title: Overexpression of p16INK4 is a reliable marker of human papillomavirus-induced oral high-grade squamous dysplasia.
abstract: OBJECTIVE
  Human papillomavirus (HPV) infection has been implicated in the development of high-grade squamous dysplasia and carcinoma of the oral cavity in the absence of other known risk factors such as smoking. HPV-induced oral dysplasia or carcinoma may be a unique tumor entity in terms of biologic behavior and treatment decisions. In detecting such cases, most reported studies have used techniques that are less sensitive than DNA amplification. Recent reports have suggested that overexpression of the p16INK4 protein is a surrogate marker of HPV-induced high-grade dysplasia or carcinoma. However, the correlation between expression of p16INK4 and the presence of HPV DNA as determined by polymerase chain reaction (PCR) amplification has not been previously reported. The purpose of this research was to determine if immunohistochemistry for p16 would serve as a marker of HPV-associated high-grade oral squamous dysplasia.
  
  
  METHODS
  Archival formalin-fixed, paraffin-embedded tissue sections from 41 cases of high-grade oral squamous dysplasia were randomly selected. Expression of p16INK4 protein was assessed by immunohistochemical analysis (16P04 Neomarkers, Fremont, CA). Strong and diffuse nuclear staining restricted to the dysplastic region in the epithelium was scored as positive for protein expression, whereas focal or weak nuclear or cytoplasmic staining was scored as negative. The presence of HPV was determined by microdissection, DNA extraction, and PCR DNA amplification using elongated primers that align with corresponding sequences of the L1 region of 23 mucosotropic HPV genotypes. The HPV type was determined by direct sequencing of the PCR product. Normal squamous epithelium was used as an internal negative control, and cases of severe cervical high-grade squamous dysplasia were used as a positive control for immunohistochemical staining and PCR.
  
  
  RESULTS
  The results of immunohistochemical analysis for overexpression of p16INK4 were positive in 6 of the 41 tissue sections. The results of PCR DNA amplification were also positive for these 6 sections. HPV-16 was identified in 5 of the positive cases; in the other case, the viral strain could not be determined.
  
  
  CONCLUSIONS
  Immunohistochemical detection of p16INK4 is a technically simple and potentially reliable assay for diagnosing cases of HPV-induced oral high-grade squamous dysplasia. Detecting such lesions may influence future therapeutic decisions.
authors.affiliation: Oral and Maxillofacial Surgery, University of Kentucky College of Dentistry, Lexington, KY 40536-0297, USA. llcunn2@email.uky.edu
authors.forename: Larry L
authors.initials: LL
authors.lastname: Cunningham
authors.forename: Giulia M
authors.initials: GM
authors.lastname: Pagano
authors.forename: Mengtao
authors.initials: M
authors.lastname: Li
authors.forename: Rahul
authors.initials: R
authors.lastname: Tandon
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Holm
authors.forename: Dean K
authors.initials: DK
authors.lastname: White
authors.forename: Subodh M
authors.initials: SM
authors.lastname: Lele
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor; D002294:Carcinoma, Squamous Cell / Q000201:enzymology / Q000209:etiology / Q000821:virology*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis*; D015344:DNA Probes, HPV; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D052162:Human papillomavirus 16 / Q000472:pathogenicity*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D009062:Mouth Neoplasms / Q000201:enzymology / Q000209:etiology / Q000821:virology*; D030361:Papillomavirus Infections / Q000150:complications; D011230:Precancerous Conditions / Q000201:enzymology / Q000209:etiology / Q000821:virology*
chemical_list: D014408:Biomarkers, Tumor; D019941:Cyclin-Dependent Kinase Inhibitor p16; D015344:DNA Probes, HPV; D004279:DNA, Viral