www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Comparative
study
between
biopsy
and
brushing
sampling
methods
for
detection
of
human
papillomavirus
in
oral
and
oropharyngeal
cavity
lesions
夽
,
夽夽
Marise
da
Penha
Costa
Marques
a,b,∗,
Ivo
Bussoloti
Filho
a,c,
Lia
Mara
Rossi
a,d,
Maria
Antonieta
Andreoli
e,
Natália
Oliveira
Cruz
aaFaculdadedeCiênciasMédicas,SantaCasadeSãoPaulo(FCMSCSP),SãoPaulo,SP,Brazil
bHospitalUniversitárioClementinoFragaFilho,UniversidadeFederaldoRiodeJaneiro(UFRJ),RiodeJaneiro,RJ,Brazil cDepartmentofOtorhinolaryngology,IrmandadedaSantaCasadeMisericórdiadeSãoPaulo,SãoPaulo,SP,Brazil dFaculdadedeMedicinadeJundiaí,Jundiaí,SP,Brazil
eInstitutoNacionaldeCiênciaeTecnologiadasDoenc¸asAssociadasaoPapilomavírus,SãoPaulo,SP,Brazil
Received13April2014;accepted8October2014 Availableonline8September2015
KEYWORDS
Polymerasechain reaction; Human
papillomavirusDNA tests;
Mouth; Oropharynx
Abstract
Introduction:Manyepidemiologicalstudieshavesuggestedthathumanpapillomavirus(HPV), especially type16,isinvolvedinthegenesisofsquamous cellcarcinomaoftheoral cavity andoropharynx,especiallyinyoung,non-smokingpatients;thus,itsdetectioninlesionsinthis regionisimportant.
Objective:Toclarifythecapacityofthebrushingsamplingmethodtodetectthepresenceof HPVinoralororopharyngeallesionsthroughpolymerasechainreaction(PCR)testing,andto comparetheresultswiththoseobtainedbybiopsy.
Methods:Prospective studyofadult patientswithoral ororopharyngeallesionsassessed by PCR,comparing biopsyspecimenswithsamplesobtainedbythebrushingmethod.Thestudy wasapprovedbytheResearchEthicsCommitteeoftheinstitution.
Results:A totalof35 samplepairs wereanalyzed,but 45.7%ofthebrushingsamples were inadequate(16/35)and,thus,only19pairscouldbecompared.Therewasagreementofresults
夽
Pleasecitethisarticleas:MarquesMPC,BussolotiFilhoI,RossiLM,AndreoliMA,CruzNO.Comparativestudybetweenbiopsyandbrushing samplingmethodsfordetectionofhumanpapillomavirusinoralandoropharyngealcavitylesions.BrazJOtorhinolaryngol.2015;81:598---603.
夽夽
Institution:FaculdadedeCiênciasMédicasdaSantaCasadeSãoPaulo(FCMSCSP),SãoPaulo,SP,Brazil. ∗Correspondingauthor.
E-mail:mariseorl@globo.com(M.P.C.Marques).
http://dx.doi.org/10.1016/j.bjorl.2015.08.007
in94.7%(18/19)ofthepairs,withHPVidentifiedin16ofthem.HPVDNAwasdetectedin8.6% (3/35)ofbiopsyand5.7%(2/35)ofbrushingsamples.
Conclusion: Therewasnostatisticallysignificantdifferencebetweenthetwomethods,butthe brushingsamplingmethodshowedahighernumberofinadequatesamples,suggestingthatit isanunreliablemethodforsurveillance.
© 2015Associac¸ãoBrasileira de Otorrinolaringologiae CirurgiaCérvico-Facial. Publishedby ElsevierEditoraLtda.Allrightsreserved.
PALAVRAS-CHAVE
Reac¸ãodepolimerase emcadeia;
TestesdeDNApara papilomavírus humano; Boca; Orofaringe
Estudocomparativoentrebiópsiaeescovadonapesquisadopapilomavírushumano emlesõesdecavidadeoraledeorofaringe
Resumo
Introduc¸ão: Muitosestudosepidemiológicosindicamaparticipac¸ãodopapilomavírushumano, especialmenteotipo16,nacarcinogênesedostumoresespinocelularesdascavidadeorale oro-faríngea,principalmenteemjovensenãofumantes,sendoportantoimportantesuadetecc¸ão naslesõesdestaregião.
Objetivo: Elucidarahabilidadedoescovadoemdetectaropapilomavírushumano,pelareac¸ão em cadeiadapolimerase,naslesõesoraiseorofaríngeas,comparandoosresultados comos obtidosporbiópsia.
Método: Estudo prospectivo depacientes com lesõesorais e orofaríngeas, pela reac¸ão em cadeiadapolimerase,noqualforampareadososresultadosdeamostrasobtidasporescovado eporbiópsia.ApesquisafoiaprovadapeloComitêdeÉticaemPesquisadainstituic¸ão. Resultado: Foramanalisados35paresdeamostras,porémestavaminapropriadasparaanálise 45,7%(16/35)dasamostrasobtidasporescovado,eportanto,somente19parespuderamser comparados.Em94,7%dospareshouveconcordânciadosresultados,sendoencontradoo papi-lomavírus humano--- 16em um destes pares. Oácido desoxirribonucleico dopapilomavírus humanofoidetectadoem8,6%(3/35)dasbiópsiaseem5,7%(2/35)dosescovados.
Conclusão:Não houvediferenc¸a estatística entre osmétodos, mas como houveum grande númerodeamostrasobtidasporescovadoinapropriadas,esteparecenãoserconfiávelparao rastreamento.
©2015Associac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial.Publicado por ElsevierEditoraLtda.Todososdireitosreservados.
Introduction
Squamouscellcarcinoma(SCC)comprisesmorethan80%of themouthandoropharynxcarcinomasanditsincidencein theheadandneckhasbeenincreasingoverthelastthirty years,especiallyinnon-smokersandpatientsyoungerthan 45 years of age.1---4 Syrjänen et al. (1983) were the first
tosuggest thathuman papillomavirus(HPV) couldalsobe
involvedinthiscarcinogenesisasitisincervicalcarcinoma;
sincethen,manystudieshavebeenperformedtoestablish
theprevalenceofHPV inthemouthandoropharynx,both
inpatientswithandwithoutlesions.1,2,5,6
Forthesereasons,it appearsimportanttoestablishan
affordableandreliable surveillancemethodfor clinicalor
subclinical infection with high-risk HPV in oral and
orop-haryngealmucosafor headandneckSCCprevention.HPV
detectionmethodsinSCCofthemouthandoropharynxshow
broad variations in sensitivity and specificity, with
preva-lencerangingbetween0%and78%;thus,itisveryimportant
tochooseamethodthathashighsensitivityandspecificity
forHPVdetection.
Currently, the most often used method is reverse
hybridizationwithdegenerateprimerslabeledwithbiotin
found incommercial kits, which allows thegenotyping of
mosttypesofhighandlow-riskHPV.Therearemanyfactors
thatcanaffectviraldetection,suchaslesionlocation,
pres-enceorabsenceofkeratinization,typeofsamplecollected,
andcollection procedure (howthe sample wascollected,
preserved,andextracted),inadditiontothemethodsused
indetection.2,7---9
Biopsy remains the preferred method for obtaining
otopharyngeallesionmaterial,since,inadditionto
provid-ingamoredetailedmorphologicalstudy,thebiopsysample
allowstherecoveryofbasallayercells,wheretheHPVcould
befound in itslatent form.3,10 However,it is a relatively
expensivemethod, asit requires thepresenceof a
physi-cianandsurgical material,whicharenotalwaysavailable
intheserviceunit.
Thisstudyaimedtocompare,throughpolymerasechain
reaction (PCR) and linear array hybridization, HPV
pres-enceinmaterialcollectedbythebrushingsamplingmethod
viabilityofthebrushingsamplingforcollectionofmaterial
frommouthandoropharynxlesions.
Methods
Thiswasaprospectivecross-sectionalstudyof35volunteers
withoralororopharyngeallesionswithindicationforbiopsy,
consecutivelytreatedattheotorhinolaryngologyclinicofa
generalhospital,fromAprilof2012 toDecemberof2012,
whometthe following inclusion criteria: individuals aged
>21yearsofage,withwhiteorred,vegetating,infiltrating,
and/orulceratedlesionsintheoralororopharyngealcavity
lastingmorethan15days.Theexclusioncriteriawere
clin-icalcontraindicationtothesurgicalprocedureandantiviral
use.HPVscreeningresultswerecomparedinthematerial
obtained by brushing sampling method and biopsy of the
samelesion.
TheprojectwasapprovedbytheHumanResearchEthics
Committee,registeredunderNo.192/09.Beforebeing
sub-mittedtomaterialcollection,theselectedpatientssigned
theinformedconsentandansweredaquestionnaireon
epi-demiologicaldatathat included age,gender, tobaccoand
alcoholconsumption,numberofsexualpartnersduringtheir
lifetimeandinthelastsixmonths,typeofpartner,andthe
durationandlocationofthelesion.
Collectionofthebiologicalsampleswasperformedbythe
sameprofessionalin asurgicalenvironment,usingaseptic
techniquesandlocoregionalinfiltrativeanesthesiawith1%
lidocaine.Materialcollectionwasfirstperformedusingthe
brushingsampling method, byrubbing a CytobrushPlusTM
brushoverthelesionusingthreeforward---backward
move-ments, followed by the biopsy performed with a scalpel
blade,avoiding areas of necrosisand,wheneverpossible,
includingtissueadjacenttothelesion.
The brush with the material was stored in a cryovial
containing0.9% aqueoussalinesolution,whichwas
imme-diatelyfrozen inliquidnitrogen at−170◦C.Subsequently,
thebiopsywasperformedandthematerialwasdividedinto
threefragments:thefirstwasplacedina10%formaldehyde
buffered aqueous solutionfor anatomopathological
analy-sis, the second was used in this research, and the third
was stored at the Biobank. The fragments were placed
in separate and dry cryotubesand immediately frozen in
the same container. All samples were transportedto the
freezertogether,remainingfrozenat−80◦Cuntiltheywere
processedbytheMolecularBiologyLaboratory.
Sampleswereprocessedaccordingtoexistingbiosecurity
standards.Thein-housemethodwasusedforDNA
extrac-tion, in which the sample was digested with proteinase
K,followedbypurificationwithphenol/chloroform/isoamyl
alcohol (25:24:1, Invitrogen) and quantified in a
Nan-odrop1000spectrophotometer(ThermoScientific).
ThequalityoftheobtainedDNAwasverifiedby
perform-ingPCRofhuman-globinwiththePCO3/PCO4primerwith
110-bpamplicon(Saiki etal.).Bothpositiveand negative
samplesforhuman-globinweregenotypedusingaLinear
ArrayHybridizationkit(RocheDiagnostics),whichallowsthe
identificationof37typesofHPVofhighandlowriskthrough
linearreversehybridization.
Thestatisticalanalysisofthisstudywasdescriptive,with
thehelpofmeasuresoflocation,andtheresultsareshown
inTable1.The‘‘z’’testwasusedforquantitativevariables.
The nullhypothesis wasnosignificant differencebetween
thetwoproportions,withasignificancelevelof0.05.The
XLSTAT2013.4.02programwasusedtotestthetwo
propor-tions,witharight-tailedone-sided,95%confidenceinterval
forthedifferencebetweenproportions.
The literature review was conductedonline, using the
databases of the US National Library of Medicine of the
National Institutes ofHealth (PubMed)and the Biblioteca
Virtual em Saúde (LILACS), using the following subject
descriptors: polymerase chain reaction, human
papillo-mavirus,oralmucosa,oropharyngealmucosa,oropharynx,
detection,brushing,andbiopsy.
Results
Atotalof35individualswereevaluated,26menandnine
women,withanapproximatemaletofemaleratioof3:1.
Agerangedfrom37to77years,withameanof54years.
Allindividualsdeclaredtheywereheterosexuals.Regarding
socialhabits,11(31.4%)hadneverconsumedalcohol
regu-larly,18(51.4%)werenotcurrentusers,andsix(17.1%)still
consumed;four(11.4%)hadneversmoked,11(31.4%)were
ex-smokers,and20(57.1%)werecurrentsmokers.
SCCwasidentifiedin21(60%)of 35lesions(21/35); of
these,15(71.4%)weremoderatelydifferentiated(15/21),
three (14.3%) were well-differentiated (3/21), and three
(14.3%)werepoorlydifferentiated(3/21).Oftheremaining
lesions(14/35),five(35.7%)receivedan
anatomopatholog-icaldiagnosisofpapillomatosis,three(21.4%)ofsquamous
cellpapilloma;three(21.4%)ofulceratedchronic
inflamma-toryprocesses,two(14.3%)oflymphoma,andone(7.2%)of
fibroepithelialpolyp.
Asfor lesionlocation,ten(28.6%) werelocatedonthe
tongue,of whichsixwereatthebaseof thetongue;nine
(25.7%)inthepalatinetonsil;seven(20%)inthesoftpalate;
three(8.6%)inthecheekmucosa;twoonthemouthfloor
(5.7%),andoneeach(2.9%)inthefollowinglocations:
ante-riorpillar,uvula,lowerlip,andoropharynxmucosa.
Allsamplesobtainedthroughbiopsywere100%positive
for-globin;incontrast,insamplesobtainedbythe
brush-ingsamplingmethod,positivitywas54.3%(19/35).Nineteen
pairswerecompared;in18,therewasagreement
concern-ing the presence or absence of HPV DNA. Of the biopsy
samples,threelesionswerepositiveforHPVDNA:type
HPV-16 in a patient also submitted to the brushing sampling
method,typeHPV-6inapatientwithbaseofthetongue
lym-phomaandHPV-11inanoropharynxpapilloma.Forbrushing
sampling cases, HPV DNA was isolatedfrom two cases of
moderatelydifferentiatedpalatinetonsilSCC(Table1).
Statisticalanalysis
Of the 35 samples obtained by the brushing sampling
method, only 19 were analyzed, which were positive for
the-globinreaction,andofthese,twowerepositivefor
HPV (2/19),withaproportionof0.105263. Asfor the
tis-sue samples obtained by biopsy, 100% were positive for
-globin, withthreebeingpositiveforHPV,witha
propor-tion of 0.085714. Only a pair of positive samples showed
Table1 CharacteristicsofthesamplesandresultsobtainedbyPCRofthe-globingene,viralDNAdetection,genotypingby lineararrayhybridizationofthematerialobtainedbybiopsy,andbrushingsamplingmethods.
Case Characteristicsoflesions Brushingsample Tissuesample
-Globin DNAHPV -Globin DNAHPV
1 TongueSCC Yes No Yes No
2 PalatinetonsilSCC Yes No Yes No
3 OralmucosaSCC Yes No Yes No
4 PalatinetonsilSCC Yes HPV-52 Yes No
5 Ulceratedinflammatoryprocessofpalatinetonsil Yes No Yes No
6 Papillomatosisoforalcheekmucosa Yes No Yes No
7 BaseofthetongueSCC Yes No Yes No
8 Ulceratedinflammatoryprocessofthemouthfloor No No Yes No
9 Hodgkin’slymphomaofbaseofthetongue No No Yes HPV-6
10 PalatinetonsilSCC No No Yes No
11 Softpalatepapillomatosis No No Yes No
12 Softpalatepapillomatosis No No Yes No
13 MouthfloorSCC No No Yes No
14 Lower-lipSCC No No Yes No
15 SCCofthebaseofthetongue No No Yes No
16 Ulceratedinflammatoryprocessoftheoralcheekmucosa No No Yes No
17 TongueSCC Yes No Yes No
18 Softpalatepapillomatosis No No Yes No
19 Palatinetonsilpapillomatosis No No Yes No
20 SCCofthebaseofthetongue No No Yes No
21 Oropharyngealsquamouspapilloma No No Yes HPV-11
22 Non-Hodgkinlymphomaofpalatinetonsil Yes No Yes No
23 SoftpalateSCC Yes No Yes No
24 TongueSCC Yes No Yes No
25 PalatinetonsilSCC No No Yes No
26 PalatinetonsilSCC Yes No Yes No
27 PalatinetonsilSCC Yes HPV-16 Yes HPV-16
28 SCCofthebaseofthetongue Yes No Yes No
29 Tonguesquamouspapilloma No No Yes No
30 Fibroepithelialpolypofthesoftpalate Yes No Yes No
31 Squamouspapillomaoftheanteriorpillar Yes No Yes No
32 SCCoftheuvula Yes No Yes No
33 SoftpalateSCC Yes No Yes No
34 TongueSCC Yes No Yes No
35 SoftpalateSCC No No Yes No
HPV,humanpapillomavirus;DNA,deoxyribonucleicacid;SCC,squamouscellcarcinoma.
sample obtained by the brushing method. The XLSTAT 2013.4.02programwasusedtotestthetwoproportions.The
z-test for twoproportions wasright-tailed andone-sided, witha95%confidenceinterval.Thedifferencebetweenthe proportionswas0.020andthez(observedvalue)was0.230, withz (criticalvalue)of 1.645and p-value (one-sided)of 0.409;showingthattherewasnostatisticallysignificant dif-ference between the proportionsof HPV in both types of samples.
Discussion
Headandnecktumorshave aglobal incidenceof460,000 newcasesperyear,leadingto228,000deathsestimatedin 2014,accordingtotheNational CancerInstituteof Brazil. HPV-16isresponsiblefor25%ofallsquamouscellcarcinomas
oftheheadandneck,anditispresentin45---90%ofallcases oforopharynxtumorsandapproximately24%oflarynxand oralcavitytumors.3
Forthisstudy,thenumberofcaseswasestablishedbased
onthe literature,where the prevalence of HPV in mouth
andpharynxlesionsisapproximately36%(p=0.36),withan
estimatederrorof3%todefinethesamplesizerequiredfor
theanalysis.11---17
In Brazil, in individuals without lesionsand with small
samples up to 100 individuals, the prevalence of HPV
ranged from 0% to 12%,18---21 whereas in other countries,
theprevalencerangedfrom0%22 to97%,23 suggestingthat
theprevalenceoforalandoropharyngealinfectionbyHPV
in the Brazilian population is low; however, a
popula-tionscreeningstudy isnecessarytobetterunderstandthe
Brazilianpopulation.Inthisstudy,thehigh-riskHPVtypes,
HPV-16andHPV-52,andthelow-risktypes,HPV-6and
HPV-11,wereidentified,withanoverallprevalenceof11.4%.The
prevalenceofHPVinoralandoropharyngeallesionsisvery
wide-ranging,varying from0%24 to77.8%9 andin national
studiesrangingfrom0%7to75%19;itwasnotpossibleto
reli-ablycomparetheresults,asthestudieshadverydifferent
methodologies.
TheprevalenceofHPVishigherinpatientswhohadother
sexuallytransmitteddiseases,25withalargenumberoforal
sexpartners a predictive factor for oral HPV detection.23
However,contrarytowhathasbeenexpounded,study
sub-jectswhohadmorethantwentysexualpartnersdidnothave
HPVDNAdetectedintheirlesions.
Todate,thereisnoconsensusonthebestsample
collec-tiontechniqueforpatientswithoralandoropharynxlesions;
thedevelopment newstudies arerequired, and thus it is
importanttocomparethemethods.
When the brushing and biopsy sampling methods were
comparedin thisstudyinsampleswithmouthor
orophar-ynxSCC, therewasno statistical differencebetween the
methods,with94.7%(18/19)agreementinpairsofsamples.
Termineetal.,inasimilarstudy,butnotinoropharyngeal
lesions,observed thatthefrequency of detectionthrough
brushingandbiopsysamplingmethodsalsoshowedno
statis-ticallysignificantdifference,butthebiopsymethodshowed
tobemoreaccurateinhigh-riskHPVdetection.26
Lawtonetal.assessedthreesamplingmethodsand
con-cluded that mouth-rinsing, when used alone is the best
method, but positivity can be increased when combined
withothermethodsformaterialcollection.27 Jarboeetal.
evaluatedthe effectiveness of the hybrid capture in two
oral brushing and oral rinsing samples, finding greater
detection in brushed samples than in those from
mouth-rinsing.28 Read et al. compared three methods, and the
mouth-rinsingmethodshowedhigherdetectionsensitivity,
especiallyinthoseindividualswhohadbrushedtheirteeth
beforerinsing.23
Even though the brushing sampling method was
per-formedaccordingtostandardizedproceduresandfollowing
previously established protocols, 45.7% of the samples
(16/35)wereinadequateforHPVscreening,suggestinglow
sensitivity of the collection method,or incapacity of the
brush,intendedforuseingynecologicalcollection,toobtain
materialfromthemouthandoropharynxlesions,orbecause
themediuminwhichsampleswereplacedwasnotableto
preservethem.Perhapsthe choiceoffixation and
preser-vation solutions to be used with the brushing sampling
method,suchasPreservCytTMsolution,2DigeneTMsolution,29
andphosphate-bufferedsaline(PBS)2,15,26,29---31mayresultin
bettersamplesforcytologicalanalysisthanthoseplacedin
salinesolution,asthatusedinthisresearch.Thecomparison
betweenthefixationsolutionsofthemouthandoropharynx
materialscollectedthroughthebrushingsamplingmethod
couldclarifythisdoubt.
These results confirm the involvement of HPV in oral
andoropharyngeallesions,but furtherstudiesareneeded
to detail the collection method, with the investment in
specifickits thatcandetect scarcecells, whichwillallow
betteridentification ofHPV intheseHPV-related oraland
oropharyngeallesions,resultinginimprovedpreventionand
treatment.
Conclusions
There was nostatistical difference between the brushing
andbiopsy methodsfor detectionofviralDNAinoral and
oropharyngeallesions,butthelargenumberofinadequate
samplesobtainedbybrushingsuggeststhismethodis
ineffi-cientwhenobtainingsamplesfromthistypeoflesion.
Funding
ThisstudywasfundedbyFAPESP.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
ToProf.Dr.LuísaLinaVillaandMs.MariaCecíliaCosta.
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