Immunohistochemical Evaluation of E6/E7 HPV Oncoproteins Staining in Cervical Cancer.
OBJECTIVE
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.


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. E6-Specific 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.


CONCLUSIONS
Our results revealed a useful staining protocol for the immunohistochemical evaluation of E6/E7 oncoprotein expression in uterine cervical cancer, as well as in HPV-positive 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.
Department of Obstetrics and Gynaecology, Ludwig Maximilian University Munich, Munich, Germany.
Annika
A
Stiasny
Department of Obstetrics and Gynaecology, Ludwig Maximilian University Munich, Munich, Germany.
Christina
C
Kuhn
Department of Pathology, Ludwig Maximilian University Munich, Munich, Germany.
Doris
D
Mayr
ENT-Clinic, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner Fresenius Stiftung University Hospital, Erlangen, Germany.
Christoph
C
Alexiou
ENT-Clinic, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner Fresenius Stiftung University Hospital, Erlangen, Germany.
Christina
C
Janko
ENT-Clinic, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner Fresenius Stiftung University Hospital, Erlangen, Germany.
Irmi
I
Wiest
Department of Obstetrics and Gynaecology, Ludwig Maximilian University Munich, Munich, Germany udo.jeschke@med.uni-muenchen.de.
Udo
U
Jeschke
Department of Obstetrics and Gynaecology, Ludwig Maximilian University Munich, Munich, Germany.
Bernd
B
Kost
D004268:DNA-Binding Proteins / Q000032:analysis; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D009856:Oncogene Proteins, Viral / Q000032:analysis*; D050725:Papillomavirus E7 Proteins / Q000032:analysis; D002583:Uterine Cervical Neoplasms / Q000821:virology*
D004268:DNA-Binding Proteins; C052603:E6 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins
E6; E7; HPV; cervical cancer