenucleiwithinthe

selectedareaswasachieved(20,30,40,50,60,max).Thehighest

numberofanalysablenucleirangedfrom65to384.Resultsusing

differentcellcountswerecompared,todetermineifithad

influ-encedthefinalscoring.Inthesecases,thefinalresultwasobtained

byagreementoftwopathologists,expertsinﬂuorescence

interpre-tation.

4. Results

4.1. Cellnumberdistribution

Cellnumbers withintheanalysed areas rangedfrom987to

127,933,witha meanof35,483.26;thestandarddeviationwas

25,844.91.Furthermore,50% ofthesamplescontained between

16,793and46,304cells.

4.2. Generalresults(Table4)

Ofthe176totalcases, 24wereinadequate(12 fortechnical

issuesand12forcellnumbers<10,000),85(48.3%)wereconﬁrmed

asequivocal,23 and44 wereclassiﬁedasnegative(13.1%) and

positive(25.0%),respectively.

In comparison tothe gold standard method of FISH the 85

equivocalcasesconﬁrmedasequivocal,67 caseswerecorrectly

reclassiﬁedaspositiveornegative,whilst5caseswerediscordant

comparedtoFISH.Thesecaseswereclassiﬁedasampliﬁedby

dig-italanalysisandnotampliﬁedbyFISH.Ofallourclinicalrecords

(176)thedigitalanalysisof62casesisconcordantwiththeFISH

(5)

Table4

CharacteristicsofslidesclassiﬁcationafterHER2immunohistochemistrydigital analysis(176cases). Characteristic N % DiagnosticCategories Inadequate 24 14% Negative(1+/0) 23 13% Positive(3+) 44 25% Equivocal(2+) 85 48% Total 176 100% TypeofSpecimen SurgicalSample 132 75% CoreBiopsy 44 25% Total 176 100%

SuitabilityforIHCdigitalanalysis

Inadequate 24 14% Adequate 152 86% Total 176 100% StainingDistributiona Homogeneous 139 85% Heterogeneous 25 15% Total 164 100%

ConcordanceRatebetweenIHC DigitalAnalysisandFISHtestb

FISHconcordant 62 41%

FISHdiscordant 5 3%

Equivocal(2+) 85 56%

Total 152 100%

a_Excluded₁₂_cases_for_the_{impossibility}_to_evaluate_the_{distribution.} b_Excluded₂₄_inadequate_cases.

withFISHﬁndingsTheseﬁvecaseswillbedescribedindetailinthe

discussion.

The24samples(14%ofcases)thatwereinadequatefordigital

analysiswereexcludedfromsomeofthefollowinginvestigations,

decreasingtheclinicalrecordsto152.

4.3. Sensitivityandspeciﬁcityofthedigitalmethod(Table5)

InthecomparisonbetweenFISHanalysisanddigitalanalysis

weexcludedthe85equivocal(2+)casesandalsothe24

inade-quateones.Thereforetheanalysisisfocusedon67cases,ofwhich

39showedampliﬁcation byFISH,while 28werenot ampliﬁed.

WiththedigitalIHCevaluation,inmostcasestherewas

concor-dancewiththeFISHevaluation(62casesof67),exceptforﬁve

cases(falsepositives)forwhichtheinstrumentidentiﬁed

hyper-expressionwhereitwasnotampliﬁed(positivepredictivevalueof

thedigitalmethodVPP=89%[95%CI78–95%]).Thesensitivityand

speciﬁcityofthedigitalmethodwere100%(95%CI[93–100%])and

82%(95%CI[71–90%]),respectively.

Table5

ComparisonbetweenFISHAnalysisandDigitalAnalysis(excluded85equivocaland 24inadequatecases).

DigitalIHCAnalyis

Positive(3+) Negative(1+) Total

FISHAnalyis Ampliﬁed 39 0 39

NotAmpliﬁed 5 23 28

Total 44 23 67

4.4. Comparisonbetweentypesofspecimen(Table6)

Therecordsincluded132(75%)surgicalspecimensand44(25%)

biopsies.Thewerenostatisticaldifferencesbetweenthe

propor-tionsofinadequatesamplesinbiopsiesandinsurgicalspecimens

(18%vs12%,p=0.47).However,therewereanumberofsamples

thatremainedequivocalafterdigitalanalysis,andmoreofthem

werefoundamongsurgicalspecimensthaninbiopsies.

Evaluat-ingtheconcordancepercentage withFISHanalysiswefoundit

tobeslightly higheramongbiopsysamplesbut notstatistically

signiﬁcant(50%vs38%,p=0.27).

4.5. Comparisonbetweenstainingdistributions(Table7)

Among the analysis of 164 samples (12 were excluded for

theimpossibilitytoevaluatethedistribution),139(85%)showed

homogeneousstaining.Furthermore, inboth homogeneousand

heterogeneousstaining categories,there wasa highnumber of

equivocalcases.Focusingonthenon-equivocalcases,in

homoge-neoussamples,thepercentageofFISH-discordantcaseswas2%(1

case in58),whileinheterogeneouscases,whichwerefewerin

number,thediscordantcasepercentagewas17%(p=0.005).The

singlehomogenousdiscordantcasewasabiopsy,whileallfour

heterogeneousdiscordantcasesweresurgicalspecimens.

4.6. Comparisonamongthreeyears(Table8)

Wedividedtheﬁnalresultsintothethreeyearsofthestudy,and

examinedthemseparately.Thenumberofcasesclassiﬁedas

equiv-ocaldecreasedovertime,from50%oftotalcasesin2012to42%

in2014.Thepercentageofcaseswithnon-equivocalﬁnalresults,

concordantwithFISH,increasedfrom22%in2012to44%in2014.

However,asmallpercentageofdiscordantcaseswaspresent

dur-ingallyears.Thefractionofcasesscoredasinadequatefortechnical

reasonsdecreasedfrom12%to5%duringthethreeyearsexamined

(Image2).

Table6

DigitalAnalysisaccordingtoTypeofSpecimens.

Characteristic SurgicalSample CoreBiopsy p-value p-trend

DiagnosticCategories 0,43 Inadequate 16(12%) 8(18%) 0,47 Negative(1+/0) 16(12%) 7(16%) 0,69 Positive(3+) 32(24%) 12(27%) 0,84 Equivocal(2+) 68(52%) 17(39%) 0,19 Total 132 44

ConcordanceRatebetweenDigitalAnalysisandFISHtesta _Surgical_Sample _Core_Biopsy _p-value _p-trend

FISHconcordant 44(38%) 18(50%) 0,27 0,44

FISHdiscordant 4(3%) 1(3%) 1

Equivocal(2+) 68(59%) 17(47%) 0,31

Total 116 36

(6)

Table7

DigitalAnalysisaccordingtoStainingDistributions.

Characteristic Homogeneous Heterogeneous p-value p-trend

DiagnosticCategories p=0.83 Inadequate 10(8%) 2(8%) 1 Negative(1+/0) 21(16%) 2(8%) 0,45 Positive(3+) 37(29%) 7(28%) 1 Equivocal(2+) 71(55%) 14(56%) 1 Total 139 25

ConcordanceRatebetweenDigitalAnalysisandFISHtest* _Homogeneous _{Heterogeneous} _p-value _p-trend

FISHconcordant 57(44%) 5(22%) 0,07 <0.001

FISHdiscordant 1(1%) 4(17%) 0.005

Equivocal(2+) 71(55%) 14(61%) 0,77

Total 129 23

* _Inadequate_cases_were_excluded.

Table8

Resultcomparison:2012–2014.

D-sightresults 2012 2013 2014

InadequatecellN◦_<_10.000 ₆ _12% ₂ _3% ₄ _6%

Inadequatefortechnicalpitfall 6 12% 3 5% 3 5%

Equivocal 26 50% 33 52% 26 42%

FISHconcordant 11 22% 24 38% 27 44%

FISHdiscordant 2 4% 1 2% 2 3%

Totalamount 51 100.0% 63 100.0% 62 100.0%

4.7. FISH

Ofthe49casesanalysedbyFISHwiththedigitalprocedure,41

showednovariationinresultsbasedonthedifferentnumbersof

totalnucleicounted.Theremainingeightcasesdidshow

differ-encesbetweenresultsobtainedfromcountingtheﬁrst20nuclei

andfromoneothertotalcountcategory.Moreover,weobserved

thatonlythe40-nucleicountsgavethecorrectresultinallcases.

5. Discussion

WecomparedtheresultsoftraditionalHer2IHCevaluationwith

aspeciﬁcsoftwarepackage(VISIAImaging)forHer2IHCanalysis,

usingFISHasgoldstandard.Toclearlyinvestigateiftheapplication

ofdigitalIHCanalysissoftwarecouldleadtoadecreaseofFISHtest

andmorerapid,reproducibleandreliableIHCresults,wediscuss

eachstepseparately.

5.1. Cellnumberdistribution

Aminimumof10,000cellsappearedtobeanidealcut-offfor

reliabilityofthisanalyticdata,conﬁrmingearlieranecdotal

evi-dence.Indeed,morethat75%ofcasesinthisstudyprovidedhigher

cellnumbers,and50%ofsamplescontainedbetween16,000and

46,000cells.Therefore,weconsidertheanalysiswehaveconducted

tobereliable.Resultfromcaseswithfewercellnumbershouldnot

beconsidered.

5.2. Generalresults

Somesamplesmaynotbesuitablefordigitalanalysis,dueto

characteristicsofthesample,asin thecase oflow cellnumber

previouslydescribed,ortotechnicallimitations.Itisimportantto

separatethesetworeasons,astheﬁrstisanindependentvariable

(becausewecannotdeterminethenumberofcancercellspresent

inasample)whiletheotherisdeterminedbythequalityofthe

immunohistochemicalassay.Wehavedemonstratedthatresults

in35%ofallequivocalcases,withevaluationbyopticalmicroscopy,

couldbebetterdeﬁnedbydigitalanalysisavoidingFISH.Inaroutine

useperspectivethissystemwilldecreasebyathirdthe

ﬂuores-cenceconﬁrmingtest.Thediagnosticcontributionthatthedigital

methodbringsonthismatterisundoubtedlyrelevant.

5.3. Sensitivityandspeciﬁcityofthedigitalmethod

Digitaltestappearedtohave100%sensitivity,duetotheabsence

offalsenegatives.However,thepresenceofFISH-discordantcases

(7)

cannotbeignored.Inthewholesetofsamplestherewereﬁve

false-positiveswhichdecreasedthetestspeciﬁcityto82%.

5.4. Comparisonbetweentypesofspecimen

Theinadequatepercentageissimilarinbothcategories,evenif

itwasslightlyhigherinbiopsies.ConsideringtheﬁveFISH

discor-dantcasesindetail,fourofthemweresurgicalspecimens,while

onewasabiopsy.Biopsiesrepresentasmallernumberofsamples

comparedtosurgicalspecimens,buthaveaslightlyhigher

frac-tionofinadequatesamples.Thisisinherenttothesamples,which

presentcomplexchallengestobothpre-analyticstandardization

andimmunohistochemicalmethods.Furthermore,mostbiopsies

samplescontainevidentartefactsofover-orunder-stainingdue

tosamplehandling,oftenfocused onbordersorseen in

small-dimensionsamples.Thedifferencebetweenbiopsiesandsurgical

samplesimmunohistochemistry couldbe of great inﬂuence for

somemarkersandthereisvarianceevenforHer2[13].This

dif-ferencehasbeenreported alsoinotherstudies onothertissue

samples[14].Guidelinesrecommendalargegroupofsituationsin

whichIHCtestonasinglebiopsysampleisnotadequateandnew

Her2testonthesurgicalspecimenmustbedone[1].Thecertainty

ofcorrecttissuehandlingandofIHCmethoddecreasestherisks

ofalteredstainingandimprovesdigital analysis.Arecentstudy

underlinedthefactthatapercentageofsamplesscoredas

posi-tive(3+)bythepathologist,usingopticalmicroscopy,werefound

nottobeampliﬁedafterFISHtest[15].Thisshowsthatthereare

fewsampleswithhighmembranestainingthatdonothavegene

ampliﬁcation.Allthesefactorleadtodiscrepancieswhenabiopsy

isanalysed,evenwithautomateddigitalsoftware.

5.5. Comparisonbetweenstainingdistribution

Heterogeneous cases present different staining intensities

withinthesamesample.

Frequentlyitisunclearifvariationinintensityisduetofocal

hyperexpressionorisrelated toartefacts (id:delaysinﬁxation,

overﬁxation,automatic stainermalfunction,ortootherreasons

relatedtopreanalyticalandanalyticalphases[10,16]).Inall

het-erogeneouscasesweselectedtwoareaswithdifferentialstaining

containingcomparablecellnumbers.ConsideringtheﬁveFISH

dis-cordantcases,fourofthemaresurgicalspecimensthatpresenta

heterogeneousstainingdistribution.Heterogeneouscasesshould

beinterpretedwithcaution.

5.6. Comparisonamongtheyears

Duringthethreeyearsofthisstudy,thequalityofthe

immuno-histochemicalassay changed, due toa strictstandardisation of

pre-analysisprocessesandincreasedcontrolofthe

immunohisto-chemicalmethods.Coldischemiaandformalinﬁxationtimewere

carefully standardized as suggested by internationalguidelines

[1,9],antibodyspeciﬁcitywasperiodicallytestedonpositiveand

negativecontroltissue, andtheanalytic activitywassubmitted

toexternalqualitycontrol(NordiQC).Particularlyusefulwasthe

standardizationofhaematoxylincounterstaining.Agood-quality

sampleis essential toperforma reliabledigital analysis.Lower

samplequalityrisksinaccuratedigitalresults.

5.7. FISH

Weobservedthatlimitingthetotalevaluatednucleito20could

leadtodoubtfulclassiﬁcations,butalsotoextendthecounttoan

excessivelyhighnucleinumberdidnotimprovethereliabilityof

results.Wesuggestthat40istheidealnucleinumberforareliable

count.

6. Conclusion

ThisanalysissoftwareofimmunohistochemistryHer2slidehas

signiﬁcantvalueandutilityintheprognostic-predictiveevaluation

ofbreast cancer.Itgives resultsneededtopatienttherapyvery

rapidly,avoidingtheuseofFISHonathirdoftotalcases.Digital

analysisisalsosimpletoapply,highlysensitive,andreproducible

whenproperlyarranged.However,therisksofmisclassiﬁcation,

although low, are not entirelyremovable, and we believe that

heterogeneous-stainingcasesshouldbeconﬁrmedbyFISHtesting.

Whiletheidealsamplefordigitalanalysisisasurgicalspecimen,

iftheonlyavailablesampleisabiopsy,itwillbeimportanttobe

certainofitscorrectprocessingandstaining.Ifdoubtsonits

han-dlingarepresent,FISHmayalsoberequired.IntheanalysisofFISH,

areliablycorrectevaluationmayrequireincreasingthenumberof

nucleiexaminedto40.Theapplicationofthisanalysissoftwarecan

leadtoanimprovedinterpretationofequivocalcases,decreasing

theneedforFISHevaluation,andincreasingdiagnosticcertainty

andreproducibility.

Conﬂictofinterest

Theauthorshavenoconﬂictofinteresttodeclare.

Acknowledgementsandfundinginformation

ThisworkwassupportedbygrantA.MenariniDiagnosticS.R.L.

TheEnglishinthisdocumenthasbeencheckedbyatleasttwo

professionaleditors, both nativespeakersof English.For a

cer-tiﬁcate,pleasesee:http://www.textcheck.com/certiﬁcate/185gVo.

TheauthorsthankAnaMariaGheorghe-Guttaforherprecious

con-tributionincheckingtheEnglishaccuracyoftheﬁnalversionofthe

manuscript. References

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