title: Identification of transcriptionally active HPV infection in formalin-fixed, paraffin-embedded biopsies of oropharyngeal carcinoma.
abstract: Human papillomavirus (HPV) oncogenic activity is the result of viral oncogene E6 and E7 expression in infected cells. Oncogene expression analysis is, however, not part of the routine diagnostic evaluation of HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) since it requires fresh tumor tissue. We compared the diagnostic accuracy of several methods commonly employed for HPV characterization in OPSCC with the results of the newly available HPV E6/E7 mRNA in situ hybridization (ISH) on formalin-fixed, paraffin-embedded biopsy samples, in order to establish if the latter should be introduced in the diagnostic routine to increase accuracy when fresh tissue is not available. p16 immunostain, DNA ISH for high-risk HPV genotypes, SPF LiPA amplification and genotyping, and HPV16 E6 amplification were performed on 41 consecutive OPSCC samples. Twenty (48.7%) cases were positive by mRNA ISH; sensitivity and specificity were 100% and 90% for p16, 90% and 100% for DNA ISH, 70% and 76% for SPF10 LiPA, 90% and 76% for E6 amplification. A diagnostic algorithm considering p16 immunostain as first step followed by either high-risk HPV DNA ISH or HPV16 E6 amplification in p16-positive cases correctly characterized 90% of mRNA-positive and all mRNA-negative cases; combining the 3 tests correctly identified all cases. While no stand-alone test was sufficiently accurate for classifying HPV-associated OPSCC, the high sensitivity and specificity of the established combination of p16 immunostain, DNA ISH, and HPV16 DNA amplification suggests that the introduction of labour- and cost-intensive mRNA ISH, is not necessary in the diagnostic routine of oropharyngeal tumors.
authors.affiliation: Department of Molecular Medicine, Unit of Pathology, University of Pavia, via Forlanini 16, 27100 Pavia, Italy; Department of Pathology IRCCS Policlinico S. Matteo Foundation, via Forlanini 16, 27100 Pavia, Italy. Electronic address: patrizia.morbini@unipv.it.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Morbini
authors.affiliation: Department of Pathology IRCCS Policlinico S. Matteo Foundation, via Forlanini 16, 27100 Pavia, Italy. Electronic address: p.aberizzi@smatteo.pv.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Alberizzi
authors.affiliation: Clinical Epidemiology and Biometric Unit, IRCCS Policlinico S. Matteo Foundation, P.le Golgi 5, 27100 Pavia, Italy. Electronic address: ctinelli@smatteo.pv.it.
authors.forename: Carmine
authors.initials: C
authors.lastname: Tinelli
authors.affiliation: Department of Oncology, IRCCS Policlinico S. Matteo Foundation, P.le Golgi 5, 27100 Pavia, Italy. Electronic address: c.paglino@smatteo.pv.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: Paglino
authors.affiliation: Department of Otolaryngology, University of Pavia and IRCCS Policlinico S. Matteo Foundation, P.le Golgi 5, 27100 Pavia, Italy. Electronic address: g.bertino@smatteo.pv.it.
authors.forename: Giulia
authors.initials: G
authors.lastname: Bertino
authors.affiliation: Pediatric Hematology/Oncology, IRCCS Policlinico S. Matteo Foundation, P.le Golgi 5, 27100 Pavia, Italy. Electronic address: p.comoli@smatteo.pv.it.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Comoli
authors.affiliation: Department of Oncology, IRCCS Policlinico S. Matteo Foundation, P.le Golgi 5, 27100 Pavia, Italy. Electronic address: p.pedrazzoli@smatteo.pv.it.
authors.forename: Paolo
authors.initials: P
authors.lastname: Pedrazzoli
authors.affiliation: Department of Otolaryngology, University of Pavia and IRCCS Policlinico S. Matteo Foundation, P.le Golgi 5, 27100 Pavia, Italy. Electronic address: marco.benazzo@unipv.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Benazzo
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000032:analysis*; D001706:Biopsy; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D005557:Formaldehyde; D005838:Genotype; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods; D008297:Male; D008875:Middle Aged; D009959:Oropharyngeal Neoplasms / Q000473:pathology* / Q000821:virology*; D030361:Papillomavirus Infections / Q000821:virology*; D010232:Paraffin; D014158:Transcription, Genetic
chemical_list: D014408:Biomarkers, Tumor; D004279:DNA, Viral; D005557:Formaldehyde; D010232:Paraffin
keywords: Human papillomavirus; In situ hybridization; Oropharyngeal cancer; mRNA in situ hybridization; p16