Abstract 1115: Protein overexpression is not associated with increased gene copy number of epidermal growth factor receptor (EGFR) in penile carcinoma
Authors: Silva Alice MT , Cunha Isabela W. , Neves Jose I. , Ferreira Severino S. , Rocha Rafael M. , Guimarães Gustavo C. , Buim Marcilei EC , Cubilla Antonio , Lopes Ademar , Soares Fernando A. 
Published: June 2012

Abstract
Squamous cell carcinoma of the penis affects mainly people with poor hygiene habits in undeveloped countries, accounting for up to 20% of all malignancies in men. EGFR is a tyrosine-kinase receptor that has its expression levels increased in diverse tumors, especially in carcinomas. This study has aimed at evaluating the gene alterations associated with cases with high protein expression levels.
Immunohistochemistry (IHC) against EGFR was performed in 195 penile carcinoma samples selected from the files of AC Camargo Hospital, Brazil. Cases showing strong and complete membrane staining in more than 10% of the tumor cells were considered positive and any other staining pattern was considered negative. All positive cases and a fraction of negative cases were submitted to dual-color fluorescence in situ hybridization (FISH). Reactions were carried out using fluorescenet-labeled probes for EGFR locus and chromosome 7 centromere (Zytovision™) in samples overexpressing EGFR. Cases with 2 signals of each probe were considered non-altered, those with more than 2 signals of each probe were considered polissomyc and those with more EGFR signals compared to centromere signals were considered amplified.
In this series, 67 (49,7%) penile carcinoma samples overexpressed EGFR by IHC and protein overexpression was significantly associated with greater risk of recurrence in univariate analysis (p=0,031). Negative cases represented 50,3% of the sample, but only 9 (5%) showed no membrane staining at all. The other 45% of the negative sample (83 cases) showed little to moderate incomplete membrane staining. Regarding FISH, 65 cases (45,8%) were uninterpretable and, out of 72 valid cases, 49 (68,1%) were non-altered cases, 17 (23,6%) were polissomyc, and 6 (8,3%) cases presented EGFR amplification. Considering IHC results, 50 were positive. Out of those, 33 (66%) had no gene number alteration, 12 (24%) were polissomies and 5 (10%) were amplified. Negative cases represented 22 valid cases out of which 16 (72,1%) showed no gene number alteration, 5 (22,8%) showed polissomy of cromossome 7 and 2 cases (9,1%) had an amplification of EGFR gene.As both positive and negative samples in IHC showed similar results in FISH analysis, it seems that EGFR overexpression, although associated with worse prognosis, is not associated either with gene copy number nor with polyssomy of chromosome 7 in penile tumors. Further studies concerning mutational analysis and clinical data are needed to explain this high and frequent protein besides being useful for identifying patients who might benefit from EGFR-target therapy. Moreover, the high number of uninterpretable cases in FISH seems to be related to technical artifacts due to the higher quantity of cytokeratin levels blocking probe penetration in cell cytoplasm and nuclei of these tumor cells.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1115. doi:10.1158/1538-7445.AM2011-1115