Bovine alphaherpesvirus type 5 (BoHV-5) is one of the main agents responsible for meningoencephalitis in cattle in Brazil, causing significant economic losses. It is known that other viruses of the Herpesviridae family such as Bovine alphaherpesvirus type 1, Swine alphaherpesvirus type 1, and the Human alphaherpesvirus types 1 and 2 encode genes homologous to BoHV-5, with recognized action in the control of apoptosis. The objective of this work was to express the BoHV-5 US3 gene in a baculovirus-based expression system for the production of the serine/threonine kinase protein and to evaluate its activity in the control of apoptosis in vitro. A recombinant baculovirus derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) containing the US3 gene and a deletion in the baculovirus anti-apoptotic p35 gene was constructed using the Bac-to-Bac™ system. This recombinant baculovirus was used to evaluate the anti-apoptotic activity of the recombinant US3 protein in insect cells comparing with two other AcMNPV recombinants, one containing a functional copy of the AcMNPVanti-apoptotic p35 gene and an AcMNPV p35 knockout virus with the anti-apoptotic iap-3 gene from Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV). We found that the caspase level was higher in insect cells infected with the US3-contanining recombinant virus than in cells infected with the AcMNPV recombinants containing the p35 and iap-3 genes. These results indicate that the BoHV-5 US3 protein kinase gene is not able to block apoptosis in insect cells induced by the infection of a p35 knockout AcMNPV.

BACKGROUND: Penile squamous cell carcinoma (PSCC) is a rare tumor. In specific regions of developing countries, however, it may represent one the most frequent male tumors. Presence of lymph node metastasis (LNM) is the main factor significantly associated with reduced survival, and molecular markers to predict it have been extensively pursued. HER2 is an oncogene part of the EGFR family, often overexpressed in PSCC. HER2 overexpression is associated with poor prognosis and presence of LNM in breast tumors, and it is generally coamplified with topoisomerase IIα (TOP2A), a neighboring gene that is a target for anthracyclines. MATERIAL AND METHODS: We evaluated the frequency of HPV infection in 84 PSCC samples.The presence or absence of HPV DNA was established using generic primers (GP5+/GP6+) specific for the L1 gene of several HPVs. Genotyping of positive samples was done by dot blot hybridization using radioactive probes. Expression of HER2 and TOP2a was evaluated through immunohistochemistry and automatedly quantified using ScanScope XT, Aperio. Sixty cases were also submitted to a triple-color fluorescence in situ hybridization (FISH) assay using probes for the genes of HER2 and TOP2a, and also for the chromosome 17 centromere. Cases showing two signals of each probe were considered non-altered, those showing more than two signals of each probe were considered polysomy and those showing more HER2 or TOP2a signals compared to centromere signals were considered amplified. RESULTS: HPV infection, characterized by detection of viral DNA on the samples, was observed in 22.6% (19) of the cases. Among the positive HPV samples, 51.3% were infected by HPV16, 21.6% by HPV18, and 2.7% by both subtypes concomitantly. In agreement with studies that showed better prognosis of tumors with HPV infection, 5-year and 10-year survival of HPV-positive patients was, respectively, 79.1% and 70.8%, whereas of HPV-negative patients was 60.2% and 38.7% (p=0,018). Immunohistochemistry showed nuclear positivity for TOP2a in 74.2% (106) cases and cytoplasmic expression of HER2 was seen in 20,3% (29) of cases, which was associated with higher histologic grade (p <0,001) and lower overall survival in multivariate analysis (RR=2,966; IC 95% [1,6-5,2]; p<0,001). No membrane staining for HER2 was observed. There was not an association between expression of HER2 and TOP2a (p=0.970), nor with these markers and HPV infection (p=0.177 and p=0.336, respectively). FISH analysis revealed 55 out of 60 cases without gene alterations (91.7%), and five cases (8,3%) presenting polysomy of chromosome 17. No amplification of HER2 or TOP2a genes was observed.CONCLUSIONS: Overexression of TOP2a was frequent and cytoplasmic HER2 associates with poor outcome of PSCC patients. HPV infection seem to have a positive impact in patients’ survival, as reported for other tumors. Citation Format: Alice M. Silva, Lara Termini, Jose Ivanildo Neves, Isabela Cunha, Ademar Lopes, Gustavo Guimaraes, Fernando A. Soares. Evaluation of HER2 and topoisomera II alpha (TOP2a) expression and gene number in penile carcinoma according to HPV status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5296. doi:10.1158/1538-7445.AM2013-5296

BACKGROUND: Although rare in developed regions, penile carcinoma is a serious concern for undeveloped countries such as Brazil, where it can represent up to 10% of all malignancies. HER2 is a well-known growth factor membrane receptor that has its expression level usually associated with aggressive disease. Recently, some studies have shown that soluble fractions of HER2 intracellular domain are found in tumors cytoplasm and they still activate intracellular pathways related to cell proliferation and survival. MATERIAL AND METHODS: Immunohistochemistry against HER2 was carried out in 195 penile carcinoma samples selected from the files of AC Camargo Hospital, Brazil, using the monoclonal CB11 antibody (Novocastra™), which recognizes specifically HER2 intracellular domain. Cases were classified as positive or negative according to membrane and to cytoplasmic staining. All statistical analyses were performed by the SPSS for Windows 18.0, SPSS Inc. Comparison between category variables was performed by the Pearson chi-square test or the Fisher's exact test. Survival rates were calculated using the Kaplan-Meier method and the curves were compared by means of the log-rank test. In all statistical tests, the alpha error was set at 5%. RESULTS: None of the cases showed membrane staining for HER2. However, a clear and strong cytoplasm staining was observed in 28 cases (14,9%), as shown in figure 1. The staining was limited to the tumor cells and does not represent background or artifact. Univariate analyses showed that overexpression is associated with higher histologic grade (p <0,001) and lower overall survival (p=0,007). Multivariate analyses showed that HER2 in the cytoplasm is an independent risk factor for death (RR=2,966; IC 95% [1,6-5,2]; p <0,001). CONCLUSIONS: Corroborating with recent findings that cytoplasmic fragments of HER2 can be active kinases, the present study suggests that the presence of HER2 in tumor cytoplasm is a prognostic marker in penile carcinoma. We have also demonstrated that, although no membrane staining was seen, presence of HER2 in cytoplasm is an independent risk factor for death in this neoplasm. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5577. doi:1538-7445.AM2012-5577