Expression of immunohistochemical markers in non-oropharyngeal head and neck squamous cell

13 Pages·2017·3.62 MB·English

RESEARCHARTICLE
Expression of immunohistochemical markers
in non-oropharyngeal head and neck
squamous cell carcinoma in Ghana
OseiOwusu-Afriyie1,2*,W.K.B.A.Owiredu1,KwabenaOwusu-Danquah3,RitaLarsen-
Reindorf4,PeterDonkor5,EmmanuelAcheampong1,SolomonE.Quayson6
1 DepartmentofMolecularMedicine,SchoolofMedicalScience,KwameNkrumahUniversityofScienceand
Technology,Kumasi,Ghana,2 DepartmentofPathology,SchoolofMedicalSciencesKomfoAnokye
TeachingHospital,Kumasi,Ghana,3 DepartmentofMedicallaboratorytechnology,FacultyofAlliedHealth,
KwameNkrumahUniversityofScienceandTechnology,Kumasi,Ghana,4 DirectorateofDental,Eye,Ear,
Nose&Throat,KomfoAnokyeTeachingHospital,Kumasi,Ghana,5 DepartmentofMaxillofacialSurgery,
a1111111111 DentalSchool,KNUST,Kumasi,Ghana,6 DepartmentofPathology,UniversityofGhanaMedicalSchool,
Accra,Ghana
a1111111111
a1111111111
*[email protected]
a1111111111
a1111111111
Abstract
OPENACCESS
Background
Citation:Owusu-AfriyieO,OwireduWKBA,Owusu-
DanquahK,Larsen-ReindorfR,DonkorP, Headandneckcancersincludecarcinomasoftheoralcavity,larynx,sinonasaltractand
AcheampongE,etal.(2018)Expressionof
nasopharynx.StudiesonmolecularexpressionofprognostictumourmarkersinGhanaare
immunohistochemicalmarkersinnon-
scarce.Thepurposeofthisstudywastodeterminetheexpressionofp53,p16,EGFR,
oropharyngealheadandnecksquamouscell
carcinomainGhana.PLoSONE13(8):e0202790. Cyclin-D1andHER2amongpatientswithnon-oropharyngealheadandnecksquamouscell
https://doi.org/10.1371/journal.pone.0202790 carcinoma(HNSCC).
Editor:AamirAhmad,UniversityofSouthAlabama
MitchellCancerInstitute,UNITEDSTATES
Methodology
Received:August24,2017
Accepted:August9,2018 Tissuemicroarraysfrom154histologicallyconfirmednon-oropharyngealHNSCCatthe
KomfoAnokyeTeachingHospitalfrom2006–2014wereconstructedusingduplicatecores
Published:August23,2018
ofrepresentativeandviableareasfromtumours.ExpressionofEGFR,p53,p16,Cyclin-D1
Copyright:©2018Owusu-Afriyieetal.Thisisan
andHER2wasevaluatedusingimmunohistochemistry.
openaccessarticledistributedunderthetermsof
theCreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal Results
authorandsourcearecredited.
Fornon-oropharyngealHNSCC,majorityofthecases(66.2%;102/154)hadstageIVdis-
DataAvailabilityStatement:Allrelevantdataare
ease.EGFRwasthemostexpressedmolecularmarker(29.4%;25/85)followedbyp53
withinthepaperanditsSupportingInformation
files. (24.0%;29/121),p16(18.3%;23/126)andCyclin-D1(10.0%;12/120).HER2wasnot
expressedinanyofthecases.Therewasasignificantly(p=0.022)higherexpressionof
Funding:Theauthorsreceivednospecificfunding
forthiswork. Cyclin-D1intumoursoftheoralcavity(19.6%;9/46)thaninthoseofthelarynx(4.7%;2/43)
andnose(3.2%;1/31).TumoursinstagesI–IIIweremorefrequentlypositiveforp16
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. (28.6%;12/42)thantumoursinstageIV(13.1%;11/84).
PLOSONE|https://doi.org/10.1371/journal.pone.0202790 August23,2018 1/13
Expressionofimmunohistochemicalmarkersinnon-oropharyngealheadandnecksquamouscellcarcinoma
Conclusion
Expressionofp53,EGFR,p16andCyclin-D1innon-oropharyngealHNSCCinGhanais
largelysimilartowhathasbeenreportedinpublishedstudiesfromothercountries.
Introduction
Globally,headandneckcancers(HNCs)areamongthetoptenmalignancies[1].HNCs
includecarcinomasofthelarynx,oralcavity,nasopharynxandsinonasaltract[2]andmajority
ofHNCsaresquamouscellcarcinomas(SCC).Theincidenceofheadandnecksquamouscell
carcinoma(HNSCC)differwidelyinAfrica,from0.8per100,000inGhanato11.1per
100,000inSouthAfrica[3,4].Moreover,previousstudiesinGhanahavealsoshownthat
tumoursofthepharynx,larynxandoralcavityformedthelargestgroupofHNCsandmostof
thepatientspresentedwithlate(stageIV)disease[5,6].Moleculartargetedtherapyseeksto
blocktumourgrowthandspreadbyinterferingwithmolecularcarcinogenicpathways[7].
Numerousgenesincludingtumoursuppressorslikecyclin-dependentkinaseinhibitors,TP53
andoncogeneslikecyclinfamily,epidermalgrowthfactorreceptor(EGFR)andrasgeneshave
beenimplicatedasplayingaroleinHNCs[8,9].
Mutationinp53gene(TP53)accountsfor45–70%ofHNSCC,andstrategiestargeting
TP53geneandp53proteinmaystoporreversetheprocessoftumorigenesis[10].Thepredic-
tivevalueofHumanpapillomavirus(HPV)innon-oropharynxHNCsisprincipallyunstud-
ied,possibleduetothefarlowerdetectionratesofHPV-positivetumoursatthesesites[11].
HigherexpressionofEGFRinoralsquamouscellcarcinomashavealsobeencorrelatedwith
largertumoursandlatestage,andhence,apoorprognosis[12,13].Inlaryngealsquamouscell
carcinoma,anassociationofCyclin-D1overexpressionwithnodalmetastasesandlatestaged
diseasehasbeenreported[14–16].TheroleoftheHER2inHNCsisnotwelldefined.More-
over,itisaproto-oncogene,withhomologytotheEGFRsequenceanditsoverexpressionhas
beenobservedinmultipletypesofcancerandusedintreatmentmeasuresagainstcancer[17].
Basedonmolecularstudies,moleculartargetedtherapiesareadvancinggloballywhichseeks
toblocktumourgrowthandspreadbyinterferingwithmolecularcarcinogenicpathwaysin
headandneckcancer.However,studiesonmolecularexpressionoftheseprognostictumour
markersinGhanaarescarce.Totheauthorbestknowledge,thereisnopublishedstudyon
expressionofmolecularmarkersinheadandneckcancersamongGhanaians.Duetothelack
ofmolecularstudies,tumourstage,patient’sageandperformancestatusstillremainthebasis
fortreatmentsdecisions.Therefore,identificationofappropriatemarkersthatcouldbe
employedasprognosticindicesofthedisease,andtheidentificationofpotentialcandidates
formoleculartargetedtherapywouldaidindevelopingmoresuitableandeffectivetreatment
strategiesfornon-oropharyngealsquamouscellcarcinoma.Theobjectiveofthisstudyisto
investigatetheexpressionofp53,p16,EGFRandCyclin-D1amongpatientswithnon-oropha-
ryngealsquamouscellcarcinoma(HNSCC).
Materialsandmethods
Studydesignandsetting
Thestudywasaretrospectivecaseseriesusingarchivalpathologicalspecimenstodetermine
themolecularcharacteristicsofhistologicallyconfirmedinvasiveSCCofnon-oropharyngeal
(oralcavity,larynx,sinonasaltractandnasopharynx)tissue.ItwasconductedattheKomfo
PLOSONE|https://doi.org/10.1371/journal.pone.0202790 August23,2018 2/13
Expressionofimmunohistochemicalmarkersinnon-oropharyngealheadandnecksquamouscellcarcinoma
AnokyeTeachingHospital(KATH)andKNUST,Kumasi,Ghana.TheKATHOncologyCen-
teristhesecondofonlytwonationalcancercentresinGhanaandthereferraldestinationof
allcancercasesseenatthehospital.Generally,cancerpatientsarereferredforneo-adjuvant
chemotherapy,adjuvantchemotherapy,concurrentchemo-radiationorradiotherapy.After
treatment,allpatientsarefollowedupatthecentre.Thecancercentrehasaclinic-basedregis-
try,whichstartedin2004.Ontheaverageabout400cancercasesareseeneveryyear.
Ethicalissues
ThestudyprotocolwasregisteredbytheResearchandDevelopmentunitoftheKomfoAno-
kyeTeachingHospital,Kumasi(RD/CR12/130)andapprovedbytheCommitteeonHuman
Research,PublicationandEthicsoftheSchoolofMedicalSciences,KwameNkrumahUniver-
sityofScienceandTechnology,Kumasi(CHRPE/AP/353/14).Participantsprovidedinformed
consentandtheiranonymitywaspreserved.Duringthewholeprojectperiod,eachpatientwas
encryptedandgivenanID-number.Aftercompletionofthestudy,allpatientinformationcol-
lectedinconnectionwiththestudywasstoredintheKwameNkrumahUniversityofScience
andTechnologyarchives.
Samplingpopulationandsize
Aconveniencesamplingof154formalin-fixedparaffin-embeddedblockswereretrievedfrom
hospitalarchivedpathologicalspecimensandthesespecimenswerecollectedbeforetreatment.
CasesubjectswereconsecutivepatientswhohadbeendiagnosedwithHNSCCatEye,ENT,
andOralMaxillofacialclinicsoftheKomfoAnokyeTeachingHospitalfromJune2006toMay
2014andreceivedtreatmentattheOncologyCentreofthehospital.
Datacollectiontools
Datawerecollectedusingapre-formatteddatacollectionform.Foreachcase,thecorrespond-
inginformationonage,gender,tumoursize,histologicaltype,tumourgrade,tumourstage,
nodesandmetastaseswerecollectedfromclinicalrecordfolders.Theclinico-pathologicalfea-
tureswereclassifiedaccordingtotheTNMsystem[18]
TwoPathologistreviewedthehistologyofallcasesofHNSCCusedinthisstudy.High-
throughputmolecularpathologywasdonusingaconstructedtissuemicroarrays(TMAs).The
mostrepresentativeareasofviabletumourcells(mostaggressiveinvasivefront)werecarefully
selectedandmarkedonthehematoxylinandeosin(HE)slidesforthematchingdonorblocks
andsampledforthetissuemicroarraycollectorblocks.TheTMAswereassembledusingatis-
sue-arrayinginstrument(BeecherInstruments).Briefly,a0.6mm-diameterneedlewasusedto
harvestthemarkedtissueareasfromthecorrespondingformalin-fixed,paraffin-embedded
tissueblocks.Weusedtwo0.6-mmcoresofviabletumourtissueeachfromdifferentareasthat
weremostrepresentative,afteralloriginalsectionsofthetumourhavebeenreviewedand
takenintoconsiderationtheheterogeneityofthetumour.Twelve(12)arrayblockswerecon-
structedtoincludeallcoresamples.Thisisbecausesinglesectionmightnotbeenoughrepre-
sentationoftheexpressionofamarkerthroughoutthewholetumourtissue,astumourcells
areregardedasheterogeneous.Multiple4-μmsectionswerecutwithaMicronmicrotome
(HM355S)andstainedwithspecificantibodiesforimmunohistochemistry(IHC)[19–21].
Deparaffinizingandrehydrating
Slideswerepre-treatedonaheatblockfor15–20seconds.Owingtothetranslationalnatureof
thisstudy,onlymanualIHCstainingprocedureswerefollowedasmuchaspossible,and
PLOSONE|https://doi.org/10.1371/journal.pone.0202790 August23,2018 3/13
Expressionofimmunohistochemicalmarkersinnon-oropharyngealheadandnecksquamouscellcarcinoma
performedonstandardwholetissuesectionsfromparaffin-embeddedarchivaltissue.Inorder
toremovetheparaffinwaxfromthetissueandallowwater-solubledyestopenetratethesec-
tions,allslidesweredeparaffinizedandrehydratedingradedxylene/ethanolbathsbeforedif-
ferentialantigenretrieval.Briefly,allslideswerewashedinthreechangesofxylenefor3
minutesbeforeabsoluteethanolandintwochangesofethanolfor3minuteswithconcentra-
tions95%and70%respectively.Slideswerethenrinsedunderrunningwaterandrinsedin
phosphate-bufferedsaline(PBS)for2x5minutes.SlideswereheldinPBSuntilreadytoper-
formantigenretrievaltopreventdehydration.
Antigenretrieval,enzymeblockingandstaining
Heat-inducedepitoperetrievalwasperformed.Allslidesandretrievalbuffer(CitrateBuffer
pH6.0)wereheldinCoplinjarsinawaterbathsetto92˚Candincubatedfor25minutes.
Slideswerethentakenoutofjarsandallowedtocoolonthebenchfor20minutesbeforewash-
inginPBSforanother5minutes.
Anin-housestainingprotocol,whichhasbeenoptimizedforeachoftheantibodies,was
usedasdescribed.Toavoidtissuedryingleadingtonon-specificbindingandultimatelyhigh
backgroundstaining,allincubationsweredoneoutinahumidifiedchamber.Sectionswere
washedinperoxidaseblockingsolution(3%hydrogenperoxide/caseininPBS)atroomtem-
peraturefor10minutestoblockoffendogenousperoxidasesandrinsedinPBSfor5minutes
withgentleagitationpriortoprimaryantibodyincubationtopreventtissuedestructionandto
eliminatefalse-positivereactionsproducedinthisway.Afterwardsslideswerewashedin
serumblockingsolution(10%horseserumincasein)for30minutesatroomtemperatureand
washedwithinPBSsolution.Usually,noserumblockingisneededifantibodydiluent(eg.
casein)isused.Thistoeffectivelyminimizenon-specificstainingastheprotein(casein)used
asablockcannotberecognizedbyanyofthesubsequentantibodiesandcansaturateandneu-
tralizechargedsitesthusenablingtheprimaryantibodytobindtotheantigenicsiteonly.Sec-
tionswerethenincubatedinprimaryantibodyatappropriatedilutioninPBSwithcaseinfor1
houratroomtemperatureorovernight.SectionswerethenrinsedinPBSfor3x5min.Hereaf-
ter,sectionswereincubatedinbiotinylatedsecondaryantibodyinPBSfor30minutesatroom
temperatureandthenrinsedinPBSfor3x5min.Next,sectionswereincubatedinABC-Perox-
idasesolutionfor30minutesatroomtemperatureandthenrinsedinPBSfor3x5min.Fur-
ther,sectionswereincubatedinperoxidasesubstratesolution(DAB(Sigma-Aldrich,
Darmstadt,Germany)for30minutesatroomtemperatureandthenrinsedindouble-distilled
waterfor5min.Acounterstain,hemotoxylinwasthenappliedtoallsectionsandrinsedin
double-distilledwaterfor2–5minutes.Finally,thesectionsweredehydratedbywashingin
95%ethanolfor1minute,100%ethanolfor2x3min,clearedinxylenefor2x5minandcover-
slipwithmountingmedium.Anegativecontrolwheretheprimaryantibodywasomittedwas
includedforallantibodiesusedandshowednostaininginallcasesofIHCstaining[22].
Scoring
Allstainedsectionswereexaminedbythepathologistswithoutknowledgeofclinicaloutcome.
Thetwoblindedpathologistsscoredindependentlyandsemi-quantitativelyviablepartsof
eachanonymizedcorebylightmicroscopy.Theextentandlocationofimmunohistochemical
stainingforindividualmolecularmarkerswereassessedusingtheimmunoreactivescore(IRS)
toevaluatetheproportionofcellsexpressingmolecularmarkerofinterestandtheintensityof
staining.Thescoringwassemi-quantitativeaccordingtoextensivelyusedprotocols[23,24].
Percentageofthecancerareastainedinhigh-powerfieldswasexamined.Stainingintensityof
epithelialtumourcellswasscoredasfollows:0(negative);1(weak);2(intermediate);and3
PLOSONE|https://doi.org/10.1371/journal.pone.0202790 August23,2018 4/13
Expressionofimmunohistochemicalmarkersinnon-oropharyngealheadandnecksquamouscellcarcinoma
(strong).Percentageofpositivecellsexaminedwasscoredas0(negative),1(<10%),2(11–
50%),3(51–80%),and4(>80%).ThetwoscoresweremultipliedandtheIRS(valuesfrom
0–12)wasdetermined:0–3asnegative,values4–6aspositive,andmultiplicationvalues8,9,
12asstronglypositive[25].Acorewasscoredasmissingifthecorewasmissingorconsidered
tobeofinadequatequalitytoscorebybothobservers.Consequently,allreportedmarker
expressionwerebasedontheevaluationofthreedistincttissuecores[26,27].
Statisticalanalysis
DatawereenteredandanalysedusingSPSS.Exploratoryanalyseswasdonetoobtaindescrip-
tivestatistics.Correlationsbetweenthebiologicalmarkersandthedifferentcategoricalvari-
ableswereassessedwithPearson‘schi-squaretest.Associationswithp<0.05wereconsidered
significant.
Results
Theageofpatientsrangedfrom15–98yearswithmean59.1years.Onehundredandeight
(70.1%)ofthepatientsweremales.Thecommonestprimarysitewasthelarynx(41.3%)fol-
lowedbyoralcavity(36.1%).Thehistologicalgradeofthetumourswasmostlymoderatelydif-
ferentiatedorpoorlydifferentiatedgrade.Morethanhalfofthepatients(66.2%)hadclinical
stageIVand38.3%hadnonodalinvolvement.Twelvehaddistantmetastases[Table1].
Table2showsthefrequencydistributionofexpressionofthemolecularmarkers.Testing
wasunsuccessfulin18.2to43.5percentofallcases.Intheremainingcases,moderatelyor
stronglypositiveexpressionvariedfrom1%forHER2,10%forCyclin-D1and18.3%forp16
to48.3%forEGFR.
Figs1and2showsthemicrographsoftheimmunochemistryforthepositiveexpressionof
p16andp53.
Amongthe87patientsthatweresuccessfullytested,(42;48.3%)showedstrongormoderate
expressionofEGFR.TherewasnostatisticallysignificantassociationofEGFRexpressionwith
age,gender,orclinico-pathologicalfeatures.Therewasasignificantly(p=0.022)higher
expressionofCyclin-D1intumoursoftheoralcavity(19.6%)thaninthoseofthelarynx
(4.7%)andnose(3.2%),butnosignificantcorrelationofexpressionofCyclin-D1expression
withage,gender,orclinico-pathologicalfeatureswasnoted.TumoursinstagesI-IIIwere
morefrequentlypositiveforp16(28.6%)thantumoursinstageIV(13.1%),butthedifference
wasonlyofborderlinestatisticalsignificance(p=0.049).Therewasnostatisticallysignificant
associationofp16expressionwithage,gender,orclinico-pathologicalfeatures.Nosignificant
associationbetweenp53expressionandage,gender,orclinico-pathologicalfeatureswas
observed[Table3].
AsshowninTable4,amongthe72successfultestedcases,(11;15.1%)showedpositiveco-
expressionofEGFR+p53,8(11.4%)showedtwo-markerpositiveco-expression(EGFR+P16),
6(8.5%)alsoshowedtwo-markerco-expressionofEGFR+Cyclin-D1and3(4.7%)co-expres-
sionofallthreemarkers(EGFR+p53+p16).
Discussion
Headandnecksquamouscellcarcinomas(HNSCCs)arethoughttobedevelopedandtoprog-
ressthroughaseriesofgeneticalterations,suchasthosecomprisingTP53andCDKN2A.
HNSCCsalsoshownumerousabnormalitiesinthechromosomesincludingamplificationsof
region11q13,whichcontainsthecyclin-D1gene,andregion7p11,whichencodesepidermal
growthfactorreceptor(EGFR)[28].
PLOSONE|https://doi.org/10.1371/journal.pone.0202790 August23,2018 5/13
Expressionofimmunohistochemicalmarkersinnon-oropharyngealheadandnecksquamouscellcarcinoma
Table1. Age,genderandclinico-pathologicalcharacteristicsofthepatients(n=154).
Variables Frequency(n) Percentages(%)
Age(years)(Mean±SD) 59.1±16.3
AgeGroups(years)
<40 22 14.3%
41–60 56 36.4%
>60 76 49.3%
Gender
Male 108 70.1%
Female 46 29.9%
ClinicalStage
I 3 2.0%
II 16 10.4%
III 33 21.4%
IV 102 66.2%
HistologicalGrade
Welldifferentiated(G1) 17 11.0%
Moderatelydifferentiated(G2) 74 48.1%
Poorlydifferentiated(G3) 63 40.9%
Tumorgrade
T1 6 3.9%
T2 41 26.6%
T3 38 24.8%
T4 69 44.8%
NodalInvolvement
N0 59 38.3%
N1 29 18.8%
N2 42 27.3%
N3 24 15.6%
Metastasis
M0 142 92.2%
M1 12 7.8%
TumorSite
Nasal 34 22.1%
Larynx 64 41.6%
OralCavity 56 36.3%
SD=StandardDeviation
https://doi.org/10.1371/journal.pone.0202790.t001
Inourstudy,EGFRwasthemostexpressedmolecularmarker(29.4%)followedbyp53
(24.0%),p16(18.3%)andCyclin-D1(10.0%)respectively.Cyclin-D1wassignificantly
expressedintumoursoftheoralcavity(19.6%)comparedtothoseofthelarynx(4.7%)and
nose(3.2%).Positiveexpressionofp16(28.6%)washigherinstagesI-IIItumourscompared
totumoursinstageIV(13.1%).Non-oropharyngealHNSCCinourstudyconsistedofcancers
ofthelarynx,sinonasaltract,oralcavityandnasopharynx.Abouthalfofallpatientswereat
least60yearsand70.3%weremales.InastudybyRiveraetal.,[29]amongChileans,themean
agewasintheseventhdecadeforpatientswithoralcavitysquamouscellcarcinoma(OCSCC)
andpredominantlyaffectedmaleswhichissimilartoourcurrentfindingsandotherprevious
reportsforOCSCCfromSriLankaandBrazil[30,31].Theresultsofthepresentstudyalso
PLOSONE|https://doi.org/10.1371/journal.pone.0202790 August23,2018 6/13
Expressionofimmunohistochemicalmarkersinnon-oropharyngealheadandnecksquamouscellcarcinoma
Table2. Frequencydistributionofmolecularmarkersexpressionofnon-oropharyngealHNSCCpatients
(n=154).
Variables Frequency(n) Percentages(%)
p16
Positive 23 18.3%
Negative 103 81.7%
InvalidResults 28
p53
Positive 29 24.0%
Negative 92 76.0%
InvalidResults 33
EGFR
Stronglypositive 25 28.7%
ModeratelyPositive 17 19.5%
Negative 45 51.7%
InvalidResults 67
HER2
Stronglypositive 0 0.0%
ModeratelyPositive 1 1.0%
Negative 96 99.0%
InvalidResults 57
Cyclin-D1
Positive 12 10.0%
Negative 108 90.0%
InvalidResults 34
https://doi.org/10.1371/journal.pone.0202790.t002
showedthatmorethanhalfofthepatientshadclinicalstageIVwhichisconsistentwithstudies
byRiveraetal.,[29]andRodriguesetal.,fromBrazil.
EGFRwaspositivein48.3%ofthesuccessfultestedpatientsinthisstudy.Thisfindingis
similartoreportfromstudydonebyAngetalwhodemonstratedthatincreasedaggressiveness
ofthecollectivegroupoftumourswithexpressionscoresabovethemedian(50%).Dragomir
etal.,reportedthatEGFRexpressiondidnotcorrelatewiththedegreeofdifferentiationofthe
OCSCC,butwashigherexpressedinwellandmoderatelydifferentiatedtumourswhichiscon-
sistentwithourfindings[32].TheimportanceofEGFRasahigh-riskindicatorforprogressive
diseaseorpoorprognosishasbeendemonstratedinheadandneckcancers[33,34].
Theresultofthestudyfoundp53expressionin24.4%ofnon-oropharyngealHNSCC,
whichislowerthan64%reportedNylanderetal.,[35]butsimilartofindingsfromprevious
studies[36,37].Shirakietal.,[38]observedstatisticallyinsignificantassociationbetween
Cyclin-D1orp53expressionandanyclinico-pathologicalcharacteristicsintheoralcancer
patient.InpatientswithLSCC,p53expressiondoesnotseemtobeassociatedwithbiological
features.[39]andthisconfirmsfindingsfromourpresentstudy.
ProteinexpressionlevelsofCyclin-D1arerelatedtothesiteofthetumour,depthoftumour
invasionandstageofthedisease.[40,41].Inaddition,Krecickietal.,[42]foundthatCyclin-
D1wasprincipallyoverexpressedinlocallyadvancedtumours(T3–T4).Inotherstudies,cor-
relationwasdetectedbetweenoverexpressionofCyclin-D1andadvancedstageofdiseaseand
nodalmetastases[14–16].Withtheexceptionoftumoursite,noassociationwasobservedwith
nodalmetastasesandCyclin-D1expressioninourcurrentstudy.Wefoundexpressionof
Cyclin-D1in10%ofthepatientswhereasotherstudiesreportedhigherexpression[14,43].
PLOSONE|https://doi.org/10.1371/journal.pone.0202790 August23,2018 7/13
Expressionofimmunohistochemicalmarkersinnon-oropharyngealheadandnecksquamouscellcarcinoma
Fig1.Microphotographoftheimmunohistochemistryforp16positive.Fig1showsmicrophotographofthe
immunohistochemistryforpositiveexpressionofp16.
https://doi.org/10.1371/journal.pone.0202790.g001
Shirakietal.,indicatedthatitisonlythecombinationofpositiveexpressionofEGFR,p53,
andCyclin-D1thatpredictspoorprognosis[29].Theresultsfoundthatpositiveco-expression
ofallthreemarkers(EGFR+p53+p16)wasobservedin3(4.7%).Suchco-expressionofmany
Fig2.Microphotographoftheimmunohistochemistryforp53positive.Fig2showsthemicrophotographofthe
immunohistochemistryforpositiveexpressionofp53.
https://doi.org/10.1371/journal.pone.0202790.g002
PLOSONE|https://doi.org/10.1371/journal.pone.0202790 August23,2018 8/13
Expressionofimmunohistochemicalmarkersinnon-oropharyngealheadandnecksquamouscellcarcinoma
Table3. Associationbetweenage,gender,andclinico-pathologicalfeaturesandtheexpressionofmolecularmarkersamongnon-oropharyngealHNSCCpatients.
EGFR Cyclin-D1 p16 p53
Variables n Positive(%) n Positive(%) n Positive(%) n Positive(%)
Age(years)
(cid:20)60 40 21(52.5%) 65 7(10.8%) 66 1218.2%) 65 14(21.5%)
>60 47 21(44.7%) 55 5(9.1%) 60 11(18.3%) 56 15(26.8%)
Gender
Male 64 31(48.4%) 82 10(12.2%) 86 17(19.8%) 82 21(25.6%)
Female 23 11(47.8%) 38 2(5.3%) 40 6(15.0%) 39 8(20.8%)
Tumoursite
Nasal 22 8(36.4%) 31 1(3.2%)(cid:3) 31 3(9.7%) 30 8(26.7%)
Larynx 37 20(54.1%) 43 2(4.7%) 51 12(23.5%) 44 9(20.5%)
Oralcavity 28 28(50.0%) 46 9(19.6%) 44 8(18.2%) 47 12(25.5%)
TumourStage
I-III 31 14(45.2%) 41 5(12.2%) 42 12(28.6%)(cid:3) 41 10(24.4%)
IV 56 28(50.0%) 79 7(8.9%) 84 11(13.1%) 80 19(23.8%)
Tumourgrade
G3 41 18(43.9%) 48 4(8.3%) 50 7(14.0%) 51 10(19.6%)
G2 35 19(54.3%) 59 7(11.9%) 62 15(24.2%) 57 15(26.3%)
G1 11 5(45.5%) 13 1(7.7%) 14 1(7.1%) 13 4(30.8%)
Tumoursize
T1-T3 49 25(51.0%) 67 6(9.0%) 70 17(24.3%) 65 14(21.5%)
T4 38 17(44.7%) 53 6(1.3%) 56 6(10.7%) 56 15(26.8%)
Nodalinvolvement
N0/N1 49 19(38.8%) 79 7(8.9%) 72 15(20.8%) 69 14(20.3%)
N2/N3 38 23(60.5%) 41 5(12.2%) 54 8(9.3%) 52 15(28.8%)
Metastasis
M0 77 38(49.4%) 111 11(9.9%) 116 22(18.9%) 112 28(25.0%)
M1 10 4(40.0%) 9 1(11.1%) 10 1(10.0%) 9 1(11.1%)
n=numberofsuccessfullytestedpatients
(cid:3)p<0.05
https://doi.org/10.1371/journal.pone.0202790.t003
molecularmarkersagreesrelativelywellwiththefundamentalconceptofmultistepdevelop-
mentandprogressionofcancer[29].
Thisstudyhasarelativelylargeproportionofmissingcasesduetoinvalidresultsofthe
immunohistochemicalstaining,largelybecauseofmissingtissueintheTMAorontheslide.
Despiteusingplusglass,sectionsfromthesmallcoresintheTMAmayloosenfromtheslide
intheprocessofimmunohistochemicalstaining.However,acomparisonofthedistributions
ofage,gender,andclinico-pathologicalvariablesofthetotalmaterialinTable1withthoseof
thesuccessfullytestedcasesinTable3shownoobvioussignificantdifference,indicatingthat
thesuccessfullytestedcasesmaybeareasonablyrepresentativesampleofthetotalmaterial.
TwoofthecomparisonsshowninTable3showeddifferencesthatwerestatisticallysignificant
atthedefinedlevelofp<0.05;thedifferenceinCyclin-D1expressionintumoursofdifferentsites
(p=0.022),andthedifferenceinp16expressionindifferentstages(p=0.049).However,asin
thisstudywithover30statisticallytesteddifferences,thisisnotmorethancanbeexpectedto
occurbypurechanceintestingthisnumberofrandomsamplesfrompopulationsbetweenwhich
norealdifferenceexists.Thus,asawhole,ourstudyhasnotdemonstratedsignificantassociations
PLOSONE|https://doi.org/10.1371/journal.pone.0202790 August23,2018 9/13
Expressionofimmunohistochemicalmarkersinnon-oropharyngealheadandnecksquamouscellcarcinoma
Table4. CombinedpositivityofEGFRandothermarkers.
Marker Successfullytestedcases Positive(%)
EGFR 87 42(48.3%)
EGFR+p53 72 11(15.1%)
EGFR+p16 70 8(11.4%)
EGFR+p53+p16 64 3(4.7%)
EGFR+Cyclin–D1 71 6(8.5%)
EGFR+p53+p16+Cyclin–D1 60 -
https://doi.org/10.1371/journal.pone.0202790.t004
betweentheexpressionofEGFR,Cyclin-D1,p16,orp53andage,gender,tumoursite,stage,
grade,size,nodalinvolvement,ormetastasisinpatientswithnon-oropharyngealHNSCC.More-
over,thestudywasconductedwithsmallsamplesizeatasinglehospital;theresultsobtained
fromthestudycannotbegeneralizedtothepopulation.
Conclusion
Expressionofp53,EGFR,p16andCyclin-D1innon-oropharyngealHNSCCinGhanais
largelysimilartowhathasbeenreportedinpublishedstudiesfromothercountries.Further
prospectivestudiesarewarrantedtodeterminetheoutcomeofexpressionofthesemarkerson
theoverallsurvivalinnon-oropharyngealHNSCCpatients.
Supportinginformation
S1Dataset.Datasetonwhichconclusionsofthismanuscriptweremadehasbeensubmit-
tedassupportinginformation.
(XLSX)
Acknowledgments
GratitudegoestoworkersattherecordsunitandauthoritiesattheDepartmentofPathology,
KomfoAnokyeTeachingHospital,UniversityofGhanaMedicalSchool,andDepartmentof
MolecularMedicine,KNUST,Kumasi-Ghana.
AuthorContributions
Conceptualization:OseiOwusu-Afriyie.
Datacuration:OseiOwusu-Afriyie,RitaLarsen-Reindorf.
Fundingacquisition:OseiOwusu-Afriyie.
Investigation:OseiOwusu-Afriyie,SolomonE.Quayson.
Methodology:OseiOwusu-Afriyie.
Projectadministration:OseiOwusu-Afriyie,EmmanuelAcheampong.
Resources:OseiOwusu-Afriyie.
Software:OseiOwusu-Afriyie,EmmanuelAcheampong,SolomonE.Quayson.
Supervision:W.K.B.A.Owiredu,KwabenaOwusu-Danquah,RitaLarsen-Reindorf,Peter
Donkor.
Validation:PeterDonkor.
PLOSONE|https://doi.org/10.1371/journal.pone.0202790 August23,2018 10/13