Study of Prevalence of Koilocytes in Oral Squamous Cell Carcinoma

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Downloaded From IP - 223.230.126.189 on dated 21-Nov-2020 Karnataka, India [4]. a roleintheinitiationordevelopmentoftheselesions papilloma virus(HPV)havebeendocumentedtoplay factors, includingDNAviruses,especiallyhuman the mostcommonriskfactorsfororalmalignancy. Other multifactorial etiopathogenesis. Tobacco andalcoholare (OSCCs) arecharacterisedbymultiphasicand countries [1–3].Oralsquamouscellcarcinomas the rateof3to4/1000,000/yearfoundinwestern 1000,000 person/years.Theincidencerateishigherthan standardised incidenceratevariesbetween7and17/ in malesandthirdmostcommonamongfemales. Age cancer casesandranksfirstamongtheall In India,oralcancerconstitutearound9.8%oftotal INTRODUCTION T. Smitha Study ofPrevalence ofKoilocytesinOralSquamousCellCarcinoma Vol. 4,No.2,June2016,pp-64-70 Indian JournalofMednodentand Allied Sciences *Corresponding authoremail id: [email protected] Rajiv GandhiCollegeofDental Sciences,Bengaluru,Karnataka,India 2 1 Original Research Principal &Professor(Director), Professor, DepartmentofOralPathology, VS DentalCollegeandHospital,K.R.Road, V.V. Puram,Bengaluru-560004, KEYWORDS: presence ofkoilocytesisreliableforthedetectionHPVinroutinehistopathologyOSCCs. the determinationofpresencekoilocytesandwereanalysed. OSCC. Haematoxylinandeosin-stainedslidesweresubmittedtoexamineunderlightmicroscopy, specificallyfor on 60paraffin-embeddedtissuespecimensthatwereobtainedfrompatientswithahistopathologicaldiagnosisof study wastoverifytheprevalenceofkoilocytesinOSCCs. and isacceptedaspathognomonic(characteristicofaparticulardisease)HPV infection. of HPVinfection.Koilocytesareepithelialcellscharacterisedbyperinuclearhaloessurroundingcondensednuclei affect theoralcavity. The mostknownviralcytopathiceffect iskoilocytosis, consideredtobeamajorcharacteristic oropharyngeal carcinomas.Oralsquamouscellcarcinoma(OSCC)represents90%ofallmalignanttumoursthat case–control studiesandsystematicreviewindicatethatHPVisanindependentriskfactorfororal (HPV) astheetiologicalfactorforcervicalcancer, theresearchhasbeenrobustinthisfield.Morerecentdatafrom Ever sinceDr. HaraldzurHausenwonthenobleprizeinmedicine2008fordiscoveringhumanpapillomavirus ABSTRACT 1 *, C.V. Mohan Oralsquamouscellcarcinoma,cancer, HPV, DNA Virus, Oralcarcinogenesis 2 3 , S.Hemavathy Professor andhead,Department ofOralPathology, DentalCareandResearchCentre,Sri 3 promised areatgreaterriskofacquiringoralHPV of sexualpartnersandthosewhoareimmunocom- practise oral-genitalsex,thosewhohavehadanumber to childtransmissionorbyautoinoculation.Personswho frequently bynon-sexualdirecttransmission,mother is acquiredprimarilybysexualtransmission,orless squamous cellcarcinoma(SCC)[5].OralHPVinfection cell transformationandtosubsequentdevelopmentof susceptible toadditionalgeneticalterationsleading genomic instabilityinthesecells,makingthem HPV-cytopositive oropharyngealepitheliuminduces high-risk HPVinfectioninasubsetofsubjectswith benign ormalignantepithelialneoplasms.Persistent association withotherill-definedfactorscaninduce form, asubclinicalformorinwhich is epitheliotropic,anditmaybepresentinalatent HPV Methodology: Conclusion: A retrospectiveanalysiswasconducted DOI :10.5958/2347-6206.2016.00010.8 Theresultssuggestthatthe I ndian Aim: J ournals.com A productofDiva The aimofthis Enterprises Pvt. Ltd. 64 www.IndianJournals.com Members Copy, Not for Commercial Sale

Downloaded From IP - 223.230.126.189 on dated 21-Nov-2020 patients withahistopathologicaldiagnosisofOSCC. embedded tissuespecimensthatwereobtainedfrom a retrospectiveanalysiswasconductedon60paraffin- not available. To predictthepresenceofHPV infection, of viralparticleswhenmolecularbiologymethodsare diagnosis, anditisausefulmethodfortheobservation most commonlyusedmethodfororalpathology Histopathological analysisunderlightmicroscopyisthe (Reid (characteristic ofaparticulardisease)HPVinfection Koilocytosis isacceptedaspathognomonic vacuolation) surroundingcondensednuclei. cells characterisedbyperinuclearhaloes(cytoplasmic cervical intraepithelialneoplasia.Theyareepithelial HPV infection.Koilocytesarecommonlypresentin 1956 isalong-recognised,pathognomonicfeatureof Koilocytosis firstdescribedbyLeopoldoKoss oral keratinocytes supported bytheabilityofhighriskHPVtoimmortalise importance ofHPVinfectioninoralcarcinogenesisis implicated inthedevelopmentofOSCC[7].The and inOSCC;particular, HPV-16 havebeen prevalent inpotentiallymalignantoralepitheliallesions, HPV genotypes,inparticularHPV-16 and-18,are and occasionallyregressspontaneously[6].High-risk multiple orclustered,areusuallypainlessandchronic surface. The lesions,canbesingle, or ‘cauliflower’-like exophytic, sessileorpedunculated,ofhavingfiliform of oralwartssharethecharacteristicsbeing disease), collectivelytermedoralwarts.Thefourtypes acuminatum andfocalepithelialhyperplasia(heck papilloma, verrucavulgaris(commonwart),condyloma HPV-associated benignorallesions,squamouscell uncommonly high-risktypeshavebeenfoundinthe infection. Manyofthelow-riskgenotypesand Pathology andMicrobiology, V.S. DentalCollege, specimens retrievedfromDepartmentofOral tissue sectionsobtainedfromthebiopsy This retrospectivehistologicalstudywasconductedon MATERIALSMETHOD AND ninJunlo endn n lidSine 65 Indian JournalofMednodent and Allied Sciences et al. , 1982)[9–11]. in vitro Study ofPrevalenceKoilocytesinOralSquamousCellCarcinoma [8]. et al. in (27/60) presentedkoilocytosis, andwereidentifiedas OSCCs wereanalysed.Forty-fivepercentofthecases requisition recordsofthe60exophyticlesions Concomitantly, thedemographicdatafrom the RESULTS (Figure 4). in thecellcytoplasmsurroundingnucleus was determinedbythepresenceofahaloorvacuole the neoplasticarea).Theidentificationofkoilocytes neoplastic andnonareas(justsurrounding 10 highpowerfields(HPF)wereseeninboth considered significantifmorethanfivekoilocytesper In thisstudy, thepresenceofkoilocytesintissuewas Koilocyticnuclearchange:pyknosis– 2. Koilocyticcytoplasmicvacuolisation:perinuclear 1. as follows: of koilocytosisonthoseestablishedbyReid changes andwebasedsimplifiedhistologicaldefinitions grading andcellulardetailsespeciallyforthekoilocytic eosin. Analysis wascarriedoutwith emphasisonthe SCC sampleswerestainedwithhaematoxylinand µm-thick sectionsobtainedfromtheparaffin-embedded SCCs wereincludedinthisstudy. Forthisstudy, 4- verrucous carcinomaorotherhistologicalvariantsof histopathological diagnosis(Figures1–3).Nocasesof verrucous orpapillaryOSCCsconfirmedby Committee. Thesampleswereselectedfromexophytic getting theapprovalbyInstitutionalEthics archives oftheDepartmentOralPathologyafter Bangalore. Thespecimenswereretrievedfromthe atypia [10]. up amajorityofthecellswithassociatednuclear terms, largecellswithperinuclearclearingtake cycle checkpoint(Cho caused byHPV-influenced activityattheG2cell known that‘binucleation’involvesmultilobules acentric. Binucleationmaybeapparent.Itisnow staining properties.Thenucleusisfrequently material thatmaybeirregularinsize,shapeand haloes surroundingcellnuclei. et al. , 2005).Insimple et al . (1982) www.IndianJournals.com Members Copy, Not for Commercial Sale

Downloaded From IP - 223.230.126.189 on dated 21-Nov-2020 or centralposition. nuclei beingseen.Thewereeitherinaperipheral very variable,withrounded,irregularandsickleshaped The shapesofthenucleiwithinkoilocyteswerealso percentages. within thekoilocytesasshownin Table 4in in Table 3andascorefortheextentofvacuolation for thenuclearmorphologyofkoilocytesasshown each section,anditwasgradedaspercentilescores The degreeofkoilocytosiswasassessedseparatelyin Table 2:Koilocytepositivityindifferent grades ofOSSC which istabulatedin Table 2. SCCs andin2casesofpoorlydifferentiatedSCCs, carcinomas, in11 cases ofmoderatelydifferentiated present inthe21casesofwell-differentiated Koilocytosis wasfoundin5–7perHPFand the floorofmouth. hard palate,2casesoftongueand1casewasinvolving was buccalmucosawith8cases,4casesinvolvedthe site with11 cases,andthesecondmostprevalentsite K+ cases,gingivobuccalsulcuswasthemostprevalent records. Thetumourlocationvaried.Inthepositive patients didnothaveanyhistoryofhabitsintheir with bothtobaccoplusalcoholhabit(23).Three in thissubgroup,mostofthetumourswereassociated smokeless tobaccoabuserswerehighinnumber(11) 82 years(meanage=48.9)intheK+patients. Although, and 14patientwerefemale,agesrangingfrom33to The patients’recordsshowed13patientsweremale Table 1:Prevalence ofkoilocyteinOSSCcases Table 1. positive casesofkoilocytosis(K+)asshownin 66 orydfeetae C 2/5 Poorly differentiatedSCC Moderately differentiatedSCC Well-differentiated SCC Negative Positive Koilocyte Number ofCases 33/60 27/60 T. Smitha,C.V. Mohan,S.Hemavathy 11/24 14/31 7). Binucleationwasseeninfewcases(Figure8). nuclei andpaleabundantcytoplasmwereseen(Figure carcinomas, alteredepithelialcellwithvesiculated In moderatelydifferentiatedandpoorly clear cytoplasmanddarkstainednucleus(Figure6). keratin formationsurroundedbykoilocytesexhibiting but notinallofthekoilocytes.Focalareasshowed shaped nuclei)wasidentifiedinamajorityofcases (Figure 5).Pyknosis(intenselystainedandirregularly perinuclear clearingalongwithabundantcytoplasm changes inthecellwithsmalldarknucleiand In well-differentiatedcarcinomas,koilocytesshowed Table 4:Extent ofvacuolationinkoilocytes Table 3:Nuclear morphologyofkoilocytes with relativelysmallbutirregular andhyperchromatic Koss evidence ofHPVinfection[12]. intermediate cellsandprovidethemostreliable changes predominantlyaffectsuperficialand of thehostepithelialcellaskoilocytes[11]. These differentiation factorsexpressedwithinvariouslayers the productivecycleofHPVisintimatelylinkedwith that caninfectepithelialcells.Insquamousepithelium, HPVs aresmalldouble-stranded,circularDNAviruses DISCUSSION hp iar-nuae,t vltitd19 Bizarre-angulated, toovaltwisted Shape Size Grade 3 Grade 2 Grade 1 Staining et al . coinedthetermkoilocyteforlargecells ecito % 21 cell periphery a thinrimofcytoplasmaround 47 Extensive clearing,leavingonly 32 to halfofcytoplasm Perinuclear clearingextending rim roundthenucleus Slight clearingtoformaclear Description ecito % 23 58 often pyknotic Dense, homogeneous,appearance Larger thannormal Description Vol.No. 2,June2016 4, www.IndianJournals.com Members Copy, Not for Commercial Sale

Downloaded From IP - 223.230.126.189 on dated 21-Nov-2020 proposed bySyrjänen precancerous lesionsandoralcarcinomawasfirst A possibleinvolvementofHPVinthedevelopment [16]. mutations, down-regulationofp16andrBup-regulation related oropharyngealcancerischaracterisedbyp53 regulation andp16up-regulation.Bycontrast,tobacco- degradation, retinoblastomaprotein(rB)down- The biologyofHPV-positive canceristypifiedbyp53 susceptible tomutationsandcancerformation[16]. pathways, respectively, renderinginfectedcells ( oncoproteins E6andE7,whichtargetthep53pRB the immortalisingandtransformingpropertiesofHPV The oncogenicnatureofhighriskHPVsisbecause SCC and20–72%of(OPSCC)oropharyngealSCC. 70% ofanogenitalcancers,5%non-oropharyngeal established initiatorsofover90%cervicalcancers, types HPV16and18whicharebothwell- proliferation ofepithelium,andthehigh-riskoncogenic such asHPV 6andHPV 11, responsibleforbenign different HPVsubtypes,includingthelow-risktypes oncovirus andisepitheliotropic.Thereareover120 From abiologicalpointofview, HPV isaDNA viral-laden nucleifromHPVlesions[15]. contribute tokeratinocytefragilityandthereleaseof apoptosis [13,14].Cytoplasmicvacuolisationcould by theE6oncoprotein,whichisknowntoinhibit and high-riskHPVinfections;koilocytosisispromoted clinical biopsies,koilocytosisisobservedinbothlow- virion assemblyoccurexclusivelyinthenucleus.In particularly becausebothHPVDNAreplicationand of thecytoplasmicvacuolehasremainedunclear, by alarge perinuclearvacuole.However, thegenesis an acentric,hyperchromaticnucleusthatisdisplaced koilocytes aresquamousepithelialcellsthatcontain meaning holloworcavityasitsdesignation.These koilocytic atypiafromtheGreekword‘Koilos’ space fordescriptivepurposes,theycoinedtheterm As thenucleusseemtobesuspendedinanempty nuclei surroundedbyclearandtransparentcytoplasm. ninJunlo endn n lidSine 67 Indian JournalofMednodent and Allied Sciences retinoblastoma protein et al. Study ofPrevalenceKoilocytesinOralSquamousCellCarcinoma (1983);basedonlight ) tumoursuppressor OSCC inIndiahasbeenreported asrangingfrom20% around 45%,andtheoverallprevalenceofHPVin In ourstudy, theprevalenceofHPV infectionwas Younger age,goodperformancestatus,fewer 5. Immunologicresponsemayplayaroleinthe 4. TheabsenceoffieldcancerizationinHPV-positive 3. HPV-positive tumourshavehigherradiosensitivity, 2. HPV-positive tumoursmayharbourfeweror 1. better prognosis? Why doesHPVpositiveoropharyngealcancerhavea HPV negativecancerpatients. and disease-freesurvivalcomparedwithpatients positive cancerhaveasignificantlyimprovedoverall Several studieshaveshownthatpatientswithHPV- diagnosis ofHPVinOSCC[19]. koilocytosis canbereliablyusedasmarkersforthe positive andnegativecases,whichindicatedthat differences forkoilocytesbetweenHPV(16,18) In arecentstudy, therewasstatisticallysignificant associated orallesions[18]. was consideredasapathognomonicsignofHPV- hyperkeratosis, acanthosisandparakeratosis,koilocytes aspects, suchaskoilocytes,dyskeratosis,papillomatosis, [17]. InastudybasedonHPV-related histopathological precancerous lesionsanduterinecervixcarcinoma biopsies thatwereidenticaltothosefoundin HPV alterations(koilocytosis)in35%oftheOSCC microscopy examination,theyobservedcytopathic survival [20]. cancer patientsmayalsocontributetoimproved comorbidities ofHPV-positive oropharyngeal antigens). immune responsedirectedtoviralspecifictumour HPV positivetumours(duetothestimulationof improved responsetoradio-andchemotherapyin tumours. radiation. probably duetointactapoptoticresponse associated withbetterresponsetotherapy. different geneticalterations,whichcanbe www.IndianJournals.com Members Copy, Not for Commercial Sale

Downloaded From IP - 223.230.126.189 on dated 21-Nov-2020 anterior region,andpalatalregionrespectively Figure 1,2,3:clinicalphotomicrographoforalcavityshowinggrowthinrightmandibularvestibule,maxillary 68 Figure 5:Koilocyteswithperinuclear clearing Figure 4:Koilocytes T. Smitha,C.V. Mohan,S.Hemavathy surrounded bykoilocytes Figure 6:Focalareasshowedkeratinformation Figure 7: Altered epithelialcellwithvesiculatednuclei Figure 8:Binucleation Vol.No. 2,June2016 4, www.IndianJournals.com Members Copy, Not for Commercial Sale

Downloaded From IP - 223.230.126.189 on dated 21-Nov-2020 in Western India.Chocolatewala 33.6% inEasternIndia,67%SouthIndiaand15% also showsregionalvariation.Ithasbeenreportedas to 50%inIndia.TheprevalenceofHPVOSCC 6 CameronJE,HagenseeME.OralHPVcomplications [6] FellarL,KhammissaR, Wood N,LemmarJ.Epithelial [5] Vidal L,Gillison ML.HumanpapillomavirusinHNSCC: [4] Rajendranand R,ShivapathasundharamB,editors. [3] AdelsteinD,RidgeJA,GillisonML, [2] Parkin DM,BrayF, FerlayJ,PisaniP. Globalcancer [1] REFERENCES incorporated inhistopathologicalreportingofOSCC. predicting HPVinfectioninOSCC,andthishastobe the useoflightmicroscopicfeaturekoilocytefor of koilocytosis–45%inOSCCs.Thus,thestudypoints In thisstudy, therewasasignificanthighprevalence CONCLUSION the futureforpatientswithOSCCs. report. Thiswillallowforpossibletargetedtherapyin the statusmustbeincorporatedintopathology aetiological factorinoralcancers,it’s ouropinionthat is agrowingbodyofevidencethatHPVasan histopathological diagnosisroutinelyofOSCC. As there At presenttime,HPVstatusisnotreportedinthe for HPV18inOSCCcases[21]. prevalence of17.6–41.8%forHPV16and0–47.3% ninJunlo endn n lidSine 69 Indian JournalofMednodent and Allied Sciences 2008;5:126–31. in HIV-infected patients.CurrHIV/AIDSRep 16. Agents Cancer2009;4:16.DOI:10.1186/1750-9378-4- maturation andmolecularbiologyoforalHPV. Infect Clin North Am 2008;22:1125–42. recognition ofadistinctdiseasetype.HematolOncol NY, USA:Elsevier;2009. Shafers textbookoforalpathology. 6thed.New York, Head Neck2009;31(11):1393–422. national cancerinstitutestateofthesciencemeeting. neck squamouscellcarcinoma&HPV: summaryof statistics, 2002.CACancerJClin2005;55(2):74–108. Study ofPrevalenceKoilocytesinOralSquamousCellCarcinoma et al. et al. reporteda Headand [17] [16] ZG, Liu LN, Zhao TT, Li [15] [14] [13] [12] [11] [10] FornatoraM,Jones AC, KerpelS,FreedmanP. Human [9] SugermanPB,ShillitoeEJ.Thehighriskhuman [8] ShettyK,LeighJ.Malignanttransformationofhuman [7] chemical evidencesuggesting humanpapillomavirus Nuutinen J.Morphologicalandimmunohisto- Syrjänen K,S,LambergM,Pyrhönen 2014;5[Jun (12)]:3956–69. associated headandneckcarcinomas.Oncotarget Zaravinos A. An updatedoverviewofHPV- World JGastroenterol2005;11:931–7. of apoptosisbythepapillomavirusE6oncogene. Biosci 2003;28:337–48. oncoproteins: implicationsfortumourprogression.J “high risk”humanpapillomavirusesE6andE7 Chakrabarti O,KrishnaS.Molecularinteractionsof apoptosis. CancerLett2002;188:15–24. human papillomavirusoncoproteinsE6andE7in of P,role F.Finzer The Rösl Aguilar-LemarroyA, Livingstone; 2003.pp.707–53. cytopathology. 2nded.London:Churchill In: Gray W, McKeeGT, editors.Diagnostic Herrington S.Thepathogenesisofcervicalneoplasia. 2009;101[(Oct (8)]:1351–6. papilloma virusinbreastcancer. BrJCancer Mann L, Lawson JS,Glenn WK, HengB, Ye Y, Tran B,Lutze- Cancer 1982;50:377–87. papillomavirus infectionandcervicalmalignancy. Evidence ofanassociationbetweensubclinical GD, SmithJP. Genital wartsandcervicalcancer. I. Reid R,StanhopeCR,HerschmanBR,BoothE,Phibbs Endod 1996;82:47–56. potential. OralSurgMedPatholRadiol (koilicytic dysplasia):anentityofunknownbiologic papillomavirus-associated oralepithelialdysplasia 47. against acausalrelationship.OralDis1997;3:130– papillomaviruses andoralcancer:evidencefor 6. review oftheliterature.OralOncolExtra2005;41:272– of thetongueinHIVpopulation:casereportand papilloma virallesionsintosquamouscellcarcinoma et al. Koilocytesindicatearoleforhuman Han Y, FanDM.Regulation www.IndianJournals.com Members Copy, Not for Commercial Sale

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