Expression of p16 protein identifies a distinct entity of tonsillar carcinomas associated with human papillomavirus

Abstract

Recent analyses of head and neck squamous cell carcinomas revealed frequent infections by oncogenic human papillomavirus (HPV) type 16 in tonsillar carcinomas. Concerning involvement of risk factors, clinical course of the disease, and prognosis there are strong indications arguing that the HPV-positive tonsillar carcinomas may represent a separate tumor entity. Looking for a surrogate marker, which in further epidemiological studies could replace the laborious and expensive HPV detection and typing we analyzed p16 protein expression in 34 tonsillar carcinoma for correlation to HPV status and load of viral DNA. p16 has been shown to be of diagnostic value for clinical evaluation of cervical dysplasia. We found 53% of the tested tonsillar carcinomas to be HPV-positive. Fifty-six percent of all tumors tested were immunohistochemically positive for the p16 protein. In 16 of 18 of the HPV-positive carcinomas diffuse p16 expression was observed. In contrast, only one of the HPV-negative carcinomas showed focal p16 staining (P < 0.001). As determined by laser-assisted microdissection and quantitative real-time polymerase chain reaction, p16 expression correlated with the presence of HPV-DNA in the individual tumor specimens. Clinical outcome analysis revealed significant correlation of p16 expression with increased disease-free survival (P = 0.02). These data indicate that p16 is a technically simple immunohistological marker, applicable for routine pathological histology, and its prognostic value for survival is fully equivalent to HPV-DNA detection.

Figure 1.

Immunohistochemical staining for p16 (antibody 16P04). A: Clear and distinct positive staining for p16 is seen in nuclei and cytoplasm of tumor cells of a HPV-positive tonsillar carcinoma. B: In the HPV-negative tonsillar carcinoma all tumor cells are p16-negative. C: Strong positive staining of tumor cells of a HPV-positive carcinoma is surrounded by p16-negative nondysplastic epithelium. D: The pattern of the p16 staining of the tumor cells of the cervical carcinoma is similar to the HPV-positive tonsillar carcinoma. E: In cases with HPV-positive tonsillar carcinomas areas with dysplastic cell epithelium were p16-positive distinct to normal epithelium. F: Crypt epithelium adjacent to HPV-positive tumor shows p16 immunoreactivity in the superficial crypt epithelium (inset: higher magnification of the crypt epithelium).

Figure 2.

Disease-free survival curves were generated by the Kaplan-Meier method and compared using the log-rank test. Probability of the disease-free survival was significantly better in p16-positive patients (immunoreactivity of >25% of the tumor cells were scored as p16-positive, <25% as p16-negative).