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1
Branford, White Harriet A. "Heterogeneity in Ewing sarcoma." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:7af59b69-e68f-41af-b5f0-8a7d278f6fd7.
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Ewing sarcoma, an aggressive primary bone and soft tissue tumour is characterised by the expression of the chimeric transcription factor EWS-FLI1 in 90% of patients. This alters expression of many genes including activation of the Insulin Growth Factor (IGF) pathway via IGFBP3 supression. Phase I/II trials with an IGF-1 inhibitor have demonstrated tumour regression in a modest number of Ewing sarcoma patients. The aim of this thesis was to identify mechanisms contributing to the heterogeneity of resistance in Ewing sarcoma following inhibition with OSI-906, a dual kinase inhibitor of IGF-1 (IGF-1R) and Insulin (IR) receptors. The hypothesis was that mechanisms of resistance relate to heterogeneity of responses to signalling pathway activation and inhibition. Through selection, disruption of the pathway would identify subpopulations of cells both sensitive and resistant in their response allowing for interrogation of resistance mechanisms. A genome wide approach was taken to model the resistance profile of cell lines. Through developing a method of unbiased quantification, a panel of validated Ewing sarcoma cell lines (EuroBoNet) were imaged and segmented to assess the responses of biomarkers on signalling pathway activation. Heterogeneity was confirmed between cell lines. The application to diagnostic biopsies led to the identification of prognostic classifiers and cellular subpopulations with clinical prognostic significance. The distribution of Ki67 was found to be predictive of survival and cells with lower levels of CD99 in the cytoplasm were most discriminative. Parallel sequencing strategies (RNA-seq, whole exome sequencing, and aCGH/ SNP array) for genome-wide screening was carried out for point mutations, copy number changes and rearrangements. Systematic detection was used to characterise genomic rearrangements and functional validation performed. Resistant clones, formed via ENU mutagenesis of cell lines, were sequenced in order to demonstrate the resistance profile of OSI-906. In summary heterogeneity of Ewing sarcoma at the genomic and proteomic level can influence the signalling dependency of tumours and response to inhibitors. Genomic and proteomic profiling of tumour cells may be relevant to future developments of novel therapies.
2
Vallurupalli, Mounica. "Identifying Targetable Liabilities in Ewing Sarcoma." Thesis, Harvard University, 2014. http://etds.lib.harvard.edu/hms/admin/view/62.
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Background: Despite multi-modality therapy, the majority of patients with metastatic or recurrent Ewing sarcoma (ES), the second most common pediatric bone malignancy, will die of their disease. ES tumors express aberrantly activated ETS transcription factors through translocations that fuse the EWS gene to ETS family genes FLI1 or ERG. The aberrant activation of ETS transcription factors promotes malignant transformation and proliferation. While, FLI1 or ERG cannot be readily targeted, there is an opportunity to deploy functional genomics screens, to develop novel therapeutic approaches by identifying targetable liabilities in EWS/FLI1 dependent tumors.
Materials and Methods: We performed a near whole-genome pooled shRNA screen in a panel of five EWS/FLI1 dependent Ewing sarcoma cell lines and one EWS/ERG cell line to identify essential genes. Essential genes were defined as those genes whose loss resulted in reduced viability selectively in ES cells compared to non-Ewing cancer cell lines. Essential hits were subsequently validated with genomic knockdown and chemical inhibition in vitro, followed by validation of the on-target effect of chemical inhibition. Next, we determined the in vivo effects of small-molecule inhibition on survival and tumor growth in NOD scid gamma (NSG) mice with established subcutaneous ES xenografts.
Results: Top hits in our screen that could be readily targeted by small-molecule inhibitors, and thus have potential for rapid clinical validation, were selected for further investigation. These hits included IKBKE, CCND1 and CDK4. IKBKΕ, a non-canonical IKK with an oncogenic role in breast cancer, was one of the top kinase hits in the screen. IKBKΕ shares significant homology to TBK1, another non-canonical IKK that is essential in k-RAS dependent lung cancer. We validated IKBKE through small-molecule inhibition of IKBKE/TBK1 and shRNA based knockdown. Ewing sarcoma cell lines are sensitive to low micromolar concentrations of two IKBKE/TBK1 inhibitors (CYT387 and MRT67307). Additionally, in a panel of ES cell lines, knockdown of IKBKE resulted in decreased growth and impaired colony formation. These observations, paired with impairment of NF-κB nuclear localization following CYT387 treatment suggests that non-canonical IKK mediated signaling may be essential in Ewing sarcoma. We further validated these results through inhibition of IKBKE/TBK1 in in vivo xenograft models treated with 100 mg/kg/day of CYT387. Treatment over the course of twenty-nine days resulted in a significant increase in survival (p-value = 0.0231) and a significant decrease (p-value = 0.036) in tumor size after fifteen days of treatment.
CDK4 and CCND1 are highly expressed in Ewing sarcoma as compared to other tumor types. shRNA mediated knockdown of CDK4 and CCND1 resulted in impaired viability and anchorage independent growth. Furthermore, treatment of Ewing sarcoma cell lines with a highly selective CDK4/6 inhibitor, LEE011, resulted in decreased viability (IC50 range of 0.26-18.06 μM), potent G1 arrest in six of eight EWS/FLI1 containing Ewing sarcoma lines tested and apoptosis in a panel of four highly sensitive lines. Administration of 75 mg/kg/day and 250 mg/kg/day of LEE011 in NSG mice with Ewing xenografts resulted in significant impairment of tumor growth, (p-value <0.001 for both treatment arms), as compared to vehicle control.
Conclusions: These studies suggest a role for the targeting of IKBKE and CDK 4/6 in Ewing sarcoma, findings with immediate clinical relevance for patients with this malignancy, because small-molecule inhibitors of these proteins have already entered clinical trial for other disease indications.
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Saulnier, Olivier. "Deciphering the splicing landscape of Ewing sarcoma." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC268.
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Certains cancers peuvent être caractérisés par un facteur de transcription aberrant. C’est le cas du sarcome d’Ewing qui est caractérisé par une translocation chromosomique générant une protéine de fusion appelée EWSR1-ETS. Ces protéines de fusion ont principalement été étudiées en tant que facteur de transcription car elles ont la capacité de se fixer sur des séquences de type répétition de GGAA dans le génome et d’activer la transcription de nombreux gènes. Les fusions EWSR1-ETS ont aussi la capacité de recruter les protéines du complexe du remodelage de la chromatine afin d’augmenter l’accessibilité aux régions riches en répétition de GGAA et donc promouvoir un programme transcriptionnel aberrant. Cette propriété dépend majoritairement de la partie EWSR1 et de son domaine de faible complexité qui lui permet, notamment, d’interagir avec de nombreuses protéines. Les fusions EWSR1-ETS ont aussi été impliquées dans la régulation de l’épissage alternatif ; mais à ce jour cette fonction reste peu décrite et est principalement attribuée à la partie EWSR1. Cependant, il a récemment été montré que la protéine ERG (qui est très homologue à FLI1) contrôle la stabilité des ARN messagers. Ces observations nous ont mené à tester le rôle potentiel de ERG (et par conséquent de FLI1) dans l’épissage alternatif afin de mieux décrire les mécanismes impliqués dans la régulation de l’épissage alternatif induite par les protéines de fusion EWSR1-ETS dans le sarcome d’Ewing. Ce travail a permis d’identifier une nouvelle fonction des protéines de la sous-famille ERG (ERG, FLI1 et FEV) dans la régulation de l’épissage alternatif. Nous avons montré que les protéines ERG interagissent avec RBFOX2, un régulateur de l’épissage et que ERG et RBFOX2 induisent une régulation de l’épissage similaire suggérant, ainsi, un mécanisme de collaboration. Nos résultats démontrent que ERG interagit avec RBFOX2 par son extrémité C-terminale. De manière intéressante, ce domaine est retenu dans les fusions EWSR1-ETS. Nous avons donc confirmé que les fusions EWSR1-ETS étaient aussi capable d’interagir avec RBFOX2 et d’induire un programme d’épissage alternatif commun. Cependant, au contraire de la collaboration observée pour ERG et RBFOX2, nous avons montré que les fusions EWSR1-FLI1 ont un rôle opposé sur le programme d’épissage de RBFOX2. Nous avons également montré que EWS-FLI1 induisait l’épissage alternative du gène ADD3 ce qui a pour conséquence la répression du phénotype mésenchymateux des cellules du sarcome d’Ewing. Notre travail a permis d’identifier de nouveaux mécanismes afin de mieux comprendre comment la dérégulation de l’épissage alternatif par des facteurs de transcriptions oncogéniques influent sur la biologie du sarcome d’EwingCancer can be characterized by abnormal fusion transcription factors. These transcription factors may have gain of function, neomorphic DNA binding properties or aberrant transcriptional activity. This is the case for Ewing sarcoma, which is characterized by a chromosomal translocation EWSR1-ETS. Ewing sarcoma fusion oncoproteins have been mostly studied as aberrant transcription factors due to their ability to specifically bind GGAA repeat sequences and to activate de novo enhancers. In addition, its ability to recruit chromatin-remodeling proteins, to induce chromatin opening and to drive an aberrant transcriptional program is a neomorphic property of the EWSR1 moiety that depends on its low complexity domain. EWSR1-ETS fusions have recently been implicated in alternative splicing regulation but to date this function is mainly attributed to the EWSR1 part. However, ERG protein, a member of the ETS transcription factor family, has been lately shown to control post-transcriptional processes such as mRNA stability. Considering these observations, we decided to challenge this view by studying ERG as a bona fide splicing regulator. This work highlights a new function of ERG subfamily proteins (ERG, FLI1 and FEV) in alternative splicing regulation. We have shown that ERG proteins interact with the master splicing regulator RBFOX2 to similarly regulate a common splicing program. We demonstrated that this new function is mediated via protein-protein interaction through the C-terminal domain of ERG. Because this domain remains in EWSR1-ETS fusions, we demonstrated that EWSR1-FLI1 protein is still able to bind RBFOX2 as expected. In addition, EWSR1-FLI1 induces massive changes of the splicing landscape of Ewing sarcoma and regulates an RBFOX2-dependent splicing program. However, in contrast to the collaborative effect observed for ERG, we found that EWSR1-FLI1 antagonizes RBFOX2-splicing function by repressing RBFOX2 binding to its pre-mRNAs targets. Importantly, we have found that mis-splicing of ADD3 by EWS-FLI1 leads to the repression of the mesenchymal phenotype of Ewing sarcoma cells. Our study provides direct evidence to understand how splicing dysregulation by an oncogenic transcription factor impacts on Ewing sarcoma biology
4
Donahue, Andrew, and Abigail Cruz. "Ewing-like Sarcoma – Hiding in PA view." Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/asrf/2019/schedule/102.
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Ewing-like sarcomas (ELS) are a heterogenous group of neoplasms that typically occur in the bone and soft tissue of pediatric and young adult patients. ELS share various degrees of morphological, immunohistochemical, molecular, and clinical similarity with Ewing sarcomas. However, these tumors lack the pathognomonic molecular hallmark of Ewing sarcoma, which is defined as translocation between a gene of the RNA-binding TET family (EWSR1 or FUS) with a gene of the ETS-transcription family (FLI1, ERG, ETV1, ETV4, or FEV). Accurate classification and distinction from classical Ewing sarcomas is important for patient management. A subset of ELS harboring the BCOR-CCNB3 fusion has been described recently – the majority of which that have been reported to date are bone-based tumors, though there have been cases of discrete soft tissue-based tumors. We herein present a case of ELS harboring the BCOR-CCNB3 translocation occurring in a pediatric patient presenting with a large abdominal mass discovered on chest CT after failed outpatient treatment for pneumonia with effusion.
This patient was a 14-year-old Caucasian boy with a past medical history significant for obesity and three episodes of pneumonia since 6-years-old. Imaging showed a large heterogeneous mass at the posterior left upper quadrant of the abdomen protruding through the posterior aspect of the left hemidiaphragm causing atelectasis. The mass abuts the inferior leftward aspect of the descending thoracic aorta and also protrudes between the 11th and 12th posterior lateral left rib. Pathology revealed this mass to be an Ewing-like sarcoma with a BCOR-CCNB3 fusion. Patient was treated with chemotherapy and radiation. This case demonstrates the importance of determining an accurate diagnosis to provide specific management.
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Becker, Ricardo Gehrke. "Controle local nos tumores da família Ewing: resultados do primeiro estudo do grupo colaborativo brasileiro (EWING I)." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2016. http://hdl.handle.net/10183/164733.
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O sarcoma de Ewing é uma neoplasia maligna agressiva que acomete ossos e tecidos moles com maior frequência em crianças e adolescentes. O tratamento consta de quimioterapia de indução, seguida pelo controle local da doença (cirurgia, cirurgia associada à radioterapia, ou apenas radioterapia), e quimioterapia de consolidação. A introdução da quimioterapia possibilitou aumento significativo na sobrevida dos pacientes nas últimas décadas. Por outro lado, o impacto da modalidade de controle local ainda não está bem estabelecido. Estudos observacionais têm demonstrado superioridade do tratamento cirúrgico em relação à radioterapia isolada, no entanto, são limitados os estudos prospectivos que confirmam esta diferença. O objetivo deste estudo é avaliar o impacto da modalidade de tratamento local nos desfechos oncológicos, bem como descrever o perfil clínico-epidemiológico de portadores de sarcoma de Ewing ósseo não-metastático. Os dados foram coletados em 15 instituições no período entre 2003 e 2010 e fazem parte do primeiro estudo do Grupo Colaborativo Brasileiro para Tratamento dos Tumores da Família Ewing (EWING I). Dos 73 pacientes incluídos, 47 foram tratados com cirurgia isolada, 13 receberam cirurgia associada à radioterapia, e 13 apenas radioterapia. O seguimento médio foi de 4,5 anos (2,3 até 6,7 anos) e a sobrevida geral e livre de eventos foi de 63,3 e 62,1 por cento em 5 anos, respectivamente. A falha do tratamento local foi de 0 (zero) por cento para a modalidade de cirurgia associada à radioterapia, 6,5 por cento para cirurgia isolada, e 10 por cento para radioterapia (p=0,5). A sobrevida dos pacientes submetidos à radioterapia isolada foi significativamente inferior à sobrevida dos tratados com cirurgia e com cirurgia associada a radioterapia (30,8 versus 71,7 versus 64,1 por cento, respectivamente). Concluiu-se que não houve diferença em termos de falha local de acordo com a modalidade de tratamento empregada, no entanto houve diferença significativa em termos de sobrevida. Apesar dos resultados cirúrgicos superiores, a radioterapia isolada ainda apresenta papel fundamental no tratamento de casos selecionados.Ewing sarcoma is a small round cell malignancy of bone and soft tissue that usually occurs in children and adolescents. Current treatment includes induction chemotherapy, local control of the primary tumor (surgery, surgery plus radiotherapy, or radiotherapy) and consolidation chemotherapy. The introduction of chemotherapy has improved significantly the oncologic outcomes in Ewing sarcoma. On the other hand, the impact of the local control modality has not been established. Surgery alone or in combination with radiation has traditionally been considered a good choice for resectable ES, while unresectable tumors have been treated with definitive radiotherapy. Despite the results from a few trials and observational studies, there is no consistent knowledge about the local control modality in ES outcomes. The present study aims to evaluate the impact of the local control modality in the oncologic outcomes, as well as to describe the clinical features of the patients with localized Ewing sarcoma of the bone. The data were collected between 2003 and 2010 in 15 hospitals and were part of the first Brazilian Collaborative Group for the Treatment of the Ewing Sarcoma Family Tumors (EWING 1). From 73 patients (median age 12.8 years old), 47 were treated with surgery, 13 with surgery plus radiotherapy, and 13 with definitive radiotherapy. Median follow up was 4.5 years (2.3 to 6.7 years) and the overall and event-free survival 63.3 and 62.1 percent in 5 years, respectively. The local control failure was 0 percent for surgery plus radiotherapy, 6.5 percent for surgery, and 10 percent for radiotherapy (p=.5). The survival of the patients treated with radiotherapy was significantly worse than those treated with surgery and surgery plus radiotherapy (30.8 versus 71.7 versus 64.1 percent, respectively). In conclusion, there was no significant difference in local failure according to the modality of treatment, but there was significant difference in survival rates. Despite the better outcomes in individuals treated with surgery, the radiotherapy modality has still an important role in selected patients.
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Lagares, Tena Laura María. "Caveolina-1 en la progresión metastásica del Sarcoma de Ewing." Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/145475.
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El sarcoma de Ewing (SE) es el segundo tumor óseo maligno infantil más frecuente y presenta una alta incidencia de enfermedad metastásica. Este tipo de tumores presentan una translocación génica característica que da origen a una proteína de fusión, normalmente EWS/FLI1. Esta proteína de fusión actúa como factor de transcripción aberrante, regulando la expresión de diferentes genes implicados en la iniciación, mantenimiento y progresión del tumor. Nuestro grupo describió como uno de estos genes diana a caveolina 1 (CAV1), describiendo además su papel determinante en el fenotipo maligno del SE, en la tumorigénesis, en la angiogénesis y en la resistencia a apoptosis inducida por quimioterapia.
Para investigar el papel concreto de CAV1 en el proceso metastásico de este sarcoma, se creó un modelo de baja expresión de CAV1 en líneas celulares de SE y se determinaron cambios en su capacidad migratoria, invasiva y metastásica. En los ensayos in vitro se halló una menor capacidad migratoria de las células con silenciamiento de CAV1 y una reducción en la expresión de metaloproteinasa 9 (MMP-9) y en la actividad de MMP-2, ambas MMPs implicadas en el proceso de invasión. La regulación de la actividad de MMP-2 parece estar relacionada con la posible regulación que ejerce CAV1 en la función de la MMP de membrana tipo 1 (MT1-MMP), proteína fundamental para la activación de MMP-2. Por otro lado, en este estudio se propone que CAV1 promueve la expresión de MMP-9 transcripcionalmente a través de la regulación de la activación de la vía de señalización de ERK1/2 (Extracellular signal-regulated kinase 1/2). En nuestro modelo, ERK1/2 activo translocaría al núcleo donde activaría los factores de transcripción responsables de la activación del promotor de MMP-9. Por otro lado, la fosforilación de RSK2 (Ribosomal S6 kinase 2) por parte de ERK1/2 en el citoplasma, produciría la activación de RSK2 que a su vez activaría la proteína ribosomal rpS6, uno de los responsables de la iniciación de la traducción, por lo que también podría estar influyendo a la producción de MMP-9 a este nivel.
Según nuestros resultados, CAV1 estaría influyendo en la capacidad migratoria de las células de SE mediante dos mecanismos: 1) a través de la activación de la vía de ERK1/2 y 2) mediante la unión a diferentes proteínas con dominio SH2 a través de la fosforilación de CAV1 en la tirosina 14. ERK1/2 influye en la regulación de la capacidad migratoria de una forma de pendiente o independiente de RSK1.
Además, en los ensayos de metástasis experimental in vivo las células con inhibición de CAV1 presentaron una menor incidencia de metástasis pulmonar, hecho que correlacionó con una disminución en la expresión de SPARC (Secreted protein acidic and rich in cysteine), una proteína de adhesión importante en procesos metastásicos. En resumen, nuestros resultados evidencian la importancia de CAV1 en el proceso metastásico del SE.Ewing’s sarcoma (ES) is the second most common bone tumor in childhood and occurs with a high incidence of metastatic disease. Such tumors have a characteristic gene translocation that gives rise to a fusion protein, most commonly EWS/FLI1. This fusion protein acts as an aberrant transcription factor regulating the expression of different target genes involved in the initiation, maintenance and progression of the tumor. Our group described caveolin 1 (CAV1) as one of these target genes, describing its role in the malignant phenotype, tumorigenicity and resistance to chemotherapy-induced apoptosis of ES cell lines. To investigate the specific role of CAV1 in the metastatic process of this sarcoma, we established a model of low expression of CAV1 in cell lines of ES. Then, we measured changes in their migratory capacity, invasiveness and metastatic potential. In vitro, we found a lower migratory capability of CAV1 knockdown cells and a reduction in MMP-9 expression and MMP-2 activity. The regulation of MMP-2 activity seems to be related to the possible regulation that CAV1 exerts on the function of MT1- MMP, an essential protein for the activation of MMP-2. On the other hand, we suggest that CAV1 promotes the expression of MMP-9, both transcriptionaly and post-transcriptionaly, through regulating ERK1/2 signaling pathway. In our model, activated ERK1/2 would translocate to the nucleus where it would activate the transcription factors responsible for MMP-9 promoter activation. At the cytoplasm, activated ERK1/2 would phosphorylate and activate RSK2, which, in turn, would promote rpS6 activation, leading to protein translation initiation. Our results indicate that CAV1 is regulating migratory capability of ES cells by two different mechanisms; 1) through ERK1/2 pathway activation, 2) by linking several proteins bearing SH2 domains trough phosphorylated Tyr14 of CAV1. ERK1/2 seems to regulate cell migration in both RSK1-dependent and independent manner. In addition, experimental metastasis assays in vivo showed that, CAV1 knockdown cells had a lower incidence of pulmonary metastasis, a fact that correlated with a decrease in the expression of SPARC, a major adhesion protein in metastatic processes. In summary, our results demonstrate the importance of CAV1 in the metastatic process on ES tumors.
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Jonker, Anneliene. "Synthetic Lethality and Metabolism in Ewing Sarcoma : Knowledge Through Silence." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA11T039/document.
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Le sarcome de Ewing est la seconde tumeur pédiatrique de l’os la plus fréquente. Elle est caractérisée par une translocation chromosomique résultant à la fusion de EWSR1 avec un membre de la famille ETS. Chez 85% des patients, cette fusion conduit à l’expression de la protéine chimérique EWS-FLI1 qui est l’oncogène majeur de ce sarcome. Ce dernier agit principalement par son action transcriptionelle sur des cibles qui lui sont propres. Au niveau thérapeutique, le sarcome d’Ewing est traité par chimiothérapie, chirurgie locale et par radiothérapie. La survie à long terme des patients est de l’ordre de 70%, mais beaucoup plus basse pour les patients métastatiques et quasi nulle lors d’une récidive. Parmi maintes caractéristiques, certains cancers présentent une dérégulation énergétique. L’influence d’EWS-FLI1 sur cet aspect n’a fait l’objet d’aucune étude dans le contexte du sarcome d’Ewing. Nous avons donc étudié par profilage métabolomique des cellules de sarcome d’Ewing en présence ou en absence d’EWS-FLI1. En comparant ces deux conditions, des modulations du profil énergétique relatif au cycle de Krebs, des précurseurs de le glycosylation ainsi que des métabolites de la voie de la méthionine et du tryptophane ont été observés. En parallèle, grâce à un crible de banque de shRNAs réalisé dans des conditions expérimentales similaires à l’étude métabolomique (lignée d’Ewing avec ou sans EWS-FLI1), nous avons pu identifier des gènes présentant des caractéristiques « synthétique létales », c'est-à-dire tuant uniquement les cellules du sarcome d’Ewing en présence de son oncogèneEwing sarcoma, the second most commonly occurring pediatric bone tumor, is most often characterized by a chromosomal translocation between EWSR1 and FLI1. The gene fusion EWS-FLI1 accounts for 85% of all Ewing sarcoma and is considered the major oncogene and master regulator of Ewing sarcoma. EWS-FLI1 is a transcriptional modulator of targets, both directly and indirectly. Ewing sarcoma is aggressively treated with chemotherapy, localized surgery and radiation and has an overall survival of about 70%, however, survival for metastasis or relapsed cases remains low. One of the cancer hallmarks, metabolic deregulation, is most likely partly dependent on EWS-FLI1 in Ewing sarcoma cells. In order to get a better understanding of Ewing sarcoma biology and oncogenesis, it might be of high interest to investigate the influence of EWS-FLI1 in Ewing sarcoma cells. We therefore performed a global metabolic profiling of Ewing sarcoma cells with or without inhibition of EWS-FLI1. Several changes in the energy metabolism were observed throughout this study; the observed changes were consistent with an energy profile that moved from a cancer cell energy metabolism towards the energy metabolism of a more normal cell upon EWS-FLI1 inhibition, primarily based on the TCA cycle. Levels of TCA intermediates, glycosylation precursors, methionine pathway metabolites and amino acids, especially changes in the tryptophan metabolic pathway, were altered upon EWS-FLI1 inhibition. Parallel to this study, we performed a high-throughput synthetic lethality screen, in order to not only identify essential genes for cell survival and proliferation, but also to identify new synthetic lethal targets that could specifically target Ewing sarcoma cells carrying the EWS-FLI1 fusion gene
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Dionísio, Fernando Carrasco Ferreira. "Avaliação da reprodutibilidade intra e interobservador da segmentação manual de sarcomas ósseos em imagens de ressonância magnética." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/17/17158/tde-10042018-165710/.
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Os sarcomas ósseos representam uma proporção significativa de tumores na faixa etária pediátrica, ainda apresentando um quadro desafiador devido a sua significativa taxa de morbimortalidade. Pesquisas para o desenvolvimento de novas modalidades terapêuticas e para o desenvolvimento de métodos que identifiquem características da doença que possam permitir melhor estratificação dos pacientes através de dados clinicamente relevantes para individualizar as condutas clínicas são necessárias. Dentro deste contexto surge o conceito de radiômica, que visa extrair dados clinicamente relevantes a partir de imagens médicas. Entretanto, para colocar a radiômica em prática, é necessário selecionar, nas imagens médicas, as áreas de interesse referentes às patologias estudadas, e este processo se denomina segmentação. O objetivo primário deste estudo foi avaliar a reprodutibilidade intra e inter-observador da segmentação manual de sarcomas ósseos em imagens de ressonância magnética (RM). Como objetivo secundário, foi avaliada a capacidade da segmentação semiautomática em reduzir o tempo necessário para segmentação, mantendo similaridade com a segmentação manual. O estudo foi realizado de forma retrospectiva com inclusão de pacientes com diagnóstico de osteossarcoma ou sarcoma de Ewing confirmado por estudo histopatológico e que tivessem imagens de RM realizadas no Hospital Universitário de nossa Instituição realizadas previamente a qualquer intervenção terapêutica. Três médicos radiologistas, de forma independente e às cegas em relação as demais segmentações e em relação ao resultado histopatológico, realizaram a segmentação manual dos contornos destes tumores utilizando o software 3DSlicer, permitindo que fosse realizada avaliação da reprodutibilidade interobservador. Um dos radiologistas realizou uma segunda segmentação manual dos mesmos casos, possibilitando a avaliação da reprodutibilidade intraobservador, e, ainda, uma terceira segmentação foi realizada, utilizando metodologia semiautomática, disponível no software mencionado. Para a análise estatística, foi utilizado o coeficiente de similaridade de Dice (DICE), a distância Hausdorff (DH), comparações de volumes e análises dos intervalos de tempo necessários para realização das segmentações. Os parâmetros avaliados demonstraram haver boa reprodutibilidade intraobservador, com DICE variando entre 0,83 a 0,97; e distância Hausdorff variando entre 3,37 a 28,73 mm. Também foi demonstrada boa reprodutibilidade interobservador com DICE variando entre 0,73 a 0,97; e distância Hausdorff variando entre 3,93 a 33,40 mm. A segmentação semiautomática demonstrou boa similaridade em relação à segmentação manual (DICE variando entre 0,71 a 0,96 e DH variando entre 5,38 a 31,54 mm), havendo redução significativa do tempo necessário para segmentação. Entre todas as situações comparadas, os volumes não apresentaram diferenças estatisticamente significativas (p-valor>0,05).Bone sarcomas represent a significant proportion of tumors in the pediatric age group and they still are a challenge due to their significant morbidity and mortality rates. Reseaches are important for the development of new therapeutic modalities and for the development of methods that identify features that allow better stratification of the patients with theses diseases for individualization of their treatments. In this context emerges the concept of radiomics, which is the process of extraction of clinically relevant data from medical images. It is important to segment the areas of interest im medical images for the pratice of this process. The primary objective of this study was to evaluate the intra- and interobserver reproducibility of manual segmentation of bone sarcomas on magnetic resonance imaging (MRI). As a secondary objective, it was evaluated if the semiautomatic segmentation could be similar to manual segmentation and if the semiautomatic method could reduce the time required for segmentation. The study was performed retrospectively with the inclusion of patients with osteosarcoma or Ewing sarcoma confirmed by histopathological study and who had MRI performed at the University Hospital of our Institution prior to any therapeutic intervention. Three radiologists, independently and blindly in relation to the other segmentations and in relation to the histopathological results, performed the manual segmentation of the contours of these tumors using 3DSlicer software, allowing an interobserver reproducibility evaluation. One of the radiologists performed a second manual segmentation of the same cases, allowing the evaluation of intraobserver reproducibility. A third segmentation was performed, using semi-automatic methodology, available in the mentioned software. For the statistical analysis, Dice similarity coefficient (DICE), Hausdorff distance (DH), comparisons between volumes and time intervals for segmentations were used. The parameters evaluated demonstrated a good intraobserver reproducibility, with DICE ranging from 0.83 to 0.97 and Hausdorff distance ranging from 3.37 to 28.73 mm. Good interobserver reproducibility was also demonstrated with DICE ranging from 0.73 to 0.97 and Hausdorff distance ranging from 3.93 to 33.40 mm. Semiautomatic segmentation demonstrated good similarity to manual segmentation (DICE ranging from 0.71 to 0.96 and HD ranging from 5.38 to 31.54mm), and there was significant reduction in the time required for segmentation. Among all the situations compared, the volumes did not present significant statistical differences (p-value> 0.05).
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Figuerola, Bou Elisabet. "Polycomb and KDM6A Roles in the Epigenetic Dynamics of Ewing Sarcoma Tumorigenesis." Doctoral thesis, Universitat de Barcelona, 2019. http://hdl.handle.net/10803/668710.
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Ewing sarcoma (ES) is a highly aggressive tumour of the bone and soft tissue that typically affects children, adolescents, and young adults. The fusion protein EWSR1‐FLI1 is the main genetic alteration found that acts as a pioneer factor during early ES tumorigenesis. EWSR1– FLI1 binds GGAA microsatellites causing remodelling of enhancers and genome reprogramming. Among the proteins cooperating with EWSR1‐FLI1 at enhancers, our group has described that the Polycomb subunit RING1B co‐localizes genome‐wide and promotes oncogene recruitment and transcriptional activation of key enhancer genes.
In this thesis, we show that RING1B is a critical factor for ES tumour growth and, together with other E3 ubiquitin ligases, is deregulated by the neddylation pathway inhibitor MLN4924 (pevonedistat). Treatment of ES cell lines with the inhibitor promotes RING1B loss from EWSR1‐FLI1–activated targets as well as eviction of EWSR1‐FLI1, thereby deregulating gene expression. Lack of neddylation caused by the drug appears to be the mechanism behind the in vitro and in vivo degradation of RING1B.
We also study how introduction of EWSR1‐FLI1 to a putative cell‐of‐origin deregulates distribution of the histone repressive mark H3K27me3. Although the global levels of this histone modification are maintained, we observe a perturbed distribution. Specifically, gain or loss of H3K27me3 occurs in some EWSR1‐FLI1–repressed or –activated regions, respectively, which coincide with enrichment of its writer, EZH2, and its eraser, KDM6A, in a transformed ES cell line. Finally, we demonstrate that targeting the tumorigenic distribution of H3K27me3 by combined inhibition of EZH2 (with GSK126) and KDM6A (with GSKJ4) causes a synergic cytotoxic response in ES cell lines.
Altogether, our data provide further insight into the epigenetic mechanisms underlying EWSR1‐FLI1–mediated transformation and reveal new targets for future clinical trials.
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Horbach, Leonardo. "Avaliação de alvos moleculares envolvidos na resistência tumoral de sarcoma de Ewing." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2017. http://hdl.handle.net/10183/171020.
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O Sarcoma de Ewing (ES) é um raro tumor de ossos e tecidos moles com uma característica translocação cromossomal, a fusão EWS/FLI-1, que atua sobre diversos processos oncogênicos. O desenvolvimento da resistência à quimioterapia é comum no tumor e continua como uma das principais causas na falha do tratamento. O objetivo desse estudo foi avaliar a expressão de genes após a indução de resistência em linhagens celulares de ES. Foi selecionado um conjunto de genes (CCAR1, TUBA1A, POLDIP2, SMARCA4 e SMARCB1) a partir da mineração da literatura em resistência tumoral para duas drogas utilizadas na terapia de ES, doxorrubicina e vincristina. Descrevemos a expressão de cada gene selecionado antes e após as linhagens SK-ES-1 serem submetidas a um protocolo de indução de resistência para ambos os fármacos, que obteve êxito ao induzir as células à resistência. A expressão relativa dos níveis de mRNA foi avaliada e foi encontrada em maior expressão para os genes SMARCA4, SMARCB1 e POLDIP2, e em menor expressão para os genes TUBA1A e CCAR1, quando comparadas às linhagens de controle não-resistentes de cada quimioterápico. Os resultados sugerem o envolvimento de mecanismos de reparo de dano ao DNA, remodelamento de cromatina via SWI/SNF, atividade de microtúbulos e atividade spliceossomal nos processos de resistência quimioterápica em ES.Ewing Sarcoma (ES) is a rare bone and soft tissue tumor with a characteristic chromosomal translocation, the fusion protein EWS/FLI-1, that drives several oncogenic processes. The development of resistance to chemotherapy is common and remains as the main cause of treatment failure. The goal of this study was to evaluate the expression of selected genes in ES cell lines after induction of resistance. A set of genes (CCAR1, TUBA1A, POLDIP2, SMARCA4 and SMARCB1) was data mined from tumoral resistance literature for two drugs used in ES therapy, doxorubicin and vincristine. We describe the expression of each selected gene before and after SK-ES-1 cell lines were exposed to a drug resistance inducing protocol for doxorubicin and vincristine. Cell lines were successfully induced to be resistant to doxorubicin and vincristine. The relative mRNA expression levels were upregulated for genes SMARCA4, SMARCB1 and POLDIP2 and downregulated for genes TUBA1A and CCAR1, when comparing resistant and non-resistant ES cell lines for each drug. The results suggest involvement of repair pathways, SWI/SNF chromatin remodeling, microtubule and spliceosomal activity processes in drug resistance mechanisms in ES.
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Twardziok, Monika [Verfasser]. "Mechanism of action of Viscum album L. extracts in Ewing sarcoma / Monika Twardziok." Berlin : Freie Universität Berlin, 2016. http://d-nb.info/1081367091/34.
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Ng, King Pan. "The mechanism of the transcription activation mediated by the Ewing sarcoma activation domain /." View abstract or full-text, 2008. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202008%20NG.
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Zaganjori, Ersida. "Il planning chirurgico nei tumori ossei: il caso studio del sarcoma di Ewing." Bachelor's thesis, Alma Mater Studiorum - Università di Bologna, 2021.
Find full textAbstract:
Il sarcoma di Ewing è un tumore che colpisce prevalentemente in età pediatrica e se non curato in tempo può espandersi in molte parti del corpo, tra cui le vie aeree. Molti giovani pazienti che manifestano il sarcoma sono costretti a farsi asportare parti del corpo di vitale importanza e spesso vivono in condizioni difficili come ad esempio senza un polmone. Proprio per questo motivo è importante intervenire subito e in maniera precisa perché, come molti altri tumori, se non eliminati completamente con il tempo presentano delle recidive.
Il planning chirurgico è molto utilizzato in questo ambito in quanto ci permette di eseguire una ricostruzione tridimensionale degli organi interessati dalle masse tumorali. Questa pianificazione facilita la visualizzazione, la manipolazione e l’analisi dei dati ottenuti con diverse tecniche di imaging. Le tecniche più recenti prevedono la simulazione in 3D dei piani di taglio consentendo di effettuare il planning preoperatorio e la ricostruzione tridimensionale delle porzioni di ossa da rimuovere per ottimizzare il gesto chirurgico. L’elevata accuratezza che ci permette di avere l’utilizzo del planning chirurgico ci aiuta sia in fase preoperatoria nello studio del caso e nella progettazione di eventuali protesi e dispositivi personalizzati al paziente che in fase intra-operatoria durante la resezione del sarcoma.
In questa tesi verranno dapprima introdotte le conoscenze relative alla fisiopatologia del sarcoma di Ewing, i percorsi di diagnosi e di trattamento di questa tipologia di carcinoma. Nell’ultimo capitolo sono state affrontate le ultime evoluzioni della tecnologia che stanno contribuendo ad un sostanziale miglioramento della prognosi di questa patologia molto aggressiva ma per fortuna poco diffusa.
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Wu, Hue-Tsi. "The WNT signalling pathway in Ewing sarcoma/primitive neuroectodermal tumour : an immunohistochemical investigation." Master's thesis, University of Cape Town, 2011. http://hdl.handle.net/11427/11479.
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Includes abstract.Includes bibliographical references.
The WNT pathway is a major developmental pathway that plays an important role in the development of many tumours, including neuroectodermal and bone tumours. Ewing sarcoma (ES) / primitive neuroectodermal tumour (PNET) shows varying degrees of neuroectodermal differentiation and is the second commonest bone malignancy in childhood. A recent study on ES cell lines using RT-PCR analysis and biological response assays suggests that an intact WNT pathway exists in ES and that addition of exogenous WNT ligands enhances cell motility. Based on this we hypothesize that the WNT pathway may play a role in the biology of ES/PNET and we aim to investigate this by immunohistochemical stains on archival tissue.
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Johnson, Kirsten M. "Characterization of length-dependent GGAA-microsatellites in EWS/FLI mediated Ewing sarcoma oncogenesis." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1523384027382108.
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Sáinz, Jaspeado Miguel Guillermo. "Regulación del aporte vascular en el Sarcoma de Ewing: Papel de la Caveolina-1." Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/107820.
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Como otros tumores sólidos, el sarcoma de Ewing (SE) requiere de un aporte vascular adecuado que permita nutrir y oxigenar a las células tumorales. En el SE, la vascularización tumoral se encuentra caracterizada principalmente por los procesos de vasculogenesis, mimetismo vascular y angiogénesis. Considerando la participación de CAV1 en el aporte vascular, nos planteamos demostrar que CAV1 juega un papel importante en el desarrollo vascular de la enfermedad.
Estableciendo tres nuevos modelos de baja expresión de CAV1 en células se SE (RDES, SKES1 y TC71), fue posible observar la reducción en el desarrollo tumoral en xenoinjertos, y a su vez esta reducción fue relacionada con la disminución en la densidad micro-vascular. Este trabajo demuestra el efecto directo del silenciamiento de CAV1 sobre el proceso angiogénico en el SE, regulando la respuesta de la célula endotelial ante las señales producidas por la célula tumoral. Nuestros resultados mostraron que las células endoteliales presentan mayor migración ante el estímulo de las señales producidas por la célula tumoral. Mediante el análisis de expresión de diferentes factores utilizando RT-PCR, fue posible determinar que la expresión de bFGF era afectada en los modelos analizados como efecto del silenciamiento de CAV1. Para determinar el mecanismo molecular a través del cual CAV1 regula la angiogénesis en el sarcoma de Ewing, partimos del análisis de expresión de 3 miembros de la familia Eph descritos como pieza clave en el desarrollo vascular. La expresión del receptor EphA2 en los modelos de baja expresión de CAV1 mostró una disminución en los clones, donde el silenciamiento de CAV1 conlleva la redistribución y reducción EphA2. Los análisis mostraron además una interacción entre ambas proteínas.
Establecida la relación EphA2/CAV1, analizamos el efecto de la estimulación de EphA2 a partir de EfnA1 y confirmamos que las células con baja expresión de CAV1 presentaban una respuesta menor. La estimulación resultó en el incremento de la fosforilación de AKT, sugiriendo que los efectos podrían depender de la actividad quinasa del receptor. Para respaldar esto, las células RDES y TC71 fueron transfectadas con un dominante negativo de EphA2 que no modificaba la expresión de CAV1 (EphA2-Kd). Los modelos EphA2-Kd reprodujeron los resultados observados en los modelos de baja expresión de CAV1. La estimulación del modelo TC71-EphA2-Kd con la proteína recombinante EfnA1, mostró tanto la activación de AKT como la sobreexpresión de bFGF, reproduciendo en cierta medida lo observado por el silenciamiento de CAV1 y destacando la importancia de la actividad dependiente de quinasa de EphA2 en el proceso angiogénico de la enfermedad.
Considerando que CAV1 es necesario para el correcto funcionamiento de las rutas de señalización controladas por EphA2 en el SE, decidimos determinar la implicación de CAV1 y EphA2 en el mimetismo vascular de la enfermedad. Se decidió analizar el papel independiente de quinasa del receptor EphA2 como una posible pieza clave en el proceso de mimetismo vascular en el SE. Nuestros resultados mostraron que el silenciamiento de CAV1 reduce el desarrollo de vasos miméticos in vivo e impide la formación tubular in vitro. Además, el efecto de bloquear la actividad quinasadependiente del receptor EphA2 mostró que la actividad quinasa del receptor no afecta al proceso de mimetismo vascular en esta entidad tumoral. Para confirmar la participación de EphA2 en el proceso de formación de vasos miméticos, se transfectó un mutante que carece de toda la región citoplasmática del receptor EphA2 (ΔCyto), eliminando así sus funciones dependientes e independientes de quinasa. Los resultados mostraron que el bloqueo total del receptor tenía un efecto negativo en la formación de estructuras tubulares. Estos resultados confirman la importancia de la actividad prooncogénica del receptor EphA2 en el sarcoma de Ewing.We established low CAV1 expressing models by knocking down CAV1 in TC71, RDES and SKES1 Ewing sarcoma (ES) cells by stably transfecting a previously validated shRNA construct. In vivo studies showed a decrease in tumor volume from the CAV1 knocked-down clones when compared with controls. This correlated with a reduction in microvascular density (MVD) and higher levels of necrosis. Conditioned media from CAV1 knocked-down cells showed reduced capability to promote migration of endothelial cells with no changes in proliferation. Different pro-angiogenic factors were analyzed by RT-PCR in the models and, downregulation of bFGF was observed in all four. Results suggested that CAV1 was indirectly affecting bFGF expression. EphA2 expression was observed in ES cell lines and tumor samples. CAV1 knocked-down cells showed a reduction in EphA2 phosphorylation as well as a displacement from the membrane to the cytoplasm. Furthermore, we showed that the CAV1/EphA2 interaction was necessary for Eph-mediated signaling. To further support these results, RDES and TC71 cells were stably transfected with a dominant negative of EphA2 that did not alter the expression of CAV1 (EphAh2-kd). EphA2-Kd models were able to replicate the effects observed in the CAV1 knocked-down models, where the stimulation of the TC71-EphA2-Kd model with the recombinant protein EfnA1, showed both the activation of AKT and the overexpression of bFGF. These results replicate the effect of silencing CAV1 and highlighted the importance of the kinase-dependent activity of EphA2 in the angiogenic process in ES. We decided to analyze the kinaseindependent role of EphA2 as a possible key in the process of vascular mimicry in ES. Our results showed that the silencing of CAV1 reduces the development of mimetic vessels in vivo and prevents the tubular formation in vitro. In addition, our results showed that the kinase-dependent activity of the receptor does not affect the process of vascular mimicry in ES. Moreover, we stably transfected a mutant that lacks all the cytoplasmic region of EphA2, blocking its kinase-dependent and –independent activities. Results showed a negative effect on the formation of tubular structures. These results confirm the importance of the pro-oncogenic activity of EphA2 in ES.
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Vieira, Gabriela Maciel. "Efeitos da inibição das quinases ROCK no potencial invasivo de linhagens celulares de sarcoma de Ewing." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-05012017-115653/.
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O sarcoma de Ewing (SE) é um tumor caracterizado por possuir células indiferenciadas, de origem neuroepitelial. É o segundo tumor ósseo maligno mais comum em crianças e adolescentes. O tratamento consiste na aplicação de quimioterapia neoadjuvante, seguida da cirurgia e a quimioterapia adjuvante ou radioterapia. Contudo, a disseminação deste tumor é muito intensa, e mesmo em pacientes onde a metástase não ocorreu, podem haver células tumorais circulantes. Além disso, a sobrevida de 5 anos que cerca de 70% dos pacientes que não apresentam metástase no momento do diagnóstico atingem, cai para 25% nos pacientes que apresentam metástase. A migração e invasão celular dos tecidos vizinhos e vasos sanguíneos, a adesão celular e a proliferação são processos essenciais para que a metástase tumoral aconteça. Dentre as reguladoras da mobilidade, adesão celular e proliferação estão as GTPases da família Rho, que possuem entre suas quinases efetoras, ROCK1 e ROCK2. Assim, as proteínas ROCK induzem inúmeras respostas celulares que envolvem a regulação de muitas proteínas associadas ao citoesqueleto. Estudos evidenciam que respostas sinalizadas por ROCK agravam fenótipos associados ao câncer e outras doenças. O presente trabalho teve como objetivo verificar a expressão dos genes ROCK1 e ROCK2 em amostras tumorais e linhagens celulares de SE, e os efeitos in vitro da inibição farmacológica de ambas as quinases. O estudo da expressão gênica de ROCK1 e ROCK2 em amostras de pacientes com SE (n=18) revelaram uma hipoexpressão de ambos genes, tanto nas amostras de pacientes com SE quanto nas linhagens celulares SK-ES-1 e RD-ES. Não foi encontrada nenhuma relação das expressões gênicas com os dados clínicos. Contudo, a presença do gene de fusão EWS-FLI1 parece estar associada com menor expressão de ROCK1. Verificou-se que a proliferação celular, a capacidade clonogênica e o ciclo celular não se alteraram significativamente nas linhagens celulares de SE após os diferentes tratamentos com as drogas GSK429286 (inibidor de ROCK1), SR3677 (inibidor de ROCK2) e Hidroxifasudil (pan-inibidor). Porém, apesar de não significativo, observou-se um tênue aumento na migração e invasão celular após o tratamento com as drogas. Nossos dados indicam que ROCK1 e ROCK2 podem possuir um papel na tumorigênese do SE, e ainda estar relacionadas com os mecanismos de migração e invasão celular, processos importantes para a metástase tumoral.Ewing\'s sarcoma (ES) is characterized by undifferentiated cells of neuroepithelial origin. It is the second most common malignant bone tumor in children and adolescents. Standard treatment consists of preoperative chemotherapy, followed by surgery and postoperative chemotherapy and/or radiotherapy. However, the spread of the tumor is very high and even in patients where the metastasis has not occurred, there may be circulating tumor cells. Furthermore, the 5-year survival which about 70% of patients without metastases at diagnosis achieve, falls to 25% in patients with metastases. Migration and invasion into adjacent tissues and blood vessels, cell adhesion and proliferation are essential for tumor metastasis to occur. Among the regulators of the processes are the Rho family of GTPases, which have as their effector kinases, ROCK1 and ROCK2. Thus, ROCK induce numerous cellular responses involving the regulation of many proteins associated with the cytoskeleton. Studies show that responses regulated by ROCK aggravate phenotypes associated with cancer and other diseases. This study aimed to verify the expression of ROCK1 and ROCK2 genes in ES tumor samples and to evaluate the effects of their pharmacological inhibition in vitro. Gene expression analysis showed lower expression of ROCK in both, patient samples (n=18) and the SK-ES- 1 and RD-ES cell lines. There was no relation of gene expression with clinical data. Nonetheless, the presence of EWS-FLI1 fusion appears to be associated with lower expression of ROCK1. Cell proliferation, clonogenic capacity and the cell cycle were not affected after different treatments with GSK429286 (ROCK1 inhibitor), SR3677 (ROCK2 inhibitor) and Hydroxyfasudil (pan-inhibitor). However, although not significant, there was a tenuous increase in cell migration and invasion following treatment with the drugs. Our data suggest that ROCK1 and ROCK2 might have a role in ES tumorigenesis and may be related im part to migration and invasion cell mechanisms, important processes for tumor metastasis.
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Machado, Lucas Faria Abrahão. "Pesquisa de biomarcadores como fator prognóstico nos tumores da família do sarcoma de Ewing." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/5/5140/tde-10112017-115117/.
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INTRODUÇÃO: Os tumores da família do sarcoma de Ewing (TFSE) compreendem um espectro de neoplasias de células neuroectodérmicas dos ossos e partes moles de comportamento biológico agressivo e prognóstico reservado, caracterizadas por translocações envolvendo um dos genes da família TET/FET e um dos genes da família ETS, mais comumente EWSR1 e FLI1. Com o avanço da medicina personalizada, cresce a demanda por biomarcadores em TFSE que tenham valor como fatores prognósticos e potencial para futuras terapias-alvo específicas. Este estudo propôs biomarcadores, incluindo proteínas relacionadas à supressão tumoral, proliferação celular, metabolismo energético, atividade imune, vias de reparo do DNA e células tronco. MÉTODOS: A expressão imuno-histoquímica dos biomarcadores MTAP, p16, STAG2, p53, USP22, PTEN, RKIP, Ciclina D1, MCTs (1, 2 e 4), CD147, CA IX, GLUT1, BRACHYURY, PD-L1, OCT4 e SALL4 foi analisada em uma série bem caracterizada de 113 TFSE através de amostras em tissue microarrays (TMA). Os perfis de expressão foram então associados aos parâmetros clínico-patológicos dos pacientes e à sobrevida global para uma análise do impacto no prognóstico. RESULTADOS: A hiperexpressão de p53 mostrou associação estatisticamente significativa com menor sobrevida global (p < 0,001), doença metastática no diagnóstico (p = 0,017) e idade acima de 20 anos (p = 0,04). A perda de expressão de MTAP (p = 0,039) e de Brachyury (p = 0,008) também se associaram significativamente com menor sobrevida global. Em relação às características clínicas dos pacientes, doença metastática no diagnóstico e etnia não-branca foram associados a um pior prognóstico. CONCLUSÕES: Os biomarcadores p53, MTAP e Brachyury foram identificados como fatores independentes relacionados ao prognóstico. A utilização destes biomarcadores como fator prognóstico nos TFSE pode auxiliar na estratificação de risco dos pacientes e até mesmo estimular o desenvolvimento de drogas-alvo específicasINTRODUCTION: The Ewing sarcoma family of tumors (ESFT) comprises a spectrum of neoplasms of neuroectodermal cells of the bones and soft tissues with an aggressive biological behavior and poor outcome, characterized by translocations involving one of the genes of the TET/FET family and one of the genes of the ETS family, most commonly EWSR1 and FLI1. With the progress of personalized medicine, there is a great demand for biomarkers in ESFT that could have prognostic values and the potential for future targeted therapies. This study proposed the evaluation of protein expression of different classes of biomarkers, including proteins related to tumor suppression, cell proliferation, energy metabolism, immune activity, DNA repair pathways and stem cells. METHODS: Immunohistochemical expression of the biomarkers MTAP, p16, STAG2, p53, USP22, PTEN, RKIP, Cyclin D1, MCTs (1, 2 and 4), CD147, CA IX, GLUT1, BRACHYURY, PD-L1, OCT4 and SALL4 was analyzed in a well-characterized series of 113 ESFT in a tissue microarray (TMA) platform. Expression profiles were then associated with patients\' clinical-pathological parameters and overall survival for analysis of the prognostic impact. RESULTS: p53 hyperexpression showed a statistically significant association with lower overall survival (p <0.001), metastatic disease at diagnosis (p = 0.017) and age over 20 years (p = 0.04). Loss of MTAP (p = 0.039) and Brachyury (p = 0.008) were also significantly associated with lower overall survival. Regarding the clinical characteristics of the patients, metastatic disease at diagnosis and non-white ethnicity were associated with a worse prognosis. CONCLUSIONS: The biomarkers p53, MTAP and Brachyury were identified as independent factors related to the prognosis. The use of these biomarkers as a prognostic factor in ESFT may aid in the risk stratification of patients and even stimulate the development of specific targeted drugs
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Hamid, Mustafa Issa. "Contributions of Fli1a and Hox13 During Zebrafish Pectoral Fin Development and Implications for Ewing Sarcoma." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/40913.
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Álvarez, San Nicolás Jordi. "Cirugía de salvamento de extremidad en sarcoma de Ewing supervivencia, complicaciones y factores de riesgo." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/310613.
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INTRODUCCIÓN:La introducción de pautas de quimioterapia supuso el aumento de la supervivencia en pacientes con sarcoma de Ewing (ES),con el consecuente desarrollo de las técnicas locales de tratamiento como la cirugía de salvamento de extremidad (LS).La supervivencia y eventos adversos han sido ampliamente estudiada en series generales de ES, así como las complicaciones locales en técnicas LS en series generales de tumores óseos. Sin embargo,no existen series exclusivas de ES tratados mediante LS, a pesar de ser la técnica de manejo local actual más frecuente. Este estudio va dirigido a analizar la supervivencia, los eventos oncológicos y las complicaciones locales en ES tratados localmente mediante LS.
MATERIAL Y MÉTODOS:se han revisado los casos de ES estudiados en el Hospital de la Santa Creu i Sant Pau de Barcelona entre los años 1984 y 2008.Se han seleccionado casos tratados mediante terapia multimodal y localmente mediante LS. Se han revisado las características personales y de tratamiento de cada paciente así como los eventos adversos.
RESULTADOS:90 pacientes ES han sido valorados, siendo 50 tratados mediante terapia multimodal y técnicas de LS. La edad media es de 20 años. La supervivencia global es del 68.8% y la libre de evento del 60.6%.La respuesta al tratamiento previo, la localización pélvica y la edad han resultado factores pronósticos. Se han desarrollado metástasis en un 36%, siendo la respuesta al tratamiento previo,la localización pélvica y la recidiva local factores independientes de riesgo.La frecuencia de aparición de recidiva local es del 14%. La edad, los márgenes de resección y la localización axial se han asociado a peor control local, sin ser las diferencias significativas en el análisis multivariante. En un 26.3% de los casos reconstruidos se han producido complicaciones mecánicas.Son factores independientes predictivos de fracaso mecánico el tamaño tumoral y la reconstrucción empleada.En un 7.9% de los casos reconstruidos se ha producido infección postoperatoria,sin hallar factores predictivos de riesgo.Se ha realizado amputación secundaria en un 6%,sin hallar factores predictivos de riesgo
DISCUSIÓN:la supervivencia y los eventos oncológicos en ES tratados mediante LS es similar a la de series previas generales de tratamiento.La diseminación al diagnóstico no supone un factor pronóstico,a diferencia de las series generales.Este hecho se explica por la selección positiva de los casos que tratados mediante LS.La diseminación a distancia tras LS presenta una frecuencia similar que en los estudios generales previos de ES.Los factores predictivos para la aparición de metástasis son similares a los pronósticos para supervivencia a lo que se añade la presencia de recidiva local.La frecuencia de recidiva local en ES tras LS es similar a la presente en casos ES tratados mediante cualquier técnica quirúrgica.No se han hallado factores independientes predictivos de aparición de recidiva local.La frecuencia de fracaso mecánico es similar a series previas de LS en diferentes tipos de tumores.Las reconstrucciones mediante aloinjerto osteoarticular y tumores de mayores a 10 cm presentan un mayor riesgo de fracaso mecánico. El riesgo de infección es similar a otros estudios de LS, siendo más frecuente en reconstrucción mediante prótesis,localización en tibia y en casos irradiados preoperatoriamente.La amputación secundaria es más frecuente en casos localizados en tibia y se relaciona con la infección local,sin hallar diferencias significativas en el análisis multivariante.
CONCLUSIONES: la supervivencia y la presencia de eventos oncológicos locales o a distancia en ES tratados mediante LS es similar al de series de tratamiento general previas.Los factores de riesgo asociados no son diferentes, a excepción de la presencia de metástasis al diagnóstico.La presencia de complicaciones no oncológicas locales y la necesidad de amputación son similares a otras series de LS,así como los factores predictivos de riesgo relacionados.The use of chemotherapy has increased survival in patients with Ewing’s sarcoma (ES), with the consequent development of local surgery techniques as limb salvage (LS). Survival and adverse events have been widely studied in general ES series,as well as local complications in LS series. However, there is not specific series of ES cases treated by LS, despite of being the most used local technique. Survival, oncological events and local complications in ES locally treated by LS were studied in this study. MATERIAL AND METHODS: ES patients treated at Hospital de la Santa Creu i Sant Pau in Barcelona between 1984 and 2008 were included in this study. Cases treated by multimodal therapy and locally by LS were selected. Patient’s characteristics, type of treatment and adverse events were analyzed. RESULTS: Ninety patients were studied. Fifty of them were treated by multimodal therapy and LS. Mean age was 20 years. Overall survival was 68.8% and event free survival was 60.6%. Response to chemotherapy, pelvic location and age were found as prognostic factors. Metastasis developed in 36% of the patients. Response to chemotherapy, pelvic location and local recurrence were independent risk factors. Local recurrence was found in 14% of cases. Age, resection margins and axial location were associated with worse local control, but the differences were not significant in multivariate analysis. 26.3% of reconstructed cases developed mechanical complications. Tumor size and type of reconstruction were found as independent risk factors for mechanical failure. Postoperative infection was found in 7.9% of reconstructed cases. Secondary amputation was made in 6%, finding no independent risk factors. DISCUSSION: Survival and oncological adverse events in ES treated by LS are similar to previous data and studies with general treatment and other local technics. However, dissemination at diagnosis is not a prognostic factor because of positive selection in ES treated by LS. Metastatic spread after LS has a similar frequency as in previous general studies. Local recurrence is a risk factor for metastasis appearance, and other predictive risk factors are similar to prognostic factors in previous general ES studies. Local recurrence in ES treated byLS is similar to ES treated by any surgical technique. Independent predictive risk factors were not found for local recurrence. Mechanical failure in ES treated byLSis similar to previous series of LS with different types of tumor. Osteoarticular allograft reconstructions and tumor size bigger than 10 cm have an increase risk of mechanical failure. Infection in ES treated by LS is similar to previous LS studies, being more frequent in prosthetic reconstructions, tibial location and patients treated with radiotherapy before surgery. Secondary amputation is more frequent in tibial tumors and is associated with local infection, without significant differences in multivariate analysis. CONCLUSIONS: Survival and oncological adverse events in ES treated by LS are similar to previous ES studies. Prognosis factors are no different, except for the presence of metastasis at diagnosis. Rates of local non oncological complications and secondary amputation are similar to previous LS studies, as well as all the predictive risk factors.
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Shaw, Debra Jayne. "Molecular Analysis of the Ewing Sarcoma Protein (EWS) and the Survival Motor Neurone protein (SMN)." Thesis, University of Plymouth, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.519440.
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Orth, Martin Franz [Verfasser], and Thomas [Akademischer Betreuer] Grünewald. "Systematic multi-omics profiling of Ewing sarcoma cell lines / Martin Franz Orth ; Betreuer: Thomas Grünewald." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2021. http://d-nb.info/1233600656/34.
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Barbi-Gonçalves, José Carlos. "Sarcoma de Ewing e tumor neuroloectodermico primitivo osseo : correlação histopatologica, imun-histoquimica e fatores prognosticos." [s.n.], 1998. http://repositorio.unicamp.br/jspui/handle/REPOSIP/313595.
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Orientador: Eliane Maria Ingrid AmstaldenDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Os objetivos desse trabalho, foram avaliar o perfil morfológico e imunohistoquímico e sua relação com o prognóstico nos Sarcoma de Ewing/PNET. Foram estudados 32 pacientes ( 16 pacientes de cada sexo), todos caucasóides, portadores de tumor de pequenas células redondas, diagnosticados como sarcoma de Ewing e PNET, num período compreendido entre 1989 e 1997. Clinicamente foram avaliados quanto à idade, sexo, topografia da lesão, presença de metástase ao diagnóstico e estadiamento do tumor. O estudo histopatológico foi avaliado quanto a: atipia celular, presença de rosetas de Homer-Wright, necrose tumoral espontânea, e Úldice mitótico. Também foi avaliada a presença de glicogênio intracitoplasmático e o padrão de distribuição das fibras de reticulina. No estudo imunohistoquímico, foram utilizados um painel com 12 marcadores para avaliação de diferenciação celular: mesenquimal, epitelial, neural, glial, e leucocitária, glicoproteÚla da membrana celular, além do gem que controla a apoptose celular . Utilizou-se o método estatístico de CoxMantel, e sobrevida de Kaplan Meier, para avaliação de fatores prognósticos. A idade dos pacientes variou de 02 a 36 anos com média de 15,11 anos. A localização topográfica do tumor teve a seguinte distribuição: no esqueleto apendicular (71,87%), e no esqueleto axial (28,13%) o qual mostrou tendência ao comprometimento do prognóstico. A maioria dos tumores era composto por células uniformes, redondas e escuras, o padrão atípico foi observado em 18,75%, a formação de rosetas em 21,87%, e necrose tumoral espontânea em 65,62%. A maioria dos casos mostrou um índice mitótico menor que 1 ( 87,5%). Presença de glicogênio foi encontrada em 53,12% das biópsias, e estava ausente as fibras de reticulina envolvendo as células tumorais. Os resultados da imuno-histoquímica foram os seguintes: : Vim (75% ); Cam 5.2 (9,37%); NSE (34,37%);S-100 (25%); Leu-7 (9,37%); Sin (21,87%); NF (3,12%); COR (43,75%); OFAP (12,5%); LCA (ausente); CD99 ( 71,87% ); Bcl-2 (6,25%). Os fatores significantes de pior prognóstico, foram dois marcadores de diferenciação neural: um morfológico, caracterizado pela presença de rosetas de Homer- Wright e outro imuno-hitoquímico, representado pela expressão do NSE. Com esses marcadores foi construído um índice chamado de Índice de Diferenciação Neural ( IDN ). Esse índice foi graduado com valores de O à lI, onde o grau O significa a ausência dos marcadores, o grau I a presença de pelo menos um desses marcadores, e o grau 11 com a presença dos dois marcadores neurais. Este índice mostrou-se muito sensível com o aumento diretamente proporcional da sua graduação com piora prognostica (p=O,0028). a - Teoricamente, a presença de metástase ao diagnóstico, tem sido um fator clínico que compromete a sobrevida. Embora sem significância estatística (p= 0,2312), todos os nossos pacientes portadores de metástase ao diagnóstico, foram a óbito nos primeiros 20 meses do acompanhamento da doença. Além da metástase ao diagnóstico, outros fatores tais como, necrose tumoral espontânea, e a 10caIização no esqueleto axial, teIIl sido de relevância prognóstica na literatura. Com esses dados criamos o LG.S. (Índice global de sobrevida), que associa esses fatores com o LD.N. O LG.S. é graduado de O à III, onde o grau O, é a completa ausência desses fatores, e de I à III, pela somatória gradativa de cada um desses fatores. Notamos significância prognóstica na aplicação desse índice (P=0,005). CONCLUSÕES: 1 - A diferenciação neural caracterizada pela presença de rosetas de Homer Wright e positividade ao N.S.E., demonstraram estreita correlação com redução ,de sobrevida, tornando fundamental a sua pesquisa nas avaliações histopatológicas do sarcoma de EwIDgfPNET. 2 - O Índice de Diferenciação Neural ( IDN), foi definitivo em mostrar a piora do prognóstico à medida do aumento da sua graduação. 3 - A correlação prognóstica observada na aplicação do Índice Global de Sobrevida ( IGS), que agrupa: metástase presente ao diagnóstico, necrose tumoral espontânea e a localização topográfica no esqueleto axial, mostra-se promissora nos estudos dos sarcomas de Ewing/PNET.
Abstract: The purpose of this paper, is to evaluate the morphologic and immunohistochemical features and their role as prognostic factors in Ewing sarcoma. We studied consecutive 32 patients (16 patient of each sex), of Caucasian origin, with diagnosis of Ewing sarcoma and PNET, by light microscopy in a period between 1989 and 1997. Clínical presentation studied were age, seXo the lesion site, presence of metastasis at diagnosis and tumor stage. On the histopathologic study we evaluated: cellular atipia, Homer- Wright rosettes, spontaneous tumor necrosis, and the mitotic index, as well as the presence of intracellular glycogen and the pattern of distribution of the reticulin fibers. In an immunohistochemical study, we used a panel with 12 markers for the evaluation of cellular differentiation ( mesenquimal, epithelial, neural, glial, and leukocyte, glicoprotein ofthe cellular membrane), and agem that controls the cellular apoptose. For statistical evaluation we used the Kaplan-Meier survival curve and the multivariate Cox proportional Hazard Mantel. The patients' age ranged ttom 02 to 36 y.ears with average of 15,11 years. The site ofthe tumor had the following distribution: we found 71,87% in the appendicular skeleton, and 28,17% in the axial skeleton. Most ofthe tumors were composed ofuniform, round and dark cells, the atypical pattem was observed in 18,75%, the rosettes formation in 21,87%,.and spontaneous tumoral necrosis in 65,62%. Most ofthe cases showed an mitotic index less than 1 (87,5%). The glycogen was found in 53,12% ofthe biopsies, the reticulin fibers involving the tumors cells could not be found. Differentiation markers express as following : Vim (75%); Cam-5.2 (9,37%); ENS (34,37%) ;S-100 (25%); Leu-7 (9,37%); Sin (21,87%); NF (3,12%); CGR (43,75%); GFAP (12,5%); LCA (absent); CD99 (71,87%). Only 6,25% was positive for Bcl-2. In univariate ana1ysis of survival the following variables to be significant: Presence of rosettes and positivity for NSE. Since these two variables are both markers for neural differentiation, we joint them together in a new index called a Neural Differentiation Index, (NDI). Cases without Homer- Wright rosettes and absence of NSE we classified "O", in presence of one ofthe two markers was equivalent "I ", and in presence of both we classified "TI". In order to evaluate the importance of a new index we performed a multivariate analysis of survival (Cox model) comparing a NDI as well known prognostic factors much a tumor topography, presence spontaneous tumoral necrosis and presence of metastasis at diagnosis. The Cox model applied to 100 data sets by bootstrap resampling with replacement ofthe original 32 patients. The NDI proved to be the most important prognostic factors since it entered in 94% ofCox model followed by topography at the axial skeleton in 47%, presence of metastasis at diagnosis 31 %, and spontaneous tumoral necrosis in 28%. CONCLUSIONS: 1 The degree of neural differentiation characterized by the presence of Homer- Wright rosettes and NSE expression, is a nnportant prognostic factor with worsens survival. 2. NDI is to be a more important prognostic factor and show worsening of the prognosis as increased its graduation. 3. We suggest that NDI should be evaluate together with established prognostic factors like metastasis, necrosis and tumor site, as axial skeleton, ( Global Survival Index ) in the future studies on the Ewing sarcoma/PNET.
Mestrado
Mestre em Anatomia Patologica
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Denton, Nicholas Lee Denton. "Modulation of tumor associated macrophages enhances oncolytic herpes virotherapy in preclinical models of Ewing sarcoma." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1523892800897524.
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Heisey, Daniel A. R. "TARGETED THERAPIES FOR EWSR1-FLI1 TRANSLOCATED EWING FAMILY OF TUMORS." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/5950.
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The EWSR1-FLI1 t(11;22)(q24;q12) translocation is the pathognomonic genomic alteration in 85% of the Ewing Family of Tumors (EWFT) a malignancy of the bone and the surrounding tissue, predominantly affecting children and adolescents. This translocation results in the formation of a chimeric oncoprotein which acts as an aberrant transcription factor that is currently not pharmaceutically druggable, driving the need for more effective targeted therapies. The EWSR1-FLI1 translocation induces a variety of changes including dysregulation of the epigenome and altered gene expression to drive tumorigenesis, and consequently contributes to the hypersensitivity of EWFT to several classes of chemotherapeutics. We sought to exploit these intrinsic sensitivities by employing a matched pair of cell lines derived from the same patient with Ewing sarcoma prior to and following chemotherapy, a panel of Ewing sarcoma cell lines, and several patient-derived xenografts (PDX) collected at the time of relapse or autopsy, which led us to the development of two novel combination targeted therapies for EWFT.
In our matched pair of EWFT cell lines, we found sensitivity to the Poly(ADP-ribose Polymerase (PARP) inhibitor olaparib was diminished following chemotherapy, despite a predicted sensitivity. In addition, we discovered increased expression of the antiapoptotic protein BCL-2 in the chemotherapy-resistant cells, conferring apoptotic resistance to olaparib. We found that EWS-FLI1 increases BCL-2 expression; however, inhibition of BCL-2 alone is insufficient to sensitize EWFT cells to olaparib, revealing a dual necessity for BCL-2 and BCL-XL (BCL2L1) in EWFT survival. These data reveal BCL-2 and BCL-XL act together to drive olaparib mediated apoptotic resistance in Ewing sarcoma and identify a novel, rational combination therapy using olaparib and the BCL-2/BCL-XL inhibitor navitoclax.
In addition, using high throughput drug screening we have identified a novel epigenetic susceptibility in EWFT to GSK-J4 (GlaxoSmithKline), an inhibitor of lysine 27 of histone 3 (H3K27) demethylases: ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) and Jumonji D3 (JMJD3). Treatment with GSK-J4 leads to a decrease in H3K27 acetylation (H3K27ac) and ultimately, the silencing of EWS-FLI1 gene targets.
We sought to sensitize GSK-J4-mediated inhibition of EWS-FLI1 targets by blocking RNA polymerase II activity using the Cyclin Dependent Kinase 7 (CDK7) inhibitor THZ1. By targeting CDK7-mediated transcription we were able to sensitize EWFTs to H3K27 demethylase inhibition. We therefore propose co-targeting of H3K27 demethylases and CDK7 acts as a surrogate EWS-FLI1 inhibitor. Given the difficulties targeting EWS-FLI1, these strategies may present viable clinical therapies.
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Neto, José Luiz de Sá. "Performance diagnostica da RM na avaliação de reações periosteais em sarcomas ósseos utilizando a radiografia convencional como padrão de referência." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/17/17158/tde-01022016-143217/.
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Objetivo: Avaliar o desempenho diagnóstico da ressonância magnética (RM) na avaliação da presença de reação periosteal (RP) em sarcomas primários de ossos longos, utilizando a radiologia convencional (RC) como padrão de referência. Materiais e Métodos: Estudo retrospectivo com aprovação do Comitê de Ética Institucional. A partir do Sistema de Informática da Radiologia (RIS) e do Sistema de Arquivo de Imagens do HCRP identificamos retrospectivamente os casos de 42 pacientes consecutivos (idade média de 22 anos, sendo 20 homens, 22 mulheres) que realizaram tratamento de osteossarcoma ou sarcoma de Ewing no HCRP e cujos exames de RM e RC estavam disponíveis para reavaliação. Foram incluídos apenas casos de tumores de ossos longos e com confirmação histopatológica. Três radiologistas musculoesqueléticos avaliaram retrospectivamente a presença ou ausência de RP e identificaram para cada caso a ausência ou presença de cada um dos padrões de RP agressiva nas imagens da RC e da RM: Triângulo de Codman, multilamelada e espiculada. A classificação dos Radiologistas foi realizada de forma independente e às cegas em relação ao diagnóstico definitivo e em relação as demais avaliações realizadas. As leituras das imagens de RM e RC realizadas pelo mesmo observador tiveram um intervalo de pelo menos três meses entre elas. Usamos as leituras dos três observadores para avaliar a confiabilidade interobservador por meio do coeficiente Kappa. Finalmente uma análise consensual das leituras realizadas pelos três observadores tanto para a RC quanto para a RM foi utilizada para calcular o desempenho diagnóstico da ressonância magnética na detecção de RP e na classificação de cada padrão agressivo de RP. O radiologista com maior tempo de experiencia fez a classificação consensual da presença ou ausência de cada subtipo de reação periosteal nos casos em que houve discordância entre os observadores. 8 Resultados: A concordância interobservador para a detecção de RP foi quase perfeita para a RC e substancial a quase perfeita para a RM. A concordância interobservador para a classificação dos diferentes padrões de RP foi moderada a substancial para a presença do triângulo de Codman. A avaliação dos padrões de RP multilamelada e espiculada foi menos consensual entre os radiologistas tanto para RC quanto para a RM. O diagnóstico por RM e RC e a classificação de cada RP agressiva foram estatisticamente associadas com o diagnóstico por RC (p <0,05). A RM mostrou alta especificidade e valor preditivo negativo elevado, mas moderada sensibilidade na detecção de reações periosteais quando comparada com a RC. Conclusão: Nossos resultados sugerem que a detecção de RP por meio da RM e da RC apresenta alta reprodutibilidade. A RM mostrou alta especificidade e sensibilidade moderada para a identificação de RP em comparação com a RC. No entanto, a reprodutibilidade da classificação em diferentes padrões de RP agressivas foi relativamente baixo, tanto para RC quanto para RM em relação às RPs multilameladas e espiculadas.Objective - To evaluate the diagnostic performance of magnetic resonance imaging (MR) assessing the presence of periosteal reaction (PR) in primary long bone sarcomas using conventional radiography (CR) as the reference standard. Materials and methods After institutional board approval, we retrospectively reviewed the MRI and conventional radiographies of 42 consecutive patients (mean age 22 years, 20 men, 22 women). We included only cases of osteosarcoma or Ewings sarcoma in the long bones with histopathological confirmation. Three experienced musculoskeletal radiologists retrospectively evaluated the presence or absence of periosteal reaction and each pattern of aggressive periosteal reaction in radiographs and MR imaging: Codman triangle, multi layered and spiculated. Radiologists classification was blinded and independent. The readings of the MR images and CR performed by the same observer had an interval of at least three months between them. We use the readings of three observers to assess the interobserver reliability through the Kappa coefficient. Finally a consensus analysis of readings performed by the three observers for both CR and MR was used to calculate the diagnostic performance of MR in detecting PR and classification of each PR aggressive pattern. The radiologist with greater experience made the consensus score of the presence or absence of each periosteal reaction subtype where there was disagreement among observers. 10 Results The interobserver agreement for the detection of PR was almost perfect for the CR and substantial to almost perfect for the MR. The interobserver agreement for the classification of different patterns of PR was moderate to substantial for the presence of Codman triangle. The evaluation of PR multilammelated standards and spiculated was less consensus among radiologists as well for CR as to MR. The diagnostic and the classification of each aggressive PR with MR imaging and with CR were statistically associated with the diagnosis by CR (p <0.05). MR showed high specificity and high negative predictive value, but moderate sensitivity in detecting periosteal reactions when compared to the CR. Conclusions Our results suggest that detection of PR through MR and CR has high reproducibility. MRI showed high specificity and moderate sensitivity for the identification of PR compared to CR. However, the reproducibility of the classification into different patterns of aggressive PRs was relatively low for both CR and for MR regarding the multilamellated and spiculated PRs.
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Toni, Elisa Cristina de. "Microssatélites (GGAA)n no promotor do gene NR0B1 em pacientes afetados e não afetados pelo sarcoma de Ewing." Pontifícia Universidade Católica do Rio Grande do Sul, 2012. http://hdl.handle.net/10923/1410.
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Previous issue date: 2012Ewing´s sarcoma is a highly aggressive tumor of bone and soft tissues. It affects mainly children and young adults. In about 88% to 95% there is the occurrence of a translocation between the EWS gene (22q12 locus) and two members of ETS transcription factors family: FLI1 (11q24 locus) or ERG (21q22). The most common translocation involves gene EWS and FLI1. This translocation leads to the formation of EWS/FLI1chimeric aberrant transcription factor. The EWS/FLI1 regulates the NR0B1 gene promoter through a direct binding to GGAA microsatellites sequences. Our objective was to identify and describe the molecular structure of GGAA motifs in NR0B1 promoter in unrelated Ewing's Sarcoma patients and healthy subjects from South Brazilian population. Were identified 21 different alleles in the 224 subjects. All alleles had at least 4 to 5 consecutive GGAA motifs. The 24. 2, corresponding to (GGAA)7A(GGAA)7A(GGAA)10 sequence, was the most frequent in our population, being present in 50. 4% of subjects. Allele 24. 2 could be associated to Ewing's sarcoma development since it was significantly more frequent among patients. Differences in the global configuration of NR0B1 promoter GGAA microsatellites (size, sequence, amount and position of GGAA repeats and single 'A' base insertions) could result in differences in Ewing's sarcoma susceptibility. These results would provide insights for the understanding of tumorigenesis.
O sarcoma de Ewing é um tumor altamente agressivo que afeta ossos e tecidos moles. Ele acomete principalmente crianças e adultos jovens. Em torno de 88% a 95% dos casos verifica-se a ocorrência de uma translocação entre o gene EWS (lócus 22q12) e dois membros da família do fator de transcrição ETS: o FLI1 (11q24) ou o ERG (21q22). A translocação mais frequente envolve os genes EWS e FLI1. Esta translocação leva à formação do fator de transcrição quimérico aberrante EWS/FLI1. O fator EWS/FLI1 regula o promotor do gene NR0B1 através de uma ligação direta a sequências de microssatélites GGAA. Nosso objetivo foi identificar e descrever a estrutura molecular de motivos GGAA no promotor de NR0B1 em pacientes com Sarcoma de Ewing não relacionados e não afetados da população sul brasileira. Foram identificados 21 alelos diferentes em 224 indivíduos estudados. Todos os alelos tiveram, pelo menos, de 4 a 5 motivos consecutivos GGAA. O alelo 24. 2, correspondente a sequência (GGAA)7A(GGAA)7A(GGAA)10, foi o mais freqüente em nossa população, estando presente em 50,4% de todos os indivíduos. O alelo 24. 2 pode estar associado ao desenvolvimento do Sarcoma de Ewing, dado que sua frequência foi significativamente mais alta entre afetados. Diferenças na configuração global dos microssatélites GGAA no promotor de NR0B1 (em relação à tamanho, sequência, quantidade e posição das repetições GGAA e inserções de base única ‘A’) podem resultar em diferente susceptibilidade ao sarcoma de Ewing. Tais resultados poderão fornecer subsídios para uma melhor compreensão da tumorigênese.
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Mateo, Lozano Silvia. "Sarcoma de Ewing: nuevas aproximaciones terapéuticas y búsqueda de dianas biológicas del oncogén EWS/FLI-1." Doctoral thesis, Universitat Autònoma de Barcelona, 2007. http://hdl.handle.net/10803/3571.
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La familia de tumores del sarcoma de Ewing (ESFT) incluye un grupo heterogéneo de neoplasias caracterizadas por la presencia de células redondas de pequeño tamaño con mínima evidencia morfológica de diferenciación. ESFT es el segundo tumor óseo más frecuente, afectando fundamentalmente a niños y adolescentes. A pesar del uso de terapias agresivas combinando quimioterapia, radioterapia y cirugía, la supervivencia de pacientes con metástasis es aproximadamente del 30%, mientras que en ausencia de enfermedad metastásica alcanza valores del 70 %. Debido a esto, son necesarias nuevas aproximaciones terapéuticas dirigidas a reducir la morbilidad relacionada con el tratamiento y a mejorar el índice de supervivencia. Afortunadamente, los ESFT presentan una diana molecular perfecta que resulta de la translocación cromosómica t(11;22)(q24;q12), que ocurre en aproximadamente un 95% de los casos, e involucra al gen EWS y a un miembro de la familia de factores de transcripción ETS, fundamentalmente FLI-1 o ERG. La translocación más común genera la formación de la oncoproteína de fusión EWS/FLI-1 que actúa como factor de transcripción y regula de forma aberrante la expresión de genes diana, favoreciendo el proceso tumorigénico. De esta forma la inactivación de la proteína de fusión EWS/FLI-1 se convierte en una estrategia atractiva, no sólo debido su papel fundamental en la tumorigénesis de ESFT, sino también por su especificidad en células transformadas. En este estudio se evaluaron diferentes estrategias dirigidas a reducir los niveles expresión de EWS/FLI-1 in vitro e in vivo. La primera estrategia utilizada para inhibir los niveles de expresión de la proteína de fusión EWS/FLI-1 se basó en el uso de la rapamicina, un antifúngico e inmunosupresor con propiedades anticancerígenas. La rapamicina inhibió la vía de señalización de mTOR/p70s6K y la proliferación de células de ESFT. Estos resultados sugirieron el uso de esta droga como agente citostático en el tratamiento de este tipo de tumores. La segunda aproximación terapéutica se basó en la inhibición simultánea de EWS/FLI-1 a nivel transcripcional y post-transcripcional. El tratamiento combinado de oligonucleótidos antisentido y rapamicina resultó en un incremento en la muerte de células de ESFT, a través de un proceso que involucra la restauración de la vía de señalización pro-apoptótica del TGF?1/TGF?-RII. In vivo, la administración del tratamiento combinado causó un retraso en el crecimiento de los tumores. Estos datos aportan la base para una mayor exploración del potencial del tratamiento combinado como una nueva estrategia en el tratamiento de este tipo de tumores. Los análisis moleculares mostraron que ESFT presentan alteraciones en proteínas reguladoras del ciclo celular, incluyendo la sobreexpresión de proteínas quinasas ciclina-dependientes (CDK2) y la pérdida o baja expresión de sus inhibidores. Basándonos en ésto, la tercera estrategia se basó en la reversión de alguna de estas alteraciones, mediante el uso de la roscovitina, un potente inhibidor de la actividad quinasa de las CDKs. El tratamiento con roscovitina resultó en un incremento de los niveles de apoptosis en células de ESFT in vitro e in vivo, por un mecanismo dependiente de la activación de caspasas. Estos resultados sugieren que la roscovitina puede ser un agente terapéutico efectivo en el tratamiento de ESFT, sola o en combinación con otras drogas potencialmente sinérgicas. Con el objetivo de identificar y evaluar nuevas proteínas que interactúan con EWS/FLI-1 y contribuir de esta forma a la comprensión de los mecanismos de transformación, identificamos como una diana transcripcional directa de EWS/FLI-1 a la caveolina-1, proteína involucrada en gran variedad de procesos celulares, tales como endocitosis, homeostasis del colesterol, transducción de señales y tumorigénesis. Los resultados de este trabajo mostraron que caveolina-1 juega un papel determinante en la tumorigénesis de células de ESFT y su inhibición podría permitir el desarrollo de nuevas estrategias terapéuticas moleculares dirigidas a mejorar el tratamiento de los pacientes de ESFT.
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Heinen, Tiago Elias. "Potencial terepêutico de inibidores de TRK no tratamento de sarcoma de Ewing : um estudo celular e molecular." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2015. http://hdl.handle.net/10183/150631.
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O sarcoma de Ewing (SE) é um dos mais agressivos tipos de câncer pediátrico. Apesar dos significativos avanços no tratamento dessa doença, ainda há uma grande necessidade no aumento das taxas de cura, redução da toxicidade quimioterápica e redução da resistência ao tratamento. Tem sido proposto que SE provém de precursores neuronais, podendo ter sua fisiologia afetada, pois, por neurotrofinas (NTs). Examinamos a influência de receptores de NTs (Trks) em SE. Foram avaliadas a expressão proteica de NTs (NGF e BDNF) e seus receptores (TrkA e TrkB, respectivamente) em amostras de tumores de pacientes com SE, e a expressão de mRNA nas linhagens celulares RD-ES e SK-ES-1. O tratamento das linhagens com o pan-inibidor de Trks (K252a) modificou a morfologia celular e diminuiu a expressão de mRNA de NGF, TrkA, BDNF e TrkB. Ainda, a inibição de Trks diminuiu drasticamente a proliferação e capacidade clonogênica celular. Efeitos sinérgicos foram observados quando as células foram tratadas em conjunto com baixas doses de quimioterápicos, tanto em células selvagens de SE, quanto nas quais induzimos quimiorresistência. Esse estudo sugere, pela primeira vez, que a inibição de Trks reduz a proliferação e sobrevivência celular em SE, além de aumentar a sensibilidade ao tratamento quimioterápico.Ewing's sarcoma (ES) is one of the most aggressive types of pediatric cancer. Despite significant advances in the treatment of this disease, there is still a great need in increasing cure rates, reducing chemotherapy toxicity and treatment resistance. It has been proposed that ES might derive from neuronal precursors and may be influenced, therefore, by neurotrophins (NTs). We have examined the influence of Trk neurotrophin receptors in ES. Protein expression of NTs (NGF and BDNF) and their receptors (TrkA, and TrkB, respectively) was detected in tumor samples from patients with ES, and mRNA expression was analyzed in the RD-ES, SK-ES-1 cell lines. Treating cells with a Trk Pan-inhibitor (K252a) altered cell morphology and decreased the mRNA expression of NGF, TrkA, BDNF, and TrkB. In addition, Trk inhibition dramatically decreased cell proliferation and clonogenic capacity. Synergistic effects were observed when cells were treated in combination with low doses of cytotoxic chemotherapeutics, both in normal ES cells and cells in which chemoresistance was induced. The results suggest for the first time that Trk inhibition can reduce the proliferation and survival of ES cells and sensitize them to cytotoxic chemotherapy.
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Santos, Michel Pinheiro dos. "Avaliação in vitro do potencial terapêutico da associação de quimioterápicos clássicos com butirato sódico e zoledronato em linhagens celulares de Sarcoma de Ewing." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2013. http://hdl.handle.net/10183/87183.
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Introdução: o sarcoma de Ewing é um dos tipo mais agressivos de câncer pediátrico. Esse tipo de câncer é um tumor primitivo neuroectodérmico, grupo que inclui ainda outros tumores pediátricos como o meduloblastoma e o neuroblastoma. Apesar de avanços significativos desde o surgimento da quimioterapia, ainda há necessidade de aumento dos índices de cura, redução da toxicidade da quimioterapia e redução da resistência ao tratamento em pacientes com essa doença. Inibidores da acetilação de histonas (HDACIs ou HDIs) e bifosfonatos têm um futuro promissor no tratamento de câncer, especialmente quando utilizados conjuntamente ou em associação com outros agentes citotóxicos, como antineoplásicos clássicos. No entanto, os efeitos destes tratamentos combinados ainda não haviam sido devidamente estudados em Sarcoma de Ewing. Objetivos: este estudo se propôs a avaliar, in vitro, os efeitos do inibidor da acetilação de histonas, butirato sódico (NaB), do bifosfonato, zoledronato (ZA), da associação destes dois agentes e de combinações dos mesmos com antineoplásicos clássicos sobre a proliferação, viabilidade e sobrevivência celular em sarcoma de Ewing. Métodos: as linhagens celulares de sarcoma de Ewing, SK-ES-1 e RD-ES, foram tratadas com NaB, ZA, doxorrubicina, etoposídeo ou vincristina e com diferentes combinações destes agentes. O crescimento tumoral in vitro, incluindo parâmetros de proliferação e viabilidade celular, foi analisado pelos métodos de contagem celular por exclusão com azul de tripan e MTT. Os efeitos tardios (sobrevivência) também foram estudados através da determinação da formação de colônias (ensaio colonogênico). Resultados: a combinação de NaB e ZA teve um efeito citotóxico sinérgico 72h após o tratamento, persistindo durante 10-14 dias após o tratamento, em ambas as linhagens celulares testadas. Todas as combinações entre NaB ou ZA e os antineoplásicos clássicos testados apresentaram efeitos citotóxicos sinérgicos 72h após os tratamentos em ambas linhagens celulares, com a exceção das seguintes associações: NaB + VCR e ZA + Doxo, que apresentaram apenas efeito aditivo nas células RD-ES, quando comparados com cada um dos agentes em monoterapia. Estes efeitos “agudos” observados em ambas as linhagens celulares de sarcoma de Ewing foram confirmados pelo ensaio clonogênico. Conclusão: os dados obtidos sugerem que o uso combinado de bifosfonatos e HDIs e a associação destes agentes com quimioterápicos clássicos representam promissoras alternativas no tratamento de sarcoma de Ewing e proporcionam a base para novos estudos.Background: Ewing sarcoma, often referred to as Ewing’s sarcoma family tumors, is a peripheral primitive neuroectodermal tumor. Ewing sarcoma is the second most common solid bone and soft tissue malignancy of children and young adults. Despite significant advances in cancer chemotherapy, there is still need for increased rates of cure, reduction of toxicity of chemotherapy and reduced resistance to treatment in patients with this disease. Histone deacetylase inhibitors (HDACIs or HDIs) and bisphosphonates have a promising future in the treatment of cancer as targeted anticancer drugs, especially when used together or in combination with other cytotoxic agents. However, the effects of these combined treatments have not yet been properly evaluated in Ewing sarcoma. Objective: In the present study, we evaluated the in vitro cytotoxic effects (on cellular proliferation, viability, and survival) elicited by the co-treatment of sodium butyrate (NaB) and zoledronic acid (ZA) alone or in combination with three anti-cancer drugs strongly recommended to treat Ewing sarcoma (doxorubicin, etoposide and vincristine) in two human cell lines. Methods: two Ewing sarcoma cell lines, SK-ES-1 and RD-ES, were treated with NaB, ZA, doxorubicin, etoposide, vincristine and with different combinations of these drugs. The proliferation and cell viability were analyzed by counting cell in a hemocytometer, by exclusion of trypan blue and by MTT assay. The survival and proliferation of cells were also studied by clonogenic assay. Results: our results demonstrate that the combination of NaB and ZA has a synergistic cytotoxic effect at 72h after treatment, persisting for 10-14 days post-treatment, in both cell lines tested. All combinations between NaB or ZA and classical antineoplastic drugs demonstrated a synergistic cytotoxic effect at 72h post-treatment in SK-ES-1 and RD-ES cells, with the exception of NaB plus VCR, and ZA plus Doxo, which showed only an additive effect in RD-ES cells when compared to each agent alone. These acute effects observed in both Ewing sarcoma cells were confirmed by the clonogenic assay. Conclusion: These data suggest that HDIs and bisphosphonate co-treatment in combination with classical chemotherapeutic drugs is a promising therapeutic venue the treatment of Ewing sarcoma, and provide a basis for further study in this field.
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Zambelli, Diana <1979>. "Inibizione di IGF1R: analisi comparativa del ruolo terapeutico dell'Insulin-like Growth Factor 1 Receptor nel sarcoma di Ewing." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/993/.
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Ventura, Selena <1982>. "Identificazione di fattori trascrizionali associati al differenziamento neurale in cellule di sarcoma di Ewing: ruolo di NF-kB." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/5253/.
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Il sarcoma di Ewing (ES) è un tumore maligno pediatrico dell’apparato scheletrico; è associato a una traslocazione specifica codificante la proteina di fusione EWS-FLI1 e all’alta espressione di CD99, una glicoproteina di membrana fisiologicamente coinvolta in diversi processi biologici. EWS-FLI1 e CD99, sono riportati avere ruoli divergenti nella modulazione della malignità e del differenziamento di ES.
CD99 inoltre è riportato modulare il pathway di MAPK, il quale interagendo con molteplici fattori di trascrizione partecipa a processi di proliferazione e differenziamento.
In questo studio abbiamo investigato in due linee cellulari di ES silenziate per CD99 (TC-71shCD99 e IOR/CARshCD99) l’attività basale di diversi fattori trascrizionali quali: NF-kBp65, AP1, Elk-1, E2F e CREB. L’unico fattore trascrizionale statisticamente significativo è risultato essere NF-kBp65 e abbiamo valutato il suo ruolo nel differenziamento neurale di cellule di ES e la relazione con EWS-FLI1 e CD99.
L’attività trascrizionale di NF-kB è stata valutata attraverso gene reporter assay in linee cellulari di ES a diversa espressione di CD99, EWS-FLI1 e NF-kB stesso. Il differenziamento neurale è stato valutato come espressione di βIII-Tubulin in immunofluorescenza.
Il silenziamento di CD99 induce una down-modulazione dell’attività trascrizionale di NF-kB, mentre il knockdown di EWS-FLI1 ne induce un’aumento.
Inoltre, il silenziamento di EWS-FLI1 non è in grado di contrastare la riduzione dell’attività di NF-kB osservata dopo silenziamento di CD99, suggerendo un ruolo dominante del CD99 nel signaling di NF-kB. Cellule deprivate di CD99 ma non di EWS-FLI1, mostrano un fenotipo differenziato in senso neurale, fenotipo che viene perso quando le cellule sono indotte a sovraesprimere NF-kB. Inoltre, in cellule CD99 positive, il silenziamento di NF-kB induce un leggero differenziamento neurale.
In conclusione, questi dati hanno evidenziato il ruolo di NF-kB nel differenziamento di cellule di ES e che potrebbe essere un potenziale target nel ridurre la progressione di questo tumore.Ewing sarcoma (ES) is a pediatric high-grade malignant bone tumor, associated with a specific chromosomal translocation encoding the EWS-FLI1 fusion protein and high expression of CD99, a membrane protein physiologically involved in several biological processes. EWS-FLI1 and CD99 were reported to have divergent roles in modulating ES malignancy and neural differentiation. CD99 was also found to modulate MAPK pathway which can cooperate with several transcription factors in proliferation and differentiation processes. In this study we investigated in two different cell lines silenced for CD99 (TC-71shCD99 and IOR/CARshCD99 models) the basal activity of different transcription factors such as: NF-kBp65, AP1, Elk-1, E2F and CREB. The only transcription factor statistically significant was nuclear factor-kappa B (NF-kB) and we investigated its role on neural differentiation in ES cells and its relationship with EWS-FLI1 and CD99. Transcriptional activity of NF-kB was evaluated by gene reporter assay in ES cell lines modified for the expression of CD99, EWS-FLI1 and NF-kB itself. Neural differentiation was detected by evaluating βIII-Tubulin using immunofluorescence microscopy. CD99 silencing was found to decrease NF-kB activity. On the contrary, EWS-FLI1 knockdown increased NF-kB transactivation. EWS-FLI1 silencing was not able to rescue the reduction of NF-kB activity observed after CD99 deprivation, suggesting that CD99 is dominant in NF-kB signaling. Cells deprived of CD99 but not of EWS-FLI1 showed a neural differentiated phenotype, which was lost when cells were induced to overexpress NF-kB. In contrast, in CD99 positive cells, silencing of NF-kB induced a weak neural differentiation. Our findings support the concept that NF-kB activity plays a role in the differentiation of ES cells, identifying NF-kB as a potential target for reducing Ewing tumor progression.
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Orihuela, Ninahuaman Shirley Lidia. "Seguimiento con estudios radiológicos en pacientes con sarcoma de Ewing esquelético tratados entre 2000-2010 en el INEN." Bachelor's thesis, Universidad Nacional Mayor de San Marcos, 2014. https://hdl.handle.net/20.500.12672/13098.
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Objetivo: Establecer la utilidad de los estudios radiológicos en el Seguimiento de pacientes con Sarcoma de Ewing Esquelético tratados durante el 2000-2010 en el INEN.
Metodología: Se realizó un estudio descriptivo, observacional, retrospectivo y transversal. Se calculó la estadística descriptiva y para la asociación de variables la Prueba de la Chi cuadrada y el Odss Ratio (OR).
Resultados: La edad promedio fue 13,9 años, los pacientes fueron del sexo masculino en el 53,8%. Las lesiones se encontraron con mayor frecuencia en los huesos de las extremidades inferiores con 69,2% y 15,4% en los huesos del tórax; el 53,8% de las lesiones tuvieron tamaños entre 5 y 10 cm y el 46,2% restante, mayor de 10 cm. La radiografía se realizó en el 76,9% de los casos y la TAC como estudio inicial fue realizado en el 23,1% de los casos. La infiltración medular se manifestó en el 38,5%; la lesión lítica, la reacción perióstica y la ruptura cortical se manifestaron en todos los casos antes del tratamiento y después del tratamiento, en el 15,4% de los casos; cada una, presentándose una mezcla de ellas. Otra de las características radiológicas fue el compromiso de partes blandas que se manifiesta en el 84,6% de los casos en el pretratamiento, y en el 15,4% de los casos, en el posttratamiento. Para determinar la extensión de la enfermedad antes del tratamiento tenemos a la radiografía de tórax con 100%, tomografía de tórax con 46,2%, la ecografía de abdomen y pelvis con 76,9% y la tomografía de abdomen y pelvis con el 30,8%. Para determinar la extensión de la enfermedad después del tratamiento encontramos a la radiografía de tórax con 92,3%, tomografía de tórax con 69,2%, la tenemos a la radiografía de tórax con el 92,3% , la ecografía de abdomen y pelvis con 61,5% y la tomografía de abdomen y pelvis con el 23,3%. Los tratamientos aplicados fueron quimioterapia, radioterapia y cirugía en el 69,2% y la quimioterapia y radioterapia en el 30,8%. En los casos en los que recibieron quimioterapia, ésta duró 350,9 días como promedio. El tratamiento de quimioterapia, radioterapia y cirugía fue efectivo en el 81,8% y el tratamiento de quimioterapia y radioterapia en el 18,2%. El tamaño de la lesión se redujo en 6,4 cm después del tratamiento, el 95% de las reducciones oscilaron entre 3,7 a 9 cm. Sólo en un caso (7,7%) persistió la enfermedad en os pacientes con Sarcoma de Ewing esquelético; en los 12 casos restantes no hubo recurrencia. El único caso de recurrencia recibió el tratamiento de quimioterapia y radioterapia. Con el tratamiento de quimioterapia, radioterapia y cirugía no hubo recurrencias.
Conclusiones: En el 15,4% de los casos, en el posttratamiento se presentó una combinación de las características radiológicas como lesión lítica, reacción perióstica,ruptura cortical y compromiso de partes blandas. La radiografía y tomografía fueron útiles en la determinación de la extensión de la enfermedad.Trabajo académico
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Alhaddad, Anna. "Etude de la vectorisation des siRNA par les nanoparticules de diamant photoluminescentes dans un modèle cellulaire de sarcome d’Ewing. Investigation de leur trafic cellulaire grâce à leurs propriétés optiques." Thesis, Paris 11, 2012. http://www.theses.fr/2012PA114811.
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Les siRNA se sont des agents actifs et spécifiques couramment utilisés en thérapie génique. L’intérêt d’utiliser des nanoparticules en tant que vecteurs des siRNA est leur capacité à protéger ces derniers de la dégradation par les nucléases et également de leur permettre d’être délivrés au niveau de leur cible thérapeutique. Afin d’appuyer cette théorie, ce travail s’est concentré sur un modèle cellulaire de sarcome d’Ewing, dans le but de mettre au point un nouveau système galénique pour le transport de siRNA formé des nanoparticules bifonctionnelles de diamants fluorescentes recouvertes par des polymères cationiques. Ces nano-vecteurs sont capables d’induire efficacement l’inhibition de l’expression de l’oncogène EWS-Fli1 dans les cellules en culture s’ils transportent des siRNA dirigés contre ce gène. Par ailleurs, les nanodiamants, grâce à leurs propriétés de fluorescence stable et intense, ont constitué des outils de détection permettant de suivre leurs voies de pénétration, leur biodistribution cellulaire, ainsi que la cinétique de libération des siRNA dans le cytoplasme. Enfin, un modèle de nanodiamants fonctionnalisés par le polyéthylenimine a été choisi pour la poursuite des travaux biologiques en raison de son efficacité de vectorisationSiRNA are powerful and commonly used agent for the specific inhibition of gene expression. They need to be vectorized by nanoparticles to facilitate cell penetration and their protection from degradation in biological media. At first, cationic nanodiamonds coated with cationic polymers were developed and were able to adsorb siRNA on their surface. Using antisense siRNA against the oncogene EWS-Fli1, nanodiamonds allowed to efficiently induce the inhibition of expression of the oncogene EWS-FLI1 in cultured Ewing sarcoma cells. As a second goal of this study, the fluorescence of red color center created in the nanodiamonds was used to follow their pathways, their cellular biodistribution and the kinetics of release of siRNA into the cytoplasm. In conclusion, nanodiamonds functionalized by polyethylenimine showed a better transfection efficiency and were chosen for further biological studies
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Claveau, Sandra. "Fluorescent nanodiamonds as siRNA vectors : in vitro efficacy evaluation and high-content/high-resolution quantifications of their distribution in vivo." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS119/document.
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Le Sarcome d'Ewing est un cancer pédiatrique rare, principalement dû à l'expression de l'oncogène de jonction EWS-Fli1, et dont les traitements médicamenteux ont peu évolué au cours des dernières décennies. Nous nous intéressons à une nouvelle approche thérapeutique utilisant des siRNA, ciblant spécifiquement l'oncogène EWS-Fli1, et permettant l'inhibition de la croissance tumorale. Durant mon travail de thèse, j'ai utilisé des nanocristaux de diamant issus soit de détonation (DND), soit de synthèse haute pression-haute température (NDHPHT) pour vectoriser les siRNA, accrochés par interaction électrostatique. Pour ce faire, les NDs ont été rendus cationiques par différentes méthodes: (i) hydrogénation assistée par plasma, (ii) par recuit thermique, ou (iii) par traitement chimique pour les DNDs, ou (iv) greffage covalent d'un polymère cationique sur des NDHPHT (COP-NDHPHT).Mes travaux ont comporté deux axes: (i) étude in vitro des complexes ND:siRNA (caractérisations physico-chimiques des NDs et étude de l'efficacité d'inhibition de l'oncogène par les complexes); (ii) distribution tissulaire de COP-NDHPHT, injectés dans des souris, grâce à des NDHPHT fluorescents, contenant des défauts azote-lacune. Pour les détecter individuellement dans des coupes d'organes de souris portant une tumeur xénogreffée sous-cutanée, nous avons développé un système d'imagerie en épifluorescence à grande ouverture numérique, et résolu en temps afin de rejeter l'autofluorescence tissulaire (de durée de vie plus courte que celle des NDs). Nous avons quantifié le nombre, l'état d'agrégation et la localisation cellulaire de ces vecteurs (grâce à un marquage histopathologique imagé simultanément) 24h après injection. Les NDs ont été clairement détectés dans les différents organes, dont la tumeur, ouvrant la voie à un contrôle de la progression tumorale grâce au siRNAEwing Sarcoma is a rare pediatric cancer, caused in the majority of the cases by the expression of the fusion oncogene EWS-Fli1. Current treatments have not much evolved over the past decades. We are investigating a new therapy based on siRNA specifically targeting the oncogene and inhibiting the tumor growth. During my PhD thesis, I have tested different types of synthetic nanodiamonds (ND) used to vectorize siRNA electrostatically bound at their surface: ND produced by detonation (DND) or by High Pressure-High Temperature synthesis (NDHPTH). Their surfaces have been cationized by various processes: (i) plasma or (ii) thermal hydrogenation, (ii) chemical treatment, or (iv) covalent grafting of a copolymer (COP-NDHPHT).My PhD work included two main axis: (i) in vitro study of ND:siRNA complexes (NDs physico-chemical characterization and oncogene inhibition efficacy by the complexes); (ii) tissue distribution of COP-NDHPHT, injected into mice, using fluorescent NDHPHT containing nitrogen-vacancy defects. To detect them individually in sections of mouse organs carrying a subcutaneous xenograft tumor, we developed an epifluorescence imaging system with large numerical aperture and resolved in time to reject tissue autofluorescence (of a shorter lifetime than NDs). We quantified the number, the aggregation state and the cell localization (thanks to simultaneous histopathological imaging) of these vectors 24 hours after injection. NDs have been clearly detected in different organs, including the tumor, paving the way for tumor progression control with siRNA
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Silva, Déborah Soares Bispo Santos. "Haplótipos de diferentes SNPs no interior do gene EWS em indivíduos afetados e não-afetados pelo sarcoma de Ewing." Pontifícia Universidade Católica do Rio Grande do Sul, 2012. http://hdl.handle.net/10923/1305.
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Previous issue date: 2012Ewing’s sarcoma was first described by James Ewing in 1921 and it is the second most common bone tumor in children and young adults. Both chromosomal breakage and translocation occur in this sarcoma. The EWS gene is localized in chromosome 22 and is involved in this translocation. However, little is known about this gene breaking region and what sequences could be involved in higher chromosomal break susceptibility. In this study we aimed to investigate three SNPs in the EWS gene breaking region in a healthy subjects’ population and in Ewing’s sarcoma patients. Genotyping was performed by TaqMan® assay for allelic discrimination using Real-Time PCR System. We conducted analysis of allelic and genotypic frequencies, as well as association and transmission disequilibrium tests. According to our results, the control group showed similar and different genotypes distribution of all SNPs when compared to other populations studied by different projects, which shows how important it is to know the frequencies of our population. To test the hypothesis that some SNP, SNParrangement or haplotype could influence in the susceptibility to develop Ewing’s sarcoma, we compared affected with non-affected individuals using association studies. The results showed one significant difference: a higher presence of homozygote T-rs4820804 in Ewing’s Sarcoma patients. Transmission Disequilibrium Test (TDT) was performed to compare data from Ewing’s Sarcoma patients and from their families but no statistically significant result was found. In conclusion, we find that the TT-rs4820804 EWS genotype can be associate with Ewing’s sarcoma and that the rs4820804 SNP can be a candidate to understand the EWS breakage susceptibility.
O sarcoma de Ewing foi primeiramente descrito por James Ewing em 1921 e é o segundo tumor ósseo mais frequente em crianças, adolescente e adultos jovens. Neste sarcoma, é comum ocorrer a quebra e a translocação cromossômica. Dentre os genes envolvidos nesta translocação está o gene EWS, localizado no cromossomo 22. Entretanto, pouco se sabe a respeito da região de quebra deste gene e quais sequências poderiam levar a uma maior susceptibilidade a quebra cromossômica. Sendo assim, o objetivo deste trabalho foi investigar três polimorfismos de base única (SNPs) presentes na região de quebra do gene EWS, em uma população de indivíduos saudáveis e em pacientes afetados pelo Sarcoma de Ewing. A genotipagem para os SNPs selecionados foi realizada usando TaqMan SNP Genotyping Assay pelo sistema de PCR em tempo real. Nós realizamos análises de frequências alélicas e genotípicas, assim como um estudo de associação e de desequilíbrio de transmissão.A comparação das frequências alélicas e genotípicas entre as populações deste estudo e entre populações de projetos já publicados mostrou particularidades entre as populações, revelando a importância de se conhecer tais frequências na população de estudo. Para testar a hipótese de que algum SNP, haplótipo ou combinação específica de SNPs poderia influenciar na susceptibilidade ao Sarcoma de Ewing, comparamos afetados com não-afetados realizando estudos de associação cujos resultados mostraram uma única diferença significativa: a maior incidência no genótipo TT-rs4820804 entre os afetados pelo Sarcoma de Ewing. O Teste de Desequilíbrio de Transmissão (TDT) comparou os dados dos pacientes afetados e os dados de seus familiares, mas nenhum resultado significativo foi encontrado. Em conclusão, o genótipo TT-rs4820804 pode estar associado ao Sarcoma de Ewing e o SNP rs4820804 pode ser candidato para auxílio do entendimento da susceptibilidade de quebra do gene EWS.
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Sawitzki, Fernanda Rosa. "Haplótipos de diferentes SNPs no interior do gene FLI1 em indivíduos afetados e não-afetados pelo sarcoma de Ewing." Pontifícia Universidade Católica do Rio Grande do Sul, 2012. http://hdl.handle.net/10923/1313.
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Previous issue date: 2012In this study the SNPs rs640098, rs491714, rs611307 into the FLI1 gene were genotyped in a sample of 201 subjects from southern Brazilian population, in 24 Ewing’s sarcoma patients (geographically matched with control group) and 54 of their family members, including parents and siblings. We performed association studies comparing genotypic frequencies of rs640098, rs491714, rs611307 into the FLI1 gene, and all possible genotype combinations between Ewing’s Sarcoma patients and control group. Of the three SNPs investigated individually, only one of them showed a significant result when compared to the control group; our non-combined analysis revealed a significantly higher presence of homozygote A-rs497714 among Ewing’s Sarcoma patients (p=0. 0065; Chi square Test). In all other tested clusters, we always noticed a higher rate of homozygote A-rs497714 among Ewing’s Sarcoma patients independent of the other SNP-arrangements and/or haplotype combinations. In addition, we performed transmission disequilibrium tests comparing data from Ewing’s Sarcoma patients and from their families (parents and siblings), but no statistically significant result was found. In conclusion, the present study provides evidence statistically founded that the AA-rs497714 FLI1 genotype can associated with Ewing's sarcoma. And that this polymorphism can be clinically useful as a potential genetic marker to the prognostic of risk to develop this cancer or to provide insights into FLI1 chromosome breakage context of tumorigenesis.
Neste estudo, os SNPs rs640098, rs491714, rs611307 no gene FLI1 foram genotipados em uma amostra de 201 indivíduos da população do sul do Brasil, e em 24 pacientes portadores de Sarcoma Ewing (geograficamente comparado com grupo controle) e 54 de seus familiares, incluindo pais e irmãos. Realizamos estudos de associação, comparando as freqüências genotípicas do rs640098 e rs491714 e rs611307 no gene FLI1, e todas as combinações possíveis entre o genótipo de pacientes Sarcoma de Ewing e grupo controle. Dos três SNPs investigados individualmente, apenas um deles apresentou um resultado significativo quando comparado com o grupo controle; nossa análise não-combinada revelou uma presença significativamente maior de homozigoto A-rs497714 entre os pacientes de Sarcoma Ewing (p = 0,0065; Chi quadrado). Em todos os outros grupos testados, foi notada uma maior taxa de homozigoto A-rs497714 entre os pacientes com Sarcoma de Ewing, independentemente dos outros arranjos e / ou combinações de SNP e haplótipos. Além disso, foram realizados testes de desequilíbrio de transmissão, comparando dados de pacientes portadores de Sarcoma de Ewing e de suas famílias (pais e irmãos), mas nenhum resultado estatisticamente significativo foi encontrado. Em conclusão, o presente estudo fornece evidências estatisticamente fundada de que o genótipo AA-rs497714 FLI1 pode associado ao sarcoma de Ewing. E que este polimorfismo pode ser clinicamente útil como um potencial marcador genético para o prognóstico de risco para desenvolver este câncer ou para fornecer insights no contexto de quebras cromossômicas de tumorigênese no gene FLI1.
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Toni, Elisa Cristina de. "Microssat?lites (GGAA)n no promotor do gene NR0B1 em pacientes afetados e n?o afetados pelo sarcoma de Ewing." Pontif?cia Universidade Cat?lica do Rio Grande do Sul, 2012. http://tede2.pucrs.br/tede2/handle/tede/5436.
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Previous issue date: 2012-03-30Ewing?s sarcoma is a highly aggressive tumor of bone and soft tissues. It affects mainly children and young adults. In about 88% to 95% there is the occurrence of a translocation between the EWS gene (22q12 locus) and two members of ETS transcription factors family: FLI1 (11q24 locus) or ERG (21q22). The most common translocation involves gene EWS and FLI1. This translocation leads to the formation of EWS/FLI1chimeric aberrant transcription factor. The EWS/FLI1 regulates the NR0B1 gene promoter through a direct binding to GGAA microsatellites sequences. Our objective was to identify and describe the molecular structure of GGAA motifs in NR0B1 promoter in unrelated Ewing's Sarcoma patients and healthy subjects from South Brazilian population. Were identified 21 different alleles in the 224 subjects. All alleles had at least 4 to 5 consecutive GGAA motifs. The 24.2, corresponding to (GGAA)7A(GGAA)7A(GGAA)10 sequence, was the most frequent in our population, being present in 50.4% of subjects. Allele 24.2 could be associated to Ewing's sarcoma development since it was significantly more frequent among patients. Differences in the global configuration of NR0B1 promoter GGAA microsatellites (size, sequence, amount and position of GGAA repeats and single 'A' base insertions) could result in differences in Ewing's sarcoma susceptibility. These results would provide insights for the understanding of tumorigenesis.
O sarcoma de Ewing ? um tumor altamente agressivo que afeta ossos e tecidos moles. Ele acomete principalmente crian?as e adultos jovens. Em torno de 88% a 95% dos casos verifica-se a ocorr?ncia de uma transloca??o entre o gene EWS (l?cus 22q12) e dois membros da fam?lia do fator de transcri??o ETS: o FLI1 (11q24) ou o ERG (21q22). A transloca??o mais frequente envolve os genes EWS e FLI1. Esta transloca??o leva ? forma??o do fator de transcri??o quim?rico aberrante EWS/FLI1. O fator EWS/FLI1 regula o promotor do gene NR0B1 atrav?s de uma liga??o direta a sequ?ncias de microssat?lites GGAA. Nosso objetivo foi identificar e descrever a estrutura molecular de motivos GGAA no promotor de NR0B1 em pacientes com Sarcoma de Ewing n?o relacionados e n?o afetados da popula??o sul brasileira. Foram identificados 21 alelos diferentes em 224 indiv?duos estudados. Todos os alelos tiveram, pelo menos, de 4 a 5 motivos consecutivos GGAA. O alelo 24.2, correspondente a sequ?ncia (GGAA)7A(GGAA)7A(GGAA)10, foi o mais freq?ente em nossa popula??o, estando presente em 50,4% de todos os indiv?duos. O alelo 24.2 pode estar associado ao desenvolvimento do Sarcoma de Ewing, dado que sua frequ?ncia foi significativamente mais alta entre afetados. Diferen?as na configura??o global dos microssat?lites GGAA no promotor de NR0B1 (em rela??o ? tamanho, sequ?ncia, quantidade e posi??o das repeti??es GGAA e inser??es de base ?nica A ) podem resultar em diferente susceptibilidade ao sarcoma de Ewing. Tais resultados poder?o fornecer subs?dios para uma melhor compreens?o da tumorig?nese.
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Roth, Laura [Verfasser], Stefan [Akademischer Betreuer] Burdach, Uta [Gutachter] Behrends, and Stefan [Gutachter] Burdach. "Contribution of HOXD10, HOXD11 and HOXD13 to malignancy of Ewing sarcoma / Laura Roth ; Gutachter: Uta Behrends, Stefan Burdach ; Betreuer: Stefan Burdach." München : Universitätsbibliothek der TU München, 2020. http://d-nb.info/1205879846/34.
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Keller, Juliane [Verfasser], and Torsten [Akademischer Betreuer] Kluba. "Inhibition of the Hedgehog pathway in combination with cytostatics as potential therapeutic option in Ewing Sarcoma / Juliane Keller ; Betreuer: Torsten Kluba." Tübingen : Universitätsbibliothek Tübingen, 2020. http://d-nb.info/1204422354/34.
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Marchetto, Aruna [Verfasser], and Thomas [Akademischer Betreuer] Grünewald. "Deciphering the role of the developmental transcription factor SOX6 in tumorigenesis and progression of Ewing sarcoma / Aruna Marchetto ; Betreuer: Thomas Grünewald." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2020. http://d-nb.info/121236287X/34.
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Ertl, Miriam Verena Manuela [Verfasser], Stephanie E. [Akademischer Betreuer] Combs, Stephanie E. [Gutachter] Combs, and Stefan [Gutachter] Burdach. "HOX genes in pathogenesis of Ewing sarcoma / Miriam Verena Manuela Ertl ; Gutachter: Stephanie E. Combs, Stefan Burdach ; Betreuer: Stephanie E. Combs." München : Universitätsbibliothek der TU München, 2018. http://d-nb.info/1170321402/34.
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Terracciano, Mario <1983>. "Cell death mechanisms triggered by monoclonal antibodies against CD99 in Ewing sarcoma: Cross-talk between MDM2, IGF-1R and Ras/MAPK." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6560/.
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CD99 is a 32 kDa transmembrane protein whose high expression characterizes Ewing sarcoma (ES), a very aggressive pediatric bone tumor. In addition to its diagnostic value, CD99 has therapeutic potential since it leads to rapid and massive ES cell death when engaged with specific antibodies. Here a novel mechanism of cell death triggered via CD99 is shown, leading, ultimately, to the appearance of macropinocytotic vescicles. Anti-CD99 mAb 0662 induces MDM2 ubiquitination and degradation, which causes not only a p53 reactivation but also the IGF-1R induction and its subsequent internalization; CD99 results internalized together with IGF-1R inside endosomes, but then the two molecules display a different sorting: CD99 is degraded, while IGF-1R is recycled on the surface, causing, as a final step, the up-regulation of RAS-MAPK. High-expressing CD99 mesenchymal stem cells show mild Ras induction but no p53 activation and escape cell death, but in presence of EWS/FLI1 mesenchymal stem cells expressing CD99 show a stronger Ras induction and a p53 reactivation, leading to a significant cell death rate. We propose that CD99 triggering in a EWS/FLI1-driven oncogenetic context creates a synergy between RAS upregulation and p53 activation in ES cells, leading to cell death. Moreover, our data rule out possible concerns on toxicity related to the broad CD99 expression in normal tissues and provide the rationale for the therapeutic use of anti-CD99 MAbs in the clinic.
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Ertl, Miriam Verena Manuela Verfasser], Stephanie [Akademischer Betreuer] [Combs, Stephanie E. [Gutachter] Combs, and Stefan [Gutachter] Burdach. "HOX genes in pathogenesis of Ewing sarcoma / Miriam Verena Manuela Ertl ; Gutachter: Stephanie E. Combs, Stefan Burdach ; Betreuer: Stephanie E. Combs." München : Universitätsbibliothek der TU München, 2018. http://nbn-resolving.de/urn:nbn:de:bvb:91-diss-20180829-1380365-1-1.
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Silva, D?borah Soares Bispo Santos. "Hapl?tipos de diferentes SNPs no interior do gene EWS em indiv?duos afetados e n?o-afetados pelo sarcoma de Ewing." Pontif?cia Universidade Cat?lica do Rio Grande do Sul, 2012. http://tede2.pucrs.br/tede2/handle/tede/5433.
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Previous issue date: 2012-03-30Ewing s sarcoma was first described by James Ewing in 1921 and it is the second most common bone tumor in children and young adults. Both chromosomal breakage and translocation occur in this sarcoma. The EWS gene is localized in chromosome 22 and is involved in this translocation. However, little is known about this gene breaking region and what sequences could be involved in higher chromosomal break susceptibility. In this study we aimed to investigate three SNPs in the EWS gene breaking region in a healthy subjects population and in Ewing s sarcoma patients. Genotyping was performed by TaqMan? assay for allelic discrimination using Real-Time PCR System. We conducted analysis of allelic and genotypic frequencies, as well as association and transmission disequilibrium tests. According to our results, the control group showed similar and different genotypes distribution of all SNPs when compared to other populations studied by different projects, which shows how important it is to know the frequencies of our population. To test the hypothesis that some SNP, SNParrangement or haplotype could influence in the susceptibility to develop Ewing s sarcoma, we compared affected with non-affected individuals using association studies. The results showed one significant difference: a higher presence of homozygote T-rs4820804 in Ewing s Sarcoma patients. Transmission Disequilibrium Test (TDT) was performed to compare data from Ewing s Sarcoma patients and from their families but no statistically significant result was found. In conclusion, we find that the TT-rs4820804 EWS genotype can be associate with Ewing s sarcoma and that the rs4820804 SNP can be a candidate to understand the EWS breakage susceptibility.
O sarcoma de Ewing foi primeiramente descrito por James Ewing em 1921 e ? o segundo tumor ?sseo mais frequente em crian?as, adolescente e adultos jovens. Neste sarcoma, ? comum ocorrer a quebra e a transloca??o cromoss?mica. Dentre os genes envolvidos nesta transloca??o est? o gene EWS, localizado no cromossomo 22. Entretanto, pouco se sabe a respeito da regi?o de quebra deste gene e quais sequ?ncias poderiam levar a uma maior susceptibilidade a quebra cromoss?mica. Sendo assim, o objetivo deste trabalho foi investigar tr?s polimorfismos de base ?nica (SNPs) presentes na regi?o de quebra do gene EWS, em uma popula??o de indiv?duos saud?veis e em pacientes afetados pelo Sarcoma de Ewing. A genotipagem para os SNPs selecionados foi realizada usando TaqMan SNP Genotyping Assay pelo sistema de PCR em tempo real. N?s realizamos an?lises de frequ?ncias al?licas e genot?picas, assim como um estudo de associa??o e de desequil?brio de transmiss?o. A compara??o das frequ?ncias al?licas e genot?picas entre as popula??es deste estudo e entre popula??es de projetos j? publicados mostrou particularidades entre as popula??es, revelando a import?ncia de se conhecer tais frequ?ncias na popula??o de estudo. Para testar a hip?tese de que algum SNP, hapl?tipo ou combina??o espec?fica de SNPs poderia influenciar na susceptibilidade ao Sarcoma de Ewing, comparamos afetados com n?o-afetados realizando estudos de associa??o cujos resultados mostraram uma ?nica diferen?a significativa: a maior incid?ncia no gen?tipo TT-rs4820804 entre os afetados pelo Sarcoma de Ewing. O Teste de Desequil?brio de Transmiss?o (TDT) comparou os dados dos pacientes afetados e os dados de seus familiares, mas nenhum resultado significativo foi encontrado. Em conclus?o, o gen?tipo TT-rs4820804 pode estar associado ao Sarcoma de Ewing e o SNP rs4820804 pode ser candidato para aux?lio do entendimento da susceptibilidade de quebra do gene EWS.
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Sawitzki, Fernanda Rosa. "Hapl?tipos de diferentes SNPs no interior do gene FLI1 em indiv?duos afetados e n?o-afetados pelo sarcoma de Ewing." Pontif?cia Universidade Cat?lica do Rio Grande do Sul, 2012. http://tede2.pucrs.br/tede2/handle/tede/5435.
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Previous issue date: 2012-03-30In this study the SNPs rs640098, rs491714, rs611307 into the FLI1 gene were genotyped in a sample of 201 subjects from southern Brazilian population, in 24 Ewing s sarcoma patients (geographically matched with control group) and 54 of their family members, including parents and siblings. We performed association studies comparing genotypic frequencies of rs640098, rs491714, rs611307 into the FLI1 gene, and all possible genotype combinations between Ewing s Sarcoma patients and control group. Of the three SNPs investigated individually, only one of them showed a significant result when compared to the control group; our non-combined analysis revealed a significantly higher presence of homozygote A-rs497714 among Ewing s Sarcoma patients (p=0.0065; Chi square Test). In all other tested clusters, we always noticed a higher rate of homozygote A-rs497714 among Ewing s Sarcoma patients independent of the other SNP-arrangements and/or haplotype combinations. In addition, we performed transmission disequilibrium tests comparing data from Ewing s Sarcoma patients and from their families (parents and siblings), but no statistically significant result was found. In conclusion, the present study provides evidence statistically founded that the AA-rs497714 FLI1 genotype can associated with Ewing's sarcoma. And that this polymorphism can be clinically useful as a potential genetic marker to the prognostic of risk to develop this cancer or to provide insights into FLI1 chromosome breakage context of tumorigenesis.
Neste estudo, os SNPs rs640098, rs491714, rs611307 no gene FLI1 foram genotipados em uma amostra de 201 indiv?duos da popula??o do sul do Brasil, e em 24 pacientes portadores de Sarcoma Ewing (geograficamente comparado com grupo controle) e 54 de seus familiares, incluindo pais e irm?os. Realizamos estudos de associa??o, comparando as freq??ncias genot?picas do rs640098 e rs491714 e rs611307 no gene FLI1, e todas as combina??es poss?veis entre o gen?tipo de pacientes Sarcoma de Ewing e grupo controle. Dos tr?s SNPs investigados individualmente, apenas um deles apresentou um resultado significativo quando comparado com o grupo controle; nossa an?lise n?o-combinada revelou uma presen?a significativamente maior de homozigoto A-rs497714 entre os pacientes de Sarcoma Ewing (p = 0,0065; Chi quadrado). Em todos os outros grupos testados, foi notada uma maior taxa de homozigoto A-rs497714 entre os pacientes com Sarcoma de Ewing, independentemente dos outros arranjos e / ou combina??es de SNP e hapl?tipos. Al?m disso, foram realizados testes de desequil?brio de transmiss?o, comparando dados de pacientes portadores de Sarcoma de Ewing e de suas fam?lias (pais e irm?os), mas nenhum resultado estatisticamente significativo foi encontrado. Em conclus?o, o presente estudo fornece evid?ncias estatisticamente fundada de que o gen?tipo AA-rs497714 FLI1 pode associado ao sarcoma de Ewing. E que este polimorfismo pode ser clinicamente ?til como um potencial marcador gen?tico para o progn?stico de risco para desenvolver este c?ncer ou para fornecer insights no contexto de quebras cromoss?micas de tumorig?nese no gene FLI1.
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Eckenrode, Joseph Michael. "DEVELOPMENT OF MITHRAMYCIN ANALOGUES WITH IMPROVED EFFICACY AND REDUCED TOXICITY FOR TREATMENT OF ETS-DEPENDENT TUMORS IN EWING SARCOMA AND PROSTATE CANCER." UKnowledge, 2019. https://uknowledge.uky.edu/pharmacy_etds/107.
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Introduction: Genetic rearrangements in Ewing sarcoma, prostate, and leukemia cells result in activation of oncogenic ETS transcription factor fusions. Mithramycin (MTM) has been identified as an inhibitor of EWS-FLI1 transcription factor, a gene fusion product responsible for oncogenesis in Ewing sarcoma. Despite preclinical success, a phase I/II clinical trial testing MTM therapy in refractory Ewing sarcoma was terminated. Liver and blood toxicities resulted in dose de-escalation and sub-therapeutic exposures. However, the promise of selectively targeting oncogenic ETS transcription factors like EWS-FLI1 prompted us to undertake the discovery of more selective, less toxic analogues of MTM. MTM is a potent inhibitor of ubiquitous SP1 transcription factor, likely inducing non-specific toxicity. In collaboration with two medicinal chemistry groups, two semi-synthetic efforts were implemented to develop novel analogues of MTM. The first effort utilized the biosynthetic product mithramycin SA (MTMSA) to modify C3-side chain. The second effort utilized an oxime linker directly formed on MTM’s C3-side chain (MTM-oxime; MTMox). Here I present the pharmacological assessment of over 75 novel MTM analogues towards selectively targeting oncogenic ETS transcription factors, like EWS-FLI1, over ubiquitous transcription factors, like SP1.
Methods: Novel MTM analogues were evaluated for selective cytotoxicity against ETS fusion-dependent cell lines. Selectively cytotoxic analogues were evaluated for inhibitory effects on several gene promoters in TC-32 reporter cell lines, a Ewing sarcoma cell line dependent on EWS-FLI1, transfected with luciferase reporter vector. Cloned reporter vectors incorporated NR0B1 (EWS-FLI1 binding), β-actin (SP1 binding) and CMV (non-specific) gene promoters. Furthermore, gene (mRNA) and protein expression changes of EWS-FLI1 and SP1, as well as regulated target genes, namely NR0B1, VEGFA and BCL-2 were evaluated with MTM analogue treatments. The MTM analogues with most selective activity in vitro were administered to mice by intravenous bolus dose for pharmacokinetic analysis. The MTM analogues with highest systemic exposure from each semi-synthetic effort, namely MTMSA-Trp-A10 and MTMox-24, were further evaluated. Metabolic stabilities in whole blood, plasma, and tumor cell matrices, and across multiple species were compared with MTM. Moreover, intrinsic hepatic clearances were estimated using mouse liver microsomes. Tumor and liver distributions were estimated in tumor bearing mice. Additionally, the effect of organic anionic transporter polypeptides (OATP) on distribution of MTM was investigated. Maximum tolerated doses were evaluated for lead MTM analogues, having both selective activities in vitro and high systemic exposure, compared to MTM. Complete blood cell counts and plasma alanine aminotransferase activity were measured to evaluate dose-dependent blood and liver toxicities, respectively. ETS fusion-dependent and non-dependent cell lines were implanted subcutaneously into immunocompromised mice for efficacy studies. Average tumor volumes and survival were tracked for mice receiving treatment, compared to MTM and vehicle treatment.
Results: Evaluation of MTM analogues from both semi-synthetic efforts revealed that conjugation of MTM C3-side chain with tryptophan (Trp) and/or phenylalanine (Phe) improved selective cytotoxicity against ETS fusion-dependent cell lines. This was highlighted by MTMSA-Trp-A2 (also refer to as MTMSA-Phe-Trp) and MTMSA-Trp-A10 (also refer to as MTMSA-Trp-Trp), with selective indices of 19.1 and 15.6, respectively, compared to MTM (1.5). Similarly, MTMox-23 (also refer to as MTMox-Phe-Trp) and MTMox-20 (also refer to as MTMox-Trp) had selectivity indices of 4.6 and 4.5, respectively. These selectively cytotoxic MTM analogues inhibited EWS-FLI1-mediated transcription 10-fold more effectively than both non-specific CMV-mediated and SP1-mediated (via β-actin promoter) transcription in TC-32 reporter cell lines. Moreover, gene (mRNA) and protein expression of EWS-FLI1 and regulated gene, NR0B1, were inhibited with MTM analogue treatment (GI50, 6-hour) in TC-32 cells. Similarly, SP1 and target genes, VEGFA and BCL-2, gene (mRNA) and protein expressions were also inhibited with MTM analogue treatment (GI50, 6-hour) in TC-32 cells.
Conjugation of Trp and/or Phe to C3-side chain of MTM increased systemic exposure in vivo. Most impressively, the addition of two Trp residues, namely MTMSA-Trp-A10 and MTMox-24 (also refer to as MTMox-Trp-Trp), resulted in systemic exposure increases of 218- and 42-fold, respectively, after intravenous (IV) bolus dose. Metabolically, tryptophan/phenylalanine conjugated MTM analogues are liable to esterase activity on carboxy-methyl functional group. Very rapid de-methylation in biological matrix was observed with MTMox-24, compared to MTMSA-Trp-A10, suggesting a regiospecific effect. However, esterase activity was limited to rodent matrices and demethylation occurred at significantly diminished rates in non-human primate and human plasma. MTM analogues were not susceptible to p450-mediated metabolism, with negligible loss in mouse liver microsome assay compared to verapamil control. MTM (1mg/kg) and MTMox-24 (6mg/kg) were detected in subcutaneously implanted (flank) LL2 tumors and liver homogenates after IV bolus dose. Interestingly, MTMSA-Trp-A10 (2mg/kg) was not. Despite a 3-fold increase in systemic exposure with rifampin oral pretreatment, an OATP inhibitor, exposure of MTM was unaffected in Oatp knockout mouse model. Exposure of MTM in liver tissue was 8.4-fold higher compared to tumor tissue with low tissue clearance. This agrees with the lack of metabolism observed in liver microsomes and may provide a mechanism for clinically observed liver toxicity.
MTMSATrp-A10 had a single maximum tolerated dose (MTD) of 0.75mg/kg, compared to 1mg/kg for MTM, administered by IV bolus. In contrast, MTM-oxime analogues (MTMox-20, -23, -24 and -25) had single maximum tolerated doses of 20 – 25mg/kg. These increased tolerances are the result of additive differences in whole blood stability, cytotoxicity and systemic exposure. At a dose of 0.75mg/kg, administered every 3 days, MTMSA-Trp-A10 did not result in an efficacious result in tumor xenograft studies. These studies remain under further investigation, but the result may indicate high plasma protein binding of MTMSA-Trp-A10 and lack of free fraction available within tumor. The most selective MTM-oxime analogue in vitro, MTMox-23, significantly inhibited TC-32 (EWS-FLI1+) tumor xenograft growth (p=0.0025, day 16, one-way ANOVA multiple comparisons test) compared to MTM (p=0.1174, day 16) and extending survival for 17 days out of 48 days on study (p=0.0003, Log Rank (Mantel-Cox) single comparison test) with treatment at MTD every 3 days, compared to vehicle. Additionally, the MTM-oxime analogue with highest systemic exposure, MTMox-24, also significantly inhibited TC-32 (EWS-FLI1+) tumor xenograft growth (p=0.0003, day 21, one-way ANOVA multiple comparisons test) compared to MTM (p=0.032, day 21) and extending survival for 12 days out of 37 days on study (p=0.0004, Log Rank (Mantel-Cox) single comparison test) with treatment, compared to vehicle.
Conclusion: These studies in whole highlight the importance of exposure (pharmacokinetics; PK), toxicity and efficacy (pharmacodynamics; PD) relationships. The cytotoxicity and high systemic exposure of MTMSA-Trp-A10 directly contributes to its lower tolerated dose. However, despite a similar tolerated dose to MTM, systemic exposure remains 163-fold higher at the MTD. High systemic exposure may be attributed to high plasma protein binding, but also reduces the exposure of free MTMSA-Trp-A10 within the tumor tissue, which drives the efficacious response. In contrast, the less cytotoxic and rapidly de-methylated MTM-oxime analogues allow for 25-fold higher tolerances in mice. This unique metabolism and clearance may prevent exposures required to induced systemic blood and liver toxicities induced by MTM. Moreover, at these highly tolerated doses, the initial systemic exposure at MTD is highest among analogues tested, which resulted in an efficacious response with MTMox-23 and MTMox-24 treatment in tumor xenograft models. It remains to be determined if these PK/PD relationships can be reproduced in additional animal models, including human, without inducing toxicity. Nonetheless, these initial studies in mice demonstrate that a more selective, more tolerated analogue of MTM has potential for clinical success in treating ETS fusion-dependent tumors.
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Thurow, Helena Strelow. "Epidemiologia genômica: estudos de polimorfismos nos genes da p53 e MDM2 associados a fatores de risco para câncer." Universidade Federal de Pelotas, 2011. http://guaiaca.ufpel.edu.br/handle/123456789/1289.
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Previous issue date: 2011-07-15A proteína p53,codificada pelo gene TP53, vem sendo estudada há mais de 30 anos e já foi denominada de "guardiã do genoma.
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Brisac, Alice. "EWSR1-FLI1 et la dissémination métastatique du sarcome d'Ewing : étude des mécanismes moléculaires mis en jeu lors de la migration et de l'invasion cellulaire." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB092/document.
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Le sarcome d’Ewing est une tumeur pédiatrique de l’os très agressive, généralement causée par une translocation chromosomique aboutissant à la protéine de fusion EWSR1-FLI1 qui dérégule l’expression d’un grand nombre de gènes participant à l’oncogenèse du sarcome d’Ewing. Au laboratoire, nous avons pu mettre en évidence une hétérogénéité intra-tumorale de l’expression d’EWSR1-FLI1 aboutissant à un nouveau modèle de la biologie du sarcome d’Ewing basé sur la coexistence de cellules EWSR1-FLI1high, participant à la prolifération de la tumeur primaire, et de cellules EWSR1-FLI1low, capables de dissémination métastatique. Au cours de ma thèse, je me suis intéressée en particulier aux mécanismes moléculaires sous-jacents aux capacités migratoires et invasives des cellules EWSR1-FLI1low. Nous avons ainsi pu mettre en évidence une diminution des jonctions serrées intercellulaires associée à une augmentation des interactions cellule-matrice chez les cellules EWSR1 FLI1low. Nous avons ensuite révélé le rôle clé des MMP dans l’invasion du sarcome d’Ewing, plus spécifiquement de la MMP2 et de la MT1-MMP qui sont toutes deux réprimées par EWSR1-FLI1 et sont apparues nécessaires à l’invasion des cellules EWSR1-FLI1low. L’un des défis majeurs actuels est de parvenir à visualiser cette sous-population très minoritaire et à suivre la plasticité de l’expression d’EWSR1-FLI1. Pour cela, l’identification de marqueurs des cellules EWSR1-FLI1low est cruciale. Ceci est également important pour déterminer si les chimiothérapies actuelles, qui ciblent plutôt les cellules prolifératives, n’entraînent pas une augmentation de la proportion de ces cellules au sein de la tumeur primaire. Enfin, nos essais infructueux d’utilisation de l’embryon de poulet comme nouveau modèle de la métastase du sarcome d’Ewing in vivo ont souligné la fragilité globale et les limitations de ce type de modèle, indiquant plutôt la nécessité d’améliorer les modèles murins existants. La mise au point de modèles in vivo apparaît en effet primordiale pour mieux disséquer les mécanismes moléculaires de la métastase et permettre le test de drogues susceptibles d’inhiber ce processusEwing sarcoma is a very aggressive pediatric bone tumor generally caused by a chromosomal translocation that leads to the expression of the EWSR1-FLI1 fusion protein. This protein deregulates the expression of a number of genes involved in Ewing sarcoma oncogenesis. In the laboratory, we discovered an intra-tumor heterogeneity of EWSR1-FLI1 expression which lead us to propose a new model for Ewing sarcoma biology based on the coexistence of EWSR1-FLI1high cells, responsible for primary tumor growth, and EWSR1-FLI1low cells, with metastatic dissemination capacities. During my phD, I was especially interested in the molecular mechanisms underlying the migration and invasion capacities of EWSR1-FLI1low cells. We showed a decreased expression of intercellular junctions proteins associated with an increased expression of cell-matrix interaction proteins in EWSR1-FLI1low cells. We then revealed the key role of MMPs in Ewing sarcoma invasion, in particular of MMP2 and MT1-MMP, which are both repressed by EWSR1-FLI1 and were found necessary for invasion of EWSR1-FLI1low cells. One of the current major challenges is to visualize this very small sub-population and to track the plasticity of expression of EWSR1-FLI1. To this end, the identification of markers of EWSR1-FLI1low cell is crucial. This is also important in order to determine if current chemotherapies that primarily target highly proliferative cells do not increase the proportion of these EWSR1-FLI1low cells. Finally, our unsuccessful attempts to use the chick embryo as a new in vivo model of Ewing sarcoma metastasis indicate the need to improve the existing murine models. Indeed, the development of in vivo models appears essential for the dissection of the molecular mechanisms of Ewing sarcoma metastasis and the test of drugs susceptible to inhibit this process
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Santos, Nathalia Kersting dos. "Modulação do receptor do fator de crescimento epidérmico (EGFR) em sarcoma de Ewing : impacto na viabilidade, proliferação celular e vias de sinalização associadas a neurotrofinas." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2016. http://hdl.handle.net/10183/151460.
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A família de Tumores de Ewing pode ser compreendida como um espectro de neoplasias de células neuroectodérmicas primitivas, dentre eles se inclui uma classe menos diferenciada chamada Sarcoma de Ewing (SE), no qual o diagnóstico é mais frequente entre 11 e 20 anos. O índice de sobrevida pode chegar em 70%; todavia, estima-se que apenas 55% dos pacientes recebam um tratamento efetivo. Alguns estudos sugerem relevância do receptor do fator de crescimento epidérmico (EGFR) no processo de tumorigênese e metátase em cânceres de cabeça e pescoço, pulmão e colorretal. Algumas vias de sinalização que poderiam explicar a relação do EGFR a estes processos incluem a MAPK/ERK e PI3K/AKT. No entanto, estes complexos de sinalização podem ser ativados por outras cascatas de transdução de sinal como do sistema BDNF/TrkB. As neurotrofinas, mais recentemente, estão sendo relacionadas ao processo tumoral. O presente trabalho tem por objetivo avaliar a importância de EGF/EGFR na progressão tumoral do Sarcoma de Ewing, bem como o crosstalking entre este sistema e neurotrofinas. Linhagens celulares foram expostas a EGF ou ao inibidor da fosforilação do respectivo receptor (AG1478). A viabilidade e proliferação celular foram avaliadas em hemocitômetro. A análise do ensaio clonogênico foi feita em software ImageJ®. Para análise do ciclo celular, após exposição ao AG1478, realizou-se a avaliação por citometria de fluxo e uma provável indução de senescência foi analisada com X-Gal. Western Blotting foi padronizado para avaliação das vias MEK/ERK, PI3K/AKT, Ciclina D1, p53 e BDNF quando da exposição ao AG1478. Análises de viabilidade celular também foram feitas usando inibidores de MEK ou PI3K em combinação ao AG1478 ou não. A exposição das linhagens SK-ES-1 E RD-ES ao EGF afeta positivamente a taxa de proliferação destas e, quando essas são tratadas com o inibidor AG1478, existe um decréscimo desta taxa além de um impacto da razão de viabilidade. O índice de citotoxicidade (IC50) resultante é de 12,8uM e 9,8uM para SK-ES-1 e RD-ES, respectivamente. Foi observado que a inibição de EGFR reduziu o número e tamanho de colônias, e que a sua ativação reflete em um aumento dos parâmetros. Alterações das porcentagens populacionais nas fases do ciclo celular foram observadas. O ensaio colorimétrico mostrou aumento da percentagem de células senescentes e sugere-se envolvimento da via da ERK; AKT; Ciclinas, P53 e BDNF no efeito mediante exposição ao AG1478. A exposição ao EGF, portanto, aumenta a proliferação e a clonogenicidade de células de SE, bem como a diminuição destas quando da inibição do receptor. Também, a inibição de EGFR resulta em alterações no ciclo celular, senescência e morte celular.The family of Ewing's tumors may be understood as a spectrum of primitive neuroectodermal cell neoplasms, including a less differentiated class called Ewing's sarcoma (ES), wherein the diagnosis is most common between 11 and 20 years. The survival rate can reach 70%; however, it is estimated que only 55% of Patients receive effective treatment. Some published studies suggest relevance of epidermal growth factor receptor (EGFR) in tumorigenesis and metastatic process in head and neck, lung and colorectal tumors. Some signaling pathways, which could explain its relation with these processes, include MAPK / ERK and PI3K / AKT. However, other signal transduction cascades such as BDNF / TrkB system may activate these signaling complexes, the neurotrophins being related to the tumoral process. This study aims to assess the importance of EGF / EGFR in Ewing's Sarcoma tumor progression, and the crosstalking between this system and neurotrophins. Cell lines were exposed to EGF or the inhibitor of phosphorylation of its receptor (AG1478). The viability and cell proliferation were evaluated in hemocytometer. The analysis of the clonogenic assay was performed in ImageJ® software. For cell cycle analysis after exposure AG1478, the evaluation was performed by flow cytometry and a probable induction senescence was analyzed with X-Gal. Western Blott was standardized to evaluate the pathways MEK / ERK, PI3K / AKT, cyclin D1 and P53 when exposure to AG1478. Cell viability analyzes were also performed on exposure to MEK and PI3K inhibitor in combination or not with AG1478. Exposure of SK-ES-1 and RD-ES lines to EGF positively affects the rate of proliferation and when these are treated with the inhibitor AG1478, there is a decrease in this rate as well as an impact reason viability. The cytotoxicity index (IC50) is 12,8uM resulting 9,8uM and SK-ES-1 and RD-ES, respectively. We observed that inhibition of EGFR reduced the number and size of colonies, and their activation reflects an increase in the parameters. Changes in population percentages in the cell cycle phases were observed. The colorimetric assay showed an increased percentage of senescent cells and it is suggested involvement of the ERK pathway; AKT; Cyclin P53 and the effect upon exposure AG1478. Therefore, the EGF exposure increases the proliferation and clonogenicity of ES cells, as well as the reduction is observed when these receptor inhibited. Also, inhibition of EGFR results in changes in the cell cycle, senescence and cell death.