Análise da expressão da proteína NY-ESO-1 no melanoma cutâneo

INTRODUÇÃO: o câncer é a doença que mais mata pessoas com idade abaixo de 85 anos e é um problema de saúde pública. Os tumores podem expressar em determinada fase de seu desenvolvimento proteínas anômalas que podem ser alvo de métodos diagnósticos e de intervenções terapêuticas. A expressão de NY-ESO-1 é detectada em 20 a 40% dos melanomas. Há evidências que esta expressão é mais freqüente em tumores de estágios mais avançados e está associada a um pior prognóstico. OBJETIVOS: determinar a frequência de expressão da proteína NY-ESO-1 no melanoma cutâneo e tentar correlacioná-la com o índice de Breslow, aspectos histopatológicos do melanoma, incluindo o infiltrado linfocítico tumoral, e a morbi-mortalidade dos pacientes. MÉTODOS: o presente estudo é longitudinal de coorte retrospectiva e foi realizado de agosto de 2009 a outubro de 2015. Foram selecionados 89 melanomas de 87 pacientes do Ambulatório de Tumores do Departamento de Dermatologia da FMUSP, divididos em 3 grupos, sendo: grupo 1: 34 melanomas com índice de Breslow <= 1,0 mm; grupo 2: 29 melanomas com índice de Breslow entre 1,1 - 4,0 mm e grupo 3: 26 melanomas com índice de Breslow >= 4,0 mm. As lâminas dos exames anátomo-patológicos destes pacientes foram revisadas quanto ao diagnóstico de melanoma, seu índice de Breslow e a presença de infiltrado linfocítico tumoral. A seguir, realizou-se exame de imunohistoquímica para a determinação da presença do antígeno NY-ESO-1 em todos os 89 tumores coletados e em mais 20 nevos (11 displásicos e 9 intradérmicos) escolhidos ao acaso. Através da revisão dos dados do prontuário, foram obtidos os dados clínicos de: idade, sexo, raça, fototipo da pele, local de aparecimento do melanoma, status do linfonodo sentinela quando realizado, desenvolvimento de metástases e sobrevida dos pacientes. Os dados anátomo-patológicos do tumor analisados foram: tipo histológico, presença de ulceração, e tipo de infiltrado linfocítico tumoral. Nos melanomas que apresentavam infiltrado linfocítico tumoral, foram realizados testes imunohistoquímicos para pesquisa de células CD3+, CD8+, FoxP3+ e CD8+FoxP3+ (duplamente positivas). RESULTADOS: O antígeno NY-ESO-1 esteve presente em 19% dos melanomas cutâneos primários e não foi detectado em nenhum dos 20 nevos pesquisados. A expressão do antígeno NY-ESO-1 esteve estatisticamente relacionada a tumores com espessuras maiores. Apresentou também uma associação inversa com o tipo extensivo superficial em relação aos outros tipos histológicos. O infiltrado linfocítico tumoral dos melanomas NY-ESO-1 positivos continha menor número de células CD3+, que se encontravam isoladas ou arranjadas em pequenos grupos de até 5 células, o que contrastava significantemente com os tumores NY-ESO-1 negativos, com maior densidade de células CD3+, dispostas em grandes grupos, com 6 ou mais células. A expressão da proteína NY-ESO-1 não esteve associada à idade, ao sexo, ao fototipo, ao sítio primário do tumor, à presença de ulceração, ao status do linfonodo sentinela, ao desenvolvimento de metástases ou à sobrevida. CONCLUSÕES: Há expressão de NY-ESO-1 em uma porcentagem considerável dos melanomas, principalmente nos mais espessos. O menor número de células CD3+ no infiltrado linfocítico tumoral, acrescido ao fato destas células estarem isoladas ou em pequenos grupos, sugere que embora imunogênico, a expressão do antígeno NY-ESO-1 não resulta num estímulo eficaz do sistema imune no combate ao tumor. O desenvolvimento de uma vacina para estes pacientes poderá, no futuro, aumentar as possibilidades terapêuticas do melanoma

Papilomatose oral : considerações a propósito de um caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Carcinoma e leucemia da glândula lacrimal: a raridade num caso clinico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Endolymphatic sac tumor : A report of 2 cases with long term follow-up

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Displasia fibrosa e dor orofacial

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

Avaliação de polimorfismos funcionais nos genes de reparo XRCC1, APEX1, XPD e XPF em carcinomas de células escamosas orais

Base excision repair (BER) and nucleotide excision repair (NER) pathways play critical role in maintaining genome integrity. Polymorphisms in BER and NER genes which modulate the DNA repair capacity may affect the susceptibility and prognosis of oral cancer. This study was conducted with genomic DNA from 92 patients with oral squamous cell carcinomas (OSCC) and 130 controls. The cases were followed up to explore the associations between BER and NER genes polymorphisms and the risk and prognosis of OSCC. Four single-nucleotide polymorphisms (SNPs) in XRCC1 (rs25487), APEX1 (rs1130409), XPD (rs13181) and XPF (rs1799797) genes were tested by polymerase chain reaction – quantitative real time method. The GraphPad Prism version 6.0.1 statistical software was applied for statistical analysis of association. Odds ratio (OR), hazard ratio (HR), and their 95 % confidence intervals (CIs) were calculated by logistic regression. Kaplan-Meier curve and Cox proportional hazard model were used for prognostic analysis. The presence of polymorphic variants in XRCC1, APEX1, XPD and XPF genes were not associated with an increased risk of OSCC. Gene-environment interactions with smoking were not significant for any polymorphism. The presence of polymorphic variants of the XPD gene in association with alcohol consumption conferred an increased risk of 1.86 (95% CI: 0.86 – 4.01, p=0.03) for OSCC. Only APEX1 was associated with decreased specific survival (HR 3.94, 95% CI: 1.31 – 11.88, p=0.01). These results suggest an interaction between polymorphic variants of the XPF gene and alcohol consumption. Additionally APEX1 may represent a prognostic marker for OSCC.

Avaliação do efeito da inibição do reparo de sítios abásicos na resposta inflamatória celular

Base excision repair (BER) proteins has been associated with functions beyond DNA repair. Apurynic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein involved in a plethora of cellular activities, such as redox activation of transcription factors, RNA processing and DNA repair. Some studies have described the action of the protein 8-oxoguanine (OGG1) in correcting oxidized lesions in promoters as a step in the transcription of pro-inflammatory cytokines. Despite being especially important in redox activation of transcription factors such as nuclear factor κB (NF-κB) and AP- 1, the repair activity of APE1 has not yet been associated with the inflammatory response. In this study, experimental and bioinformatic analysis approaches have been used to investigate the relationship between inhibition of the repair of abasic sites in DNA by MX, a synthetic molecule designed to inhibt the repair activity of APE1, and the modulation of the inflammatory response. The results showed that treatment of monocytes with lipopolysaccharide (LPS) and MX reduced the expression of cytokines, chemokines and toll-like receptors, and negatively regulated biological immune processes, as macrophages activation, and NF-κB and tumor necrosis factor (TNF-α) and interferon pathways, without inducing cell death. The transcriptomic analysis suggests that LPS/MX treatment induces mitochondrial dysfunction, endoplasmic reticulum stress and activation of autophagy pathways, probably activated by impairment of cellular energy and/or the accumulation of nuclear and mitochondria DNA damage. Additionally, it is proposed that the repair activity of APE1 is required for transcription of inflammatory genes by interaction with abasic sites at specific promoters and recruitment of transcriptional complexes during inflammatory signaling. This work presents a new perspective on the interactions between the BER activity and the modulation of inflammatory response, and suggests a new activity for APE1 protein as modulator of the immune response in a redox-independent manner.

Imunoexpressão das proteínas APE-1 e XRCC-1 em carcinoma epidermoide de língua oral

DNA repair systems play a critical role in protecting the human genome from damage caused by carcinogens present in the environment. Mutations in DNA repair genes may be responsible for tumor development and resistance of malignant cells to chemotherapeutic agents. The major pathway for oxidative DNA damage repair is the base excision repair pathway. The objective of this study was to investigate the immunoexpression of APE-1 and XRCC-1, which are proteins involved in DNA base excision repair and its association with clinical and histopathological parameters in oral tongue squamous cell carcinoma (OTSCC), in order to investigate a possible prognostic value for those proteins. The expression of APE-1 and XRCC-1 was evaluated semi-quantitatively by immunohistochemistry in 50 OTSCC cases. Clinical data was collected from patients’ medical charts and histopathological grading was performed for each case. Statistical analysis (Chi-square and Fisher’s exact tests; significance of 5%) was performed to determine the association between protein expressions and clinico-pathological characteristics. APE-1 was highly expressed in nucleus and cytoplasm in 56% of cases. XRCC-1 showed overexpression only in nucleus in 60% of cases. High expression of XRCC-1 was significantly associated to clinical stages I and II (P=0.02). Both proteins were not associated to other clinical parameters or histopathological grading. Our findings demonstrate that DNA base excision repair proteins APE-1 and XRCC-1 are upregulated in OTSCC, however, they are not related to clinical and histologic parameters, except for XRCC-1 association to better clinical staging. Our results indicate that the immunohistochemical expression of these proteins has no association with prognostic parameters in this tumor.

Imunoexpressão das proteínas APE-1 e XRCC-1 em carcinoma epidermoide de língua oral

DNA repair systems play a critical role in protecting the human genome from damage caused by carcinogens present in the environment. Mutations in DNA repair genes may be responsible for tumor development and resistance of malignant cells to chemotherapeutic agents. The major pathway for oxidative DNA damage repair is the base excision repair pathway. The objective of this study was to investigate the immunoexpression of APE-1 and XRCC-1, which are proteins involved in DNA base excision repair and its association with clinical and histopathological parameters in oral tongue squamous cell carcinoma (OTSCC), in order to investigate a possible prognostic value for those proteins. The expression of APE-1 and XRCC-1 was evaluated semi-quantitatively by immunohistochemistry in 50 OTSCC cases. Clinical data was collected from patients’ medical charts and histopathological grading was performed for each case. Statistical analysis (Chi-square and Fisher’s exact tests; significance of 5%) was performed to determine the association between protein expressions and clinico-pathological characteristics. APE-1 was highly expressed in nucleus and cytoplasm in 56% of cases. XRCC-1 showed overexpression only in nucleus in 60% of cases. High expression of XRCC-1 was significantly associated to clinical stages I and II (P=0.02). Both proteins were not associated to other clinical parameters or histopathological grading. Our findings demonstrate that DNA base excision repair proteins APE-1 and XRCC-1 are upregulated in OTSCC, however, they are not related to clinical and histologic parameters, except for XRCC-1 association to better clinical staging. Our results indicate that the immunohistochemical expression of these proteins has no association with prognostic parameters in this tumor.

Imunoexpressão da proteína endonuclease apurínica/apurimidínica (APE-1) em adenomas pleomórficos e carcinomas ex-adenomas pleomórficos

Introduction: Apurinic/Apyrimidinic Endonuclease 1 (APE-1) is an essential protein for DNA base excision repair (BER) pathway and regulation of redox activities. The ability of malignant cells to recognize and repair DNA damage is an important mechanism for tumor survival, and recent studies suggest that APE-1 overexpression is related to poor prognosis in some tumors. Purpose: To analyze the immunoreactivity of APE-1 in Pleomorphic Adenomas (PA) and Carcinomas Ex Pleomorphic Adenomas (CaExPA) of salivary glands. Materials and Methods: A total of 49 tumors fixed in formalin and embedded in paraffin (33 PA and 16 CaExPA) underwent immunohistochemical study by the immunoperoxidase technique. APE-1 immunoreactivity was evaluated quantitatively by the percentage of immunopositive cells. For statistical analysis a significance level of 5% (p≤ 0.05) was adopted. Results: All cases of PA and CaExPA (n=49) were positive for APE-1, however, there was a higher expression in CaExPA, with statistically significant difference (p<0.001). There was no association between APE-1 expression and tumors of major or minor salivary gland, however, not encapsulated PA (median expression = 54.2%) showed higher expression when compared to encapsulated tumors (p=0.02). APE-1 overexpression was found mainly in cases of CaExAP with lymph node metastasis (median expression = 90.3% - p=0.002) and invasive pattern (median expression = 89.9% - p=0.003), when compared to cases without metastasis and intracapsular pattern. Conclusion: This study suggests that APE-1 is deregulated in the studied tumors. The increased expression of APE-1 is associated with the absence of complete capsule in PA and it is associated with more aggressive behavior in CaExPA.

Imunoexpressão da proteína endonuclease apurínica/apurimidínica (APE-1) em adenomas pleomórficos e carcinomas ex-adenomas pleomórficos

Introduction: Apurinic/Apyrimidinic Endonuclease 1 (APE-1) is an essential protein for DNA base excision repair (BER) pathway and regulation of redox activities. The ability of malignant cells to recognize and repair DNA damage is an important mechanism for tumor survival, and recent studies suggest that APE-1 overexpression is related to poor prognosis in some tumors. Purpose: To analyze the immunoreactivity of APE-1 in Pleomorphic Adenomas (PA) and Carcinomas Ex Pleomorphic Adenomas (CaExPA) of salivary glands. Materials and Methods: A total of 49 tumors fixed in formalin and embedded in paraffin (33 PA and 16 CaExPA) underwent immunohistochemical study by the immunoperoxidase technique. APE-1 immunoreactivity was evaluated quantitatively by the percentage of immunopositive cells. For statistical analysis a significance level of 5% (p≤ 0.05) was adopted. Results: All cases of PA and CaExPA (n=49) were positive for APE-1, however, there was a higher expression in CaExPA, with statistically significant difference (p<0.001). There was no association between APE-1 expression and tumors of major or minor salivary gland, however, not encapsulated PA (median expression = 54.2%) showed higher expression when compared to encapsulated tumors (p=0.02). APE-1 overexpression was found mainly in cases of CaExAP with lymph node metastasis (median expression = 90.3% - p=0.002) and invasive pattern (median expression = 89.9% - p=0.003), when compared to cases without metastasis and intracapsular pattern. Conclusion: This study suggests that APE-1 is deregulated in the studied tumors. The increased expression of APE-1 is associated with the absence of complete capsule in PA and it is associated with more aggressive behavior in CaExPA.

Caracterização de genes de reparo de DNA em cana-de-açúcar

Sugarcane is an important culture for Brazil that holds almost half of all worldwide productivity. Plants face many challenges, because of biotic and abiotic stresses presents in the production field, which could prevent plants from reaching their genetic potential. As consequence, those stresses can generate Reactive Oxygen Species – ROS – that can cause damages on DNA. Another consequence of stress is the early-flowering process, which contributes for a reduction on yield. In this context, the aim of this work is to characterize ScMUTM1 and ScMUTM2, two DNA glycosylases belonging to base excision repair pathway; and identify genes potentially related to stress and DNA repair in two sugarcane cultivars with contrasting flowering phenotypes. The characterization of the DNA glycosylases included the construction of vector to over express the recombinant proteins ScMUTM1 and ScMUTM2; they will be used in a near future to purification of these proteins and use in enzymatic assays. It was also made a phylogenetic reconstruction of this gene in plants and analysis of its promoter. With the phylogenetic analysis, it is possible to observe the presence of these genes grouped inside a branch with monocots and another one with dicots. This suggests that the duplication of this gene probably occurred after the separation of these two groups. The analysis of the promotor of MUTM shows of the presence of stress-related regulatory motifs at ScMUTM2 promoter, when compared with ScMUTM1. This may suggests that ScMUTM1 might be suffering sub functionalization process. After the analysis of microarrays data, it is observed an up-regulation from some stress-related genes in one of the conditions analyzed, related to early flowering process.

Caracterização de genes de reparo de DNA em cana-de-açúcar

Sugarcane is an important culture for Brazil that holds almost half of all worldwide productivity. Plants face many challenges, because of biotic and abiotic stresses presents in the production field, which could prevent plants from reaching their genetic potential. As consequence, those stresses can generate Reactive Oxygen Species – ROS – that can cause damages on DNA. Another consequence of stress is the early-flowering process, which contributes for a reduction on yield. In this context, the aim of this work is to characterize ScMUTM1 and ScMUTM2, two DNA glycosylases belonging to base excision repair pathway; and identify genes potentially related to stress and DNA repair in two sugarcane cultivars with contrasting flowering phenotypes. The characterization of the DNA glycosylases included the construction of vector to over express the recombinant proteins ScMUTM1 and ScMUTM2; they will be used in a near future to purification of these proteins and use in enzymatic assays. It was also made a phylogenetic reconstruction of this gene in plants and analysis of its promoter. With the phylogenetic analysis, it is possible to observe the presence of these genes grouped inside a branch with monocots and another one with dicots. This suggests that the duplication of this gene probably occurred after the separation of these two groups. The analysis of the promotor of MUTM shows of the presence of stress-related regulatory motifs at ScMUTM2 promoter, when compared with ScMUTM1. This may suggests that ScMUTM1 might be suffering sub functionalization process. After the analysis of microarrays data, it is observed an up-regulation from some stress-related genes in one of the conditions analyzed, related to early flowering process.

Adenocarcinomas nasossinusais: experiência do Serviço de Otorrinolaringologia do Instituto Português de Oncologia de Lisboa entre 2000 e 2014

Objetivos: Analisar dados demográficos, apresentação clínica, fatores de risco, opções terapêuticas e sobrevida de doentes com adenocarcinoma nasossinusal. Material e Métodos: Estudo retrospetivo de doentes com Adenocarcinoma Nasossinusal tratados entre 2000 e 2014, no IPOFGL. Resultados: Identificamos 33 doentes com diagnóstico de Adenocarcinoma. A idade média foi de 65.6 anos. A terapêutica mais comum foi cirurgia com radioterapia adjuvante. A sobrevida global e livre de doença aos 3 anos foi de 57.6% e 40.5%. A invasão do seio esfenoidal (p=0.038) e da base do crânio (p=0.003) influenciaram a sobrevida global. O desenvolvimento de metástases à distância teve impacto sobre a sobrevida livre de doença (p=0.01). Conclusões: Os Adenocarcinomas são tumores raros. A excisão da lesão toma um papel determinante no tratamento dos doentes. Na nossa amostra, a invasão do seio esfenoidal, da base do crânio e o desenvolvimento de metástases à distância estão associados a um pior prognóstico.

Cirurgia de catarata secundária em criança

Introdução: A catarata em idade pediátrica constitui um desafio cirúrgico. Este desafio assume maior dimensão quando a catarata pediátrica é secundária ao tratamento com radioterapia por tumor ocular. A complexidade da técnica, a maior fragilidade ou fibrose de estruturas oculares e o maior risco de complicações intra e pós operatórias são obstáculos que o cirurgião deve equacionar. Os autores apresentam um caso clinico de uma cirurgia de catarata unilateral, secundária a radiação por feixe de protões externo para tratamento de melanoma da íris, em criança com 11 anos de idade. Métodos: Criança de 8 anos, com lesão pigmentada infero-temporal no olho esquerdo. Estabeleceu-se o diagnóstico de melanoma da íris, tendo realizado radioterapia com feixe acelerado de protões com excisão e recolocação de células limbares. Após 18 meses de radioterapia, constatou-se desenvolvimento de catarata subcapsular posterior com evolução para catarata branca. Aos 11 anos, foi submetida, sob anestesia geral, a facoemulsificação microincisional. Realizaram-se 2 paracenteses de 1 mm e, através destas, efectuou-se a capsulorexis anterior, após coloração com azul tripano. Procedeu-se à realização da incisão principal (2,2mm), à faco-aspiração do cristalino opacificado e à irrigação/aspiração bimanual do córtex. Implantou-se a lente intra-ocular monobloco monofocal (SN 60WF) no saco capsular e efectuou-se capsulorexis posterior primária. Com o objectivo de excluir a presença de vítreo na câmara anterior e simultaneamente obter um efeito anti-inflamatório, injectou-se intra-camerularmente triamcinolona sem conservantes. Resultados: Não se observaram complicações intra ou pós-operatórias, imediatas ou tardias, de relevo. Com 1 ano de evolução, a acuidade visual do olho esquerdo é de 10/10. Após 4 anos de radioterapia não se observa recorrência do tumor, doença metastática nem complicações da radioterapia. Conclusões: A micro-incisão, a capsulorexis posterior primária e a aplicação de triamcinolona intracamerular asseguram maior segurança no procedimento, mais rápida recuperação visual com transparência duradoura do eixo visual e menor inflamação no pós-operatório.