Braz. j. . vol.81 número1

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Brazilian

Journal

of

OTORHINOLARYNGOLOGY

www.bjorl.org

ORIGINAL

ARTICLE

Correlation

between

vascular

endothelial

growth

factor

expression

and

presence

of

lymph

node

metastasis

in

advanced

squamous

cell

carcinoma

of

the

larynx

夽

,

夽夽

Rodrigo

Gonzalez

Bonhin

∗

_,

_Vanessa

_Brito

_Campoy

_Rocha,

Guilherme

Machado

de

Carvalho,

Alexandre

Caixeta

Guimarães,

Agrício

Nubiato

Crespo,

Carlos

Takahiro

Chone,

Eliane

M.I.

Amstalden

UniversidadeEstadualdeCampinas(UNICAMP),Campinas,SP,Brazil

Received15June2013;accepted24August2014 Availableonline20November2014

KEYWORDS Squamouscell carcinomaoflarynx; VEGF;

Immunohistochemistry; Cervicallymphatic metastases

Abstract

Introduction:Squamouscellcarcinomaisthemostcommonneoplasmofthelarynx,andits evolutiondependsontumorstaging.Vascularendothelialgrowthfactorisamarkerof angio-genesis,anditsexpressionmayberelatedtoincreasedtumoraggressiveness,asevidencedby thepresenceofcervicallymphaticmetastases.

Objectives:To evaluatethe expressionofthevascular endothelialgrowth factor markerin non-glotticadvancedsquamouscellcarcinomaofthelarynx(T3/T4)andcorrelateitwiththe presenceofcervicallymphnodemetastases.

Methods:Retrospectiveclinicalstudyandimmunohistochemicalanalysisofvascular endothe-lialgrowthfactor throughthe Germanscaleofimmunoreactivityinproducts ofnon-glottic squamouscellcarcinomas.

Results:Thisstudyanalyzed15casesofadvancednon-glotticlaryngealtumors(T3/T4),four ofwhichexhibitedcervicallymphaticmetastases.Therewasnocorrelationbetweenvascular endothelialgrowthfactorexpressionandthepresenceofcervicalmetastases.

Conclusion:Althoughvascularendothelialgrowthfactorwasexpressedinafewcases,there wasnocorrelationwiththespreadofcervicallymphmetastases.

夽 _Please_cite_this_article_as:_Bonhin_RG,_Rocha_VB,_de_Carvalho_GM,_Guimaraes_AC,_Crespo _AN,_Chone_CT,_et_al._Correlation_between vascularendothelialgrowthfactorexpressionandpresenceoflymphnodemetastasisinadvancedsquamouscellcarcinomaofthelarynx. BrazJOtorhinolaryngol.2015;81:58---62.

夽夽_Institution:_Department_of_{Otorhinolaryngology,}_Head_and_Neck,_Universidade_Estadual_de_Campinas_(UNICAMP),_Campinas,_SP,_Brazil.

∗_{Corresponding}_author.

E-mail:bonhin@yahoo.com(R.G.Bonhin).

http://dx.doi.org/10.1016/j.bjorl.2014.08.016

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PALAVRAS-CHAVE Carcinoma

epidermoidede laringe; VEGF;

Imunohistoquímica; Metástase

Correlaçãoentreexpressãodofatordecrescimentoendotelialepresençade metástaselinfáticanoscarcinomasepidermoidesavançadosdelaringe

Resumo

Introdução: Ocarcinomadecélulasescamosaséaneoplasiamaisfrequentedalaringeeseu prognóstico depende do estadiamento. A progressão da doença está relacionada a fatores intrínsecoscelularesdocâncer,nãoconhecidos.OVEGF(vascularendothelialgrowthfactor)é ummarcadordeangiogêneseesuaexpressãopodeestarrelacionadaaumamaioragressividade tumoral,evidenciadapelapresençademetástaseslinfáticascervicais.

Objetivos: AvaliaraexpressãodomarcadorVEGFemcarcinomadecélulasescamosasdalaringe avanc¸ados(T3/T4),não glóticosecorrelacionarquantoàpresenc¸ade metástaseslinfáticas cervicais.

Método: EstudoclínicoretrospectivodeanáliseimunohistoquimicadoVEGFatravésdaescala Germânicadeimunorreatividadeemprodutosdecarcinomasepidermóidesnãoglóticos.

Resultados: Analisados15casosdetumoresavançadosdelaringe(T3/T4)nãoglóticos,sendo setecompresençademetástaseslinfáticascervicais.Nãohouvecorrelaçãoentreaexpressão doVEGFeapresençademetástasescervicais.

Conclusão:OVEGFfoipoucoexpressadonoscasosestudadosenãofoiobservadasuacorrelac¸ão comadisseminac¸ãodemetástaselinfáticacervical.

Introduction

Angiogenesis is characterized by the formation of new capillary blood vessels that originate from a pre-existing vasculature.This processisessentialtoprovidenutrients, oxygen,andgrowthfactorsthat supportcellfunction and survival.Therefore,itisassociatedwithtumorgrowthand metastasis.1,2

Acomplexinteractionbetweenendothelialcells, extra-cellularproteinmatrix,andsolublefactorsinplasmaoccurs inangiogenesis.Endothelialcellsleavetheirquiescentstate whenstimulatedbyVEGF(vascularendothelialgrowth fac-tor)andinitiatethefollowingsteps:dissolutionofthebasal membrane;migrationandproliferationofendothelialcells; capillary tube formation; and maturation and survival of newlyformedvessels.Thisensurestherequiredblood sup-plyforneoplasticevolution.1,2

Squamouscellcarcinomaisthemostcommonneoplasm ofthe larynxanditsprognosisdependsonthesizeof the lesion,theleveloflocalinvasion,cervicallymphaticspread, andpresenceofdistantmetastases.1,3

Histologically,squamouscellcarcinomaofthelarynxand hypopharynx aresimilar to that of other regions and are classified into different degrees of differentiation by the neoplastic proliferation of squamouscells and the nature of tumor infiltration. Depending on the degree of kera-tinizationand cellular atypia, theyare classified aswell, moderately,orpoorlydifferentiated.3

Lymphatic drainageof the glotticregionis scarce; this decreasesthemetastaticpotentialoftumorsthatdevelopin thisregion.Thesupraglotticandsubglotticregionshaverich lymphatic drainage, with the supraglottic region draining mainlyintotheupperjugulocarotidchainlymphnodesand toalesserdegreeintothemiddlejugulocarotidchainlymph

nodeswhereasthesubglotticregiontendstodrain ipsilat-erallytothemiddleandlowerjugulocarotidlymphnodes, thatmaydraintotheanterosuperiormediastinalchain.3_For

thisreason,wedecidedtoexcludeglottaltumorsfromthe study.

Theliteraturediscussingtreatmentofthesetumorshas increasedandstudiesofnewchemotherapeuticagentsand targeted therapies have gained prominence. Examples of thisincludethepromising resultsreportedforcetuximab, amonoclonalantibody thatblocksendothelialgrowth fac-torreceptor(EGFR)usedforthetreatmentofheadandneck cancer,andbevacizumabandotherrecombinantanti-VEGF monoclonalantibodies.4,5

TheseantibodiesbindtoVEGFisoformswithhighaffinity andpreventthecytokinefrombindingwiththeendothelial cellreceptorandtriggeringthe angiogenesisprocess.The useofangiogenesis-inhibitingmonoclonalantibodiesforthe treatmentofcolorectal,kidney,andlungcancerhasalready beenapprovedbytheFDA.Forthetreatmentoflarynx/head andneckcancer,theyarestillbeingassessed.4,5

ConsideringthatVEGFplaysanimportantroleintumor angiogenesisandthatthisfactor is associatedwithtumor formation,progression,andmetastasis,itsexpressioncould berelatedtoincreasedtumoraggressivenessevidencedby cervicalmetastaticspread.2

The present studyaimedtoevaluate theexpression of VEGF markerin advanced, non-glottic squamouscell car-cinoma of the larynx (T3/T4) and correlate it with the presenceorabsenceofcervicallymphnodemetastasis.

Methods

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cohort,usingmaterials (surgicalspecimens)obtainedfrom theresectionofnon-glotticsquamouscellcarcinomaofthe larynxfromsurgeriesinatertiaryuniversityhospitalduring aspecified periodoftenyears.Theproceduresperformed withthepatientswerehemi-laryngectomyortotal laryngec-tomy,withrespectivelymphadenectomy.

Patients were divided by the presence or absence of metastases and by surgical-clinical staging. Cases of non-glottic squamous cell carcinoma of the larynx and hypopharynx were obtained from the tumor bank of the PathologicalAnatomyDepartmentofatertiaryhospital.Of these,15casesofadvancednon-glotticlarynxtumorswere selected,aswellasthreecasesoftumorsofthebaseofthe tongue,tencasesofsupraglottictumors,andtwocasesof hypopharynx/pyriformsinustumors.

Inclusionandexclusioncriteria

Tumorswhose paraffin blockswerelocatedand contained enough material for new sections were included in the survey. We required adequate tissue and data for stag-ing each lesion by its tumor location, and determining lesion size,level of invasion into adjacent tissues, lymph nodeinvolvement,anddistantmetastasis.Clinicaldataand tumorstagingwereobtainedfrompatients’records.

Allcases that werenot in accordancewiththe above-mentionedcriteriawereexcluded.

Immunohistochemicalanalysis

Immunohistochemical analysis was carried out using the avidin-biotin-peroxidase method and the VEGF antibody (type IgG1, 1:100 dilution, Santa Cruz, pretreated with citratebuffer).

Positiveandnegativemarkercontrolswerepreparedin ordertocompare withthe studiedcases.Lymphoid tissue wasusedaspositivecontrol(aspermanufacturer’s instruc-tions).

Slidereadingwascarriedoutrandomly,byaninvestigator whoneitherhadknowledgeofthepreviousdiagnosisgiven bythe pathologist norknowledge of thepatient’s clinical status,bycomparingthe slideswithpositiveandnegative controls.Scannedphotographsathighmagnification(400×) were made of highly reactive areas (hot spots), using a NikonCOOLPIX Camera 995. Afterthat, the imageswere senttothecomputer forhistologicalanalysis,which used theImagelab2000software.

Allparameterswereevaluatedinablindedmanner. Finally, thehistologicaland immunohistochemical find-ingswere correlatedwiththe TNMstage (TNM-UICC-AJCC Classification[2010]).6

ThedegreeofVEGFexpressionwasbasedontheGerman scaleof immunoreactivity.This consistsinmultiplyingthe coefficientoftheimmunomarkerexpressionintensitybythe percentagecoefficientofpositivecells.

The coefficientofthe immunomarkerexpression inten-sity was graded from 0 to 3, thus characterized: 0, no staining; 1, stained weakly; 2, stained moderately; 3, stainedstrongly.

Toobtainthepercentagecoefficientofpositivecells,the following sequence wasused: coefficient, represented by

Table 1 Distribution of 15 patients according to TNM staging.

T3 T4

N0 4 7

N1/N2/N3 2 2

thenumberofpositive(stained)andnegative(notstained) cells,wascountedonasamplespaceofatleast500cells, and stratified asfollows: coefficient of 0 ifthere wasno staining;coefficientof1iftherewas1---10%positivity; coef-ficientof2iftherewas11---50%positivity;coefficient of3 iftherewas51---80%positivity;andcoefficientof4ifthere was81---100%positivity.

The final value of the German scale of immunore-activity (coefficient of the immunomarker expression intensity×coefficient of positive cell percentage) ranged

from 0 to12, stratified as: 0 (negative), 1---4 (poor), 5---8 (moderate), and 9---12 (strong). To facilitate the analysis, the negative/weakcases were considered asgroup 1 and themoderate/strongcasesasgroup2.

Statisticalanalysis

Afterpoolingthedata,statisticalanalyseswereperformed using the IBM software SPSS Statistics®_, _using _the chi-squaredtestandFisher’sexacttest.

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

• GermanScaleofImmunoreactivity:

Negativeandweak---Group1 Moderateandstrong---Group2

• Cellproliferationindex:

Lowandslightcellproliferation---Group1 Moderateandhighcellproliferation---Group2

• TNM(TNM-UICC-AJCCClassification---2010)6

T1andT2---Group1/T3andT4---Group2 N0---Group0/N1,N2,andN3---Group1

Ethicalaspects

The study was approved by the Ethics Committee of the institution(485/11).

Results

Atotalof15caseswereevaluated,includingfourwith cer-vicalmetastases.

Thestagingofthesetumors,definedthroughTNM(2010), isdescribedinTable1.Itcanbeobservedthatonlyfourof the15caseshadcervicalmetastases.Table1furtherdetails thedistribution ofthefour caseswithcervicalmetastasis accordingtotheTclassification.

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Table2 Distributionofgroups1and2(VEGFexpression) in15cases,theircorrelationwiththepresenceofcervical metastases,andstatisticalanalysis.

n N0 N1/N2/N3

Group1

(negative/weak expressionVEGF)

11 7 4

Group2

(moderate/strong expressionVEGF)

4 4 0

VEGFvs.N0orN1/N2/N3

Chi-squaredtest 0.159 Fisher’sexacttest 0.516

VEGF,vascularendothelialgrowthfactor.

inepithelialcellsofsquamouscarcinomaswasobservedin only26.6%ofthecasesstudied.

Table2alsoshowsthecorrelationofVEGFexpressionin groups 1 and 2, according tothe presence or absenceof cervicallymphnodemetastasis,withp=0.363.

There was no correlation between the overexpression of this marker with the presence of cervical lymph node metastasis(p=0.159).

Discussion

Intheliterature,studiesevaluatingheadandnecktumors, which include not only squamous cell carcinomas of the larynx (glottic and non-glottic), but also oral cavity and pharynx tumors, have reported an increase in VEGF correlatedwithincreasedangiogenesis,withneoplastic pro-gression,andwithprognosis;theanalysestookintoaccount thepositivityofthemarker,regardlessofitsintensity.1,2,7---9

Thereis only onerecent studythat specificallyaddressed laryngealcanceranditsassociationwithVEGF.Inthiscase, thepositivityof thismarkerwashigherthan72%,10 _unlike

theresultsfoundhere,perhapsduetothesmallnumberof casesinthepresentsample.

Thereisdivergenceamongauthorsregardingthe corre-lationbetweenVEGFexpressionandthepresenceoflymph nodeanddistantmetastases.Someauthorsfound positive resultsbetweenVEGFexpressionandthepresenceof cervi-callymphnodemetastasesanddistantmetastases.2,8,9,11

The meta-analysisbyPanayiotisetal.foundthatVEGF correlates with poor survival in patients with head and neck squamous cell carcinoma; however, the correlation of VEGF overexpression withthe presence of lymphnode metastasis/distant metastasis is yet to be established.12

Boonkitticharoen also found no correlation between this markerandmetastases.13

A recent Asian study reported a close association between VEGF-C/VEGFR-3 expression and lymph node metastasesinsquamouscellcarcinomasofthelarynx.The expressionofthesemarkerswaspositiveonlyinthe analy-sisofcervicalmetastases,anddidnotcorrelatewithage, gender,Tstage,orprimarylocationofthelaryngeallesion.14

The antibody againstepithelial growthfactor receptor (EGFR) has already been approved by the FDA (Food and DrugsAdministration)forafewyearsforthetreatmentof

headandneckcarcinoma.However,itsuseinpatientswith tumorrecurrenceand/ormetastaticdiseasehasshownlittle efficacy.VEGFhasbeenimplicatedasapotentialmechanism ofresistancetoanti-EGFRtherapy.Therefore,recent clini-caltrialsseektheuseofanti-EGFRdrugsincombinationwith anti-VEGFantibodies(bevacizumab),acombinationthathas beenwelltoleratedbypatients(PhaseI/IIstudies).15---17

Currently, several phase II clinical trials have stud-ied the role of bevacizumab in tumors of the head and neck,includingitscombinationwithcetuximab,cisplatin, and radiotherapy for non-metastatic stage III/IV cancer; witherlotinibin locoregionaladvanced and/or metastatic disease; and with cetuximab, docetaxel, and cisplatin (induction) followed by cetuximab, cisplatin, and radio-therapy in previously untreated locoregional advanced disease.18

Theemergenceofthispromisingtreatmentraises impor-tantissues.Withbroad indications,theindiscriminateuse of bevacizumab can develop resistance. Therefore, stud-ies have sought to define and validate biomarkers of its efficiency. Until then, specific VEGF genotypes and arte-rialhypertension(inducedoraggravatedbythetreatment) appeartoberelatedtoabetterresponse.19_Other_markers

suchasCD105, CD34,andCD31,relatedtotheprocessof angiogenesis,maybeusefulintheidentificationofpatients whoarecandidatesformoreaggressiveanti-neoplastic ther-apy,includingtheuseofmonoclonalantibodies.20

SomeauthorsbelievethattheregulationofVEGF expres-sion can be modulated by the microRNA-206, and this pathwaycanbeusedin thefuturefor anticancertherapy relatedtothistumormarker.21

AlthoughthisstudydidnotobserveacorrelationofVEGF withcervicallymphnodemetastasis,thetargetedtherapy hasshownpromisingresultsforthetreatmentofheadand neckcancer.22

Conclusion

VEGFwasscarcelyexpressed,andnocorrelationwith cer-vicallymphmetastasisspreadwasobservedinthissample of15cases.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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