Unter Verwendung eines mathematischen Hämatopoese-Modells werden verschiedene Fragen adressiert, die im Zusammenhang mit einer möglichen Optimierung der gegenwärtigen Therapie chronischer myeloischer Leukämie (CML) stehen. Es handelt sich um ein agentenbasiertes Modell, das heißt, jede Zelle wird als einzelnes Objekt repräsentiert und gemäß festgelegter Regeln im Computer simuliert. Es werden proliferative von ruhenden Stammzellen unterschieden, wobei sich der Proliferationszustand reversibel ändern kann. Das Modell basiert auf der Annahme, dass sich normale und maligne Stammzellen in einem Wettbewerb um gemeinsame Ressourcen befinden, wobei der CML-Klon einen kompetitiven Vorteil besitzt. Es ist ungeklärt, ob Tyrosinkinaseinhibitoren wie Imatinib (IM) in der Lage sind, die Erkrankung zu heilen. Es gibt Evidenz, dass residuale leukämische Stammzellen im Knochenmark persistieren, welche in einem Ruhezustand (G0-Phase des Zellzyklus) von IM nicht eradiziert werden können. Proliferativ aktive Zellen sind der IM-Wirkung hingegen ausgesetzt. Das Modell sagt voraus, unter welchen Bedingungen eine Kombinationsstrategie von IM mit stammzellaktivierenden Substanzen Synergieeffekte hervorbringen könnte. Ein verwandtes Problem ist die Frage, in welchen Fällen nach Reduktion der Tumorlast auf ein mittels hochsensitiver Messmethoden undetektierbares Niveau ein Therapieabbruch gerechtfertigt ist. Basierend auf dem dynamischen Modell wird in dieser Arbeit ein Prädiktor vorgeschlagen, der vorhersagt, ob ein Patient nach Abbruch der Therapie einen molekularen Rückfall zu erwarten hat. Zusätzlich wird approximativ ein modellunabhängiger Prädiktor angegeben, der die Vorhersage nur auf Basis klinisch messbarer Größen gestattet.

<p>Hyperspectral imaging is widely used for obtaining optical information of light absorbers (e.g. biochemical composition) in a variety of specimens or tissues in a label-free manner. Acquiring and processing spectral data using hyperspectral imaging usually requires advanced instrumentation such as spectrometers, spectrographs or tunable color filters, which are not easily adaptable in developing instrumentation for field-based applications. Also, use of only RGB information from conventional cameras is not sufficient to obtain a reliable correlation with the actual content of the analyte of interest. We propose a new concept of ‘virtual hyperspectral imaging’ to reconstruct the full reflectance spectra from RGB image data. This allows us to use only RGB image data to determine detailed spatial distributions of analytes of interest. More importantly, it simplifies instrumentation without requiring bulky and expensive hardware. Using a data-driven approach, we apply multivariate regression to reconstruct hyperspectral reflectance image data from RGB images obtained using a conventional camera or a smartphone. </p> <p> </p> <p>In developing a reliable reconstruction matrix, it is critical to obtain a training data set of the specimen of study under the same optical geometry since the spectral reflectance and absorbance is sensitive to the detection and illumination parameters. We designed an image-guided hyperspectral system that can acquire both hyperspectral reflectance and RGB data sets under the same imaging configuration to minimize any discrepancies in the hyperspectral reflectance data acquired using different optical sensing geometries. In our technology development, a telecentric lens that is commonly used in machine vision systems but rarely in bioimaging, serves as a key component for reducing unwanted scattering in biological tissue due to its highly anisotropic scattering properties, by acting as a back-directional gating component to suppress diffuse light. We evaluate our spectrometer-less reflectance imaging method using RGB-based hyperspectral reconstruction algorithm for integration into a smartphone application for non-invasive hemoglobin analysis for anemia risk assessment in communities with limited access to central laboratory tests.</p>

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23775号 / 医博第4821号 / 新制||医||1057(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 永井 純正, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Sabe-se que TRAIL é capaz de matar células tumorais de forma seletiva e que TRAIL tem sua expressão reduzida em diversos tumores, porém pouco se sabe sobre os mecanismos responsáveis pela sua inibição. Tendo em vista que a expressão de TRAIL pode ser regulada pelo Ácido Retinóico; que PRAME é capaz de inibir a via do ácido retinóico através da proteína EZH2 e que nós observamos anteriormente que a expressão de TRAIL esta diminuída em pacientes com LMC, nós decidimos investigar a associação entre PRAME, EZH2 e TRAIL na LMC. Nós demonstramos que PRAME, mas não EZH2, tem sua expressão aumentada em células BCR-ABL+ e sua expressão está associada com a progressão da LMC. Alem disto, existe uma correlação positiva entre PRAME e BCR-ABL e negativa entre PRAME e TRAIL nestes pacientes. A inibição da expressão de PRAME ou EZH2 por RNAi induziu um aumento da expressão de TRAIL. Estes dados revelam um novo mecanismo de regulação responsável por diminuir a expressão de TRAIL, e geram novos possíveis alvos para a terapia da LMC e, possivelmente, também para outros tumores. / TRAIL was shown to selectively kill tumor cells. Not surprisingly, TRAIL is down-regulated in a variety of tumor cells, but the mechanism responsible for TRAIL inhibition remains elusive. Because TRAIL can be regulate by retinoic acid; PRAME was shown to inhibit transcription of retinoic acid receptor target genes through the polycomb protein EZH2; and we have found that TRAIL is inversely correlated with BCR-ABL in CML patients, we decided to investigate the association of PRAME, EZH2 and TRAIL in BCR-ABL-positive leukemia. Here, we demonstrate that PRAME, but not EZH2, is up-regulated in BCR-ABL cells and is associated with the progression of disease in CML patients. In addition, PRAME expression is positively correlated with BCR-ABL and negatively with TRAIL in these patients. Importantly, knocking down of PRAME or EZH2 by RNA interference restores TRAIL expression. Our data reveal a novel regulatory mechanism responsible for lowering TRAIL expression and provide the basis of alternative targets for combined therapeutic strategies for CML.

Dentre os vários tipos de leucemia, destaca-se a Leucemia Mielóide Crônica (LMC), um distúrbio mieloproliferativo em que ocorre a translocação entre o gene BCR no cromossomo 22 e o gene ABL1 no cromossomo 9. Essa translocação cria um cromossomo conhecido como Philadelphia (t 9,22)(q34;q11), ou Ph+, e a consequente formação de um produto único de proteínas BCR-ABL1. Essa proteína tem atividade de quinase constitutiva e impulsiona a proliferação descontrolada de células tronco hematopoiéticas. O surgimento de uma nova classe farmacológica no início dos anos 2000 - os inibidores de tirosina quinases, revolucionou o tratamento e o prognóstico da LMC, permitindo que esse câncer fosse tratado praticamente como uma doença crônica, com farmacoterapia oral. A droga de estréia dessa classe, o Mesilato de Imatinib, foi desenvolvida através de modelagem molecular para ser alvo-específica, mas apesar do desenvolvimento exitoso, após o início da comercialização, foram observadas falhas na ação em determinados pacientes. Há evidências de que a avaliação da relação entre a dose de imatinibe (e seus níveis sanguíneos) e a eficácia do tratamento medida através das respostas Hematológica, Citogenética e Molecular, seja uma forma de realizar o ajuste da dose reduzindo efeitos colaterais e custo do tratamento. No presente estudo foram avaliadas as concentrações séricas de imatinib, Cmin e Cmax, em 51 pacientes com Leucemia Mielóide Crônica, dos quais 33 atingiram Resposta Molecular Maior em até 12 meses de tratamento, 11 levaram mais que 12 meses para antingir, e 7 não atingiram. As concentrações séricas obtidas desses pacientes indicaram que no grupo que atingiu RMM em até 12 meses, os valores de vale (Cmin) se apresentaram com mediana de 889.2 ng/mL (721.9 e 1202.4 para primeiro e terceiro quartis respectivamente), sendo que o grupo que levou mais de 12 meses para atingir RMM, a concentração mediana observada foi de 611.0 ng/mL (493.0 e 816.0 para primeiro e terceiro quartis respectivamente), sendo essa diferença estatisticamente significativa (p < 0.05). Dessa forma demonstrou-se a importância do monitoramento das concentrações séricas de imatinib para o ajuste da dose e para a gestão do tratamento na mudança para segunda geração de inibidores de tirosina quinase. Através da análise comparativa dos dados populacionais estudados, observou-se não haver correlação significativa entre as concentrações séricas e índice de massa corpórea (IMC), peso, idade ou sexo / Among the various types of leukemia, Chronic Myeloid Leukemia (CML) stands out as a myeloproliferative disorder in which translocation occurs between the BCR gene on chromosome 22 and the ABL1 gene on chromosome 9. This translocation creates a chromosome known as Philadelphia (t 9,22) (q34; q11), or Ph +, and the consequent formation of a unique BCR-ABL1 protein product. This protein has constitutive kinase activity and drives the uncontrolled proliferation of hematopoietic stem cells. The launch of a new pharmacological class in the early 2000s - the tyrosine kinases inhibitors, revolutionized the treatment and prognosis of CML, allowing that cancer to be treated virtually as a chronic disease with oral pharmacotherapy. The newbie drug of this class, Imatinib Mesylate, was developed through molecular modeling to be target-specific, but despite the successful development, after the beginning of marketing, certain patients presented some failures in the response. There is an evidence that an assessment of the relationship between a dose of Imatinib (and its blood levels) and the efficacy of treatment from its Hematologic, Cytogenetic and Molecular Responses, is a really effective way to perform dose adjustment reducing side effects and cost of treatment. In the present study, the serum concentrations of Imatinib, Cmin and Cmax were evaluated in 51 patients with chronic myeloid leukemia, of which 33 reached major molecular response in up to 12 months of treatment, 10 took more than 12 months to achieve it, and 7 did not reach that. The serum concentrations obtained from those patients indicated that in the group that reached Major Molecular Response (MMR) within 12 months, the trough level (Cmin) presented a median of 889.2 ng / mL (721.9 and 1202.4 for first and third quartiles, respectively), and the group which took more than 12 months to reach MMR, the median concentration observed was 611.0 ng / mL (493.0 and 816.0 for the first and third quartiles respectively), and this difference was statistically significant (p < 0.05). Thus, the importance of monitoring serum imatinib concentrations for dose adjustment and treatment management in switching to second-generation tyrosine kinase inhibitors has been demonstrated. Through the comparative analysis of the population data, there was no significant correlation between serum concentrations and body mass index (BMI), weight, age or gender

Nous avons produit des CART-cells et des LT-mock ont été produits ex-vivo. L'efficacité de transduction est mesuré par l'expression CD3+/CD19+ (82% en moyenne, n=S), en cytométrie en flux. L'expression protéique de CAR a été vérifiée par western blot et en cytométrie en flux attestant de son expression membranaire. Nous avons testé la fonctionnalité de la cassette suicide iCaspase9/ AP1903 sur CART-cells après 48h de traitement à I' AP1903. Nous avons montré que plus de 90% des CART-cells entrent en apoptose (Marquage 7-AAD en cytométrie en flux). Nous avons ensuite réalisé des tests fonctionnels in-vitro des CART-cells contre des lignées LMC exprimant naturellement IL-lRAP ou contre des lignées transfectées pour exprimer la forme membranaire d'IL-lRAP. Après co­culture des CART-cells en présence des cellules cibles IL-lRAP+ nous avons montré qu'ils prolifèrent (test CFSE), qu'ils sécrètent de l'interféron y et des cytokines inflammatoires. Enfin, par marquage 7-AAD, nous avons démontré la cytotoxicité des CART-cells contre les cellules ILl-RAP+. Finalement, nous avons montré l'efficacité des CART-cells in vivo, dans un modèle de xénogreffe tumorales dans des souris immunodéficiences NSG greffées par la lignée K562-IL1RAP+ / Luciférase +. On constate une diminution de la masse tumorale 4 jours après injection des CART-cells, jusqu'à disparition totale. Cette preuve de concept laisse entrevoir des perspectives thérapeutiques alternatives dans le traitement de la LMC ou LAM, qui devront être optimiser dans des modèles murins (séquence et nombres d'injection, nombres de cellules, associations avec ITKS ou autres chimiothérapies) afin de conduire un essai clinique de phase I, pour démontrer la faisabilité et la sécurité de l'approche pour envisager une démonstration d'efficacité chez l'homme. La sécurisation par la cassette suicide permet d'envisager l'utilisation des CART-cells en situation autologue ou allogénique (Donor Lymphocytes infusion, DU) / Chronic myeloid leukemia (CML) is a myeloproliferative disorder and is characterized by the presence of a Philadelphia chromosome (Phl) encoding the BCR-ABL fusion protein with constitutive tyrosine kinase activity. The treatment by Tyrosine Kinase lnhibitors (ITK) is not curative. The problem today is ta target leukemic stem cells (HSCs) to prevent relapse of patients after stopping treatment with TKI and permanently eradicate the disease. Studies have identified the interleukin-1 accessory protein receptor (IL-lRAP) as a potential target for killing CSL (BCR-ABL1 positive) in CML or in acute myeloid leukemia (AML). We therefore propose to develop a cell therapy approach targeting IL-lRAP, using T cells (LT) redirected by a CAR (Chimeric Antigen Receptor) in the treatment of CML and AML. We produced a specific anti-lL-1RAP murine monoclonal antibody (mAb) called # A3C3. We tested the specificity of the antibody # A3C3 in flow cytometry, in Western blot, by immunohistochemistry or confocal microcopy, on positive human cell lines IL-1RAP (KU812) or IL-1RAP negative (Raji). Moreover, by using this antibody, we have demonstrated by immunohistochemistry, on 20 different normal tissues that our antibody does not recognize or very few non-specific (off-target) targets. Finally, we demonstrated that this antibody is able to detect positive IL-1RAP CSLs in primary bone marrow or peripheral blood samples of LMC patients at diagnosis or after ITK treatment. By molecular biology, we have sequenced and identified rearrangements of the heavy (lgH) and light {lgL) chains coding region for hypervariable regions of# A3C3. The coding sequence for the "single chain variable fragment" (scFv), lin king the 2 lgH and lgl chains, was cloned into a 3rd generation lentiviral skeleton securised by a suicide gene, inducible caspase 9 (iCASP9). We produced CART-cells and LT-mock ex-vivo. The transduction efficiency is measured by CD3 + / CD19 + expression (82% on average, n = 5), in flow cytometry. The functionality of the suicide cassette iCaspase9/AP1903 on CART-cells after 48h treatment with AP1903 was tested. We have shown the death of more than 90% of CART-cells (7-AAD labeling in flow cytometry). We then performed in-vitro functional tests of CART-cells against LMC lines naturally expressing IL-1RAP or against transfected lines to express the membrane form of IL-1RAP. After co-culture of CART-cells in the presence of IL-1RAP + target cells, we have shown that they proliferate (CFSE test), that they secrete interferon y and inflammatory cytokines (intracellular labeling and Cytokines Bead Array assay). They are able to degranulate (CD107a & b labeling). Finally, by 7 AAD labeling, we demonstrated the cytotoxicity of CART-cells against IL1-RAP + cells. Finally, we showed the effectiveness of CART-cells in vivo, in a tumor xenograft model in NSG immunodeficiency mice engrafted by the K562-IL1RAP+/Luciferase+ line. There is a decrease in the tumor load 4 days after injection of CART-cells, until complete disappearance.This proof of concept suggests alternative therapeutic perspectives in the treatment of CML or AML, which should be optimized in murine models (sequence and injection numbers, cell numbers, associations with ITKS or other chemotherapies) in order to Phase I clinical trial, to demonstrate the feasibility and safety of the approach to consider a demonstration of efficacy in human. Securing by the suicide cassette makes it possible to consider the use of CART-cells in an autologous or allogeneic situation (Donor Lymphocytes infusion, DU)

Dentre os vários tipos de leucemia, destaca-se a Leucemia Mielóide Crônica (LMC), um distúrbio mieloproliferativo em que ocorre a translocação entre o gene BCR no cromossomo 22 e o gene ABL1 no cromossomo 9. Essa translocação cria um cromossomo conhecido como Philadelphia (t 9,22)(q34;q11), ou Ph+, e a consequente formação de um produto único de proteínas BCR-ABL1. Essa proteína tem atividade de quinase constitutiva e impulsiona a proliferação descontrolada de células tronco hematopoiéticas. O surgimento de uma nova classe farmacológica no início dos anos 2000 - os inibidores de tirosina quinases, revolucionou o tratamento e o prognóstico da LMC, permitindo que esse câncer fosse tratado praticamente como uma doença crônica, com farmacoterapia oral. A droga de estréia dessa classe, o Mesilato de Imatinib, foi desenvolvida através de modelagem molecular para ser alvo-específica, mas apesar do desenvolvimento exitoso, após o início da comercialização, foram observadas falhas na ação em determinados pacientes. Há evidências de que a avaliação da relação entre a dose de imatinibe (e seus níveis sanguíneos) e a eficácia do tratamento medida através das respostas Hematológica, Citogenética e Molecular, seja uma forma de realizar o ajuste da dose reduzindo efeitos colaterais e custo do tratamento. No presente estudo foram avaliadas as concentrações séricas de imatinib, Cmin e Cmax, em 51 pacientes com Leucemia Mielóide Crônica, dos quais 33 atingiram Resposta Molecular Maior em até 12 meses de tratamento, 11 levaram mais que 12 meses para antingir, e 7 não atingiram. As concentrações séricas obtidas desses pacientes indicaram que no grupo que atingiu RMM em até 12 meses, os valores de vale (Cmin) se apresentaram com mediana de 889.2 ng/mL (721.9 e 1202.4 para primeiro e terceiro quartis respectivamente), sendo que o grupo que levou mais de 12 meses para atingir RMM, a concentração mediana observada foi de 611.0 ng/mL (493.0 e 816.0 para primeiro e terceiro quartis respectivamente), sendo essa diferença estatisticamente significativa (p < 0.05). Dessa forma demonstrou-se a importância do monitoramento das concentrações séricas de imatinib para o ajuste da dose e para a gestão do tratamento na mudança para segunda geração de inibidores de tirosina quinase. Através da análise comparativa dos dados populacionais estudados, observou-se não haver correlação significativa entre as concentrações séricas e índice de massa corpórea (IMC), peso, idade ou sexo / Among the various types of leukemia, Chronic Myeloid Leukemia (CML) stands out as a myeloproliferative disorder in which translocation occurs between the BCR gene on chromosome 22 and the ABL1 gene on chromosome 9. This translocation creates a chromosome known as Philadelphia (t 9,22) (q34; q11), or Ph +, and the consequent formation of a unique BCR-ABL1 protein product. This protein has constitutive kinase activity and drives the uncontrolled proliferation of hematopoietic stem cells. The launch of a new pharmacological class in the early 2000s - the tyrosine kinases inhibitors, revolutionized the treatment and prognosis of CML, allowing that cancer to be treated virtually as a chronic disease with oral pharmacotherapy. The newbie drug of this class, Imatinib Mesylate, was developed through molecular modeling to be target-specific, but despite the successful development, after the beginning of marketing, certain patients presented some failures in the response. There is an evidence that an assessment of the relationship between a dose of Imatinib (and its blood levels) and the efficacy of treatment from its Hematologic, Cytogenetic and Molecular Responses, is a really effective way to perform dose adjustment reducing side effects and cost of treatment. In the present study, the serum concentrations of Imatinib, Cmin and Cmax were evaluated in 51 patients with chronic myeloid leukemia, of which 33 reached major molecular response in up to 12 months of treatment, 10 took more than 12 months to achieve it, and 7 did not reach that. The serum concentrations obtained from those patients indicated that in the group that reached Major Molecular Response (MMR) within 12 months, the trough level (Cmin) presented a median of 889.2 ng / mL (721.9 and 1202.4 for first and third quartiles, respectively), and the group which took more than 12 months to reach MMR, the median concentration observed was 611.0 ng / mL (493.0 and 816.0 for the first and third quartiles respectively), and this difference was statistically significant (p < 0.05). Thus, the importance of monitoring serum imatinib concentrations for dose adjustment and treatment management in switching to second-generation tyrosine kinase inhibitors has been demonstrated. Through the comparative analysis of the population data, there was no significant correlation between serum concentrations and body mass index (BMI), weight, age or gender

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No. of bitstreams: 2 Tese - Adriana do Prado Barbosa - 2015.pdf: 900945 bytes, checksum: c7a5ed7dcaeb54e2967829169a9de269 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-04-10 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The treatment of chronic myeloid leukemia (CML) has changed dramatically with the advent of imatinib mesylate (IM). Besides the convenience of oral use, other benefits were achieved with the new drug, with faster therapeutic responses and increased survival, giving the CML similar characteristics as chronic diseases. In this scenario, there was another challenge, drug compliance, since a significant proportion of patients fail to ingest all the prescribed doses of imatinib. The concern was to optimize the adherence of CML patients, the hematology ambulatory at the Clinical Hospital of the Federal University of Goias (HC-UFG), led the authoress to create a film cartoon, as a pro-adhesion educational intervention model. To investigate the effectiveness of this new educational material, we used in 65 patients three adherence measures, two indirect (Morisky Test and Molecular Response [MR]) and direct (plasma dosage of IM), before and after the screening of film. In univariate analysis, from the Morisky Test, the film was striking, with increased adherent patients, which increased from 15 (23.1%) to 43 (66.1%). The results of MR showed an improvement trend after the movie, because the positive molecular response (major MR or complete MR) increased from 81.5% to 86.1%. Regarding the serum levels of IM, with daily doses of 400-800 mg IM, the premovie samples showed higher average than the post-movie (2473.16 ± 1049.55 ng/ml versus 1414.72 ± 715 73 ng/ml), with a variation coefficients interpatients of 43.4% and 50.6%, respectively. This high dispersion index found has been reported by other authors. By multivariate analysis, patients were divided into three groups. The first brought together compliant patients before and after the film with a good therapeutic response (major MR) after the intervention. It was: patients over 53 years old, females, with associated diseases before and after the treatment of CML that use more than two drugs in addition to imatinib. The second group was marked by the change of not adherence pre to adherence post-film. Its features were younger than or equal to 53, the absence of other disease before the CML, the use of less than two drugs and complete molecular response after the film. In the third group, we observed patients without molecular response before and after the educational intervention and no medication adherence after the film. They had in common their age (less than or equal to 53 years), and drug discontinuation due to adverse reactions. The last represents the set of patients resistant to the educational film, drawing attention to the fact that only one pro-adhesion method may be insufficient for all individuals. It is concluded that medication adherence was higher among patients older than 53 years, the educational film is an effective proadhesion assistance and continuing education, if combined with another method, it could help maintain or enhance the benefits achieved in this work. / O tratamento da leucemia mielóide crônica (LMC) mudou radicalmente com o advento do mesilato de imatinibe (MI). Além da comodidade do uso oral, outros benefícios foram alcançados com o novo fármaco, como respostas terapêuticas mais rápidas e aumento da sobrevida, dando `a LMC características semelhantes `as de doenças crônicas. Neste cenário, surgiu outro desafio, a adesão medicamentosa, pois uma proporção significativa de pacientes deixa de ingerir a dose prescrita de imatinibe. A preocupação em otimizar a adesão dos pacientes com LMC, do Ambulatório de Hematologia do Hospital das Clínicas da Universidade Federal de Goiás (HC-UFG), motivou a autora a criar um filme em desenho animado, como modelo de intervenção educativa pró-adesão. Para investigar a eficácia deste novo material educativo, empregou-se, em 65 pacientes, três medidas de adesão, duas indiretas (Teste de Morisky e Resposta Molecular [RM]) e uma direta (dosagem plasmática do MI), antes e depois da exibição do filme. Em análise univariada, pelo teste de Morisky, o filme foi impactante, com aumento dos pacientes aderentes, que passaram de 15 (23,1%) para 43 (66,1%). Os resultados da RM indicaram uma tendência de melhora após o filme, pois a resposta molecular positiva (RM maior ou RM completa) passou de 81,5% para 86,1%. Em relação `a dosagem sérica do MI, com doses diárias entre 400-800 mg de MI, as amostras pré-filme apresentaram média superior `as do pósfilme (2.473,16 ± 1.049,55 ng/ml versus 1.414,72 ± 715,73 ng/ml), com coeficientes de variação interpaciente de 43,4% e 50,6%, respectivamente. Este elevado índice de dispersão encontrado tem sido relatado por outros autores. Pela análise multivariada, os pacientes foram separados em três grupos. O primeiro, reuniu os pacientes aderentes antes e após o filme e com boa resposta terapêutica (RM maior) após a intervenção. Foram eles: os doentes com mais de 53 anos, do gênero feminino, com doenças associadas antes e após o tratamento da LMC e que usam mais de dois medicamentos além do imatinibe. O segundo grupo foi marcado pela mudança de não adesão pré para adesão pós-filme. Suas características foram idade menor ou igual a 53, ausência de outra doença antes da LMC, uso de menos de dois medicamentos e resposta molecular completa pós-filme. No terceiro grupo, observou-se pacientes sem resposta molecular antes e depois da intervenção educativa, bem como não adesão medicamentosa após o filme. Eles tinham em comum a idade, menor ou igual a 53 anos, e suspensão do medicamento por reação adversa. Estes últimos representam o conjunto de pacientes resistentes ao filme educacional, chamando atenção para o fato de que somente um método pró-adesão pode ser insuficiente para todos os indivíduos. Conclui-se que a adesão medicamentosa foi maior entre os pacientes maiores de 53 anos, que o filme educativo é uma intervenção pró-adesão eficaz e que a educação continuada, aliada a outro método, poderia ajudar a manter ou ampliar os benefícios conquistados neste trabalho.

Sabe-se que TRAIL é capaz de matar células tumorais de forma seletiva e que TRAIL tem sua expressão reduzida em diversos tumores, porém pouco se sabe sobre os mecanismos responsáveis pela sua inibição. Tendo em vista que a expressão de TRAIL pode ser regulada pelo Ácido Retinóico; que PRAME é capaz de inibir a via do ácido retinóico através da proteína EZH2 e que nós observamos anteriormente que a expressão de TRAIL esta diminuída em pacientes com LMC, nós decidimos investigar a associação entre PRAME, EZH2 e TRAIL na LMC. Nós demonstramos que PRAME, mas não EZH2, tem sua expressão aumentada em células BCR-ABL+ e sua expressão está associada com a progressão da LMC. Alem disto, existe uma correlação positiva entre PRAME e BCR-ABL e negativa entre PRAME e TRAIL nestes pacientes. A inibição da expressão de PRAME ou EZH2 por RNAi induziu um aumento da expressão de TRAIL. Estes dados revelam um novo mecanismo de regulação responsável por diminuir a expressão de TRAIL, e geram novos possíveis alvos para a terapia da LMC e, possivelmente, também para outros tumores. / TRAIL was shown to selectively kill tumor cells. Not surprisingly, TRAIL is down-regulated in a variety of tumor cells, but the mechanism responsible for TRAIL inhibition remains elusive. Because TRAIL can be regulate by retinoic acid; PRAME was shown to inhibit transcription of retinoic acid receptor target genes through the polycomb protein EZH2; and we have found that TRAIL is inversely correlated with BCR-ABL in CML patients, we decided to investigate the association of PRAME, EZH2 and TRAIL in BCR-ABL-positive leukemia. Here, we demonstrate that PRAME, but not EZH2, is up-regulated in BCR-ABL cells and is associated with the progression of disease in CML patients. In addition, PRAME expression is positively correlated with BCR-ABL and negatively with TRAIL in these patients. Importantly, knocking down of PRAME or EZH2 by RNA interference restores TRAIL expression. Our data reveal a novel regulatory mechanism responsible for lowering TRAIL expression and provide the basis of alternative targets for combined therapeutic strategies for CML.

Après la découverte des mutations de la calréticuline dans les néoplasmes classiques myéloproliferatifs négatifs pour le Ph1, les travaux se sont focalisés sur les deux mutations les plus fréquentes, c'est-à-dire la calréticuline del52 et l’ins5, mais il existe environ 20% de mutants rares de la calréticuline (une cinquantaine), qui ont été classés en type-1 « like » et type-2 « like », classification basée sur leur structure. Cependant il reste à déterminer si cette classification est pertinente du point de vue fonctionnel, ce qui pourrait avoir des conséquences pour la prise en charge des patients et leur traitement. Ici, nous démontrons que deux mutants rares de type-1 (del34 et del46) et un de type-2 (del19) se comportent de manière similaire aux deux mutations fondatrices de cette classification, del52 et ins5, respectivement. Ces résultats ont été validés par des expériences in vivo chez la souris. Tous les mutants de la calréticuline (del19, del34 et del46) nécessitent absolument le récepteur de la thrombopoïétine, appelé MPL, pour induire une transformation cellulaire en provoquant une activation indépendante de la thrombopoïétine de la voie MPL / JAK2-STAT, comme les mutants del52 et ins5. Dans les expériences de transplantation de moelle osseuse de souris, les mutants rares de type-1 sont associés à une progression fréquente de la maladie d’un tableau proche d’une thrombocytémie essentielle à une myélofibrose, tandis que le mutant rare de type 2 est associé à une légère thrombocytose. Du point de vue hématopoïétique, les mutants rares de type-1 provoquent une amplification au niveau des cellules souches hématopoïétiques donc à un stade précoce tandis que les mutants rares de type-2 provoquent une amplification tardive de la mégacaryopoïèse. Grâce à une modélisation protéique basée sur l'homologie des mutants de calréticuline, nous avons identifié des domaines oncogènes qui seraient potentiellement responsables de l'interaction pathologique de la calréticuline et de MPL pour conduire à une activation indépendante de la thrombopoïétine. Maintenant, ces résultats in silico doivent être absolument validés par des études structure fonction. Enfin, nous avons modélisé un nouveau mécanisme de signalisation dans la leucémie myéloïde chronique comprenant IRE-1alpha, un bras de la voie de réponse des protéines mal repliées (UPR), qui pourrait être responsable de la perte de la fonction de la p53 pendant la progression de la leucémie myéloïde chronique vers une leucémie aiguë. Un tel mécanisme pourrait être impliqué dans les autres MPN. / After the discovery of calreticulin mutations in classical Ph1- Myeloproliferative Neoplasms, extensive investigation is underway on the two most frequent mutations, i.e., del52 and ins5, but it remains that the rare calreticulin mutants, which include both type-1 like and type-2 like require a similar investigation for ascertaining whether the classification of type-1 and type-2 has a functional relevance as well as for therapeutic intervention and patient management. Here we demonstrate that type-1 like (del34 and del46) and type-2 like (del19) mutants behave similarly as del52 and ins5 mutants, respectively. Moreover, we validate our findings with in vivo experiments. All the calreticulin mutants (del19, del34 and del46) absolutely require the thrombopoietin receptor, MPL, to induce cell transformation by causing ligand independent activation of the MPL/JAK2-STAT pathway. In mouse bone marrow transplantation experiments, type-1 like mutants are associated with frequent progression from an essential thrombocythemia-like phenotype to myelofibrosis whereas type-2 like mutant is associated with mild thrombocytosis. Type-1 like mutants cause clonal amplification of early hematopoetic stem cells whereas the type-2 like mutant causes late platelet amplification. Further, by homology based protein modeling of calreticulin mutants, we have identified possible oncogenic domains responsible for pathologic interaction of CALR and MPL leading to ligand independent activation of MPL. Now they must be validated by structural-functional studies Finally, we have modelled a novel signaling mechanism in chronic myeloid leukemia comprising of IRE-1alpha, an unfolded protein response (UPR) pathway arm, which may be responsible for loss of the WT p53 function during leukemic development and progression. Such a mechanism may be involved in the other MPNs