title: RNA in-situ hybridization is a practical and effective method for determining HPV status of oropharyngeal squamous cell carcinoma including discordant cases that are p16 positive by immunohistochemistry but HPV negative by DNA in-situ hybridization.
abstract: OBJECTIVE
  Evaluation of human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma (OPSCC) has become increasingly important for prognostication and clinical trial enrollment. This assessment is confounded in OPSCCs that are p16 positive by immunohistochemistry (IHC) but HPV negative by DNA in situ hybridization (DISH). This study evaluates whether E6/E7 mRNA in situ hybridization (RISH) can detect transcriptionally active HPV in these problematic cases.
  
  
  METHODS
  Eighty-two head and neck squamous cell carcinoma cases that had previously undergone p16 IHC and HPV DISH were evaluated with two RISH platforms and a second-generation DISH probe. The study included 21 p16+/DISH+ concordant cases, 19 p16-/DISH- concordant cases, and 42 p16+/DISH- discordant cases.
  
  
  RESULTS
  RISH identified E6/E7 mRNA in 37 (88%) p16+/DISH- cases, 21 (100%) p16+/DISH+ cases, and 0 (0%) p16-/DISH- cases. RISH signals were clearly visible at low to medium magnification in 97% of positive cases, facilitating almost-perfect inter-observer reproducibility. The performance of the manual and automated RISH platforms were equivalent (kappa=0.915). Only 29% of carcinomas that demonstrated E6/E7 mRNA transcriptional activity were positive using the 2nd generation DISH probe.
  
  
  CONCLUSIONS
  HPV RISH is a highly sensitive and specific platform that can clarify the HPV status of those perplexing OPSCCs that are p16 positive by IHC but HPV negative by DISH. Moreover, it is easy to interpret, readily adaptable to the clinical laboratory, and provides direct evidence of HPV transcriptional activity. E6/E7 RISH should be considered as a first-line platform for determination of HPV status in OPSCCs.
authors.affiliation: Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, MD, USA.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Rooper
authors.affiliation: Affymetrix, Inc., Santa Clara, CA, USA.
authors.forename: Manoj
authors.initials: M
authors.lastname: Gandhi
authors.affiliation: Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, MD, USA; Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins Medical Institutions, Baltimore, MD, USA.
authors.forename: Justin A
authors.initials: JA
authors.lastname: Bishop
authors.affiliation: Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, MD, USA; Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins Medical Institutions, Baltimore, MD, USA; Department of Oncology, The Johns Hopkins Medical Institutions, Baltimore, MD, USA. Electronic address: wwestra@jhmi.edu.
authors.forename: William H
authors.initials: WH
authors.lastname: Westra
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000032:analysis; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000821:virology*; D005260:Female; D006258:Head and Neck Neoplasms / Q000378:metabolism / Q000821:virology*; D052162:Human papillomavirus 16 / Q000302:isolation & purification*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D008297:Male; D008875:Middle Aged; D009959:Oropharyngeal Neoplasms / Q000378:metabolism / Q000821:virology; D030361:Papillomavirus Infections / Q000175:diagnosis / Q000821:virology*; D012333:RNA, Messenger; D012367:RNA, Viral; D015203:Reproducibility of Results
chemical_list: D014408:Biomarkers, Tumor; D012333:RNA, Messenger; D012367:RNA, Viral
keywords: Carcinoma, Squamous cell; Cyclin-dependent kinase inhibitor p16; Head and neck neoplasms; Human papillomavirus DNA tests; Immunohistochemistry; In situ hybridization; Oncogene proteins, Viral; Papillomavirus infections; RNA, Messenger; RNA, Viral