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Background The aim of the present study was to identify the presence and frequency of human papillomavirus (HPV) nucleic acid in p16 positive oral squamous cell carcinomas (OSCC), to assess if the virus were transcriptionally active, and to assess the utility of p16 overexpression as a surrogate marker for HPV in OSCC. Methods Forty-six OSCC patients treated between 2007 and 2011 with available formalin-fixed paraffin embedded (FFPE) specimens were included. Twenty-three patients were positive for p16 by immunohistochemistry (IHC) and these were matched with 23 patients with p16 negative tumours. Laser capture micro-dissection of the FFPE OSCC tissues was undertaken to isolate invasive tumour tissue. DNA was extracted and tested for high-risk HPV types using a PCR-ELISA method based on the L1 SPF10 consensus primers, and a real-time PCR method targeting HPV-16 and HPV-18 E6 region. Genotyping of HPV positive cases was performed using a reverse line blot hybridization assay (Inno-LiPA). RNAScope® (a chromogenic RNA in situ hybridization assay), was utilized to detect E6/E7 mRNA of known high-risk HPV types for detection of transcriptionally active virus. Results HPV DNA was found in 3 OSCC cases, all of which were p16 IHC positive. Two cases were genotyped as HPV-16 and one as HPV-33. Only one of the HPV-16 cases was confirmed to harbour transcriptionally active virus via HPV RNA ISH. Conclusion We have shown that the presence of transcriptionally active HPV rarely occurs in OSCC and that p16 is not an appropriate surrogate marker for HPV in OSCC cases. We propose that non-viral mechanisms are responsible for the majority of IHC p16 overexpression in OSCC. This article is protected by copyright. All rights reserved.

CDKN2A (p16) disruption is reported as a frequent event in head and neck squamous cell carcinomas that confers poor prognosis. We investigated the frequency of different potential mechanisms of CDKN2A inactivation in oral tongue squamous cell carcinomas (OTSCC) and their impact on patient outcome. From a cohort of 153 OTSCC patients, 131 formalin fixed paraffin embedded blocks of pre-treatment primary tumours were suitable for further molecular analysis. We assessed CDKN2A (p16) levels by immunohistochemistry (IHC), promoter methylation status by methylation-sensitive high resolution melting, mutation status by Sanger sequencing, gene copy number variation by fluorescence in situ hybridisation, and correlated these with patient outcome. We found that the majority of OTSCC did not overexpress p16 (110/116, 95%), assessed by IHC. The frequency of CDKN2A mutations was 20% (21/103), homozygous loss was 7% (7/97), hemizygous loss 31% (30/97), and promoter methylation was 18% (20/113). We found no evidence of these mechanisms in 24/106 (23%) p16 IHC negative tumours. No significant correlation was identified between any potential mechanism of CDKN2A inactivation and clinical features, including smoking status and age. There was a non-significant trend for worse overall survival for p16 IHC negative patients versus positive patients (HR = 1.81, 95% CI = 0.44-7.47, p = 0.40). No relationship was found between mechanisms of CDKN2A disruption and patient outcome. In conclusion, we demonstrate that CDKN2A alteration is a frequent event in OTSCC tumourigenesis. However, no correlation was identified between different potential mechanisms of CDKN2A disruption and clinical characteristics or patient outcome.