Análise in situ de proteínas diferencialmente expressas em carcinomas epidermoides penianos por espectrometria de massas utilizando Matrix-Assisted Laser Desorption/Ionization (MALDI) / In situ analysis of protein differentially expressed in penile squamous cell carcinoma by Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry

O carcinoma de pênis (CaPe) corresponde a uma doença maligna mutilante do homem. É mais frequente em regiões economicamente desprivilegiadas, como o Norte/Nordeste do Brasil, onde frequentemente é diagnosticado como doença mais avançada. Assim, novos marcadores diagnósticos, prognósticos e preditivos de tratamentos terapêuticos ainda são necessários. Abordagens proteômicas, incluindo o MALDI Imaging, podem contribuir neste sentido. Esta técnica emergente de espectrometria de massas permite a visualização da distribuição espacial de centenas de dados moleculares diretamente da superfície de uma secção tecidual, adquiridos por razão massa/carga (m/z). Neste contexto, nosso principal propósito foi integrar dados de proteômica clássica (gel 2D e Cromatografia Líquida acoplada à Espectrometria de Massas) e de MALDI Imaging, para obter padrões diferenciais de proteínas associados com amostras de Carcinoma Epidermoide Peniano usual (relacionado ou não ao HPV) e espécimes normais, a fim de buscar possíveis biomarcadores da doença. Um total de 45 amostras de CaPe, congeladas, foram inicialmente genotipadas para a presença do HPV. Destas, 60% foram positivas para variantes virais de alto risco. A proteômica clássica (N=24) evidenciou níveis diferenciais de 35 proteínas entre amostras de CaPe e controles, e 29 entre CaPe HPV positivo versus negativo (P<0,05; ANOVA). Redes de interações demonstraram que estes perfis proteicos interagiam com clusters de proteínas relacionadas com a carcinogênese e progressão tumoral. Entre eles, se destacaram aqueles formados por proteínas antioxidantes e de adesão celular, presentes em níveis elevados em tumores HPV negativos. A partir dos interactomas, quatro alvos proteicos foram selecionados para a análise in situ por imageamento: Calreticulina, 14-3-3 sigma, Serpina B5 e Glutationa-s-transferase. A aquisição de dados do MALDI Imaging foi conduzida após a digestão in situ pela tripsina, usando uma resolução de 200 µm e faixa de 700-3500 m/z para peptídeos (N=31). Os dados de identificação do gel 2D foram então integrados aos do imageamento. A identidade proteica dos filtros foi confirmada, in silico, por meio da presença de peptídeos teóricos co-localizados com o peptídeo experimental alvo nas secções de CaPe. Não houve associação significativa entre os parâmetros clinicopatológicos e as intensidades de sinal dos alvos (P>0,05, U de Mann-Whitney). Análises não supervisionadas, realizadas a partir dos dados do MALDI Imaging, evidenciaram mapas de segmentação que coindiciram com as regiões tumorais e margens adjacentes livres de neoplasia. Entre os principais valores de m/z diferenciadores estava o pico 1413 ± 2,5 Da, abundante nas regiões tumorais, e correlacionado ao peptídeo experimental m/z 1410,86 referente à proteína Calreticulina (CRT), o. Análises estatísticas (PCA e Curva ROC) indicaram este valor de m/z como potencial biomarcador da doença. Por conseguinte, a CRT foi selecionada para a etapa de validação por imunoistoquímica em tecidos parafinados de CaPe (N=158). Níveis elevados de imunoreatividade da CRT foram associados com piores tempo de sobrevida global (Razão de Risco 2,3; IC-1,46-3,96; P<0,001) e câncer específica (Razão de Risco 4,37; IC-1,66-11,51; P=0,002) nos casos de CaPe. A presença de metástase em linfonodos foi considerado um fator prognóstico independente para o risco de morte pelo câncer (Razão de Risco ­ 14,18; CI-3,29-61,12; P <0,001). A imunoreatividade da CRT também foi capaz de predizer a presença de metástase em linfonodos (Chance de Risco: 1,006; IC- 1,0001-1,0012; p=0,044). Estes dados, em conjunto, sugerem que a CRT pode ser um potencial biomarcador prognóstico do CaPe. A estratégia de integração da proteômica clássica com o MALDI Imaging, mostrou-se uma ferramenta útil na busca de novos biomarcadores para o CaPe. Além disto, o trabalho adicionou uma visão analítica à histopatologia clássica, o que deverá inserir as técnicas utilizadas neste projeto em estudos de Anatomia Patológica, tanto em nossa instituição, quanto no contexto global.

Penile cancer (PeCa) corresponds to a mutilating malignant disease in men. It is more frequent in underprivileged socioeconomic regions (e.g., Noth, North-East of Brazil), where it is frequently diagnosed in advanced stages. Thus, new markers are still needed for early diagnosis, prognosis and prediction of therapy. Proteomic approaches, including MALDI Imaging, could assist in this effort. This emerging spatially resolved mass spectrometric technique can obtain topographical distribution of hundreds of molecules directly from the tissue section surface, mensured by mass/charge ratio (m/z). In this context, our mainly propose was to integrate classic proteomic data (2D gel and Liquid Chromatograph coupled with Mass Spectrometry) with MALDI Imaging to obtain diferential patterns of protein associated with Usual Squamous Cell Penile Carcinoma (HPV related or not) and normal specimens, to look for possible biomarkers of the disease. A total of 45 fresh-frozen PeCa samples were initially searched for HPV genotype, 60% of which were positive for high-risk HPV. Classic proteomics (N=24) demonstrated diferential levels of 35 proteins comparing PeCa and control samples, and 29 comparing HPV-positive versus HPV-negative PeCa samples (P<0.05; ANOVA). Protein networks showed that these protein profiles interact with clusters of proteins related with tumorigenesis and tumor progression processes. Among them, antioxidant and cell adhesion proteins play a critical role in HPV negative penile tumors. Based on interactome data, four protein targets were selected for in situ analyses by imaging: Calreticulin, 14-3-3 protein sigma, Serpin B5 and Glutatione-s-transferase. MALDI Imaging data acquisition of peptides was conducted after in situ trypsin digestion using a lateral resolution of 200 µm, covering the range 700- 3500 m/z (N=31). After that, 2D gel based proteomic data was integrated with Imaging data. The filter protein identities were confirmed in silico by the co-localization of theoretical triptic peptides with the experimental peptides in PeCa sections. There was no significant association between the clinical and pathological parameters and the target signal intensities (P>0.05; U de Mann-Whitney). An unsupervised clustering analysis based on MALDI Imaging data reveled segmentation maps that coincide with histological annotation for tumor and adjacent non-neoplasic regions. Among the mainly differentiating m/z values there was 1413 ± 2.5 Da. This peak was especially co-localized with tumoral regions and correlated with Calreticulin (CRT) experimental peptide (m/z 1410,86). Statistical analysis (PCA and ROC Curves) indicated this m/z value as a potencial biomarker of the disease. For this reason, CRT was selected for validation by immunohistochemistry performed on paraffin-embedded PeCa tissues (N=158). As result, CRT hiperexpression in PeCa tissue increased the risk of unfavorable overall survival (Relative Risk ­ 2.3; CI-1.46-3.96; P<0.001) and cancer specific survival (Relative Risk ­ 4.37; CI-1.66-11.51; P=0.002) in these patients. Lymph node metastasis represented an independent prognostic risk factor for death related to cancer in our patients (Relative risk ­ 14.18; CI-3.29-61.12; P <0.001). CRT immunoreactivity was also capable to predict the presence of lymph node metastases (Risk Chance ­ 1,006; CI-1.0001-1.00123; P =0.044). Taken together, our results sugest that CRT may represent a prognostic biomarker of PeCa. The strategy of integrated classic proteomic and MALDI Imaging revealed as usefull tool to search for news biomarkers of the disease. Futhermore, this work added an analytical perspective to the classical histopathology, allowing to include the techniques used in this project in future morphological studies, both in our institution and in the global context.

Proteomics analysis of tissue samples from patients with squamous cell carcinoma of the penis and positive to human papillomavirus

ABSTRACTPurpose:The aim of this study was to identify possible protein biomarkers and/or candidates for therapeutic targets in tissues of patients with SCCP, infected by HPV, applying one dimensional electrophoresis (1DE), followed by direct mass spectrometry (MS) analysis.Materials and Methods:Tissues from 10 HPV positive patients with SCCP and from 10 patients with HPV negative non-tumorous penile foreskins were analyzed applying 1D electrophoresis, followed by analysis with direct mass spectrometry (MS).Results:Sixty-three different proteins were identified in the first group and 50 in the second group. Recognition was possible for 28 proteins exclusively detected in Group 1 and 21 proteins presented only in Group 2.Conclusion:Some proteins in the first group are directly involved in the development of other types of cancer, and therefore, suitable for analysis. Complement C3 protein is a strong candidate for evaluating SCCP patients.

Human Papilloma Virus: Prevalence, distribution and predictive value to lymphatic metastasis in penile carcinoma

Objectives To evaluate the prevalence, distribution and association of HPV with histological pattern of worse prognosis of penile cancer, in order to evaluate its predictive value of inguinal metastasis, as well as evaluation of other previous reported prognostic factors. Material and Methods Tumor samples of 82 patients with penile carcinoma were tested in order to establish the prevalence and distribution of genotypic HPV using PCR. HPV status was correlated to histopathological factors and the presence of inguinal mestastasis. The influence of several histological characteristics was also correlated to inguinal disease-free survival. Results Follow-up varied from 1 to 71 months (median 22 months). HPV DNA was identified in 60.9% of sample, with higher prevalence of types 11 and 6 (64% and 32%, respectively). There was no significant correlation of the histological characteristics of worse prognosis of penile cancer with HPV status. Inguinal disease-free survival in 5 years did also not show HPV status influence (p = 0.45). The only independent pathologic factors of inguinal metastasis were: stage T ≥ T1b-T4 (p = 0.02), lymphovascular invasion (p = 0.04) and infiltrative invasion (p = 0.03). conclusions HPV status and distribution had shown no correlation with worse prognosis of histological aspects, or predictive value for lymphatic metastasis in penile carcinoma. .

Prevalence of human papillomavirus and Epstein-Barr virus DNA in penile cancer cases from Brazil

Penile cancer is a potentially mutilating disease. Although its occurrence is relatively rare worldwide, penile cancer rates can be high in developing countries. A few studies have been conducted on the involvement of human papillomavirus (HPV) in penile carcinoma, which have found HPV present in 30-70 percent of penile malignant lesions, with a higher prevalence of HPV 16 and 18. It has been assumed that cofactors, such as Epstein-Barr virus (EBV) infections, may play a role in the progression of penile neoplasia. The aim of this study was to determine HPV and EBV prevalence in 135 penile malignant lesions from Brazilian men through the use of MY09/11 polymerase chain reaction (PCR), type-specific PCR and restriction fragment length polymorphism analysis. HPV prevalence among the men tested was 60.7 percent. Of the men who tested positive, 27 presented with HPV 16 (29.7 percent), five with HPV 18 (5.5 percent), 21 with HPV 45 (23.1 percent) and nine with HPV 6 (9.9 percent). Seven mixed infections were detected (9.2 percent), while 11 cases remained untyped (13.4 percent). Regarding EBV positivity, 46.7 percent of the samples contained EBV DNA with EBV-1 as the most prevalent type (74.6 percent). More than 23 percent of the men were co-infected with both HPV and EBV, while 35 percent presented exclusively with HPV DNA and 20 percent presented only with EBV DNA. Penile carcinoma aetiology has not been fully elucidated and the role of HPV and EBV infections individually or synergistically is still controversial. Hence, more studies are needed to determine their possible role in carcinogenesis.

Carcinoma de ano, pene, vulva y vagina: ¿son enfermedades de transmisión sexual? / Anogenital neoplasms: are sexually transmitted diseases?

Introducción: La infección por virus papiloma humano (VPH) sería factor causal de cánceres de ano, pene, vulva y vagina. Objetivo: Analizar la evidencia actual en cuanto a infección por VPH y su rol carcinogénico en estas neoplasias. Metodología: Búsqueda de la literatura para identificar artículos sobre la transmisión sexual como factor de riesgo en cánceres anogenitales. Resultados: En lesiones premalignas y malignas anogenitales se encuentra en gran frecuencia el DNA de VPH, especialmente tipo 16. Se ha demostrado que la vacunación contra VPH previene el desarrollo de lesiones preinvasoras anales; en cambio, ni la vacuna ni la circuncisión parecen ser factores protectores contra cáncer de pene. Discusión: No hay estudios prospectivos que permitan establecer una relación causal entre VPH y cánceres anogenitales, lo que impide la elaboración de estrategias de prevención. El manejo de ciertos factores de riesgo sugeridos previamente en la literatura no reduce el riesgo de cáncer anogenital.

Introduction: Human papillomavirus (HPV) infection has been suggested as a causal factor of anal, penile, vulvar and vaginal cancers. Objective: To analyze current evidence about HPV infection and its carcinogenic role in these neoplasms. Methodology: Literature search to identify articles about sexual transmission as a risk factor in anogenital cancers. Results: In premalignant and malignant anogenital lesions, an important presence of HPV DNA is often found, specially type16. It has been demonstrated that HPV vaccine prevents premalignant anal lesions; however, this vaccine and circumcision do not seem to be protective against penile cancer. Discussion: There are no prospective studies that had established a causal relationship between HPV and anogenital cancers. This keeps off the development of adequate prevention strategies. Management of certain previously suggested risk factors do not reduce the risk of anogenital cancer.

Human papillomavirus and penile cancers in Rio de Janeiro, Brazil: HPV typing and clinical features

OBJECTIVE: To determine the prevalence of human papillomavirus (HPV) DNA in penile cancers in Rio de Janeiro, Brazil. MATERIALS AND METHODS: We studied, prospectively, 80 consecutive cases of patients with penile cancers who underwent surgical treatment at three different Hospitals in Rio de Janeiro between March 1995 and June 2000. Of these patients, 72 were diagnosed with invasive squamous cell carcinoma and 8 patients with verrucous carcinoma. The following parameters were observed: presence or absence of HPV DNA viral type, histological subtypes, clinical stage and overall survival. RESULTS: HPV DNA was detected in 75 percent of patients with invasive carcinomas and in 50 percent of patients with verrucous carcinomas. High risk HPVs were detected in 15 of 54 (27.8 percent) patients with HPV positive invasive tumors and in 1 of 4 (25 percent) patients with HPV positive verrucous tumors. HPV 16 was the most frequent type observed. No correlation was observed between HPV status and histological subtype (p = 0.51) as well as HPV status and stage stratification (p = 0.88). HPV status was also not significantly associated with the presence of regional metastases (p = 0.89). The overall survival was related to the presence of lymph node metastases (p < 0.0001). CONCLUSIONS: HPV infection may have contributed to malignant transformation in a large proportion of our penile cancer cases but only inguinal metastasis was a prognostic factor for survival in these patients with penile carcinoma.