Human papillomavirus (HPV) is established as causative in oropharyngeal squamous cell carcinomas (OSCCs), being detected in 50% to 80% of tumors by DNA in situ hybridization (ISH) and/or polymerase chain reaction. However, these tests do not assess viral transcription. Many consider E6/E7 messenger ribonucleic acid (mRNA) the best indicator of HPV status, but it has not been detected in situ in OSCC. We constructed tissue microarrays (TMAs) from a cohort of OSCC for which p16 immunohistochemistry and HPV DNA ISH were previously performed on whole sections. We utilized a novel, chromogenic RNA ISH assay called RNAscope to detect E6/E7 mRNA of HPV-16 and other high-risk types on these TMAs. RNA ISH results were obtained for 196 of 211 TMA cases, of which 153 (78.1%) were positive. p16 immunohistochemistry and HPV DNA ISH were positive in 79.0% and 62.4% of cases, respectively. Concordance between RNA and p16, DNA and p16, and RNA and DNA were 96.4%, 78.7%, and 83.5%, respectively. Only 7 cases (3.6%) were discrepant between RNA ISH and p16. In univariate analysis, all 3 tests correlated with better overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS) (all P<0.001). In multivariate analysis, OS correlated significantly with RNA (hazard ratio=0.39, P=0.001), DNA (0.53, P=0.03), and p16 (0.30, P<0.001), but DSS and DFS correlated significantly only with p16 (DSS: 0.36, P=0.006; DFS: 0.42, P=0.016). RNA ISH is more sensitive than DNA ISH in detecting HPV in OSCC, and it correlates strongly with p16. Although both tests were comparable, p16 more strongly stratified patient outcomes.

Human papillomavirus (HPV) is associated with oropharyngeal squamous cell carcinomas. Persistent viral infection is postulated to lead to carcinogenesis, although infection of benign adjacent epithelium is not typically observed. It is known that immune evasive tumor cells can provide an ideal niche for a virus. The B7-H1/ PD-1 cosignaling pathway plays an important role in viral immune evasion by rendering CD8? cytotoxic T cells anergic. We hypothesized that HPV-related oropharyngeal squamous cell carcinomas express B7-H1 as a mechanism for immune evasion. A tissue microarray was utilized, for which HPV E6/E7 mRNA by in situ hybridization was previously performed. Immunohistochemistry was performed to detect B7-H1 and staining was characterized by pattern, distribution, and intensity. B7-H1 was expressed by 84 of the 181 (46.4 %) cases. Both tumor cell membranous and cytoplasmic expression were present and cytoplasmic expression was identified in some peritumoral lymphocytes. Expression was analyzed in several different ways and then considered binarily as positive versus negative. Tumors expressing B7-H1 were more likely to be HPV positive (49.2 vs. 34.1 %, p = 0.08). B7-H1 expression showed no correlation with disease recurrence in the entire cohort (OR = 1.09, p = 0.66), HPV positive cohort (OR = 0.80, p = 0.69) or HPV negative cohort (OR = 2.02, p = 0.22). However, B7-H1 expression intensity did correlate with the development of distant metastasis (p = 0.03), and B7-H1 intensity of 3? (versus all other staining) showed a strong trend towards distant metastasis in the HPV positive (OR = 6.67, p = 0.13) and HPV negative (OR = 9.0, p = 0.13) cohorts. There was no correlation between B7-H1 expression and patient survival for any of the different ways in which staining was characterized, whether binarily, by distribution, intensity, or combined scores. B7-H1 is expressed in the majority of oropharyngeal squamous cell carcinomas with transcriptionally-active HPV. This suggests that B7-H1 expression by tumor cells may play a role in harboring persistent HPV infection.

Transcriptionally-active human papillomavirus appears to be rare in laryngeal, hypopharyngeal, and oral cavity squamous cell carcinomas. As such, it appears unlikely to be a 'driver' or to be clinically significant in most established tumours.

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Primary tumor burden was not associated with HPV status. Patients with HPV-positive oropharyngeal squamous cell carcinomas had a higher prevalence of neck nodal metastases and high-grade lesions.