Brazilian
Journal
of
OTORHINOLARYNGOLOGY
www.bjorl.org
ORIGINAL
ARTICLE
Tissue
expression,
serum
and
salivary
levels
of
vascular
endothelial
growth
factor
in
patients
with
HNSCC
夽
Azadeh
Andisheh-Tadbir
a,
Marzieh
Hamzavi
a,
Gita
Rezvani
a,
Mohammad
Javad
Ashraf
b,
Mohammad
Javad
Fattahi
c,
Bijan
Khademi
c,d,
Fereshteh
Kamali
a,∗aDepartmentofOralandMaxillofacialPathology,SchoolofDentistry,ShirazUniversityofMedicalSciences,Shiraz,Iran bDepartmentofPathology,SchoolofMedicine,ShirazUniversityofMedicalSciences,Shiraz,Iran
cShirazInstituteforCancerResearch,ShirazUniversityofMedicalSciences,Shiraz,Iran
dDepartmentofOtolaryngology,KhaliliHospital,ShirazUniversityofMedicalSciences,Shiraz,Iran
Received21July2013;accepted9March2014 Availableonline13August2014
KEYWORDS Vascularendothelial growthfactors; Squamouscell carcinoma; Serum; Saliva
Abstract
Introduction:Vascularendothelialgrowthfactoristhoughttobeanimportantangiogenicfactor involvedintumorgrowth,progression,andmetastasis.
Objective: The presentstudy evaluated the relationbetween tissueexpression, serum and salivarylevelsofvascularendothelialgrowthfactorinheadandnecksquamouscellcarcinomas, andtheircorrelationwithclinicopathologicfeatures.
Methods:Sampleswerecollectedfrom30patientswithheadandnecksquamouscell carcino-masand24healthyvolunteers.Immunohistochemicalanalysiswasusedfortissueexpression andenzyme-linkedimmunosorbentassaywasemployedtomeasureserumandsalivarylevels.
Results:Novascularendothelialgrowthfactorstainingwasobservedinnormaltissues,whereas vascularendothelialgrowthfactorexpressionwasseenin6patients(20%).Meanserumlevelof VEGFwas83.7±104.47inpatientsand50.04±32.94incontrols.Meansalivarylevelofvascular endothelialgrowthfactorwas174.41±115.07inpatientsand149.58±101.88incontrols.No significantdifferencewasfoundbyMann---Whitneytestbetweencontrolsandpatients(p=0.411,
p=0.944,respectively).Nocorrelationwasfoundbetweenvascularendothelialgrowthfactor tissueexpressionanditsserumandsalivarylevel.
夽
Pleasecitethisarticleas:Andisheh-TadbirA,HamzaviM,RezvaniG,AshrafMJ,FattahiMJ,KhademiB,etal.Tissueexpression,serum andsalivarylevelsofvascularendothelialgrowthfactorinpatientswithHNSCC.BrazJOtorhinolaryngol.2014;80:503---7.
∗Correspondingauthor.
E-mail:feresh876@yahoo.com(F.Kamali).
http://dx.doi.org/10.1016/j.bjorl.2014.03.001
1808-8694/©2014Associac¸ãoBrasileirade OtorrinolaringologiaeCirurgiaCérvico-Facial.Published byElsevierEditoraLtda.Allrights
Conclusion:Overexpressionofvascularendothelialgrowthfactorwasfoundinheadandneck squamouscellcarcinomapatients, suggestingitsroleinthepathogenesisofheadandneck squamouscellcarcinoma,butnorelationwasfoundbetweentissueexpression,serumlevels, andsalivarylevelsofthismarker.
© 2014Associac¸ãoBrasileira de Otorrinolaringologiae CirurgiaCérvico-Facial. Publishedby ElsevierEditoraLtda.Allrightsreserved.
PALAVRAS-CHAVE Fatoresde
crescimento endotelialvascular; Carcinomadecélulas escamosas;
Soro; Saliva
Expressãotecidual,soroeníveissalivaresdeVEGFempacientescomcarcinomade célulasescamosasdecabec¸aepescoc¸o
Resumo
Introduc¸ão:Acredita-sequeofatordecrescimentovascularendotelial(FCEV)sejaum impor-tantefatorangiogênicoenvolvidonocrescimento,naprogressãoenametástasetumoral.
Objetivo:Opresenteestudoavaliaarelac¸ãoentreaexpressãotecidualeosníveisséricose salivaresdoFCEVem carcinomasdecélulasescamosasdacabec¸aepescoc¸o(CCECPs)esua correlac¸ãocomaspectosclinicopatológicos.
Método: Foramcoletadasamostrasde30pacientescomCCECPede24voluntáriossaudáveis. Utilizamosanáliseimuno-histoquímicaparaaexpressãotecidualeELISAparadeterminac¸ãodos níveisséricosesalivares.
Resultados: Nãofoi observadacolorac¸ãopara FCEVnos tecidos normais, enquanto que foi observada expressão de FCEV em seis pacientes (20%). O nível sérico médio de FCEV foi 83,7±104,47empacientese50,04±32,94emcontroles.OnívelsalivarmédiodeFCEVfoide 174,41±115,07empacientese149,58±101,88emcontroles.Nãofoiobservadadiferenc¸a sig-nificativapelotestedeMann-Whitneyentrecontrolesepacientes(respectivamente,p=0,411, p=0,944).Nãofoiobservadarelac¸ãoentreaexpressãotecidualdeFCEVeseusníveisséricos esalivares.
Conclusão:AexpressãoelevadadeFCEVfoiobservadaempacientescomCCECP,eissosugere seupapelnapatogênesedeCCECP,masnãofoiobservadarelac¸ãoentreaexpressãotecidual eosníveisséricosesalivaresdessemarcador.
©2014Associac¸ãoBrasileira deOtorrinolaringologiaeCirurgiaCérvico-Facial.Publicadopor ElsevierEditoraLtda.Todososdireitosreservados.
Introduction
Angiogenesis is an important phenomenon in the devel-opment of tumors and metastasis, which is created by secretion of several growth factors by means of tumor cellsandtumorsurroundings.Althoughtumordevelopment is a multiple phaseprocess, angiogenesisis necessary for tumorgrowthandmetastasis.1,2Vascularendothelialgrowth factor (VEGF) is knownto be a fundamental regulator of angiogenesisthatacceleratescellularproliferation, vascu-larpermeability,andendothelialcellmigration,aswellas functioning as an apoptosis inhibitor.3 VEGF is a heparin bindingglycoproteinanditsgeneislocatedonchromosome 6.1ThisproteinisthemostimportantofVEGFfamilyandhas aroleinvascularpermeability.4InhibitionofVEGFactivity leadstodiminished growth anddecreased tumor progres-sion,whichsuggeststhatithasanimportantroleininitiation of tumor angiogenesis.5 Furthermore, it has been deter-minedthathypoxiacanincreaseVEGFexpression,andthat VEGF overexpressionleads to angiogenesisin the hypoxic areaoftumors.6
VEGF is expressed in several malignant tumors, which indicatesitsimportantroleintheprocessofangiogenesis.
Over-expression of VEGF has been found in solid tumors, suchasthoseofthecolon,kidney,breast,brain,pancreas, bladder,ovary,stomach,lung,andoralcavity.7
RegardingtheroleofVEGFintheangiogenicprocess,the aimofthisstudywastoinvestigatetheexpression,serum andsalivarylevelsofthismarkerinpatientswithheadand necksquamouscellcarcinoma(HNSCC).
Methods
In this cross-sectional study, 30 patients with HNSCC (21 malesandninefemales),whowerereferredtoKhaliliand ChamranHospitalsofShirazUniversityofMedicalSciences, wereexamined.Thecontrolgroupconsistedof24healthy individuals(16maleandeightfemales)whowerematched withthepatientsgroupintermsofageandsex.
H&Eslideswere examined inpatients withHNSCC and thosewith sufficient tissuewere selected for immunohis-tochemical(IHC) studies. Clinicalinformationof patients, includingage,sex,tumor location,tumorsize,metastasis stage and grade, as well as tobaccoconsumption habits, werecollectedfromthepatients’records.TheEthics Com-mitteeofShirazUniversityofMedicalSciencesapprovedthe studyandallpatientssignedaninformedconsent.
To prepare serum, 5cm3 of blood wasdrawn from the
patients’ veins, who were NPO for 12h, in the morn-ing before surgery. The blood samples were immediately centrifuged at 3000rpm for 10min. The serum was then separatedandstoredat−80◦Cuntilanalysis.8
Inordertocollectsaliva,unstimulatedwholesaliva sam-ples werecollectedfromthe patients,whowere NPOfor 12h,inthemorningbeforesurgery.
The patients wereasked torefrain fromeating, drink-ing,andsmokingfor 30min.Then,thepatients’lipswere cleanedandeachpatientrinsedhis/hermouthwithwater. Approximately 5---10mL saliva was collected from every patient. After centrifugation of the samples (2600×g, 15min,40◦C),theywerethenstoredat−80◦Cuntiluse.9
Serumandsalivaanalysis
Concentrationof VEGFwasmeasured usingenzyme-linked immunosorbent assay(ELISA)(Bender Med.SystemsGmbH --- Germany),accordingtothemanufacturer’sinstructions.
Immunohistochemicalstaining
IHCstainingwasperformedbyusingtheEnVision® Labeled
PeroxidaseSystem (DAKO,Carpentaria,CA,USA).All sam-pleswerefixedin10%bufferedformalinandwereembedded in paraffin. Sections with 4m thickness were prepared, deparaffinizedinxylene,rehydratedingradedalcohol,and washed with distilled water. Antigen retrieval was per-formedbyusingDAKOcytomationtargetretrievalsolution with pH=9, for 20min. Internal peroxidase activity was inhibitedby3%H2O2.Tissuesectionswerethen incubated
for30minwiththeanti-VEGFantibody(mouseanti-human; DAKO Corporation --- Denmark) at a 1:25 dilution. Normal samples were stained with the same amount of antibody used for staining tumor tissues. Omission of the primary antibodywasemployedasnegativecontrol,whilepyogenic granulomawasusedaspositivecontrol.Browncytoplasmic stainingforVEGFwasconsideredaspositive.
The stained slides were initially scanned at low mag-nification. For the slidesshowing heterogeneous staining, the regions with higher staining werestudied. Five fields wererandomlychosen,500cellswerecounted,andthe per-centageofstainingwascalculated.The extentof staining wasclassified as: 0if 0---10% oftumor cells were stained, 1if11---25%oftumorcellswerestained,2if26---50% were stained,and3ifmorethan50%werestained.
Statisticalanalysis
Afterentering thedata intothestatistical software(SPSS 18.0),thenormalityofthedatawasfirstexaminedusingthe
Table 1 Clinicopathological profile of30 headand neck squamouscellcarcinomapatients.
Clinicopathologicalprofile n(%)
Gender
Male 21(70%)
Female 9(30%)
Tumorsize
T1 12(40%)
T2 18(60%)
Regionallymphnodeinvolvement
N0 27(90%)
N1 3(10%)
Distantmetastasis
M0 30(100%)
TNMstage
I 11(36.7%)
II 16(53.3%)
III 3(10%)
Histologicalgrade
I(well-diff.) 8(26.7%)
II(moderately-diff.) 18(60%) III(poorly-diff.) 4(13.3%)
Location
Larynx 22(73.3%)
Oralcavity 8(26.7%)
Kolmogorov---Smirnovtest.Asthevariablesunderstudywere not normally distributed, Mann---Whitney, Kruskal---Wallis, andchi-squaredtestswereusedinordertocomparethetwo groups.Differenceswereconsideredsignificantatp<0.05.
Results
Descriptionofvariables
The present study was performed on 30 patients with HNSCC (21 males and nine females) with a mean age of 56.76±12.46years.Clinicopathologiccharacteristicsofthe patientsareshowninTable1.
NoVEGFstainingwasobservedinnormaltissues,whereas VEGFexpressionwasobservedinsixpatients(20%);fourhad scoresof1andtwohadscoresof2.VEGFexpressionwasnot seenaroundkeratinpearls(Figs.1and2).
TheresultsindicatedthatthetissueexpressionofVEGF betweencasesandcontrolsshowedasignificantdifference (p:0.023),suchthatitwashigherintheHNSCCgroupthan the controls. There wasno apparent correlation in VEGF expression with the clinicopathological features, such as stage,tumorsize,nodalstatus,andhistologicalgrade.
1 mm
Figure1 Cytoplasmicstainingofvascularendothelialgrowth factorinsquamouscellcarcinoma(200×).
1 mm
Figure2 Vascularendothelial growthfactorexpressionwas negativearoundkeratinpearls(100×).
No significant correlation was found by Mann---Whitney test between VEGF tissue expression and its serum and salivarylevels(p=0.517,p=0.716,respectively).The rela-tionships between serum and salivary levels of VEGF and othervariablessuchasage,sex,M,N,T,grade,andstage wereseparatelyexamined, and nosignificant relationship wasfound betweenserumor salivarylevelsandthe men-tionedvariables.
Discussion
VEGFhasbeenrecognizedasaneffectivefactorfor induc-tionofangiogenesisandisapotentmitogenforendothelial cells.10Inthepresentstudy,theexpressionofVEGFwasseen in20%oftumorsamples,whilenoneofthetissuesofcontrol group werepositive, and there wassignificant difference regardingtheexpressionofVEGF.Theincreasedexpression ofVEGFinthepresentstudyisinaccordancewiththeother studieswhichwereperformedonoralSCCandHNSCC.10---12 However,therateofexpressionofVEGFinthepresentstudy waslow comparedtoother studies,which maybedue to few patients in this study when compared toother stud-ies.The relationship betweenthe expressionof VEGFand theparameterssuchastumordifferentiation,lymphnodes metastasis, and depth of invasion has been investigated indifferent studies.13 The results regardingthe investiga-tion of relationship between the expression of VEGF and
relationship between the VEGF salivary level and clinico-pathologic factors was not investigated. In reviewing the article,thepresentauthorsdidnotfindanystudieswhich investigatedtherelationship betweenthesalivarylevelof VEGFandclinicopathologicfactors.
Conclusion
Inthepresent study,overexpressionofVEGFwasfoundin HNSCCpatients,whichsuggestsitsroleinthepathogenesis of HNSCC, but no relation was found between the tissue expression,serumlevel,andsalivarylevelofthismarker.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
The authors would like to thank the Vice-Chancellery for Researchof Shiraz University of MedicalSciences for pro-viding financial support for this study (Grant#90-5551). This manuscriptis relevant tothepost graduatethesisof Dr.MarziehHamzavi.
References
1.SulluY,GunS,AtmacaS,KaragozF,KandemirB.Poorprognostic clinicopathologicfeaturescorrelatewithVEGFexpressionbut notwithPTENexpressionin squamouscellcarcinoma ofthe larynx.DiagnPathol.2010;35:35---40.
2.SmithB,SmithGL,CarterD,SasakiCT,HafftyB.Prognostic sig-nificanceofvascularendothelialgrowthfactorproteinlevelsin oralandoropharyngealsquamouscellcarcinoma.JClinOncol. 2000;18:2046---52.
3.Khaled M,LeA, DuongH, Wu Y, ZhangQ, MessadiDV. Cor-relation between VEGF and HIF-1alpha expression in human oral squamous cell carcinoma. Exp Mol Pathol. 2004;76: 143---52.
4.MichaelJC, John D,Taro M, Lena CW.VEGF-receptor signal transduction.BiochemSci.2003;28:488---94.
5.Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS, etal.Inhibitionofvascularendothelialgrowthfactor-induced angiogenesis suppressestumour growth in vivo. NatLondJ. 1993;362:841---4.
6.ShweikiD,ItinA,SofferD,KeshetE.Vascularendothelialgrowth factorinducedbyhypoxiamaymediatehypoxia-initiated angio-genesis.NatLondJ.1992;359:843---5.
7.LiuCJ,Chang KW, LinSC,ChengHW. Presurgical serum lev-els of matrix metalloproteinase-9 and vascular endothelial growth factor in oral squamous cell carcinoma.Oral Oncol. 2009;45:920---5.
8.ShangZJ,LiJR,LiZB.Upregulationofserumandtissue vascu-larendothelialgrowthfactorcorrelateswithangiogenesisand
prognosisoforalsquamous cellcarcinoma.JOralMaxillofac Surg.2007;65:17---21.
9.BrooksMN,WangJ,LiY,ZhangR,ElashoffD,WongDT.Salivary proteinfactorsareelevatedinbreastcancerpatients.MolMed Rep.2008;1:375---8.
10.Connolly DT, Heuvelman DM, Nelson R. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.JClinInvest.1989;84:1470---89.
11.GazzarRE,MacluskeyM,WilliamsH,OgdenGR.Vascularityand expressionofvascularendothelialgrowthfactorinoral squa-mouscellcarcinoma,resectionmargins,andnodalmetastases. BrJOralMaxillofacSurg.2006;44:193---7.
12.ShangZJ,LiJR,LiZB.Circulatinglevelsofvascularendothelial growthfactorinpatientswithoralsquamouscellcarcinoma. IntJOralMaxillofacSurg.2002;31:495---8.
13.LiC,ShintaniS,TerakadoN,KlosekSK,IshikawaT,Nakashiro K.Microvesseldensityandexpression ofvascularendothelial growth factor, basic fibroblast growth factor, and platelet-derived endothelial growth factor in oral squamous cell carcinomas.IntJOralMaxillofacSurg.2005;34:559---65.
14.TseGM,ChanWH,YuKH,KingAD,WongKT,ChenGG,etal.
Strong immunohistochemicalexpressionof vascular endothe-lialgrowth factor predicts overallsurvivalin headand neck squamouscellcarcinoma.SurgOncol.2007;14:3558---65.
15.KyzasPA,StefanouD,BatistatouA,AgnantNJ.Prognostic sig-nificanceofVEGFimmunohistochemicalexpressionandtumor angiogenesisinheadandnecksquamouscellcarcinoma.J Can-cerResClinOncol.2005;131:624---30.
16.Friedrich RE, Klapdor R, Hagel C, Bartel-Friedrich S. VEGF in sera of oral and oropharyngeal squamous cell carcinoma patients.AnticancerRes.2010;30:1765---6.
17.Cheng SJ, Lee JJ, Kok SH, Chou CH, Chang HH, Yang H. Expressionofvascularendothelialgrowthfactorissignificantly associatedwithprogressionandprognosisoforalsquamouscell carcinomasinTaiwan.JFormosMedAssoc.2011;110:50---7.
18.MeyerF,SamsonE,PierreD,DuchesneT,LiuG,BairatiI.Serum prognosticmarkersinheadandneckcancer.ClinCancerRes. 2010;16:1008---15.
19.Ryuto M, Ono M, Izumi H, Yoshida S, Weich HA, Kohno K, etal.Inductionofvascularendothelialgrowthfactorbytumor necrosis factor alpha in human glioma cells. J Biol Chem. 1996;271:28220---8.
20.TelesRP,LikhariV,SocranskySS,HaffajeeAD.Salivarycytokine levelsinchronicperiodontitisandinperiodontallyhealthy sub-jects.JPeriodontalRes.2009;44:411---7.
21.TaichmanNS,CruchleyAT,FletcherLM,Hagi-PavliEP,Paleolog EM, AbramsWR,etal. Vascularendothelialgrowth factorin normalhumansalivaryglandsandsaliva:apossibleroleinthe maintenanceofmucosalhomeostasis.LabInvestJTechMethod Pathol.1998;78:869---75.
22.WuX,ZhaoH,DoKA, JohnsonMM, DongQ,HongWK,etal. Serum levelsofinsulingrowth factor (IGF-I)and IGF-binding proteinpredictriskofsecondprimarytumorsinpatientswith headandneckcancer.ClinCancerRes.2004;10:3988---95.
