www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Apoptosis
and
expression
of
argyrophilic
nucleolus
organizer
regions
in
epithelial
neoplasms
of
the
larynx
夽
,
夽夽
,
夽夽夽
Christiana
Vargas
Ribeiro
a,∗,
Anilton
Cesar
Vasconcelos
b,c,
José
de
Souza
Andrade
Filho
d,eaFaculdadeeEscolaTécnicaNovoRumo,BeloHorizonte,MG,Brazil bUniversityofCalifornia,California,USA
cDepartmentofGeneralPathology,UniversidadeFederaldeMinasGerais(UFMG),BeloHorizonte,MG,Brazil dDepartmentofPathology,FaculdadedeCiênciasMédicas,UniversidadeFederaldeMinasGerais(UFMG),
BeloHorizonte,MG,Brazil
eDivisionofPathologyandCytopathology,HospitalFelícioRocho,BeloHorizonte,MG,Brazil
Received14November2013;accepted22July2014 Availableonline27December2014
KEYWORDS Apoptosis; Larynx; Squamouscell carcinoma;
Nucleolusorganizer region;
Papilloma
Abstract
Introduction:Occurrenceofapoptosis andexpression ofproliferativemarkersarepowerful toolstoestablishaprognosisinthefollow-upofcancer.
Objective:Toevaluatethegrowthfractioninpapillomasandlaryngealsquamouscell carcino-maswiththreedegreesofdifferentiationthroughapoptosisandtheexpressionofnucleolus organizerregions.
Methods:Retrospectivestudy fromwhichparaffinmaterialwassubmittedtomicrotomyand hematoxylin---eosinandsilverstaining.Stainedslideswereusedtoquantifytheapoptoticindex andthenumberofnucleolusorganizerregionsbymorphometry.
Results:Apoptosis was significantlymore frequent inwelldifferentiated carcinomasandin papillomas,andahighergrowthfractionofexpressednucleolusorganizerregionsandcellsthat expressedagreaterthanaveragenumberofnucleolusorganizerregionsweremorefrequently notedinundifferentiatedcarcinomas.
夽 Pleasecitethisarticleas:RibeiroCV,VasconcelosAC,AndradeFilhoJS.Apoptosisandexpressionofargyrophilicnucleolusorganizer
regionsinepithelialneoplasmsofthelarynx.BrazJOtorhinolaryngol.2015;81:158---66.
夽夽Institution:ThisprojectwasdevelopedattheInstitutodeCiênciasBiológicas(ICB),UniversidadeFederaldeMinasGerais(UFMG);and
EscoladeMedicina,UniversidadeFederaldeMinasGerais(UFMG),BeloHorizonte,MG,Brazil.
夽夽夽
Casereport:aretrospectivestudyof40casesofepithelialneoplasmsofthelarynx,with3degreesoftumordifferentiation. ∗Correspondingauthor.
E-mail:christianavargas@yahoo.com.br(C.V.Ribeiro). http://dx.doi.org/10.1016/j.bjorl.2014.12.003
Conclusions: Thus,itwaspossibletoverifythatahighapoptoticindexwasassociatedwitha lowerchanceoftumordifferentiationincarcinomas,whileagreaternumberoftotal nucleo-lusorganizerregions,cellsexpressingnucleolusorganizerregionsaboveaverageandahigher growth fraction were associated with greater likelihood ofabnormal cell proliferation and increasedtumordifferentiation.
© 2014Associac¸ãoBrasileira de Otorrinolaringologiae CirurgiaCérvico-Facial. Publishedby ElsevierEditoraLtda.Allrightsreserved.
PALAVRAS-CHAVE Apoptose;
Laringe; Papiloma;
Carcinomadecélulas escamosas;
Regiãoorganizadora donucléolo
Apoptoseeexpressãoderegiõesargirófilasorganizadorasdenucléolosemneoplasias epiteliaisdalaringe
Resumo
Introduc¸ão: Aocorrência deapoptoseeaexpressãodemarcadoresproliferativos são ferra-mentaspoderosasnoestabelecimentodoprognósticodocâncer.
Objetivo: Avaliar afrac¸ão de crescimento de papilomas e carcinomas laríngeos de células escamosas, com três graus de diferenciac¸ão, através da apoptose e expressão de regiões organizadorasdenucléolo.
Método: Estudoretrospectivo,cujomaterialemblocadoem parafinafoi submetidoà micro-tomiaecolorac¸ãoemhematoxilina-eosina,epelaprata.Aslâminascoradasforamutilizadas paraquantificaroíndiceapoptóticoeonúmeroderegiõesorganizadorasdenucléolo(NORs) atravésdamorfometria.
Resultados: Aapoptosefoisignificativamentemaisfrequenteemcarcinomasbem diferenci-adoseem papilomas;enquantoqueumamaiorfrac¸ãodecrescimento,deNORsexpressose de células queexpressaram maiornúmerode NORs,forammaisfrequentesnoscarcinomas indiferenciados.
Conclusões: Foipossívelverificarqueoíndiceapoptóticoelevadoindicamenoreschancesde diferenciac¸ãotumoralnoscarcinomas,enquantoqueummaiornúmerodeNORstotaisecélulas expressandoNORsacimadamédia,eumamaiorfrac¸ãodecrescimento,determinammaiores chancesdeproliferac¸ãocelularanormalemaiordiferenciac¸ãotumoral.
©2014Associac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial.Publicado por ElsevierEditoraLtda.Todososdireitosreservados.
Introduction
Worldwide, carcinomas of the head and neck occupy the fifthpositioninincidence,andaffectmainlymalesmokers anddrinkers,andtheregionsofthelarynxandoralcavity.1 Itisestimated thatinBrazilmorethan6000newcasesof carcinomaofthelarynxoccurredin2012,andworldwideit isconsideredthesecondmostcommontumorinthe respira-torytractandthemostcommonamongcancersofthehead andneck,representing2%ofallmalignancies.2
Severalstudieshaveshownthatthenumberofnucleolar organizer regions stainedby silver (AgNORs) in malignant neoplasticcellsisoftengreaterthaninbenigntumorcells ornormalcells.3,4
Rüschoffetal.5concludedthattheguidedevaluationof thepatternofAgNORsfacilitatestheunderstandingofcell biology, since the number and area of points dissociated fromAgNORsaremainlyrelatedtotherateofproliferation, and differ markedly among cell strains, in the various degrees of differentiation, regardless of the type and size of AgNOR aggregates in the cell. Thus, the relation-ship betweenAgNOR quantificationat interphaseand cell
proliferationhasbeenwidelyinvestigatedinhumantumors, bycomparingAgNORrelativeparametersatinterphasewith thosecell proliferation markers commonly used in tumor pathology.4
Antonangeloetal.6 examinedtherelationshipbetween theexpressionofnucleolusorganizerregionsstainedby sil-ver(AgNORs) andsurvival timeinpatients withsquamous cellcarcinomaof thelung andconcludedthatthe expres-sionofAgNORswaslowerintumorsintheirearlystages,and intumorsthat wereof ahigh histologicalgrade;ahigher numberofAgNORswasassociatedwithlowersurvivalrates. Theseresultsencouragedtheuseof AgNORs’techniquein theprognosisforlungcancer.
Rüschoff et al.,5 working with normal and neoplastic cellsoftheurinarysystem,showedthatthesizeofAgNOR aggregates is related to cell differentiation, and larger AgNOR aggregateswere observed in those more differen-tiatedtumors.
withacluster ofAgNORscouldbeusedasaparameterof thekineticsofthistumor,helpingtodifferentiatepatients with the disease in both stable and progressive stages, correctly classifying 94% of patients. For all practical purposes,avalueofmorethan13%ofcellswithclustersof AgNORssuggestedprogressivedisease.
Cases of squamous cell carcinoma of the oral cavity hadtheirprognosispredictedbyquantifyingthenumberof AgNORs,despitetheoccurrenceofsomeoverlappingamong individualvalues.The meannumberofAgNORswashigher forlesionswithapoorprognosisandwithamoreaggressive character.8
Basedonthis,AgNORshavebeenwidelyusedasmarkers ofcellproliferation,showinggooddiagnosticandprognostic valueinthefieldoftumorpathology.9---11
Recent studies have described the occurrence of apo-ptosis in several types of human carcinomas, the most undifferentiated tumors being those that usually present thelowestapoptoticindex.12,13Thus,theapoptosisprocess seemstohaveacloserelationshipwithagoodprognosisfor patientswithepithelialtumorsofthelarynx.
Inthiscontext,theobjectivesofthisstudywere:(a)to determinetheapoptoticindex;(b)todeterminethenumber ofAgNORs;(c)toassesstherateofgrowthofmalignantand benignepithelialneoplasmsofthelarynx.
Methods
For this research, we conducted a study of a historical cross-sectionalcohort.Sampleswereobtainedfrompatients undergoingsurgical treatment of laryngeal tumors during the period between the years 1989 and 2002. All mate-rialusedwasprimarilyharvestedbydoctorsatthehospital with diagnostic and therapeutic purposes; at that time, there was no knowledge of the possibility of conducting thisresearch.The study wasconductedduringthe period between the years 2002---2004; at that time, we did not have norms establishing guidelines for the use of biologi-calmaterials inresearch.Since2005, withtheResolution 347/05,normshavebeenestablishedtoregulatethe stor-ageanduseofhumanbiologicalmaterialinthecontextof researchprojects.Resolution441/11isanupdatedversion of the Resolution 347/05, in which Article 15 maintained virtuallythesameprovisionsofthepreviousversion. There-fore,asthisstudywasconductedpriortothisResolution, theEthics Committeedecided thesedeterminations were notnecessary.
Initially, we undertook a survey of hospital clinical records,withtheaimofidentifyingindividualswithbenign andmalignantlaryngealneoplasms.Onehundredand forty-twopatients were found, among whom40 wereselected becausethe materialembedded in paraffin wasin better condition.Wedisregardedtheremainderbecauseofahigh incidenceofmoderately differentiatedcarcinomas,which didnot justify increasingthe sample size we hadalready obtainedandjudgedtoberepresentative.
Gender,race andagewerenotconsidered asselection criteria. Thus, based solely on the histopathologic crite-ria, these patients were divided into groups, and their carcinomas were characterized (based on the degree of keratinization)as well differentiated (grade I--- 4 cases),
moderatelydifferentiated(gradeII ---16 cases)and undif-ferentiated (grade III --- 14 cases). The patient sample included also six benign tumors that were papillomas. Well-differentiatedlesionsarethoseinwhichthereoccurs keratinizationof theindividual cellsand theformation of multiple horny or keratin beadsor pearls of varying size; therearegroups of cells actively invading theconnective tissue.Moderatelydifferentiatedlesionsretain,inpart,the characteristicsofnormalepithelium,butthecellsalready exhibit alterationsin their forms.In poorlydifferentiated lesions the resemblance to squamous epithelium is less pronounced.The characteristic shapeofthe cells maybe altered, aswell asthe typical intercellular arrangement. Thecellsshowvariationinsize,stainingstrengthandability toperformthefunctionofasquamouscell,whichisthe for-mationofkeratin;thesearedifferentandpleomorphiccells, with loss of cohesion and showing numerous and atypical mitoticfigures.
With a goal to analyze the histological grade and the apoptotic index, the samples embedded in paraffin were sectionedat4mandstainedwithhematoxylin---eosin(HE).
Other sectionswere submitted to silver impregnation for quantification of the cell proliferation index by counting NORs.Thesectionsweredewaxed,rehydratedsequentially inabsolute alcohol,70%and50% andwashedin deionized water.ThesectionswerestainedwithPlotonetal.14 tech-nique,modifiedbyAubele etal.15 The silverimpregnation wasperformed bytwopreviouslypreparedsolutions: solu-tionA(aqueoussolutionofsilvernitrate,50%)andsolutionB (formicacid1%containing2gofcolorlessgelatin).Solutions AandBweremixedataratioof2:1atthetimeofincubation ofthesections.Theincubationwasperformedinawet/dark chamberat37◦Cfor20min.Subsequently,thesectionswere
washedwithdeionizedwaterfor15minutesanddehydrated in70%alcoholandabsolutealcohol,diaphanizedinxylene andmountedwithEntelan.16
Oneslide fromeach group (papillomas,gradeI, II and IIIcarcinomas)wasselectedfor determiningtheminimum numberoffields tocalculate theapoptoticindex.Ineach slide, the highest numberof possible fields was analyzed (usinga40×objective),todeterminetheapoptoticindexes, i.e., thenumberof apoptoticcellsin thefielddivided by the total number of cells in the same field. The images were generated by a microscope connected to a camera andtransferredtoadigitizingboardinthecomputer,where themorphometrywasperformedusingaspecificcomputer program(KontronKS300version2.0).Toquantify apopto-sisintheselesions,theminimumnumberofrepresentative microscopic fields was obtained by an analysis of insta-bility of the variationcoefficient.17 Among the evaluated fields, mean values for multiple samples of five random fields were obtained, until attaining the total number of fields per slide. These mean values were considered as experimentalunitsandusedinthestatisticalanalysis.The standard deviations (SDs) and coefficientsof variationfor eachsamplesizedecreased,asthenumberoffields consid-ered increased,untilthe occurrenceof stabilization.This stabilizationoccurred when10fields perslidefor papillo-mas and 30 fields per slide for carcinomas of all degrees wereobtained.
Figure1 GradeIsquamouscellcarcinoma(A)MicrographofgradeIsquamouscarcinomacells,withthepresenceofdiffusekeratin pearls(CP)(HE,bar=100m).(B)MicrographofgradeIsquamouscarcinomacellswithvariousapoptoticcellswithcondensation ofcytoplasm(arrows)andanoikis(*)(HE,bar=10m).
offields(100×objective)wasanalyzedandthenumberof totalNORs(i.e.,thenumberclumpspernucleusinthefield divided by the totalnumber of nuclei in that same field, usingthe same methodand equipmentmentioned above) wasregistered.Thestabilizationoccurredwhen10fieldsper slideforpapillomasand15slidesforallgradesofcarcinomas wereachieved.
As to the analysis of the apoptotic index (number of apoptoticcells/totalnumberofcells×100),thecellswere observed by a single researcher, whocounted only those cellsexhibitingatleastthreeofthefollowingmorphological characteristicspeculiartotheprocess:(1)anoikis(presence of a pericellular clear halo); (2) cytoplasm condensation; (3) nuclear condensation (crescents); (4) nuclear frag-mentation; (5) cell fragmentation (apoptotic bodies); (6) phagocytosisofapoptoticbodies(‘‘cellcannibalism’’).The areasofnecrosiswereexcluded.Thecellsinapoptosis,and subsequently all the epithelial cells, were counted,in an areacorrespondingto53,333.4m2 ina totalof 10 fields
forpapillomasand30fieldsforallgradesofcarcinomas. Astotheanalysisofthecellproliferationindex(number of NORs/total number of nuclei×100) in an area corre-spondingto8.533,37m2,hecountedtheNORspresentin
eachnucleusand,inparallel,thetotalnumberofepithelial nuclei,foratotalof10fieldsforpapillomasand15forall gradesofcarcinomas.Theratiobetweenthenumberofcells expressingAgNORsaboveaverageandtheapoptoticindex wasregardedasthegrowthfractionforeachtumortype.
Means or medians were obtained for the studied morphometric parameters, in order to characterize the groups. At first, the normality of the data through the Kolmogorov---Smirnovtest wasverified.Whenthe distribu-tionwasnormal,weusedtheStudent’sttest(aparametric test) to compare the means of different injuries. When the distribution of the data did not show normality, we usedthenonparametricMann---Whitneytesttocomparethe mediansofdifferentinjuries.Alldatawereanalyzedwith a significance level of 0.05 with the GraphPad Prism 3.0 software.
Results
Of the 40 selected samples, 34 were from patients with laryngealcarcinoma,28men(82.35%)and6women(17.65%) withameanageof59.5years.Thelaryngealpapillomacases
Figure 2 Grade I squamous cell carcinoma. Micrograph of gradeIsquamous cells,withinvasionofconnectivetissueby tumorcellsintheformofverydelimitedepithelialnests(EN) (HE,bar=100m).
consistedof4men(66.66%)and2women(33.34%)witha meanageof36years.
Histologically,weobservedinthesampleswithGradeI squamouscellcarcinoma(SCC)avariablenumberofkeratin pearlsscatteredthroughoutthetissue(Fig.1A),aswellas isolatedintracellularkeratinization.Wealsonoted numer-ouscellsinapoptosisscatteredthroughthetumordisplaying condensationofthecytoplasmandanoikis(Fig.1B).The cel-lularpleomorphismvariedbetweenmildandmoderate,and thenuclearsizeshowedlittlevariability.Althoughan inva-sionofadjacenttissuebytumorcellswasnoted,theedges oftheepithelialstrandsandnestswerewelldefined(Fig.2). InGradeIISCC(SCCII),weobservedfewerkeratinpearls and a greater connective tissue invasion by tumor cells (Fig. 3). Mitoses were frequent, many of them atypical. Apoptoticcellswithcondensationofcytoplasmandanoikis (Fig.4)werepresentthroughoutthetumortissue,buttoa lesserextentthaninGradeISCC(SCCI).
Figure3 GradeIIsquamous carcinomacells.Micrographof gradeIIsquamouscarcinomacellswithconnectivetissue infil-trationbytumorcells(HE,bar=100m).
Figure4 GradeIIsquamous carcinomacells.Micrographof gradeIIsquamouscarcinomacellswithpresenceofapoptotic cellswithcondensation ofcytoplasm(arrows) andanoikis(*) (HE,bar=10m).
andalsothroughdeeperareas,suchasmuscleand glandu-lartissues.Virtuallynocellwithintracellularkeratinization wasobserved,andtherewerenokeratinpearls.Fewcells wereinapoptosis, andthecondensationofthecytoplasm andanoikis(Fig.6)occurredwithoutadefinite patternof locationandalsoinisolatedepithelial cellspresentinthe stroma.
Thepapillomapresentedexophyticprojections(Fig.7). Each projection was covered by keratinized squamous epithelium with hyperkeratosis and with cells supported bya central partof connectivetissue withblood vessels. Furthermore,weobserved cellsundergoingapoptosiswith cytoplasmic condensation and anoikis in the papilloma’s finger-like projections (Fig. 8). The apoptotic cells were distributedmostfrequentlyinthebasalandspinouslayers. AgNORswereidentifiedinmanytumortypesintheform ofbrownishorblackclumpswithvariedshape,sizeand dis-tribution.InSCCI,AgNORspresentedasisolatedclumpsor
Figure5 GradeIIIsquamouscarcinomacells.Micrographof grade IIIsquamous carcinoma cellswith apoptotic cells with nuclearhyperchromatacism(arrows)(HE,bar=10m).
Figure6 GradeIIIsquamouscarcinomacells.Micrographof gradeIIIsquamouscarcinomacellswithpresenceofafew apop-toticcellswithcondensationofcytoplasm(arrows)andanoikis (*)(HE,bar=10m).
formedclusterswithroundedorirregularshapes,fillingthe entire nucleolusor scatteredin thenucleus likesatellites (Fig.9).InSCCII,wenotedthepresenceofAgNORs form-ingroundedclumps,fillingtheentirenucleolus,orassmall satellitesscatteredthroughoutthenucleus(Fig.10).Onthe other hand, inSCC III,the AgNORsformed dark, irregular adjoiningclumps occupyingvirtually theentire nucleolus, or were isolated in the form of satellites in large nuclei (Fig.11).
Inthepapilloma samples,AgNORsexhibiteda distribu-tionsimilartothatfoundinSCCIsamples,formingsmall, isolatedclumps orasclusters,present assatellitesin the nucleusoroccupyingtheentirenucleolus(Fig.12).
Figure7 Papilloma.Micrographofpapillomawithexophytic projections extending below the surfaceofthe mucosa (HE, bar=100m).
Figure8 Papilloma.Micrographofpapillomawithcell apo-ptosiswithcondensationofcytoplasm(arrows)andanoikis(*) (HE,bar=10m).
Table1 Mediansofapoptoticindexes.
GroupsCompared p
SCCI×SCCII(0.10;0.09) 0.0127a SCCI×SCCIII(0.10;0.08) 0.0001a SCCII×SCCIII(0.09;0.08) <0.0001a Papilloma×SCCI(0.09;0.10) 0.1504a Papilloma×SCCII(0.09;0.09) 0.8783a Papilloma×SCCIII(0.09;0.08) 0.0008a
SCC,squamouscellcarcinoma. a Mann---Whitneytest.
topapilloma (p=0.8783). Finally,SCC III,when compared withpapilloma,showedalowerapoptoticindex(p=0.0008) (Table1).
In papilloma samples,the totalnumber ofAgNORs per fieldwassignificantlyhighercomparedtoSCCI(p=0.035) andsignificantlylowercomparedtoSCCII(p=0.0051)and SCC III (p<0.0001). SCC I had a lower number of total
Figure 9 Grade I squamous carcinoma cells with AgNORs. MicrographofgradeIsquamouscarcinomacellswithAgNORs intheformofisolatedlumpsorforminglargeaggregates,with roundedorirregularshapesfillingtheentirenucleusorpresent inthenucleolusassatellites(AgNOR,bar=10m).
Figure10 GradeIIsquamouscarcinomacellswithAgNORs. Micrograph of grade II squamous cell carcinoma containing AgNORsformingroundedclumpsfillingtheentirenucleolusoras smallsatellitesscatteredbythenucleus(AgNOR,bar=10m).
AgNORs per field compared with SCC II (p<0.0001) and III (p<0.0001). Likewise, SCC II showed a lower mean of total AgNORs per field compared to SCC III (p<0.0001) (Table2).
Table2 MeansoftotalnumberofAgNORs.
GroupsCompared p
SCCI×SCCII(2.29;2.76) <0.0001a SCCI×SCCIII(2.29;2.99) <0.0001a SCCII×SCCIII(2.76;2.99) <0.0001a Papilloma×SCCI(2.53;2.29) 0.035a Papilloma×SCCII(2.53;2.76) 0.0051a Papilloma×SCCIII(2.53;2.99) <0.0001a
Figure11 GradeIIIsquamouscarcinomacellswithAgNORs. MicrographofgradeIIIsquamouscarcinomacellswithAgNORs formingdarkclumpstogetherandoccupyingtheentire nucle-olus or isolated as small satellites in large nuclei (AgNOR, bar=10m).
Figure 12 Papilloma with AgNORs. Micrograph of papil-loma with AgNORs forming small clumps present in the nucleusassatellitesoroccupyingtheentirenucleolus(AgNOR, bar=10m).
Table3 NumberofcellswithAgNORsaboveaverage.
GroupsCompared p
SCCI×SCCII(1.87;2.15) <0.0001a SCCI×SCCIII(1.87;2.35) <0.0001a SCCII×SCCIII(2.15;2.35) <0.0001a Papilloma×SCCI(1.98;1.87) 0.2021a Papilloma×SCCII(1.98;2.15) 0.0053a Papilloma×SCCIII(1.98;2.35) <0.0001
SCC,squamouscellcarcinoma. aStudent’sttest.
Table 3 shows comparisons of the number of cells expressing above average AgNORs per field among the different groups of tumors. There was no significant dif-ferencebetween papilloma and SCCI(p=0.2021) groups.
Table4 Mediansofgrowthfractions.
Groupscompared p
SCCI×SCCII(13.58;22.50) <0.0001a SCCI×SCCIII(13.58;28.21) <0.0001a SCCII×SCCIII(22.50;28.21) <0.0001a Papilloma×SCCI(18.70;13.58) 0.0021a Papilloma×SCCII(18.70;22.50) 0.1595a Papilloma×SCCIII(18.70;28.21) <0.0001a
SCC,squamouscellcarcinoma. a Mann---Whitneytest.
The number of cells expressing above average AgNORs in papillomagroupwaslowerthaninSCCII(p=0.0053)andIII (p<0.0001).SCCIshowedalowernumberofcells express-ingaboveaverageAgNORsversusSCCII(p<0.0001)andIII (p<0.0001).Likewise,SCCIIshowedalowernumberofcells expressingAgNORsaboveaverageversusSCCIII(p<0.0001).
Table4showscomparisonsofgrowthfractions(numberof cellsexpressingAgNORsaboveaverageperfield/apoptotic indexes)amongdifferenttumortypes.SCCIIIhadahigher growthfractionwhencomparedwithothertypesoftumors (p<0.0001). SCC II had a higher growth fraction versus SCCI(p<0.0001),butwithnosignificantdifferenceversus papilloma(p=0.1595).Papillomagrouphadahighergrowth fractionwhencomparedwithSCCI(p=0.0021).
Discussion
that the high apoptoticindex wassignificantly associated withpoor survival,andsuggested that, inthe future, the presenceofahighdegreeofapoptosiscouldbeusedto iden-tifypatients with apoor prognosis for laryngeal cancer.23 Likewise, Alcaideetal.24 reportedthe occurrenceofhigh numbersofapoptoticcellsincolorectalcarcinomas,when comparedtoadenomasandtonormalepithelium;the apop-toticindexwasalsohigherinmetastaticdisease(stage4), whencomparedtootherstages. Thisassertion isnot sup-portedbythefindingsofthisstudy,becauseherethehigh apoptoticindex wasrelated toa lowerhistological grade andabetterprognosis.
With respecttothe total numberof AgNORsper field, theSCCIgroupshowedthelowestvalue,followedby papil-loma, Grade II and SCCIII groups. We observed the same withrespecttothenumberofcellsexpressingaboveaverage AgNORsperfield.Some studieshaveshown thatthe num-berofAgNORsinmalignantneoplasticcellsisoftengreater thaninbenignneoplasticcells,orinnormalcells.10,25---27In manytumors,thequantificationanddistributionofAgNORs in the nucleus and nucleolus have shown significant pro-gnosticvalue.28 Someauthorsfound thattumorswithhigh malignancyhave ahighernumberofAgNORscomparedto low-grade tumors, which agrees with the findings of this study.28---30
Previous studies have reported that the average of AgNORs in SCCs of the oral cavity and larynx is higher in tumors with a poor prognosis and with more aggres-sivecharacteristics,i.e.,poorlydifferentiatedlesions.28,31 Hanemann, MiyazawaandSouza28 statedthatthe method of counting AgNORs is a useful diagnostic tool for identi-fyingdifferenttypes oforalsquamousmalignanciesandis alsoaprognosticmarkerofthesecarcinomas.Furthermore, this countinghelpstodifferentiate amongpre-cancerous, benignandmalignantlesions.AccordingtoUnderwoodand Giri,32 AgNORs scatter throughout the nucleus in varying lengths,makingtheirmeasurementeasy.Thus,the quantifi-cationofAgNORsisdependentonthedegreeofdispersion or breakdown of a large relative number of NORs in the nucleus. Therefore, the so-called histologic counting of NORs --- both in benign and malignant lesions --- does not result in an absolute number, but rather, in a numerical index of their dispersion. This does not impair or neces-sarily invalidatethe usefulnessof thiscounting technique asa discriminantfactor for malignancy or transcriptional activity.
As regardstogrowthfraction,SCCIshowedthelowest value,followedbypapilloma,SCCIIandSCCIII.Considering that SCCIpresented the highest apoptotic indexand the lowestnumberofAgNORsaboveaverage,itsgrowthfraction was the lowest among malignant tumors. The papilloma groupshowedaslightlyhighergrowthfractionversusSCCI, andwasslightlysmallerthanSCCII.However,these differ-enceswerenotstatisticallysignificant.Ontheotherhand, SCCIIIshowedanextremelyhighgrowthfraction,sinceits apoptoticindexwaslowandthenumberofaboveaverage AgNORswashigh.Liuetal.33 have supportedourfindings, and stated that the degree of differentiation in human cancersgenerallyreflectsthedegreeofmalignancy,where the most undifferentiated lesion is the most aggressive, basedonananalysisofovariancarcinomas.Theseauthors suggest that microRNAs are deregulated and therefore
overexpressedinthesetumors,causing anincrease inthe rateofcellproliferation.
Karkietal.34 foundirregularlyshapedAgNORsin malig-nantcellsofpleuralandperitonealeffusions,andlargeand individual AgNORs in benign cells of the same effusions. Aroraetal.35showedthatapparentlythenumberofAgNORs pernucleusinpatientswithSCCofthelarynxincreasesas thedegree of tumor dedifferentiation increases. Further-more,withahighertumorgrade,NORsdecreaseinsizeand becomemoreirregular.Inourstudy,thepresenceoflarger, rounded,regular andlessscattered aggregates wasnoted inthenucleiofSCCI,andsmallerandirregularaggregates wereobservedinSCCIII.Therefore,ourfindingsaresimilar tothosedescribedforcancersofthelarynx35andforcells ofpleuralandperitonealeffusions.34
Conclusions
Thehighapoptoticindexindicateslowerchancesoftumor differentiationincasesofsquamouscellcarcinomasofthe larynx.
ThegreaterthenumberoftotalAgNORsandabove aver-ageAgNOR-expressing cells, thehigher the chancesof an abnormalcellproliferationandthelessdifferentiatedthe tumortendstobe.
The higher the growth fraction, defined as the ratio betweenthenumberofAgNOR-expressingcellsabove aver-ageandtheapoptoticindex,thehigherthechancesofthe tumorbeingofalessdifferentiatedtype.
Funding
ThisstudywassupportedbyFAPEMIGwhichofferedfinancial supportfortherealizationofthisproject.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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