Head and neck squamous cell carcinoma (HNSCC) has the potential for early metastasis and is associated with poor survival. Ano1 (Dog1) is an established and sensitive marker for the diagnosis of gastrointestinal stromal tumors (GIST) and has recently been identified as a Ca2+ activated Cl− channel. Although the ANO1 gene is located on the 11q13 locus, a region which is known to be amplified in different types of human carcinomas, a detailed analysis of Ano1 amplification and expression in HNSCC has not been performed. It is thus still unclear how Ano1 contributes to malignancy in HNSCC. We analyzed genomic amplification of the 11q13 locus and Ano1 together with Ano1-protein expression in a large collection of HNSCC samples. We detected a highly significant correlation between amplification and expression of Ano1 and showed that HNSCC patients with Ano1 protein expression have a poor overall survival. We further analyzed the expression of the Ano1 protein in more than 4′000 human samples from 80 different tumor types and 76 normal tissue types and detected that besides HNSCC and GISTs, Ano1 was rarely expressed in other tumor samples or healthy human tissues. In HNSCC cell lines, expression of Ano1 caused Ca2+ activated Cl− currents, which induced cell motility and cell migration in wound healing and in real time migration assays, respectively. In contrast, knockdown of Ano1 did not affect intracellular Ca2+ signaling and surprisingly did not reduce cell proliferation in BHY cells. Further, expression and activity of Ano1 strongly correlated with the ability of HNSCC cells to regulate their volume. Thus, poor survival in HNSCC patients is correlated with the presence of Ano1. Our results further suggest that Ano1 facilitates regulation of the cell volume and causes cell migration, which both can contribute to metastatic progression in HNSCC.

The incidence of human papilloma virus (HPV) induced oropharyngeal squamous cell carcinoma (OPSCC) increases in the western countries. These OPSCC show distinct molecular characteristics and are characterized by an overexpression of p16, considered a surrogate marker for HPV infection. When compared to patients with p16 negative OPSCC, patients with HPV induced p16 positive OPSCC show a significantly better prognosis, which is reported to be caused by increased radiosensitivity. The objective of the present study was to analyze the impact of p16 expression status on the prognosis of OPSCC treated by either radiotherapy (RT) or primary surgery. Results are based upon a tissue microarray (TMA) of 365 head neck squamous cell carcinomas (HNSCC) including 85 OPSCC with clinico-pathological and follow-up data. p16 positivity correlated significantly with oropharyngeal tumor localization (p < 0.001). Patients with p16 positive OPSCC exhibited a significantly better overall survival than those with p16 negative tumors (p 5 0.007). In a multivariate analysis, survival benefit of patients with p16 positive OPSCC was independent of clinico-pathological parameters such as cT and cN classification and treatment modality. The improved prognosis of p16 positive OPSCC is found after RT as well as after surgery.Although incidence of head and neck squamous cell carcinoma (HNSCC) in general has decreased progressively during the last two decades, 1 an increased incidence of oral and oropharyngeal squamous cell carcinoma (OPSCC) was reported in the United States as well as in Europe. [2][3][4] As already suggested in 1983, these OPSCC are characterized by human papilloma virus (HPV) infection. 5 Viral DNA of high risk HPV 16 can be detected in nuclei of tonsillar cancer cells, 6 and is responsible for the vast majority of HPV positive tumors. 7 These tumors arise mainly in the lingual and palatal tonsils and are characterized as being a type of HNSCC with different distinct epidemiological, molecular, and clinical characteristics. 8 The main risk factor for these HPV positive OPSCC is sexual behavior with a high number of vaginal or oral sex partners. 8,9 HPV-induced OPSCC show distinct molecular characteristics suggesting a different pathway in carcinogenesis compared to HPV negative HNSCC. 10 One particular molecular difference among others concerns p16 expression. p16 functions as a cell-cycle checkpoint regulator, a tumor suppressor gene located on chromosome 9p21.In HPV negative HNSCC p16 is frequently inactivated by deletions, mutations or promoter methylation. 11 In HPV positive OPSCC, overexpressed viral oncoprotein E7 degrades pRb, 12 which otherwise inhibits p16 transcription. 13 The resulting nuclear and cytoplasmic p16 overexpression correlates precisely to HPV positivity and is suggested to be specific for HPV positive OPSCC. 14,15 As p16 overexpression is very rarely seen in HPV negative HNSCC it is considered a surrogate marker for HPV positive OPSCC. Direct diagnosis of HPV in HNSCC is only reliably possible by in s...

p16 is the most relevant prognostic marker in OPSCC and should be considered for inclusion into the official staging system of HNSCC.

Lymph node involvement is prognostically the most determinant clinical factor for patients with head and neck squamous cell carcinomas (HNSCCs). Ultrasound of the neck and fine-needle aspiration (FNA) cytology is one of the first diagnostic procedures and the most accurate diagnostic staging tool for the neck. Patients with HPV-positive oropharyngeal carcinomas (OPSCC) show a significantly better prognosis when compared with HPV-negative OPSCC. P16 overexpression is accepted as surrogate marker for HPV-positive in OPSCC. These HPV/p16-positive OPSCC are localized either in the palatal tonsils or the base of tongue and frequently present with lymph node metastases. We analyzed the correlation and reliability of p16 expression of the FNA of the lymph node metastasis with the immunohistochemical expression of p16 of the same lymph node metastasis and its corresponding primary tumor, as it could be of importance for determining the localization and different prognosis of the primary tumor. 54 HNSCC patients were evaluated, p16 expression of the primary tumors and their lymph node metastases correlated precisely. In 25 of the 54 HNSCC patients, a FNA of the lymph node metastases was taken before the treatment. The positive cytological and immunohistochemical p16 staining correlated exactly. Of the 17 histologically p16-negative lymph node metastases 15 FNA were p16-negative, whereas two samples were p16-positive. In our view, a cytological p16 analysis of cervical lymph node metastasis can facilitate the correct localization of the primary tumor and discriminate reliably HPV-positive OPSCC from HPV-negative HNSCC with their significantly diverse prognosis.