title: Evaluation of human papilloma virus diagnostic testing in oropharyngeal squamous cell carcinoma: sensitivity, specificity, and prognostic discrimination.
abstract: OBJECTIVE
  Human papillomavirus-16 (HPV16) is the causative agent in a biologically distinct subset of oropharyngeal squamous cell carcinoma (OPSCC) with highly favorable prognosis. In clinical trials, HPV16 status is an essential inclusion or stratification parameter, highlighting the importance of accurate testing.
  
  
  METHODS
  Fixed and fresh-frozen tissue from 108 OPSCC cases were subject to eight possible assay/assay combinations: p16 immunohistochemistry (p16 IHC); in situ hybridization for high-risk HPV (HR HPV ISH); quantitative PCR (qPCR) for both viral E6 RNA (RNA qPCR) and DNA (DNA qPCR); and combinations of the above.
  
  
  RESULTS
  HPV16-positive OPSCC presented in younger patients (mean 7.5 years younger, P = 0.003) who smoked less than HPV-negative patients (P = 0.007). The proportion of HPV16-positive cases increased from 15% to 57% (P = 0.001) between 1988 and 2009. A combination of p16 IHC/DNA qPCR showed acceptable sensitivity (97%) and specificity (94%) compared with the RNA qPCR "gold standard", as well as being the best discriminator of favorable outcome (overall survival P = 0.002). p16 IHC/HR HPV ISH also had acceptable specificity (90%) but the substantial reduction in its sensitivity (88%) impacted upon its prognostic value (P = 0.02). p16 IHC, HR HPV ISH, or DNA qPCR was not sufficiently specific to recommend in clinical trials when used in isolation.
  
  
  CONCLUSIONS
  Caution must be exercised in applying HPV16 diagnostic tests because of significant disparities in accuracy and prognostic value in previously published techniques.
authors.affiliation: Department of Molecular & Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Schache
authors.forename: Triantafilos
authors.initials: T
authors.lastname: Liloglou
authors.forename: Janet M
authors.initials: JM
authors.lastname: Risk
authors.forename: Anastasia
authors.initials: A
authors.lastname: Filia
authors.forename: Terence M
authors.initials: TM
authors.lastname: Jones
authors.forename: Jon
authors.initials: J
authors.lastname: Sheard
authors.forename: Julia A
authors.initials: JA
authors.lastname: Woolgar
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Helliwell
authors.forename: Asterios
authors.initials: A
authors.lastname: Triantafyllou
authors.forename: Max
authors.initials: M
authors.lastname: Robinson
authors.forename: Philip
authors.initials: P
authors.lastname: Sloan
authors.forename: Colin
authors.initials: C
authors.lastname: Harvey-Woodworth
authors.forename: Daniel
authors.initials: D
authors.lastname: Sisson
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Shaw
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000401:mortality / Q000821:virology*; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D052162:Human papillomavirus 16 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008297:Male; D008875:Middle Aged; D009959:Oropharyngeal Neoplasms / Q000401:mortality / Q000821:virology*; D016133:Polymerase Chain Reaction; D011379:Prognosis; D012680:Sensitivity and Specificity
chemical_list: D004279:DNA, Viral