HPV DNA genotyping, HPV E6*I mRNA detection, and p16INK4a/Ki-67 staining in Belgian head and neck cancer patient specimens, collected within the HPV-AHEAD study

Cancer Epidemiol. 2021 Jun:72:101925. doi: 10.1016/j.canep.2021.101925. Epub 2021 Apr 8.


Background: The main risk factors for head and neck cancer (HNC) are tobacco and alcohol use. However, an important fraction of oropharyngeal cancer (OPC) is caused by human papillomaviruses (HPV), a subgroup with increasing incidence in several western countries.

Methods: As part of the HPV-AHEAD study, we assessed the role of HPV infection in 772 archived tissue specimens of Belgian HNC patients: 455 laryngeal (LC), 106 oral cavity (OCC), 99 OPC, 76 hypopharyngeal (HC), and 36 unspecified parts of the head and neck. All specimens were tested for HPV DNA (21 genotypes); whereof all HPV DNA-positives, all HPV DNA-negative OPCs and a random subset of HPV DNA-negatives of the other HNC-sites were tested for the presence of type-specific HPV RNA and p16INK4a over-expression.

Results: The highest HPV DNA prevalence was observed in OPC (36.4 %), and was significantly lower (p < 0.001) in the other HNCs (OCC:7.5 %, LC:6.6 %). HPV16 was the most common HPV-genotype in all HNCs. Approximately 83.0 % of the HPV DNA-positive OPCs tested HPV RNA or p16-positive, compared to about 37.5 % and 44.0 % in OCC and LC, respectively. Estimation of the attributable fraction of an HPV infection in HNC was very similar for HPV RNA or p16 in addition to DNA-positivity; with 30 % for OPC, and 3 % for OCC and LC.

Conclusion: Our study confirms the heterogeneity of HPV DNA prevalence across anatomical sites in HNC, with a predominance of HPV16 in all sites. The estimated proportion of HPV-driven HNC in Belgium, during the period 1980-2014, was 10 times higher in OPC compared to OCC and LC.

Keywords: Head and neck neoplasms; Human papillomavirus; Human papillomavirus DNA tests; mRNA; p16(INK4a).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Alphapapillomavirus / genetics*
  • Belgium
  • Cyclin-Dependent Kinase Inhibitor p16
  • DNA, Viral / genetics*
  • Female
  • Genotype
  • Head and Neck Neoplasms / therapy
  • Head and Neck Neoplasms / virology*
  • Humans
  • Ki-67 Antigen
  • Male
  • Middle Aged
  • Oncogene Proteins, Viral / genetics
  • Papillomavirus Infections / virology*
  • RNA, Messenger / isolation & purification*
  • Staining and Labeling


  • Cyclin-Dependent Kinase Inhibitor p16
  • DNA, Viral
  • E6 protein, human papillomavirus type 1
  • Ki-67 Antigen
  • Oncogene Proteins, Viral
  • RNA, Messenger