While its prognostic significance remains unclear, p16INK4a protein expression is increasingly being used as a surrogate marker for oncogenic human papillomavirus (HPV) infection in head and neck squamous cell carcinomas (HNSCC). To evaluate the prognostic utility of p16 expression in HNSCC, we prospectively collected 163 primary tumor specimens from histologically confirmed HNSCC patients who were followed for up to 9.4 years. Formalin fixed tumor specimens were tested for p16 protein expression by immunohistochemistry. HPV type-16 DNA and RNA was detected by MY09/11-PCR and E6/E7 RT-PCR on matched frozen tissue, respectively. P16 protein expression was detected more often in oropharyngeal tumors (53%) as compared with laryngeal (24%), hypopharyngeal (8%), or oral cavity tumors (4%; P<0.0001). With respect to prognosis, p16-positive oropharyngeal tumors exhibited significantly better overall survival than p16-negative tumors (log-rank test p=0.04), whereas no survival benefit was observed for non-oropharyngeal tumors. However, when both p16 and HPV DNA test results were considered, concordantly positive non-oropharyngeal tumors had significantly better disease-specific survival than concordantly negative non-oropharyngeal tumors after controlling for sex, nodal stage, tumor size, tumor subsite, primary tumor site number, smoking, and drinking (adjusted hazard ratio [HR]=0.04, 0.01–0.54). Compared with concordantly negative non-oropharyngeal HNSCC, p16(+)/HPV16(-) non-oropharyngeal HNSCC (n=13, 7%) demonstrated no significant improvement in disease-specific survival when HPV16 was detected by RNA (adjusted HR=0.83, 0.22–3.17). Our findings show that p16 immunohistochemistry alone has potential as a prognostic test for oropharyngeal cancer survival, but combined p16/HPV testing is necessary to identify HPV-associated non-oropharyngeal HNSCC with better prognosis.

Recent evidence suggests that human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) patients have better survival than HPV-negative patients. However, it is unclear if similar patterns for survival exist across different tumor sites, and whether HPV-associated prognosis is modified by type of treatment. We prospectively tested 222 histologically confirmed HNSCC primary tumors for HPV DNA by PCR and HPV E6/E7 RNA by RT-PCR prior to treatment at a large urban health center. Cox proportional hazard ratio models were constructed to assess HPV-associated differences in overall and disease-specific survival adjusting for clinical and demographic covariates. HPV detection varied significantly by primary HNSCC tumor site, from 35 % for oropharynx, to 25 % for hypopharynx, 5 % for larynx, and 3 % for oral cavity (p \ 0.0001), with HPV16 accounting for the majority (95 %) of HPV-positive tumors. The hazard-risk of overall and disease-specific death comparing HPV16-positive versus negative oropharyngeal HNSCC was reduced by 74 and 89 %, respectively (p values \ 0.05), and was independent of other prognostic indicators; no statistically significant changes in outcomes were observed for non-oropharyngeal HNSCC sites. Prediction of overall survival was better with combined DNA and RNA HPV16 positive PCR detection. There was no difference in HPV16-associated survival whether patients received either surgery or (chemo)radiotherapy as their initial treatment modality. Improved HPV-associated HNSCC survival is limited to Electronic supplementary material The online version of this article

To assess the relationship of E2 gene disruption with viral gene expression and clinical outcome in human papillomavirus (HPV) positive head and neck squamous cell carcinoma, we evaluated 31 oropharyngeal and 17 non-oropharyngeal HPV16 positive carcinomas using two PCR-based methods to test for disruption of E2, followed by Sanger sequencing. Expression of HPV16 E6, E7 and E2 transcripts, along with cellular ARF and INK4A, were also assessed by RT-qPCR. Associations between E2 disruption, E2/E6/E7 expression, and clinical outcome were evaluated by Kaplan-Meier analysis for loco-regional recurrence and disease-specific survival. The majority (n = 21, 68%) of HPV16 positive oropharyngeal carcinomas had an intact E2 gene, whereas the majority of HPV16 positive non-oropharyngeal carcinomas (n = 10, 59%) had a disrupted E2 gene. Three of the oropharyngeal tumors and two of the non-oropharyngeal tumors had deletions within E2. Detection of an intact E2 gene was associated with a higher DNA viral load and increased E2/E6/E7, ARF and INK4A expression in oropharyngeal tumors. Oropharyngeal carcinomas with an intact E2 had a lower risk of loco-regional recurrence (log-rank p = 0.04) and improved disease-specific survival (p = 0.03) compared to tumors with disrupted E2. In addition, high E7 expression was associated with lower risk of loco-regional recurrence (p = 0.004) as was high E6 expression (p = 0.006). In summary, an intact E2 gene is more common in HPV16 positive oropharyngeal than non-oropharyngeal carcinomas; the presence of an intact E2 gene is associated with higher HPV viral load, higher viral oncogene expression, and improved clinical outcome compared to patients with a disrupted E2 gene in oropharyngeal cancer.