The incidence of penile cancer (PeCa) is increasing worldwide, however, the highest rates are reported in underdeveloped countries. The molecular mechanisms that underly the onset and progression of these tumors are still unclear. Therefore, our goal was to determine the genome-wide copy number alterations and the involvement of human papiloma virus (HPV) (TP53 and RB1), inflammatory (COX2 and EGFR), and PI3K/AKT pathway (AKT1, AKT2, EGFR, ERBB3, ERBB4, PIK3CA, and PTEN) associated genes in patients with PeCa from a high incidence region in Brazil (Maranhão). HPV genotyping was performed by nest-PCR and genome sequencing, copy number alterations (CNAs) by array comparative genomic hybridization and gene copy number status, gene, and protein expression by quantitative polymerase chain reaction, reverse transcriptasequantitative polymerase chain reaction, and immunohistochemistry, respectively.HPV genotyping revealed one of the highest frequencies of HPV reported in PeCa, affecting 96.4% of the cases. The most common CNAs observed were located at the HPV integration sites, such as 2p12-p11.2 and 14q32.33, where Abbreviations: aCGH, array comparative genomic hybridization; CNA, copy number alteration; Cq, quantification cycle; FFPE, formalin-fixed paraffin-embedded; HPV, human papiloma virus;HrHPV, High risk HPV; IHCi, mmunohistochemistry; PCR, polymerase chain reaction; PeCa, penile cancer; qPCR, quantitative polymerase chain reaction; RT-qPCR, reverse transcriptase-quantitative polymerase chain reaction; TMA, tissue microarray. ADAM 6, KIAA0125, LINC00226, LINC00221, and miR7641-2, are mapped. Increased copy number of ERBB3 and EGFR genes were observed in association with COX2 and EGFR overexpression, reinforcing the role of the inflammatory pathway in PeCa, and suggesting anti-EGFR and anti-COX2 inhibitors as promising therapies for PeCa. Additionally, TP53 and RB1 messenger RNA downregulation was observed, suggesting the occurrence of other mechanisms for repression of these oncoproteins, in addition to the canonical HPV/TP53/RB1 signaling pathway. Our data reinforce the role of epigenetic events in abnormal gene expression in HPV-associated carcinomas and suggest the pivotal role of HPV driving CNAs and controlling gene expression in PeCa. K E Y W O R D Scarcinoma of the penis, genomic alterations, human papillomavirus, molecular markers
Penile cancer (PeCa) is a rare neoplasm in developed countries; however, its incidence is high in developing countries. In Brazil, the North and Northeast regions present the highest incidence of cases; in the state of Maranhão, in particular, about four penectomy procedures are conducted weekly in patients with advanced PeCa. Several risk factors have been attributed to the development of PeCa, including infection with human papillomavirus (HPV) and/or chronic inflammation. However, the genetic pathways that are involved in the malignant transformation impacted by these factors are not completely known. Gene alterations affecting the PI3K/AKT signaling pathway are commonly observed in many human cancers. Mutations in the PI3KCA gene and overexpression of EGFR are described in PeCa; however, their prognostic value and/or association with HPV and inflammatory markers are not well known. Therefore, in this study our objectives were to investigate copy number alterations (CNAs) in specific genes involved in the PI3K/AKT pathway (PIK3CA, AKT2 and PTEN) and the expression of the inflammatory associated markers COX2 and EGFR and their association with HPV infection and histopathologic parameters from the patients. Formalin-fixed, paraffin-embedded (FFPE) tissue specimens from 37 patients with penile cancer were obtained from Instituto Maranhense de Oncologia Aldenora Belo (IMOAB) and Hospital Universitário Presidente Dutra (HU-UFMA), São Luis, Maranhão. The samples were obtained at the time of the surgery; 78.9% and 21.1% of the patients underwent partial and total penectomy, respectively. Lymphadenectomy was conducted in 76.7% of the patients. The average age of the patients was 59.9+18.3 years and the average tumor size was 4.44+2.39 mm. Most of the cases were of the spinocellular carcinoma histology (48.6%), followed by epidermoid (40.5%) and squamous cell carcinoma (10.8%). Tumor grade II was observed in 62.2% of the patients, and grades I and III in 19% each. Lymphatic and perineural invasion was observed in 33.3% and 42% of the patients, respectively. DNA and RNA were isolated from the tumor tissue samples using AllPrep DNA/RNA FFPE Kit. Nested PCR genotyping assay was carried out to determine HPV subtypes of the patients. Copy number of the PIK3CA, AKT 2, and PTEN genes were accessed by TaqMan copy number assays and mRNA expression of the inflammatory associated genes EGFR and COX2 by qRT-PCR analysis. HPV infection was detected in 91.9% of the patients for 14 subtypes of HPV (6, 16, 30, 35, 44, 51, 52, 53, 56, 59, 66, 70, 73, and 74), 23.5% of whom presented with multiple HPV infection. The most frequent subtypes observed, affecting 34.4% of the patients, were the subytpes 16 and 18. Deletion of copy number was observed for all three genes analyzed in almost all the patients (90%); PIK3CA presented the highest frequency of homozygous deletion (56.3%), followed by PTEN (39.4%) and AKT2 (36.4%). No gains and/or amplifications on these genes were observed in any of the cases analyzed. EGFR and COX2 genes were observed to be overexpressed in 87% and 40% of the tumors, respectively. No association was observed between copy number and gene expression alterations of the genes analyzed and HPV infection, and the clinical-histopathologic parameters analyzed. However, patients infected with the HPV subtypes 16 and 18 presented tumors of more advanced grade (grade III). Homozygous deletion of the PIK3CA, PTEN, and AKT2 as well as overexpression of COX2 and EGFR genes were observed in the group of PeCa patients from the state of Maranhão. Although a remarkably high level of patients presented with HPV infection (92%), these genomic alterations occurred independently of this infection and of the other prognostic parameters evaluated. These findings support the involvement of alterations in the PI3K/AKT and inflammatory pathways in the etiology of PeCa, and open up opportunities of using the available or novel therapeutic strategies targeting these pathways for PeCa patients. Citation Format: Silma Regina Ferreira Pereira, Juliana Melo Macedo, Marta Regina de Castro Belfort, Elis Vanessa de Lima Silva, Leudivan Ribeiro Nogueira, José Ribamar Calixto, Ronald Coelho, Antonio Augusto Lima Teixeira Júnior, Jaqueline Diniz Pinho, André Salim Khayat, Gyl Eanes Barros Silva, Luciane Regina Cavalli. Copy number and mRNA expression alterations of the PI3K/AKT and inflammatory pathways in penile cancer patients [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B15.
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