Essential thrombocythemia (ET) is a rare myeloproliferative neoplasm affecting 43.7 out of every 100,000 people in the United States. The disease is characterized by abnormally high platelet counts, mutational abnormalities in Janus Kinase 2 (JAK2)/Calreticulin (CALR)/myeloproliferative leukemia virus oncogene (MPL), and increased megakaryocyte production and differentiation. The average age of onset for patients with ET is between 65-70 years, but recent studies have demonstrated a downward trend in the age of diagnosis. Mechanistically, ET mutations cause the dimerization of JAK and upregulation of the JAK-STAT pathways. Common treatment approaches seek to use cytoreduction and platelet inhibition to lower the risk of a thrombotic event. Hydroxyurea and low-dose aspirin have been the gold-standard of treatment for ET patients. This thesis sought to compare the current available therapy with second-line treatments and investigational treatments. Anagrelide is a key second-line treatment for ET that is used in the event of intolerance to hydroxyurea. It acts through cytoreductive mechanisms which result in a decreased platelet count. Major bleeding is a severe adverse event associated with anagrelide. Interferons are another second-line defense in the treatment of ET despite a lack of FDA approval for this indication. Interferons act directly to reduce platelet counts and, unlike other drug classes, mount an immunological response against the JAK2 stem cells to reduce the allelic burden. An immunological approach to ET may be key to the sustained treatment of the disorder without a daily dosing regimen. Despite the promise of interferons, severe adverse effects limit the adherence of many patients to this class of drugs. JAK inhibitors are an investigational drug class that acts directly through the JAK-STAT pathway. JAK inhibitors have shown little efficacy in the treatment of ET and may be better suited for treatment in combination therapies. Telomerase inhibitors are one such investigational drug class that may pair well with JAK inhibitors for the treatment of ET. All of these drug classes were compared to hydroxyurea with respect to their pharmacokinetics, pharmacodynamics, and patient evaluation. Hydroxyurea and low-dose aspirin showed superiority in comparison to other drug classes due to their low toxicity profile and minimum adverse side-effects, high oral bioavailability and wide distribution, high adherence, and production of the most uniform response to reducing thrombotic events and platelet counts. The interferon drug class shows unique potential for the treatment of ET and should be placed above the second-line treatment standard of anagrelide due to its benefits in treatment of younger and pregnant patients. Interferons are the only class of drug for the treatment of ET that did not increase the risk of drug-related leukemogenic transformations. Despite non-adherence due to side-effects and lack of an oral administration, interferons are superior to anagrelide due to their longer dosing interval and immunological attack on JAK2 stem cells. Treatment of ET with anagrelide has shown similar efficacy to hydroxyurea and interferons in platelet reduction and rivals hydroxyurea in the prevention of thrombotic risk. Despite this benefit, the risk of bleeding associated with anagrelide is a significant disadvantage. Hydroxyurea and low-dose aspirin remain the current standard of treatment for patients with ET, although new approaches may soon be available.

OBJETIVO: Investigar o efeito da expressão de RNAm dos SMADs e de microRNAs (miRNAs) que possuem o TGFB1 como alvo na expressão gênica (RNAm e proteína) de TGF-β1 e seu papel na fisiopatologia da angiogênese em pacientes com mielofibrose (MF) e trombocitemia essencial (TE). MÉTODOS: Foram incluídos 21 pacientes com MF primária (MFP), 21 com MF pós-TE (MFPTE) e 24 com TE, além de 98 indivíduos controles pareados de acordo com gênero e idade com os pacientes. As análises realizadas no sangue periférico foram: quantificação das concentrações plasmáticas e de RNAm de TGFB1, VEGFA e FGF2; quantificação de RNAm de SMADs 1 a 7 e de miRNAs 193a-5p, 369-5p, 542-5p, 590-3p, e 590- 5p; e detecção das mutações JAK2V617F (com quantificação alélica), MPLW515K/L e CALR. Em 26 biópsias de medula óssea dos pacientes, foram determinados o grau de microvasculatura (angiogênese estimada - CD34), a imunoexpressão de TGF-b1 ativo, TGF-β1 latente e c-MPL. RESULTADOS: As concentrações de TGF- β1 plasmático foram semelhantes entre os pacientes e controles, enquanto o VEGFA plasmático foi maior em todos os grupos de pacientes comparados aos seus controles. O FGF2 plasmático também foi maior em todos os grupos de pacientes, e a expressão de seu RNAm foi maior nos pacientes com TE do que em seus controles. As expressões de SMADs e de miRNAs foram semelhantes entre pacientes e controles. TGF-β1 e FGF2 plasmáticos apresentaram correlações positivas nos pacientes com MFP, e correlações negativas nos seus controles, assim como nos controles de MFPTE. Em todos os grupos estudados foi observada correlação positiva entre TGF-β1 e VEGFA plasmáticos. Além disso, foram demonstrados diferentes perfis de correlações entre a expressão gênica de TGF-β1 e os diversos SMADs e miRNAs em cada grupo de pacientes e controles. Os pacientes com MFP com maior angiogênese (de acordo com a mediana da concentração plasmática de VEGFA e FGF2) apresentaram maiores concentrações plasmáticas de TGF-β1 do que aqueles com menor angiogênese. A angiogênese medular estimada (CD34) não foi diferente entre os três grupos de pacientes estudados. Além disso, não foram encontradas correlações entre a imunoexpressão de CD34 e as expressões de RNAm de TGFB1, VEGFA e FGF2 medulares nem em leucócitos de sangue periférico, ou a concentrações plasmáticas de TGF-β1, VEGFA e FGF2. As imunoexpressões de TGF-b1 ativo, TGF-β1 latente e c-MPL foram semelhantes entre os três grupos de pacientes. As frequências das mutações avaliadas foram similares às descritas na literatura. Os pacientes com MFPTE portadores de mutação CALR apresentaram menores concentrações plasmáticas de VEGFA e FGF2 do que os JAK2V617F positivos, enquanto os pacientes com TE portadores de mutação CALR exibiram menores concentrações plasmáticas de TGF-β1 do que os portadores de JAK2V617F. CONCLUSÕES: O presente trabalho permitiu confirmar a correlação positiva entre o TGF-β1 com outros dois marcadores de angiogênese (VEGFA e FGF2). As expressões de SMADs e de miRNAs estudados foram semelhantes entre pacientes e controles, visto não haver diferenças na expressão gênica de TGF-β1. Entretanto, disparidades encontradas nas correlações entre a expressão gênica de TGF-β1 e diferentes SMADs e miRNAs nos pacientes e controles poderiam indicar que a regulação da expressão gênica de TGF-β1 nas doenças estudadas seja distinta da apresentada nos indivíduos sem essas doenças. / AIM: To investigate the effects of the expression of SMADs mRNA and microRNAs (miRNAs) that target TGFB1 in TGF-β1 gene expression (mRNA and protein) and its role in the angiogenesis pathophysiology in myelofibrosis (MF) and essential thrombocythemia (ET) patients. METHODS: Twenty-one primary MF (PMF), twenty-one MF post-ET (MPET) and twenty-four ET patients were included, besides 98 controls matched for gender and age with patients. In peripheral blood were assessed: TGF-β1, VEGFA and FGF2 plasmatic levels and mRNA quantification; SMADs 1 to 7 mRNA quantification and miRNAs 193a-5p, 369-5p, 542-5p, 590-3p, and 590-5p quantification; and detection of JAK2V617F (and allele burden), MPLW515K/L and CALR mutations. Estimated angiogenesis (microvessel grade - CD34), active TGF-b1, latent TGF-β and c-MPL immunoexpression were determined in 26 bone marrow biopsies. RESULTS: Plasmatic TGF-β1 levels were similar in patients and controls, while all the patients groups had higher plasmatic VEGFA than controls. Plasmatic FGF2 was higher in all the patients groups, and its mRNA expression was higher in ET patients than in controls. No differences in SMADs and miRNAs expression were found between patients and controls. There was a positive correlation between plasmatic TGF-β1 and FGF2 in PMF, and a negative correlation between these variables in their controls, as well as in MPET controls. In all studied groups, there was a positive correlation between plasmatic TGF-β1 and VEGF. In addition, different profiles of correlations were demonstrated between TGF-β1 gene expression and the several SMADs and miRNAs studied in each group of patients and controls. PMF patients with higher angiogenesis (according to the median of VEGFA and FGF2 plasma levels) had higher plasmatic TGF-β1 levels than those with lower angiogenesis. Estimated angiogenesis (CD34) in bone marrow biopsies were not different among PMF, MPET and ET patients. Moreover, there were no correlation between CD34 immunoexpression and TGFB1, VEGFA and FGF2 mRNA bone marrow or peripheral blood expression or plasmatic levels, as well as latent TGF-β1, active TGF-b1, and c-MPL immunoexpression were similar in patients studied groups. The frequencies of evaluated mutations were similar to previously reported. MPET patients harboring CALR mutations had lower plasmatic VEGFA and FGF2 than JAK2V617F mutated, while ET patients carrying CALR mutations had lower plasmatic TGF-β1 than JAK2V617F mutated. CONCLUSIONS: This study confirmed the positive correlation among TGF-β1 and two other markers of angiogenesis (VEGFA and FGF2). SMADs and miRNAs expressions were similar between patients and controls, since there were no differences in TGF-β1 gene expression between patients and controls. However, disparities found in the correlations between TGF-β1 gene expression and different SMADs and miRNAs in patients and controls may indicate that TGF-β1 gene expression regulation in studied diseases is distinct from those presented by individuals without these diseases.

OBJETIVO: Investigar o efeito da expressão de RNAm dos SMADs e de microRNAs (miRNAs) que possuem o TGFB1 como alvo na expressão gênica (RNAm e proteína) de TGF-β1 e seu papel na fisiopatologia da angiogênese em pacientes com mielofibrose (MF) e trombocitemia essencial (TE). MÉTODOS: Foram incluídos 21 pacientes com MF primária (MFP), 21 com MF pós-TE (MFPTE) e 24 com TE, além de 98 indivíduos controles pareados de acordo com gênero e idade com os pacientes. As análises realizadas no sangue periférico foram: quantificação das concentrações plasmáticas e de RNAm de TGFB1, VEGFA e FGF2; quantificação de RNAm de SMADs 1 a 7 e de miRNAs 193a-5p, 369-5p, 542-5p, 590-3p, e 590- 5p; e detecção das mutações JAK2V617F (com quantificação alélica), MPLW515K/L e CALR. Em 26 biópsias de medula óssea dos pacientes, foram determinados o grau de microvasculatura (angiogênese estimada - CD34), a imunoexpressão de TGF-b1 ativo, TGF-β1 latente e c-MPL. RESULTADOS: As concentrações de TGF- β1 plasmático foram semelhantes entre os pacientes e controles, enquanto o VEGFA plasmático foi maior em todos os grupos de pacientes comparados aos seus controles. O FGF2 plasmático também foi maior em todos os grupos de pacientes, e a expressão de seu RNAm foi maior nos pacientes com TE do que em seus controles. As expressões de SMADs e de miRNAs foram semelhantes entre pacientes e controles. TGF-β1 e FGF2 plasmáticos apresentaram correlações positivas nos pacientes com MFP, e correlações negativas nos seus controles, assim como nos controles de MFPTE. Em todos os grupos estudados foi observada correlação positiva entre TGF-β1 e VEGFA plasmáticos. Além disso, foram demonstrados diferentes perfis de correlações entre a expressão gênica de TGF-β1 e os diversos SMADs e miRNAs em cada grupo de pacientes e controles. Os pacientes com MFP com maior angiogênese (de acordo com a mediana da concentração plasmática de VEGFA e FGF2) apresentaram maiores concentrações plasmáticas de TGF-β1 do que aqueles com menor angiogênese. A angiogênese medular estimada (CD34) não foi diferente entre os três grupos de pacientes estudados. Além disso, não foram encontradas correlações entre a imunoexpressão de CD34 e as expressões de RNAm de TGFB1, VEGFA e FGF2 medulares nem em leucócitos de sangue periférico, ou a concentrações plasmáticas de TGF-β1, VEGFA e FGF2. As imunoexpressões de TGF-b1 ativo, TGF-β1 latente e c-MPL foram semelhantes entre os três grupos de pacientes. As frequências das mutações avaliadas foram similares às descritas na literatura. Os pacientes com MFPTE portadores de mutação CALR apresentaram menores concentrações plasmáticas de VEGFA e FGF2 do que os JAK2V617F positivos, enquanto os pacientes com TE portadores de mutação CALR exibiram menores concentrações plasmáticas de TGF-β1 do que os portadores de JAK2V617F. CONCLUSÕES: O presente trabalho permitiu confirmar a correlação positiva entre o TGF-β1 com outros dois marcadores de angiogênese (VEGFA e FGF2). As expressões de SMADs e de miRNAs estudados foram semelhantes entre pacientes e controles, visto não haver diferenças na expressão gênica de TGF-β1. Entretanto, disparidades encontradas nas correlações entre a expressão gênica de TGF-β1 e diferentes SMADs e miRNAs nos pacientes e controles poderiam indicar que a regulação da expressão gênica de TGF-β1 nas doenças estudadas seja distinta da apresentada nos indivíduos sem essas doenças. / AIM: To investigate the effects of the expression of SMADs mRNA and microRNAs (miRNAs) that target TGFB1 in TGF-β1 gene expression (mRNA and protein) and its role in the angiogenesis pathophysiology in myelofibrosis (MF) and essential thrombocythemia (ET) patients. METHODS: Twenty-one primary MF (PMF), twenty-one MF post-ET (MPET) and twenty-four ET patients were included, besides 98 controls matched for gender and age with patients. In peripheral blood were assessed: TGF-β1, VEGFA and FGF2 plasmatic levels and mRNA quantification; SMADs 1 to 7 mRNA quantification and miRNAs 193a-5p, 369-5p, 542-5p, 590-3p, and 590-5p quantification; and detection of JAK2V617F (and allele burden), MPLW515K/L and CALR mutations. Estimated angiogenesis (microvessel grade - CD34), active TGF-b1, latent TGF-β and c-MPL immunoexpression were determined in 26 bone marrow biopsies. RESULTS: Plasmatic TGF-β1 levels were similar in patients and controls, while all the patients groups had higher plasmatic VEGFA than controls. Plasmatic FGF2 was higher in all the patients groups, and its mRNA expression was higher in ET patients than in controls. No differences in SMADs and miRNAs expression were found between patients and controls. There was a positive correlation between plasmatic TGF-β1 and FGF2 in PMF, and a negative correlation between these variables in their controls, as well as in MPET controls. In all studied groups, there was a positive correlation between plasmatic TGF-β1 and VEGF. In addition, different profiles of correlations were demonstrated between TGF-β1 gene expression and the several SMADs and miRNAs studied in each group of patients and controls. PMF patients with higher angiogenesis (according to the median of VEGFA and FGF2 plasma levels) had higher plasmatic TGF-β1 levels than those with lower angiogenesis. Estimated angiogenesis (CD34) in bone marrow biopsies were not different among PMF, MPET and ET patients. Moreover, there were no correlation between CD34 immunoexpression and TGFB1, VEGFA and FGF2 mRNA bone marrow or peripheral blood expression or plasmatic levels, as well as latent TGF-β1, active TGF-b1, and c-MPL immunoexpression were similar in patients studied groups. The frequencies of evaluated mutations were similar to previously reported. MPET patients harboring CALR mutations had lower plasmatic VEGFA and FGF2 than JAK2V617F mutated, while ET patients carrying CALR mutations had lower plasmatic TGF-β1 than JAK2V617F mutated. CONCLUSIONS: This study confirmed the positive correlation among TGF-β1 and two other markers of angiogenesis (VEGFA and FGF2). SMADs and miRNAs expressions were similar between patients and controls, since there were no differences in TGF-β1 gene expression between patients and controls. However, disparities found in the correlations between TGF-β1 gene expression and different SMADs and miRNAs in patients and controls may indicate that TGF-β1 gene expression regulation in studied diseases is distinct from those presented by individuals without these diseases.

As Neoplasias Mieloproliferativas Crônicas (NMPC) Trombocitemia Essencial (TE), Mielofibrose (MF) - são desordens hematopoéticas resultantes da expansão clonal da célula tronco hematopoética alterada. Essas doenças caracterizam-se pela independência dos progenitores hematopoéticos aos estímulos dos fatores de crescimento e citocinas e pela proliferação exacerbada das células da linhagem mielóide com maturação preservada. Os mecanismos moleculares e celulares envolvidos na patogênese e progressão da TE e MF não foram ainda esclarecidos, mas o mieloacúmulo presente nessas doenças parece estar associado à alteração de proliferação e apoptose celular. Nesse contexto, os objetivos deste trabalho foram avaliar em células CD34+ da medula óssea e leucócitos dos pacientes com TE e MF: (1) a expressão de apoptomirs e de genes pró- e anti-apoptóticos pertencentes à via intrínseca e extrínseca da apoptose pela metodologia de RT-PCR em tempo real; (2) expressão de proteínas pró- e anti-apoptóticas por Western-blot; (3) o perfil de sensibilidade das células mononucleares à apoptose induzida por diferentes agentes apoptogênicos pela técnica de citometria de fluxo e (4) correlacionar os resultados obtidos com dados clínico-laboratoriais dos pacientes e com a expressão do gene PRV-1. Em TE, nas células CD34+, foi detectado aumento da expressão de a1, mcl-1, bid, bok e noxa e diminuição de bax em relação aos controles. Nos leucócitos, foi detectada a elevação da expressão de a1, bcl-2, bcl-w e bcl-xL, bad e bok e diminuição de bid e bimEL. Em comparação com o grupo controle, os pacientes com MF apresentaram maior expressão dos genes a1, bcl-w bak, bok e noxa e menor de bcl-2 nas células CD34+. Nos leucócitos dos pacientes com MF foi verificado aumento da expressão dos genes bcl-2, bcl-w e bcl-xL, bad e bok. O c-iap-1 apresentou maior expressão nas células CD34+ dos pacientes com MF e TE, enquanto que o c-iap-2 estava elevado nos leucócitos e células CD34+. Foi ainda detectada a expressão diferencial em TE e MF dos genes pertencentes a via de receptor de morte, como a maior expressão dos genes fas, fas-L, faim e dr4 nas células CD34+ dos pacientes e menor expressão do fas-L e trail nos leucócitos dos pacientes com TE e MF. Os resultados indicaram associação da expressão do gene PRV-1 com a mutação JAK2V617F e com a expressão dos genes a1, bcl-w, bik, bax, c-iap-2 e trail. Com relação à expressão proteica, BCL-XL estava aumentada e TRAIL diminuída em leucócitos de pacientes com MF enquanto que a molécula BID estava diminuída em leucócitos de pacientes com TE. Os miRNA26a, let 7d e miR15a estavam mais expressos nos leucócitos de pacientes com TE e MF. Em pacientes com TE o miR130b estava elevado e o mIR16 diminuído, enquanto que nos pacientes com MF o miR198 estava aumentado. Os linfócitos dos pacientes com TE e MF apresentaram maior resistência à apoptose estimulada por: actinomicina, etoposídeo, teniposídeo, citarabina e estaurosporina do que os linfócitos dos controles. Em conclusão, os dados obtidos sugerem a ligação da alteração do processo de apoptose celular, com a expressão do gene PRV-1 e status da mutação JAK2V617F. Esses resultados contribuem para o melhor entendimento da fisiopatologia das NMPC visto que associam a fisiopatologia da TE e MF com a resistência das células alteradas à apoptose. Essas informações serão úteis no futuro, possibilitando o desenho de novos alvos terapêuticos e a descrição de novos marcadores de prognóstico. / Chronic Myeloproliferative Neoplasms (cMPN) - Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF) and Polycythemia Vera (PV) - are clonal hematopoietic stem cell malignancies characterized by an accumulation of mature myeloid cells in bone marrow and peripheral blood. It seems that apoptotic machinery deregulation contribute to ET and PMF pathogenesis. Despite the advances in the molecular knowledge, the physiopathology of these diseases remains unknown. This study investigated cellular and molecular mechanisms involved in apoptosis process regulation in bone marrow haematopoietic progenitor CD34+ cells and leukocytes from ET and PMF patients. The specifics aims were: (1) to evaluate death receptors family members and Bcl-2 related genes expression as well apoptosis-related microRNAs by Real Time PCR; (2) apoptosis-related protein expression by Western Blot; (3) access mononuclear cells apoptosis resistance by flow cytometry and (4) to correlate the results with JAK2V617F mutation, PRV-1 gene expression and as well clinical data. In ET CD34+ cells, we found overexpression of a1, mcl-1, bid, bok e noxa and a decrease of bax compared to controls, while in leukocytes a1, bcl-2, bcl-w e bcl-xL, bad e bok expression was increased and bid and bimEL expression was lower than in controls. In PMF, a1, bcl-w bak, bok e noxa were overexpressed and bcl-2 downregulated in CD34+ cells. In PMF leukocytes bcl-2, bcl-w e bcl-xL, bad e bok mRNA levels were increased. c-iap-1 was increased in ET and PMF CD34+ cells and c-iap-2 expression elevated in ET and PMF CD34+ cells and leukocytes. Death receptor related genes showed overexpression of fas, fas-L, faim and dr4 in patients CD34+ cells and downregulation of fas-L and trail in ET and PMF leukocytes. We found differential expression of several genes between patients JAK2V617F positive and negative, as well we found correlation between gene expression and JAK2V617F allele burden, white blood cells and platelets count and splenomegaly. PRV-1 gene was overexpressed in ET and PMF leukocytes and showed correlation with JAK2V617F mutation and a1, bcl-w, bik, bax, c-iap-2 and trail gene expression. Regarding protein expression, BCL-XL was increased and TRAIL decreased in PMF leukocytes and BID was decreased in ET leukocytes. miRNA26a, let 7d e miR15a was overexpressed in ET and PMF leukocytes, while miR130b was increased only in ET and miR198 only in PMF. miR16 was downregulated in ET leukocytes comparing to controls. We also detected a resistance to apoptosis-inducers in ET and PMF lymphocytes and we observed correlation between apoptosis percentage and the expression of many studied genes. In conclusion, the results indicate the participations of Bcl-2 family genes and Death Receptor pathway genes, as well PRV-1 and JAK2V617F mutation in these disorders, which contribute to elucidate cMPN physiopathology and might lead to the discovery of new cMPN therapies and molecular markers.

As Neoplasias Mieloproliferativas Crônicas (NMPC) Trombocitemia Essencial (TE), Mielofibrose (MF) - são desordens hematopoéticas resultantes da expansão clonal da célula tronco hematopoética alterada. Essas doenças caracterizam-se pela independência dos progenitores hematopoéticos aos estímulos dos fatores de crescimento e citocinas e pela proliferação exacerbada das células da linhagem mielóide com maturação preservada. Os mecanismos moleculares e celulares envolvidos na patogênese e progressão da TE e MF não foram ainda esclarecidos, mas o mieloacúmulo presente nessas doenças parece estar associado à alteração de proliferação e apoptose celular. Nesse contexto, os objetivos deste trabalho foram avaliar em células CD34+ da medula óssea e leucócitos dos pacientes com TE e MF: (1) a expressão de apoptomirs e de genes pró- e anti-apoptóticos pertencentes à via intrínseca e extrínseca da apoptose pela metodologia de RT-PCR em tempo real; (2) expressão de proteínas pró- e anti-apoptóticas por Western-blot; (3) o perfil de sensibilidade das células mononucleares à apoptose induzida por diferentes agentes apoptogênicos pela técnica de citometria de fluxo e (4) correlacionar os resultados obtidos com dados clínico-laboratoriais dos pacientes e com a expressão do gene PRV-1. Em TE, nas células CD34+, foi detectado aumento da expressão de a1, mcl-1, bid, bok e noxa e diminuição de bax em relação aos controles. Nos leucócitos, foi detectada a elevação da expressão de a1, bcl-2, bcl-w e bcl-xL, bad e bok e diminuição de bid e bimEL. Em comparação com o grupo controle, os pacientes com MF apresentaram maior expressão dos genes a1, bcl-w bak, bok e noxa e menor de bcl-2 nas células CD34+. Nos leucócitos dos pacientes com MF foi verificado aumento da expressão dos genes bcl-2, bcl-w e bcl-xL, bad e bok. O c-iap-1 apresentou maior expressão nas células CD34+ dos pacientes com MF e TE, enquanto que o c-iap-2 estava elevado nos leucócitos e células CD34+. Foi ainda detectada a expressão diferencial em TE e MF dos genes pertencentes a via de receptor de morte, como a maior expressão dos genes fas, fas-L, faim e dr4 nas células CD34+ dos pacientes e menor expressão do fas-L e trail nos leucócitos dos pacientes com TE e MF. Os resultados indicaram associação da expressão do gene PRV-1 com a mutação JAK2V617F e com a expressão dos genes a1, bcl-w, bik, bax, c-iap-2 e trail. Com relação à expressão proteica, BCL-XL estava aumentada e TRAIL diminuída em leucócitos de pacientes com MF enquanto que a molécula BID estava diminuída em leucócitos de pacientes com TE. Os miRNA26a, let 7d e miR15a estavam mais expressos nos leucócitos de pacientes com TE e MF. Em pacientes com TE o miR130b estava elevado e o mIR16 diminuído, enquanto que nos pacientes com MF o miR198 estava aumentado. Os linfócitos dos pacientes com TE e MF apresentaram maior resistência à apoptose estimulada por: actinomicina, etoposídeo, teniposídeo, citarabina e estaurosporina do que os linfócitos dos controles. Em conclusão, os dados obtidos sugerem a ligação da alteração do processo de apoptose celular, com a expressão do gene PRV-1 e status da mutação JAK2V617F. Esses resultados contribuem para o melhor entendimento da fisiopatologia das NMPC visto que associam a fisiopatologia da TE e MF com a resistência das células alteradas à apoptose. Essas informações serão úteis no futuro, possibilitando o desenho de novos alvos terapêuticos e a descrição de novos marcadores de prognóstico. / Chronic Myeloproliferative Neoplasms (cMPN) - Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF) and Polycythemia Vera (PV) - are clonal hematopoietic stem cell malignancies characterized by an accumulation of mature myeloid cells in bone marrow and peripheral blood. It seems that apoptotic machinery deregulation contribute to ET and PMF pathogenesis. Despite the advances in the molecular knowledge, the physiopathology of these diseases remains unknown. This study investigated cellular and molecular mechanisms involved in apoptosis process regulation in bone marrow haematopoietic progenitor CD34+ cells and leukocytes from ET and PMF patients. The specifics aims were: (1) to evaluate death receptors family members and Bcl-2 related genes expression as well apoptosis-related microRNAs by Real Time PCR; (2) apoptosis-related protein expression by Western Blot; (3) access mononuclear cells apoptosis resistance by flow cytometry and (4) to correlate the results with JAK2V617F mutation, PRV-1 gene expression and as well clinical data. In ET CD34+ cells, we found overexpression of a1, mcl-1, bid, bok e noxa and a decrease of bax compared to controls, while in leukocytes a1, bcl-2, bcl-w e bcl-xL, bad e bok expression was increased and bid and bimEL expression was lower than in controls. In PMF, a1, bcl-w bak, bok e noxa were overexpressed and bcl-2 downregulated in CD34+ cells. In PMF leukocytes bcl-2, bcl-w e bcl-xL, bad e bok mRNA levels were increased. c-iap-1 was increased in ET and PMF CD34+ cells and c-iap-2 expression elevated in ET and PMF CD34+ cells and leukocytes. Death receptor related genes showed overexpression of fas, fas-L, faim and dr4 in patients CD34+ cells and downregulation of fas-L and trail in ET and PMF leukocytes. We found differential expression of several genes between patients JAK2V617F positive and negative, as well we found correlation between gene expression and JAK2V617F allele burden, white blood cells and platelets count and splenomegaly. PRV-1 gene was overexpressed in ET and PMF leukocytes and showed correlation with JAK2V617F mutation and a1, bcl-w, bik, bax, c-iap-2 and trail gene expression. Regarding protein expression, BCL-XL was increased and TRAIL decreased in PMF leukocytes and BID was decreased in ET leukocytes. miRNA26a, let 7d e miR15a was overexpressed in ET and PMF leukocytes, while miR130b was increased only in ET and miR198 only in PMF. miR16 was downregulated in ET leukocytes comparing to controls. We also detected a resistance to apoptosis-inducers in ET and PMF lymphocytes and we observed correlation between apoptosis percentage and the expression of many studied genes. In conclusion, the results indicate the participations of Bcl-2 family genes and Death Receptor pathway genes, as well PRV-1 and JAK2V617F mutation in these disorders, which contribute to elucidate cMPN physiopathology and might lead to the discovery of new cMPN therapies and molecular markers.

Les néoplasies myéloprolifératives (NMP) sont des hémopathies myéloïdes clonales, chroniques et prolifératives. Les plus fréquentes sont la polyglobulie de Vaquez et la thrombocytémie essentielle. Elles s’accompagnent de risques importants de thrombose (artérielles et veineuses) et de transformation en pathologies plus agressives (myélofibrose secondaire et leucémie aigüe). Les thromboses peuvent être la situation diagnostique de ces maladies, ou survenir au cours de la prise en charge. Le sujet de cette thèse est d’étudier la relation clinique entre NMP et thrombose. Dans un contexte de survenue de thrombose veineuse idiopathique, sans antécédent NMP, nous nous sommes intéressés à la recherche de mutation clonale chez les patients comme moyen diagnostique d’une NMP. Nous avons ainsi exploité la cohorte EDITH du CIC en prenant les patients ayant expérimentés un puis une récurrence thrombotique. A l’inverse, nous avons constitué une base de données (OBENE) des patients pris en charge pour une NMP au CHRU de Brest.Nous avons ensuite exploité cette base, en analysant la fréquence et l’impact des arythmies cardiaques auriculaires, la balance bénéfice-risque à l’utilisation des NACO, l’impact des statines sur la réduction du risque de thrombose ainsi que la fréquence et l’impact de la nonadhérence aux traitements dans les PV et TE.NMP et thromboses sont liées, il est donc nécessaire d’approfondir les connaissances de leur physiopathologie spécifique pour améliorer la prévention et le traitement des épisodes. Cette thèse amène quelques réponses àcertaines questions mais elle est surtout le point de départ de réflexion commune entre les praticiens et biologistes intéressés par ces domaines. / The myeloproliferative neoplasms (MPN) are clonal myeloid, chronic and proliferative disorders. The most frequent are polycythemia vera and essential thrombocythemia. The more frequent complications are thromboses (arterial and venous) and phenotypic evolutions (secondary myelofibrosis and acute leukemia). Thromboses can be a situation of diagnosis or observed during the followup of a MPN. This thesis is focused on the clinical link between MPN and thromboses.In a context of idiopathic venous thromboses (first event or recurrence), without medical history of MPN, we have tested patients for the most frequent MPN clonal mutations. So, we have used the informations and patients of the dedicated EDITH cohort.On the other hand, we have constituted a MPN database (OBENE) of the patients diagnosed for MPN in our Hospitalcentre. By this way, we have analysed the frequency and impact of atrial arrhythmias, the benefit-risk balance of the use of DOAC, the impact of statins to reduce the thrombotic risk and the frequency and impact of the treatment nonadherence in this population.MPN and thromboses are linked, so it is necessary to increase our knowledge of their physiopathology to improve prevention and treatment of the events. This thesis brings some answers to some questions but, she is almost the starting point of common reflexion between clinicians and biologists interested in these domains.

As Neoplasias mieloproliferativas (NMP) representam um vasto grupo de doenças clonais hematológicas malignas com três elementos principais: Policitemia vera (PV), Trombocitemia essencial (TE), e Mielofibrose Primária (MFP). JAK2 é uma proteína citoplasmática com atividade de tirosina quinase com função na transdução de várias vias na hematopoiese. A identificação da mutação do gene JAK2 (JAK2V617F) nas PV, TE e MFP representa um importante avanço para a compreensão da biologia destas NMPs. Variações marcantes na frequência desta mutação são observadas entre os diferentes estudos e acredita-se que um dos fatores responsáveis por estas diferenças seja a sensibilidade do método utilizado. Atualmente, diversas técnicas para detecção de JAK2V617F têm sido utilizadas, testadas e validadas quanto à sua sensibilidade e especificidade, entre elas: PCR RFLP (Restriction Fragment Lenght Polymorphysm), ARMS PCR (Amplification-Refractory Mutation System), HRM (High-Resolution Melt Analysis) e Sequenciamento pela técnica de Sanger. Neste estudo foram realizadas todas as metodologias citadas anteriormente para a detecção da mutação de JAK2V617F em amostras de sangue de 136 pacientes (PV=20; MFP=20; TE=28; suspeita de NMP=68). Os resultados obtidos foram concordantes para as quatro técnicas empregadas nos pacientes com PV e MFP, já nos pacientes com TE as metodologias PCR-ARMS e PCR-HRM detectaram a mutação JAK2V617F em 67,8% enquanto o PCR-RFLP e o Sequenciamento pela técnica de Sanger foi 71,4% e 64,2% respectivamente. Nos casos onde houve suspeita diagnóstica de NMP também foram encontradas discordâncias entre as metodologias PCR-RFLP (4,4%) e PCR-HRM (1,5%) quando comparadas ao PCR-ARMS (3%) e o Sequenciamento (3%). O PCR-ARMS foi considerado nesse estudo como a melhor técnica para a detecção da mutação JAK2V617F, devido o menor risco de contaminação cruzada durante a reação, baixo tempo de execução, além da sua capacidade de determinação da carga alélica de JAK2, importante para o acompanhamento do paciente / Myeloproliferative neoplasms (MPN) represent a large group of clonal hematologic malignant diseases with three main members: Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Mielofibroses (PMF). JAK2 is a cytoplasmic tyrosine kinase protein and is important in different signal transduction pathways. Identification of JAK2V617F mutation in PV, ET and PMF is an important advance for understanding the biology of MPN. Differences in the frequency of this mutation are reported among different studies and it is believed that technical sensitivity could be the major reason for this variability. Currently, several techniques for detection of JAK2V617F have been developed, tested and validated for their sensitivity and specificity, including: PCR-RFLP (Restriction Fragment Lenght Polymorphysm), PCR-ARMS (Amplification Refractory Mutation System), PCR-HRM (High-Resolution Melt analysis) and Sanger Direct Sequencing. The present study, evaluated all four molecular diagnostic methods mentioned above blood samples from 136 patients (PV=20; MFP=20; ET=28 and other MPN=68). Comparable results were observed for PV and PMF when all technics were applied. Patients with diagnosis of ET JAK2V617F mutations were detected in 67.8% when PCR-ARMS and PCR-HRM were used whilst PCR-RFLP and direct sequencing detected 71.4% and 64.2% respectively. In 68 patients with suspicion of MPN discordant results were seen between PCR-RFLP (4.4%) and PCR-HRM (1.5%) when compared to PCR-ARMS (3%) and direct sequencing (3%) related to JAK2V617F frequency. In conclusion PCR-ARMS was considered the most reliable methodology for JAK2V617F detection by presenting the lowest risk for cross contamination, less laborious, and the ability in determining allele burden that is becoming an important tool for risk stratification

As Neoplasias mieloproliferativas (NMP) representam um vasto grupo de doenças clonais hematológicas malignas com três elementos principais: Policitemia vera (PV), Trombocitemia essencial (TE), e Mielofibrose Primária (MFP). JAK2 é uma proteína citoplasmática com atividade de tirosina quinase com função na transdução de várias vias na hematopoiese. A identificação da mutação do gene JAK2 (JAK2V617F) nas PV, TE e MFP representa um importante avanço para a compreensão da biologia destas NMPs. Variações marcantes na frequência desta mutação são observadas entre os diferentes estudos e acredita-se que um dos fatores responsáveis por estas diferenças seja a sensibilidade do método utilizado. Atualmente, diversas técnicas para detecção de JAK2V617F têm sido utilizadas, testadas e validadas quanto à sua sensibilidade e especificidade, entre elas: PCR RFLP (Restriction Fragment Lenght Polymorphysm), ARMS PCR (Amplification-Refractory Mutation System), HRM (High-Resolution Melt Analysis) e Sequenciamento pela técnica de Sanger. Neste estudo foram realizadas todas as metodologias citadas anteriormente para a detecção da mutação de JAK2V617F em amostras de sangue de 136 pacientes (PV=20; MFP=20; TE=28; suspeita de NMP=68). Os resultados obtidos foram concordantes para as quatro técnicas empregadas nos pacientes com PV e MFP, já nos pacientes com TE as metodologias PCR-ARMS e PCR-HRM detectaram a mutação JAK2V617F em 67,8% enquanto o PCR-RFLP e o Sequenciamento pela técnica de Sanger foi 71,4% e 64,2% respectivamente. Nos casos onde houve suspeita diagnóstica de NMP também foram encontradas discordâncias entre as metodologias PCR-RFLP (4,4%) e PCR-HRM (1,5%) quando comparadas ao PCR-ARMS (3%) e o Sequenciamento (3%). O PCR-ARMS foi considerado nesse estudo como a melhor técnica para a detecção da mutação JAK2V617F, devido o menor risco de contaminação cruzada durante a reação, baixo tempo de execução, além da sua capacidade de determinação da carga alélica de JAK2, importante para o acompanhamento do paciente / Myeloproliferative neoplasms (MPN) represent a large group of clonal hematologic malignant diseases with three main members: Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Mielofibroses (PMF). JAK2 is a cytoplasmic tyrosine kinase protein and is important in different signal transduction pathways. Identification of JAK2V617F mutation in PV, ET and PMF is an important advance for understanding the biology of MPN. Differences in the frequency of this mutation are reported among different studies and it is believed that technical sensitivity could be the major reason for this variability. Currently, several techniques for detection of JAK2V617F have been developed, tested and validated for their sensitivity and specificity, including: PCR-RFLP (Restriction Fragment Lenght Polymorphysm), PCR-ARMS (Amplification Refractory Mutation System), PCR-HRM (High-Resolution Melt analysis) and Sanger Direct Sequencing. The present study, evaluated all four molecular diagnostic methods mentioned above blood samples from 136 patients (PV=20; MFP=20; ET=28 and other MPN=68). Comparable results were observed for PV and PMF when all technics were applied. Patients with diagnosis of ET JAK2V617F mutations were detected in 67.8% when PCR-ARMS and PCR-HRM were used whilst PCR-RFLP and direct sequencing detected 71.4% and 64.2% respectively. In 68 patients with suspicion of MPN discordant results were seen between PCR-RFLP (4.4%) and PCR-HRM (1.5%) when compared to PCR-ARMS (3%) and direct sequencing (3%) related to JAK2V617F frequency. In conclusion PCR-ARMS was considered the most reliable methodology for JAK2V617F detection by presenting the lowest risk for cross contamination, less laborious, and the ability in determining allele burden that is becoming an important tool for risk stratification

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