Exome Sequencing with Validations and Expression of p16/CDKN2A Shows no Association with HPV in Oral Cancers

Asian Pac J Cancer Prev. 2022 Jan 1;23(1):191-200. doi: 10.31557/APJCP.2022.23.1.191.


Purpose: Despite many studies attributing HPV infection to oropharyngeal tumorigenesis, its involvement in non-oropharyngeal cancers is ambiguous. We have evaluated the mutation profile of p16 along with protein expression and correlated it with the HPV status in oral cancers.

Methods: Somatic mutations in p16 were studied by exome sequencing (n=25) and validated by Sequenom Mass spectrometry (n=50). Expression of p16 was studied by immunohistochemistry (IHC) and correlated with HPV16/18 status evaluated by PCR, and IHC (n=221) in oral cancers.

Results: Out of 25 oral cancer patients' samples sequenced by Exome sequencing, p16 mutations were found in 4 samples (16%). All the p16 mutations were identified in patients with cancers in the site of gingivobuccal complex and not tongue subsite. All the 4 patients with p16 mutations had failed treatment, and showed a significantly poor disease-free survival. Insilico analysis of the types of p16 mutations showed mutated, truncated p16 protein having an increased intrinsic disorder, and all the mutations involved truncation post arginine. Validation of the p16 mutations by mass spectrometry showed 8/50 (16%) of patients harbouring pArg80Ter mutation, of which 7/8 (87.5%) had failed treatment. Overexpression of p16 in >70% of the tumour cells was found in 21.4% (26/121) OSCC patients, 6.75% (5/74) OPML patients and p16 expression was significantly correlated (p=0.001; χ2 = 25.601) to the grade. All the samples were studied for HPV presence by PCR and IHC. We found that none of the p16 positive tumours showing expression in >70% of the tumour cells harbored HPV both by PCR as well as IHC.

Conclusion: Our study emphasises the importance of p16 in oral cancers, and shows that oral cancer is not HPV associated and p16 expression is not a surrogate marker for HPV.

Keywords: HPV; P16 mutation; oral cancer; p16 expression; p16 surrogate marker.

MeSH terms

  • Alphapapillomavirus*
  • Biomarkers, Tumor / genetics
  • Cyclin-Dependent Kinase Inhibitor p16 / genetics*
  • Exome Sequencing
  • Genes, p16*
  • Humans
  • Immunohistochemistry
  • Mouth Neoplasms / genetics*
  • Mouth Neoplasms / virology
  • Mutation / genetics*
  • Papillomavirus Infections / complications
  • Papillomavirus Infections / genetics


  • Biomarkers, Tumor
  • CDKN2A protein, human
  • Cyclin-Dependent Kinase Inhibitor p16