Evaluation of a combined triple method to detect causative HPV in oral and oropharyngeal squamous cell carcinomas: p16 Immunohistochemistry, Consensus PCR HPV-DNA, and In Situ Hybridization

Background: Recent emerging evidences identify Human Papillomavirus (HPV) related Head and Neck squamouscell carcinomas (HN-SCCs) as a separate subgroup among Head and Neck Cancers with different epidemiology,histopathological characteristics, therapeutic response to chemo-radiation treatment and clinical outcome.However, there is not a worldwide consensus on the methods to be used in clinical practice. The endpoint of thisstudy was to demonstrate the reliability of a triple method which combines evaluation of: 1. p16 proteinexpression by immunohistochemistry (p16-IHC); 2. HPV-DNA genotyping by consensus HPV-DNA PCR methods(Consensus PCR); and 3 viral integration into the host by in situ hybridization method (ISH). This triple method hasbeen applied to HN-SCC originated from oral cavity (OSCC) and oropharynx (OPSCC), the two anatomical sites inwhich high risk (HR) HPVs have been clearly implicated as etiologic factors. Methylation-Specific PCR (MSP) wasperformed to study inactivation of p16-CDKN2a locus by epigenetic events. Reliability of multiple methods wasmeasured by Kappa statistics.Results: All the HN-SCCs confirmed HPV positive by PCR and/or ISH were also p16 positive by IHC, with the lattershowing a very high level of sensitivity as single test (100% in both OSCC and OPSCC) but lower specificity level(74% in OSCC and 93% in OPSCC).Concordance analysis between ISH and Consensus PCR showed a faint agreement in OPSCC ( = 0.38) and amoderate agreement in OSCC ( = 0.44). Furthermore, the addition of double positive score (ISHpositive andConsensus PCR positive) increased significantly the specificity of HR-HPV detection on formalin-fixed paraffinembedded (FFPE) samples (100% in OSCC and 78.5% in OPSCC), but reduced the sensitivity (33% in OSCC and60% in OPSCC). The significant reduction of sensitivity by the double method was compensated by a very highsensitivity of p16-IHC detection in the triple approach.Conclusions: Although HR-HPVs detection is of utmost importance in clinical settings for the Head and Neck Cancerpatients, there is no consensus on which to consider the ‘golden standard’ among the numerous detection methodsavailable either as single test or combinations. Until recently, quantitative E6 RNA PCR has been considered the‘golden standard’ since it was demonstrated to have very high accuracy level and very high statistical significance