title: Sensitive HPV detection in oropharyngeal cancers.
abstract: BACKGROUND
  Human papillomaviruses (HPV) are the aetiological agents of certain benign and malignant tumours of skin and mucosae; the most important of which is cervical cancer. Also, the incidence of ano-genital warts, HPV-anal cancer and oropharyngeal cancers are rising. To help ascertain a useful PCR detection protocol for oropharyngeal cancers, we directly compared three commonly used primer sets in detection of HPV from different clinical samples.
  
  
  METHODS
  We compared PGMY09/11, MY09/11 and GP5+/6+ primers sets in PCRs of 34 clinically diagnosed samples of genital warts, cervical brushings (with associated histological diagnosis) and vulval biopsies. All negative samples were subsequently tested using the previously reported PGMY/GP PCR method and amplicons directly sequenced for confirmation and typing. An optimised PCR protocol was then compared to a line blot assay for detection of HPV in 15 oropharyngeal cancer samples.
  
  
  RESULTS
  PGMY09/11 primers detected HPV presence in more cervical brushing (100%) and genital wart (92.9%) samples compared to MY09/11 (90% and 64.3%) and GP5+/6+ (80% and 64.3%) primer sets, respectively. From vulval biopsies, HPV detection rates were: MY09/11 (63.6%), GP5+/6+ (54.5%) and PGMY09/11 (54.5%). PGMY/GP nested PCR demonstrated that HPV was present, and direct sequencing confirmed genotypes. This nested PCR protocol showed detection of HPV in 10/15 (66.7%) of oropharyngeal cancer samples.
  
  
  CONCLUSIONS
  PGMY09/11 primers are the preferred primer set among these three for primary PCR screening with different clinical samples. MY09/11 and GP5+/6+ may be used (particularly for cervical samples) but demonstrate lower detection rates. A nested PCR approach (i.e. a PGMY-GP system) may be required to confirm negativity or to detect low levels of HPV, undetectable using current primary PCR methods, as demonstrated using oropharyngeal cancer samples.
authors.affiliation: Dept of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK. dmw24@cam.ac.uk
authors.forename: David M
authors.initials: DM
authors.lastname: Winder
authors.forename: Siolian L R
authors.initials: SL
authors.lastname: Ball
authors.forename: Katie
authors.initials: K
authors.lastname: Vaughan
authors.forename: Nashat
authors.initials: N
authors.lastname: Hanna
authors.forename: Yin Ling
authors.initials: YL
authors.lastname: Woo
authors.forename: Jürgen-Theodor
authors.initials: JT
authors.lastname: Fränzer
authors.forename: Jane C
authors.initials: JC
authors.lastname: Sterling
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Stanley
authors.forename: Holger
authors.initials: H
authors.lastname: Sudhoff
authors.forename: Peter K C
authors.initials: PK
authors.lastname: Goon
mesh_terms: D003218:Condylomata Acuminata / Q000821:virology; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D005260:Female; D006801:Humans; D009959:Oropharyngeal Neoplasms / Q000821:virology*; D030361:Papillomavirus Infections / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014846:Vulvar Neoplasms / Q000821:virology; D002578:Uterine Cervical Dysplasia
chemical_list: D017931:DNA Primers; D004279:DNA, Viral