Spelling suggestions: "subject:"lymphoproliferative neoplasias""
1 |
Rôle de la calréticuline dans les néoplasmes myéloprolifératifs / Role of calreticulin in myeloproliferative neoplasmEl khoury, Mira 23 November 2016 (has links)
Les néoplasmes myéloprolifératifs (NMPs) classiques BCR-ABL négatifs regroupent la Polyglobulie de Vaquez, la Thrombocytémie Essentielle et la Myélofibrose Primaire. Ce sont des pathologies malignes clonales entraînées par la signalisation constitutive de la voie JAK2/STAT en raison de mutations somatiques acquises qui affectent trois gènes, JAK2, CALR et MPL. Il s’agit des mutations “motrices” de la maladie responsable du syndrome myéloprolifératif et du phénotype. Cependant CALR n’est pas une molécule de signalisation mais une chaperonne du réticulum endoplasmique. En utilisant des lignées dépendantes de facteurs de croissance soit murines (Ba/F3) soit humaines (UT-7), des cellules primaires de patients et des modèles murins nous avons montré que les mutants CALRdel52 et CALRins5 avaient acquis de nouvelles propriétés qui en font des molécules de signalisation en induisant: - une indépendance aux facteurs de croissance uniquement lorsque MPL, le récepteur de la thrombopoïétine est exprimé ; - une phosphorylation constitutive de JAK2, des STAT1, 3 et 5 et une activation faible des voies PI3K/AKT et ERK1/2, suggérant une activation de MPL/JAK2 par les mutants CALR différente de celle induite par JAK2V617F. De manière intéressante un mutant de la CALR ayant une délétion entière de l’exon 9 n’est pas transformant, suggérant que l’activité oncogénique est liée à la présence de la nouvelle séquence C-terminale. L’activation de JAK2 uniquement par MPL en présence des mutants CALR pourrait expliquer le phénotype mégacaryocytaire/plaquettaire de ces NMPs. Cette activation de MPL au contraire de celle exercée par JAK2V617F a lieu non seulement à la membrane mais aussi dans le cytoplasme.Les modèles murins ont montré que les mutants CALR étaient responsables de la maladie et que celle-ci était dépendante de MPL, validant les résultats obtenus sur les lignées.Nous avons également montré que contrairement à JAK2V617F, les mutants de la CALR induisent chez l’homme une dominance clonale très tôt au niveau du compartiment des cellules souches. L’ensemble de ces résultats contribue à une meilleure compréhension du rôle des mutations CALR dans les NMPs. La démonstration que les molécules mutées sont présentes à la surface cellulaire ouvre la voie à des immunothérapies ciblant le nouveau peptide C-terminal. / Classical BCR-ABL negative myeloproliferative neoplasms (MPNs) include three disorders: Polycythemia Vera, Essential Thrombocythemia and Primary Myelofibrosis. They are clonal malignant diseases driven by the constitutive JAK2/STAT signaling pathway due to acquired somatic mutations affecting three genes: JAK2, CALR and MPL. These are the "driver" mutations of the disease responsible of the myeloproliferation and of the disease phenotype. However, CALR is not a signaling molecule, but a chaperonne of the endoplasmic reticulum. Using murine (Ba/F3) and human (UT-7) cell lines dependent on growth factors and primary patient cells and mouse model, we have shown that the CALRdel52 and CALRins5 mutants have acquired new signaling properties and induce:- growth factor independence only when MPL, the thrombopoietin receptor, is expressed;- constitutive phosphorylation of JAK2, of STAT1, 3 and 5 and a low activation of the PI3K/AKT and ERK1/2 pathways, suggesting an activation of MPL/JAK2 by a different manner than JAK2V617F. Interestingly, a CALR mutant deleted for the entire exon 9 has not transformation properties suggesting that the oncogenic activity is related to the presence of the new C-terminal sequence. This JAK2 activation only by MPL in presence of CALR mutants could explain the megakaryocytic/platelet phenotype of these MPNs.The use of a mouse modeling using retroviral vectors and bone marrow transplantation has shown that CALRdel52 and ins5 were really the drivers of the disease and that in vivo the thrombocytosis was dependent of MPL validating the results obtained in vitro.In addition, we have shown that in human, CALR mutants induce a clonal dominance early in the stem cell compartment in ET. This is in sharp contrast with JAK2V617F in ET. Overall, these results contribute to a better comprehension of the role of CALR mutations in MPNs. Furthermore, the demonstration that the CALR mutants are expressed at the cell surface open the way to the development of new immunotherapy targetting the new C-terminus peptide.
|
2 |
Expressão das moléculas da via Hippo em neoplasias mieloproliferativas / Expression of Hippo pathway molecules in myeloproliferative neoplasmsCacemiro, Maira da Costa 23 August 2018 (has links)
As neoplasias mieloproliferativas (NMP) são doenças hematológicas caracterizadas pela proliferação aumentada e acúmulo de células mieloides maduras de uma ou mais séries hematopoéticas: granulocítica, eritrocítica, megacariocítica ou mastocítica. Os pacientes com NMP podem apresentar mutações como a JAK2V617F, MPL e CALR, cujas descrições foram fundamentais para o início da elucidação da fisiopatologia das NMP. As células neoplásicas das NMP apresentam resistência à apoptose e proliferação celular exacerbada. Sabe-se ainda que essas doenças são consideradas oncoinflamatórias e pré-leucêmicas. Apesar de todos esses conhecimentos sobre os mecanismos moleculares e celulares envolvidos na patogênese das NMP, não há até a presente data tratamentos eficazes que curam ou alteram a história natural de progressão dessas desordens para LMA. Pelo exposto, foi aqui investigada a potencial participação dos membros da via de sinalização Hippo na fisiopatologia das NMP BCR-ABL1 negativas mais frequentes, a policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose primária (MF). A via de sinalização Hippo foi descrita como supressora de tumor e é uma das responsáveis pela regulação da proliferação, diferenciação e morte celular. Foi analisada, ainda nesse estudo, a correlação dos níveis de expressão dos genes da via Hippo com o perfil de citocinas plasmáticas dos pacientes com PV, TE e MF, a associação com o \"status mutacional\" e com a expressão dos genes que regulam a apoptose celular pela via intrínseca. Os principais achados nos pacientes com PV foram a diminuição da expressão dos genes supressores de tumor LATS2, MST1 e MST2 acompanhada pela presença de elevada concentração de citocinas pró-inflamatórias e fenótipo de resistência a apoptose. Na TE, os dados relevantes foram a detecção da diminuição da expressão dos genes supressores de tumor LATS1, LATS2 MST1 e SAV1, e o observação da relação entre alta expressão dos genes SAV1 e MOB1B e a mutação da CALR. Em MF destaca-se a redução da expressão dos genes SAV1 e TAZ da via de sinalização Hippo e do gene AURKB do ciclo celular. Em conclusão, os dados indicam que a diminuição da expressão dos genes supressores de tumor da via Hippo contribui para a fisiopatologia e para o fenótipo de resistência das células neoplásicas à apoptose das NMP. / Myeloproliferative neoplasms (MPN) are hematological disorders characterized by increased proliferation of mature myeloids cells of one or more hematopoietic series: granulocytic, erythrocytic, megakaryocytic or mastocytic. Patients with MPN may present mutations such as JAK2V617F, MPL and CALR, which were essential descriptions for the beginning of pathophysiological elucidation of MPN. The NMP neoplastic cells show resistance to apoptosis and exacerbated cell proliferation. It is known that these entities are considered also oncoinflamatory and pre-leukaemic. Despite all this knowledge about the molecular and cellular mechanisms involved in the pathogenesis of MPN, there are no effective treatments that cure or alter the natural history of progression of these disorders to AML. For the above, a participatory potential of the Hippo signaling pathway members in the pathophysiology of the most frequent negative BCR-ABL1 MPN, the polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) were investigated. The Hippo signaling pathway has been described as tumor suppressor and is responsible for the regulation of proliferation, differentiation and cell death. The correlation analysis of the expression levels of the Hippo pathway genes with the plasma cytokine profile of patients with PV, ET and PMF, the association with the mutational status and the expression of the genes that regulate intrinsic cellular apoptosis were performed. The main findings in patients with PV were decreased expression of the tumor suppressor genes LATS2, MST1 and MST2 accompanied by the presence of high concentration of proinflammatory cytokines and phenotype of resistance to apoptosis. In ET, relevant data were the detection of decreased expression of the tumor suppressor genes LATS1, LATS2 MST1 and SAV1, and the observation of the relationship between high expression of the SAV1 and MOB1B genes and the mutation of the CALR. In PMF, stands out the reduction of the SAV1 and TAZ gene expression of the Hippo signaling pathway and the AURKB gene of the cell cycle. In conclusion, the data indicate that decreased expression of the tumor suppressor genes of the Hippo pathway contributes to the pathophysiology and neoplastic cell resistance phenotype to the apoptosis of MPN.
|
3 |
Expressão de Galectinas-1 e 3 em Neoplasias Mieloproliferativas / Galectin-1 and 3 expression in Myeloproliferative NeoplasmsMoura, Lívia Gonzaga 26 October 2012 (has links)
Doenças Mieloproliferativas Crônicas são desordens hematológicas malignas caracterizadas pela alteração na célula-tronco hematopoética e independência ou hipersensibilidade dos progenitores hematopoéticos a citocinas. Em 2008 a OMS renomeou esse grupo como Neoplasias Mieloproliferativas (NMPs), no qual estão inclusas as entidades nosológicas Policitemia Vera (PV), Trombocitemia Essencial (TE) e Mielofibrose Primária (MFP), doenças alvo desse estudo. Apesar dos avanços no diagnóstico das NMPs e nos mecanismos envolvidos com a fisiopatologia dessas doenças, sua patogênese permanece desconhecida. Alterações na maquinaria apoptótica parecem estar envolvidas em na fisiopatologia das NMP e por isso a compreensão dos mecanismos de regulação da apoptose e a interferência das galectinas-1 e 3 nesse processo, em pacientes com NMPs, é relevante para a busca de novos alvos terapêuticos. Neste contexto, os objetivos deste trabalho foram: avaliar em leucócitos de sangue periférico e células tronco hematopoéticas CD34+ de medula óssea dos pacientes com PV, TE e MFP os níveis de expressão das LGALS1 e LGALS3 e a concentração de galectina-3 plasmática. Foram determinadas as correlações dos níveis de expressão de LGALS1 e LGALS3 e da concentração da galectina-3 plasmática com os níveis de expressão do RNAm das moléculas reguladoras da apoptose e com os dados clínico-laboratoriais dos pacientes como a concentração de hemoglobina, percentagem de hematócrito, porcentagem de alelos mutados JAK2V617F, contagem de leucócitos e esplenomegalia. A expressão de LGALS1 estava diminuída em células CD34+ em PV e MFP e em leucócitos de sangue periférico de pacientes com MFP. Os pacientes de TE apresentaram aumento na expressão de LGALS3 em leucócitos de sangue periférico e alta concentração de galectina-3 no plasma. Houve correlação entre os níveis de expressão de LGALS1 e a porcentagem de alelos mutados e a contagem de leucócitos, em pacientes com PV. Foi detectada a correlação entre os níveis de expressão de LGALS3 a porcentagem de alelos mutados e o tamanho do baço, em pacientes com MFP. Com relação aos genes reguladores da apoptose, foram observadas correlações entre os níveis de expressão de LGALS1 e BCL-2 em células CD34+ de pacientes PV e entre LGALS3 e A1, MCL-1, BAX e C-FLIP em leucócitos de de pacientes com TE. Os resultados obtidos indicam que as NPM apresentam expressão diferencial de LGALS1 e LGALS3 e sugerem a associação entre a expressão de galectinas e o status da mutação JAK2V617F, principalmente em pacientes com MFP / Chronic myeloproliferative diseases are haematological malignant disorders characterized by the presence of an altered haematopoietic stem cell and independence or hypersensibility of their hematopoietic progenitors to cytokines. In 2008, WHO renamed this group of diseases as Myeloproliferative Neoplasms (MPN) in which is included Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). There have been advances concerning the knowledge about the mechanisms involved in MPN pathophysiology, however their pathogenesis remains unknow. Deregulation in apoptotic machinery seems to be involved in MPN pathophysiology. Fully understanding of apoptotic machinery and the influence of galectin-1 and 3 in this process in NMP patients might unveil novel targets for manipulation. The aims of the present study were to evaluate in leukocytes and CD34+ hematopoietic stem cells from PV, ET and PMF patients the LGALS1 and LGALS3 expression levels, the Galectin-3 plasma levels and to correlate LGALS1 and LGALS3 expression levels with galectin-3 plasma levels, apoptosis-related genes expression, JAK2 mutation status and clinic-laboratorial parameters. PV and PMF patients showed decreased expression levels of LGALS1 in CD34+ cells and also decreased LGALS1 expression levels in PMF leukocytes. ET patients presented an increased expression level of galectin-3 in leukocytes and plasma. We detected the correlations between LGALS3 gene expression with JAK2 allele burden and with leukocytes number in PV patients. We also observed in PMF patients the correlation between LGALS3 expression levels with JAK2 allele burden and spleen size. We also detected the correlation between LGALS1 expression levels BCL-2 gene expression in PV CD34+ HSC cells and between LGALS3 expression and A1, MCL-1, BAX and C-FLIP gene expression in TE leukocytes. Taken together, the results suggest the LGALS1and LGALS3 differential expression in NMP and the relation between JAK2V617F status with galectins expression, especially in PMF patients.
|
4 |
Identificação da família BCL2 como alvo terapêutico no tratamento das neoplasias mieloproliferativas associadas à mutação da JAK2V617F / BCL2 family as potential therapeutical targets in the treatment of JAK2V617F- associated myeloproliferative neoplasmsLeal, Cristina Tavares 01 September 2017 (has links)
As neoplasias mieloproliferativas (NMPs) negativas para o rearranjo t(9;22)/BCRABL1, incluindo Policitemia Vera (PV), Trombocitemia Essencial (TE) e Mielofibrose Primária (MFP), são doenças hematopoéticas clonais e estão frequentemente associadas à mutação JAK2V617F. Apesar dos avanços no conhecimento da fisiopatologia após a descoberta da mutação JAK2V617F e do desenvolvimento de inibidores da JAK2, o tratamento permanece não curativo. Sabe-se que as célulastronco mais primitivas nas NMPs são responsáveis pela iniciação da doença e que a expansão dos precursores mieloeritróides contribui para o fenótipo clínico. Dados recentes obtidos com ensaios in vitro mostram que as proteínas da família BCL2, reguladoras da apoptose mitocondrial, desempenham um papel relevante na patogênese das NMPs. Acreditamos que a expressão anômala de BCL2 nas células progenitoras hematopoéticas (CPH) das NMPs pode contribuir para a patogênese desse grupo de doenças. Avaliamos a expressão gênica, por meio de PCR em Tempo Real, da família BCL2 (genes antiapoptóticos BCL-xL e BCL2 e o pró-apoptótico BIM) nas diferentes subpopulações de progenitores hematopoéticos murinos (de um modelo condicional knockin de expressão heterozigótica condicional da Jak2V617F) e de pacientes portadores de NMPs bem como sua contribuição para o fenótipo da doença e resposta ao inibidores da JAK2 (com a droga ruxolitinibe) e/ou inibição da família BCL2 (com o inibidor de BCL2 obatoclax). Não encontramos diferença de expressão basal dos genes BCL2, BCL-xL e BIM nas células CD34+ bem como nas subpopulações de células CD34+38-/+ de pacientes com NMPs, independente da presença da mutação JAK2V617F, em relação às células CD34+ e subpopulações CD34+38-/+ dos controles (p>0.05). Nas células CD34+ de pacientes com TE encontramos aumento de expressão de BCL2 em relação às células CD34+ pacientes com MFP (p=0.03). No modelo transgênico de camundongos Jak2 wt/VF (que apresentam uma NMP semelhante à PV) e Jak2 wt/wt (controles), comparamos a expressão diferencial dos genes da família Bcl2 em precursores hematopoéticos imaturos (LSKs) e progenitores mieloides mais maduros (MPs). A expressão do BclxL em MPs de camundongos wt/VF foi maior em relação à subpopulação de células LSKs e em relação as duas subpopulações de células dos controles (p=0.0011). Não houve diferença significativa de expressão do Bcl2 nas subpopulações de células LSKs e MPs de animais wt/VF e wt/wt (p=0.12). Observou-se menor expressão de Bim em LSKs em relação às células MPs dos animais mutados (p=0.026), diferença essa não observada entre os controles Jak2 wt/wt. O tratamento isolado com inibidor de JAK2 ou de BCL2 resultou em aumento de expressão do Bim nas CPH (LSKs e MPs) de camungongos Jak2 wt/VF em relação aos animais Jak2 wt/wt. Este aumento da expressão de Bim foi ainda mais evidente após o tratamento das células com a combinação das duas drogas quando comparadas às células não tratadas ou tratadas com um dos dois inibidores, sendo maior em animais doentes do que em animais controles (p<0.0001). A análise do efeito do tratamento com os inibidores de JAK2 e BCL2 na indução de apoptose por meio de citometria de fluxo (marcação com anexina/7-AAD) revelou que as células LSKs foram mais resistentes à apoptose tardia do que as células MPs independentemente da mutação da JAK2 (p<0.05). O tratamento com obatoclax resultou em indução de apoptose diferentemente do que foi observado com o tratamento com ruxolitinibe (p=0.594) nas células MPs de animais Jak2 wt/VF. Ademais, o tratamento combinado com ruxolitinibe e obatoclax resultou no aumento da apoptose nas células MPs dos animais com fenótipo de PV (Jak2 wt/VF) em relação aos animais Jak2 wt/wt (p=0.05). Em conclusão, demonstramos que a resistência à apoptose nas NMPs ocorre desde as CPH iniciadoras da doença. Nossos resultados sugerem que a modulação da apoptose mitocondrial pode ser uma nova estratégia terapêutica para pacientes com NMP em combinação aos inibidores de JAK2, na medida em que atua tanto nas CPH que iniciam a doença como nos MPs, responsáveis pelos sinais e sintomas de mieloproliferação. / Myeloproliferative Neoplasms (MPNs) negative for t(9;22)/BCR-ABL1 rearrangement, including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF), are clonal hematopoietic diseases and are often associated with the JAK2V617F mutation. Despite advances in the pathophysiology knowledge after the discovery of the JAK2V617F mutation and the development of JAK2 inhibitors, treatment remains non-curative. It is known that MPN primitive stem cells are essential for the initiation of the disease and that the expansion of the myeloeritroid precursors contributes to the clinical phenotype. Recent data, obtained with in vitro assays, showed that BCL2 family proteins, regulators of mitochondrial apoptosis, play a relevant role in the pathogenesis of MPNs. We believe that the anomalous expression of BCL2 in hematopoietic progenitor cells (HPCs) of MPNs may contribute to their pathogenesis. We evaluated BCL2 family (antiapoptotic genes BCL-xL and BCL2 and the pro-apoptotic BIM) gene expression by real-time PCR in different subpopulations of hematopoietic progenitors from a conditional Jak2V617F knockin murine model and from patients with MPNs as well as their contribution to the disease phenotype and response to JAK2 inhibitors (with ruxolitinib) and/or to the inhibition of the BCL2 family (with the BH3-mimetic obatoclax). We found no difference in the basal expression of the BCL2, BCL-xL and BIM in CD34+ cells as well as in subpopulations of CD34+ 38-/+ cells from patients with MPNs, regardless of the presence of the JAK2V617F mutation. In CD34+ cells obtained from patients with ET, we found an increase of BCL2 expression when compared to CD34+ cells with PMF (p=0.03). In the Jak2 wt/VF transgenic mice (that develop a MPN similar to PV) and Jak2 wt/wt controls, we compared the differential expression of Bcl2 family genes in immature hematopoietic precursors (LSKs) and more mature myeloid progenitors (MPs). Expression of Bcl-xL in MPs of wt/VF mice was greater when compared to LSKs and to the two progenitor subpopulations of control cells (p=0.0011). There was no significant difference in Bcl2 expression between the subpopulations of LSKs and MPs from wt/VF and wt/wt animals (p=0.12). Lower Bim expression in LSKs than in MPs was observed in samples from JAK2-mutated animals (p=0.026). Such difference was not observed between the Jak2 wt/wt subpopulations. Treatment with JAK2 or BCL2 inhibitors alone resulted in increased Bim expression in LSKs and MPs of the Jak2 wt/VF mice when compared to Jak2 wt/wt animals. This increase in Bim expression was even more evident when these cells were treated with the combination of the two drugs as compared to single treatment with one of the two inhibitors, being higher in mutaded than control animals (p<0.0001). The analysis of apoptosis by flow cytometry (annexin / 7-AAD labeling) revealed that LSK cells were more resistant to late apoptosis than MP cells regardless of the JAK2 mutation (p<0.05). Treatment with obatoclax resulted in greater apoptosis induction than it was observed with ruxolitinib treatment (p=0.594) on MP cells of Jak2 wt/VF animals. In addition, the combined treatment with ruxolitinib and obatoclax resulted in increased apoptosis in MP cells of animals with the PV phenotype (Jak2 wt/VF) as compared to the Jak2 wt/wt animals (p=0.05). In conclusion, we demonstrated that resistance to apoptosis in MPNs occurs at the level of the hematopoietic progenitors that initiate the disease. Our results suggest that modulation of mitochondrial apoptosis may be a new therapeutic strategy for MPN patients in combination with JAK2 inhibitors, as it acts on both the disease initiating and more mature progenitors, responsible for the clinical findings of myeloproliferation.
|
5 |
Impacto da análise molecular da mutação JAK2V617F no diagnóstico de neoplasias mieloproliferativas crônicas de acordo com os critérios da OMS 2016Pedrazzani, Fabiane Spagnol January 2016 (has links)
As neoplasias mieloproliferativas (NMPs) são um grupo de doenças derivadas de uma transformação clonal de célula tronco hematopoiéticas no qual a linhagem celular mielóide é predominantemente expandida no sangue periférico. As NMPs Philadelphia-negativas incluem policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose primária (MFP) que compartilham muitas características hematológicas, clínicas e evolutivas. A mutação da JAK2 (JAK2V617F) está presente em cerca de 95% dos pacientes com PV, entre 50 a 70% com TE e 40 a 50% com MFP. No entanto, os testes moleculares para diagnóstico são muitas vezes um desafio devido ao alto custo e a disponibilidade de equipamentos especializados. Objetivo: Verificar o impacto do teste molecular da mutação JAK2V617F para o diagnóstico de NMPs nos pacientes atendidos no Hospital de Clínicas de Porto Alegre. Métodos: Foram avaliados 87 pacientes com suspeita de NMPs. As amostras de sangue periférico foram analisadas para a mutação JAK2V617F pelo método genético molecular de PCR alelo-específico e os resultados correlacionados com os dados clínico-laboratoriais. Para estabelecimento do diagnóstico, foram utilizados os critérios da Organização Mundial da Saúde (OMS) de 2016. Resultados: Dos 87 pacientes avaliados, 27,6% foram diagnosticados como PV, 39,1% como TE, 4,6% como MFP e 28,7% não contemplavam os critérios para o diagnóstico NMPs. A comparação da utilização do teste da mutação JAK2V617F mostrou que, apenas 41,7% dos pacientes com PV sem utilizar o teste, teriam sido diagnosticados comparados a 91,7% utilizando este teste como um dos critérios no diagnóstico final (p = 0,004). Na TE e na MFP, este critério não foi estatisticamente significativo. Conclusão: O teste molecular para a mutação de JAK2V617F no nosso hospital teve um impacto significativo no diagnóstico dos pacientes com PV, mostrando ser uma ferramenta importante para o diagnóstico final desta NMP. / Myeloproliferative neoplasms (MPNs) are a group of disorders derived from a clonal transformation of stem cell on which myeloid cell lineage is predominantly expanded in the peripheral blood. Philadelphia-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) which share many hematological, clinical, and evolutionary characteristics. The JAK2 mutation (JAK2V617F) is present in about 95% of patients with PV, between 50 to 70% with ET and 40 to 50% PMF. However, the molecular diagnostic tests are often a challenge due to the high cost and the availability of specialized equipment. Objective: To verify the impact of molecular testing of the JAK2V617F mutation for the diagnosis of MPNs in patients attended at Hospital de Clinics, Porto Alegre. Methods: A total of 97 patients were evaluated with suspected of MPNs. The peripheral blood samples were analyzed for the JAK2V617F mutation by the molecular genetic allelespecific PCR method and the results correlated with the clinical-laboratory data. To establish the diagnosis, the 2016 World Health Organization (WHO) criteria were used. Results: Of the 87 patients evaluated, 27.6% were diagnosed as PV, 39.1% as ET, 4.6% as PMF and 28.7% did not meet criteria for MPNs diagnosis. Comparison of the use of the JAK2V617F test showed that only 41.7% of patients with PV without the mutation test were diagnosed compared to 91.7% using this test as one of the criteria for the final diagnosis (p = 0.004). In the ET and the PMF, this criterion was not statistically significant. Conclusion: The molecular test for the JAK2V617F mutation in our hospital had a significant impact in the diagnosis of patients with PV, showing to be an important tool for the final diagnosis of this MPN.
|
6 |
Modélisation des néoplasmes myéloprolifératifs grâce aux cellules souches induites à la pluripotence (IPSC) / Modeling of myeloproliferative neoplasms thanks to an induced pluripotent stem cell model (IPSC)Secardin, Lise 25 November 2016 (has links)
Les néoplasmes myéloprolifératifs (NMP) sont hémopathies malignes aboutissant à la surproduction d'une ou plusieurs lignées myéloïdes. Elles sont dues à l'acquisition de mutations sur l'axe de signalisation MPL/JAK2 incluant des mutations de JAK2V617F, de MPL et plus récemment de la calréticuline (CALR), dont les deux principales sont CALRdel52 et CALRins5. Ces mutations de signalisations peuvent être accompagnées de mutations de l'épigénétique, les plus importantes étant des mutations dans TET2. Le but de cette thèse était d'étudier le rôle des mutations de TET2 et de la calrdel52 dans les NMP grâce à une technologie de cellules souches induites à la pluripotence (IPSC). Dans la première partie j'ai pu démontrer que TET2 joue un rôle dans le processus de reprogrammation, vraisemblablement de manière indépendante de son activité catalytique. Dans la seconde partie, j'ai démontré que CALRdel52 joue un rôle dans les MPN en provoquant une hypersensibilité et une pousse indépendante de la TPO des progéniteurs mégakaryocytaires ainsi qu'une hyperprolifération des mégacaryocytes, liées à l'activation constitutive de stat3 et de ERK. J'ai également démontré une pousse indépendante du GCSF des granulocytes. Ce travail a donc permis de mettre en lumière le rôle du facteur épigénétique TET2 dans le processus de reprogrammation ainsi que le rôle de CALRdel52 dans les MPN dans un contexte d'expression endogène. / Myeloproliferative neoplasms (NMP) are hematological malignancies that lead to an ovrproduction of one or more myeloid lineages. They are driving by mutations in MPLl/jak2 signaling pathway, mainly JAK2V617F, MPL, and more recently calreticulin (CARL), with two main mutations being calrdel52 and calrins5. These signaling mutations are sometimes associated with epigenetic mutations, the major one being in tet2. The objective of my thesis was to study the role of TET2 and CALRdel52 in MPN thanks to an induced pluripotent stem cells (IPSC) model. In the first part i demonstrated the role of TET2 in reprogramming process, probably independently of the catalytic domain. In the second part i demonstrated that CALRdel52 induced a TPO hypersensitivity and a TPO indenpendant growth of the megakaryocytic progenitors as well as a hyperproliferation of the megakaryocytes. This phenotype is associated with a constitutive activation of stat3 and ERK. A G-CSF independent growth of the granulocyte was also demonstrated. In conclusion this work underline the role of an epegenetic factor, TET2, in the reprogramming process and demonstrate the role of CALRdel52in MPN with an endogenous expression model.
|
7 |
The Mevalonate Pathway: A Potential Therapeutic Target for JAK2-driven Myeloproliferative NeoplasmsGriner, Lori Nicole 01 January 2013 (has links)
The Mevalonate Pathway: A Potential Therapeutic Target for JAK2-driven Myeloproliferative Neoplasms
Lori Nicole Griner
Abstract
Myeloproliferative neoplasms (MPNs) are diseases of hematopoietic stem cell origin and are characterized by uncontrolled growth of cells of the myeloid compartment. The Philadelphia chromosome negative classical MPNs, including polycythemia vera, essential thrombocythemia, and myelofibrosis, are diseases of dysregulated JAK2 signaling. In fact, the majority of MPN patients have activating mutations in JAK2 (e.g JAK2-V617F), a tyrosine kinase that contributes to the growth and survival of myeloid cells. While MPNs were first described over sixty years ago, a significant need remains to develop therapeutic strategies for them. Inhibitors of JAK2 are currently being developed, and one inhibitor, ruxolitinib, was recently approved for certain MPN patients. Ruxolitinib has made profound impacts on improving splenomegaly and constitutional symptoms in MPN patients, but it and other JAK2 inhibitors have not significantly reduced the JAK2 mutant allele burden, and thus such inhibitors have not induced remission in these patients. The current consensus in the MPN field supports JAK inhibition for the treatment of patients, but a further understanding of MPNs and JAK2 signaling, as well as improved JAK2 inhibitors, may be necessary for treating MPN patients.
The work described in this dissertation has uncovered novel requirements for JAK2-V617F-driven signaling and transformation. We demonstrate that JAK2-V617F co-localizes with lipid rafts, cholesterol-rich microdomains within the plasma membrane that function to serve as platforms for signaling complex formation. Signaling complex formation is a necessary component for dysregulated signaling induced by JAK2-V617F. We provide evidence that cholesterol altering-lipid raft disrupting agents attenuate JAK2-V617F-driven signaling. We also show that cholesterol-lowering statins are effective at downregulating JAK2 signaling and inducing apoptosis in JAK2-V617F-driven cell lines. Importantly, we show that statins, inhibitors of the mevalonate pathway, inhibit the growth of primary MPN cells, while the same statin doses have no effect on healthy controls. Impressively, we demonstrate that statins cooperate with multiple JAK inhibitors, including ruxolitinib, to inhibit cell growth and induce apoptosis of JAK2-V617F-driven cells.
This report establishes statin-mediated inhibition of the mevalonate pathway as a potential approach to improve MPN therapeutics. We propose future studies with statins and JAK2 inhibitors in the treatment of MPNs.
|
8 |
Meilensteine in der Verlaufskontrolle von Patienten mit JAK2 p.V617F positiver myeloproliferativer Neoplasie nach StammzelltransplantationEdelmann, Anja 30 June 2014 (has links) (PDF)
Das Ziel der vorliegenden Arbeit war die Quantifizierung JAK2 p.V617F mutierter Allele zur Verlaufskontrolle von Patienten mit JAK2 p.V617F positiven MPN nach allogener Stammzelltransplantation (SCT). Dabei sollte insbesondere untersucht werden, ob sich frühzeitig nach SCT ein höheres Rezidivrisiko der MPN vorhersagen lässt und zu welchen Zeitpunkten molekulare Untersuchungen nach SCT sinnvoll sind.
Wir analysierten retrospektiv den Krankheitsverlauf von 30 Patienten. Dafür verwendeten wir die ARMS-QPCR und WTB-AS QPCR als zwei allel-spezifische Amplifikationsmethoden und untersuchten 142 Proben der ersten Kohorte (n=14) und 32 Proben einer zweiten Kohorte (n=16) im direkten Vergleich.
Aus unseren Ergebnissen konnten folgende Rückschlüsse gezogen werden:
1. Die beiden allel-spezifischen Amplifikationsmethoden ARMS-QPCR und WTB-AS QPCR zur Quantifizierung der JAK2 p.V617F Mutation sind vergleichbar.
2. Als Ausgangsmaterial sind antikoaguliertes Vollblut oder auch Beckenkammbiopsien gleichermaßen geeignet.
3. Der Nachweis von > 1% JAK2 p.V617F Allele 28 Tage nach allogener SCT ist assoziiert mit einem signifikant höheren Rezidivrisiko einer JAK2 positiven MPN und einem schlechteren Gesamtüberleben.
|
9 |
Etude de la calréticuline dans les syndromes myéloprolifératifs : de la détermination de la charge allélique aux mécanismes de dégradation des variants protéiques / Study of calreticulin in myeloproliferative neoplasms : from allelic burden determination to mechanisms of variant proteins degradationMansier, Olivier 14 December 2017 (has links)
Des mutations dans le gène de la calréticuline (CALR), codant pour une protéine résidente du réticulum endoplasmique (RE), ont été découvertes récemment dans les syndromes myéloprolifératifs (SMP). Elles sont associées à augmentation de prolifération cellulaire portant spécifiquement sur la lignée mégacaryocytaire. Ceci est le résultat d’une activation constitutive de la signalisation des voies JAK-STAT et MAP Kinases, consécutive à l’interaction des protéines mutantes CALR avec le récepteur à la thrombopoïétine. Plusieurs études ont montré la faible expression de ces protéines mutées dans les cellules, mais aucune n’a déterminé l’impact de leur expression sur l’homéostasie du RE ni les acteurs mis en jeu dans leur élimination. Dans ce travail, nous avons montré que l’expression des protéines CALR mutées ne perturbe pas sensiblement l’équilibre du RE et ne modifie pas la sensibilité des cellules à l’apoptose induite par un stress du RE. Nous avons ensuite démontré dans différents modèles, y compris des cellules engagées dans la différenciation mégacaryocytaire, que les faibles niveaux intracellulaires de variants protéiques CALR n’étaient pas liés à une sécrétion accrue dans le milieu extracellulaire ni à un défaut transcriptionnel. Cette faible expression est en fait la conséquence d’une dégradation mettant en jeu principalement la voie ERAD-protéasome. Dans ce processus, la reconnaissance de motifs glycans n’est pas impliquée, mais EDEM3 semble avoir un rôle majeur puisque son extinction augmente l’expression des formes mutées de CALR. La modulation de cette dégradation pourrait constituer une approche thérapeutique innovante dans les SMP. / Mutations in the calreticulin gene (CALR), encoding for an endoplasmic reticulum (ER) resident protein, have recently been discovered in myeloproliferative neoplasms (MPN). They are associated with an increased cell proliferation, specifically in the megakaryocytic lineage. This is the result of a constitutive activation of the JAK-STAT and MAP kinase pathways, following the interaction of mutant calreticulin proteins with the thrombopoietin receptor. Several studies have demonstrated that these mutated proteins are faintly expressed in cells, but none have determined the impact of their expression on ER homeostasis, nor addressed the actors at play in their degradation. In this work, we showed that the expression of mutated CALR proteins does not significantly disturb ER equilibrium, nor does it change the cellular sensitivity to ER stress-induced apoptosis. We next demonstrated in different models including cells committed towards megakaryocytic differentiation that the poor intracellular levels of variant CALR proteins are neither due to enhanced secretion into the extracellular medium, nor to transcriptional defects. This low-level expression is mainly the result of increased degradation, involving the ERAD-proteasome pathway. In this process, the recognition of glycan motifs is not engaged, but EDEM3 seems to be a key component as its extinction increases the expression levels of variant forms of CALR. Modulating this degradation process could represent a therapeutic option for MPN patients.
|
10 |
Impacto da análise molecular da mutação JAK2V617F no diagnóstico de neoplasias mieloproliferativas crônicas de acordo com os critérios da OMS 2016Pedrazzani, Fabiane Spagnol January 2016 (has links)
As neoplasias mieloproliferativas (NMPs) são um grupo de doenças derivadas de uma transformação clonal de célula tronco hematopoiéticas no qual a linhagem celular mielóide é predominantemente expandida no sangue periférico. As NMPs Philadelphia-negativas incluem policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose primária (MFP) que compartilham muitas características hematológicas, clínicas e evolutivas. A mutação da JAK2 (JAK2V617F) está presente em cerca de 95% dos pacientes com PV, entre 50 a 70% com TE e 40 a 50% com MFP. No entanto, os testes moleculares para diagnóstico são muitas vezes um desafio devido ao alto custo e a disponibilidade de equipamentos especializados. Objetivo: Verificar o impacto do teste molecular da mutação JAK2V617F para o diagnóstico de NMPs nos pacientes atendidos no Hospital de Clínicas de Porto Alegre. Métodos: Foram avaliados 87 pacientes com suspeita de NMPs. As amostras de sangue periférico foram analisadas para a mutação JAK2V617F pelo método genético molecular de PCR alelo-específico e os resultados correlacionados com os dados clínico-laboratoriais. Para estabelecimento do diagnóstico, foram utilizados os critérios da Organização Mundial da Saúde (OMS) de 2016. Resultados: Dos 87 pacientes avaliados, 27,6% foram diagnosticados como PV, 39,1% como TE, 4,6% como MFP e 28,7% não contemplavam os critérios para o diagnóstico NMPs. A comparação da utilização do teste da mutação JAK2V617F mostrou que, apenas 41,7% dos pacientes com PV sem utilizar o teste, teriam sido diagnosticados comparados a 91,7% utilizando este teste como um dos critérios no diagnóstico final (p = 0,004). Na TE e na MFP, este critério não foi estatisticamente significativo. Conclusão: O teste molecular para a mutação de JAK2V617F no nosso hospital teve um impacto significativo no diagnóstico dos pacientes com PV, mostrando ser uma ferramenta importante para o diagnóstico final desta NMP. / Myeloproliferative neoplasms (MPNs) are a group of disorders derived from a clonal transformation of stem cell on which myeloid cell lineage is predominantly expanded in the peripheral blood. Philadelphia-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) which share many hematological, clinical, and evolutionary characteristics. The JAK2 mutation (JAK2V617F) is present in about 95% of patients with PV, between 50 to 70% with ET and 40 to 50% PMF. However, the molecular diagnostic tests are often a challenge due to the high cost and the availability of specialized equipment. Objective: To verify the impact of molecular testing of the JAK2V617F mutation for the diagnosis of MPNs in patients attended at Hospital de Clinics, Porto Alegre. Methods: A total of 97 patients were evaluated with suspected of MPNs. The peripheral blood samples were analyzed for the JAK2V617F mutation by the molecular genetic allelespecific PCR method and the results correlated with the clinical-laboratory data. To establish the diagnosis, the 2016 World Health Organization (WHO) criteria were used. Results: Of the 87 patients evaluated, 27.6% were diagnosed as PV, 39.1% as ET, 4.6% as PMF and 28.7% did not meet criteria for MPNs diagnosis. Comparison of the use of the JAK2V617F test showed that only 41.7% of patients with PV without the mutation test were diagnosed compared to 91.7% using this test as one of the criteria for the final diagnosis (p = 0.004). In the ET and the PMF, this criterion was not statistically significant. Conclusion: The molecular test for the JAK2V617F mutation in our hospital had a significant impact in the diagnosis of patients with PV, showing to be an important tool for the final diagnosis of this MPN.
|
Page generated in 0.1299 seconds