Purpose: Human papillomavirus-16 (HPV16) is the causative agent in a biologically distinct subset of oropharyngeal squamous cell carcinoma (OPSCC) with highly favorable prognosis. In clinical trials, HPV16 status is an essential inclusion or stratification parameter, highlighting the importance of accurate testing.Experimental Design: Fixed and fresh-frozen tissue from 108 OPSCC cases were subject to eight possible assay/assay combinations: p16 immunohistochemistry (p16 IHC); in situ hybridization for highrisk HPV (HR HPV ISH); quantitative PCR (qPCR) for both viral E6 RNA (RNA qPCR) and DNA (DNA qPCR); and combinations of the above.Results: HPV16-positive OPSCC presented in younger patients (mean 7.5 years younger, P ¼ 0.003) who smoked less than HPV-negative patients (P ¼ 0.007). The proportion of HPV16-positive cases increased from 15% to 57% (P ¼ 0.001) between 1988 and 2009. A combination of p16 IHC/DNA qPCR showed acceptable sensitivity (97%) and specificity (94%) compared with the RNA qPCR "gold standard", as well as being the best discriminator of favorable outcome (overall survival P ¼ 0.002). p16 IHC/HR HPV ISH also had acceptable specificity (90%) but the substantial reduction in its sensitivity (88%) impacted upon its prognostic value (P ¼ 0.02). p16 IHC, HR HPV ISH, or DNA qPCR was not sufficiently specific to recommend in clinical trials when used in isolation.Conclusions: Caution must be exercised in applying HPV16 diagnostic tests because of significant disparities in accuracy and prognostic value in previously published techniques.

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Background. The concept that a patient could develop cancer twice was first put forward by Billroth. Second primary neoplasms are a particular feature of head and neck cancer. Methods. This study examines the records of 3436 patients with squamous cell carcinoma of the head and neck, of whom 274 subsequently developed a second neoplasm. Results. The actuarial second primary rate was 9.1% at 372 months, and median time to presentation for the second tumor was 36 months. Second tumors were more likely to occur in male patients younger than 60 years at the time of their index tumor, and who had laryngeal and oral cavity index tumors. Patients whose index tumor was small at diagnosis had a greater chance of developing a second tumor as did those with no cervical lymph node metastases to the neck. Radiotherapy to the index tumor was not associated with an increased risk of developing a second tumor. The commonest sites for second tumors were the head and neck (50%) and the lung (34%), and 86% were squamous cell carcinomas. The tumor‐specific mortality for those who developed a second primary tumor was 20% after 15 years compared with 44% for patients who did not develop a second primary tumor. The 5‐year survival for patients who developed a secondary tumor from the time of its diagnosis was 26%. Conclusions. Second primary tumors in the head and neck of patients with cancer are not uncommon. If the second tumor occurs in the head and neck region, the prognosis is reasonably good. Cancer 1995;75:1343‐53.