Braz. j. . vol.80 número4

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Brazilian

Journal

of

OTORHINOLARYNGOLOGY

www.bjorl.org

ORIGINAL

ARTICLE

Histologic

correlation

of

expression

of

Ki-67

in

squamous

cell

carcinoma

of

the

glottis

according

to

the

degree

of

cell

differentiation

夽

Rodrigo

Gonzalez

Bonhin

a,∗

,

Guilherme

Machado

de

Carvalho

a

,

Alexandre

Caixeta

Guimarães

a

_,

_Carlos

_Takahiro

_Chone

a

_,

_Agrício

_Nubiato

_Crespo

a

_,

Albina

Messias

de

Almeida

Milani

Altemani

b

_,

_Eliane

_M.I.

_Amstalden

b

a_Discipline_of_{Otorhinolaryngology,}_Head_and_Neck,_Universidade_Estadual_de_Campinas_(UNICAMP),_Campinas,_SP,_Brazil b_Department_of_Pathology,_Universidade_Estadual_de_Campinas_(UNICAMP),_Campinas,_SP,_Brazil

Received9February2013;accepted11March2014 Availableonline11June2014

KEYWORDS Carcinoma, squamouscell; Glottis;

Biologicalmarkers; Cellproliferation; Headandneck neoplasms

Abstract

Introduction:Squamouscellcarcinomaisthemostcommonneoplasmofthelarynxandglottis, anditsprognosisdependsonthesizeofthelesion,leveloflocalinvasion,cervicallymphatic spread, andpresenceofdistantmetastases.Ki-67(MKI67) isaprotein presentinthecore, whosefunctionisrelatedtocellproliferation.

Aim:ToevaluatetheexpressionofmarkerKi-67insquamouscellcarcinomaofthelarynxand glottisanditscorrelationtopathologicalfindings.

Methods:ExperimentalstudywithimmunohistochemistryanalysisofKi-67,calculatingthe per-centageofthecellproliferationindexinglotticsquamouscellcarcinomas.

Results:Sixteencaseswereanalyzed,withsixwell-differentiatedand10poorly/moderately differentiatedtumors.Therewasacorrelationbetweencellproliferationindexanddegreeof celldifferentiation,withhigherproliferationinpoorly/moderatelydifferentiatedtumors. Conclusion:Thecellproliferationindex,asmeasuredbyKi-67,maybeusefulinthe character-izationofhistologicaldegreeinglotticsquamouscelltumors.

夽 _Please_cite_this_article_as:_Bonhin_RG,_de_Carvalho_GM,_Guimarães_AC,_Chone_CT,_Crespo_AN,_Altemani_AM,_et_al._Histologic_correlation

ofexpressionofKi-67insquamouscellcarcinomaoftheglottisaccordingtothedegreeofcelldifferentiation.BrazJOtorhinolaryngol. 2014;80:290---5.

∗_{Corresponding}_author.

E-mail:bonhin@yahoo.com(R.G.Bonhin). http://dx.doi.org/10.1016/j.bjorl.2014.05.016

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PALAVRAS-CHAVE Carcinomadecélulas escamosas;

Glote; Marcadores biológicos; Proliferac¸ãode células;

Neoplasiasdecabec¸a epescoc¸o

Correlac¸ãohistológicadaexpressãodoKi-67noCarcinomaEpidermoideGlóticode

acordocomgraudediferenciac¸ãocelular

Resumo

Introdução: O carcinoma de células escamosas é a neoplasia mais frequente da laringe e daregião glótica,seuprognósticodependedotamanhodalesão,doníveldeinvasãolocal, disseminaçãocervicallinfáticaedaexistênciademetástasesàdistância.Ki-67(MKI67)éuma proteínapresentenonúcleo,cujafunçãoestárelacionadacomaproliferaçãocelular. Objetivos: AvaliaraexpressãodomarcadorKi-67emcarcinomadecélulasescamosasdalaringe glóticosecorrelacionarsuaexpressãocomosachadosanatomopatológicos.

Método: TrabalhoexperimentaldeanáliseimunohistoquímicadoKi67atravésdocálculo per-centualdoíndicedeproliferaçãocelularemprodutosdecarcinomasepidermóidesglóticos. Resultados: Dezesseis casos foram analisados, sendo seis bem diferenciados e dez pouco/moderadamentediferenciados.Houvecorrelaçãoentreoíndicedeproliferação celu-lareograudediferenciaçãocelular,sendoaproliferaçãomaiornospouco/moderadamente diferenciados.

Conclusão:Oíndicedeproliferac¸ãocelular,medidopeloKi-67podeserútilnacaracterizac¸ão dograuhistológicoemtumoresglóticosdecélulasescamosas.

Introduction

Squamouscellcarcinomaisthemostcommonneoplasmof thelarynxandglotticregion,anditsprognosisdependson the size of the lesion, level of local invasion, lymphatic spread,andpresenceofdistantcervicalmetastases.1---5_The

behaviorofthesquamouscellcarcinomaismarkedbythe degree of cell proliferation and differentiation, and this indexcanbederivedbymeasuringKi-67.6---10

Histologically, the epidermoid or squamous cell carci-nomaintheseregionsissimilartotheothers.Itisdefined bytheneoplasticproliferationofsquamouscellsin differ-entdegreesofdifferentiationandtheirinfiltrativenature. Depending on the power of keratinization and cellular atypia,theyareclassifiedasfollows:well,moderately,or poorlydifferentiated.1

Thewell-differentiatedtypeischaracterizedbyits sim-ilarity with normal squamous epithelium, high power of keratinization,andverymildcellatypia;themoderately dif-ferentiatedtypecontainsdistinctnuclearpleomorphismand mitoticactivityispresent,includingatypicalfigures.Inthis type,keratinizationismuchlower.1,2_In_the_poorly

differen-tiatedtype,thereisapredominanceofimmaturecells,with numerousmitoticfiguresandvirtuallyabsentkeratinization. The levelofinvasionmaybelimitedtotheepithelium, respectingthebasallamina,whichiscalledinsitu.Whenit affectsonlythelaminapropria,itisconsidered microinva-siveorsuperficiallyinvasive.2

Asforthefranklyinvasivecarcinoma,itmanifestsasthe destruction of the basal lamina, clearly extending to the underlying tissues,possiblyaccompanied by stromal reac-tion. Perineural invasion and angiolymphatic invasion are signs of increased aggressiveness and change in staging, respectively.3

Broders proposed a grading system for squamous cell carcinomas.11,12 _The _{classification} _established _in ₁₉₂₀

and subsequently reviewed in 1925 was based on the

Table1 Broders’classification.

Broders’histologicalclassification

Parameter Characteristics

GradeI 0---25%undifferentiatedcells GradeII 25---50%undifferentiatedcells GradeIII 50---75%undifferentiatedcells GradeIV 75---100%undifferentiatedcells

fundamental principleof cellulardifferentiation, and was completely dissociated from clinical history. Carcinomas were divided into four grades, ranging from I to IV. Car-cinomas of grade I had up to 25% undifferentiated cells. GradeIItumorshad25---50%undifferentiatedcells.GradeIII tumorsconsistedof50---75%undifferentiatedcellsandGrade IVtumorshad75---100%undifferentiatedcells(Table1).11,12

The histopathological classification of malignancy pro-posedby theWorldHealthOrganization (WHO, 2005) was basedonthedegreeofcelldifferentiationandallowedthe classificationof this malignancy into threecategories, as well-,moderatelyandpoorlydifferentiated(Table2).2,11---13

The well-differentiated tumor is characterized by sim-ilarity with normal squamous epithelium, high power of keratinization,and very mild cell atypia; the moderately differentiatedcontainsdistinctnuclearpleomorphism, pres-enceof mitotic activity,includingatypical figures.In this type, keratinization is much lower.2 _In _the _poorly

differ-entiatedtype,immaturecellspredominatewithnumerous mitotic figures and keratinization is virtually absent. Regardingthe power ofinvasion, itcan belimited tothe epithelium,respectingthebasal lamina,when itis called insitu.Whenitonlyaffectsthelaminapropria,itis consid-eredmicroinvasiveorsuperficiallyinvasive.2

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Table2 ClassificationrecommendedbytheWorldHealth Organization(WHO).

Histologicalclassification---WHO

Parameter Characteristics

Poorlydifferentiated Predominanceofimmaturecells Abundanttypicalandatypical mitoses

Minimalkeratinization Moderately

differentiated

Somedegreeofpleomorphismand mitoticactivity

Littlekeratinization

Well-differentiated Tissuearchitecturesimilartothe normalpatternofsquamous epithelium

underlyingtissues, possiblyaccompanied bystromal reac-tion. Perineuralinvasion and angiolymphatic invasion are signs of increased aggressiveness and change in staging, respectively.3

Themeasurementofmitoticfigures,acommonpractice intheevaluationoftumorsregardingtheirgrowthrateand degreeof aggressiveness,is farfromitsdesired accuracy, sinceitisnotpossibletoknow,throughhistologicalsections routinelystainedwithH&E,whichareexactlythecellsthat enteredthecelldivisioncycle.Thisisduetothefactthat thephasesG1,S,andG2ofthecellcyclearenotidentified byopticalmicroscopy(Fig.1).12

Ki-67(MKI67)isaproteinpresentinthenucleus,whose function is related to cell proliferation. This protein is onlyexpressedinthecelldivisioncycle:interphase(G1,S, G2),prophase,metaphase,anaphase,andtelophase,andis absentintheG0phase,whenthecellisquiescent; there-fore,itisanexcellentmarkerofcelldivision.11---13

The monoclonal antibody Ki-67 is used todemonstrate theexpression ofthisnuclearprotein.Thisantibody binds toitsspecific-antigen(MKi67)presentinthenuclei of pro-liferatingcells,stainingthemandhighlightingallcellsthat haveenteredthecelldivisioncycle.12,13

Through optical microscopy it is possible tocount the numberof cells per fieldor ‘‘hot spot’’ areas, which are stained, and to calculate the proliferation index (Ki-67

DNA synthesis

S

G1

G M

G0 Cell growth and protein synthesis

Quiescence and differentiation

Division

Figure1 Phasesofcellcycle.G1,S,G---presenceofMKI67.

index). The analysis of this index is what allows for the characterization of thedegree of tumor cell proliferation (growth rate) and consequently, itsdegree of aggressive-ness,increased risk of newtumor clones, and mutations, whichhindertumorrestrictionbytheimmunesystem.12,13

ThisstudyaimedtoevaluatetheexpressionoftheKi-67 markeringlotticsquamouscellcarcinomaofthelarynxand correlateitregardingpathologicalfindingsandcell prolifer-ationcharacteristics.

Materials

and

methods

This wasan experimental clinical study performed witha cross-section in a historicalcohort, using resection speci-mensfromglotticsquamouscellcarcinomaofpatientsfrom atertiarycareuniversityhospital.Thepatientswere submit-tedtohemi-laryngectomy,totallaryngectomy,andremoval ofthevocalcordswiththeirrespectivelymphadenectomy.

To facilitate the statistical analysis of glottic tumors, they were divided into well-differentiated and moder-ately/poorlydifferentiated.

Cases of glottic squamouscell carcinoma tumors were obtained from the database of the Pathological Anatomy Departmentofatertiaryuniversityhospitalintheperiodof 1994---2004.Thissampleincluded99casesintotal:85cases weremoderately/poorlydifferentiatedtumors,ofwhich41 cases were products of surgical specimens and 44 cases werebiopsiesthatwerenottreatedsurgically (chemother-apy/radiotherapy).Fourteencaseswerewell-differentiated tumors,witheightcases representingproductsof surgical specimensandsixbiopsycasesthatwerenottreated surgi-cally(chemo/radiotherapy).

The study only included cases that had surgical speci-mens,excludingbiopsycases,astheywouldbringbiastothe studybyincludingrandomtumorfragments.Ofeight well-differentiatedproductsofsurgicalspecimens,twocasesdid nothaveparaffinblocksavailablefornewsections.

Therefore, the study was conducted with six cases of well-differentiatedglotticlaryngealtumors,and10casesof moderately/poorly differentiated glottic laryngeal tumors wererandomlyselectedtocomprisethecontrolgroup.The groupsdidnothavethesamenumberofcases,astherewere notenoughcasesofwell-differentiatedtumorstocomplete thegroup.

Theinclusion criteriawereglottic laryngealtumorsfor which paraffin blocks were identified and that contained enoughmaterialfornewsections.Clinicaldataandtumor stagingwereobtainedfrommedicalrecords(Table3).

Thestudywasapprovedbytheethicscommitteeofthe institution(no.485/11).

Immunohistochemical analysiswas performed with the avidin---biotin---peroxidase method and used the following antibody:Ki-67(CloneMIB-1,IgG1,1:150,Dakobrand, pre-treatedTris---EDTA).

Positiveandnegativecontrolsofthemarkerwereused, inordertocomparethemwiththestudiedcases.Lymphoid tissuewasusedaspositivecontrol.

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Table3 Clinicaldataofstudypatients.

Clinicaldata Group1 Group2 Obs

TNM T1N0M0---fourcases T1N0M0---threecases

T2NOMO---onecase T2N0M0---fivecases T3N0M0---onecase T2N1M0---onecase

T3N0M0---onecase Alcoholconsumption 50%(3cases)

Smoking 67%(4cases)

Gender 4M:2F 10M:0F

Age 53(37---73) 58(39---70)

Preop.clinicalneck Negative 1positivecase(N1)

Margins 50%positive 100%negative

Vascularinvasion 100%negative 80%negative 2cases+

Neuralinvolvement 100%negative 90%negative 1case+

Extracapsularextension 100%negative 100%negative

Preop.radiotherapy 0cases 1case

Postop.radiotherapy 50%(3cases) 20%(2cases)

Preop.chemotherapy 0cases 0cases

Postop.chemotherapy 0cases 0cases

Total 6patients 10patients

Group1,well-differentiatedglotticSCC;Group2,CECmoderately/poorlydifferentiatedglotticSCC;Preop.,preoperative;Postop., postoperative.

controls. Pictures scanned at high magnification (400×) weretakenfromhighlyreactive areas(hotspots),usinga Nikoncamera(Coolpix995).Subsequently,theimageswere uploaded to the computer for histological analysis, using the software Imagelab 2000. All parameters were blindly evaluated.

The evaluation of the cell proliferation rate was per-formed using commonpercentage ratio,asit already has been used in literature.8,9 _Five _areas _of _greater _intensity

ofimmunomarkerexpression(hotspots)wereselected.At least 500 cells were counted, and the ratiobetween the numberofcellsthathadapositivelystainednucleusandthe totalnumberofcellswascalculated.Thepercentageratio wasstratifiedasfollows:<5%---lowcellproliferationrate; between5%and10%---mildcellproliferationrate;between 10%and30%---moderatecellproliferationrate;and>30% ---highcellproliferationrate.

To simplify and better analyze the data, they were groupedasfollows:

Cellproliferationindex:

--- Low/mildcellproliferation. --- Moderate/highcellproliferation.

Histologicaltype:

--- Glottic,well-differentiated.

--- Glottic,moderately/poorlydifferentiated.

Statisticalanalysis

Afterpoolingofthedata,thestatisticalanalysiswascarried outwithIBMSPSSStatistics®_software,_using_the_Chi-squared

testandFisher’sexacttest.

Results

A total of 16 cases were evaluated, with six well-differentiated and 10 moderately/poorly differentiated cases.

Among the well-differentiated glottic tumors (n=6 cases),fourcaseshadlow/mildcellproliferation andtwo caseshadmoderate/highcellproliferation.Amongthe mod-erately/poorlydifferentiatedglottictumors(n=10cases), onecasehadlow/mildcellproliferationandninecaseshad moderate/highcellproliferation.

Inthecorrelationanalysisofcelldifferentiationdegree versuscellproliferation index,statistical significance was demonstrated(p<0.05)whencomparingthegroupsthrough two-tailedFisher’sexacttest,withp=0.036.

Table4 Distributionofcasesaccordingtohistologicaltypeandcellproliferation.

Histologicaltype Total

Well-differentiated Moderately/poorlydifferentiated

Ki-67 Low/mild 4 1 5

Moderate/high 2 9 11

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Consideringthehistologicaltypeasthegoldstandardto assesscelldifferentiationinlaryngealcarcinomas,thecell proliferation index had a sensitivity of 0.9, specificity of 0.67,positive predictive value of 0.82,and negative pre-dictivevalueof0.8(Table4).

Discussion

Thecellproliferationindexwaspositivelycorrelatedwith thedegreeofcelldifferentiation,andwashigherin moder-ately/poorlydifferentiatedcases.Thisfindingcorroborates theideathatthemoreundifferentiatedtheneoplasm,the poorer the control of the cell division process and the greater the proliferation.8 _This _evaluation _showed

statis-ticalsignificancebetweentumor typecorrelationandcell proliferationindex(p<0.05).

The studies by Rodrigues et al. found no correlation betweentheimmunohistochemicalexpressionofKi-67and prognosticvalue;7,14,15_however,_it_appears_that_Ki-67

over-expression associated with the idea of mitotic disarray, uncontrolledcellgrowth,andproliferationwouldbe associ-atedwithprognosticfactorsandapproachprioritizationof thesepatients.

An interesting fact was pointed out in a Canadian retrospective study on the clinical use of Ki-67, which demonstratedthatitsoverexpressioniscorrelatedwithhigh risk of cervical recurrenceafter radiotherapy and, there-fore,suchcases shouldundergomoretargeted treatment atearlierstages.16

Otherhead andneck tumorsites,suchastheoral cav-ity,alsoprovideevidencethatover expression ofmarkers suchasp53andKi-67 constituteagreaterrisk andpoorer prognosisforthistypeofsquamouscellcarcinoma.17

In a study with animals exposed to tobacco, only the developmentofbenignlesionswasobserved,withno pro-gression to malignant tumors. Histological evaluation of these animals showed absenceof p53marking, but Ki-67 positivityassociated with the tobacco-induced lesion was observed.18

Whenassessingtheclinicalprognosisofpatientswith epi-dermoidcarcinomasofthelarynx,itwasobservedthatthe Ki-67markerassociatedwithKi-S11isanimportanttoolto aidinclinicalmanagementandprognosticanalysis.Patients withlow cellproliferationrateassessed bythesemarkers have a lower recurrence rate with statistical significance (p<0.05)and improved five-year survival when compared tothegroupwithhighcellproliferationrate.19

Regarding the clinical application of Ki-67, it was also shownthatthegroupofpatientswithexpressionconsidered tobepositivehadhigherrateoflocalrecurrence,evenafter radiotherapy.Itissuggestedthatthesepatientshavea per-sonalizedandmoredetailedfollow-upprotocol.16_However,

inanothergroup,itwasidentifiedthatpatientswithglottic tumorsinvolvingtheanteriorcommissureandshowingover expressionof Ki-67had abetteroutcome andhad tumors consideredtoberadio-sensitive,withsignificantrelevance ofresults.20

The literature also indicates the methodology used in the anatomopathological evaluation of advanced epider-moidcarcinomaofthelarynx.Intheaforementionedstudy, theheterogeneityinKi-67expressionwasdemonstratedin

areasoftumor cellactivityand,therefore,anassessment methodologyshouldbe well-established,andthe variabil-ity of previous studies that correlated the expression of immunomarkers withprognostic factors can be explained bysuchcharacteristic.21

Theevaluationofcarcinomaprecursorlesions (hyperpla-sia, carcinomain situ)wasalsoperformed instudieswith immunomarkerssuchasKi-67andp-16.Oftheselesions, car-cinomainsitutendstohaveahigherpositivitythanisolated hyperplasias.22

Although the present study found a significant corre-lation between cell proliferation degree and histological tumordifferentiation,thereweretwocases(18%)inwhich the rate of cell proliferation was moderate/high even in well-differentiated tumors. Considering that even some well-differentiated tumors showed high cell proliferation index, these tumors possibly exhibit a more aggressive behavior,aswiththeincreasedpopulation oftumor cells, thechanceofmitoticdisarrayisgreater.

Newclonesthatmaybemoreaggressiveduetomutations andthathinderrestrictionbytheimmunesystemmayhave betterresponsetochemotherapyandradiotherapy.Future studies that assess the behavior of well-differentiated tumorswithhighrateofcellproliferationmayassistinthe managementofthesepatients.

It is noteworthy that although this study had a sam-ple of 16 patients, limited by the difficulty in obtaining surgical materialfromwell-differentiated glotticcases, it emphasized theanalysis ofthis specific regionof the lar-ynx,differentfromotherstudieswithlargersamplesthat included patients with carcinomas in any region of the larynx.Therefore,thepresentstudy corroboratesthe cor-relation between increased expression of Ki-67 in more undifferentiatedtumors,8 _and_demonstrates_that_this

cor-relationalsoappliesspecificallytolaryngealglottictumors. The authors believe that the association between the expression of Ki-67 with clinical criteria and prognosis remainsunclear.Thisassociationisstillquitecontroversial in themedical literature,in both clinical and experimen-talstudies.Therelevanceofusingimmunomarkersisclear; however,furtherstudiesarenecessarytobetterdefinethe patientsinwhichimmunomarkersshouldbeusedinclinical practice.

Conclusion

Thisstudydemonstratedthattherateofcellproliferation measuredbyKi-67maybeusefulincharacterizingthe histo-logicalgradeinglotticsquamouscelltumorsofthelarynx. Furtherstudiesareneededtoevaluatethebehaviorof well-differentiated tumors with high rate of cell proliferation todefinethebesttherapeuticapproachinthesepatients. Otherfactorsmustbeinvolvedregardingthepathogenesis andtumorprogressionofthisdiseaseintheglottis.

Funding

(6)

Conflict

of

interest

Theauthorsdeclarenoconflictsofinterest.

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