title: Detection of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma using droplet digital polymerase chain reaction.
abstract: The incidence of oropharyngeal squamous cell carcinoma caused by oncogenic HPV (HPV-OPSCC) is rising worldwide. HPV-OPSCC is commonly diagnosed by RT-qPCR of HPV-16 E6 and E7 oncoproteins or by cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1 (p16) immunohistochemistry (IHC). Droplet digital PCR (ddPCR) has been recently reported as ultra-sensitive and highly precise method of nucleic acid quantification for biomarker analysis. We aimed to validate this method for the detection of HPV-16 E6 and E7 in HPV-OPSCC.
  
  
  
  Participants were recruited from January 2015-November 2015 at initial presentation to the University of Alberta Head and Neck Oncology Clinic. RNA was extracted, purified and quantified from prospectively collected participant tissues, and ddPCR was performed with fluorescent probes detecting HPV-16 E6 and E7. Results from ddPCR were compared with p16 IHC performed by clinical pathology as standard of care.
  
  
  
  Head and neck tissues were prospectively obtained from 68 participants including 29 patients with OPSCC, 29 patients with non-OPSCC and 10 patients without carcinoma. 79.2% of patients with OPSCC were p16 positive. The sensitivity and specificity of ddPCR HPV E6/E7 compared with p16 IHC in OPSCC was 91.3 and 100%, respectively. The amount of target RNA used was ≤1 ng, 20-50 times lower than reported by other for RT-qPCR HPV E6/E7.
  
  
  
  The ddPCR of HPV E6/E7 is a novel and highly specific method of detecting HPV-16 in OPSCC. Cancer 2016;122:1544-51. © 2016 American Cancer Society.
authors.affiliation: Department of Surgery, Division of Otolaryngology-Head and Neck Surgery University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Vincent L
authors.initials: VL
authors.lastname: Biron
authors.affiliation: Department of Surgery, Division of Otolaryngology-Head and Neck Surgery University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Morris
authors.initials: M
authors.lastname: Kostiuk
authors.affiliation: Department of Surgery, Division of Otolaryngology-Head and Neck Surgery University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Andre
authors.initials: A
authors.lastname: Isaac
authors.affiliation: Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Puttagunta
authors.affiliation: Department of Surgery, Division of Otolaryngology-Head and Neck Surgery University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: O'Connell
authors.affiliation: Department of Surgery, Division of Otolaryngology-Head and Neck Surgery University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Harris
authors.affiliation: Department of Surgery, Division of Otolaryngology-Head and Neck Surgery University of Alberta, Edmonton, Alberta, Canada.
authors.forename: David W J
authors.initials: DW
authors.lastname: Côté
authors.affiliation: Department of Surgery, Division of Otolaryngology-Head and Neck Surgery University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Hadi
authors.initials: H
authors.lastname: Seikaly
mesh_terms: D000328:Adult; D000368:Aged; D002294:Carcinoma, Squamous Cell / Q000821:virology*; D016022:Case-Control Studies; D015331:Cohort Studies; D005260:Female; D006258:Head and Neck Neoplasms / Q000821:virology*; D052162:Human papillomavirus 16 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009856:Oncogene Proteins, Viral / Q000235:genetics; D009959:Oropharyngeal Neoplasms / Q000821:virology*; D050725:Papillomavirus E7 Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011446:Prospective Studies; D012367:RNA, Viral / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D000077195:Squamous Cell Carcinoma of Head and Neck; D055815:Young Adult
chemical_list: C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012367:RNA, Viral; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16
keywords: droplet digital polymerase chain reaction (ddPCR); human papillomavirus; human papillomavirus oncogenes E6 and E7 (HPV E6/E7); human papillomavirus type 16 (HPV-16); oropharyngeal cancer; squamous cell carcinoma