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

Abstract

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; χ 2 = 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.

The Potential Association between E2F2, MDM2 and p16 Protein Concentration and Selected Sociodemographic and Clinicopathological Characteristics of Patients with Oral Squamous Cell Carcinoma

“…These discrepancies could be due to such factors as differences in sampling techniques, preparation and detection methods. In our study, the median concentration of p16 in the study group was higher in the tumour tissue than in the margin samples, which seemed to confirm the results of studies carried out with the use of the bioinformatics instruments and IHC [ 48 , 49 , 50 , 51 , 52 ]. Interestingly, it has been speculated that the p16 concentration could be higher in OSCC patients with HPV infection [ 53 ].…”

“…Consequently, multiple studies have attempted to evaluate the utility of p16 as a surrogate marker for HPV infection in OSCC. A large cross-sectional study found no HPV DNA or E6 mRNA in 1260 OSCC samples [ 51 ]. Similar to this study, other authors reported no association between the level of p16 and HPV infection, either during virus detection performed by ISH or PCR [ 45 , 47 , 55 , 56 , 57 , 58 , 59 ].…”

Prevalence of high-risk human papillomavirus in oral squamous cell carcinoma with or without chewing habits

“…The absence of a significant association between p16 and chewing habits in our study is strengthened by the findings of some recent work [ 53 ]. Another study concluded that p16 had no role as a surrogate marker [ 54 ]. In a larger sample size setting, with most of the population being chewers, p16 and HR-HPV were not associated with chewing habits [ 55 ].…”

Human Papillomavirus–Attributable Head and Neck Cancers in India—A Systematic Review and Meta-Analysis

“… 41 Another study has shown that p16 expression in OC was associated with a higher tumor grade with 21.4% overexpression; however, none of the p16-positive tumors were associated with HPV. 42 Studies with only p16INK4a as an HPV detection method in HNC were not included, as many studies have found discordance between p16 and HPV positivity. The reported attributable fraction concerning p16INK4a positivity reflects maximum estimates because some positive tumors for p16INK4a might not have been caused by HPV infection.…”