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Stiasny, Annika; Kuhn, Christina; Mayr, Doris; Alexiou, Christoph; Janko, Christina; Wiest, Irmi; Jeschke, Udo and Kost, Bernd (2016): Immunohistochemical Evaluation of E6/E7 HPV Oncoproteins Staining in Cervical Cancer. In: Anticancer Research, Vol. 36, No. 6: pp. 3195-3198

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Background/Aim: High-risk human papillomavirus (HPV) subtypes (i.e. 16 and 18) lead to uterine cervical cancer as well as HPV-positive oropharyngeal cancer (OSCC), a form of head and neck cancer. The induction of HPV-induced cancer is driven by virus-specific oncoproteins E6 and E7. E6 protein of HPV types 16 and 18 interacts with the E3 ubiquitin protein ligase, resulting in ubiquitination and proteolysis of tumor protein p53. E7 inactivates retinoblastoma protein (Rb) by phosphorylation followed by an increase of free eukaryotic transcription factor E2F (E2F) in the cell. This leads to an increase of cyclin-dependent kinase inhibitor p16, that is used as an immunohistochemical marker of HPV-associated OSCC. Unfortunately, p16 is not exclusively increased by E7 oncoprotein in carcinogenesis. Therefore, the aim of this study was to develop an immunohistochemical approach for the direct detection of E6/E7 oncoproteins in uterine cervical cancer as well as in OSCC. Material and Methods: Paraffin sections of uterine cervical cancer and 130 were analyzed. Immunohistochemical staining protocols were evaluated with tissue slides from patients with cervical dysplasia (CIN III) and squamous epithelial carcinoma tissue with HPV infection. Liver and placental tissues were used as negative controls. E6Specific antibody (Biorbyt) was used as primary antibody. The polymer staining method and diaminobenzidine were applied for further development. Panels of E7-specific antibodies were tested. Again, the polymer staining method and diaminobenzidine were applied for further development. Results: E6-Specific antibody revealed specific and intense staining after pre-incubation of tissue slides with citrate buffer solution. Only the E7 antibody obtained from Chemicon showed intense and specific staining in patients with CIN III and squamous epithelial carcinoma tissue. Pre-incubation with proteinase K diminished non-specific reaction. Conclusion: Our results revealed a useful staining protocol for the immunohistochemical evaluation of E6/E7 oncoprotein expression in uterine cervical cancer, as well as in HPVpositive oropharyngeal cancer. Advantages of this method compared to mRNA in situ hybridization of E6/E7 are the much lower costs, as well as the broader applicability in pathological practice.

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