Abstract
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. 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. 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 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.
Uterine cervical cancer is the second most common malignant tumor in women worldwide, which means that there are about 530,000 new cases and more than 270,000 such tumor-associated deaths every year (1). The main cause of uterine cervical cancer is persistent infection with high-risk human papillomavirus (HR-HPV) (2); specifically HPV subtypes 16 and 18 are responsible for about 70% of all cases (2, 3). Apart from uterine cervical cancer, HPVs are also responsible for 25 to 60% of oral squamous cell carcinomas (OSCCs), a form of head and neck cancer (4, 5). At the moment, over 170 HPV types are known (6) and it is highly likely that infection with 15 types (namely types16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82) leads to cancer. Therefore, these 15 types are called carcinogenic or high-risk types (7). Human papillomaviruses are small double-stranded DNA viruses, their genome contains barely 8,000 base pairs arranged as a circle, containing six ‘early’ genes (E6, E7, E1, E2, E4, E5) and two ‘late’ genes (L1, L2) (4). As HPVs do not contain genes coding for replication enzymes, they are dependent on host cells for their replication (4). The replication of the viral genes E6 and E7 leads to cellular expression of E6 and E7 oncoproteins which interferes with the cell cycle (8). E6 oncoprotein links to E6- associated protein (E6-AP) forming a complex which selectively binds to p53, leading to its ubiquitin-dependent proteolytic degradation (9). p53 acts as a tumor suppressor, inducing cell-cycle arrest or apoptosis in cases of DNA damage (10). As a result of the E6-dependent degradation of p53, it can no longer fulfil its tasks (11). p53 is not the only tumor suppressor affected by HPV oncproteins. The retinoblastoma protein (Rb) blocks cell-cycle progression from G1 to S phase by binding and inactivating transcription factors of the free eukaryotic transcription factor E2F family (12, 13). E7 oncoprotein interacts with Rb and leads to its ubiquitin-dependent degradation (14, 15). Furthermore, E7 leads to increased phosphorylation of Rb, but only a small proportion of phosporylated Rb can form a complex with E2F, hence the E2F–Rb interaction is disturbed and E2F is no longer inactivated (16, 17). Consequently, the amount of cyclin-dependent kinase p16 is increased and this is what is used as an immunohistochemical marker for the presence of HPV infection concerning OSCC (18, 19).
In routine practice, the detection of E6/E7 is based on either in situ hybridization (20) or PCR (21) methodology due to unspecific staining results of antibodies used in former studies (22). Therefore, we tested a variety of different antibodies and different unmasking, antigen retrieval and staining protocols. The aim of this study was the establishment of an immunohistochemical staining procedure for a safe and simple detection of E6/E7 at the protein level in HPV-infected tumor tissue.
Materials and Methods
Specimens. For the evaluation of the immunohistochemical staining, paraffin-embedded tissue slides (4 μm thickness) from patients with normal control cervical tissue (10 cases), cervical intraepithelial neoplasia (CIN; 10 CIN III cases) and from patients with uterine cervical carcinoma (10 cases) were used. The CIN III cases were considered as being HPV-positive. Placenta (10 cases) and liver (10 cases) were obtained from the Department of Pathology and used as tissues for providing negative staining results.
Immunohistochemistry. The formalin-fixed paraffin-embedded sections (3 μm) were dewaxed in xylol, the endogenous peroxidase was inhibited by 3% methanol/H2O2 and rehydrated in a descending ethanol gradient. The slides were pretreated in Citrate Buffer (100°C, pH: 6.0) to unmask the antigen. Afterwards, non-specific binding of the primary antibodies was blocked by using the appropriate blocking solution. Incubation with the primary antibodies followed (Table I). Incubation with the secondary antibody and following steps of the convenient detection system and color development were carried out according to the manufacturer protocol. Finally, the slides were counterstained by hemalaun (2 min), dehydrated in a rising ethanol gradient and covered.
Evaluation of staining. The immunoreactive score (IRS) used examines the intensity and distribution of antigen expression and is calculated by multiplying the percentage of positively stained cells (0: no staining; 1<10% of the cells; 2: 11-50%; 3: 51- 80%, 4>81%) with the cell's intensity of staining (0: none; 1: weak; 2: moderate; 3: strong). Two independent investigators examined the sections using a Leitz Diaplan microscope (Leitz, Wetzlar, Germany). The concordance was 98%. In case of different staining evaluation, reevaluation was made by both investigators until an agreement was reached (2% of all cases). Positive controls were carried out with tissues of CIN (10 CIN III cases). Negative controls were performed by replacement of the primary antibody and alternative incubation of the slides with IgG rabbit or mouse control antibodies (Biogenex, San Ramen, USA). In addition, specificity of the staining was evaluated by using control tissue that should not express the E6/E7 antigen (placental and liver tissue).
Antibodies used for the study.
Results
E6 immunohistochemistry. Concerning the detection of E6 oncoprotein, the best results were obtained by using the E6 antibody by Abcam (Table I). By adhering to this procedure, the expression of E6 oncoprotein was shown by intense staining of the cytoplasm of the tumor cells (Figure 1A). As can be seen in Figure 1A, only the cytoplasm of the tumor cells is stained and not the surrounding connective tissue, hence the staining is very selective. It was even possible for us to show that there is a graduation of the expression of the oncoprotein. Within this study, we identified cases of uterine cervical carcinoma with intense staining of E6 (Figure 1A) but also with lower expression of this oncoprotein (Figure 1B). Staining in carcinoma tissue was more intense compared to staining of CIN III (Figure 1C). No expression of the oncoprotein in a sample of non-dysplastic cervix and consequently no staining are shown in Figure 1D. For the establishment of the staining procedure, uterine cervical carcinoma tissue was used (Figure 1E). Liver tissue served for negative control staining (Figure 1F) and as isotype control, we used the same cervical carcinoma as used for staining establishment (Figure 1G).
E7 immunohistochemistry. The evaluation of the E7 antibodies revealed intense and specific expression with the antibody obtained from Chemicon (Table I). By following this instruction, in cases of expression of E7 oncoprotein, the cytoplasm of the tumor cells is stained. We identified uterine cervical carcinoma cases with intensively stained cytoplasm (Figure 2A) and, in addition, cases with lower intensity (Figure 2B). CIN III cases exhibited a less intense staining compared to tumor cases (Figure 2C). There was no expression of E7 oncoprotein found in a non-dysplastic cervix sample (Figure 2D). As positive control tissue tissue from conization of uterine cervix was used (Figure 2E), as negative control, placental tissue was used (Figure 2F), and as isotype control, we used the same cervical carcinoma as used for staining establishment (Figure 2G).
Uterine cervical carcinoma showing intense staining of E6 by using the Abcam antibody (A) but also lower expression of this oncoprotein in another specimen (B). Staining in carcinoma tissue was more intense compared to staining of cervical dysplasia (CIN) grade III (C). No expression of the oncoprotein and consequently no staining were seen in all samples of non-dysplastic cervix (D). For the establishment of the staining procedure, uterine cervical carcinoma tissue was used (E). Liver tissue served for negative control staining (F), and as isotype control, the same cervical carcinoma, as used for staining establishment (G) was used. Magnification, ×25 in all cases.
E7 oncoprotein in uterine cervical carcinoma cases is reflected in intensively stained cytoplasm (A), but there were also cases with lower intensity (B). Cases of cervical dysplasia (CIN) grade III showed less intense staining compared to tumor cases (C). There was no expression of E7 oncoprotein in all samples of non-dysplastic cervix (D). As positive control tissue, tissue from conization of uterine cervix was used (E), as negative control tissue, placenta was obtained (F), and as isotype control, the same cervical carcinoma as used for staining establishment (G) was used. Magnification, ×25 in all cases.
Discussion
In this study, we were able to establish a fast and simple method for the detection of the HPV-related oncoproteins E6/E7 in uterine cervical cancer tissue by using immunohistochemistry. As a result, a useful immunohistochemical evaluation protocol for the detection of the HPV oncoproteins E6 and E7 was established. The best results were obtained with the E6 antibody from Abcam and the E7 antibody from Chemicon. The advantage of this procedure is the possibility of immunohistochemical evaluation of E6/E7 in routine pathology by following our staining protocols. An additional advantage of the immunohistochemical evaluation is that this method is easier to apply and less expensive in comparison to in situ mRNA hybridization. We are aware that in situ mRNA hybridization might be the best way to detect HPV, but on the other hand it is too complicated to be used for routine detection of HPV oncoproteins (23) compared to a simple immunohisto-chemistry protocol.
In summary, we showed that E6 and E7 oncoproteins expressed in uterine cervical carcinoma and CIN III carcinoma in situ tissues can be easily detected by immunohistochemistry using the E6 antibody from Abcam and the E7 antibody from Chemicon.
- Received March 15, 2016.
- Revision received May 2, 2016.
- Accepted May 8, 2016.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.