Detection of Human Papillomavirus-16 in Fine-NeedleAspirates to DetermineTumor Originin Patients with Metastatic Squamous Cell Carcinoma of the Head and Neck ShahnazBegum, 1 MauraL.Gillison, 2 TheresaL.Nicol, 1 andWilliamH.Westra 1,2,3 Abstract Purpose: Patientswithheadandnecksquamouscellcarcinoma(HNSCC)oftenclinicallypres- entwithmetastasestoregionallymphnodes.Fine-needleaspirationofneckmassesisroutinely usedtoestablishthepresenceofmetastaticcarcinomaandinturntoinitiateasubsequentworkup todeterminethesiteoftumororigin.Humanpapillomavirus(HPV)16isanimportantetiologic agentforHNSCCsthatarisefromtheoropharynxbutlesssofortumorsfromnon-oropharyngeal sites.HPV16detectionthusprovidesastrategyforlocalizinganimportantsubsetofHNSCCs,but thisapproachhasnotbeenappliedtofine-needleaspirationspecimens. ExperimentalDesign: Wedid in situ hybridizationforHPV16on77consecutiveaspiratedneck massesdiagnosedasmetastaticsquamouscellcarcinoma.P16immunohistochemistrywasalso donebecausep16overexpressionmayserveasasurrogatemarkerofHPV-associatedHNSCC. Results: HPV16wasdetectedin13ofthe77(17%)aspirates.Bysiteoforigin,HPV16was detectedin10of19metastasesfromtheoropharynxbutinnoneof46metastasesfromother sites(53%versus0%; P < 0.0001).HPV16wasnotdetectedin2branchialcleftcystsmisdiag- nosedasmetastaticsquamouscellcarcinoma,butitwasdetectedin3of10metastasesfrom occultprimarytumors.P16expressionwasassociatedwiththepresenceofHPV16:12of 13HPV16-positivemetastasesexhibitedp16expression,whereasonly4of62HPV16-negative metastaseswerep16positive(92%versus6%; P < 0.0001).P16expressionalsocorrelatedwith siteoftumororigin:13of19oropharyngealmetastaseswerep16positive,whereasonly1of46 non-oropharyngealmetastaseswasp16positive(68%versus2%; P < 0.0001). Conclusions: HPV16statuscanbedeterminedintumorcellsaspiratedfromthenecksofpatients withmetastaticHNSCC.Itspresenceisareliableindicatoroforiginfromtheoropharynx. Oncogenic human papillomavirus (HPV), particularly type HPV16,hasbeenestablishedasacausativeagentfor f25%of headandnecksquamouscellcarcinoma(HNSCC;refs.1–3). AstheroleofHPV16inheadandnecktumorigenesisbecomes better understood, these HPV16-related cancers are becoming increasingly recognized as a biologically distinct subgroup of HNSCC with a characteristic clinical profile. Accordingly, detection of HPV16 may have several practical clinical applications. For example, HPV16 DNA can be detected in the saliva and serum of patients with HNSCC and may have clinical usefulness for cancer screening and cancer surveillance (4–7). The presence HPV16 in HNSCC has been correlated withimprovedsurvivalandmayserveasausefulbiomarkerof prognosis (1, 8–10). Additionally, patients with HPV-induced HNSCC may benefit from future HPV-targeted treatment strategies (e.g., therapeutic HPV vaccines; ref. 11). Another distinctive feature of HPV16-related HNSCC is its strongtendencytoarisefromtheoropharynx.Accordingly,HPV DNA detection may be exploited to pinpoint primary tumor origininpatientswhopresentwithmetastaticHNSCC(12,13). DiscerningsiteoftumororiginforpatientswithHNSCCisnot always straightforward. Indeed, most patients with HNSCC already have metastatic spread to regional lymph nodes at the timeofpresentation(14),13%ofpatientspresentwithaneck massasthefirstandonlyclinicalmanifestation(15),and3%to 9% of the primary tumors elude detection despite a thorough clinical, radiographic, endoscopic, and histopathologic evalua- tion (16). We previously did HPV16 analysis on surgically excisedcervicallymphnodesharboringmetastaticHNSCCand foundthatHPV16isonlydetectedinthosetumorsarisingfrom the oropharynx (17). This finding points to HPV16 as an attractive biomarker for discerning tumor origin and sets the stagefortheapplicationofthisstrategytocellsaspiratedfrom neckmassesduringtheinitialstagesoftheclinicalevaluation. PatientsandMethods Cases. Study approval was obtained from The Johns Hopkins Medical Institutions Internal Review Board. The cytopathology files of Authors’Affiliations: Departmentsof 1 Pathology, 2 Oncology,and 3 Otolaryn- gology-HeadandNeckSurgery,TheJohnsHopkinsMedicalInstitutions,Baltimore, Maryland Received7/11/06;revised10/18/06;accepted10/24/06. Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage charges.Thisarticlemustthereforebeherebymarked advertisement inaccordance with18U.S.C.Section1734solelytoindicatethisfact. Requests for reprints: WilliamH.Westra,TheJohnsHopkinsHospital,Room 2242,TheWeinbergBuilding,401NorthBroadway,Baltimore,MD21231-2410. Phone:410-955-2163;Fax:410-955-0115;E-mail:wwestra@jhmi.edu. F 2007AmericanAssociationforCancerResearch. doi:10.1158/1078-0432.CCR-06-1690 www.aacrjournals.org ClinCancerRes2007;13(4)February15,2007 1186