www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Downregulation
of
Notch4
---
a
prognostic
marker
in
distinguishing
oral
verrucous
carcinoma
from
oral
squamous
cell
carcinoma
夽
M.K.
Harishankar
a,
A.
Mathan
Mohan
b,
A.
Vinod
Krishnan
b,
Arikketh
Devi
a,∗ aSRMUniversity,SchoolofBioengineering,DepartmentofGeneticEngineering,Kattankulathur,IndiabKarpagaVinayagaInstituteofMedicalandDentalSciences,DepartmentofOralandMaxillofacialSurgery,OralCancer Foundation,Kancheepuram,India
Received20July2017;accepted26September2017 Availableonline31October2017
KEYWORDS
Oralverrucous carcinoma; Oralsquamouscell carcinoma; Notch4;
Prognosticmarker
Abstract
Introduction:Oralverrucouscarcinomaisaspecialformofwell-differentiatedsquamouscell carcinoma which possesses specificclinical, morphologic andcytokinetic features that dif-fer from other types oforal cancers and hence diagnosis requires immense experience in histopathology. Hence itiscertainly importantto distinguish such alesion from otheroral tumorsastreatmentstrategiesvarywidelybetweenthem.
Objective: Insearchofacriticaldiagnosticmarkerindistinguishingoralverrucouscarcinoma fromoralsquamouscellcarcinoma,Notch4receptor,oneofthekeyregulatorymoleculesof theNotchsignaling familyhasbeenaberrantlyactivatedintheprogressionofseveraltypes oftumors. Howeverits functioninoral verrucous carcinomaremains unexplored. Thusthe presentstudyaimsindeterminingthedifferentialexpressionpatternofNotch4inoralverrucous carcinomaandoralsquamouscellcarcinoma.
夽 Pleasecitethisarticleas:HarishankarMK,MohanAM,KrishnanAV,DeviA.DownregulationofNotch4---aprognosticmarkerin
distin-guishingoralverrucouscarcinomafromoralsquamouscellcarcinoma.BrazJOtorhinolaryngol.2019;85:11---6.
∗Correspondingauthor.
E-mail:adevipradeep@gmail.com(A.Devi).
PeerReviewundertheresponsibilityofAssociac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial. https://doi.org/10.1016/j.bjorl.2017.09.005
1808-8694/©2017Associac¸˜aoBrasileiradeOtorrinolaringologiaeCirurgiaC´ervico-Facial.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
Methods:Tenpatientsreportedpositivefororalcancer(5patientswithoralverrucous carci-nomaand5patientswithoralsquamouscellcarcinoma).Fivenormaltissuesampleswerealso obtainedandevaluatedforclinicopathologicalparametersandimmunohistochemistry,western blottingandrealtimepolymerasechainreactionforNotch4expression.
Results:Ourresultsreveal thattheexpressionofNotch4wasconsiderablyhighinoral squa-mouscellcarcinomalesionscomparedtonormaltissue,whereasinoralverrucouscarcinoma, irrespectiveoftheclinicopathological features,complete regulac¸ão descendenteofNotch4 wasobserved.
Conclusions:ThesepreliminaryfindingsstronglysupportthefactthatNotch4isdownregulated inoralverrucouscarcinomaandcouldbeconsideredasasuitableprognostic markerin dis-tinguishingoralverrucouscarcinomafromoral squamouscellcarcinoma.This distinguishing marker canhelpinimproving therapeuticoptions inpatientsdiagnosedwithoral verrucous carcinoma.
© 2017 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). PALAVRAS-CHAVE Carcinomaverrucoso oral; Carcinomadecélulas escamosasoral; Notch4; Marcadorprognóstico
Regulac¸ãodescendentedeNotch4---ummarcadorprognósticonadistinc¸ãoentre carcinomaverrucosooralecarcinomadecélulasescamosasoral
Resumo
Introduc¸ão: Ocarcinomaverrucosodecavidadeoraléumaformaespecialdecarcinomade célulasescamosasbemdiferenciadaquetemcaracterísticasclínicas,morfológicase citocinéti-casespecíficas quediferemdeoutrostiposdecânceresorais.Poressarazão,odiagnóstico requergrandeexperiênciaemhistopatologia.Portanto,écertamenteimportantedistingui-lo deoutrostumoresorais,poisasrespectivasestratégiasdetratamentovariammuito.
Objetivo:Em busca de um marcadorde diagnóstico críticona distinc¸ãoentre ocarcinoma verrucosoeocarcinomadecélulasescamosasdecavidadeoral,oreceptorNotch4,umadas principaismoléculasreguladorasdafamíliadesinalizadoresNotch,foiativadodemaneira anor-malnaprogressãodeváriostiposdetumores.Noentanto,suafunc¸ãonocarcinomaverrucoso permaneceinexplorada.Assim,opresenteestudotemcomoobjetivodeterminaropadrãode expressãodiferencialdeNotch4nocarcinomaverrucosoedecélulasescamosasdecavidade oral.
Método: Dezpacientestiveramresultadopositivoparacânceroral(cincopacientescom car-cinomaverrucosoe cincopacientescomcarcinoma decélulasescamosas) ecincoamostras normaisforamtambémobtidas.Alémdaavaliac¸ãodosparâmetrosclínico-patológicos,foram feitosanáliseimuno-histoquímica,WesternBlotereac¸ãodepolimeraseemcadeiaemtempo realparaaexpressãodeNotch4.
Resultados: NossosresultadosrevelamqueaexpressãodeNotch4foiconsideravelmentealta emcarcinomasdecélulasescamosasemcomparac¸ãocomostecidosnormais,enquantoqueno carcinomaverrucoso,independentementedascaracterísticasclínico-patológicas,observou-se
regulac¸ãodescendentecompletadeNotch4.
Conclusão:EssesachadospreliminaresapoiamfortementeofatodequeNotch4estava regu-ladoparabaixonocarcinomaverrucosooralepoderiaserconsideradoummarcadorprognóstico adequadoparadistinguirentrecarcinomaverrucosoecarcinomadecélulasescamosasde cavi-dadeoral.Essemarcadordistintivopodeajudaramelhorarasopc¸õesterapêuticasempacientes comdiagnósticodecarcinomaverrucosooral.
© 2017 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Publicado por Elsevier Editora Ltda. Este ´e um artigo Open Access sob uma licenc¸a CC BY (http:// creativecommons.org/licenses/by/4.0/).
Introduction
Oral Verrucous Carcinoma (OVC),typically representing a rarevariantofwelldifferentiatedsquamouscellcarcinoma isconsideredtobeanon-invasiveformoftumorwithspecific
clinical,morphologicandcytokineticfeatures.1,2Ingeneral, lymphnodeanddistantmetastasisarerareinOVCbutthe large size and involvement of bone structures,renders it locallyaggressiveifnottreatedproperly.3ThoughOVChas uniquepathologicalcharacteristics,it’sverychallengingfor
thepathologisttodifferentiateamongoralcancersubtypes becauseaccuratediagnosis requires adequatetumor sam-ples aswellasexperienced clinicians.4 Treatment of OVC stillremainscontroversialbecauseoftheirextensivenature mimicking an invasive cancer and hence identification of a critical diagnostic marker which could discriminate the componentsofOralSquamousCellCarcinoma(OSCC)from OVCiscrucialtoevaluatetheclinicalsignificanceofOVC.5 Therefore, the need of the hour is to identify a definite markerforOVCwhichcouldbeusedeffectivelytodiagnose andtreatOVC.
Notchsignalingpathwayisoneofthecelltocell commu-nicationssignalingpathways thatpromotesavastarrayof regulatory functions suchas cell proliferation, differenti-ationandapoptosis.6Theelevatedexpressionofthenotch signalingmolecules(Notch1---4receptors,Deltalike1,Delta like 3, Delta like 4, Jagged 1 and Jagged 2 ligands) has been considered to be one of the critical event in sev-eral malignancies.7---9 Importantly, accumulative evidence hasshownthatconstitutiveactivationof Notch4receptor, one of thekey receptormolecules of theNotch signaling familyhasbeenassociatedwithseveralcancer pathogene-sis.However,itsfunctionasoncogeneortumorsuppressor geneiscellcontextspecific.10,11
In our previous study, we found that Notch4 plays an importantroleinthepathobiologyofOSCCandhencethis studyhasbeentargetedtoanalyzetheexpressionofNotch4 amongOVCandOSCC.12 To achievethis,theexpressionof Notch4wasanalyzedonthetumorsectionsofOVCandOSCC withvariedclinicopathologicalparameters.Thepurposeof thestudywastodeterminethedifferentialexpression pat-ternofNotch4betweenthemajorsubtypesoforalcancer suchthatareliablediagnosticmarkercouldbeestablished fortheimprovedtreatmentofOVCpatients.
Materials
and
methods
Patientssamplewithclinicopathological
parameters
A total of 15 post-surgical oral cancer samples which includes5samplesreportedpositiveforOSCCand5samples withOVCwerecollectedalong with5normaloral mucosa samplesfromindividualswhounderwentsurgeryforbenign oral and maxillofacial conditionsfrom the Departmentof OralandMaxillofacialSurgery,KarpagaVinayagaInstituteof DentalSciences,India.Allthesamplesweredividedintotwo parts:onepartwasfixedin10%bufferedformaldehyde solu-tionandtheotherpartwasfrozenimmediatelyandstoredin −80◦Cuntiluse.Informationonthevariousclinical param-eters (gender, age, site of tumor and TNM staging) were obtainedfrommedicalrecords.Thestudywasapprovedby theInstitutionalEthicalCommittee(490/IEC/2013).
Immunohistochemicalanalysis
Immunohistochemicalanalysisfor thetumorsectionswere performed as previously described.12 Primary antibodies usedwereasfollows:Notch4(sc-8646),GAPDH(sc-47724). After incubating the samples with HRP conjugated sec-ondaryantibodies,theslideswereexamined underalight
microscopeandtheresultswerecategorizedashigh, mod-erateandmildandnegativeexpressionbasedonhighversus lowantigenexpression.Alltheantibodieswerepurchased fromSantaCruzBiotechnology,USA.
Immunoblotting
Westernblotanalysiswasperformedontotalproteins har-vestedfrom the tissue sectionsusing lysis buffer. Briefly, the separated proteins were transferred to nitrocellulose membrane(AmershamProtran,GEHealthcareLifeSciences, Germany)andblockedwith3%BSAin TrisBufferedSaline with0.1%Tween 20 (TBST)followed by overnight incuba-tion withanti-Notch4 (sc-8646) or anti-GAPDH (sc-47724) at4◦C.The membranewasincubated withsuitable horse radishperoxidase-labeledsecondaryantibodiesat37◦Cfor 1h.TheblotswerethendevelopedusingDAB(Sigma)asper manufacturer’s protocol. Densitometric analysis was per-formedontheblots.
RT-PCRanalysis
Total RNA was isolated from frozen tissue samples using Trizol (Merck) and quantified using the Nanodrop system (Nanodrop lite spectrophotometer, Thermo Sci-entific). cDNA was prepared from 1g of total RNA using M-MuLV reverse transcriptase (New England Bio-labs Inc.). Prepared cDNA was subsequently subjected to PCR amplification using the following primers: Notch4: forward: 5-CCACTAGGCGAGGACAGCATT-3; reverse: 5-CAACTCCATCCTCATCAACTTCTG-3;  actin: forward: 5-AGAGCTACGAGCTGCCTGAC-3; reverse: 5 -GGATGCCACAGGACTCCA-3. The amplified PCR products werevisualizedusingagarosegelelectrophoresisby ethid-iumbromidestaining.Allthesampleswerenormalizedwith actinusingdensitometricanalysis.
Statisticalanalysis
Statistical analysis of the expression of Notch4 between OVC and OSCC patients were analyzed by Student’s t-test using the Graph pad online software (www.graphpad.com/quickcalcs/ttest1). All the experi-mentswererepeatedthriceandthe statisticaltestswere performedatasignificantlevelofp<0.05.
Results
The clinicalcharacteristics of the OVCand OSCCpatients have been summarized in Table 1. The mean age of the OVCandOSCCgroupswere65.2±3.05yearsand63.8±5.19 years,respectively.Maletofemaleratioforboththegroups was4:1 and the primarysite of the tumors wasconfined tobuccal mucosa (3/5 in both the groups). Thus, all the majorclinicalparametersforboththegroupsselectedfor the study were almost identical. Although the etiological factorsofOVCstillremainscontroversial,inourstudyallthe patientswerereportedwithhistoryoftobaccousage(data notshown).On performingimmunolocalizationfor Notch4 ontumor sections it was visualized that the protein was
Table1 Clinicalparametersofsubtypesoforalcancer(OVCandOSCC)withtheexpressionofNotch4representedashigh, moderate,mildandnegativeexpressionbasedontheintensityoftheproteinsvisualizedbyimmunolocalizationexperiments (p<0.05wasconsideredstatisticallysignificant).
Sampleno Age(years) Sex Primarysiteoftumor Clinicaldiagnosis Notch4expression p-Value
OVC1 61 Male Buccalmucosa Verrucouscarcinoma Mild
OVC2 70 Male Alveolarmucosa Verrucouscarcinoma Mild
OVC3 64 Male Buccalmucosa Verrucouscarcinoma Negative
OVC4 57 Male Palate Verrucouscarcinoma Mild
OVC5 72 Female Buccalmucosa Verrucouscarcinoma Negative 0.003
OSCC1 68 Female Buccalmucosa Squamouscellcarcinoma High OSCC2 70 Male Alveolarmucosa Squamouscellcarcinoma Moderate OSCC3 52 Male Buccalmucosa Squamouscellcarcinoma Moderate
OSCC4 49 Male Ca-Tongue Squamouscellcarcinoma High
OSCC5 80 Male Buccalmucosa Squamouscellcarcinoma High
Figure1 ImmunohistochemicalexpressionofNotch4proteininspecimensofnormalmucosaandsubtypesoforalcancer(OVC andOSCC)(20×magnification).(A)ImmunoreactivityofNotch4innormalmucosaoftheoralcavity.(B)ImmunoreactivityofNotch4 inOVC.(C)ImmunoreactivityofNotch4inOSCC.
membranousand cytoplasmic and itsexpression was cer-tainlyhighinOSCCtumorscomparedtothatofthenormal mucosa whereas very low expression was observed in all thesamplesofOVCirrespectiveoftheclinicalparameters (Fig.1).Inaddition,westernblottingandRT-PCRdataalso showedthatNotch4wasabundantlypresentinOSCCwhile verypoor expression wasseen inOVC cases(Fig.2). Fur-ther,thedensitometricanalysisoftheblotsconfirmedthat Notch4wassignificantlydownregulatedinOVCsuggestingits importanceintheprognosisofthetumorsubtypes(Fig.3).
Discussion
Oral verrucous carcinomas are slow-growing, exophytic, well-demarcatedhyperkeratoticlesionsconsideredtobea rarevariantofsquamouscellcarcinomaswithanoccurrence rateof 2---12% amongall types of oral cancer.13 However, OSCCis a verycommon neoplasm of the oral cavity rep-resenting almost 90% of the tumors of the oral cavity.14 In general, OSCC is considered to be a more aggressive formof tumor,oftenleadingtometastasis whichishighly uncommon inOVC. In addition,thehistopathological fea-tureofOVCremainsdistinctfromthatofotherconventional carcinomas.15 Thoughdistinct,anaccurate histopathologi-caldiagnosis requiresa skillfulpathologist anda clinician
Notch4
1 0.118±0.0006
GAPDH
Figure2 Totalproteins isolatedfromOVCandOSCCtissue samples wereanalyzedby westernblottingusinganti-Notch4 antibody.TheblotsweredevelopedusingDAB,showingahigh expressionofNotch4inOSCCsampleswhereasverypoor expres-sionwasobserved inOVCsamples. Alltheexperiments were performedintriplicatesandthedatawerefurtheranalyzedby densitometrywithGAPDHservingasnormalizer.
witha sufficientbiopsy sample withdeepinfiltrating por-tions of lesions.16 Hence the optimal treatment for OVC stillremainscontroversialduetodifficultiesinappropriate classification of lesions and also the ability to mimic the invasive OSCC in its biologic behavior.17 However,a clear
1.2 1 0.8 0.6 0.4 0.2 0 OSCC OVC
Subtypes of oral cancer
F o ld activ a tion ∗∗∗ Notch4
Figure3 RT-PCRanalysisofNotch4indifferentsubtypesof oralcancer(OSCCandOVC)showingadownregulationofNotch4 inOVC samples.Densitometric analysis was performed using ImageJ 1.47vsoftware andthe valueswere normalized to  Actin(***p<0.001,Student’st-test).
identificationofthetumorsubtypesisofgreatimportance astreatmentstrategiesgreatlyvaryamongthetwogroups. Innumerable studies in the past have been performed in order to identify the active molecule involved in the pathogenesis of OVC. Mohtashametal. (2013) performed histochemical analysis of p53, Ki-67, MMP-2, MMP-9 and showedthattheseproteinscouldbeusedinidentifyingthe tumor invasive front that distinguishes OSCC from OVC.18 Similarly,severalproteinssuchasBcl-XRetinoblastoma(Rb) oncogeneand CyclinD1 werealsofound tohavea differ-entialexpression inOVC.19,20 HoweverOgawaetal.(2004) observednoobvious differencesinp53proteinexpression between VC and well-differentiated SCC in proliferative activityoftumorcells.16Hencethereliabilityofthese pro-teinmarkersisquestionableduetolackofuniformityinits expressionpatternamongindividuals.21 Thusthe differen-tialdiagnosisofOVCremainsdifficultandrequirescareful examinationofthetumors.
Notch signaling pathway, one of the key cell commu-nication pathways has an important role in maintaining thebalancebetweencellproliferation,differentiationand apoptosis.22 Apart from its role in regulating biological behavior of normal cells, members of the notch fam-ily (Notch1, Notch2, Notch3 and Notch4 receptors) has alsobeen reported toinduce severaltypes of cancer and might be considered as a potential therapeutic target in oncology.23,24 In ourprevious study we demonstrated that Notch4 was upregulated in the late stages of OSCC sug-gestingitasapotentialmetastaticmarker.12 However,its biological functionas oncogeneor tumor suppressorgene is purely based on cell context. For example, Clementz et al. (2011) and Nagamatsu et al. (2014) demonstrated independently the oncogenic role of Notch4 in promoting breastmalignancyandsuggesteditasapotential therapeu-ticoptionintreatingmetastaticpatients.10,25 FurtherDing etal. (2010) reportedthe upregulation of Notch4in Sali-varyAdenoidCyctic Carcinoma (SACC) andits keyrolein inducingSACCmetastasis.11SimilarlyNotch4hasbeen con-sideredasacandidatehistochemicalmarkerinidentifying hepatocellularcarcinoma.26 Whereasinsomecancertypes suchasrenalcellcarcinoma,downregulationofNotch4has beenreported.27Henceit’simpossibletogeneralizetherole of Notch4 in progression of cancer. Till now we know of
noreportsthat elucidate the tissue-specificexpression of Notch4in OVC andhence thecurrent study wasdesigned toidentifyitspotential rolein OVC pathogenesis.Results fromthepresent studycorrelatedwithourprevious study onthefactthatexpressionofNotch4washighinOSCC sam-ples.InterestinglycompletedownregulationofNotch4was observed in OVC patient samples confirming the cell and diseasecontext specific role of Notch4 withinthe closely relatedoral cancer subtypes.Togetherthisstudy provides newinsightinbringingoutamolecularapproachin differ-entiatingOVCandOSCC.
Conclusions
In summary, this study was majorly focused on deriving a key diagnostic marker that could be more specific for OVC.Hence ourfindingsconfirm that depletionof Notch4 expression in OVC tissue samples could be considered as a valuable prognosticmarker in differentiating OVC from OSCC,asOSCClesionsmaynotbedistinguishedclinicallyor maycoexistwithOVC.Althoughourresultsstronglysupports thesignificanceof Notch4 asa reliableprognosticmarker forOVCstillelaboratestudiesmustbeperformedinorder toderivetheregulatoryfactorsthattriggersthe downregu-lationofNotch4suchthatanoveltherapeuticapproachfor OVCcouldbeattained.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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