Introduction: Chronic inflammation plays an important role on the transformation and tumor progression during oral carcinogenesis. There is a great number of chronic inflammatory lesions (CIL) in the oral cavity which are related to dysplastic processes of the epithelium, immune response and changes on the collagen deposition. Objectives: To investigate the presence of dysplasia and to histologically grade them in the CIL of traumatic cause, as well as toaccessthe density of mast cells and different types of collagen fibers in cases of epithelial dysplasias and oral squamous cell carcinomas (OSCC). Material and Method: Initially, 183 CIL cases were evaluated as to the presence of dysplasia and also classified according to its degree of epithelial dysplasia. Among those lesions, 45 CIL cases were selected and divided into two groups: group 1 (15 cases of mild dysplasia), group 2 (15 cases of moderate/severe dysplasia). The control group was composed by 15 cases of OSCC.They were stained with toluidine blue in order to quantify the mast cells and picrosirius red to semi-quantify the collagen type fibers. Results: The mast cells were detected in all groups presenting a mean of 6,76 cells/mm2, 10.82 cells/mm2 and 19.18 cells/mm2 in the control, group 1and 2 respectively. Regarding the collagen fibers, type III was more prevalent on groups 2 and control while type I fibers were more abundant on group 1. Conclusion: Oral chronic inflammatory lesions showed dysplastic changes in most analyzed cases. The results suggests an active participation of mast cells in the stage of tumor transformation, since it was detected a higher density onthe dysplasia cases when compared to the OSCC cases. Nevertheless, the gradual change of collagen type fibers indicates that collagenproducing cells become altered during the stages of dysplasia (tumor transformation). / Introdução: A inflamação crônica tem um papel importante na transformação e progressão tumoral durante a carcinogênese oral. Muitas lesões inflamatórias crônicas (LIC) da cavidade oral estão relacionadas a processos displásicos do epitélio, à resposta imune e à mudança na deposição do colágeno. Objetivos: Investigar a presença de displasia e graduá-las histologicamente nas LIC de origem traumática, como também, avaliar a densidade de mastócitos e de diferentes tipos de fibras colágenas nas LIC com displasias epiteliais e comprar aos casos de carcinomas de células escamosas (CCE). Material e Métodos: Inicialmente 183 LIC foram avaliadas quanto à presença de displasia e classificadas em relação ao grau. Em seguida, 45 casos foram divididos em: Grupo controle (CCE), Grupo 1 (displasia leve- DL), Grupo 2 (displasia moderada/severa- DM/S). Foram corados com Azul de Toluidina para quantificar os mastócitos e Picrosirius Red para avaliação dos tipos de fibras colágenas I e III. Resultados: As LIC foram mais frequentes em mulheres (n=107) com idade de 36,6 anos. O sítio mais afetado foi a mucosa do lábio inferior (29,7%), já a lesão mais frequente foi o fibroma traumático (39,2%). A displasia leve esteve presente em 56,3% da amostra. Os mastócitos foram evidenciados nos três grupos: grupo controle (6,76 mastócitos/mm), grupo 1 (10,82 mastócitos/mm2) e grupo 2 (19,18 mastócitos/mm2).Quando analisadas as fibras colágenas, observouse no grupo controle e no grupo 2 que as fibras tipo III foram mais prevalentes, já no grupo 1 prevaleceu-se as fibras tipo I. Conclusão: Lesões inflamatórias crônicas orais apresentaram alterações displásicas na maior parte dos casos. O estudo sugere uma participação dos mastócitos na fase de transformação tumoral. E a alteração gradativa dos colágenos tipo I e III indica alteração das células produtoras de colágeno, durante transformação tumoral.

INTRODUÇÃO: Alterações genéticas múltiplas são comuns durante a carcinogênese e, nesse panorama, o gene supressor tumoral TP53 é um dos mais associados à transformação maligna. Recentemente, dois genes similares ao TP53, foram identificados, o TP73 e o TP63. O TP63 situa-se no cromossomo 3q e tem papel comprovado no desenvolvimento epidérmico, sendo detectado em vários tecidos humanos. Inúmeros trabalhos relacionaram a expressão da proteína p63 com carcinomas do trato aerodigestivo superior. OBJETIVOS: O presente estudo objetivou avaliar a expressão Imunoistoquímica da proteína p63 e seu valor prognóstico nos carcinomas epidermóides da laringe. CASUÍSTICA E MÉTODOS: Foram estudados retrospectivamente 127 pacientes submetidos a laringectomia total no Instituto Nacional de Câncer, Rio de Janeiro, entre 1998 e 2000. Houve 111 doentes masculinos e 16 femininos, 69 brancos e 58 não-brancos, com idade entre 36 a 93 anos, média de 59 e mediana de 58 anos. Dezenove tumores eram glóticos, 16 supraglóticos e 92 acometiam mais de um local, correspondendo a 15%, 13% e 72% respectivamente. Quanto ao estadiamento clínico, dois casos eram do estádio I (1,6%), 21 do II (16,5%), 82 do III (64,6%) e 22 do IV (17,3%). Noventa e seis pacientes (75,6%) receberam radioterapia complementar. A técnica imunoistoquímica, com anticorpos monoclonais do clone 4A4, foi utilizada para estudar a expressão da p63. O percentual de células imunocoradas positivamente foi estimado conforme os seguintes escores: 0: ausência de imunocoloração; 1: imunocoloração em < 30% das células neoplásicas; 2: imunocoloração em > 30% e < 70% das células neoplásicas; e 3: imunocoloração em > 70% das células neoplásicas. Foram observados 62 casos do escore 3 (+++), 60 do escore 2 (++), 4 do 1 (+) e 1 caso sem expressão (0), correspondendo respectivamente a 48,8%, 47,2%, 3,1% e 0,8% da amostra. Através de análises uni e multivariadas, a imunoexpressão da proteína p63 e os outros fatores de provável impacto prognóstico foram avaliados quanto ao grau de associação aos eventos recidiva e óbito. RESULTADOS: A análise multivariada identificou a imunoexpressão da proteína p63 e o envolvimento da hipofaringe como preditivas para ocorrência de recidiva e óbito pelo carcinoma. A sobrevida global foi de 73,9% em 24 meses e de 59,5% em 60 meses. A sobrevida livre de recorrência foi de 77,2% e 75,1%, e a sobrevida relacionada ao óbito pelo carcinoma foi de 79% e 67% em 24 e 60 meses respectivamente. CONCLUSÕES: Apenas a recidiva associou-se estatisticamente à expressão da proteína p63. A p63 se mostrou altamente expressa nos carcinomas epidermóides de laringe e, apesar dos poucos casos com expressão reduzida, a hipoexpressão da p63 foi preditiva de um pior prognóstico nesses doentes. / INTRODUCTION: Multiple genetic changes are common during carcinogenesis and, in this scene, the gene TP53 is one of the most associated with malignant transformation. Recently, two related genes to the TP53, were identified, the TP73 and TP63. The TP63 stands on the chromosome 3q and has a proven role in the epidermal development, being detected in several human tissues. Many work linked the expression of p63 protein with carcinomas of the upper aero-digestive tract. OBJECTIVE: The objective of this study was to evaluate the immunohistochemical expression of p63 protein and its prognostic value in squamous cell carcinomas of the larynx. CASUISTRY AND METHODS: The p63 expression has been examined in 127 patients who were submitted to total laryngectomy, with or without adjuvant radiotherapy, in the Brazilian National Cancer Institute, between 1998 and 2000. There were 111 male patients and 16 female, 69 white and 58 non-white, aged between 36 to 93 years, average 59 and median of 58 years. Nineteen tumors were glottic, 16 supraglottic and 92 affected more than one place, corresponding to 15%, 13% and 72% respectively. As to the clinical staging, two cases were stage I (1.6%), 21 of the II (16.5%), 82 of the III (64.6%) and 22 of the IV (17.3%). Ninety-six patients (75.6%) received complementary radiotherapy. The immunohistochemical technique with the use of monoclonal antibodies of clone 4A4, has been used to study the expression of p63. The percentage of positive cells was estimated as the following scores: 0: no immunostaining; 1: immunostaining in <30% of neoplastics cells; 2: immunostaining in >30% and <70% of neoplastics cells; And 3: immunostaining in >70% of neoplastics cells. Sixty two cases were observed in score three, 60 in score two 4 in score one (+) and 1 case without expression (0), corresponding respectively to 48.8%, 47.2%, 3.1% and 0.8% of the sample. Through uni and multivariate analysis, the immunoexpression of p63 protein and the other factors likely to impact prognosis were evaluated on the degree of association to recurrence and death. RESULTS: The multivariate analysis identified the immunoexpression of protein p63 and the involvement of the hypopharynx as statistically significant for the risk of recurrence and death by cancer. The overall survival was 73.9% in 24 months and 59.5% at 60 months. CONCLUSIONS: The disease-free survival was 77.2% and 75.1%, and the disease-specific survival was 79% and 67% at 24 and 60 months respectively. The p63 protein was highly expressed in squamous cell laryngeal carcinomas. In spite of few cases with reduced expression, p63 protein underexpression was statistically associated to the recurrence and may have a negative impact upon prognosis.

Presente estudo teve como objetivo avaliar a utilização da tecnologia de banda estreita com filtros ópticos (TBE) no rastreamento do carcinoma espinocelular do esôfago (CEC), utilizando como método comparativo a cromoscopia com a solução de Lugol. Trata-se de um estudo prospectivo de teste de diagnóstico, para o qual foram avaliados 129 pacientes do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP), com diagnóstico de carcinoma epidermóide de cabeça e pescoço, em programa de rastreamento de tumores secundários, no período de agosto de 2006 a fevereiro de 2007. Os exames de endoscopia convencional, TBE e a cromoscopia com Lugol foram realizados consecutivamente em um mesmo procedimento, e as lesões encontradas foram registradas e submetidas a biópsias. Foram calculados para cada método valores da sensibilidade, especificidade, acurácia, valores preditivos positivos e negativos, valores de verossimilhança positivo e negativo. Foram diagnosticados nove carcinomas superficiais (7%), sendo cinco carcinomas in situ e quatro carcinomas intramucosos, todos detectados pelo TBE e pelo Lugol, porém apenas seis foram diagnosticados pelo exame convencional e destes, nenhum foi menor ou igual a 10 mm. A tecnologia de bandas estreitas com filtros ópticos (TBE) sem magnificação de imagem apresentou resultados superponíveis a cromoscopia com Lugol, método atualmente de escolha para rastreamento do CEC esofágico em grupos de pacientes de alto risco, portadores de tumores de cabeça e pescoço / Background and study aims: The aim of this study was to compare narrow band imaging (NBI) without magnification and chromoendoscopy with Lugols solution for detecting superficial esophageal squamous cell carcinoma in patients with head and neck cancer. Patients and methods: This is a prospective observational study of 129 patients with primary head and neck tumors consecutively referred to the Gastrointestinal Endoscopy Unit of Hospital das Clínicas, São Paulo University Medical School (FMUSP), Brazil, between August 2006 and February 2007. Conventional examinations, NBI and Lugol chromoendoscopy were consecutively performed, and the detected lesions were mapped, recorded and sent for biopsy. The results of the three methods were compared regarding sensitivity, specificity, accuracy, positive predictive value, negative predictive value, positive likelihood value and negative likelihood value. Results: Of the 129 patients, nine (7%) were diagnosed with carcinomas, five of which were in situ and four intramucosal. All carcinomas were detected through NBI and Lugol chromoendoscopy. Only six lesions were diagnosed by conventional examination, all of which were larger than 10 mm. Conclusions: Narrow-band imaging technology with optical filters has high sensitivity and high negative predictive value for detecting superficial esophageal squamous cell carcinomas and produces results comparable to those obtained with 2.0% Lugol chromoendoscopy in patients with head and neck cancer

A programação epigenética do genoma por metilação do DNA, modificação das histonas e remodelamento da cromatina é crucial para o desenvolvimento e o crescimento normal dos mamíferos e alterações nesses mecanismos contribuem diretamente para a transformação maligna. Em função do papel relevante da metilação do DNA na carcinogênese, da importância e da necessidade de identificação de marcadores moleculares em tumores de cabeça e pescoço e dos resultados já obtidos pelo grupo, o presente trabalho teve por objetivo geral investigar o perfil de metilação de ilhas CpG em carcinomas primários de cabeça e pescoço (CECP), bem como identificar e iniciar estudos funcionais de biomarcadores candidatos para diagnóstico e prognóstico desses tumores. Alguns genes previamente descritos com padrões anormais de metilação em neoplasias humanas (CDH1, CDH13, DAPK, CDKN2A, RASSF1A, SOCS3 e TIMP3), além de outros dois genes (MX1 e SLC15A3) identificados em nosso estudo anterior, foram analisados em amostras normais e margens cirúrgicas de CECP por meio da técnica de pirosequencimento de DNA após tratamento com bissulfito de sódio. As análises estatísticas não mostraram diferenças significativas para a maioria dos genes analisados, com exceção do gene SLC15A3, que apresentou diferença significativa (p<0,05) entre tumores e amostras de sangue de doadores saudáveis. Diferentemente, os resultados obtidos com a metodologia de PCR-MSP em nosso trabalho anterior mostraram que o gene MX1 está metilado em CECP. Os estudos funcionais do MX1, por RNA de interferência, ensaios de migração e citometria de fluxo mostraram que seu produto contribui para migração e proliferação celular, e talvez para resistência à apoptose. Os resultados sugeriram que o nível de expressão do MX1 pode ser um preditor do potencial metastático em carcinoma epidermóide / The genome epigenetic programming by DNA methylation, histone modification and chromatin remodeling is crucial for normal growth and development in mammals and changes in these mechanisms contributes directly to malignant transformation. Regarding the role of DNA methylation in carcinogenesis, the importance and necessity for the identification of molecular markers in head and neck tumors, and the results already obtained by the group, this study was aimed at investigating the methylation profile of CpG islands in primary head and neck squamous cell carcinomas (HNSCC), and to identify and initiate functional studies of candidate biomarkers for diagnosis and prognosis of these tumors. Therefore, some genes previously described with abnormal methylation pattern in humans tumors (CDH1, CDH13, DAPK, CDKN2A, RASSF1A, and TIMP3 SOCS3), and two other genes (MX1 and SLC15A3) identified in our previous study were analyzed in normal samples, surgical margins, and in HNSCC by pyrosequencing after sodium bisulfite treatment. The statistical analysis showed no significant differences for most of analyzed genes, except for the SLC15A3, which showed significant difference (p <0.05) between tumors and blood samples from healthy donors. However, our earlier results showed a higher frequency of MX1 hypermethylation in primary HNSCC using the PCR-MSP methodology. To gain a better understanding of the role MX1 in cancer biology we investigated whether a downregulation of MX1 by interference RNA contribute to apoptosis resistance and cell migration during cancer development. Wound healing and flow cytometry assays were performed to determine changes in cell motility, death and cell cycle in SCC cells. The results indicated that low levels of MX1 could regulate the cell cycle, increase proliferation, and enhance tumor cell migration in HNSCC cell lines, but it might not contribute to apoptosis resistance. It also suggests that the level of MX1 expression may be a predictor of metastatic potential in HNSCC

En pathologie tumorale, l’étude du micro-environnement tumoral doit prendre en compte différents modes de communication cellulaire : contacts directs entre membranes plasmiques, émission et réception de cytokines et enfin émission et internalisation d’objets biologiques plus complexes comme les microvésicules et les exosomes qui peuvent être assimilés à de véritables organites extra-cellulaires. Le virus d’Epstein-Barr (EBV) participe à l’oncogenèse de plusieurs affections malignes humaines d’origine épithéliale (carcinomes nasopharyngés ou NPC) ou lymphocytaire (lymphomes post-transplantation). Dans ces tumeurs, les cellules malignes qui sont infectées de façon latente par EBV libèrent des exosomes et des microvésicules qui contiennent des protéines et des acides nucléiques d’origine virale. L’étude de ces éléments doit permettre de mieux comprendre les interactions hôte-tumeur et de mettre en évidence de nouveaux biomarqueurs utiles pour le diagnostic précoce et la surveillance de la maladie sous traitement. Le premier objectif de ma thèse consistait à étudier la sécrétion par les cellules malignes d’une famille de microARN viraux appelés miR-BART et leur diffusion dans le sang périphérique chez les sujets porteurs de tumeurs associées à EBV. Pour la première fois j’ai mis en évidence une sécrétion d’exosomes porteurs de miR-BART par les cellules de NPC en culture in vitro. J’ai également montré que les miR-BART, particulièrement miR-BART7, sont détectables dans le plasma de sujets porteurs de NPC. Contrairement à ce qui se passe in vitro les miR-BART plasmatiques ne sont pas transportés par des exosomes. Des données obtenues chez la souris montrent qu’ils peuvent être transportés par des complexes extra-cellulaires que l’on peut précipiter au moyen d’anticorps anti-ago2. Nous cherchons à confirmer ces données sur des échantillons de plasma provenant de patients porteurs de NPC. Ces données pourront guider à l’avenir l’utilisation des miR-BART circulants comme source de biomarqueurs.Le deuxième volet de ma thèse avait pour but d’étudier les modifications du protéome des exosomes induites par une oncoprotéine du virus d’Epstein-Barr appelée LMP1 (latent membrane protein 1). J’ai montré que la LMP1, lorsqu’elle est exprimée dans les cellules lymphocytaires ou épithéliales, infectées ou non par EBV, induit la libération de la protéine PARP1 dans le milieu extra-cellulaire. Cette PARP1 extra-cellulaire n’est pas associée aux exosomes ni aux microvésicules mais à des nano-objets non-vésiculaires contenant notamment des histones et de l’ADN. Nous avons désignés ces objets sous le terme de complexes ADN-protéines extra-cellulaires. Nous ne savons presque rien de la biogenèse de ces complexes ; nous pensons qu’ils ne proviennent pas uniquement de cellules en apoptose. En revanche, des expériences préliminaires suggèrent que la présence de PARP1 dans ces complexes coïncide avec une activation permanente de la PARP1 induite dans les cellules productrices par l’expression de l’oncoprotéine LMP1. Cette hypothèse est en cours de vérification grâce à des expériences menées sur des lignées cellulaires exprimant différentes formes sauvages ou mutées de la LMP1. Ces données sur l’activation de la PARP1 et sur sa sécrétion induite par la LMP1 auront des retombées intéressantes pour notre compréhension des mécanismes d’oncogenèse et d’auto-immunité liés à l’infection par le virus d’Epstein-Barr. / The study of tumoral microenvironment should take into account different modes of intercellular communications: direct contacts between extracellular membranes, secretion and uptake of cytokines and finally emission and uptake of complex biological objects like exosomes and microvesicles.Epstein-Barr virus (EBV) is associated with several human malignancies of epithelial origin (Nasopharyngeal carcinoma or NPC) or of lymphoïd origin (post-transplant lymphoproliferative disorder or PTLD). In these tumors, malignant cells are latently infected by EBV and release exosomes and microvesicles containing viral nucleic acids and proteins. Studying them will enable a better understanding of tumor-host interactions and the discovery of new markers which could be useful for early diagnostic and the follow-up of the disease under treatment.The first aim of this thesis was to study the release by malignant cells of EBV microRNAs belonging to the BART family and their blood diffusion in patients bearing NPC tumors. For the first time, I’ve shown that exosomes released by NPC cells in vitro contain EBV miR-BART microRNAs. Moreover, ebv-miR-BART7 can be detected in the plasma of NPC patients. Unlike what is observed in vitro, circulating BART microRNAs are not carried by exosomes. Recent data from studies in xenografted mice show that they are carried by extra-cellular complexes which can be immunoprecipitated by anti-Ago2 antibodies. We are currently trying to confirm these data in plasma from NPC patients. This work will ease the use of miR-BARTs as potential biomarkers.The second aim was to study the proteome modifications induced by the EBV Latent Membrane Protein 1 protein (LMP1). I’ve shown that LMP1 expression in lymphoid or epithelial cells infected or not by EBV induces the release of PARP1 in the extra-cellular space. This extra-cellular PARP1 is not carried by exosomes or microvesicles but is embedded in non-vesicular nano-objects containing histones and DNA. We have called these objects “DNA-proteins complexes”. We don’t know how they are produced and released by cells. We think that they are not only secreted by apoptotic cells. Recent data show that this release of extra-cellular PARP1 is associated with PARP1 activation by LMP1 oncoprotein expression. We are trying to prove this hypothesis using cell lines expressing wild type or mutated LMP1. The release and the activation of PARP1 induced by LMP1 expression will help to understand the mechanisms of EBV-associated oncogenesis and auto-immunity.

Distant brain metastases from oral squamous cell carcinomas (OSCC) are extremely rare. Here we describe a case of a 53-year-old man with a primary OSCC who referred to the neurosurgical department because of epileptic seizures. MR imaging revealed an enhancing lesion in the right parietal lobe. A craniotomy with tumor removing was performed. Histopathological examination verified an invasive, minimally differentiated metastasis of the primary OSCC. The patient refused whole brain radiation therapy and died from pulmonary metastatic disease 10 months after the neurosurgical intervention without any cerebral recurrence. To the authors’ knowledge, only two similar cases have been previously reported.

Overall survival of the metastatic forms of epithelial derived cancers, especially head and neck squamous cell carcinomas (HNSCC), has not significantly improved even with the application of aggressive combined modality approaches incorporating radiation and chemotherapy. Cumulative evidence implicates the epidermal growth factor receptor (EGFR) as an important therapeutic target in HNSCC. We have previously demonstrated that the combination of lovastatin, a potent inhibitor of the mevalonate pathway, with EGFR tyrosine kinase inhibitors induced robust synergistic cytotoxicity. However, the use of high dose statins in our clinical trial was associated with significant toxicities including higher than anticipated rate of muscle pathologies. Our goal was to uncover novel downstream targets of the mevalonate pathway that may enhance the efficacy or limit toxicities of this novel combination therapeutic approach. In this study we have demonstrated that GGTI-298, an inhibitor of protein geranylgeranylation, through its ability to disrupt the actin cytoskeleton, inhibits EGFR dimerization and cellular trafficking. This novel mechanism targeting the EGFR has clinical implications as GGTI-298 in combination with tarceva, a clinically relevant EGFR inhibitor, showed enhanced cytotoxicity and inhibitory effects on EGFR activation and its downstream signaling.

Thesis (Ph.D.); Submitted to Iowa State Univ., Ames, IA (US); 19 Dec 2004. / Published through the Information Bridge: DOE Scientific and Technical Information. "IS-T 2692" Satoshi Matsuzaki. US Department of Energy 12/19/2004. Report is also available in paper and microfiche from NTIS.

Le syndrome de Gorlin est une maladie rare caractérisée par de nombreuses anomalies du développement. Ces manifestations cliniques, dues à des mutations d'un acteur essentiel de la voie de signalisation sonic hedgehog, incluent aussi une hyper-radiosensibilité et une forte prédisposition à développer des carcinomes basocellulaires. Etant donné l'implication de défaut de la réparation de l'ADN au niveau des affections liées à l'hyper-radiosensibilité, nous avons décidé d'étudier l'effet des mutations du gène PTCH1 sur la réponse aux dommages de l'ADN afin de mieux comprendre les mécanismes cellulaires et moléculaires conduisant au phénotype Gorlin.Cette étude permet de mettre en évidence une défaillance globale des systèmes de réparation des dommages de l'ADN dans les fibroblastes issus de patients Gorlin par rapport à des fibroblastes normaux. Elle met notamment en exergue un écroulement de la réparation par excision de bases (BER) responsable de la réparation des dommages oxydatifs. / The Gorlin syndrome is a rare genetic disorder characterized by several developmental abnormalities. Due to mutations in PTCH1, a key player of the sonic hedgehog signaling pathway, clinical manifestations also includes hyper-radiosensitivity and an increased predisposition to the development of basal cell carcinomas. Given the implication of DNA repair system defects in hyper-radiosensitivity pathologies, we decided to study the effect of PTCH1 mutations on the DNA damage response in order to better understand the cellular and molecular mechanisms leading to Gorlin's phenotype.This study demonstrate a global failure of the DNA damage repair systems in Gorlin fibroblasts with respect to controls. It highlights in particular the collapse of the base excision repair pathway (BER) responsible for the repair of oxidative DNA damage.

Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2015-01-21T10:06:51Z No. of bitstreams: 0 / Made available in DSpace on 2015-01-21T10:06:51Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP