Systemic sclerosis (SSc) – is one of the most complicated and fatal systemic diseases, and the lack of effective therapy is very evident. The tyrosine kinase inhibitor imatinib mesylate (IM) was shown to inhibit TGF-β and PDGF signaling pathways and prevent the development of dermal fibrosis upon challenge with bleomycin in murine model of SSc. The aim of therapy is not only to stop disease progression, but even induce regression of preexisting fibrosis. On other hand, blocking TGF-β and PDGF signaling in angiogenesis might worsen the vascular manifestations of SSc. We found important to evaluate effectiveness of IM for the treatment of pre-established tissue fibrosis and to exclude that the anti-fibrotic effects of IM are complicated by inhibitory effects on endothelial cell functions. Aim of the study: assess the effect of IM on the process of fibrosis and endothelium in experimental models of systemic sclerosis and cell cultures. Objectives of the study: assess the effectiveness of IM on murine models of established fibrosis; evaluate if IM has an effect on basal functions of endothelial cells; assess effect of IM on the process of angiogenesis. We have shown that IM exerts potent antifibrotic effects in two different models of SSc. Imatinib was effective for prevention of fibrosis and for treatment of established dermal fibrosis. We’ve demonstrated that IM does not inhibit major functions of endothelial cells. Thus, IM might not augment further the preexisting vascular... [to full text] / Sisteminė sklerozė (SSc) – viena sunkiausių ir fatališkiausių autoimuninių sisteminių reumatinių ligų, o bazinių vaistų stygius, šiai ligai gydyti, itin didelis. Į onkologinę klinikinę praktiką įdiegtas tirozinkinazių inhibitorius – imatinibo mezilatas(IM). IM blokuoja TGF-β ir PDGF intraląstelinio signalo perdavimą ir taip sąlygoja fibrozės prevenciją SSc pelių modelyje. Mums buvo svarbu išsiaiškinti, ar imatinibas gali turėti įtakos ne tik prevencijai, bet ir susiformavusiai fibrozei. Be to TGF-β ir PDGF blokavimas angiogenezėje, galėtų riboti daug žadančio fibrozės inhibitoriaus IM naudojimą gydant SSc. Darbo tikslas: įvertinti imatinibo mezilato poveikį fibrozės procesui ir endoteliui sisteminės sklerozės eksperimentiniuose modeliuose ir ląstelių kultūrose. Darbo uždaviniai: įvertinti imatinibo efektyvumą neuždegiminiame SSc modelyje ir patikrinti imatinibo mezilato efektyvumą uždegiminiame suformuotos fibrozės modelyje; ištirti, ar terapinės imatinibo mezilato koncentracijos daro neigiamą poveikį gyvybinėms endotelio funkcijoms; įvertinti imatinibo mezilato poveikį angiogenezės etapams. Mūsų gauti duomenys rodo, kad: IM ne tik sustabdė bet ir paskatino jau egzistuojančios (bleomicino sukeltos) odos firbrozės regresiją; IM ryškiai sumažino poodžio ir odos storį, bei normalizavo miofibroblastų skaičių Tsk-1 pelėse; IM neturėjo poveikio endotelio ląstelių bazinėms funkcijoms; IM neturėjo neigiamo poveikio angiogenezės etapams.

L'érythropoïèse est le processus aboutissant à la production des hématies à partir d'une cellule souche hématopoïétique. La différenciation érythroïde implique des changements morphologiques en partie liés à la perte d'expression membranaire du récepteur à activité tyrosine kinase de classe III, c-Kit. En réponse à son ligand, le SCF, c-Kit est activé puis internalisé et dégradé par la voie du protéasome, via l'ubiquitine E3-ligase c-Cbl, ou par la voie lysosomale suite à une endocytose. Dans la première partie de ce travail, nous avons pu mettre en évidence qu'en absence de SCF et en réponse à un inhibiteur de tyrosine kinase, l'imatinib, les érythroblastes cultivés ex vivo perdent l'expression membranaire de c-Kit et accélèrent leur entrée en différenciation terminale. Au vu de ces observations, nous avons cherché à comprendre les mécanismes impliqués. Sur un modèle de cellules érytholeucémiques dépendantes de l'érythropoïétine, mais exprimant de manière endogène c-Kit, nous avons montré que l'imatinib induit une internalisation du récepteur ainsi que sa dégradation par la voie lysosomale et de manière indépendant de c-Cbl. De plus, nous avons montré que cet effet est réversible et que l'imatinib ne bloque pas la réexpression de c-Kit après son internalisation en réponse au SCF. Des marquages métaboliques ont permis de montrer que l'imatinib ne modifie ni la synthèse ni la maturation de c-Kit et que le profil phospho-tyrosine des cellules traitées à l'imatinib est globalement inchangé. Enfin, nous avons montré que la fixation de l'imatinib à la poche catalytique de c-Kit est indispensable à son internalisation, et par conséquent à sa dégradation. Il apparait donc que l'imatinib lève l'auto-inhibition de c-Kit, qui semble nécessaire pour son maintien à la membrane. Dans la seconde partie de ce travail, nous nous sommes intéressés aux changements morphologiques subis par les nucléoles, lieu de la biogenèse des ribosomes, au cours de différenciation des érythroblastes. L'étude de la taille et du potentiel prolifératif des cellules, ainsi que l'analyse morphologique des nucléoles, nous a permis de confirmer que la réduction de taille des cellules est contemporaine d'un ralentissement de leur prolifération ainsi que de la réduction du volume et de la surface du composé granulaire (CG), " matrice " du nucléole. En microscopie électronique, nous montrons la persistance des CG en fin de maturation. Enfin, nous avons également étudié l'évolution des nucléoles dans un contexte pathologique de syndromes myélodysplasiques de faible risque, qui se caractérisent par une hématopoïèse inefficace. Nous observons que les cellules pathologiques immatures ont des CG plus volumineux que les cellules normales immatures, et qu'au cours de la différenciation, la morphologie des nucléoles est identique entre les cellules normales et pathologiques. En conclusion, ce travail a permis de décrire 1) le mécanisme d'internalisation d'un récepteur à activité tyrosine kinase de classe III, c-Kit par l'imatinib et 2) la morphologie du nucléole au cours de la différenciation érythroïde normale et pathologique des syndromes myélodysplasiques de faible risque.

Les modifications physiopathologiques induites par l'obésité sont susceptibles de modifier la pharmacocinétique d'un grand nombre de médicaments. Toutes les étapes peuvent-être touchées : modification des compartiments de l'organisme, de la fonction rénale et de l'expression des protéines du métabolisme. L'intensité des variations induites reste difficile à anticiper en l'absence d'études spécifiques, ce qui pose également la question du choix du meilleur estimateur de poids dans l'expression du schéma posologique du médicament chez le sujet obèse. Nous avons évalué l'impact de différents descripteurs de poids sur les variabilités inter-individuelles de trois médicaments couramment utilisés en diabétologie, dans la douleur et en cancérologie. Une modélisation a été faite en pharmacocinétique de population (Monolix®) avec une évaluation des descripteurs de poids (poids de masse maigre, poids idéal, poids total, surface corporelle...). Pour la metformine (n=105 patients), molécule fortement hydrophile, CL/F et V/F augmentent avec le poids. Le poids de masse maigre est le meilleur descripteur de poids pour les deux paramètres et permet d'abaisser de manière importante la variabilité inter-individuelle. Dans le cas de la morphine (n=31), aucun descripteur n'est corrélé à CL/F ou V/F dans une population de sujets atteints d'obésité morbide. L'analyse de l'imatinib (n=54), de lipophilie plus importante, et de son métabolite actif montre que le poids idéal est la covariable la plus performante. Son impact n'explique cependant qu'une part minime de la variabilité de CL/F et ne remet pas en cause le principe d'une dose fixe. Les résultats confirment l'intérêt des méthodes de pharmacocinétique de population dans l'évaluation des covariables morphologiques, et indiquent qu'il n'existe pas de descripteur de poids universel. Ces résultats doivent nous encourager à évaluer systématiquement l'impact de l'obésité avec des modèles de population. Dans certains cas, des études de confirmation comparant différents schémas devraient-être mises en place.

The survival for high-grade glioma patients is poor and the treatment may cause severe side effects. A common obstacle in the treatment is chemoresistance. To improve the quality of life and prolong survival for these patients prognostic biomarkers and new approaches for chemotherapy are needed. To this end, a strategy to evade chemoresistance was evaluated by combining chemotherapeutic drugs with agents inhibiting resistance mechanisms identified by a bioinformatic analysis (paper I). The prognostic value of 13 different proteins was analyzed in this thesis (papers II-IV). Two of them, p38 mitogen-activated protein kinase (MAPK) and protein tyrosine phosphatase non-receptor type 6 (PTPN6, also known as SHP1) were analyzed for their potential as targets in combination chemotherapy (in paper III and IV, respectively).   We found that: PTPN6 expression and methylation status may be important for survival of anaplastic glioma patients, p38 MAPK phosphorylation may be a potential negative prognostic biomarker for high-grade glioma patients and FGF2 expression may be a potential negative prognostic biomarker for proneural glioma patients. PTPN6 may be a useful target for combination chemotherapy with cisplatin, melphalan or bortezomib in high-grade gliomas. The following drug combinations; camptothecin combined with an EGFR or RAC1 inhibitor, imatinib combined with a Notch or RAC1 inhibitor, temozolomide combined with an EGFR or FAK inhibitor and vandetanib combined with a p38 MAPK inhibitor may be useful combination chemotherapy for high-grade gliomas.

Orientador: Célia Regina Nogueira / Resumo: A Leucemia Mielóide Crônica (LMC) é uma doença malígna, clonal, da célula tronco hematopoética. A descoberta do cromossomo filadélfia e subsequente identificação do gene BCR-ABL, levaram a compreensão da biologia da doença que culminou com o desenvolvimento de drogas alvo-específicas, assim como o de métodos moleculares para o monitoramento da doença. O foco atual das pesquisas em LMC está voltado para o maior entendimento dos mecanismos moleculares e epigenéticos que levam a resistência terapêutica e progressão da doença. Estudos recentes demonstram que a expressão de micro-RNAs específicos modula oncogenes e genes supressores envolvidos no desenvolvimento de neoplasias. Ao encontro a esta tendência, propomos um estudo que procurou identificar o perfil de micro-RNAs dos pacientes bons respondedores aos tratamentos de primeira linha para LMC. Avaliamos o perfil de micro-RNAs, de 41 pacientes com LMC que atingiram resposta citogenética completa (ausência do cromossomo Filadélfia) após o tratamento com inibidor de tirosinaquinase e transplante alogênico de células progenitoras hematopoéticas, por meio do sistema de micro-RNA PCR arrays (TaqMan® Human Micro-RNA Array A e B). Identificamos uma assinatura de micro-RNA distinta entre os grupos tratados, apesar de se encontrarem no mesmo patamar de resposta clínica e citogenética. Palavras-chave: Leucemia Mielóide Crônica, micro-RNA, imatinibe, transplante, qPCR array. / Abstract: Chronic Myeloid Leukemia (CML) is a malignant clonal hematopoietic stem cell disease. The discovery of the Philadelphia chromosome and subsequent identification of the gene BCR-ABL have led to understanding the biology of the disease and the development of target-specific drugs, as well as molecular methods for monitoring the disease. The current focus of research in the CML is facing the greatest understanding of the molecular and epigenetic mechanisms that lead to therapy resistance and disease progression. Recent studies show that the expression of specific micro-RNAs modulates oncogenes and tumor suppressor genes involved in cancer development.We are proposing a new study in the literature that aims to identify the profile of micro-RNAs of patients good responders to first-line treatments for CML.Evaluated the profile of micro-RNAs in 41 CML patients who achieved a complete cytogenetic response (absence of Philadelphia chromosome) after treatment with tyrosine kinase inhibitor and allogeneic bone marrow transplantation, through the micro-RNA- PCR arrays (TaqMan® Human Micro-RNA Array A e B). We identified a distinct micro-RNA signature between the treated groups, despite being on the same level of cytogenetic and clinical response.Key Words: Chronic Myeloid LeuKemia, micro-RNA, imatinib, bone marrow transplantation, qPCR array. / Mestre

A leucemia mieloide crônica (LMC) é uma expansão clonal da célula progenitora hematopoiética, traduzindo-se por hiperplasia mielóide, leucocitose, neutrofilia, basofilia e esplenomegalia. O cromossomo Filadélfia é característico da doença, sendo produto da translocação t(9;22) (q34;q11), resultando na fusão dos genes ABL e BCR. Esta fusão gera um gene híbrido que produz uma proteína com elevada atividade tirosinoquinase que tem um papel central da patogenia da LMC. O mesilato de imatinibe (MI) é um derivado da fenilaminopirimidina que inibe a proteína-tirosina quinase ABL in vitro e in vivo. O MI interage com transportadores de membrana de efluxo, como o ATP binding cassette B1 (ABCB1). Polimorfismos no gene ABCB1 têm sido associados com alteração na sua funcionalidade e podem estar envolvidos na resposta ao tratamento farmacológico. Este estudo tem por objetivo investigar a relação dos polimorfismos C1236T, C3435T e G2677T/A no gene ABCB1 com marcadores de resposta ao tratamento com MI, em indivíduos com LMC, e determinar os fatores de predisposição de resposta ao MI. Foram incluídos 118 pacientes portadores de LMC. Foram constituídos dois grupos: Grupo 1 com 70 pacientes com resposta citogenética completa com a dose padrão de MI (400 mg/dia de MI) por até 18 meses e, Grupo 2 com 48 pacientes sem resposta citogenética completa com a dose inicial de 400 mg/dia de MI ou que perderam esta resposta ao longo do tratamento . Amostras de sangue foram obtidas para: quantificação de BCR-ABL1, extração de DNA genômico e análise citogenética de banda G. As análises dos polimorfismos foram realizadas por PCR-RFLP. A resposta ao tratamento foi avaliada segundo os critérios da European LeukemiaNet. A distribuição da frequência dos genótipos dos polimorfismos C1236T, C3435T e G2677T/A foi similar nos dois gêneros e entre brancos e não brancos. Não houve influência dos polimorfismos estudados no risco de desenvolvimento da LMC e na resposta ao MI. O haplótipo ABCB1 1236CT/2677GT/3435CT (para os polimorfismos C1236T/G2677T/C3435T no gene ABCB1) foi encontrado em 51,7% dos pacientes com resposta molecular maior (P=0,010). Houve tendência a maior frequência de pacientes portadores de genótipos 1236 CT e TT no grupo de respondedores (86,7%) quando foi analisada a resposta molecular completa (p=0,069). O mesmo aconteceu no grupo de não respondedores quando foi considerado o polimorfismo C1236T. Houve tendência a maior frequência de resposta molecular completa em portadores de genótipo 2677 GT+TT+TA nos dois grupos (respondedores P=0,074 e não respondedores P=0,076). Em conclusão, os genótipos e haplótipos para os polimorfismos ABCB1 C1236T, C3435T e G2677T/A estão associados com a resposta molecular em portadores de LMC respondedores ao tratamento com MI. / The chronic myeloid leukemia (CML) is a clonal expansion of the hematopoietic progenitor cell, representing myeloid hyperplasia, leukocytosis, neutrophilia, basophilia and splenomegaly. The Philadelphia chromosome is peculiar on the disease, being the result of the translocation t(9; 22) (q34; q11), leading on the fusion of the ABL and BCR genes. This merger creates a hybrid gene that produces a protein with high activity of tyrosine kinase that is the main pathogenesis of CML. The Imatinib mesylate (IM) is a fenilaminopirimidine derivative which inhibits the ABL protein-tyrosine kinase in vitro and in vivo. The MI interacts with membrane efflux transporters, such as ATP binding cassette B1 (ABCB1). Polymorphisms in the ABCB1 gene have been associated with changes in its functionality and may be involved on the response to drug treatment. This study aims to investigate the relationship of C1236T, C3435T and G2677T / A polymorphisms in ABCB1 gene with response markers for MI treatment in individuals with CML, and to determine the predisposing factors of response to MI. 118 patients with CML were included and divided in two groups. Group 1: 70 patients with complete cytogenetic response and a standard dose of IM (400 mg / day IM) for up to 18 months. Group 2: 48 patients without a complete cytogenetic response and initial dose of 400 mg / day IM or whith response lost throughout the treatment. Blood samples were obtained for: quantification of BCR-ABL1, genomic DNA extraction and band G cytogenetic analysis. The analysis of the polymorphisms were performed by PCR-RFLP. The treatment response was evaluated according to European LeukemiaNet criteria. The frequency distribution of genotypes of C1236T, C3435T and G2677T / A polymorphisms were similar in both sexes and between whites and nonwhites. The polymorphisms studied had no influence on the CML development or MI response. The haplotype ABCB1 1236CT/2677GT/3435CT (for C1236T/G2677T/C3435T polymorphisms in the ABCB1) was found in 51.7% patients with major molecular response (P = 0.010). There was a tendency for higher frequency of patients with 1236 CT and TT genotypes in the responders group (86.7%) when the molecular response was analyzed (p = 0.069). The same happened in the nonresponders group when the C1236T polymorphism was considered. There was a tendency for a higher frequency of complete molecular response in patients with 2677 GT + TT + TA genotype in both groups (responders P = 0.074 and nonresponders P = 0.076). In conclusion, genotypes and haplotypes for ABCB1 C1236T, C3435T and G2677T / A polymorphisms are associated with molecular response in patients with CML that respond to MI treatment.

Orientador: Katia Borgia Barbosa Pagnano / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T22:22:44Z (GMT). No. of bitstreams: 1 Silveira_RosanaAntunesda_D.pdf: 5849811 bytes, checksum: 2e4127fa41a0f3f36ca9540a1ea2cb63 (MD5) Previous issue date: 2013 / Resumo: O uso de inibidores de tirosinoquinases (TKIs) tem demonstrado eficácia no tratamento da Leucemia Mieloide Crônica (LMC). Porém, o fenômeno da resistência é um importante problema na prática clínica e ainda não está completamente elucidado. Além da presença de mutações no domínio quinásico (DQ) do gene BCR-ABL, diversos mecanismos moleculares foram relacionados à resistência aos TKIs. Adicionalmente, alguns estudos têm sido realizados com o objetivo de identificar assinaturas de expressão gênica associadas à resistência citogenética primária ao imatinibe (IM). Até o momento, a expressão de transcritos sem potencial codificador, mais especificamente de RNAs não codificadores longos (lncRNAs), ainda não foi investigada neste aspecto. LncRNAs, transcritos especialmente de regiões intrônicas, têm sido apontados recentemente como possíveis reguladores da expressão gênica em células normais e cancerosas. O objetivo geral deste estudo foi avaliar possíveis mecanismos de resistência ao tratamento com TKIs em pacientes com LMC, através da pesquisa de mutações no DQ do gene BCR-ABL e da identificação de genes codificadores e lncRNAs intrônicos diferencialmente expressos entre pacientes responsivos (R) e não responsivos (NR) ao IM e dasatinibe. A pesquisa de mutações realizada pela técnica de sequenciamento direto revelou que 37% (26/71) dos pacientes avaliados apresentavam mutações, sendo as mais frequentes T315I (25%), M244V (18%) e E255K/V (14%). As taxas de Sobrevida Global (SG) (p = 0,008), Sobrevida Livre de Progressão (p = 0,03) e Sobrevida Livre de Evento (SLE) (p = 0,006) foram menores para os pacientes com mutações. SG (p = 0,04) e SLE (p = 0,03) foram significativamente menores para os pacientes com a mutação T315I. A identificação de perfis de expressão gênica por microarranjos foi realizada a partir de amostras de células mononucleares de sangue periférico de 7 e 21 pacientes, respectivamente, para os estudos envolvendo dasatinibe e IM. Duas plataformas de microarranjos (Agilent Technologies) foram utilizadas neste estudo; a primeira com sondas que mapeiam exclusivamente em genes codificadores de proteína e a segunda, customizada, com 44.000 elementos que mapeiam em regiões intrônicas e exônicas de mesmo locus. As análises estatísticas foram realizadas com as técnicas Significance Analysis of Microarrays e patient leave-one-out cross-validation. Genes codificadores de proteína e lncRNAs diferencialmente expressos foram identificados a partir de comparações de três subgrupos de amostras de R e NR, pré e pós-tratamento. Em seguida, cada conjunto de transcritos diferencialmente expressos foi analisado através do programa Ingenuity Pathway Analysis (IPA) para a identificação de funções biológicas, redes gênicas e vias canônicas significativamente enriquecidas. Entre os transcritos encontrados como diferencialmente expressos entre R e NR estão ABCF2, PAWR, ncCD44, ncTNFAIP8 e ncFOXO3A no estudo com dasatinibe e LEF1, BCL2, FYN, ncPXN, ncNCAM1 e ncRASEF no estudo com IM. Porém, no estudo com IM, os conjuntos de transcritos diferencialmente expressos não distinguiram totalmente os casos R e NR. O presente trabalho identificou transcritos diferencialmente expressos entre amostras de pacientes R e NR tratados com dasatinibe e MI e sugere que lcnRNAs possuem um importante papel nos mecanismos moleculares de resistência. Futuras investigações serão necessárias para a confirmação destes achados / Abstract: The use of tyrosine kinase inhibitors (TKIs) has demonstrated efficacy in treating chronic myeloid leukemia (CML). However, resistance is an important problem in clinical practice and it has not yet been completely elucidated. Besides mutations within the BCR/ABL kinase domain (KD), diverse molecular mechanisms have been proposed to account for resistance to TKIs. Furthermore, some studies have been performed in order to identify gene expression signatures associated with primary cytogenetic resistance to imatinib (IM). So far, non-protein-coding RNAs expression, more specifically long non-coding RNAs (lncRNAs) expression, has not been investigated in this aspect. LncRNAs, especially those transcribed from intronic regions, have recently been suggested as potential regulators of gene expression in normal and cancer cells. The aim of this study was to evaluate possible mechanisms of resistance to TKI treatment in CML patients by performing BCR-ABL mutation analysis and identifying differentially expressed protein-coding genes and intronic lncRNAs in responder (R) and non-responders (NR) patients to IM and dasatinib. Mutation analysis performed by direct sequencing identified mutations in 37% (26/71) of the analyzed patients; the three most frequently detected mutations were T315I (25%), M244V (18%) and E255K/V (14%). Overall survival (OS) (p = 0.008), progression-free survival (p = 0.03) and event-free survival (EFS) (p = 0.006) rates were worse for patients harboring mutations. OS (p = 0.04) and EFS (p = 0.03) were significantly worse for T315I patients. Microarray expression profiling was performed on peripheral blood mononuclear cells from 7 and 21 patients, respectively, for the dasatinib and IM studies. Two array platforms (Agilent Technologies) were used in this study: the first one with probes exclusively mapping to protein-coding genes; the second, a custom-designed 44K oligoarray containing probes for intronic and exonic regions from the same genomic locus. Statistical analyses were performed with Significance Analysis of Microarrays and patient leave-one-out cross-validation techniques. Differentially expressed protein-coding genes and lncRNAs were identified by comparing three subgroups of pre- and post-treatment samples from R and NR. Next, each set of differentially expressed transcripts was analyzed by using the Ingenuity Pathway Analysis (IPA) software for identifying significantly enriched biological functions, gene networks and canonical pathways. Amongst the transcripts found as differentially expressed between R and NR are ABCF2, PAWR, ncCD44, ncTNFAIP8, and ncFOXO3A in the dasatinib study and LEF1, BCL2, FYN, ncPXN, ncNCAM1, and ncRASEF in the IM study. Nevertheless, in the IM study, the sets of differentially expressed transcripts were not capable of entirely distinguishing between responder and non-responders cases. In the present study differentially expressed transcripts between responder and non-responders' samples treated with dasatinib and IM were identified, and it suggests that lncRNAs play an important role in the molecular mechanisms of resistance. Further investigations are necessary to confirm these findings / Doutorado / Ciencias Basicas / Doutora em Clínica Médica

Orientador: Carmen Silvia Passos Lima / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T21:59:05Z (GMT). No. of bitstreams: 1 Numa_IramaiaAngelicaNeri_M.pdf: 4547636 bytes, checksum: 22548046dff40a3bb4e586230e3f13b7 (MD5) Previous issue date: 2012 / Resumo: O mesilato de imatinibe (MI) é o tratamento de escolha para pacientes com leucemia mielóide crônica (LMC) em fase crônica, mas a resposta ao medicamento é variável em indivíduos distintos. A CYP3A4 é a principal enzima responsável pelo metabolismo hepático do MI. O alelo variante G do polimorfismo CYP3A4 A-290G codifica menor quantidade de enzima do que o alelo selvagem A, mas o papel do referido polimorfismo em pacientes com LMC tratados com MI é desconhecido. Os objetivos deste trabalho foram os de avaliar a eficácia, a toxicidade a sobrevida livre de progressão (SLP) e global (SG) de pacientes com LMC durante a administração de MI e verificar se estes parâmetros são alterados pela variabilidade interindividual no metabolismo do fármaco, relacionada ao polimorfismo CYP3A4 A-290G. Foram avaliados 100 pacientes com LMC em FC precoce atendidos no Centro de Hematologia e Hemoterapia da UNICAMP. O diagnóstico da LMC, o exame hematológico, o cariótipo, a pesquisa do gene BCR-ABL e os genótipos do polimorfismo CYP3A4 A-290G foram realizados por métodos convencionais. Os pacientes receberam o MI na dose de 400mg e a resposta ao tratamento foi avaliada segundo os critérios do European Leukemia Net. Identificamos respostas hematológicas, citogenética e molecular similares às previamente descritas. A taxa de resposta hematológica foi de 95% ao longo do estudo. Aos doze meses, as respostas citogenética completa ou parcial foram de 72% e 11% respectivamente. Já as taxas de respostas moleculares completas e maiores aos 22 meses foram de 28% e 26%, respectivamente. A sobrevida global foi de 94% aos 92 meses bem como a sobrevida livre de progressão para as fases avançadas da doença. Observamos que pacientes com resposta citogenética completa ou parcial e molecular xiv completa ou maior apresentaram maior SLP e SG do que os demais. Cerca de 13% dos pacientes era portadores do genótipo AG do polimorfismo CYP3A4 A-290G, o qual esteve associado à obtenção de resposta molecular completa tardia e tendência à menor SLP e SG. Apesar da hipótese do alelo variante (G) exibir um fenótipo metabolizador lento associado a uma menor taxa de biotransformação do MI e portando maior risco de reações tóxicas, não observamos diferenças entre as toxicidades hematológicas e não hematológica (P= 0,28). Assim, concluímos que nossos pacientes respondem de forma similar ao MI do que os demais e que o polimorfismo CYP3A4 A-290G pode vir a funcionar como biomarcador de resposta ao fármaco / Abstract: Imatinib (IM) is widely recognized as the standard of care in the first-line treatment of CML but the response to the drug is variable in different subjects. CYP3A4 is the main enzyme responsible for the hepatic metabolism of Imatinib. The G variant allele of the polymorphism A-290G encoding least amount of enzyme than the wild-type allele, but the role of this polymorphism in CML patients treated with Imatinib is unknown. The aims of this study were to assess the efficacy, toxicity, progression-free survival (PFS) and overall survival (OS) of patients with CML during the administration of Imatinib and check if these parameters are affected y interindividual variability in drug metabolism, the polymorphism related to CYP3A4 A-290G. We evaluated 100 patients with CML newly diagnosed at Center of Hematology and Hemoterapy of UNICAMP. The diagnosis of CML, hematology, karyotyping, research the BCR-ABL gene polymorphism and CYP3A4 genotypes A-290G were performed by conventional methods. Patients received a dose of 400mg IM and treatment response was assessed according to the criteria of the European Leukemia Net responses identified hematologic, cytogenetic and molecular similar to those previously described. The hematologic response rate was 95% throughout the study. At 22 months the complete or partial cytogenetic responses were 72% and 11% respectively. The complete molecular response rates at 22 months were 28% and 26%, respectively. Overall survival (OS) was 94% at 92 months and the progression-free survival (PFS) for advanced stages of the disease. We observed that patients with partial or complete cytogenetic response and major molecular and complete PFS and OS showed higher than other. We observed that patients with partial or complete cytogenetic response and major molecular xvi and complete PFS and OS showed higher than others. About 13% of patients were of AG genotype polymorphism of the CYP3A4 -290A>G, which was associated with achieving complete molecular response and late tendency to lower PFS and OS. Despite the possibility of variant allele (G) display a slow metabolizer phenotype associated with a lower rate of biotransformation of IM and carrying greater risk of toxic reactions, no significant differences between hematological and non hematological toxicities (P= 0,28). Thus, we conclude that our patients respond similary to IM than others and that the polymorphism CYP3A4 -290A>G might function as a biomarker of response to the drug / Mestrado / Ciencias Basicas / Mestra em Clínica Médica

<p>Treatment results and clinical characteristics in adult acute lymphoblastic leukaemia (ALL) were evaluated regarding three issues: a new treatment with cytarabine up-front, stem cell transplantation and a comparison between adult and paediatric treatment protocols. All studies were conducted on a national basis. Furthermore, activity of imatinib was investigated by in vitro cytotoxicity assay. </p><p>The national protocol was evaluated in 153 adult ALL patients. A high complete remission rate, 86%, was achieved with 29% overall survival at 3-years. Favourable outcome was identified in patients < 40 years with precursor B phenotype and continuous complete remission was higher for precursor B compared to T-ALL. </p><p>Stem cell transplantation was evaluated in 187 patients. No differences in outcome between allogeneic and autologous transplantation were found, with the exception of Philadelphia-positive ALL, in which allogeneic transplantation was preferable. Limited chronic graft-versus-host disease (compared to none) resulted in superior disease free survival. </p><p>The paediatric NOPHO-92 and the Adult protocols were evaluated for 243 ALL-patients. Superior remission rate and survival were achieved for 10-18 year-olds treated according to the Paediatric protocol compared to both 15-25 and 25-40 year-olds treated according to the Adult protocol. Treatment protocol was a significant prognostic factor for patients aged 15-20 years. </p><p>Fluorometric Microculture Cytotoxicity Assey was used to analyze 15 tumour cell samples from ALL patients. High concordance was determined between in vitro sensitivity to imatinib and presence of BCR-ABL. Daunorubicin, prednisolone and cytarabine had the greatest benefit from a combination with imatinib. </p><p>The national adult treatment protocol’s results were consistent with international trials regarding precursor B ALL but may be under performing for T-ALL. Adolescents may benefit from treatment according to the Paediatric protocol. No difference in outcome between allogeneic and autologous stem cell transplantation was determined except for Philadelphia-positive patients, despite the indication of a graft-versus-leukaemia effect.</p>

Les fibromatoses agressives sont des tumeurs conjonctives rares, pouvant envahir les structures adjacentes parfois de manière très agressive et responsables de fréquentes récidives loco-régionales mais dépourvues de potentiel métastatique. Leur évolution est imprévisible. Actuellement, la stratégie de leur prise en charge est remise en cause et ces tumeurs sont de plus en plus souvent surveillées à leur diagnostic. En cas de tumeur évolutive, les traitements systémiques disponibles sont multiples mais d'efficacité variable. Aucun facteur pronostique de récidive ou d'évolutivité spontanée et aucun facteur prédictif d'efficacité des traitements médicaux n'a aujourd'hui été identifié. Les travaux de 2 premières publications ont étudié les facteurs cliniques de récidive après exérèse chirurgicale, et ont retenu le jeune âge du patient, la grande taille tumorale et sa localisation extra-abdominale comme étant de mauvais pronostic. Dans une 3ème publication, nous avons recherché si le sous-type moléculaire de mutation de CTNNB1 observé dans les fibromatoses sporadiques pouvait influencer la récidive: la mutation S45F est de moins bon pronostic que les autres. Un autre article rapporte les résultats de l'analyse des profils d'expression des miRNAs des fibromatoses qui semblent se corréler à leur pronostic mais cela doit être confirmé. Les 3 articles suivant présentent des travaux recherchant des facteurs prédictifs de réponse des fibromatoses à l'imatinib : l'expression d'aucune des cibles connues de l'imatinib n'a été retrouvée comme influençant significativement la réponse au traitement. Le rôle des variants M541L et V530I de l'exon 10 de KIT reste à déterminer