title: High-risk human papillomavirus detection in oropharyngeal, nasopharyngeal, and oral cavity cancers: comparison of multiple methods.
abstract: OBJECTIVE
  Human papillomaviruses are now recognized as an etiologic factor in a growing subset of head and neck cancers and have critical prognostic importance that affects therapeutic decision making. There is no universally accepted gold standard for high-risk HPV (hrHPV) assessment in formalin-fixed, paraffin-embedded (FFPE) tissue specimens, nor is there a clear understanding of the frequency or role of hrHPV in sites other than oropharynx.
  
  
  OBJECTIVE
  To determine the optimal assessment of hrHPV in FFPE head and neck tumor tissue specimens.
  
  
  METHODS
  In the setting of a large Midwestern referral center, assessment of hrHPV by p16 immunohistochemical staining, in situ hybridization, and polymerase chain reaction (PCR)-MassArray (PCR-MA), with L1 PGMY-PCR and sequencing to resolve method discordance, was conducted for 338 FFPE oropharyngeal, nasopharyngeal, and oral cavity tumor tissue specimens. Relative sensitivity and specificity were compared to develop a standard optimal test protocol. Tissue specimens were collected from 338 patients with head and neck cancer treated during the period 2001 through 2011 in the departments of Otolaryngology, Radiation Oncology, and Medical Oncology.
  
  
  METHODS
  Patients received standard therapy.
  
  
  METHODS
  Optimal hrHPV identification, detection, and activity in head and neck cancers.
  
  
  RESULTS
  Using combined PCR-MA with L1 PGMY-PCR and sequencing for conclusive results, we found PCR-MA to have 99.5% sensitivity and 100% specificity, p16 to have 94.2% sensitivity and 85.5% specificity, and in situ hybridization to have 82.9% sensitivity and 81.0% specificity. Among HPV-positive tumors, HPV16 was most frequently detected, but 10 non-HPV16 types accounted for 6% to 50% of tumors, depending on the site. Overall, 86% of oropharynx, 50% of nasopharynx, and 26% of oral cavity tumors were positive for hrHPV.
  
  
  CONCLUSIONS
  PCR-MA has a low DNA (5 ng) requirement effective for testing small tissue samples; high throughput; and rapid identification of HPV types, with high sensitivity and specificity. PCR-MA together with p16INK4a provided accurate assessment of HPV presence, type, and activity and was determined to be the best approach for HPV testing in FFPE head and neck tumor tissue specimens.
authors.affiliation: Cancer Biology Program, Program in the Biomedical Sciences, Rackham Graduate School, University of Michigan, Ann Arbor.
authors.forename: Heather M
authors.initials: HM
authors.lastname: Walline
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, University of Michigan, Ann Arbor.
authors.forename: Chris
authors.initials: C
authors.lastname: Komarck
authors.affiliation: Department of Pathology, University of Michigan, Ann Arbor.
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: McHugh
authors.affiliation: University of Michigan Medical School, Ann Arbor8currently with the Department of Otolaryngology/Head and Neck Surgery, St Louis University, St Louis, Missouri.
authors.forename: Serena A
authors.initials: SA
authors.lastname: Byrd
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, University of Michigan, Ann Arbor.
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Spector
authors.affiliation: University of Michigan Medical School, Ann Arbor9currently with the Department of Otolaryngology/Head and Neck Surgery, University of California, San Diego.
authors.forename: Samantha J
authors.initials: SJ
authors.lastname: Hauff
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, University of Michigan, Ann Arbor.
authors.forename: Martin P
authors.initials: MP
authors.lastname: Graham
authors.affiliation: Cancer Biostatistics, University of Michigan Comprehensive Cancer Center, Ann Arbor.
authors.forename: Emily
authors.initials: E
authors.lastname: Bellile
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, University of Michigan, Ann Arbor.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Moyer
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, University of Michigan, Ann Arbor.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Prince
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, University of Michigan, Ann Arbor.
authors.forename: Gregory T
authors.initials: GT
authors.lastname: Wolf
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, University of Michigan, Ann Arbor.
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Chepeha
authors.affiliation: Division of Medical Oncology, Department of Medicine, University of Michigan, Ann Arbor.
authors.forename: Francis P
authors.initials: FP
authors.lastname: Worden
authors.affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor.
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Stenmark
authors.affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor.
authors.forename: Avraham
authors.initials: A
authors.lastname: Eisbruch
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, University of Michigan, Ann Arbor.
authors.forename: Carol R
authors.initials: CR
authors.lastname: Bradford
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, University of Michigan, Ann Arbor.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Carey
mesh_terms: D000328:Adult; D000368:Aged; D001707:Biopsy, Needle; D016208:Databases, Factual; D005260:Female; D006258:Head and Neck Neoplasms / Q000473:pathology / Q000821:virology; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D008297:Male; D008875:Middle Aged; D009062:Mouth Neoplasms / Q000473:pathology / Q000821:virology*; D009303:Nasopharyngeal Neoplasms / Q000473:pathology / Q000821:virology*; D009959:Oropharyngeal Neoplasms / Q000473:pathology / Q000821:virology*; D027383:Papillomaviridae / Q000302:isolation & purification* / Q000472:pathogenicity; D030361:Papillomavirus Infections / Q000175:diagnosis*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018570:Risk Assessment; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000175:diagnosis*